T The immunoregulatory abilities of polymorphonuclear … · 2019. 8. 1. · The immunoregulatory...

The immunoregulatory abilities ofpolymorphonuclear neutrophils inthe course of multiple sclerosis

J. Ziçcaber,1,CA J. Paśnik,2 Z. Baj,2 L. Pokoca,3

H. Chmielewski1 and H. Tchórzewski4

Departments of 1Neurology; 2Pathophysiology;3Clinical Immunology Military Medical Academy, ul.úZeromskiego 113, 90–549 Lódź; 4Department ofClinical Immunology of Polish Mother’s HealthInstitute, Lódź, Poland

CACorresponding AuthorTe l/Fax : (+48) 42 636 52 82

THE polym orphonuclear neutroph ils (PMN) posse sssufficien t potential to affect both im m une responseand in flam m ation , however it has not been yetde scribed in th e course of m ultiple sclerosis (MS). Wehave studie d binding of fluore sce in isothiocyanate(FITC)- s tain ed TNF-a by PMN, the ex press ion ofCD11a, CD11b, and CD18 m olecule s of b 2-integr ine sand the ex press ion of CD10 (neutral endopeptidase -NEP) and of CD13 (am in opeptidase N; APN) an tigenson PMN in th ree differen t groups of MS patien ts . Thecon trol group in cluded neurological patients (OND)w ith nonin flam m atory diseases . Th e obtained resultshave proved that durin g MS ex acerbation and in thecourse of chron ic progress ive MS, PMN reveal severalform s of preactivation, in cluding s ignifican tly h igherstain ed-TNF-a binding, h igher ex press ion of CD11band CD18, as w ell as CD10 and CD13 an tigens , incom paris on w ith MS rem iss ion or OND. We suggestthat th e increased ex press ion of th es e m olecules onPMN of MS patien ts in ex acerbation of th e disease andto a low er degree in th e course of CP-MS is a r esult ofPMN prim ing, and directly prove th e PMN involve-m ent in the diseas e pathogenesis .

Key w ords : Multiple sclerosis , Neutrophils, Tumour necro-sis factor, Tumour necrosis factor receptors, Integrins,Proteases

Introduction

Multiple sclerosis (MS) is a dise ase in w hich multifocalinflammation and damage of the blood–brain barrierand myelin sheath are salient pathologic features.Overw he lming evidence demonstrates that MS is apredominantly T-cell and monocyte/macrophage-mediated autoimmune disorde r.1 ,2 Polymorphonu-clear ne utrophils (PMN) have not been considere d as ace ll population participating in it.3 PMN can howeverex pre ss immunoregulatory abilities, that has not be enye t de sc ribed in the course of MS. Activated in vitroPMN produce a number of immune mediators includ-ing cytokines like IL-1 b , IL-4, IL-6, IL-8, IL-10, IL-12,TNF, TGF-b 1.4 ,5 There fore the re gulatory functions ofthese ce lls may be postulated in the course of MS.

PMN in the pe ripheral blood (PB) of MS patientscan be primed mainly by inflammatory cytokine s likeIL-1, INF-g , or by TNF-a sec re ted by mononuclearce lls . Hypothetically PMN priming or activation in PBof MS patients may depend also on complement(especially C5a) immunologic al complex es, or certainmetabolite s of the arachidonic ac id (LTB4 , PAF).

Priming re sults in the enhanced ex pre ssion on PMNof re ceptors for chemokines and other chemotacticpeptide s,6 priming also enhance s the ex pre ssion ofCD11b/CD18 molecules on PMN cell surface 7,8 w hichresults in the indire ct ac tivation of PMN.9,10 In the

presented paper w e have suggested that PMN primingcan be obse rved in MS patients periphe ral blood.

Patients and methodsPatients (34 total; 19 w omen and 15 men, aged 22–56years) w ere se lected w ith a clinically definitive diag-nosis of MS1 1 and w ith Kurtzke Expanded DisabilityStatus Scale 12 scores 5 or fewer and categorized ashaving the re lapsing-remitting MS (RR-MS) course for atleast 5 years but currently in remission (REM) (n =13),as having either secondary CP-MS for at least 2 years(n=13) or RR-MS and currently ex pe riencing clinicalex ace rbation (REL) of the disease (n=12). Ex acerba-tions were defined as the appearance of new symptomsor significant w orsening of the old ones, attributabl e toMS, for at least 24 hours w ithout any fever.

