Dyad of CD40/CD40 Ligand Fosters Neuroinflammation at the Blood-Brain Barrier and Is Regulated via...

Dyad of CD40/CD40 ligand fosters neuroinflammation at theblood brain barrier and is regulated via JNK signaling:Implications for HIV-1 encephalitis

Servio H. Ramirez‡, Shongshan Fan‡, Holly Dykstra‡, Nancy Reichenbach‡, Luis DelValle‡,*, Raghava Potula‡, Richard P. Phipps§,#, Sanjay B. Maggirwar§, and YuriPersidsky‡‡Department of Pathology & Laboratory Medicine, Temple University School of Medicine,Philadelphia, PA, USA*Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA§Department of Microbiology & Immunology, University of Rochester School of Medicine,Rochester, NY, USA#Department of Environmental Medicine, University of Rochester School of Medicine, Rochester,NY, USA

AbstractHuman immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheralmonocytes followed by their infiltration into the central nervous system, where the release of pro-inflammatory mediators causes neurologic disease. Previously we detected high levels of solubleCD40-ligand (CD40L) in cerebrospinal fluid and plasma of HIV-infected patients with cognitiveimpairment. We now show that CD40, a receptor for CD40L, is highly expressed in brainendothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important rolefor the CD40/CD40L dyad in regulating blood-brain-barrier (BBB) functions. This concept wasfurther supported by in-vitro experiments. Exposure of primary human brain microvascularendothelial cells (BMVEC) to CD40L up-regulated the expression of adhesion molecules,ICAM-1 and VCAM-1, which caused a 4-fold increase in monocyte adhesion to BMVEC andstimulated migration across an in-vitro BBB model. Investigations into the intracellular signalingpathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40activation in the BMVEC. CD40L induced activation of mixed-lineage-kinase-3 (MLK-3) andJNK leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition inthe BMVEC prevented CD40L-mediated induction of adhesion molecules, monocyte adhesionand transendothelial migration. These new findings support the concept that the CD40/CD40Ldyad plays an important role in HIVE neuroinflammation.

KeywordsCD40; brain endothelial cells; blood brain barrier; monocyte; HIV-1 encephalitis

Corresponding author: Yuri Persidsky, Department of Pathology and Laboratory Medicine, Temple University School of Medicine,3401 N. Broad St., Philadelphia, PA 19140 [email protected], Phone: (215) 707-4353 Fax: (215) 707-2781.

NIH Public AccessAuthor ManuscriptJ Neurosci. Author manuscript; available in PMC 2011 January 1.

Published in final edited form as:J Neurosci. 2010 July 14; 30(28): 9454–9464. doi:10.1523/JNEUROSCI.5796-09.2010.

NIH

-PA Author Manuscript

NIH


NIH


IntroductionDespite the introduction of highly active antiretroviral therapy (HAART) which efficientlysuppresses viral replication and normalizes immunologic parameters, a significant numberof HIV-1 - infected patients show progressive loss of cognitive abilities. These cognitivedeficits are collectively termed HIV-associated neurocognitive disorder (HAND)(Boisse etal., 2008; Minagar et al., 2008). The pathogenesis of HAND involves activation ofmonocytes and their subsequent recruitment into the central nervous system (CNS) alteringblood brain barrier (BBB) function and resulting in HIV-1 encephalitis (HIVE) (Persidsky etal., 2006a). The effector molecules and mechanisms that regulate monocyte migration acrossthe BBB remain poorly defined. Enhanced expression of adhesion molecules on brainmicrovascular endothelial cells (BMVEC) triggered by inflammatory mediators (Mondal etal., 2004; Ramirez et al., 2008) control leukocyte trafficking into the CNS. Increasedexpression of adhesion molecules and BBB permeability has been demonstrated in HANDpatients (Eugenin et al., 2006). A disrupted BBB allows accumulation of toxic serumproteins and increased infiltration of monocytes and lymphocytes, thereby acceleratinginflammation and viral entry into the CNS. HAART fails to control BBB leakage andinflammation in HAND patients (Avison et al., 2004b; Eilers et al., 2008), in part because itdoes not reduce the high levels of CD40 ligand found in the plasma and cerebrospinal fluid(CSF) of HIV-1 infected patients (Sipsas et al., 2002; Sui et al., 2007). As demonstrated inother systems (Piguet et al., 2001; Ishikawa et al., 2005; Sitati et al., 2007), high levels ofsCD40L can regulate CNS inflammation at the level of the BBB.

CD40L (CD154) is a 33 kDa type II membrane glycoprotein from the TNFα family. CD40Lis expressed predominantly by activated leukocytes and platelets (Li, 2008). In addition tothe membrane-bound form of the protein, 31 kDa and/or 18 kDa versions of CD40L can besecreted or shed from activated cells. Either form of CD40L retains the ability to activateCD40, a 45- to 50-kDa type I membrane glycoprotein expressed at a low level in restingcells of myeloid and vascular origin (Sui et al., 2007;Mancino et al., 2008;Pluvinet et al.,2008). CD40 expression is rapidly up regulated in these cells following exposure to pro-inflammatory mediators (Sui et al., 2007;Pluvinet et al., 2008).

Elevated levels of sCD40L are found in a variety of diseases in which sCD40L is thought toinitiate or potentiate inflammation (Tsakiris et al., 2000; Heeschen et al., 2003; Devaraj etal., 2006). Inflammatory conditions increase the expression of the CD40 receptor on thesurface of endothelial cells and the shedding of the ligand (Chai et al., 2006). In HIV-1neuropathogenesis, a connection between CD40 and microglia has been established.Upregulation of CD40 expression has been detected on microglia of HIV-1-infected braintissues (D'Aversa et al., 2005). CD40L was also shown to potentiate the ability of HIV-1protein (Tat) to activate monocytes and microglia leading to the secretion of neurotoxicinflammatory mediators (Sui et al., 2007).

A role for the CD40/CD40L dyad in brain endothelium remains largely unknown. Herein,we detected high levels of CD40 on brain endothelium in patients with HIVE. Wedemonstrated that engagement of endothelial CD40 promotes adhesion and migration ofleukocytes across an in-vitro BBB model. Our studies also show that CD40 signalingconverges to the JNK signaling pathway, which was found to mediate the effects of CD40Lon the endothelial regulation of leukocyte adhesion and migration.

Ramirez et al. Page 2

J Neurosci. Author manuscript; available in PMC 2011 January 1.

NIH


NIH


NIH


Materials and MethodsReagents

Recombinant human soluble CD40L (sCD40L) was purchased from ProSpec (Rehovot,Israel). Recombinant membrane bound CD40L, CD40L(M), and corresponding controlmembranes that lack CD40L, were generated in a Baculovirus based expression system (Rayet al., 2005; Sui et al., 2007). Neutralizing antibodies to human CD40, recombinant humanTNFα and CCL2/MCP-1 were purchased from R&D Systems (Minneapolis, MN). Thefollowing inhibitors were obtained from Calbiochem (San Diego, CA): JAK inhibitor P6,JAK-3 inhibitor-VI (3′-pyridyl oxindole derivative), IKK inhibitor-X (N-(6-chloro-9H-b-carbolin-8-yl) nicotinamide), p38 inhibitor SB202190, ERK inhibitor FR180204, JNKinhibitor I ((L)-HIV-TAT48–57-PP-JBD20) and JNK inhibitor II (SP600125). Unlessspecified all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell culturePrimary brain microvascular endothelial cells (BMVEC) were supplied by Dr. MichaelBernas and Dr. Marlys Witte (University of Arizona, Tucson, AZ). BMVEC were isolatedfrom vessels of normal tissue derived from brain resections performed for the treatment ofintractable epilepsy. The BMVEC cultures were used until passage 5 and were expanded inDMEM/F-12 media supplemented with 10% heat-inactivated fetal bovine serum (FBS),endothelial cell growth supplement (ECGS, BD Bioscience, Franklin Lakes, NJ), heparin(1mg/ml, Sigma), amphotericin B (2.5µg/ml, Invitrogen, Carlsbad, CA), penicillin (100U/ml, Invitrogen) and streptomycin (100µg/ml, Invitrogen). Prior to experimentation, BMVECmonolayers were placed with media containing the above supplements but lacking ECGSand heparin. Under these conditions the BMVEC cultures were routinely evaluated for thepresence of endothelial markers and barrier formation.

Primary human monocytes were supplied by the Human Immunology Core at the Universityof Pennsylvania (Philadelphia, PA). The cells were isolated by countercurrent centrifugalelutriation and maintained in DMEM containing heat inactivated 10% FBS, penicillin (100U/ml), streptomycin (100 U/ml), and L-glutamine (2mM)(Ramirez et al., 2009) and wereused within 24 hrs of isolation.

