Research ArticleA Simple Method for Establishing Adherent Ex Vivo ExplantCultures from Human Eye Pathologies for Use in SubsequentCalcium Imaging and Inflammatory Studies
Andrea Facskoacute2 Marko Hawlina1 and Goran Petrovski23
1 Eye Hospital University Medical Centre 1000 Ljubljana Slovenia2Department of Ophthalmology Faculty of Medicine University of Szeged Koranyi fasor 10-11 Szeged 6720 Hungary3 Stem Cells and Eye Research Laboratory Department of Biochemistry and Molecular Biology Apoptosis andGenomics Research Group of the Hungarian Academy of Sciences University of Debrecen Szeged 6720 Hungary
Correspondence should be addressed to Goran Petrovski petrovskigoranmedu-szegedhu
Received 24 April 2014 Revised 26 June 2014 Accepted 10 July 2014 Published 4 September 2014
Academic Editor Kai Kaarniranta
Copyright copy 2014 Sofija Andjelic et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
A novel simple and reproducible method for cultivating pathological tissues obtained from human eyes during surgery wasdevelopedusing viscoelasticmaterial as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of culturedcells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies Anterior lens capsule-lensepithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes(fvERMs) fromhuman eyeswere used in the studyWehereby show calcium signaling in aLC-LECs bymechanical and acetylcholine(ACh) stimulation and indicate presence of ACh receptors in these cells Furthermore an ex vivo study model was established formeasuring the inflammatory response in fvERMs and aLC-LECs upon TNF120572 treatment
1 Introduction
Human eye diseasemodeling requires well established ex vivocell cultures Such cultures allow studying diseases of interestat a cellular level using multiple techniques In addition theyprovide possibility to grow primary human eye cells with thepurpose of repairing a defect and eventually transplantingthem back to the patient in an autologous or heterologousmanner
An important condition for growing ex vivo eye explantcultures is to have an adherent environment We developed asimple method for attaching eye tissue explants to the surfaceof a Petri dish by using surgical grade viscoelastic materialotherwise routinely used in ophthalmic surgery [1]
Human anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and fibrovascular epiretinalmembranes (fvERM) from proliferative diabetic retinopathy(PDR) were cultured adherently under viscoelastic materialThe single-layered LECs underlying the aLC aremetabolically
the most active part of the lens and are responsible forsustaining physiological health of the tissue ERMs are acollection of cells and extracellular matrix that occur inthe inner vitreal surface of the central retina They havecontractile properties and can lead to visual disturbanceand metamorphopsia (distorted vision) due to their effecton the underlying retina FvERMs represent the final anddevastating stage of PDR and form due to heavy hypoxiaretinal ischemia and unbalanced glucose metabolism theresult of which is a state of chronic inflammation [2 3]
Cells growing out of cultured aLCs and fvERM explantswere studied functionally by examining intracellular calcium[Ca2+]i signaling under adherent culture conditions Calciumsignaling plays an important role in the regulation of cellfunction affecting every aspect of the cellsrsquo life and death [4]We hereby show free [Ca2+]i changes upon mechanical andacetylcholine (ACh) stimulation in cultured cells obtainedfrom human aLCs under adherent conditions and indicate
Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 232659 10 pageshttpdxdoiorg1011552014232659
2 Journal of Immunology Research
presence of ACh receptors in these cells In addition theinflammatory nature of fvERMs and aLC-LECs as well astheir relation to tumor necrosis factor alpha (TNF120572) andangiogenesis is addressed here
2 Methods
21 Tissue Collection and Processing All tissue collectioncomplied with the guidelines of the Helsinki Declaration andwas approved by the National Medical Ethics Committeeof Slovenia all patients signed an informed consent formbefore surgery which was performed at the Eye HospitalUniversity Medical Centre (UMC) Ljubljana Slovenia Alto-gether 11 patientswere included in this studymdash6 cultureswereanalyzed for mechanical stimulation and 5 cultures for AChstimulationwith the patientsrsquo age ranging from70 to 92 years
The aLC explants consisted of a monolayer of LECsattached to the basal lamina andwere obtained fromunevent-ful cataract surgeries due to progredient cataract Lenses weredissected so that the aLCs (ie basal lamina and associatedLECs) were isolated from the fiber cells that form the bulkof the lens FvERMs were obtained from patients undergoingvitrectomy due to intravitreal hemorrhage in PDR
All explants were obtained from single patients and wereusually placed in a single dish accordingly Immediately afterisolation the excised human eye explants were placed insterile tubes filled with DMEMF12 (D8437 Sigma-AldrichAyrshire UK) supplemented with 10 fetal calf serum (FCS)(PAA Laboratories GmbH Pasching Austria) and trans-ported from the operating room to the research departmentin the same building The explants were then transferred toempty cell culture glass bottom Petri dishes (Mattek CorpAshland MA USA 35 cm in diameter) or tissue culture 12-well plates (TPP Sigma Germany) by using microdissectingtweezers (WPI by Dumont MedBiologie Germany) TheaLC explants were placed into the culture dish so that theconcave side with the LECs was on the top and orientedupwards The time of culturing ranged from 6 to 48 days
22 Tissue FixationAdherence by Viscoelastic Material Forobtaining adherent conditions careful removal of theremaining medium from the tissue cultures was performedby a micropipette and then viscoelastic (HEALON OVDAbbottMedical Optics USA)was added on top of the explantto allow for flattening or ldquoironingrdquo of the tissue onto thesurface of the Petri dish (Figure 1)
For ex vivo cultivation under adherent conditionsDMEMF12 supplemented with 10 FCS was then addedslowly with the micropipette not to disturb or remove theviscoelastic cover on top of the explants The micropipettetip was positioned close to the culture dish surface but faraway from the explant so that the medium arrived softly incontact with the viscoelastic and did not move the explantfrom its location The culture dishes were then kept in a CO
2
incubator (Innova CO-48 New Brunswick Scientific EdisonNJ USA) at 37∘C and 5 CO
2
The culture dish was kept in the incubator withoutmoving for 2-3 days in order to allow the cells to attach and
Explant tissue+ viscoelastic
Viscoelastic
Explanttissue
Petri dish
Cell outgrowth
Growth
Figure 1 Method for adherent ex vivo cultivation of human eyetissue explants in a cell culture Petri dish
start proliferating out of the explant Duringmedium changethe medium was removed gently and a fresh one was addedsubsequently by a micropipette from the opposite side of theexplant in the dish the pipette tip being close to the surfaceof the dish all the time The viscoelastic dissolved over timeand got replaced by newmediummdashtime by which the explantwas fully attached to the surface of the culture dish
23 Light Microscopy and Calcium Imaging The prolifera-tion and migration of the cells were recorded throughouttheir continued growth using inverted light microscope(Axiovert S100 Carl Zeiss AG Oberkochen Germany)The same microscope was used for [Ca2+]i measurementsImage acquisition was carried out by a 12-bit cooled CCDcamera SensiCam (PCO Imaging AG Kelheim Germany)The software used for the acquisition was WinFluor (writtenby J Dempster University of Strathclyde Glasgow UK)Microscope objectives used were 4x010 Achroplan 10x030Plan-Neofluar 40x050 LD A-plan and 63x125 oil Plan-Neofluar (Zeiss)
The excitation filters usedweremounted on a Lambda LS-10 filter wheel (Sutter Instruments Co CA USA) and had awavelength of 360 and 380 nm (Chroma Technology CorpBellows Falls VT USA) Excitation with the 360 nm filter(close to the Fura-2 isosbestic point) allowed observation ofthe cellsrsquo morphology and of the changes in the concentrationof the dye irrespective of the changes in [Ca2+]i while the360380 nm ratio allowed visualization of the [Ca2+]i changesin the cytoplasm Image acquisition timing and filter wheeloperation were all controlled by WinFluor software via aPCI6229 interface card (National Instruments Austin TXUSA) The light source used was XBO-75W (Zeiss) Xe arclamp The light intensity was attenuated when necessarywith grey filters with optical densities 05 1 and 2 (ChromaTechnology Corp Bellows Falls VT USA) The criteria forselecting the region for imagingwere the presence of adherentcells and good cell morphology both assessed by observationof transilluminated and 360 nm fluorescence images Indi-vidual image frames were acquired every 500ms resulting inframe cycles being 1 second long (two wavelengths)
For [Ca2+]i monitoring the cell cultures were loadedwith the acetoxymethyl (AM) ester of Fura-2 (Fura-2 AM
Journal of Immunology Research 3
500120583m
(a)
LC
1000 120583m
(b)
Figure 2 Examples of attached human eye explants with the growing cells (a) anterior lens capsule (aLC) in a 12-well plate (b) fibrovascularepiretinal membrane (fvERM) cells growing attached to and independently from the tissue explant in a cell culture Petri dish