The control group consiste d of patients w ith otherne urologic al diseases (OND) (14 total; nine w omenand five men, aged from 24 to 37 years), includingthose w ith vasomotor headache (n=8) and ischialgia(n=6).

Sample collection

The studies we re performed on PMN of the periph-eral venous blood, collec ted into heparin-containingtubes (10 U/ml).

0962-9351/98/050335-04 $9.00 © 1998 Carfax Publishing Ltd 335

Research Paper

Mediators of Inflammation, 7, 335–338 (1998)

TNF labelling with fluorescein isothiocyanate(FITC)

The synthesis and biological analysis of the TNFmolecules w ere pe rformed at the Department ofBioorganic Chemistry, Lódź, Poland (Patent No.168858). TNF staining was done w ith FITC (Serva),according to Shirakaw a e t a l.13 100 mg of FITC and100 m l of 0.2 M carbonate buffe r (pH = 9.2) w ereadded to 400 m g of TNF, diluted in 400 m l of PBS andthen, incubated for 6 h at 4°C. The labelled TNF w asseparated from the non-labe lled w ith FITC by gelfiltration on a 3.5 cm3 column, filled w ith SephadexG-25. TNF labelled FITC (TNF-FITC) w as sterilized byfiltration, using a 0.22 m m filter and then, stored at4°C. The staining e ffic iency was measured, taking intoaccount the absorbency at 280 nm, in comparisonw ith the absorbency at 495 nm. The inve stigationsw ere performed w ithin 10 days after the TNF labe llingdue to re latively low stability of the labe lling.

Evaluation of TNF-FITC binding to PMN

One hundred m l of the w hole blood w as incubatedw ith TNF-FITC at the concentration of 1000 ng/ml for2 h at 4°C. The erythrocytes w e re lysed, using 1 ml ofFACS Lysing Solution (Bec ton Dickinson) for 15 min atroom te mperature. Cells we re centrifuged for 5 min at400 3 g , then w ashed w ith 3 ml of PBS andsuspended in 100 m l of PBS. The fluore scence inten-sity of PMN w as measured using a flow cytometryFACScan (Becton Dickinson) and Lysis II softw are.Each measurement w as re peated four times andpresented here as a mean fluore scence intensity(MFI). Specificity of the binding of TNF-FITC to PMNw as evaluated in blocking ex pe riment by pre incuba-tion of the PMN w ith non-stained TNF in ex cess for45 min before TNF-FITC binding (Fig. 1).

Evaluation of the expression of CD11a, CD11band CD18 molecules of LFA-1 and Mac-1intregine and CD10 and CD13 Ag of neutralendopeptidase (NEP) and aminopeptidase N(APN) on PMN

The dete rmination of molecule ex pre ssions on thesurface of PMN was performed in the w hole blood.Techniques, generally used for immunofluore scentlabelling of the cells in w hole blood collec ted onheparin, w ere applied. One hundred m l of blood w asmix ed and incubated at room te mperature w ithappropriate quantitie s of monoclonal antibodies,provided by Dako (Denmark). CD11a, CD11b andCD18 antibodies w e re used. A double-step stainingprocedure w as used for the evaluation of CD10 andCD13 Ag ex pre ssion. Mouse immunoglobulins anti-CD10 and CD13 (Dako) were used as a first s tep.Rabbit anti-mouse IgG polyclonal immunoglobulins Gstained w ith R-phycoerythrin were used as a second-ary antibody. Mouse IgG2a, stained w ith RPE, w asused as a ne gative control. Erythrocytes w ere e limi-nated by an addition of lysing solution (BectonDickinson) into the blood samples. Afte r a short timeof incubation and rinsing, the cells w ere suspended inphysiological buffe red saline (PBS). FACscan flowcytomete r w ith a 488 nm argon lase r (Becton Dick-inson) and Lysis II softw are w ere used. The re sultsw ere ex pressed as the value s of MFI of the labe lledsurface antigens.