Immunofluorescence staining and image analysisEvaluation of CD40 expression was performed on frozen brain tissue from seven HIV-1infected patients (four with severe encephalitis(Persidsky et al., 2006b) and three withoutevidence of encephalitis), and four seronegative age-matched controls were provided by theNational NeuroAIDS Consortium (Washington, DC) (Table 1). Indirectimmunofluorescence was performed on serial sections cut at 5 µm thick. Monoclonalantibodies to human CD68 (diluted 1:200, Abcam, Cambridge, MA), CD163 (1:200, SantaCruz Biotech, Santa Cruz, CA), ICAM-1 and VCAM-1 (diluted 1:200, R&D Systems) orpolyclonal antibodies to human CD40 (diluted 1:50, Abcam) together with rhodamineconjugated Ulex europeus agglutinin 1 lectin (UEA-1, 2µg/ml, Vector Laboratories,Burlingame, CA), were placed on sections overnight at 4°C. Tissue sections were rinsed andsecondary antibodies conjugated to Alexa-488 (diluted 1:250, Invitrogen) were then addedfor 1 hr. The slides were mounted with Prolong antifade reagent containing DAPI(Invitrogen).

We performed semi-quantitative image analysis to determine the up-regulation of CD40 inHIVE. For each case, images from 5 fields were acquired with equal acquisition parametersunder 20× objective magnification for both CD40 and UEA-1 using a Coolsnap EZ CCDcamera (Photometrics, Tucson, AZ) coupled to a Nikon i80 Eclipse (Nikon, Tokyo, Japan).



NIH


NIH


NIH


Up to 20 vessels (with diameters between 30–60µm) per field were identified using theUEA-1 label and then matched to the corresponding CD40 staining. The integrated intensity(average optical densities from all the pixels measured) was determined by placing a definedrectangular box (10µm × 100µm) region of interest within the CD40 labeled vessels usingAxiovision imaging software (Zeiss, Thornwood, NY). For display purposes, Axiovisionwas also used to pseudo-color the acquired fluorescence images.

Flow cytometrySurface expression of adhesion molecules was measured in BMVEC by flow cytometry.Briefly, following various treatments, 2 × 106 cells were placed in staining solution (2%BSA in PBS with 0.5% NaN3) containing fluorophore-conjugated antibodies to CD40 (anti-CD40-Phycoerythrin, R&D Systems), CD54 (anti-ICAM-1-allophycocyanin, BDBiosciences) and CD106 (anti-VCAM-1- FITC, BD Biosciences) for 30 min on ice. Cellswere then washed and fixed in 2% methanol-free formaldehyde (Thermo Scientific,Rockford, IL) in 1× PBS. Acquisition and analysis of the labeled cells were then performedusing a FACSCalibur flow cytometer (BD Biosciences). Acquisition parameters and gatingwere controlled by CellQuest software (BD Biosciences). Data analysis was performed withFlowJo software (Tree Star Inc, Ashland, OR). The data represent the mean fluorescenceintensity (MFI) of gated populations (as determined by isotype-matched controls; seeSupplemental Figure S1A and S1B) of at least 10,000 events recorded in a single experimentwhich was repeated at least three times.

Western blotBMVEC monolayers were treated and lysed for either total protein or nuclear protein. Celllysates were prepared using the CelLytic-M cell lysis reagent (Sigma) containing proteaseinhibitor cocktail (Sigma) and phosphatase inhibitor cocktail set I (Calbiochem). Proteinsfrom the nuclear fraction were isolated using the CelLytic nuclear extraction kit (Sigma).The protein content in the lysate/fraction was then determined using the BCA protein assay(Thermo Scientific). Protein lysate/fraction containing 20µg of protein was mixed with 2×Laemmli (sample loading) buffer containing β-mercaptoethanol and then boiled for 5 min.The proteins were then resolved by SDS-PAGE (4–20% precast gels; Thermo Scientific),followed by electrophoretic transfer to nitrocellulose membranes. The following primaryantibodies diluted in 1× TBS/0.1% tween 20 were used to detect target proteins: anti-CD40(diluted 1:500), anti-β tubulin (diluted 1:2000), anti-lamin A/C (diluted 1:2000) fromAbcam, anti-ERK1/2 (diluted 1:1000), anti-p-ERK1/2 thr202/tyr204 (diluted 1:1000), anti-JNK (1:500), anti-p-JNK thr183/tyr185 (diluted 1:250), anti-MLK3 (diluted 1:500), anti-p-MLK3 thr277/ser281 (diluted 1:250), anti-cJun (diluted 1:1000), and anti-p-cJun ser63(diluted 1:500) all purchased from Cell Signaling Technology (Danvers, MA). Allantibodies (except for β tubulin, 1 hr at room temperature) were incubated with themembranes overnight at 4°C under gentle shaking. Bound primary antibodies were exposedto the corresponding species-specific peroxidase-conjugated secondary antibody (diluted1:5000, Thermo scientific) for 1 hr at room temperature, and detected using the supersignalWest-Femto chemluminescent substrate (Thermo scientific), acquired on a G:Box ChemiHR16 (Syngene, Frederick, MD) gel documentation system.

Transcription factor ELISAEvaluation of DNA binding by activated cJun was performed using the TransAM cJuntranscription factor assay (Active Motif, Carlsbad, CA). BMVEC (2 × 106) were exposed toincreasing concentrations of sCD40L or anisomysin (control) for 30 min followed by lysisand extraction of the nuclear fraction. Nuclear proteins (10µg) were allowed to bind tooligonucleotide coated 96-well plates. The bound proteins were quantitated using antibodies



NIH


NIH


NIH


described above and measuring absorbance at 450 nm in a Spectramax M5 (MolecularDevices, Sunnyvale, CA).

Adhesion and migration assaysQuantitative adhesion and migration assays were performed as previouslydescribed(Ramirez et al., 2008). For adhesion and migration assays, BMVEC were seededon collagen type I coated 96-well plates (adhesion) or on 3-micron 24-well tissue cultureinserts (FluoroBlok, BD Bioscience) at a density of 2.5 × 104 cells/well. Confluentmonolayers were then exposed to the indicated experimental treatments. Freshly isolatedhuman monocytes at 5 × 106 cells/ml were labeled with 5µM of the fluorescent tracer,calcein-AM (Invitrogen). All treatments were removed from the endothelial cells prior toadding monocytes at 2.5 × 105 cells/well or insert.

For adhesion assays, the monocytes and endothelial cells were incubated together for 15 minand then rinsed 3 times with 1× PBS to eliminate non-adherent monocytes. For migrationassays, the monocytes were placed for 2 hrs in the upper chamber of the tissue culture insertsystem, while chemoattractant, CCL2/MCP-1 (50 ng/ml), was added to the lower chamberto stimulate migration. The fluorescence of adherent monocytes or migrated monocytes wasmeasured using a Spectramax M5 fluorescence plate reader (Molecular Devices). Theamount of migrated monocytes was determined from external standards of known numbersof labeled monocytes. The results for adhesion are represented as the mean ± SEM foldadhesion (number of adherent monocytes for each experimental condition divided by thebasal adhesion of the untreated control). The results for migration are shown as the averagefold migration ± SEM; the fold migration is derived from the number of migratedmonocytes for each experimental condition divided by the number of migrated monocytes inthe untreated, no chemoattractant control.

Statistical analysisThe values shown in all figures and those mentioned in the text represents the average ±SEM of experiments that were conducted multiple times (as indicated). Statisticalsignificance (P < 0.05) was determined by performing unpaired two-tailed Student’s t test orANOVA utilizing Prism v5 software (GraphPad Software Inc., La Jolla, CA).

