Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake

Activated platelets express CD40L on their plasma membrane and release the soluble fragment sCD40L. The interaction between platelet surface CD40L and endothelial cell CD40 leads to the activation of endothelium contributing to atherothrombosis. Few studies have directly demonstrated an increased expression of platelet CD40L in conditions of in vivo platelet activation in humans, and no data are available on its relevance for endothelial activation. We aimed to assess whether platelets activated in vivo at a localized site of vascular injury in humans express CD40L and release sCD40L, whether the level of platelet CD40L expression attained in vivo is sufficient to induce endothelial activation, and whether platelet CD40L expression is inhibited by aspirin intake. We used the skin-bleeding-time test as a model to study the interaction between platelets and a damaged vessel wall by measuring CD40L in the blood emerging from a skin wound in vivo in healthy volunteers. In some experiments, shed blood was analyzed before and 1 h after the intake of 500 mg of aspirin. Platelets from the bleeding-time blood express CD40L and release soluble sCD40L, in a time-dependent way. In vivo platelet CD40L expression was mild but sufficient to induce VCAM-1 expression and IL-8 secretion in coincubation experiments with cultured human endothelial cells. Moreover, platelets recovered from the bleeding-time blood activated endothelial cells; an anti-CD40L antibody blocked this effect. On the contrary, the amount of sCD40L released by activated platelets at a localized site of vascular injury did not reach the concentrations required to induce endothelial cell activation. Soluble monocyte chemoattractant protein-1, a marker of endothelium activation, was increased in shed blood and correlated with platelet CD40L expression. Aspirin intake did not inhibit CD40L expression by platelets in vivo. We concluded that CD40L expressed by platelets in vivo in humans upon contact with a damaged vessel wall activates endothelium; aspirin treatment does not inhibit this mechanism.     

IgG-complex stimulated platelets: A source of sCD40L and RANTES in initiation of inflammatory cascade

Platelets are a crucial element in maintenance of hemostasis. Other functions attributable to platelets are now being appreciated such as their role in inflammatory reactions and vascular remodeling. Platelets have been reported to bind immunological stimuli like IgG-complexes and the understanding that platelets may participate in immunological reactions has been speculated for nearly 50 years. In previous observations, we demonstrated that platelets could bind and internalize aggregated IgG-complexes without inducing platelet aggregation or granule release. To characterize this observation further, we tested the hypothesis that aggregated IgG-complexes do not activate platelets. To this end, platelets were stimulated with IgG-complexes or thrombin as a positive control and evaluated for activation by aggregation, expression of surface markers and production of cytokines. Activation with thrombin resulted in aggregation, expression of high levels of CD62P (P-selectin) expression and activation of the fibrinogen receptor, α_IIbβ₃. Furthermore, stimulation with thrombin resulted in significant amounts of sCD40L (CD154) and RANTES (CCL5). However, platelets stimulated with IgG-complexes resulted in no aggregation and low levels of CD62P expression. Surprisingly, platelets stimulated with aggregated IgG-complexes released similar amounts of sCD40L and RANTES as platelets activated by thrombin. These data suggest that platelets are capable of secreting inflammatory molecules in response to IgG-complexes.     

TNF-alpha blockade down-regulates the CD40/CD40L pathway in the mucosal microcirculation: a novel anti-inflammatory mechanism of infliximab in Crohn's disease

The CD40/CD40 ligand (CD40L) pathway is involved in Crohn's disease (CD) pathogenesis. In the patients' circulation, soluble CD40L (sCD40L) levels are elevated and surface CD40L is increased in platelets and T cells, whereas in the intestine CD40 is overexpressed in the microvasculature and CD40L in platelets and T cells. The therapeutic effects of infliximab in CD are attributed to its systemic anti-TNF-alpha action, but because TNF-alpha modulates both CD40 and CD40L, we investigated whether infliximab affects the CD40/CD40L pathway in the intestine. Eighteen CD patients were evaluated before and after infliximab therapy. Plasma sCD40L was measured by ELISA and platelet and peripheral blood T cell (PBT) CD40L expression by flow cytometry. Microvascular CD40 and VCAM-1 expression were assessed in mucosal biopsies by immunohistochemistry and by flow cytometry in human intestinal microvascular endothelial cells (HIMEC). Cell cultures were performed in the presence and absence of infliximab. Infliximab treatment significantly reduced plasma sCD40L levels and eliminated CD40 and VCAM-1 from mucosal microvessels. In vitro infliximab prevented TNF-alpha-induced CD40 and VCAM-1 expression by HIMEC, and reduced PBT, but not platelet, surface CD40L expression and sCD40L release. In addition, infliximab decreased T cell-induced VCAM-1 expression in HIMEC by down-regulating CD40L in T cells and promoting T cells apoptosis. These findings point to a novel mechanism of action of infliximab, i.e., the disruption of CD40/CD40L-dependent cognate interactions between intestinal microvessels and T cells. Thus, in addition to neutralizing TNF-alpha and inducing T cell death, the therapeutic effects of infliximab in CD appear to be also mediated by inhibition of vascular inflammation in the gut.     