Invitrogen-Molecular Probes Carlsbad CA USA) intracel-lular calcium indicator For loading Fura-2 AM in dimethylsulfoxide (DMSO) was suspended in 3mL of medium (highglucose medium with FBS) or physiological saline with (inmM) NaCl (1318) KCl (5) MgCl2 (2) NaH2PO4 (05)NaHCO3 (2) CaCl2 (18) HEPES (10) glucose (10) pH724 to the final working concentration of 2120583M (aLC) Theloading was done in the incubator at 37∘C for 30min (aLC)After loading the cell cultures were washed twice for 7minwith the medium or physiological saline The final workingconcentration of Fura-2 and the time of incubationwashingwere larger for larger eye explants (it depended on the explantsize)
Fura-2 dye has two excitation (absorption) peaks (340and 380 nm) an isosbestic point at 360 nm and one emissionpeak at 510 nm Its absorption and fluorescent propertieschange in accordance with Ca2+ binding (low [Ca2+]imdashhigh absorption at 380 nm high [Ca2+]imdashhigh absorptionat 340 nm while the absorption is not Ca2+ dependent atthe isosbestic point of 360 nm) The absorptive properties ofFura-2 allow the use of ratio imaging (360380 ratio) whichconsiderably reduces the effects of uneven dye loading leak-age of the dye and photobleaching as well as problems asso-ciated with measuring [Ca2+]i in cells of unequal thickness
24 Mechanical and Acetylcholine (ACh) Stimulation To testresponses to mechanical stimuli a tip of a glass micropipettemounted on a MP-285 micromanipulator (Sutter NovatoCA USA) was used Prior to use the tip of the pipette washeat-polished until it rounded up
The agonist acetylcholine (ACh Sigma USA)was appliedin 10 120583M concentration which was enough to induce gt90maximal [Ca2+]i response according to the data by Collisonet al [12] The agonist application as well as its washout fromthe bath was driven simply by the hydrostatic pressure of a35 cm of water column and controlled manually by a luer-lock stopcock (WPI) and applied through a polyethyleneplastic tubing (inner diameter 2mm) attached to the coarsemicromanipulator The excess bathing solution was removedby a suction line
25 Secretion of Inflammatory Cytokines by ELISA Theexpanded fvERM cells were plated onto 6-well plates ata density of 2 times 105 cells per well in triplicates Similarplating was carried out in case of the aLC-LECs until propercell number was achieved for cytokine measurements After24 hrs the medium was changed and the cells were treatedwith 100 ngmL recombinant human TNF120572 (PreprotechRocky Hill NJ USA) for additional 24 hours The secretedcytokines IL-6 and IL-8 were analyzed by commercialELISA kit (RampD Germany) according to the manufacturerrsquosprotocolThree independent experiments were performed onthree different outgrowing cells from both fvERM and aLC
3 Results
31 HumanEye Explant Tissues Adhere to the Cell CultureDishunder a Gravitational Force of Viscoelastic Material Novelsimple and reproducible method for ex vivo cultivation ofhuman explant tissues (aLCs and fvERMs) was establishedusing viscoelastic material (Figure 1)
The cells started proliferating out of the explants in 2-3days (Figure 2)Themethod for attachment of human eye tis-sue explants to the 12-well plates is shown in Figure 2(a)mdashtheaLC explant and the cells are flattened under the gravitationalforce of the viscoelastic material The fvERM cells grew outof the explants within 24 hours and continued proliferatingindependently throughout the study period (for more than 6months) (Figure 2(b))
32 Mechanical Stimulation and ACh Induce Rise in [1198621198862+]119894in
the aLC-LECs The functionality of the aLC-LECs attachedunder the viscoelastic was examined during mechanicalstimulation and application of agonist ACh both of whichinduced rise in the [Ca2+]i Representative examples of 6explant cultures were analyzed for mechanical stimulationcontaining 27 cells being stimulated (mostly the cells on theglass surface and some on the aLC) similarly representativeexamples of 5 explant cultures were analyzed for ACh stim-ulation Figure 3 shows the calcium signaling upon agonistACh stimulation of the aLC explant-cultured cells The oscil-lations of [Ca2+]i are clearly visible here with each cell having
4 Journal of Immunology Research
10120583m
(a)
40 60 80 100 120500
540
580
620
660
360
380
(s)(b)
Figure 3 Calcium signaling upon agonist ACh stimulation of the aLC explant-cultured cells (a) The traces (b) represent the time coursesof the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest (ROI) shown on the BampW image (a) in the samecolors (explant growth time 28 days)
its own frequency of oscillation (Figure 3(b) upper part) 50cells were analyzed here out of which 15 (30) had oscillatingresponse with average of 166 plusmn 44 sec from minimum tominimum Accommodation can be observed for the greentrace as the interval between the two maxima decreases withtime while a time delay of 2-3 sec in the [Ca2+]i propagationcan be seen (Figure 3(b) lower part) needed for the [Ca2+]ito reach its first maximum for different ROIs of the same cell(blue and red) No mechanically stimulated cell respondedat oscillation manner The transient responses to mechanicalstimulation were usually comparable to those elicited byACh
The calcium signaling upon mechanical stimulation of asingle cell of the aLC explant culture showed [Ca2+]i propaga-tion as well (Figure 4)mdashin the example shown 26 cells hadresponse with two peaks the first one being bigger than theother and the time interval between the peak maxima being25 and 26 sec the rest of the cells had no or very small calciumincreaseThe blue ROI represents the stimulation site and thered ROI represents the more distal site There is a delay ofaround 5 sec in the time needed for the [Ca2+]i to reach itsmaximum at two selected ROIs The increases in [Ca2+]i inthe cells surrounding themechanically stimulated cell suggestthe involvement of intercellular connections
The intercellular dendrite connection strength uponmechanical fluid movement for the nonattached dendritesin aLC explant culture could also be observed (Figure 5)Indeed a confirmation that the [Ca2+]i changes are notdependent on themechanical effect of fluidmovement but onACh is shown by the fact that [Ca2+]i increase occurs muchlater (119905 = 101 s) in comparison to the dendritic movementdependent on the mechanical effect of fluid movement (119905 =39ndash47 s) as visible on Figure 5(c)
The [Ca2+]i dynamics upon mechanical stimulation offvERMs has been previously described by our group [5]which is a proof of the viability and functionality of these cells
33 Measurement of ProinflammatoryAngiogenic FactorsSecreted by the fvERM Outgrowing Cells upon TNF120572 Treat-ment The outgrowing cells from the fvERMs showed basalexpression of the proinflammatory cytokine interleukin-(IL-) 6 ex vivo which was further enhanced by TNF120572stimulation Similar enhancement was noted in the proin-flammatory cytokine release of IL-8 upon TNF120572 stimulation(Figure 6(a)) In the case of aLC-LECs there were no basalIL-6 and IL-8 responses and TNF120572-induced IL-8 secretion(Figure 6(b))
4 Discussion
Anovel simple and reproduciblemethod for creating adher-ent conditions for human eye explants and ex vivo cellularexpansion using viscoelastic material as well as studies oncalcium dynamics and inflammation is established here Theoutgrowing cells over time migrate out of the explants andgrow adherently onto the surface of the cell culture dishshowing signs of continuous proliferation
Alternative adherence methods for tissues explants canbe the use of dry surface concentrated serum drop orthe fibrin-glue methodmdashthe latter being used mostly forin vivo purposes The advantage of the viscoelastic methodis in avoiding extreme conditions such as dryness andserum stimulants yet preserving natural architecture of thetissue and standard nutritional conditions for the cells Theviscoelastic is an inert substance having viscous elasticand gravitational properties which force the graft to attachto a surface The viscoelastic HEALON OVD is used inophthalmic surgical procedures to maintain deep anteriorchamber which facilitates manipulation inside the eye withreduced trauma to the corneal endothelium and other oculartissues
Two tissue types are used here to establish adherent exvivo explant cultures aLCs containing LECs and fvERMsTissue and cell adherence allow measurement of the [Ca2+]i
Journal of Immunology Research 5
10120583m
(a)
40 60 80 100 120380
400
420
440
360
380
(s)(b)
10120583m 10120583m 10120583m
10120583m 10120583m 10120583m
t = 50 s t = 56 s t = 58 s
t = 61 s t = 67 s t = 74 s
(c)
Figure 4 Calcium signaling upon mechanical stimulation of a single cell of the aLC explant culture showing the [Ca2+]i propagation andinvolvement of intercellular connections The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i andcorrespond to the regions of interest (ROI) shown in the BampW image (a) in the same colors (c) A series of the 360380 ratio images at thetime points are indicated The values for 119877 are color coded with bluegreen representing low ratio values and yellowred representing highratios (explant growth time 14 days)
upon mechanical or pharmacological stimulation givingadvantage of having less noise from cellularmovementwithinthe cell culture dish
Precise regulation of the [Ca2+]i levels is critical formain-taining normal cellular function fluctuations ofwhich can actas signals for numerous physiological or pathological eventsImbalance in the [Ca2+]i levels may lead to development ofcataract in the lens [6ndash9] Our results indicate an increase inthe [Ca2+]i upon mechanical stimulation and application of