ResultsCD40 is upregulated in brain endothelium from patients with HIVE

In order to establish clinical relevance of CD40 expression in BMVEC in the context ofHIV-1 infection, we evaluated the CD40 levels in the frontal cortex derived from sevenHIV-1 infected patients of which four cases were determined to be representative of severeHIVE(Persidsky et al., 1999; Persidsky et al., 2006b), whereas no neuropathologic changeswere detected in three other cases (Table 1). As a negative control, the CD40 levels werealso examined in frontal cortex obtained from seronegative patients. Since the degree ofneurologic deficit in HIV-infected individuals is strongly correlated with the number ofactivated macrophages and microglia within the basal ganglia and frontal lobes(Persidsky etal., 1999; Avison et al., 2004b), we first verified the severity of HIVE by detecting CD68positive cells in the perivascular spaces. While very few CD68 cells were found in controlcases, HIVE brains featured mononuclear cell infiltration (Fig. 1A; additionalhistopathology in supplemental figure S2A). Next we examined CD40 expression bystaining with anti-CD40 antibodies and double-labeling with the lectin, UEA-1, to identifythe microvasculature. Control brains demonstrated minimal level of CD40 expression(Figure 1 B, case 8); however, CD40 expression was readily detectable in endothelial cells(arrowheads) in brain tissues affected by severe HIVE (Figure 1B, cases 1, 2, 3, and 4) and



NIH


NIH


NIH


to a lesser extent in tissue from HIV-1 infected patients without HIVE (data not shown).Consistent with previous observations(D'Aversa et al., 2002), astrocytes and microglia insevere HIVE cases were positive for CD40 (Figure 1C, cases 1 and 4), and noimmunostaining was detected in controls (Figure 1C, case 8). The increase in CD40immunostaining seen in HIVE cases paralleled the up-regulation of adhesion molecules inbrain endothelium, VCAM-1(Supplemental Figure S2B) and to a less degree ICAM-1 (datanot shown) as compared to controls. This new finding suggested that CD40 plays a role inpromoting leukocyte infiltration across the BBB.

Next we assessed CD40 expression in brain endothelium. For each case, up to 5 fields wereacquired under 20× objective magnification for both CD40 and UEA-1. Up to 20 vessels perfield were identified using UEA-1 staining and then matched to the corresponding CD40labeling. The integrated intensity was measured by placing a defined rectangular region ofinterest on the vessel. We found that CD40 expression was up-regulated over 2 fold inHIV-1 patients (without HIVE) and over 5 fold in cases of HIVE when compared to theseronegative controls (Figure 1D, P < 0.01). These data suggest that CD40 expression isindeed increased in brain endothelium during HIVE, and levels appear elevated even inHIV-1 infection without evidence of encephalitis.

CD40L promotes monocyte adhesion to and migration across human BMVECIncreased expression of CD40 can potentially promote leukocyte adhesion to and migrationacross endothelium. Indeed, augmented CD40L levels have been found in inflammatoryconditions including HIV-1 CNS infection(Sui et al., 2007; Rizvi et al., 2008). However, theability of CD40L to mediate monocyte adhesion to and/or migration across brainendothelium has not been studied. Therefore, we sought to determine whether CD40L couldaffect monocyte adhesion to BMVEC monolayers. We conducted immunoblot analyses andfound detectable amounts of CD40 in the lysates of BMVEC, but not in negative controlcells (HeLa) (Figure 2A).

Next, we performed monocyte adhesion assays in which monolayers of BMVEC wereexposed to two forms of CD40L; membrane-bound CD40L, CD40L(M); this form ofCD40L mimics CD40L that is expressed on the surface of activated cells), and solubleCD40L (sCD40L; similar to CD40L that is shed or released by activated cells). Doses ofCD40L(M) were similar to those previously validated in cell culture (Ray et al., 2005; Ryanet al., 2005). Soluble CD40L was applied in concentrations used by otherinvestigators(Kehry and Castle, 1994). Adhesion assays revealed that exposure toCD40L(M) induced a 5-fold increase in the number of monocytes attaching to the BMVECmonolayers (P < 0.01, Figure 2B). This effect of CD40L(M) was specific to CD40L, at alldoses. Exposure to equivalent amounts of control membranes that lack CD40L resulted inmonocyte attachment comparable to untreated BMVEC. Similarly, sCD40L produced ~3.5-fold increase in monocyte adhesion over untreated BMVEC (Figure 2C). Increases inmonocyte adhesion were completely blocked following co-administration of CD40neutralizing antibodies, but not by addition of isotype-matched non-immune serum (Figure2B and 2C).

Analogous to sCD40L(Sipsas et al., 2002; Sui et al., 2007), high levels of TNFα are alsopresent in the blood of HIV-1 infected patients especially in those with greater BBBimpairment(Sharief et al., 1992). We sought to determine whether TNFα augments theability of CD40L to induce monocyte adhesion. Pretreatment of BMVEC with TNFαinduced monocyte adhesion by almost 4-fold; this was further enhanced following co-administration of sCD40L, suggesting additive effect of these two pro-inflammatorymediators on monocyte adhesion (Figure 2C).



NIH


NIH


NIH


Subsequently, we tested whether CD40 engagement in BMVEC would promote monocytepassage (migration) across BMVEC monolayers. BMVEC were treated with bothCD40L(M) and sCD40L, after which fluorescently labeled human monocytes were placed inthe upper chamber and allowed to migrate across the BMVEC monolayer. Addition ofCD40L caused a dose-dependent increase in monocyte passage across BMVEC monolayers(up to 2-fold, P < 0.01, Figure 2D and 2E). We also used CCL2/MCP-1 as a chemoattractantimplicated in HIVE pathogenesis(Persidsky et al., 1999). Application of CCL2/MCP-1 tothe lower chamber of BBB construct increased monocyte migration by 2.5-fold compared tomodels without chemokine addition. This effect of CCL2/MCP-1 was more intense (over 4fold increase for the highest concentration tested) in BMVEC monolayers pretreated witheither CD40L configuration (Figure 2D and 2E). As in the case of the adhesion, CD40Lmediated transendothelial migration was also significantly blocked by CD40 neutralization.Our data suggest that the CD40/CD40L interaction in BMVEC may play an important rolein controlling trans-BBB migration of monocytes during HIVE

CD40L promotes expression of adhesion molecules in BMVECTo better understand how CD40 engagement regulates monocyte migration across the BBB,we determined whether CD40L treatment caused increased expression of adhesionmolecules on BMVEC. BMVEC were exposed to sCD40L for 24 hrs, either alone ortogether with CD40 neutralizing antibodies. The cells were stained using antibodies toVCAM-1, ICAM-1 or isotype-matched non-immune antibodies. Treatment of BMVEC withsCD40L resulted in a 2-fold increase in cell surface expression of VCAM-1 (Figure 3),whereas, nearly a 4 to 5 fold increase in ICAM-1 expression was observed in BMVEC underthese conditions. These effects of sCD40L were completely reversed by pre-incubation ofthe cells with neutralizing antibodies to CD40. These data indicate that CD40 stimulation byCD40L promoted adhesion molecule expression.

CD40L stimulates expression of adhesion molecules via activation of JNK-dependentsignaling events

To understand the molecular basis for the observed induction of ICAM-1 and VCAM-1, wedetermined whether sCD40L might activate specific signaling pathways, and therebypromote sCD40L-mediated effects on adhesion molecule production by BMVEC. Weexplored putative signaling molecules previously identified as targets for stimulation byCD40L including Janus kinase (Jak)(Klein et al., 2003), IκB-α kinase (IKK)(Schwabe et al.,2001), JNK and mitogen - activated protein kinase (MAPK) family members ERK1/2 andp38(Yu net al., 2004; Chen et al., 2006; Li and Nord, 2009) that determine activation oftranscription factors such as Signal Transducers and Activators of Transcription (STAT),Nuclear factor κB (NF-κB), and cJUN, respectively. We used specific cell-permeablepharmacological and biological inhibitors of relevant signaling molecules in order to dissectCD40L induced increased expression of adhesion molecules. BMVEC were pretreated withthe inhibitors for 30 min then co-incubated with sCD40L for 24 hrs after which ICAM-1 andVCAM-1 expression was detected by FACS analysis. The JAK/STAT inhibitor (pyridone 6,10µM) and JAK3/STAT3 inhibitor VI (3′-pyridyl oxindole based compound, 1µM)produced no decrease in expression of either ICAM-1 or VCAM-1 (Figure 4). Inhibition ofthe IKK/NFκB pathway with the potent IKK ATP-competitor, N-(6-chloro-9H-b-carbolin-8-yl)nicotinamide (1µM) only marginally diminished VCAM-1 surface levels without effecton ICAM-1 expression. Inhibition of p38 (by FHPI, 5µM) and of ERK1/2 (by FR180204,10µM) failed to prevent CD40L- inducted up-regulation of adhesion molecules (Figure 4).These results were in stark contrast to the effects of a JNK inhibitor (cell permeable JNK-binding domain peptide) that caused a near complete inhibition of ICAM-1 and VCAM-1.These data indicate that enhanced expression of adhesion molecules by signaling eventstriggered by the CD40L/CD40 dyad is sensitive to inhibition of JNK.



NIH


NIH


NIH


CD40L activates JNK and ERK1/2 but not p38To test whether JNK-dependent signaling was activated by CD40L in BMVEC, weexamined the ability of sCD40L to promote the phosphorylation and thus activation ofmixed-lineage kinase-3 (MLK3), this molecule targets JNK. As determined by immunoblotanalyses, rapid phosphorylation of MLK3 at residues Thr-277 and Ser-281 occurred insCD40L-exposed BMVEC (Figure 5A). This effect indicates activation of MLK3 becauseautophosphorylation at Thr-277 and Ser-281 is an obligatory intermediate step of MLK3activation, following its dimmerization in response to specific stimuli(Du et al., 2005).

