CD40 expressed by human brain endothelial cells regulates CD4+ T cell adhesion to endothelium

Recent evidence suggests that interactions between CD40 on antigen presenting cells (APC) and CD40L on T cells generate signals that result in the activation of APC. In this study, the expression and function of CD40 was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). Results revealed constitutive expression of CD40 on untreated HBMEC. Stimulation with TNF-alpha, IFN-gamma, LPS or combination of TNF-alpha and IFN-gamma significantly upregulated CD40. The majority of CD40 molecules were localized on the apical surface of EC. Incubation of HBMEC with soluble CD40L resulted in increased expression of the adhesion molecules E-selectin, VCAM-1 and ICAM-1. Consequently, the adhesion of both resting and anti-CD3 activated CD4+ T lymphocytes to CD40L treated HBMEC was significantly increased compared to unstimulated EC. The expression of CD40 by cerebral endothelium, and endothelial cell activation following binding of CD40 to its ligand, CD40L, suggest a potential mechanism by which activated CD40L expressing T cells could enhance adhesion and migration of inflammatory cells across the blood-brain barrier (BBB) to sites of inflammation in the human central nervous system (CNS).     

Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance.

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.     

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.     

CD40 promotion of amyloid beta production occurs via the NF-kappaB pathway

The CD40 receptor is a member of the tumor necrosis factor (TNF) super-family of trans-membrane receptors. Interaction of CD40 with its ligand CD40L mediates a broad range of immune and inflammatory responses in the periphery and in the central nervous system. Recently it has been suggested that CD40/CD40L interaction is involved in amyloid precursor protein (APP) processing and Alzheimer's disease (AD)-like pathology in transgenic mouse models of AD. We have previously shown that pharmacologically inhibiting CD40/CD40L interaction improves memory deficits in the PSAPP AD mouse model. We have also recently shown that CD40 deficiency mitigates amyloid deposition in APPsw and PSAPP mouse models. In the present report, using human embryonic kidney cells (HEK293) over-expressing both the APPsw mutation and CD40, we demonstrate that CD40/CD40L interaction directly increases the production of APP metabolites (Abeta 1-40, Abeta 1-42, CTFs, sAPPbeta and sAPPalpha). The results also show that CD40/CD40L interaction affects APP processing via the NF-kappaB pathway. Using NFkappaB inhibitors and SiRNAs to silence diverse elements of the NFkappaB pathway, we observe a reduction in levels of both Abeta 1-40 and Abeta 1-42. Taken together, our results further suggest that CD40L stimulation may be a key component in AD pathology and that elements of the NF-kappaB pathway may be suitable targets for therapeutic approaches against AD.     

Differentiated Th1 autoreactive effector cells can induce experimental autoimmune encephalomyelitis in the absence of IL-12 and CD40/CD40L interactions.

IL-12 plays a critical role in the priming of Th1 responses to bacterial/parasitic antigens and autoantigens. Several studies have demonstrated a dependency on CD40/CD40L interactions and IL-12 for maintenance of both antibacterial/parasitic and autoreactive Th1 cells in vivo. However, it is still unclear if fully differentiated Th1 effectors require continued stimulation by IL-12. We demonstrate that the proliferative response and IFN-gamma production by a fully differentiated T cell line specific for myelin oligodendrocyte glycoprotein are completely independent of IL-12 and CD40/CD40L interactions. The capacity of this line to adoptively transfer experimental autoimmune encephalomyelitis is also independent of IL-12 and CD40/CD40L. These results have important implications regarding the therapeutic usefulness of blockade of IL-12 or the CD40/CD40L pathway for treatment of autoimmune disease.     