Results

The MFI of the PMN incubated w ith FITC-TNF w ashighest in the group w ith ex ace rbation of RR-MS(REL) (Table 1) and the value w as significantly higher(P

CP-MS.2 The obtained data suggest that re lapsing-remitting MS follow s cycles of immunologic al activa-tion as a result of Th-1 response (ex ac erbation), w hichis then followed by a suppressor re sponse thatdow nregulates inflammation (remiss ion).1 ,2 In CP-MSthere is continuous low -grade inflammation w ith noobvious ex acerbations or remissions.

The involvement of TNF in immunopathologic alproce sse s in MS has alre ady be en know n for quitesome time .14 This cytokine is also one of the mostpotent priming factors for PMN in vitro .7,1 5 Wesuggest that se rum TNF of patients w ith acuteex ace rbations of RR-MS or in low er degree in thecourse of CP-MS can be the main priming fac tor ofPMN.

TNF in v itro stimulates the ex pre ssion of many ofthe PMN surface molecules . Simultaneously TNFstimulates its ow n receptors ex press ion (TNF-R) onPMN.16 This has been confirmed in our study as theinc re ased TNF binding to PMN in the course of acuteex ace rbations of MS. We have obse rved also thesignificant inc re ase of the CD10, CD13 Ag andCD11b/CD18 molecule ex press ion on PMN of MSpatients, mainly in the course of ex ac erbation com-pared w ith MS remission and w ith OND groups. Thismay be a s ign of priming of PMN in MS ex acerbationand to a low er degree in the course of CP-MS.

Podikoglou e t a l.3 have show n that the typic alfunctions of PMN, like adhere nce , chemotax is, pha-gocytosis or bactericidal action have be en s ignifi-cantly diminished in PB of MS patients . The re sultsthat w e have obtained in our study correspond w ithGoto e t a l..1 7 In some earlie r obse rvations Aoki e ta l.18 and Guarnieri e t a l.1 9 have show n that theinc re ased intracellular neutral proteinase and medul-las ine concentrations can be the symptom of PMN

activation in the blood of patients w ith acute ex ac er-bation of MS.

PMN contain large amounts of proteinases , likeNEP and APN, in their intracellular granules.2 0,21 Theincre ased ex pre ssion on in v itro stimulated PMN is aresult of rapid translocation of an intracellular pool tothe cell surface .22 ,2 3 The incre ased ex pression ofCD11b/CD18–Mac -1 molecules probably results fromtheir rapid shift from internal granule s to the surfaceof primed PMN. Such proce ss has not be en observedin CD11a/CD18 molecules of LFA-1 integrine ex pre s-sion of in vitro primed PMN.24,25 In our studies w ehave not noticed the inc reased ex press ion of CD11amolecule on PMN of MS patients w ith acute ex acerba-tions or in the course of CP-MS. In our opinion, the secan suggest PMN priming in patients w ith active MS.Activated PMN can produce ox ygen and nitrogenspecie s, some matrix degrading metalloprote inasesand some cytokine s.5 ,26 Priming is a pre liminaryevent be fore receptor-induced stimulation of PMN. Itis then a sign of these cells’ hype rreac tivity. PMNpriming features obse rved in the PB of patients w ithactive MS suggest that these cells may participate inthe MS immunopathology.

References1. Hohlfe ld R, Me inl E, Webe r F, e t a l. The role of autoimmune T

lymphocyte s in the pathogene s is of multip le sc le rosis . Ne u ro lo g y 1995:45; (suppl 6); S33–S38.

2. Giovannoni G, Hartung HP. The immunopathogene sis of multip lesc le rosis and Guillain-Barré syndrome. Cur r Opin Ne uro l 1996: 9;165–177.

3. Podikoglou DG, Lianou PE, Tsakanikas CD, Papavass iliou JT. Poly-morphonuclear leukoc yte functions and multip le sc le ros is. Ne u ro lo g y1994: 44; 129–132.