To test whether downstream kinases known to be activated by MLK3 were also involved insCD40L-induced expression of adhesion molecules, cell lysates were analyzed for activationof JNK and other MAPKs, namely p38 and ERK1/2. As shown in Figure 5B and C,immunoblots revealed enhanced phosphorylation of JNK (including both JNK1 and JNK2isoforms of JNK) and ERK1/2, indicating activation of these kinases. However, under theseconditions, phosphorylation of p38 MAPK was not detected (Figure 5D), suggesting thiskinase does not mediate CD40L signaling in BMVEC.

To verify whether CD40L activates JNK, additional experiments were performed in whichBMVEC were exposed to sCD40L and then examined for the phosphorylation of cJUN atsites commonly targeted by JNK. Since phosphorylation of cJUN at Ser-63 rapidly triggersits translocation, we prepared nuclear extracts and subjected them to immunoblot analyses.Prompt accumulation of cJUN was observed in BMVEC treated with sCD40L (Figure 5E).Moreover, cJUN protein detected in the nucleus was found predominantly phosphorylated atSer-63, further suggesting activation of the JNK.

Finally, we tested whether the increased phosphorylation of cJUN in sCD40L treatedBMVEC resulted in higher DNA-binding activity of cJUN containing complexes oftranscription factors (largely referred to as the AP-1 family). Nuclear extracts were preparedand the DNA-binding activity of AP-1 was measured. AP-1 DNA binding activity wasrapidly stimulated in sCD40L-treated cells (Figure 5F), indicating activation of AP-1.

Inhibition of JNK signaling blocks monocyte adhesion and trans-endothelial migrationstimulated by sCD40L

To test whether activation of JNK signaling mechanisms by sCD40L in BMVEC isbiologically significant, we determined whether JNK inhibition could decrease the monocytemigration induced by sCD40L. BMVEC exposure to sCD40L led to a 3.3-fold increase inthe number of monocytes adhering to the BMVEC monolayers (P < 0.01, Figure 6A).Increased adhesion was reduced by 2-fold and 1.6-fold in the cells exposed to sCD40Ltogether with a JNK biological inhibitor (JBD peptide) or a pharmacological inhibitor(SP600125), respectively. To verify whether JNK signaling indeed plays a crucial role inregulation of monocyte adhesion, we also employed a positive control, TNFα. Treatment ofBMVEC monolayers with TNFα increased monocyte adhesion more than 5 fold, which wasreduced by 60% following treatment with JNK inhibitors consistent with previouslypublished data(Ramirez et al., 2008).

Next, we tested whether JNK inhibition in endothelial cells could prevent monocyte passage(migration) across BMVEC monolayers activated with sCD40L. sCD40L applicationincreased migration by 2.2-fold, which was reduced by 30% following addition of JNKinhibitors (Figure 6B). We used CCL2/MCP-1 as a relevant chemoattractant (applied to thelower chamber of BBB models). In the presence of CCL2/MCP-1, sCD40L treatmentincreased migration nearly 5-fold. Remarkably, as seen without chemoatractant, JNKinhibitors reduced transendothelial migration by almost 70% (P < 0.01). Thus, JNKinhibitors significantly attenuated monocyte adhesion and transendothelial migration in



NIH


NIH


NIH


CD40-activated endothelial monolayers. Additionally, JNK inhibitors were also effective atblocking the synergistic effect seen in adhesion/migration when sCD40L was added in thepresence of a second inflammatory factor, TNFα or chemoattractant, CCL2/MCP-1.

To ensure that the effects by JNK inhibitors seen in the functional assays are due to JNKinhibition and not that of broad inhibition on other kinases (like ERK); lysates from cellstreated with sCD40L or in combination with JNK inhibitor were evaluated. The blots inFigure 5G, show the nuclear accumulation of phosphorylated c-jun in the CD40L treatedalong with its significant reduction in cells that were co-incubated with CD40L and JNKinhibitor. In contrast, ERK activation by CD40L was unaffected by the presence of JNKinhibitors (Figure 5H). Therefore adhesion and transendothelial migration stimulated byCD40/CD40L are mediated by JNK and not by other kinases (like ERK).

DiscussionCD40/CD40L interactions in endothelial cells have been implicated in several pathologicconditions including atherosclerosis, allograft rejection, Alzheimer disease and chronicinflammation reviewed previously in (Phipps, 2008) and (Town et al., 2001). Using humanbrain tissues affected by HIVE (HIV Encephalitis), we found prominent up-regulation ofCD40 in brain endothelium. CD40 expression levels paralleled the severity of HIVE,expression of adhesion molecules and immune cell infiltration into the neuropil. SinceCD40L levels are increased in the blood of HIV-1 infected patients, we investigated whetherCD40-CD40L interactions at the BBB promoted neuroinflammation. Exposure of BMVECto sCD40L led to a 4-fold increase in monocyte adhesion to BMVEC monolayers andparalleled the up-regulation of adhesion molecules. Using BBB models we demonstratedthat sCD40L pretreatment of BMVEC produced a 4-fold increase in monocyte migrationacross endothelial monolayers in response to a relevant chemoattractant, CCL2/MCP-1.Next, we defined sCD40L/CD40 signaling in BMVEC, demonstrating fast activation ofERK1/2 and JNK1 in response to CD40L. Interestingly, only inhibition of JNK1 reversedthe induction of ICAM-1 and VCAM-1. Activation of the JNK1 pathway by sCD40L led toan increase in phosphoryation, nuclear translocation and DNA binding activity of thetranscription factor c-Jun. Suppression of JNK1 prevented the enhanced monocyte adhesionto and transendothelial migration across BMVEC monolayers exposed to sCD40L. Takentogether, up regulation of CD40 in HIVE brain tissue, and increased monocyte adhesion andtransmigration across BMVEC exposed to CD40L, accompanied by an increase in adhesionmolecules, point to an important role for the CD40/CD40L dyad in BBB disruption duringneuroinflammation.

CD40 belongs to the TNF receptor family and is expressed in immune cells(Chen et al.,2006). CD40 is also detected in non-immune cells like those comprising the vascular wall,in particular, in endothelial cells(Urbich and Dimmeler, 2004). Activation of CD40 resultsfrom engagement to its natural ligand CD40L. sCD40L has similar induction characteristicsto the trans-membranous form. Triggering of CD40/CD40L interaction initiates multiplesignaling cascades that lead to the release of key pro-inflammatory mediators includingIL-1, IL-6, IL-8, IL-10, TNFα, MIP-1α and CCL2/MCP-1. Co-stimulatory molecules(including its own receptor CD40) and adhesion molecules (ICAM-1, VCAM-1, E-selectin)are induced by the CD40-CD40L dyad(Elgueta et al., 2009). In terms of the endothelium,activation of CD40 is a critical component of inflammatory response and a strongcontributor to chronic inflammation in cardiovascular disease(Hassan et al., 2009).

In the CNS, a number of studies focusing on neurons and glial cells point to the importanceof CD40 in the pathobiology of chronic neuroinflammation (Tan et al., 1999a; Town et al.,2001:Calingasan, 2002 #1264). For example, in studies related to Alzheimer’s disease (AD),



NIH


NIH


NIH


exposure of β-Amyloid (Aβ) peptides to cultured microglia induces the expression of CD40which is further elevated with the co-addition of interferon-γ (Tan et al., 1999b). Moreimportantly, the upregulation of microglial CD40 by Aβ1–42 and in combination withsoluble CD40L, results in significant neuronal loss (Tan et al., 1999b). In-vivo studies havealso shown that the β-amyloid plaque burden and microgliosis are markedly reduced inTgAPPsw (AD mouse model) mice crossed with CD40L knock-out mice (Tan et al., 2002b).Therefore, interruption of the CD40-CD40L dyad directly attenuates the neuroinflammationassociated with the pathology of AD (comprehensively reviewed in (Tan et al., 2002a).

Thus far, the inflammatory contribution of CD40-CD40L in the brain have primary centeredon neurons, astrocytes and microglial cells; while the effects of CD40 on brain endothelialcells remains largely unexplored. The involvement of CD40 in the brain vasculature isunderscored in a study by Togo and colleagues where strong CD40 positive immunolabelingof cerebral vessels is observed in post-mortem tissue from AD cases and other neurologicaldiseases (Togo et al., 2000). The studies reported here provide the first evidence of CD40upregulation in the brain vasculature in viral encephalitis (HIVE) neuroinflammation.