B cells play a cooperative role via CD40L-CD40 interaction in T cell-mediated experimental autoimmune neuritis in Lewis rats

The expression of co-stimulatory molecules CD40 and CD40L was examined over the course of experimental autoimmune neuritis (EAN) induced in Lewis rats by immunization with bovine peripheral nerve myelin. In draining lymph nodes, highest level of CD40L expression was seen on day 7 post immunization (p.i.), i.e. before onset of clinical signs of EAN, while CD40 expression was increased on day 14 p.i., i.e. at peak of clinical disease. In contrast, both CD40 and CD40L expressing cells in sciatic nerves, a target organ of EAN, peaked on day 14 p.i., large numbers of both expressing cells were mainly detected on day 14-21 p.i. After co-culture with EAN rat B cells bearing CD40, P0 peptide 180-199-specific T cell line cells exhibited a rapid down-regulation of CD40L expression. Furthermore, EAN rats had enhanced P0 peptide 180-199-specific antibody responses on day 14 p.i., which might have contributed to their aggravated EAN and further demonstrated the role of antibodies in EAN. The results indicate that CD40L-CD40 interactions are involved in the initiation of the antigen-specific T cell responses associated with the generation and development of EAN, and may mediate autoantibody production in EAN. Evidently, B cells play a cooperative role via CD40L-CD40 interaction in T cell-mediated EAN of Lewis rats.     

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT - PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P<0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder, an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.     

Cytokine regulation of CD40 expression in fetal human astrocyte cultures

CD40 can participate in inflammatory processes after binding its cognate ligand (CD40L). We found that fetal human astrocytes constitutively express CD40 mRNA and protein. Upon incubating cultures with proinflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) or with lipopolysaccharide (LPS), CD40 expression was increased. No change in CD40 expression was noted in astrocyte cultures incubated with IL-6, HIV or gp41. Astrocytes also showed increased release of proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 after incubation with CD40L peptide. These observations suggest a role for CD40 in central nervous system (CNS) inflammation and that CD40/CD40L autocrine or paracrine pathways may mediate this role.     

Neutrophil CD40 enhances platelet-mediated inflammation

INTRODUCTION: CD40 is a transmembrane protein expressed on monocytes, macrophages, endothelial cells, and platelets. Platelets are the richest source of soluble CD40 ligand (sCD40L) and interact with monocytes and endothelial cells via CD40. While CD40 was recently reported to be present on neutrophils, the detailed mechanism of its interaction with platelets via CD40-CD40L has not been examined. MATERIALS AND METHODS: The existence of neutrophil CD40 was verified by real-time PCR and western blot. Platelet sCD40L release was measured by ELISA. Neutrophil superoxide generation was measured by chemiluminescence and confocal microscopy. The neutrophil-platelet conjugates were measured by flow cytometry. RESULTS AND CONCLUSION: The presence of neutrophils enhances stimulation-induced platelet release of sCD40L. The addition of platelets leads to an enhancement of neutrophil superoxide and reactive oxygen species (ROS) generation. The specificity of the CD40-CD40L pathway in the neutrophil-platelet interaction was confirmed by using recombinant soluble CD40L (rsCD40L) and an anti-CD40L antibody. The involvement of the PI3 kinase/Akt pathway in neutrophil superoxide production was revealed by using LY294002 in isolated neutrophils/platelets experiments, as well as during whole blood aggregation-mediated neutrophil-platelet conjugation. N-acetylcysteine, a scavenger of ROS, eliminates both neutrophil superoxide generation and sCD40L release from activated platelets. These data suggest that activated neutrophils release ROS in a PI3 kinase-dependent manner, contributing to platelet activation and further sCD40L release in a redox-controlled positive feed-back loop. In conclusion, our results define a new pathway by which platelets and neutrophils interact and modulate each other's function, and may be relevant in understanding acute thrombo-inflammatory processes.     

Death receptor-mediated apoptosis in human malignant glioma cells: modulation by the CD40/CD40L system

CD40, a TNF-R-related cell surface receptor, is shown here to be expressed by glioma cells in vitro and in vivo. Glioma cell lines expressing low levels of CD40 at the cell surface resist cytotoxic effects of CD40L. CD40 gene transfer sensitizes glioma cells to CD40L. Inhibition of protein synthesis potentiates cell death which involves CD40 clustering and caspases 8 and 3 processing. CD40-transfected LN-18 cells acquire resistance to CD95L. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis. Bispecific CD40xCD95 antibodies specifically kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling.     

Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis.