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Po lym o rpho nu cle a r n eu tro phils a nd m u ltiple s c le ro s is

Mediators of Inflammation · Vol 7 · 1998 337

Table 1. Mean fluorescence intensity (MFI) of fluorescein isothiocyanate (FITC) labelled TNF binding to polymorphonuclear neutrophils(PMN) and the CD10, CD13 Ag, and CD11a, CD11b, CD18 molecule expression on PMN in patients with relapsing-remitting MS duringexacerbation (REL) (1), in chronic progressive MS (CP-MS) (2) during remission (REM) of the disease (3) and in the other neurologicaldiseases (OND) (4)

MFI of TNF-FITCbinding

CD10expression

CD13expression

CD11aexpression

CD11bexpression

CD18expression

1. REL 134.6 ± 17.3 range: 52.7 ± 10.7 range: 138.6 ± 20.8 range: 70.7 ± 12.0 range: 942 ± 56 range: 280.9 ± 39.9 range:(n = 12) 115.8–151.6 38.7–69.8 126.4–168.8 51.5–94.7 827–1010 210.1–347.1

1 vs. 2 P < 0.01 1 vs. 2 P < 0.05 1 vs. 2 P < 0.05 1 vs. 3 P < 0.01 1 vs. 2 P < 0.01 1 vs. 3 P < 0.011 vs. 3 P < 0.01 1 vs. 3 P < 0.01 1 vs. 3 P < 0.01 1 vs. 3 P < 0.01 1 vs. 4 P < 0.011 vs. 4 P < 0.01 1 vs. 4 P < 0.01 1 vs. 4 P < 0.01 1 vs. 4 P < 0.01

2. CP-MS 119.1 ± 14.5 range: 42.9 ± 13.9 range: 124.1 ± 29.6 range: 70.4 ± 9.1 range: 743 ± 114 range: 277.2 ± 30.9 range:(n = 12) 98.6–144.2 29.4–76.2 87.2–174.2 58.2–81.2 622–968 221.2–308.7

2 vs. 3 P < 0.01 2 vs. 3 P < 0.05 2 vs. 3 P < 0.01 2 vs. 3 P < 0.012 vs. 4 P < 0.05 2 vs. 4 P < 0.01

3. REM 112.0 ± 15.2 range: 23.8 ± 2.6 range: 115.2 ± 13.6 range: 56.5 ± 13.3 range: 483 ± 166 range: 197.5 ± 42.1 range:(n = 13) 89.4–141.2 20.4–27.6 89.4–140.8 31.2–84.1 278–760 162.1–286.4

3 vs. 4 P < 0.05 3 vs. 4 P < 0.05 3 vs. 4 P < 0.01

4. OND 110.4 ± 7.5 range: 37.8 ± 13.6 range: 106.9 ± 17.1 range: 69.7 ± 14.7 range: 610 ± 100 range: 184.5 ± 45.7 range:(n = 14) 94.5–121.6 20.6–67.2 80.2–148.2 52.7–94.6 460–728 127.4–274.2

6. Mc Coll SR, Beause igle D, Gilbe rt C, Naccache PH. Priming of the humanneutrophil respiratoty burst by granuloc yte-mac rophage colony s timulat-ing factor and tumor nec rosis factor-a involves regulation at a pos t-c e llsurfac e rec eptor leve l: enhanceme nt of the e ffe ct of age nts w hichdirectly ac tivate G prote ins. J Im m u n o l 1990: 145; 3047–3052.

7. Asman B, Gustafs son A, Bergstrom K. Priming of neutrophils w ith tumornec rosis factor-a me asured as Fc g rece ptor-mediated respiratory burstcorre late s w ith increase d complement re ceptor 3 me mbrane de nsity. In tJ Clin Lab Re s 1996: 26; 236–239.

8. Condliffe AM, Chilvers ER, Hasle tt C, Drans fie ld I. Priming diffe rentiallyre gulate s neutrophil adhesion molec ule ex pres sion/func tion. Im m u no l-o g y 1996: 89; 105–111.