During HIVE, viral infection of blood-borne or resident CNS macrophages induces reactiveastrocytosis, microglial nodules, BBB dysfunction and infiltration of monocytes into theperivascular spaces(Boisse et al., 2008). Chronic inflammation in HIVE causes disruption inneuronal networks leading to cognitive deficits(Kaul et al., 2001). The findings presentedhere demonstrate the abundant up-regulation of CD40 in the vasculature of brain tissuesaffected by HIVE. CD40 levels detected on brain endothelium parallel over expression ofVCAM-1 and ICAM-1(Persidsky et al., 1997). Our findings complement early studieswhere a marked increase of CD40 was found in activated microglia in HIVE brain(D'Aversaet al., 2005). It is possible that shed or secreted sCD40L by HIV-1 infected cells in the brainexacerbates and perpetuates inflammatory responses by acting on and up-regulating CD40on the brain endothelium. Cognitive impairment is clearly associated with BBBimpairment(Avison et al., 2004a) and increased levels of CD40L in CSF and serum(Sui etal., 2007).

Activation of the endothelium is a central component of immune-mediated inflammatorydiseases of the CNS. In the case of HIVE and multiple sclerosis, immune access to the CNSis a required step in the induction of chronic neuroinflammation(Minagar et al., 2002; Trebstet al., 2003). To evaluate whether CD40L activates brain endothelial cells to allow forincreased monocyte-endothelial interaction, we performed adhesion assays using primaryhuman BMVEC. We showed that CD40L greatly increased adhesion of monocytes toendothelial cells. Similarly, transendothelial migration was also enhanced in a dose-dependent manner with CD40L. In the presence of a secondary inflammatory mediator suchas TNFα or in CCL2/MCP-1 driven chemotaxis, sCD40L produces synergistic effectsleading to greater adhesion and transendothelial migration. Higher adhesion of resting andactivated T-cells to primary BMVEC exposed to sCD40L alone or in combination withTNFα has been shown previously(Omari and Dorovini-Zis, 2003). In addition to increases incytokine production, activation of the CD40/CD40L promoted expression of adhesionmolecules, ICAM-1 and VCAM-1 (Elgueta et al., 2009). Our analysis of ICAM-1 andVCAM-1 indicates sCD40L dose-dependent induction of both adhesion molecules. Theseresults differ from an early report using BMVEC, that showed a marginal increase inVCAM-1 after CD40 activation with anti-CD40 antibodies and a high VCAM-1 expressionafter BMVEC had been exposed to both the HIV-1 virus and CD40 activation (Moses et al.,1997). Perhaps the manner in which CD40 was activated may account for some of thedifferences since our studies used the CD40L to activate the receptor.



NIH


NIH


NIH


Intracellular signaling events triggered by the CD40/CD40L system have been characterizedin immune cells(Elgueta et al., 2009). In the brain, however, the focus on the CD40 pathwayactivation has centered on microglia, with little known about CD40 signaling in the brainendothelium(Chen et al., 2006). We explored the pathways that are activated by CD40/CD40L and the potential role in neuroinflammation via enhanced expression of ICAM-1 andVCAM-1. Although CD40 lacks intrinsic enzymatic activity, its trimerization with CD40Lpromotes interaction mainly with the TNF receptor-associated factors (TRAFs). Directly andvia the TRAFs, CD40 initiates multiple and diverse signaling cascades involved ininflammation including: the JAK/STAT, IKK/NFκB, p38 MAPK, ERK1/2, and JNK1/AP1(Elgueta et al., 2009). In the current report, we have systematically inhibited the knownpathways that participate in CD40 mediated inflammatory response in BMVEC activated bysCD40L and detected a nearly complete inhibition of the two adhesion molecules withJNK-1 inhibitors.

Analysis of phosphorylation status showed that not only JNK1 but also ERK1/2 was rapidlyactivated by sCD40L. Interestingly, the p38 MAPK appeared active in the BMVEC underresting conditions; however, it was not further activated/phoshorylated by sCD40L. Thisobservation contrasts to a previous study implicating that p38 MAPK activation wasinvolved in cytokine production after CD40 stimulation(Mathur et al., 2004). Our data aredifferent from results obtained using coronary endothelial cells where CD40L increasedphosphorylation of MAPK p38 and ERK1/2 supporting important tissue differences betweenbrain and endothelial cells from other tissues(Chen et al., 2008). Since ERK1/2 inhibitiondid not prevent adhesion molecule up-regulation by CD40, we investigated JNK1 activationof its downstream effectors. JNK1 contributes to the inflammatory response via AP-1transcription factors. Our analysis demonstrates that the AP1 transcription factor c-JUN hadincreased phosphorylation, nuclear translocation and DNA binding activity after CD40activation. Using HUVEC, Xia and colleagues reported that the sCD40L-induced sheddingof soluble ICAM-1 and VCAM-1 was a JNK-1 and p38 MAPK mediated phenomenon(Xiaet al., 2009). Our data also suggest that signal integration from CD40 in the BMVEC mayoccur via MLK3. Thus sCD40L engagement of CD40 generates signaling that activatesMLK3→JNK1→c-JUN leading to the induction of ICAM-1 and VCAM-1 in the BMVEC.If JNK1 inhibition blocks adhesion molecule expression by sCD40L, then monocyteadhesion and transendothelial migration would also be affected. The results presented heredemonstrate that inhibition of JNK drastically reduces monocyte adhesion to and migrationof monocytes across CD40 activated brain endothelial cells. Overall, our findings supportthe key concept that sCD40L is an important regulator of brain endothelial activation thatcould potentiate the progression of HIV-1 driven neuroinflammation and HAND.Uncovering the signaling mechanism has significant implications beyond HIV-1 CNSinfection, given the observed up-regulation of CD40L in Alzheimer’s disease, multiplesclerosis, stroke and numerous inflammatory conditions outside of the brain(Ishikawa et al.,2005; Desideri et al., 2008; Elgueta et al., 2009; Rezai-Zadeh et al., 2009).

The BBB is responsible for maintaining the delicate neuronal environment optimal forsynaptic communication. Therefore, it may also be important to understand whethersCD40L breaches barrier function by effects on the tight junction complexes. Supportingthis idea is the ability of sCD40L to decrease transendothelial electrical resistance, ameasure of barrier “tightness” (unpublished results). Thus, in conjunction with its pro-inflammatory inducing ability, CD40 may also acts as a BBB modulator.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.



NIH


NIH


NIH


AcknowledgmentsHuman BMVEC were provided by Drs. Marlys H. Witte and Michael Bernas through contract with the Universityof Arizona Medical Center (Tucson, AZ). The National Institutes of Health (NIH) National NeuroAIDSConsortium is acknowledged for brain tissue specimens used in this study. This study was supported (in part) byresearch funding from NIH: to RO1MH65151, RO1AA015913 (to YP), RO1NS054578 (to SBM, RPP),PO1MH64570, RO1MH56838 and RO1NS066801 (to SBM), RO1DE011390 (to RPP and SBM), RO1HL078603,RO1ES01247 (to RPP).

ReferencesAvison MJ, Nath A, Greene-Avison R, Schmitt FA, Greenberg RN, Berger JR. Neuroimaging

correlates of HIV-associated BBB compromise. J Neuroimmunol 2004a;157:140–146. [PubMed:15579291]

Avison MJ, Nath A, Greene-Avison R, Schmitt FA, Bales RA, Ethisham A, Greenberg RN, Berger JR.Inflammatory changes and breakdown of microvascular integrity in early human immunodeficiencyvirus dementia. J Neurovirol 2004b;10:223–232. [PubMed: 15371152]

Boisse L, Gill MJ, Power C. HIV infection of the central nervous system: clinical features andneuropathogenesis. Neurol Clin 2008;26:799–819. x. [PubMed: 18657727]

Borda JT, Alvarez X, Mohan M, Hasegawa A, Bernardino A, Jean S, Aye P, Lackner AA. CD163, amarker of perivascular macrophages, is up-regulated by microglia in simian immunodeficiencyvirus encephalitis after haptoglobin-hemoglobin complex stimulation and is suggestive ofbreakdown of the blood-brain barrier. Am J Pathol 2008;172:725–737. [PubMed: 18276779]

Chai H, Yan S, Wang H, Zhang R, Lin PH, Yao Q, Chen C. CD40 ligand increases expression of itsreceptor CD40 in human coronary artery endothelial cells. Surgery 2006;140:236–242. [PubMed:16904975]