It is well known that microglial cells perform a key role in mediating inflammatory processes, which are associated with neurodegenerative diseases such as multiple sclerosis (MS). In this study, we report that CD40 expression on microglia is greatly enhanced by a low dose (10 U/ml) of IFN-gamma. We also find that ligation of microglial CD40 by CD40L triggers a significant production of TNF-alpha. Activation of microglia by ligation of CD40 in the presence of IFN-gamma results in cultured cortical neuronal injury, which is markedly attenuated by blockade of the CD40 pathway or neutralization of TNF-alpha. Finally, we find significant levels of IFN-gamma and TNF-alpha in the medium of co-cultured activated CD4+ T cells and microglial cells, showing that microglia can supply the CD40 receptor to activated CD4+ T cells and suggesting that this cellular interaction is a key event in MS pathophysiology.     

Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy

CD40 ligand (CD40L) is mainly expressed on activated platelets and CD4+T cells, and it can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L). Most sCD40L is derived from activated platelets. A previous paper revealed that the platelet number of patients with influenza virus-associated encephalopathy (IE) was correlated with the outcome. We determined the utility of sCD40L as a predictor for the prognosis of IE. We measured the serum concentration of sCD40L and the platelet number on the day of hospitalization in 34 patients with IE, 16 with influenza virus-associated febrile seizures (IFS), 19 with influenza virus infection without complications (Flu), and 7 with Epstein-Barr virus (EBV) infection. The serum sCD40L concentrations in IE and IFS were significantly lower than those in controls, Flu, and EBV infections. Serum sCD40L concentrations in the IE group were 0.70+/-0.43 ng/ml for deceased patients, 1.73+/-1.36 ng/ml for those with sequelae, and 3.85+/-2.91 ng/ml for those without sequelae. There was no significant difference in platelet number between IE patients with and without sequelae, while the platelet number of deceased patients with IE was significantly lower than in controls, Flu, and IFS. Serum sCD40L concentration on the day of hospitalization was more correlated with the outcome of IE than platelet number. Our findings suggest that the serum sCD40L concentration during acute IE is important for predicting the prognosis at an early stage.     

Expression of OX40 and OX40 ligand (gp34) in the normal and myasthenic thymus

OBJECTIVES: To examine the expression of OX40, an activated memory T-cell marker, and its ligand (OX40L), a set of molecules for T-cell-B-cell interaction, and other lymphocyte activation markers in the thymuses of myasthenia gravis (MG) and controls. MATERIAL AND METHODS: We studied the expression of OX40, OX40L, IL-2Ralpha and HLA-DR in the thymic tissues of MG and controls using immunocytochemistry and flowcytometry. RESULTS: In both hyperplastic thymus of MG and control thymus, OX40+ cells were scattered mainly in the medulla with much fewer OX40L+ cells being distributed in the corticomedullary junctions. IL-2Ralpha and HLA-DR were expressed in the medulla at higher frequencies as compared with OX40 in controls as well as MG. In contrast, the numbers of OX40+ cells around the germinal centers (GC) were significantly greater than those of control thymuses, and some mononuclear cells in GC were OX40L+. A considerable number of OX40+ cells were seen in the thymic tissues adjacent to thymomas. OX40+ cells were CD4+ CD8- or CD4+ CD8+ and were mostly HLA-DR-. (The coexpression of OX40 and IL-2Ralpha on activated CD4+ T cells was previously reported.) CONCLUSION: OX40, expressed in a fraction of activated CD4+ T cells, may be upregulated in thymic tissues adjacent to GC and thymoma in MG, and OX40 may interact with OX40L in GC to enhance anti-acetylcholine receptor antibody production in MG.     

Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with secondary progressive MS (SPMS) had constitutive CD154 expression on CD4 and CD8 T cells in blood. Constitutive CD154 expression was not observed in patients with relapsing-remitting MS (RRMS) or clinically isolated syndromes (CIS) suggestive of demyelinating disease. After ex vivo activation CD154 was, however, expressed on a higher percentage of T cells from patients with CIS or RRMS than from healthy control subjects. These results suggest involvement of CD154 in the pathogenesis of MS, and the shift from a relapsing-remitting to a secondary progressive disease course may be associated with constitutive, systemic CD154 expression.     