Why do B cells produce CD40 ligand?

The CD40-CD40 ligand (CD40L) interaction is one of the most important receptor-ligand interactions that occurs during a T dependent immune response. However, while CD40L is expressed on a range of cell types including activated T cells and B cells, dendritic cells granulocytes, macrophages and platelets, only CD40L on T cells is considered by most immunologists when planning experiments or analysing data. The current theory professes that T cells expressing CD40L can provide signals to B cells that induce proliferation, immunoglobulin class switching, antibody secretion, rescue from apoptosis at different times during the life of a B cell and also has a role in the development of germinal centres and the survival of memory B cells. However, the whole story is more complex than presently understood as human and mouse B cells express CD40L on their surface following activation and can release a soluble form of the ligand. This paper hypothesizes how CD40L on B cells may regulate antibody responses and the development of germinal centres.     

Internalization of CD40 regulates its signal transduction in vascular endothelial cells

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Platelets contribute to allograft rejection through glutamate receptor signaling

Platelets recruit leukocytes and mediate interactions between leukocytes and endothelial cells. Platelets have been long described as markers of transplant rejection, but the contribution of platelets to transplant rejection has not been critically examined. We demonstrate in this study that following T cell initiation of allograft rejection, platelets contribute to T cell recruitment and increased plasma inflammatory mediators and accelerate T cell-meditated skin graft rejection. Prior work from our laboratory has shown that platelets secrete glutamate when activated, which then induces platelet thromboxane production by signaling through platelet-expressed ionotropic glutamate receptors. Glutamate receptor antagonists therefore represent, to our knowledge, novel inhibitors of platelet-accelerated inflammation. We have found that plasma glutamate is increased in mice that receive skin grafts and that mice treated with glutamate receptor antagonists have improved graft survival and decreased plasma thromboxane, platelet factor 4 (CXCL4), and IFN-γ. Taken together, our work now demonstrates that subsequent to T cell initiation of skin graft rejection, platelets contribute to further T cell recruitment and that by blunting glutamate-mediated platelet activation, graft survival is improved.     

CD40-40L signaling in vascular inflammation

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.     

Monoclonal antibody to CD9 inhibits platelet-induced human endothelial cell proliferation

Platelets are anucleate cytoplasmic fragments derived from bone marrow megakaryocytes, and endothelial cells constitute the barrier between bloodstream and adjacent tissues. Although platelets are thought to regulate the biological functions of endothelial cells, the molecular mechanisms involved are poorly understood. With human umbilical vein endothelial cells and freshly isolated platelets, we established an in vitro model of platelet-induced endothelial cell proliferation. Platelets stimulated endothelial cell proliferation in a dose-dependent manner and transwell experiments with semi-permeable membranes suggested that direct cell-to-cell contacts were required. We developed mAbs against platelets and selected a mAb that blocks their proliferative effect. We purified the antigen by immunoprecipitation and identified it by Q-TOF MS analysis as the tetraspanin CD9. Since both platelets and endothelial cells expressed CD9 strongly on their surfaces we carried out a pre-treatment experiment that showed that CD9 molecules on the endothelial cells participate in the mitogenic effect of the platelets. The inhibitory effect of our mAb was comparable to that of a well-known functional anti-CD9 mAb. These results suggest that the tetraspanin CD9 plays an important role in endothelial regeneration.     

Regulation of CD40L expression by cyclic AMP: contrasting proinflammatory and inhibitory actions

CD40L expression is well recognized to be of critical importance in initiation of the immune response. Because cAMP mediates actions of bronchodilators commonly used in asthma, the effects of cAMP in regulating the immune response are of major importance. Cyclic AMP was found to either inhibit or markedly increase CD40L expression dependent upon the mechanisms of T cell activation. Cyclic AMP inhibited CD40L expression induced by TCR activation. In contrast, cAMP enhanced CD40L induced by CD2-mediated T cell activation or by calcium-dependent mechanisms. While neither CD28 costimulation nor exogenous IL-2 or IL-4 prevented cAMP inhibition in TCR activated cells, addition of calcium ionophore to TCR activation prevented any inhibitory effects and caused cAMP to increase CD40L expression. Actions of cAMP to increase CD40L expression appeared independent of PKC and were not a reflection of generalized cellular activation since neither CD25 nor CD69 expression was affected. The markedly contrasting actions of cAMP to decrease or increase CD40L expression, an important control point in the immune response, could be relevant to actions of commonly used medications including bronchodilators.     