Chen C, Chai H, Wang X, Jiang J, Jamaluddin MS, Liao D, Zhang Y, Wang H, Bharadwaj U, ZhangS, Li M, Lin P, Yao Q. Soluble CD40 ligand induces endothelial dysfunction in human and porcinecoronary artery endothelial cells. Blood 2008;112:3205–3216. [PubMed: 18658029]

Chen K, Huang J, Gong W, Zhang L, Yu P, Wang JM. CD40/CD40L dyad in the inflammatory andimmune responses in the central nervous system. Cell Mol Immunol 2006;3:163–169. [PubMed:16893496]

D'Aversa TG, Weidenheim KM, Berman JW. CD40-CD40L interactions induce chemokine expressionby human microglia: implications for human immunodeficiency virus encephalitis and multiplesclerosis. Am J Pathol 2002;160:559–567. [PubMed: 11839576]

D'Aversa TG, Eugenin EA, Berman JW. NeuroAIDS: contributions of the human immunodeficiencyvirus-1 proteins Tat and gp120 as well as CD40 to microglial activation. J Neurosci Res2005;81:436–446. [PubMed: 15954144]

Desideri G, Cipollone F, Necozione S, Marini C, Lechiara MC, Taglieri G, Zuliani G, Fellin R,Mezzetti A, di Orio F, Ferri C. Enhanced soluble CD40 ligand and Alzheimer's disease: evidenceof a possible pathogenetic role. Neurobiol Aging 2008;29:348–356. [PubMed: 17123665]

Devaraj S, Glaser N, Griffen S, Wang-Polagruto J, Miguelino E, Jialal I. Increased monocytic activityand biomarkers of inflammation in patients with type 1 diabetes. Diabetes 2006;55:774–779.[PubMed: 16505242]

Du Y, Bock BC, Schachter KA, Chao M, Gallo KA. Cdc42 induces activation loop phosphorylationand membrane targeting of mixed lineage kinase 3. J Biol Chem 2005;280:42984–42993.[PubMed: 16253996]

Eilers M, Roy U, Mondal D. MRP (ABCC) transporters-mediated efflux of anti-HIV drugs, saquinavirand zidovudine, from human endothelial cells. Exp Biol Med (Maywood) 2008;233:1149–1160.[PubMed: 18535159]

Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism andfunction of CD40/CD40L engagement in the immune system. Immunol Rev 2009;229:152–172.[PubMed: 19426221]



NIH


NIH


NIH


Eugenin EA, Gamss R, Buckner C, Buono D, Klein RS, Schoenbaum EE, Calderon TM, Berman JW.Shedding of PECAM-1 during HIV infection: a potential role for soluble PECAM-1 in thepathogenesis of NeuroAIDS. J Leukoc Biol 2006;79:444–452. [PubMed: 16507710]

Hassan GS, Merhi Y, Mourad WM. CD154 and its receptors in inflammatory vascular pathologies.Trends Immunol 2009;30:165–172. [PubMed: 19282242]

Heeschen C, Dimmeler S, Hamm CW, van den Brand MJ, Boersma E, Zeiher AM, Simoons ML.Soluble CD40 ligand in acute coronary syndromes. N Engl J Med 2003;348:1104–1111. [PubMed:12646667]

Ishikawa M, Vowinkel T, Stokes KY, Arumugam TV, Yilmaz G, Nanda A, Granger DN. CD40/CD40ligand signaling in mouse cerebral microvasculature after focal ischemia/reperfusion. Circulation2005;111:1690–1696. [PubMed: 15795333]

Kaul M, Garden GA, Lipton SA. Pathways to neuronal injury and apoptosis in HIV-1-associateddementia. Nature 2001;410:988–993. [PubMed: 11309629]

Kehry MR, Castle BE. Regulation of CD40 ligand expression and use of recombinant CD40 ligand forstudying B cell growth and differentiation. Semin Immunol 1994;6:287–294. [PubMed: 7865800]

Kim WK, Alvarez X, Fisher J, Bronfin B, Westmoreland S, McLaurin J, Williams K. CD163 identifiesperivascular macrophages in normal and viral encephalitic brains and potential precursors toperivascular macrophages in blood. Am J Pathol 2006;168:822–834. [PubMed: 16507898]

Klein B, Tarte K, Jourdan M, Mathouk K, Moreaux J, Jourdan E, Legouffe E, De Vos J, Rossi JF.Survival and proliferation factors of normal and malignant plasma cells. Int J Hematol2003;78:106–113. [PubMed: 12953803]

Li H, Nord EP. IL-8 amplifies CD40/CD154-mediated ICAM-1 production via the CXCR-1 receptorand p38-MAPK pathway in human renal proximal tubule cells. Am J Physiol Renal Physiol2009;296:F438–F445. [PubMed: 18667484]

Li N. Platelet-lymphocyte cross-talk. J Leukoc Biol 2008;83:1069–1078. [PubMed: 18198208]Mancino A, Schioppa T, Larghi P, Pasqualini F, Nebuloni M, Chen IH, Sozzani S, Austyn JM,

Mantovani A, Sica A. Divergent effects of hypoxia on dendritic cell functions. Blood2008;112:3723–3734. [PubMed: 18694997]

Mathur RK, Awasthi A, Wadhone P, Ramanamurthy B, Saha B. Reciprocal CD40 signals throughp38MAPK and ERK-1/2 induce counteracting immune responses. Nat Med 2004;10:540–544.[PubMed: 15107845]

Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C. The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associateddementia, Alzheimer disease, and multiple sclerosis. J Neurol Sci 2002;202:13–23. [PubMed:12220687]

Minagar A, Commins D, Alexander JS, Hoque R, Chiappelli F, Singer EJ, Nikbin B, Shapshak P.NeuroAIDS: characteristics and diagnosis of the neurological complications of AIDS. Mol DiagnTher 2008;12:25–43. [PubMed: 18288880]

Mondal D, Pradhan L, Ali M, Agrawal KC. HAART drugs induce oxidative stress in humanendothelial cells and increase endothelial recruitment of mononuclear cells: exacerbation byinflammatory cytokines and amelioration by antioxidants. Cardiovasc Toxicol 2004;4:287–302.[PubMed: 15470276]

Moses AV, Williams SE, Strussenberg JG, Heneveld ML, Ruhl RA, Bakke AC, Bagby GC, NelsonJA. HIV-1 induction of CD40 on endothelial cells promotes the outgrowth of AIDS-associated B-cell lymphomas. Nat Med 1997;3:1242–1249. [PubMed: 9359699]

Omari KM, Dorovini-Zis K. CD40 expressed by human brain endothelial cells regulates CD4+ T celladhesion to endothelium. J Neuroimmunol 2003;134:166–178. [PubMed: 12507785]

Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD. Blood-brain barrier: structural components andfunction under physiologic and pathologic conditions. J Neuroimmune Pharmacol 2006a;1:223–236. [PubMed: 18040800]

Persidsky Y, Stins M, Way D, Witte MH, Weinand M, Kim KS, Bock P, Gendelman HE, Fiala M. Amodel for monocyte migration through the blood-brain barrier during HIV-1 encephalitis. JImmunol 1997;158:3499–3510. [PubMed: 9120312]



NIH


NIH


NIH


Persidsky Y, Heilman D, Haorah J, Zelivyanskaya M, Persidsky R, Weber GA, Shimokawa H,Kaibuchi K, Ikezu T. Rho-mediated regulation of tight junctions during monocyte migrationacross the blood-brain barrier in HIV-1 encephalitis (HIVE). Blood 2006b;107:4770–4780.[PubMed: 16478881]

Persidsky Y, Ghorpade A, Rasmussen J, Limoges J, Liu XJ, Stins M, Fiala M, Way D, Kim KS, WitteMH, Weinand M, Carhart L, Gendelman HE. Microglial and astrocyte chemokines regulatemonocyte migration through the blood-brain barrier in human immunodeficiency virus-1encephalitis. Am J Pathol 1999;155:1599–1611. [PubMed: 10550317]

Phipps RP. CD40: Lord of the endothelial cell. Blood 2008;112:3531–3532. [PubMed: 18948578]Piguet PF, Kan CD, Vesin C, Rochat A, Donati Y, Barazzone C. Role of CD40-CVD40L in mouse

severe malaria. Am J Pathol 2001;159:733–742. [PubMed: 11485931]Pluvinet R, Olivar R, Krupinski J, Herrero-Fresneda I, Luque A, Torras J, Cruzado JM, Grinyo JM,

Sumoy L, Aran JM. CD40: an upstream master switch for endothelial cell activation uncovered byRNAi-coupled transcriptional profiling. Blood 2008;112:3624–3637. [PubMed: 18669876]