CD40 engagement stimulates IL-12 p70 production by human microglial cells: basis for Th1 polarization in the CNS

The APC-derived cytokine interleukin (IL)-12 polarizes CD4 T cells towards the pro-inflammatory Th1 phenotype and has been shown to be crucial for the development of EAE in rodents. In this study we demonstrate that production of IL-12 by human adult CNS-derived microglial cells can be triggered by cell contact with activated T cells. Microglial activation and IL-12 production can be blocked by anti-CD154 mAbs. IL-12 production could also be induced by direct engagement of CD40 on microglia using a CD40 agonist. IL-12 secretion by microglia is significantly reduced by TNF and IFN-gamma antagonists showing that the IL-12 production is subject to regulation by auto- and paracrine stimuli.     

CD40-CD40 ligand interactions in human microglia induce CXCL8 (interleukin-8) secretion by a mechanism dependent on activation of ERK1/2 and nuclear translocation of nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP-1)

CXCL8 is a CXC chemokine that recruits leukocytes to sites of inflammation. Expression of CXCL8 in the CNS has been demonstrated in neuroinflammatory diseases, including human immunodeficiency virus (HIV-1) encephalitis, but the mechanism of secretion of this chemokine is not fully understood. CD40 is a 50-kDa protein on the surface of microglia, and we have previously shown that it is increased in expression in HIV-1-infected brain tissue as well as by interferon-gamma (IFNgamma) in tissue culture. We examined the expression and regulation of CXCL8 in cultured human fetal microglia after ligation of CD40 with soluble trimeric CD40 ligand (sCD40L) as well as the expression of CXCL8 on microglia in HIV encephalitic brain tissue sections. Treatment of cultured microglia with IFNgamma + sCD40L resulted in significant induction of CXCL8. This expression was mediated by activation of the ERK1/2 MAPK pathway, as demonstrated by ELISA and Western blot using a specific inhibitor (U0126). Gel shift analyses demonstrated that NFkappaB and AP-1, but not C/EBPbeta, mediate microglial CXCL8 production. We also found increased colocalization of CXCL8 with CD68/CD40-positive cells in HIV encephalitic brain tissue compared with HIV-infected nonencephalitic and normal tissue. Thus, CD40-CD40L interactions facilitate chemokine expression, leading to the influx of inflammatory cells into the CNS. These events can lead to the pathology that is associated with neuroinflammatory diseases.     

重症肌无力患者外周血CD4+T细胞OX40的表达及作用机制研究

目的 分析重症肌无力(MG)患者外周血CD4+T细胞协同刺激分子OX40表达及其对FoxP3+CD4+CD25+调节性T细胞(Treg)的调控作用,初步探讨OX40在MG免疫学发病中的作用机制.方法 以流式细胞技术检测42例MG患者及38名健康对照的外周血OX40+CD4+T细胞、FoxP3+CD4+CD25+Treg表达水平,比较OX40表达在MG患者不同临床疾病状态、Osserman分型、临床绝对评分、胸腺病理类型等情况下的差异,并分析OX40对FoxP3+CD4+CD25+Treg细胞的影响.结果 (1) MG患者外周血OX40+CD4+T细胞占淋巴细胞百分比高于健康对照组(P<0.01).(2)MG患者OX40+CD4+T细胞百分比在发作或加重期高于缓解期(P＜0.05);在临床绝对评分呈中、重度患者OX40+CD4+T细胞百分比高于轻度患者(均P＜0.05);Osserman Ⅱ、Ⅳ型患者OX40+CD4+T细胞百分比高于Ⅰ型患者(均P＜0.05);胸腺增生及胸腺瘤患者OX40+CD4+T细胞百分比高于胸腺正常患者(P＜0.05,P＜0.01).(3)MG患者外周血OX40+CD4+T细胞百分比与FoxP3+CD4+CD25+Treg细胞百分比呈负相关(r=-0.843,P=0.01).结论 协同刺激分子OX40参与MG发病,可能通过抑制FoxP3+CD4+CD25+Treg细胞生成发挥作用.     

Differences of soluble CD40L in sera and plasma: implications on CD40L assay as a marker of thrombotic risk

INTRODUCTION: Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results. METHODS: We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP). RESULTS: Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis. CONCLUSIONS: Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.