ACh to aLC-LECs Previously mechanical stimulation hadbeen used to induce [Ca2+]i rise in cultured bovine LECs [10]Such stimulation of a single cell within a confluent layer wasshown to initiate cell-to-cell calcium signaling Contractionsin human aLECs attached to the surgically isolated capsulescould also be mechanically induced [11]
The increase in [Ca2+]i suggests involvement of inter-cellular connections between the LECs studied ex vivo Inhuman aLECs ACh binds to M1 muscarinic receptors (M1
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
2 Journal of Immunology Research
presence of ACh receptors in these cells In addition theinflammatory nature of fvERMs and aLC-LECs as well astheir relation to tumor necrosis factor alpha (TNF120572) andangiogenesis is addressed here
2 Methods
21 Tissue Collection and Processing All tissue collectioncomplied with the guidelines of the Helsinki Declaration andwas approved by the National Medical Ethics Committeeof Slovenia all patients signed an informed consent formbefore surgery which was performed at the Eye HospitalUniversity Medical Centre (UMC) Ljubljana Slovenia Alto-gether 11 patientswere included in this studymdash6 cultureswereanalyzed for mechanical stimulation and 5 cultures for AChstimulationwith the patientsrsquo age ranging from70 to 92 years
The aLC explants consisted of a monolayer of LECsattached to the basal lamina andwere obtained fromunevent-ful cataract surgeries due to progredient cataract Lenses weredissected so that the aLCs (ie basal lamina and associatedLECs) were isolated from the fiber cells that form the bulkof the lens FvERMs were obtained from patients undergoingvitrectomy due to intravitreal hemorrhage in PDR
All explants were obtained from single patients and wereusually placed in a single dish accordingly Immediately afterisolation the excised human eye explants were placed insterile tubes filled with DMEMF12 (D8437 Sigma-AldrichAyrshire UK) supplemented with 10 fetal calf serum (FCS)(PAA Laboratories GmbH Pasching Austria) and trans-ported from the operating room to the research departmentin the same building The explants were then transferred toempty cell culture glass bottom Petri dishes (Mattek CorpAshland MA USA 35 cm in diameter) or tissue culture 12-well plates (TPP Sigma Germany) by using microdissectingtweezers (WPI by Dumont MedBiologie Germany) TheaLC explants were placed into the culture dish so that theconcave side with the LECs was on the top and orientedupwards The time of culturing ranged from 6 to 48 days
22 Tissue FixationAdherence by Viscoelastic Material Forobtaining adherent conditions careful removal of theremaining medium from the tissue cultures was performedby a micropipette and then viscoelastic (HEALON OVDAbbottMedical Optics USA)was added on top of the explantto allow for flattening or ldquoironingrdquo of the tissue onto thesurface of the Petri dish (Figure 1)
For ex vivo cultivation under adherent conditionsDMEMF12 supplemented with 10 FCS was then addedslowly with the micropipette not to disturb or remove theviscoelastic cover on top of the explants The micropipettetip was positioned close to the culture dish surface but faraway from the explant so that the medium arrived softly incontact with the viscoelastic and did not move the explantfrom its location The culture dishes were then kept in a CO
2
incubator (Innova CO-48 New Brunswick Scientific EdisonNJ USA) at 37∘C and 5 CO
2
The culture dish was kept in the incubator withoutmoving for 2-3 days in order to allow the cells to attach and
Explant tissue+ viscoelastic
Viscoelastic
Explanttissue
Petri dish
Cell outgrowth
Growth
Figure 1 Method for adherent ex vivo cultivation of human eyetissue explants in a cell culture Petri dish
start proliferating out of the explant Duringmedium changethe medium was removed gently and a fresh one was addedsubsequently by a micropipette from the opposite side of theexplant in the dish the pipette tip being close to the surfaceof the dish all the time The viscoelastic dissolved over timeand got replaced by newmediummdashtime by which the explantwas fully attached to the surface of the culture dish
23 Light Microscopy and Calcium Imaging The prolifera-tion and migration of the cells were recorded throughouttheir continued growth using inverted light microscope(Axiovert S100 Carl Zeiss AG Oberkochen Germany)The same microscope was used for [Ca2+]i measurementsImage acquisition was carried out by a 12-bit cooled CCDcamera SensiCam (PCO Imaging AG Kelheim Germany)The software used for the acquisition was WinFluor (writtenby J Dempster University of Strathclyde Glasgow UK)Microscope objectives used were 4x010 Achroplan 10x030Plan-Neofluar 40x050 LD A-plan and 63x125 oil Plan-Neofluar (Zeiss)
The excitation filters usedweremounted on a Lambda LS-10 filter wheel (Sutter Instruments Co CA USA) and had awavelength of 360 and 380 nm (Chroma Technology CorpBellows Falls VT USA) Excitation with the 360 nm filter(close to the Fura-2 isosbestic point) allowed observation ofthe cellsrsquo morphology and of the changes in the concentrationof the dye irrespective of the changes in [Ca2+]i while the360380 nm ratio allowed visualization of the [Ca2+]i changesin the cytoplasm Image acquisition timing and filter wheeloperation were all controlled by WinFluor software via aPCI6229 interface card (National Instruments Austin TXUSA) The light source used was XBO-75W (Zeiss) Xe arclamp The light intensity was attenuated when necessarywith grey filters with optical densities 05 1 and 2 (ChromaTechnology Corp Bellows Falls VT USA) The criteria forselecting the region for imagingwere the presence of adherentcells and good cell morphology both assessed by observationof transilluminated and 360 nm fluorescence images Indi-vidual image frames were acquired every 500ms resulting inframe cycles being 1 second long (two wavelengths)
For [Ca2+]i monitoring the cell cultures were loadedwith the acetoxymethyl (AM) ester of Fura-2 (Fura-2 AM
Journal of Immunology Research 3
500120583m
(a)
LC
1000 120583m
(b)
Figure 2 Examples of attached human eye explants with the growing cells (a) anterior lens capsule (aLC) in a 12-well plate (b) fibrovascularepiretinal membrane (fvERM) cells growing attached to and independently from the tissue explant in a cell culture Petri dish
Invitrogen-Molecular Probes Carlsbad CA USA) intracel-lular calcium indicator For loading Fura-2 AM in dimethylsulfoxide (DMSO) was suspended in 3mL of medium (highglucose medium with FBS) or physiological saline with (inmM) NaCl (1318) KCl (5) MgCl2 (2) NaH2PO4 (05)NaHCO3 (2) CaCl2 (18) HEPES (10) glucose (10) pH724 to the final working concentration of 2120583M (aLC) Theloading was done in the incubator at 37∘C for 30min (aLC)After loading the cell cultures were washed twice for 7minwith the medium or physiological saline The final workingconcentration of Fura-2 and the time of incubationwashingwere larger for larger eye explants (it depended on the explantsize)
Fura-2 dye has two excitation (absorption) peaks (340and 380 nm) an isosbestic point at 360 nm and one emissionpeak at 510 nm Its absorption and fluorescent propertieschange in accordance with Ca2+ binding (low [Ca2+]imdashhigh absorption at 380 nm high [Ca2+]imdashhigh absorptionat 340 nm while the absorption is not Ca2+ dependent atthe isosbestic point of 360 nm) The absorptive properties ofFura-2 allow the use of ratio imaging (360380 ratio) whichconsiderably reduces the effects of uneven dye loading leak-age of the dye and photobleaching as well as problems asso-ciated with measuring [Ca2+]i in cells of unequal thickness
24 Mechanical and Acetylcholine (ACh) Stimulation To testresponses to mechanical stimuli a tip of a glass micropipettemounted on a MP-285 micromanipulator (Sutter NovatoCA USA) was used Prior to use the tip of the pipette washeat-polished until it rounded up
The agonist acetylcholine (ACh Sigma USA)was appliedin 10 120583M concentration which was enough to induce gt90maximal [Ca2+]i response according to the data by Collisonet al [12] The agonist application as well as its washout fromthe bath was driven simply by the hydrostatic pressure of a35 cm of water column and controlled manually by a luer-lock stopcock (WPI) and applied through a polyethyleneplastic tubing (inner diameter 2mm) attached to the coarsemicromanipulator The excess bathing solution was removedby a suction line
25 Secretion of Inflammatory Cytokines by ELISA Theexpanded fvERM cells were plated onto 6-well plates ata density of 2 times 105 cells per well in triplicates Similarplating was carried out in case of the aLC-LECs until propercell number was achieved for cytokine measurements After24 hrs the medium was changed and the cells were treatedwith 100 ngmL recombinant human TNF120572 (PreprotechRocky Hill NJ USA) for additional 24 hours The secretedcytokines IL-6 and IL-8 were analyzed by commercialELISA kit (RampD Germany) according to the manufacturerrsquosprotocolThree independent experiments were performed onthree different outgrowing cells from both fvERM and aLC
3 Results
31 HumanEye Explant Tissues Adhere to the Cell CultureDishunder a Gravitational Force of Viscoelastic Material Novelsimple and reproducible method for ex vivo cultivation ofhuman explant tissues (aLCs and fvERMs) was establishedusing viscoelastic material (Figure 1)
The cells started proliferating out of the explants in 2-3days (Figure 2)Themethod for attachment of human eye tis-sue explants to the 12-well plates is shown in Figure 2(a)mdashtheaLC explant and the cells are flattened under the gravitationalforce of the viscoelastic material The fvERM cells grew outof the explants within 24 hours and continued proliferatingindependently throughout the study period (for more than 6months) (Figure 2(b))