Ramirez SH, Heilman D, Morsey B, Potula R, Haorah J, Persidsky Y. Activation of peroxisomeproliferator-activated receptor gamma (PPARgamma) suppresses Rho GTPases in human brainmicrovascular endothelial cells and inhibits adhesion and transendothelial migration of HIV-1infected monocytes. J Immunol 2008;180:1854–1865. [PubMed: 18209083]

Ramirez SH, Potula R, Fan S, Eidem T, Papugani A, Reichenbach N, Dykstra H, Weksler BB, RomeroIA, Couraud PO, Persidsky Y. Methamphetamine disrupts blood-brain barrier function byinduction of oxidative stress in brain endothelial cells. J Cereb Blood Flow Metab. 2009

Ray DM, Akbiyik F, Bernstein SH, Phipps RP. CD40 engagement prevents peroxisome proliferator-activated receptor gamma agonist-induced apoptosis of B lymphocytes and B lymphoma cells byan NF-kappaB-dependent mechanism. J Immunol 2005;174:4060–4069. [PubMed: 15778364]

Rezai-Zadeh K, Gate D, Town T. CNS infiltration of peripheral immune cells: D-Day forneurodegenerative disease? J Neuroimmune Pharmacol 2009;4:462–475. [PubMed: 19669892]

Rizvi M, Pathak D, Freedman JE, Chakrabarti S. CD40-CD40 ligand interactions in oxidative stress,inflammation and vascular disease. Trends Mol Med 2008;14:530–538. [PubMed: 18977174]

Ryan EP, Pollock SJ, Murant TI, Bernstein SH, Felgar RE, Phipps RP. Activated human Blymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibodyproduction. J Immunol 2005;174:2619–2626. [PubMed: 15728468]

Schwabe RF, Schnabl B, Kweon YO, Brenner DA. CD40 activates NF-kappa B and c-Jun N-terminalkinase and enhances chemokine secretion on activated human hepatic stellate cells. J Immunol2001;166:6812–6819. [PubMed: 11359840]

Sharief MK, Ciardi M, Thompson EJ, Sorice F, Rossi F, Vullo V, Cirelli A. Tumour necrosis factor-alpha mediates blood-brain barrier damage in HIV-1 infection of the central nervous system.Mediators Inflamm 1992;1:191–196. [PubMed: 18475460]

Sipsas NV, Sfikakis PP, Kontos A, Kordossis T. Levels of soluble CD40 ligand (CD154) in serum areincreased in human immunodeficiency virus type 1-infected patients and correlate with CD4(+) T-cell counts. Clin Diagn Lab Immunol 2002;9:558–561. [PubMed: 11986259]

Sitati E, McCandless EE, Klein RS, Diamond MS. CD40-CD40 ligand interactions promote traffickingof CD8+ T cells into the brain and protection against West Nile virus encephalitis. J Virol2007;81:9801–9811. [PubMed: 17626103]

Sui Z, Sniderhan LF, Schifitto G, Phipps RP, Gelbard HA, Dewhurst S, Maggirwar SB. Functionalsynergy between CD40 ligand and HIV-1 Tat contributes to inflammation: implications in HIVtype 1 dementia. J Immunol 2007;178:3226–3236. [PubMed: 17312171]

Tan J, Town T, Mullan M. CD40-CD40L interaction in Alzheimer's disease. Curr Opin Pharmacol2002a;2:445–451. [PubMed: 12127879]

Tan J, Town T, Paris D, Placzek A, Parker T, Crawford F, Yu H, Humphrey J, Mullan M. Activationof microglial cells by the CD40 pathway: relevance to multiple sclerosis. J Neuroimmunol 1999a;97:77–85. [PubMed: 10408982]

Tan J, Town T, Paris D, Mori T, Suo Z, Crawford F, Mattson MP, Flavell RA, Mullan M. Microglialactivation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science 1999b;286:2352–2355. [PubMed: 10600748]



NIH


NIH


NIH


Tan J, Town T, Crawford F, Mori T, DelleDonne A, Crescentini R, Obregon D, Flavell RA, MullanMJ. Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat Neurosci 2002b;5:1288–1293. [PubMed: 12402041]

Togo T, Akiyama H, Kondo H, Ikeda K, Kato M, Iseki E, Kosaka K. Expression of CD40 in the brainof Alzheimer's disease and other neurological diseases. Brain Res 2000;885:117–121. [PubMed:11121537]

Town T, Tan J, Mullan M. CD40 signaling and Alzheimer's disease pathogenesis. Neurochem Int2001;39:371–380. [PubMed: 11578772]

Town T, Laouar Y, Pittenger C, Mori T, Szekely CA, Tan J, Duman RS, Flavell RA. Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology. Nat Med2008;14:681–687. [PubMed: 18516051]

Trebst C, Staugaitis SM, Tucky B, Wei T, Suzuki K, Aldape KD, Pardo CA, Troncoso J, Lassmann H,Ransohoff RM. Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of thecentral nervous system (CNS). Neuropathol Appl Neurobiol 2003;29:584–595. [PubMed:14636165]

Tsakiris DA, Tschopl M, Wolf F, Labs KH, Jager KA, Marbet GA. Platelets and cytokines in concertwith endothelial activation in patients with peripheral arterial occlusive disease. Blood CoagulFibrinolysis 2000;11:165–173. [PubMed: 10759010]

Urbich C, Dimmeler S. CD40 and vascular inflammation. Can J Cardiol 2004;20:681–683. [PubMed:15197419]

Xia M, Ling W, Zhu H, Ma J, Wang Q, Hou M, Tang Z, Guo H, Liu C, Ye Q. Anthocyanin attenuatesCD40-mediated endothelial cell activation and apoptosis by inhibiting CD40-induced MAPKactivation. Atherosclerosis 2009;202:41–47. [PubMed: 18495129]

Yu Q, Kovacs C, Yue FY, Ostrowski MA. The role of the p38 mitogen-activated protein kinase,extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transductionpathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+T cell memory responses. J Immunol 2004;172:6047–6056. [PubMed: 15128788]



NIH


NIH


NIH


Figure 1.Up-regulation of CD40 expression in microvessels in HIV-1 infected brain tissue. Frontalcortex brain tissue specimens were obtained from seven cases of HIV-1 encephalitis (HIVE)of different severity (moderate to severe according to previously described criteria, reference1) along with HIVE cases and seronegative age-matched controls (n=4). Serial frozensections (5 µm thick) were cut and stained for CD68 (monocyte-macrophage marker; cyan-colored). On serial sections, cerebral vessels were identified by double staining with Ulexeuropaeus agglutinin 1 (UEA-1, red) and antibodies to CD40 (green). (A) Infiltration ofmonocytes (a feature of HIVE) was confirmed by staining of CD68 (V – vessel, PS –perivascular space). (B) While no staining for CD40 was seen in controls, there was asubstantial increase in CD40 levels in HIVE (arrowheads). (C) Higher magnificationdemonstrated minimal-to-no staining for CD40 in controls and up-regulation of CD40 inHIVE. In addition, glial cells were also CD40 positive in HIVE (arrowheads) but not incontrols. Stained sections were observed by immunofluorescent microscopy (objective 10×and 40×), and digital images were acquired by a cooled CCD camera. (D) Semi quantitativeassessments of CD40 levels were performed as described in Materials and Methods. CD40was up regulated 2.5 – fold and 5-fold in HIV+ and severe HIVE cases, respectively, ascompared to controls. Data are shown as mean ± SEM. * P < 0.01. Original magnification:panels A, B x100; panel C × 400.



NIH


NIH


NIH


Figure 2.Brain endothelial cells exposed to CD40L increase monocyte adhesion and transendothelialmigration. (A) Basal expression of CD40 was found in BMVEC, but was undetectable inresting HeLa cells. Analysis of adhesion was performed with a fluorescence-based assayusing primary human BMVEC and monocytes as described in the methods section. Afterformation of monolayers, the BMVEC were treated with the indicated test conditions orwith TNFα (20ng/ml) for 24 hrs. Treatments were removed and fluorescent labeledmonocytes were added to the BMVEC. (B) Adhesion assay demonstrating endothelialactivation and subsequent monocyte adhesion induced by increasing concentrations ofmembrane-bound CD40L [shown as CD40L(M)]. Control membrane [CTR(M)] prepslacking CD40L had no appreciable induction in adhesion. Co-incubation of CD40L(M) withneutralizing antibodies (Neut. AbCD40) to CD40 blocked the effects of CD40L. (C)Adhesion assays with soluble CD40L (sCD40L) also shows a dose response in adhesionwhich is blocked by the presence of CD40 neutralizing antibodies. Transendothelialmigration assays were performed as describe in the methods. The indicated treatments wereadded for 24hrs and then removed followed by monocyte addition and chemotaxis towardsthe relevant chemokine CCL2/MCP-1 (50 ng/ml). (C) A dose-dependent increase inmonocyte migration was found after addition of CD40L(M) and sCD40L (D) addition toBMVEC monolayers. The effects of either form of CD40L were specific since the degree ofmigration induced by CD40L was eliminated by antibody neutralization. Of note, non-chemoattractant driven migration also showed an increase in basal migration when BMVECwere stimulated with either membrane bound or soluble CD40L. Data shown as mean +SEM. * P < 0.01.