Regulation of CD40L expression on natural killer cells by interleukin-12 and interferon γ: its role in the elicitation of an effective antitumor immune response

Effector cell functions are regulated by a number of positive signals for the mediation of antitumor immunity. The CD40 and CD40 ligand (CD40L) interaction has been implicated in the generation of effective cell-mediated and humoral immune responses, where cytokines have been shown to play a significant role in the expression of these molecules. Our earlier studies have shown that spontaneous regression of a rat histiocytoma transplanted s.c. is mediated by CD8⁺ CD3⁻ NK cells. The CD40-CD40L mediation during tumor regression was of interest. Tumor-transplanted animals showed enhanced expression of CD40L on natural killer (NK) and T cells, when compared to cells from normal animals. CD40 expression on AK-5 tumor cells was also induced after s.c. transplantation. Administration of anti-(interleukin-12) (anti-IL-12) and anti-(interferon γ) (anti-IFNγ) antibodies in tumor-bearing animals showed down-regulation of the expression of CD40L on NK and T cells with simultaneous inhibition of cytotoxic acitivity of NK cells, cytokine release and the production of antitumor antibody. Naive NK cells, when co-cultured with fixed AK-5 cells, were induced to express CD40L. CD40L expression modulated the immune response exerted by NK cells, in part by the activation of nuclear factor kB (NF-kB). Furthermore, the signaling via CD40L through the use of anti-CD40L antibody promoted the in vitro activation of cytotoxic as well as NF-kB binding activity in NK cells from tumor-transplanted animals. These observations demonstrate that the expression of CD40L by the effector cells is regulated by IL-12 and IFNγ, and could effectively modulate the NK-cell-mediated immune response during the regression of AK-5 tumor. Received: 1 June 2000 / Accepted: 27 July 2000     

Role of B7-CD28/CTLA-4 costimulation and NF-kappa B in allergen-induced T cell chemotaxis by IL-16 and RANTES

The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NF kappa B activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-kappa B.     

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappaB

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.     

Increased levels of soluble CD40L in African tick bite fever: possible involvement of TLRs in the pathogenic interaction between Rickettsia africae, endothelial cells, and platelets

The pathophysiological hallmark of spotted fever group rickettsioses comprises infection of endothelial cells with subsequent infiltration of inflammatory cells. Based on its ability to promote inflammation and endothelial cell activation, we investigated the role of CD40L in African tick bite fever (ATBF), caused by Rickettsia africae, using different experimental approaches. Several significant findings were revealed. 1) Patients with ATBF (n = 15) had increased serum levels of soluble CD40 ligand (sCD40L), which decreased during follow-up. 2) These enhanced sCD40L levels seem to reflect both direct and indirect (through endothelial cell activation involving CX3CL1-related mechanisms) effects of R. africae on platelets. 3) In combination with sCD40L, R. africae promoted a procoagulant state in endothelial cells by up-regulating tissue factor and down-regulating thrombomodulin expression. 4) Although the R. africae-mediated activation of platelets involved TLR2, the combined procoagulant effects of R. africae and sCD40L on endothelial cells involved TLR4. 5) Doxycycline counteracted the combined procoagulant effects of R. africae and sCD40L on endothelial cells. Our findings suggest an inflammatory interaction between platelets and endothelial cells in ATBF, involving TLR-related mechanisms. This interaction, which includes additive effects between sCD40L and R. africae, may contribute to endothelial inflammation and hypercoagulation in this disorder.     

Involvement of nuclear factor κB in platelet CD40 signaling

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.     

Molecular cloning and sequencing of 25 different rhesus macaque chemokine cDNAs reveals evolutionary conservation among C,CC, CXC,AND CX3C families of chemokines

The interaction between CD40L (CD154) on T cells and CD40 on antigen-presenting cells induces expression of accessory molecules that facilitate immune activation. Therefore, CD40L may have utility as an adjuvant for the development of potent antigen-specific immune responses following vaccination. As there was no information about the feline homologue of CD40L or its function, we generated stable cell lines expressing cDNAs encoding the feline CD40L homologue. As a preliminary to investigating the use of CD40L as an adjuvant for vaccination of the domestic cat, we tested the biological activity of the feline cytokine molecule in vitro. We demonstrated that cells expressing feline CD40L induced proliferation of feline peripheral blood mononuclear cells (PBMC) and that purified B cells could be induced to proliferate in response to feline CD40L.