32 Mechanical Stimulation and ACh Induce Rise in [1198621198862+]119894in
the aLC-LECs The functionality of the aLC-LECs attachedunder the viscoelastic was examined during mechanicalstimulation and application of agonist ACh both of whichinduced rise in the [Ca2+]i Representative examples of 6explant cultures were analyzed for mechanical stimulationcontaining 27 cells being stimulated (mostly the cells on theglass surface and some on the aLC) similarly representativeexamples of 5 explant cultures were analyzed for ACh stim-ulation Figure 3 shows the calcium signaling upon agonistACh stimulation of the aLC explant-cultured cells The oscil-lations of [Ca2+]i are clearly visible here with each cell having
4 Journal of Immunology Research
10120583m
(a)
40 60 80 100 120500
540
580
620
660
360
380
(s)(b)
Figure 3 Calcium signaling upon agonist ACh stimulation of the aLC explant-cultured cells (a) The traces (b) represent the time coursesof the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest (ROI) shown on the BampW image (a) in the samecolors (explant growth time 28 days)
its own frequency of oscillation (Figure 3(b) upper part) 50cells were analyzed here out of which 15 (30) had oscillatingresponse with average of 166 plusmn 44 sec from minimum tominimum Accommodation can be observed for the greentrace as the interval between the two maxima decreases withtime while a time delay of 2-3 sec in the [Ca2+]i propagationcan be seen (Figure 3(b) lower part) needed for the [Ca2+]ito reach its first maximum for different ROIs of the same cell(blue and red) No mechanically stimulated cell respondedat oscillation manner The transient responses to mechanicalstimulation were usually comparable to those elicited byACh
The calcium signaling upon mechanical stimulation of asingle cell of the aLC explant culture showed [Ca2+]i propaga-tion as well (Figure 4)mdashin the example shown 26 cells hadresponse with two peaks the first one being bigger than theother and the time interval between the peak maxima being25 and 26 sec the rest of the cells had no or very small calciumincreaseThe blue ROI represents the stimulation site and thered ROI represents the more distal site There is a delay ofaround 5 sec in the time needed for the [Ca2+]i to reach itsmaximum at two selected ROIs The increases in [Ca2+]i inthe cells surrounding themechanically stimulated cell suggestthe involvement of intercellular connections
The intercellular dendrite connection strength uponmechanical fluid movement for the nonattached dendritesin aLC explant culture could also be observed (Figure 5)Indeed a confirmation that the [Ca2+]i changes are notdependent on themechanical effect of fluidmovement but onACh is shown by the fact that [Ca2+]i increase occurs muchlater (119905 = 101 s) in comparison to the dendritic movementdependent on the mechanical effect of fluid movement (119905 =39ndash47 s) as visible on Figure 5(c)
The [Ca2+]i dynamics upon mechanical stimulation offvERMs has been previously described by our group [5]which is a proof of the viability and functionality of these cells
33 Measurement of ProinflammatoryAngiogenic FactorsSecreted by the fvERM Outgrowing Cells upon TNF120572 Treat-ment The outgrowing cells from the fvERMs showed basalexpression of the proinflammatory cytokine interleukin-(IL-) 6 ex vivo which was further enhanced by TNF120572stimulation Similar enhancement was noted in the proin-flammatory cytokine release of IL-8 upon TNF120572 stimulation(Figure 6(a)) In the case of aLC-LECs there were no basalIL-6 and IL-8 responses and TNF120572-induced IL-8 secretion(Figure 6(b))
4 Discussion
Anovel simple and reproduciblemethod for creating adher-ent conditions for human eye explants and ex vivo cellularexpansion using viscoelastic material as well as studies oncalcium dynamics and inflammation is established here Theoutgrowing cells over time migrate out of the explants andgrow adherently onto the surface of the cell culture dishshowing signs of continuous proliferation
Alternative adherence methods for tissues explants canbe the use of dry surface concentrated serum drop orthe fibrin-glue methodmdashthe latter being used mostly forin vivo purposes The advantage of the viscoelastic methodis in avoiding extreme conditions such as dryness andserum stimulants yet preserving natural architecture of thetissue and standard nutritional conditions for the cells Theviscoelastic is an inert substance having viscous elasticand gravitational properties which force the graft to attachto a surface The viscoelastic HEALON OVD is used inophthalmic surgical procedures to maintain deep anteriorchamber which facilitates manipulation inside the eye withreduced trauma to the corneal endothelium and other oculartissues
Two tissue types are used here to establish adherent exvivo explant cultures aLCs containing LECs and fvERMsTissue and cell adherence allow measurement of the [Ca2+]i
Journal of Immunology Research 5
10120583m
(a)
40 60 80 100 120380
400
420
440
360
380
(s)(b)
10120583m 10120583m 10120583m
10120583m 10120583m 10120583m
t = 50 s t = 56 s t = 58 s
t = 61 s t = 67 s t = 74 s
(c)
Figure 4 Calcium signaling upon mechanical stimulation of a single cell of the aLC explant culture showing the [Ca2+]i propagation andinvolvement of intercellular connections The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i andcorrespond to the regions of interest (ROI) shown in the BampW image (a) in the same colors (c) A series of the 360380 ratio images at thetime points are indicated The values for 119877 are color coded with bluegreen representing low ratio values and yellowred representing highratios (explant growth time 14 days)
upon mechanical or pharmacological stimulation givingadvantage of having less noise from cellularmovementwithinthe cell culture dish
Precise regulation of the [Ca2+]i levels is critical formain-taining normal cellular function fluctuations ofwhich can actas signals for numerous physiological or pathological eventsImbalance in the [Ca2+]i levels may lead to development ofcataract in the lens [6ndash9] Our results indicate an increase inthe [Ca2+]i upon mechanical stimulation and application of
ACh to aLC-LECs Previously mechanical stimulation hadbeen used to induce [Ca2+]i rise in cultured bovine LECs [10]Such stimulation of a single cell within a confluent layer wasshown to initiate cell-to-cell calcium signaling Contractionsin human aLECs attached to the surgically isolated capsulescould also be mechanically induced [11]
The increase in [Ca2+]i suggests involvement of inter-cellular connections between the LECs studied ex vivo Inhuman aLECs ACh binds to M1 muscarinic receptors (M1
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
Journal of Immunology Research 3
500120583m
(a)
LC
1000 120583m
(b)
Figure 2 Examples of attached human eye explants with the growing cells (a) anterior lens capsule (aLC) in a 12-well plate (b) fibrovascularepiretinal membrane (fvERM) cells growing attached to and independently from the tissue explant in a cell culture Petri dish
Invitrogen-Molecular Probes Carlsbad CA USA) intracel-lular calcium indicator For loading Fura-2 AM in dimethylsulfoxide (DMSO) was suspended in 3mL of medium (highglucose medium with FBS) or physiological saline with (inmM) NaCl (1318) KCl (5) MgCl2 (2) NaH2PO4 (05)NaHCO3 (2) CaCl2 (18) HEPES (10) glucose (10) pH724 to the final working concentration of 2120583M (aLC) Theloading was done in the incubator at 37∘C for 30min (aLC)After loading the cell cultures were washed twice for 7minwith the medium or physiological saline The final workingconcentration of Fura-2 and the time of incubationwashingwere larger for larger eye explants (it depended on the explantsize)
Fura-2 dye has two excitation (absorption) peaks (340and 380 nm) an isosbestic point at 360 nm and one emissionpeak at 510 nm Its absorption and fluorescent propertieschange in accordance with Ca2+ binding (low [Ca2+]imdashhigh absorption at 380 nm high [Ca2+]imdashhigh absorptionat 340 nm while the absorption is not Ca2+ dependent atthe isosbestic point of 360 nm) The absorptive properties ofFura-2 allow the use of ratio imaging (360380 ratio) whichconsiderably reduces the effects of uneven dye loading leak-age of the dye and photobleaching as well as problems asso-ciated with measuring [Ca2+]i in cells of unequal thickness
24 Mechanical and Acetylcholine (ACh) Stimulation To testresponses to mechanical stimuli a tip of a glass micropipettemounted on a MP-285 micromanipulator (Sutter NovatoCA USA) was used Prior to use the tip of the pipette washeat-polished until it rounded up
The agonist acetylcholine (ACh Sigma USA)was appliedin 10 120583M concentration which was enough to induce gt90maximal [Ca2+]i response according to the data by Collisonet al [12] The agonist application as well as its washout fromthe bath was driven simply by the hydrostatic pressure of a35 cm of water column and controlled manually by a luer-lock stopcock (WPI) and applied through a polyethyleneplastic tubing (inner diameter 2mm) attached to the coarsemicromanipulator The excess bathing solution was removedby a suction line
25 Secretion of Inflammatory Cytokines by ELISA Theexpanded fvERM cells were plated onto 6-well plates ata density of 2 times 105 cells per well in triplicates Similarplating was carried out in case of the aLC-LECs until propercell number was achieved for cytokine measurements After24 hrs the medium was changed and the cells were treatedwith 100 ngmL recombinant human TNF120572 (PreprotechRocky Hill NJ USA) for additional 24 hours The secretedcytokines IL-6 and IL-8 were analyzed by commercialELISA kit (RampD Germany) according to the manufacturerrsquosprotocolThree independent experiments were performed onthree different outgrowing cells from both fvERM and aLC