NIH


NIH


NIH


Figure 3.Increased surface expression of ICAM-1 and VCAM-1 on brain endothelial cells aftersCD40L exposure. FACS analyses of adhesion molecules ICAM-1 and VCAM-1 wereperformed in BMVEC exposed to increasing concentrations of sCD40L. Induction ofadhesion molecules ICAM-1 and VCAM-1 was observed after sCD40L exposure for 24 hrs.The up regulation of adhesion molecules was also dose-dependent, and was prevented byintroduction of neutralizing anti-CD40 antibodies.



NIH


NIH


NIH


Figure 4.sCD40L induction of ICAM-1 and VCAM-1 in BMVEC is JNK dependent. FACS analyseswere performed to determine which pathway was responsible for the upregulation ofadhesion molecules during CD40 activation. Endothelial cells were co-incubated withsCD40L and cell permeable inhibitors to JAK1/3, IKKα, ERK1/2, and JNK1. The followinginhibitors were introduced for 30min following co-incubation with sCD40L (10ng/ml): (A)JAK/STAT inhibitor pyridone 6 (10µM), (B) JAK3/STAT3 inhibitor VI (1µM) a 3′-pyridyloxindole based compound, (C) IKKα inhibitor N-(6-chloro-9H-b-carbolin-8-yl)nicotinamide(1µM), (D) p38 inhibitor FHPI (5µM), (E) ERK1/2 inhibitor FR180204 (10µM) and (E) thecell permeable JNK-binding domain peptide inhibitor. Histograms on the left side indicateVCAM-1 expression. Histograms on the right indicate ICAM-1. The histrograms are asingle representation of at least 3 independently performed experiments showing similarresults. The filled area under the curve shows the IgG isotype control, the green lineindicates the expression after sCD40L exposure and the orange line indicates expressionafter sCD40L plus respective inhibitor.



NIH


NIH


NIH


Figure 5.Activation of CD40 in BMVEC leads to ERK1/2 and JNK-1 activation but not p38 MAPK.Western blots are shown for the phosphorylated/activated status of ERK1/2, JNK1 and p38MAPK after CD40 activation. (A) Western blots for mixed-lineage kinase 3 (MLK3),upstream activators of JNK1, show increases in p-MLK3 after 5 min stimulation of sCD40L.BMVEC were exposed to sCD40L for 5, 15 and 30 min, lysed and western blots performed.(B) Phosphorylation of JNK1 (thr183/tyr185) is shown along with densitometry valuescalculated from the ratio of phospho JNK1/total JNK; the values are expressed as foldchange relative to untreated control . (C) Phosphoryation status of ERK1/2 (thr202/tyr204)is shown. (D) The unchanged phosphorylation status of p38 MAPK (thr180/tyr182) is



NIH


NIH


NIH


shown. Corresponding blots for total ERK1/2, JNK1 and p38 MAPK protein are shownbelow the blots probed with the phospho-specific antibody. β-tubulin was used as loadingcontrol (A–D). (E) Activation of c-JUN was determined by assessing the increase in c-JUNnuclear transclocation and phosphorylation of ser63. (F) The DNA binding activity ofactivated c-JUN complexes was determined using an ELISA based transactivation assay.Nuclear extracts from sCD40L treated BMVEC were prepared for the indicated time points.The results are shown as the average ± SEM (n=3) optical density measurements taken at450nm; (*) statistical significance (P<0.05). (G) Activation and nuclear translocation of c-JUN after stimulation by sCD40L is inhibited by the presence of JNK inhibitor II (2µM;SP600125). The unchanging levels of lamin A/C serve as a control for nuclear fractionloading. (H) In contrast to c-JUN, JNK inhibitor II (2µM; SP600125) does not block theactivation of ERK after CD40L activation (cytosolic fraction shown).



NIH


NIH


NIH


Figure 6.Inhibition of JNK in BMVEC attenuates CD40 mediated monocyte migration. The effect onadhesion and transendothelial migration of monocytes was evaluated in BMVEC exposed toJNK inhibitors co-incubated with sCD40L (100ng/ml). (A) Adhesion assays of untreated,sCD40L treated, and TNFα treated endothelial cells exposed to JNK1 inhibitor, JNK bindingdomain peptide (10µM) and pharmacological JNK1 inhibitor, SP600125 (2µM), for 24hours. Prior to assay, all treatments were removed and calcein-AM labeled monocytes wereadded to the BMVEC and allowed to adhere for 15 min, unattached cells were rinsed and thefluorescence was measured. The data are represented as mean ± SEM fold difference, whichis the adhesion value from treated BMVEC divided by the basal adhesion value from



NIH


NIH


NIH


untreated cells. (B) Transendothelial migration of monocytes was performed across sCD40Lstimulated BMVEC monolayers in the presence of JNK inhibitors. All treatments (as above;except no TNFα) were removed prior to monocyte introduction and where indicatedchemotaxis was performed towards CCL2/MCP-1 (50ng/ml). The data are represented asfold difference (mean ± SEM) of migration, which is the value from migration of treatedcells divided by the basal migration of untreated cells without chemoattractant. All datacollected were from at least three independent experiments performed in triplicate.



NIH


NIH


NIH


NIH


NIH


NIH



Tabl

e 1

Clin

ical

dat

a fo

r hum

an b

rain

tiss

ue sa

mpl

es

Cas

enu

mbe

rH

IV-1

infe

ctio

nA

geG

ende

rV

iral

Loa

d#O

ther

syst

emic

dis

ease

s

Cas

e 1

HIV

E se

vere

*37

M75

0,00

0Pu

lmon

ary

aspe

rgill

osis

Cas

e 2

HIV

E se

vere

44M

389,

120

Cac

hecx

ia, K

apos

i sar

com

aof

lym

ph n

odes

, chr

onic

hepa

titis

Cas

e 3

HIV

E se

vere

32M

1,09

8,94

7B

acte

rial b

ronc

hopn

eum

onia

Cas

e 4

HIV

E se

vere

42M

6940

Bac

teria

l bro

ncho

pneu

mon

ia

Cas

e 5

HIV

-1‡

51M

65Pu

lmon

ary

embo

lus,

pulm

onar

y as

perg

illom

a

Cas

e 6

HIV

-143

M25

2,60

4Pn

eum

ocys

tis c

arin

iipn

eum

onia

, dis

sem

inat

edC

MV

, liv

er st

eato

sis

Cas

e 7

HIV

-147

M38

61H

epat

itis C

, mic

rono

dula

rci

rrho

sis,

foca

l seg

men

tal

glom

erul

oscl

eros

is

Cas

e 8

Sero

nega

tive

cont

rol

33F

N/A

Cys

tic fi

bros

is, s

eptic

emia

Cas

e 9

Sero

nega

tive

cont

rol

55M

N/A

Bro

ncho

pneu

mon

ia

Cas

e 10

Sero

nega

tive

cont

rol

46M

NA

Hep

atiti

s C, c

irrho

sis,

athe

rosc

lero

sis

Cas

e 11

Sero

nega

tive

cont

rol

52M

NA

Stat

us p

ost a

ortic

val

vere

pair,

myo

card

ial i

nfar

ct,

bron

chop

neum

onia

# Vira

l loa

d –

copi

es p

er m

l.

* Seve

re H

IVE

is d

efin

ed a

s exp

ress

ion

of H

LA-D

R o

n 80

% to

90%

of m

icro

glia

; HIV

-1 in

fect

ion

of ra

mifi

ed m

icro

glia

; for

mat

ion

of 1

to 2

mic

rogl

ial n

odul

es a

nd 2

5 to

50

infil

tratin

g m

acro

phag

es p

er fi

ve

× 10

-pow

er fi

elds

; and

abu

ndan

t mul

tinuc

leat

ed c

ells

(as d

escr

ibed

26)

‡ No

sign

ifica

nt n

euro

path

olog

ic c

hang

es w

ere

foun

d.


Date post:	27-Nov-2023
Category:	Documents
Upload:	lsuhsc
View:	0 times
Download:	0 times

Dyad of CD40/CD40 Ligand Fosters Neuroinflammation at the Blood-Brain Barrier and Is Regulated via...

Documents