9. Monk PN, Barke r MD, Partr idge LJ. Multip le signalling pathw ays in theC5a-induce d ex pres sion of adhe sion rece ptor Mac-1. Bio c him Bio phy sAc ta 1994: 1221; 323 –329.

10. Jage ls MA, Chambers JD, Arfors KE, Hugli TE. C5a and tumor ne cros isfac tor-a induc ed leukocytos is oc curs inde npendently of b 2 integrins andL-se lectin: diffe rential e ffec ts on neutrophil adhesion mole cule ex pres-sion in v ivo. Blo o d 1995: 85; 2900–2909.

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14. Hartung HP, Arche los JJ, Zie lasek J, e t a l. Circulating adhes ion molec ulesand inflammatory mediators in demye lination: a review. Ne u ro lo g y1995: 45 (suppl 6); S22 –S32.

15. Kle in JB, Sche rzer JA, Harding G, Jacobs AA, McLe ish KR. TNF-astimulates incre ased plasma membrane guanine nucleotide bindingprote in activ ity in polymorphonuclear leukocyte s. J Leu ko c y te Bio l1995: 57; 500–509.

16. Lantz M, Bjornbe rg F, Olsson I, Richte r J. Adhe renc e of neutrophilsinduces re lease of soluble tumor necrosis factor receptor forms . JIm m u no l 1994: 152; 1362–1369.

17. Goto I, Shinno N, Kuroiw a Y. Prote olytic e nzyme activitie s in mono-nuclear ce lls and granuloc ytes of patients w ith various ne urologicaldisorders . J Ne u ro l Sci 1983: 59; 323–329.

18. Aoki Y, Miyatake T, Shimizu N, Yoshida M. Medullas in activity ingranulocytes of patients w ith multiple sc le rosis. Ann Ne uro l 1984: 15;245–249.

19. Guarnie ri B, Lolli F, Amaducc i L. Polymorphonuclear neutral proteaseactivity in multip le sc le rosis and othe r dise ases . An n Ne u ro l 1985: 18;620–622.

20. Casale L, Cardozo C, Kalb T, Les se r M. Quantitation of endopeptidase24.11 and endopeptidase 24.15 in human blood leukocytes . En zy m ePro te in 1994/95: 48; 143–148.

21. Riemann D, Kehle n A, Thie le K, Lohn M, Langner J. Induc tion ofaminope ptidase-N/CD13 on human lymphocytes afte r adhes ion tofibroblast-like synoviocyte s, endothe lial ce lls, e pithe lial ce lls, and mono-cytes/macrophages . J Im m un o l 1997: 158; 3425–3432.

22. We rfe l T, Sonntag G, Weber MH, Gotze O. Rapid incre ases in themembrane ex pres sion of neutral endopeptidase (CD10), aminopeptidaseN (CD13), tyrosine phosphatase (CD45) and Fc g -RIII (CD16) uponstimulation of human periphe ral leukocyte s w ith human C5a. JIm m u no l 1991: 147; 3909–3914.

23. Connely JC, Chamble rs R, Holiday D, Chittende n K, Johnson AR. Up-re gulation of neutral e ndope ptidase (CALLA) in human neutrophils bygranulocyte-macrophage colony-s timulating factor. J Leu ko c y te Bio l1993: 53; 685–690.

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25. Zeman K, Kantorski J, Paleolog E, Fe ldmann M, TchÛrzew ski H. The roleof re ceptors for tumor necros is factor-a in the induction of humanpolymorphonuclear neutrophil chemiluminesc ence . Im m u n o l Le tt1996: 53; 45 –50.

26. We iss SJ. Tissue destruction by neutrophils. N Eng l J Med 1989: 320;365–376.

ACKNOWLEDGEMENTS. The author s w ould like to thank Professor Wojc iechStec and Dr Boúze na Szymańska from the Department of BioorganicChemistry Polish Academy of Sc ience , Lódź.

This w ork w as supporte d by a Grant No. 4 PO5A 100 09 from the StateCommittee for Sc ie ntific Rese arch, Poland (KBN).

Received 27 May 1998;accepted in revised form 31 August 1998

J. Ziçcabe r et al.

338 Mediators of Inflammation · Vol 7 · 1998

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