3 Results
31 HumanEye Explant Tissues Adhere to the Cell CultureDishunder a Gravitational Force of Viscoelastic Material Novelsimple and reproducible method for ex vivo cultivation ofhuman explant tissues (aLCs and fvERMs) was establishedusing viscoelastic material (Figure 1)
The cells started proliferating out of the explants in 2-3days (Figure 2)Themethod for attachment of human eye tis-sue explants to the 12-well plates is shown in Figure 2(a)mdashtheaLC explant and the cells are flattened under the gravitationalforce of the viscoelastic material The fvERM cells grew outof the explants within 24 hours and continued proliferatingindependently throughout the study period (for more than 6months) (Figure 2(b))
32 Mechanical Stimulation and ACh Induce Rise in [1198621198862+]119894in
the aLC-LECs The functionality of the aLC-LECs attachedunder the viscoelastic was examined during mechanicalstimulation and application of agonist ACh both of whichinduced rise in the [Ca2+]i Representative examples of 6explant cultures were analyzed for mechanical stimulationcontaining 27 cells being stimulated (mostly the cells on theglass surface and some on the aLC) similarly representativeexamples of 5 explant cultures were analyzed for ACh stim-ulation Figure 3 shows the calcium signaling upon agonistACh stimulation of the aLC explant-cultured cells The oscil-lations of [Ca2+]i are clearly visible here with each cell having
4 Journal of Immunology Research
10120583m
(a)
40 60 80 100 120500
540
580
620
660
360
380
(s)(b)
Figure 3 Calcium signaling upon agonist ACh stimulation of the aLC explant-cultured cells (a) The traces (b) represent the time coursesof the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest (ROI) shown on the BampW image (a) in the samecolors (explant growth time 28 days)
its own frequency of oscillation (Figure 3(b) upper part) 50cells were analyzed here out of which 15 (30) had oscillatingresponse with average of 166 plusmn 44 sec from minimum tominimum Accommodation can be observed for the greentrace as the interval between the two maxima decreases withtime while a time delay of 2-3 sec in the [Ca2+]i propagationcan be seen (Figure 3(b) lower part) needed for the [Ca2+]ito reach its first maximum for different ROIs of the same cell(blue and red) No mechanically stimulated cell respondedat oscillation manner The transient responses to mechanicalstimulation were usually comparable to those elicited byACh
The calcium signaling upon mechanical stimulation of asingle cell of the aLC explant culture showed [Ca2+]i propaga-tion as well (Figure 4)mdashin the example shown 26 cells hadresponse with two peaks the first one being bigger than theother and the time interval between the peak maxima being25 and 26 sec the rest of the cells had no or very small calciumincreaseThe blue ROI represents the stimulation site and thered ROI represents the more distal site There is a delay ofaround 5 sec in the time needed for the [Ca2+]i to reach itsmaximum at two selected ROIs The increases in [Ca2+]i inthe cells surrounding themechanically stimulated cell suggestthe involvement of intercellular connections
The intercellular dendrite connection strength uponmechanical fluid movement for the nonattached dendritesin aLC explant culture could also be observed (Figure 5)Indeed a confirmation that the [Ca2+]i changes are notdependent on themechanical effect of fluidmovement but onACh is shown by the fact that [Ca2+]i increase occurs muchlater (119905 = 101 s) in comparison to the dendritic movementdependent on the mechanical effect of fluid movement (119905 =39ndash47 s) as visible on Figure 5(c)
The [Ca2+]i dynamics upon mechanical stimulation offvERMs has been previously described by our group [5]which is a proof of the viability and functionality of these cells
33 Measurement of ProinflammatoryAngiogenic FactorsSecreted by the fvERM Outgrowing Cells upon TNF120572 Treat-ment The outgrowing cells from the fvERMs showed basalexpression of the proinflammatory cytokine interleukin-(IL-) 6 ex vivo which was further enhanced by TNF120572stimulation Similar enhancement was noted in the proin-flammatory cytokine release of IL-8 upon TNF120572 stimulation(Figure 6(a)) In the case of aLC-LECs there were no basalIL-6 and IL-8 responses and TNF120572-induced IL-8 secretion(Figure 6(b))
4 Discussion
Anovel simple and reproduciblemethod for creating adher-ent conditions for human eye explants and ex vivo cellularexpansion using viscoelastic material as well as studies oncalcium dynamics and inflammation is established here Theoutgrowing cells over time migrate out of the explants andgrow adherently onto the surface of the cell culture dishshowing signs of continuous proliferation
Alternative adherence methods for tissues explants canbe the use of dry surface concentrated serum drop orthe fibrin-glue methodmdashthe latter being used mostly forin vivo purposes The advantage of the viscoelastic methodis in avoiding extreme conditions such as dryness andserum stimulants yet preserving natural architecture of thetissue and standard nutritional conditions for the cells Theviscoelastic is an inert substance having viscous elasticand gravitational properties which force the graft to attachto a surface The viscoelastic HEALON OVD is used inophthalmic surgical procedures to maintain deep anteriorchamber which facilitates manipulation inside the eye withreduced trauma to the corneal endothelium and other oculartissues
Two tissue types are used here to establish adherent exvivo explant cultures aLCs containing LECs and fvERMsTissue and cell adherence allow measurement of the [Ca2+]i
Journal of Immunology Research 5
10120583m
(a)
40 60 80 100 120380
400
420
440
360
380
(s)(b)
10120583m 10120583m 10120583m
10120583m 10120583m 10120583m
t = 50 s t = 56 s t = 58 s
t = 61 s t = 67 s t = 74 s
(c)
Figure 4 Calcium signaling upon mechanical stimulation of a single cell of the aLC explant culture showing the [Ca2+]i propagation andinvolvement of intercellular connections The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i andcorrespond to the regions of interest (ROI) shown in the BampW image (a) in the same colors (c) A series of the 360380 ratio images at thetime points are indicated The values for 119877 are color coded with bluegreen representing low ratio values and yellowred representing highratios (explant growth time 14 days)
upon mechanical or pharmacological stimulation givingadvantage of having less noise from cellularmovementwithinthe cell culture dish
Precise regulation of the [Ca2+]i levels is critical formain-taining normal cellular function fluctuations ofwhich can actas signals for numerous physiological or pathological eventsImbalance in the [Ca2+]i levels may lead to development ofcataract in the lens [6ndash9] Our results indicate an increase inthe [Ca2+]i upon mechanical stimulation and application of
ACh to aLC-LECs Previously mechanical stimulation hadbeen used to induce [Ca2+]i rise in cultured bovine LECs [10]Such stimulation of a single cell within a confluent layer wasshown to initiate cell-to-cell calcium signaling Contractionsin human aLECs attached to the surgically isolated capsulescould also be mechanically induced [11]
The increase in [Ca2+]i suggests involvement of inter-cellular connections between the LECs studied ex vivo Inhuman aLECs ACh binds to M1 muscarinic receptors (M1
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
4 Journal of Immunology Research
10120583m
(a)
40 60 80 100 120500
540
580
620
660
360
380
(s)(b)
Figure 3 Calcium signaling upon agonist ACh stimulation of the aLC explant-cultured cells (a) The traces (b) represent the time coursesof the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest (ROI) shown on the BampW image (a) in the samecolors (explant growth time 28 days)
its own frequency of oscillation (Figure 3(b) upper part) 50cells were analyzed here out of which 15 (30) had oscillatingresponse with average of 166 plusmn 44 sec from minimum tominimum Accommodation can be observed for the greentrace as the interval between the two maxima decreases withtime while a time delay of 2-3 sec in the [Ca2+]i propagationcan be seen (Figure 3(b) lower part) needed for the [Ca2+]ito reach its first maximum for different ROIs of the same cell(blue and red) No mechanically stimulated cell respondedat oscillation manner The transient responses to mechanicalstimulation were usually comparable to those elicited byACh
The calcium signaling upon mechanical stimulation of asingle cell of the aLC explant culture showed [Ca2+]i propaga-tion as well (Figure 4)mdashin the example shown 26 cells hadresponse with two peaks the first one being bigger than theother and the time interval between the peak maxima being25 and 26 sec the rest of the cells had no or very small calciumincreaseThe blue ROI represents the stimulation site and thered ROI represents the more distal site There is a delay ofaround 5 sec in the time needed for the [Ca2+]i to reach itsmaximum at two selected ROIs The increases in [Ca2+]i inthe cells surrounding themechanically stimulated cell suggestthe involvement of intercellular connections
The intercellular dendrite connection strength uponmechanical fluid movement for the nonattached dendritesin aLC explant culture could also be observed (Figure 5)Indeed a confirmation that the [Ca2+]i changes are notdependent on themechanical effect of fluidmovement but onACh is shown by the fact that [Ca2+]i increase occurs muchlater (119905 = 101 s) in comparison to the dendritic movementdependent on the mechanical effect of fluid movement (119905 =39ndash47 s) as visible on Figure 5(c)
The [Ca2+]i dynamics upon mechanical stimulation offvERMs has been previously described by our group [5]which is a proof of the viability and functionality of these cells
33 Measurement of ProinflammatoryAngiogenic FactorsSecreted by the fvERM Outgrowing Cells upon TNF120572 Treat-ment The outgrowing cells from the fvERMs showed basalexpression of the proinflammatory cytokine interleukin-(IL-) 6 ex vivo which was further enhanced by TNF120572stimulation Similar enhancement was noted in the proin-flammatory cytokine release of IL-8 upon TNF120572 stimulation(Figure 6(a)) In the case of aLC-LECs there were no basalIL-6 and IL-8 responses and TNF120572-induced IL-8 secretion(Figure 6(b))
4 Discussion
Anovel simple and reproduciblemethod for creating adher-ent conditions for human eye explants and ex vivo cellularexpansion using viscoelastic material as well as studies oncalcium dynamics and inflammation is established here Theoutgrowing cells over time migrate out of the explants andgrow adherently onto the surface of the cell culture dishshowing signs of continuous proliferation
Alternative adherence methods for tissues explants canbe the use of dry surface concentrated serum drop orthe fibrin-glue methodmdashthe latter being used mostly forin vivo purposes The advantage of the viscoelastic methodis in avoiding extreme conditions such as dryness andserum stimulants yet preserving natural architecture of thetissue and standard nutritional conditions for the cells Theviscoelastic is an inert substance having viscous elasticand gravitational properties which force the graft to attachto a surface The viscoelastic HEALON OVD is used inophthalmic surgical procedures to maintain deep anteriorchamber which facilitates manipulation inside the eye withreduced trauma to the corneal endothelium and other oculartissues
Two tissue types are used here to establish adherent exvivo explant cultures aLCs containing LECs and fvERMsTissue and cell adherence allow measurement of the [Ca2+]i
Journal of Immunology Research 5
10120583m
(a)
40 60 80 100 120380
400
420
440
360
380
(s)(b)
10120583m 10120583m 10120583m
10120583m 10120583m 10120583m
t = 50 s t = 56 s t = 58 s
t = 61 s t = 67 s t = 74 s
(c)
Figure 4 Calcium signaling upon mechanical stimulation of a single cell of the aLC explant culture showing the [Ca2+]i propagation andinvolvement of intercellular connections The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i andcorrespond to the regions of interest (ROI) shown in the BampW image (a) in the same colors (c) A series of the 360380 ratio images at thetime points are indicated The values for 119877 are color coded with bluegreen representing low ratio values and yellowred representing highratios (explant growth time 14 days)
upon mechanical or pharmacological stimulation givingadvantage of having less noise from cellularmovementwithinthe cell culture dish
Precise regulation of the [Ca2+]i levels is critical formain-taining normal cellular function fluctuations ofwhich can actas signals for numerous physiological or pathological eventsImbalance in the [Ca2+]i levels may lead to development ofcataract in the lens [6ndash9] Our results indicate an increase inthe [Ca2+]i upon mechanical stimulation and application of
ACh to aLC-LECs Previously mechanical stimulation hadbeen used to induce [Ca2+]i rise in cultured bovine LECs [10]Such stimulation of a single cell within a confluent layer wasshown to initiate cell-to-cell calcium signaling Contractionsin human aLECs attached to the surgically isolated capsulescould also be mechanically induced [11]
The increase in [Ca2+]i suggests involvement of inter-cellular connections between the LECs studied ex vivo Inhuman aLECs ACh binds to M1 muscarinic receptors (M1
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
Journal of Immunology Research 5
10120583m
(a)
40 60 80 100 120380
400
420
440
360
380
(s)(b)
10120583m 10120583m 10120583m
10120583m 10120583m 10120583m
t = 50 s t = 56 s t = 58 s
t = 61 s t = 67 s t = 74 s
(c)
Figure 4 Calcium signaling upon mechanical stimulation of a single cell of the aLC explant culture showing the [Ca2+]i propagation andinvolvement of intercellular connections The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i andcorrespond to the regions of interest (ROI) shown in the BampW image (a) in the same colors (c) A series of the 360380 ratio images at thetime points are indicated The values for 119877 are color coded with bluegreen representing low ratio values and yellowred representing highratios (explant growth time 14 days)
upon mechanical or pharmacological stimulation givingadvantage of having less noise from cellularmovementwithinthe cell culture dish
Precise regulation of the [Ca2+]i levels is critical formain-taining normal cellular function fluctuations ofwhich can actas signals for numerous physiological or pathological eventsImbalance in the [Ca2+]i levels may lead to development ofcataract in the lens [6ndash9] Our results indicate an increase inthe [Ca2+]i upon mechanical stimulation and application of
ACh to aLC-LECs Previously mechanical stimulation hadbeen used to induce [Ca2+]i rise in cultured bovine LECs [10]Such stimulation of a single cell within a confluent layer wasshown to initiate cell-to-cell calcium signaling Contractionsin human aLECs attached to the surgically isolated capsulescould also be mechanically induced [11]
The increase in [Ca2+]i suggests involvement of inter-cellular connections between the LECs studied ex vivo Inhuman aLECs ACh binds to M1 muscarinic receptors (M1
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
6 Journal of Immunology Research
10120583m
(a)
360
380
(s)
570
550
530
510
490
470
450
70 80 90 100 110 120
(b)
10120583m 10120583m
10120583m 10120583m
10120583m 10120583m
t = 30 s t = 39 s
t = 47 s t = 81 s
t = 119 st = 101 s
(c)
Figure 5 The intercellular dendrite connection strength upon mechanical fluid movement for the nonattached dendrites in aLC explantcultures The traces (b) represent the time courses of the 360380 ratio (119877) proportional to [Ca2+]i and correspond to the regions of interest(ROI) shown in the BampW image(a) in the same colors (c) A series of the 360380 ratio images at the time points are indicatedThe values for119877 are color coded with bluegreen representing low ratio values and yellowred representing high ratios (explant growth time 21 days)
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
Journal of Immunology Research 7
Control fvERM + TNF120572
fvERM
IL-6IL-8
(pg
mL2lowast105
cells
)
3000
2500
2000
1500
1000
500
0
(a)
Control
1400
1200
1000
800
600
400
0
200
aLC-LEC + TNF120572
aLC-LEC
(pg
mL2lowast105
cells
)
(b)
Figure 6 Cytokine secretion by aLC-LEC and fvERM outgrowing cells upon TNF120572 treatment
mAChR) and induces a rise in [Ca2+]i [12ndash14] The originof ACh in the lens is not clear however its presence cancertainly affect cells of the immune system which possessmembrane bound mAChR and nicotinic (nAChR) receptorsthat can regulate their function [15ndash18] Choline acetyltrans-ferase (ChAT) enzyme expression in CD4+ and CD8+ T-cells has been previously shown suggesting that lymphocytespossess all of the necessary biochemical machinery to pro-duce this neurotransmitter thereby regulating their functionin an autocrine manner [19] In general according to thedata obtained in mammalian models it has been proposedthat cholinergic activity increases as a result of direct contactbetween T-cell receptor (TCR)CD3 molecules CD4 andCD8 coreceptors and other accessory molecules [17] Exper-imental data obtained by means of in vitro models and inabsence of neuronal innervation have shown ChAT produc-tion in B-cells macrophages and dendritic cells from miceproduction of this enzyme appears to be upregulated by Toll-like receptor (TLR) activation a pathway acting via MyD-88[19]MoreoverNeumann et al in 2007 [20] showed in humanleukocytes that antagonists of the nicotinic and muscarinicreceptors (tubocurarine and atropine resp) could signif-icantly decrease the phagocytic functions of granulocytesbut did not change the migration of these cells whereas inJurkat cells (the human helper T-lymphocyte leukemic line)exposure to oxotremorine-M (Oxo-M) a cholinergic agonistcould significantly increase the synthesis of IL-2 which couldbe related to the transcriptional factor activator protein-1 (AP-1) and mitogen-activated protein kinases (MAPK)[21] Experiments with MOLT-3 cells (the human T-cellleukemia line) showed involvement of the protein kinaseC (PKC) signaling pathway-MAPK cyclic adenosine 3101584051015840-monophosphate (cAMP) and calcineurin in the synthesis ofACh [18] There are findings suggesting that photoreceptorouter segments (OS) communicate via neurotransmitterssuch as ACh and SLURP-1 while RPE cells may receivethese signals through nAChRs1205727 in their microvilli [22]
It cannot be ruled out however that other cells includingLECs can be activated by ACh in such a manner Indeedevidence that RPE cells can express nAChRs similar to howother epithelial cells do has been related to cell developmentdeath migration and angiogenesis [23] The nAChR1205727 isresponsible for the inhibition of macrophage TNF release viathe parasympathetic anti-inflammatory pathway [24] thusopening up new avenues for the design of experimental anti-inflammatory therapeutics in different segments of the eye
The adherent aLC-LEC cultures can be used for inflam-matory studies as well Inhibition of aldose reductase (AR)for example can prevent lipopolysaccharide- (LPS-) inducedinflammatory response in human LECs [25] such as syn-thesis of large quantities of bioactive inflammatory medi-ators nitric oxide prostaglandins TNF-120572 IL-1 IL-6 andIFN-120574 [26ndash28] Ocular tissues can be exposed to variousproinflammatory factors released due to injury infection ordisease [29ndash31] The lens can also be exposed to such factorsappearing in the aqueous humor during bacterial infections[32ndash34] It was shown that incubation of human LECs withcytokines such as TNF120572 increases the activation of nuclearfactor kappa-light-chain-enhancer of activated B-cells (NF-120581B) and causes cytotoxicity [35 36] as well as apoptosis inhuman LECs (HLECs) Preventing NF-120581B activation by ARinhibitors should therefore rescueHLECs from cell death andinflammation [36 37] Transforming growth factor- (TGF-)120572 and TGF-120573 (2) mRNA can also be synthesized by humancataract LECs in situ while IL-8 mRNA can be synthesized invitro [38] IL-1 IL-6 and basic fibroblast growth factor (b-FGF) can be produced in vivo by residual LECs followingcataract surgery which can cause postoperative inflammationand LEC proliferation IL-1 and TGF120573may participate in thepostoperative inflammation by increasing PGE2 synthesis byresidual LECs [39] The role of these cytokines which can besynthesized by the LECs in vitromay therefore be significantin studying proliferation of LECs after cataract surgery whichcan eventually lead to inflammation and secondary cataract
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
8 Journal of Immunology Research
[40] It was revealed that the expression of TNF120572 gene inLECs is more extended compared to that of IL-1120572 in lenscapsule samples obtained from cataract surgery [35]
Cell death studies using terminal deoxynucleotidyltransferase- (TdT-) mediated dUTP nick-end labeling(TUNEL) of LECs in capsulotomy specimens found necroticcell death caused by damage during or soon after cataractsurgery Loss of cells from the lens epithelium by apoptosisor other mechanisms of cell death does not seem to play amajor role in age-related cataract formation [41]
Proper phenotypization of the cell surface markers ofex vivo cultured cells growing out of human fvERMs fromPDR gives possibility to study their role and function inimmunity The cell adhesion molecules (CAMs) and inte-grins profile are meaningful in structuring the cell-basedtissue integrity and immune processes Application of high-throughput screening by angiogenic protein arrays allowsmeasuring the angiogenic potential of fvERM outgrowingcells under presence or absence of proinflammatory factorTNF120572 [5]
Presence of TNF120572 in the vitreous is important markerfor PDR [42 43] High levels of IL-6 IL-8 and TNF120572have been measured in the vitreous of PDR patients [2 3]giving support to the role of inflammatory cytokines inangiogenesis in PDR Increased secretion of IL-6 and IL-8 was also measured in our fvERM outgrowing cells uponTNF120572 stimulation using the ELISA method Understandingtheir role can provide important diagnostic and therapeutictargets for the treatment and prevention of inflammation andangiogenesis in PDR
Fourteen main TNF120572-inducible proteins have beenreported in the literature in relation to immune responseamong them being the pentraxin-related protein 3 (PTX3)a known marker for rapid primary local activation of innateimmunity and inflammation ICAM-1 expression increasedupon TNF120572 proinflammatory stimulus in primary hRPEcells similar to the fvERM outgrowing cells giving a link tothe functionwhich these activated cells may play in leukocyteadhesion [44ndash50] Endothelin 1 (ET-1) molecule secretedby endothelial cells when stimulated by proinflammatorycytokines increases in the vitreous of patients with PDR[51] also detected in the fvERMs upon TNF120572 treatmentin our previous study [5] IL-1120573 concentrations are higherin the vitreous of patients with PDR compared to non-PDR and controls indicating that there could be a mini-mal acute inflammatory activity present in the early stagesof retinopathy which progressively increases in the mostadvanced stages of the disease In comparison the level ofIL-1Ra which is an anti-inflammatory cytokine was foundto be significantly higher in the controls compared to thoseof PDR [51] The process of complement C5a activation leadsto release of cytokines reactive oxygen species proteolyticenzymes and other proinflammatory molecules [52]
Recently the extracellular high-mobility group box-1(HMGB1) was reported as proinflammatory cytokine [53ndash56]playing a role in angiogenesis [53 57ndash59] and it was detectedin the vitreous of patients with PDR together with MCP-1 and sICAM-1 [60] HMGB1 and the soluble receptor foradvanced glycation-end products (RAGE) are also expressed
by vascular endothelial and stromal cells in fvERM fromPDR suggesting a role for the HMGB1RAGE signaling axisin the progression of PDR [53 57ndash59] A significantly elevatedlevel of five novel cytokines including sCD40L GM-CSFIFN1205722 IL-12p40 and MCP-3 in the vitreous of PDR patientspreviously not associated with the disease was also recentlyreported [61]
In conclusion providing adherent inert conditions for exvivo cultivation and expansion of cells from different tissuesis crucial for establishing disease models Using viscoelasticmaterial as a novel and simple method for achieving tissueand cell adherence can empower studies on intracellularcalcium dynamics upon mechanical stimulation calciumsignaling and intercellular communication upon ACh stim-ulation as well as inflammatory studies in as little backgroundnoise and artifacts as possible void of detachment-associatedcell death and associated inflammation Future studies oncell functionality and homeostasis using calcium imaging andinflammation screening widen the possibilities for develop-ment of pharmacological and cell-based therapies that areattractive approach for treating eye diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work has been supported by the TAMOP-422A-111KONV-2012-0023 Grant project implemented throughthe New Hungary Development Plan and cofinanced by theEuropean Social Fund and the Slovenian Research Agency(ARRS) program P3-0333 and a personal cofinancing grantwas given to G Petrovski from ARRS
References
[1] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronaterdquo Journal ofClinical Ophthalmology vol 2 no 1 pp 21ndash30 2008
[2] U E Koskela S M Kuusisto A E Nissinen M J Savolainenand M J Liinamaa ldquoHigh vitreous concentration of IL-6 andIL-8 but not of adhesion molecules in relation to plasma con-centrations in proliferative diabetic retinopathyrdquo OphthalmicResearch vol 49 no 2 pp 108ndash114 2013
[3] J Zhou S Wang and X Xia ldquoRole of intravitreal inflamma-tory cytokines and angiogenic factors in proliferative diabeticretinopathyrdquo Current Eye Research vol 37 no 5 pp 416ndash4202012
[4] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007
[5] Z Vereb X Lumi S Andjelic et al ldquoFunctional and molec-ular characterization of ex vivo cultured epiretinal membranecells from human proliferative diabetic retinopathyrdquo BioMedResearch International vol 2013 Article ID 492376 14 pages2013
[6] L Liu C A Paterson and D Borchman ldquoRegulation ofsarcoendoplasmic Ca2+-ATPase expression by calcium in
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
Journal of Immunology Research 9
human lens cellsrdquo Experimental Eye Research vol 75 no 5 pp583ndash590 2002
[7] G Duncan and I M Wormstone ldquoCalcium cell signalling andcataract role of the endoplasmic reticulumrdquo Eye vol 13 part 3bpp 480ndash483 1999
[8] G C Churchill M M Lurtz and C F Louis ldquoCa2+ regulationof gap junctional coupling in lens epithelial cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 281 no 3 pp C972ndashC981 2001
[9] K Yawata M Nagata A Narita and Y Kawai ldquoEffects of long-term acidification of extracellular pH on ATP-induced calciummobilization in rabbit lens epithelial cellsrdquo Japanese Journal ofPhysiology vol 51 no 1 pp 81ndash87 2001
[10] G C Churchill M M Atkinson and C F Louis ldquoMechanicalstimulation initiates cell-to-cell calcium signaling in ovine lensepithelial cellsrdquo Journal of Cell Science vol 109 part 2 pp 355ndash365 1996
[11] S Andjelic G Zupancic D Perovsek andMHawlina ldquoHumananterior lens capsule epithelial cells contractionrdquo Acta Ophthal-mologica vol 89 no 8 pp e645ndashe653 2011
[12] D J Collison R A Coleman R S James J Carey and G Dun-can ldquoCharacterization of muscarinic receptors in human lenscells by pharmacologic and molecular techniquesrdquo InvestigativeOphthalmology and Visual Science vol 41 no 9 pp 2633ndash26412000
[13] D J Collison and G Duncan ldquoRegional differences in func-tional receptor distribution and calcium mobilization in theintact human lensrdquo Investigative Ophthalmology and VisualScience vol 42 no 10 pp 2355ndash2363 2001
[14] J D Rhodes and J Sanderson ldquoThe mechanisms of calciumhomeostasis and signalling in the lensrdquo Experimental EyeResearch vol 88 no 2 pp 226ndash234 2009
[15] Y Horiuchi R Kimura N Kato et al ldquoEvolutional study onacetylcholine expressionrdquo Life Sciences vol 72 no 15 pp 1745ndash1756 2003
[16] T Fujii and K Kawashima ldquoAn independent non-neuronalcholinergic system in lymphocytesrdquo Japanese Journal of Phar-macology vol 85 no 1 pp 11ndash15 2001
[17] K Kawashima and T Fujii ldquoThe lymphocytic cholinergic sys-tem and its contribution to the regulation of immune activityrdquoLife Sciences vol 74 no 6 pp 675ndash696 2003
[18] K Kawashima T Fujii Y Moriwaki H Misawa and KHoriguchi ldquoReconciling neuronally and nonneuronally derivedacetylcholine in the regulation of immune functionrdquo Annals ofthe New York Academy of Sciences vol 1261 no 1 pp 7ndash17 2012
[19] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[20] S Neumann M Razen P Habermehl et al ldquoThe non-neuronalcholinergic system in peripheral blood cells effects of nicotinicand muscarinic receptor antagonists on phagocytosis respira-tory burst and migrationrdquo Life Sciences vol 80 no 24-25 pp2361ndash2364 2007
[21] Y Okuma and Y Nomura ldquoRoles of muscarinic acetylcholinereceptors in interleukin-2 synthesis in lymphocytesrdquo JapaneseJournal of Pharmacology vol 85 no 1 pp 16ndash19 2001
[22] H Matsumoto K Shibasaki M Uchigashima et al ldquoLocal-ization of acetylcholine-related molecules in the retina Impli-cation of the communication from photoreceptor to retinalpigment epitheliumrdquo PLoS ONE vol 7 no 8 Article ID e428412012
[23] V Maneu G Gerona L Fernandez N Cuenca and P LaxldquoEvidence of alpha 7 nicotinic acetylcholine receptor expressionin retinal pigment epithelial cellsrdquo Visual Neuroscience vol 27no 5-6 pp 139ndash147 2010
[24] A V Osborne-Hereford S W Rogers and L C GahringldquoNeuronal nicotinic alpha7 receptors modulate inflammatorycytokine production in the skin following ultraviolet radiationrdquoJournal of Neuroimmunology vol 193 no 1-2 pp 130ndash139 2008
[25] A Pladzyk A B M Reddy U C S Yadav R Tammali K VRamana and S K Srivastava ldquoInhibition of aldose reductaseprevents lipopolysaccharide-induced inflammatory response inhuman lens epithelial cellsrdquo Investigative Ophthalmology andVisual Science vol 47 no 12 pp 5395ndash5403 2006
[26] C M Blatteis S Li Z Li C Feleder and V Perlik ldquoCytokinesPGE2 and endotoxic fever a re-assessmentrdquo Prostaglandins andOther Lipid Mediators vol 76 no 1ndash4 pp 1ndash18 2005
[27] A I Ivanov and A A Romanovsky ldquoProstaglandin E2as
a mediator of fever synthesis and catabolismrdquo Frontiers inBioscience vol 9 pp 1977ndash1993 2004
[28] J de Angelo ldquoNitric oxide scavengers in the treatment of shockassociated with systemic inflammatory response syndromerdquoExpert Opinion on Pharmacotherapy vol 1 no 1 pp 19ndash291999
[29] R L Penland M Boniuk and K R Wilhelmus ldquoVibrio ocularinfections on the US Gulf Coastrdquo Cornea vol 19 no 1 pp 26ndash29 2000
[30] L D Hazlett ldquoCorneal response to Pseudomonas aeruginosainfectionrdquo Progress in Retinal and Eye Research vol 23 no 1pp 1ndash30 2004
[31] R A Sack I Nunes A Beaton and C Morris ldquoHost-defensemechanism of the ocular surfacesrdquo Bioscience Reports vol 21no 4 pp 463ndash480 2001
[32] G N Palexas G Sussman and N H Welsh ldquoOcular andsystemic determination of IL-1120573 and tumour necrosis factor ina patient with ocular inflammationrdquo Scandinavian Journal ofImmunology vol 36 pp 173ndash175 1992
[33] K Nishi O Nishi and Y Omoto ldquoThe synthesis of cytokines byhuman lens epithelial cellsmdashinterleukin 1 (IL-1) tumor necrosisfactor (TNF) interleukin 6 (IL-6) and epidermal growth factor(EGF)rdquoNihon Ganka Gakkai Zasshi vol 96 no 6 pp 715ndash7201992
[34] N H Sachdev N Di Girolamo T M Nolan P J McCluskeyD Wakefield and M T Coroneo ldquoMatrix metalloproteinasesand tissue inhibitors of matrixmetalloproteinases in the humanlens implications for cortical cataract formationrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 11 pp 4075ndash40822004
[35] J Prada T Ngo-Tu H Baatz C Hartmann and U PleyerldquoDetection of tumor necrosis factor alpha and interleukin 1alpha gene expression in human lens epithelial cellsrdquo Journal ofCataract and Refractive Surgery vol 26 no 1 pp 114ndash117 2000
[36] K V Ramana B Friedrich A Bhatnagar and S K SrivastavaldquoAldose reductase mediates cytotoxic signals of hyperglycemiaand TNF-alpha in human lens epithelial cellsrdquo The FASEBJournal vol 17 no 2 pp 315ndash317 2003
[37] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo The Journal of ClinicalInvestigation vol 107 no 3 pp 255ndash264 2001
[38] O Nishi K Nishi K Wada and Y Ohmoto ldquoExpression oftransforming growth factor (TGF)-120572 TGF-120573
2and interleukin 8
messenger RNA in postsurgical and cultured lens epithelial cells
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
10 Journal of Immunology Research
obtained frompatients with senile cataractsrdquoGraefersquos Archive forClinical and Experimental Ophthalmology vol 237 no 10 pp806ndash811 1999
[39] O Nishi K Nishi M Imanishi Y Tada and E ShirasawaldquoEffect of the cytokines on the prostaglandin E2 synthesisby lens epithelial cells of human cataractsrdquo British Journal ofOphthalmology vol 79 no 10 pp 934ndash938 1995
[40] O Nishi K Nishi and Y Ohmoto ldquoSynthesis of interleukin-1 interleukin-6 and basic fibroblast growth factor by humancataract lens epithelial cellsrdquo Journal of Cataract amp RefractiveSurgery vol 22 supplement 1 pp 852ndash858 1996
[41] G J Harocopos K M Alvares A E Kolker and D C BeebeldquoHuman age-related cataract and lens epithelial cell deathrdquoInvestigative Ophthalmology amp Visual Science vol 39 no 13 pp2696ndash2706 1998
[42] G A Limb A H Chignell W Green F LeRoy and D CDumonde ldquoDistribution of TNF120572 and its reactive vascularadhesionmolecules in fibrovascularmembranes of proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 80no 2 pp 168ndash173 1996
[43] C Gustavsson E Agardh B Bengtsson and C-D AgardhldquoTNF-120572 is an independent serum marker for proliferativeretinopathy in type 1 diabetic patientsrdquo Journal of Diabetes andits Complications vol 22 no 5 pp 309ndash316 2008
[44] G A Limb C J Cole O Earley R D Hollifield W Russelland M R Stanford ldquoExpression of hematopoietic cell markersby retinal pigment epithelial cellsrdquoCurrent Eye Research vol 16no 10 pp 985ndash991 1997
[45] K F PlattsM T Benson I G Rennie RM Sharrard and R CRees ldquoCytokine modulation of adhesion molecule expressionon human retinal pigment epithelial cellsrdquo Investigative Oph-thalmology and Visual Science vol 36 no 11 pp 2262ndash22691995
[46] S G Elner V M Elner M A Pavilack et al ldquoModulation andfunction of intercellular adhesionmolecule-1 (CD54) onhumanretinal pigment epithelial cellsrdquo Laboratory Investigation vol66 no 2 pp 200ndash211 1992
[47] N Kanuga H LWinton L Beauchene et al ldquoCharacterizationof genetically modified human retinal pigment epithelial cellsdeveloped for in vitro and transplantation studiesrdquo InvestigativeOphthalmology and Visual Science vol 43 no 2 pp 546ndash5552002
[48] M V Kumar C N Nagineni M S Chin J J Hooks and BDetrick ldquoInnate immunity in the retina toll-like receptor (TLR)signaling in human retinal pigment epithelial cellsrdquo Journal ofNeuroimmunology vol 153 no 1-2 pp 7ndash15 2004
[49] H-P Heidenkummer and A Kampik ldquoIntercellular adhe-sion molecule-1 (ICAM-1) and leukocyte function-associatedantigen-1 (LFA-1) expression in human epiretinal membranesrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 230 no 5 pp 483ndash487 1992
[50] L Devine S L Lightman and J Greenwood ldquoRole of LFA-1ICAM-1 VLA-4 and VCAM-1 in lymphocyte migration acrossretinal pigment epithelial monolayers in vitrordquo Immunologyvol 88 no 3 pp 456ndash462 1996
[51] J I Patel G M Saleh P G Hykin Z J Gregor and I A CreeldquoConcentration of haemodynamic and inflammatory relatedcytokines in diabetic retinopathyrdquo Eye vol 22 no 2 pp 223ndash228 2008
[52] J Kohl ldquoAnaphylatoxins and infectious and non-infectiousinflammatory diseasesrdquo Molecular Immunology vol 38 no 2-3 pp 175ndash187 2001
[53] J R Van Beijnum W A Buurman and A W GriffioenldquoConvergence and amplification of toll-like receptor (TLR) andreceptor for advanced glycation end products (RAGE) signalingpathways via high mobility group B1 (HMGB1)rdquo Angiogenesisvol 11 no 1 pp 91ndash99 2008
[54] C J Treutiger G E Mullins A-S M Johansson et alldquoHigh mobility group 1 B-box mediates activation of humanendotheliumrdquo Journal of Internal Medicine vol 254 no 4 pp375ndash385 2003
[55] C Fiuza M Bustin S Talwar et al ldquoInflammation-promotingactivity of HMGB1 on human microvascular endothelial cellsrdquoBlood vol 101 no 7 pp 2652ndash2660 2003
[56] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010
[57] S Mitola M Belleri C Urbinati et al ldquoCutting edge extra-cellular high mobility group box-1 protein is a proangiogeniccytokinerdquo Journal of Immunology vol 176 no 1 pp 12ndash15 2006
[58] C Schlueter H Weber B Meyer et al ldquoAngiogenetic signalingthrough hypoxia HMGB1 an angiogenetic switch moleculerdquoTheAmerican Journal of Pathology vol 166 no 4 pp 1259ndash12632005
[59] E Chavakis AHainMVinci et al ldquoHigh-mobility group box 1activates integrin-dependent homing of endothelial progenitorcellsrdquo Circulation Research vol 100 no 2 pp 204ndash212 2007
[60] A M A El-Asrar M I Nawaz D Kangave et al ldquoHigh-mobility group box-1 and biomarkers of inflammation in thevitreous from patients with proliferative diabetic retinopathyrdquoMolecular Vision vol 17 pp 1829ndash1838 2011
[61] J L Bromberg-White L Glazer R Downer et al ldquoIdentifica-tion of VEGF-independent cytokines in proliferative diabeticretinopathy vitreousrdquo Investigative Ophthalmology amp VisualScience vol 54 no 10 pp 6472ndash6480 2013
