Soluble CD40 Ligand Stimulates the Pro-Angiogenic Function of Peripheral Blood Angiogenic Outgrowth Cells via Increased Release of Matrix Metalloproteinase-9

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway.     

CD40 ligand (CD40L) does not stimulate proliferation of vascular smooth muscle cells

The present study investigates the effects of CD40 ligand (CD40L) on mitogenic signalling, proliferation, and migration of cultured bovine coronary artery smooth muscle cells (SMC). A time- and concentration-dependent phosphorylation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) and the mitogen-activated protein kinase p38 (p38-MAPK) was observed upon stimulation with soluble CD40L (sCD40L). This phosphorylation was inhibited by neutralizing antibodies against the CD40 and CD40L, respectively. Activation of the phosphatidylinositol-3-phosphate (PI-3) kinase pathway by sCD40L, as determined by the measurement of Akt phosphorylation, was not detected. However, there was evidence for direct activation of the NFkappaB system (degradation of IkappaBalpha and nuclear translocation of the p65 NFkappaB subunit) by sCD40L. Accordingly, sCD40L caused a small but significant increase in DNA synthesis. However, sCD40L-induced DNA synthesis was not followed by proliferation (increase in cell number). Furthermore, sCD40L did not potentiate SMC mitogenesis induced by known mitogens such as platelet-derived growth factor-BB, thrombin or serum. The lack of cell proliferation was not caused by a concomitant induction of SMC apoptosis by sCD40L. The possible role of membrane-bound CD40L in SMC mitogenesis was also studied using different membrane preparations (platelets, lymphocytes). However, no mitogenic effects of membrane-bound CD40L were detected. Finally, sCD40L did not induce SMC migration. From these data it is concluded that CD40L activates mitogenic signalling and DNA synthesis but does not contribute to proliferation or migration of vascular SMC.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.     

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.     

Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A(2) activation: a phospholipase Cgamma-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis.     

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.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

Background

CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration.

Methods and results

Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6 dyne/cm²) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated.

Conclusion

CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.     

Ligation of microglial CD40 results in p44/42 mitogen-activated protein kinase-dependent TNF-alpha production that is opposed by TGF-beta 1 and IL-10

Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-alpha production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-alpha release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-beta1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-alpha in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-alpha release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-beta1 and IL-10.     

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.     

beta-Chemokine production in CD40L-stimulated monocyte-derived macrophages requires activation of MAPK signaling pathways

CD40 ligand is a cell surface molecule on CD4(+) T cells that interacts with its receptor, CD40, on antigen presenting cells to mediate humoral and cellular immune responses. Our previous studies demonstrated that a trimeric soluble form of CD40L (CD40LT) activates macrophages to produce beta-chemokines and decrease CCR5 and CD4 cell surface expression, thus inducing resistance to HIV-1 infection. However, the mechanism(s) by which CD40LT mediates these effects in primary macrophages remains unclear. In this report, we demonstrate that CD40LT induces synthesis of beta-chemokines through the activation of MAPK signaling pathways. Treatment of macrophages with CD40LT results in a rapid activation of p38 and ERK1/2 mitogen-activated protein kinases. Inhibitors of these MAPKs blocked beta-chemokine production, while protein kinase A and C inhibitors had little or no effect. We also provide evidence that CD40LT stimulates beta-chemokine production directly, as well as indirectly via a TNF-alpha-dependent mechanism. At the early time points, CD40LT directly stimulated beta-chemokine production, whereas at later time points the effect was mediated to some extent by TNF-alpha. In conclusion, our results suggest that CD40-CD40L interactions are important for the activation of monocyte-derived macrophage antiviral response affecting both viral replication and the recruitment of immune cells.     

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.     

Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification

Platelets, in addition to exerting hemostatic activity, contribute to immunity and inflammation. The recent report that platelets express CD40 led us to hypothesize that CD40 ligand (CD40L)-positive T cells could bind to platelets, cause their activation, and trigger granular RANTES release, creating a T cell recruitment feedback loop. Platelets were cocultured with resting or activated autologous T cells and their activation was assessed by P-selectin expression. RANTES binding to endothelial cells was assessed by confocal microscopy, and its biological activity was demonstrated by a T cell adhesion assay. CD40L-positive T cells induced platelet activation through a contact-mediated, CD40-dependent pathway resulting in RANTES release, which bound to endothelial cells and mediated T cell recruitment. Soluble CD40L induced the same events via p38, but not extracellular signal-regulated kinase, phosphorylation. These results show the existence of a novel platelet-dependent pathway of immune response amplification which brings these nonimmune cells close to the level of pathogenic relevance traditionally attributed to classical immune cells.     

Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols

Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPKalpha attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPKalpha also blunted Akt activation in response to black tea polyphenols, suggesting that p38alpha MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.     

CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection

The macrophage-expressed CD40 regulates immune responses to Leishmania major infection by reciprocal signaling through p38 MAPK and ERK1/2. CD40-induced IL-10 or IL-12 plays crucial roles in the promotion or protection from L. major infection, respectively. Because p38 MAPK and ERK1/2 are dephosphorylated by dual-specificity MAPK phosphatases (MKPs), we tested the role of CD40 in the regulation of MKPs in L. major infection. MKP-1 expression and activity increased whereas MKP-3 expression and activity decreased in virulent L. major-infected macrophages. CD40 differentially regulated the expression and activity of MKP-1 and MKP-3, which, in turn, reciprocally regulated CD40-induced p38 MAPK and ERK1/2 phosphorylation and effector functions in macrophages. Triptolide, an inhibitor of MKP-1 expression, and lentivirally expressed MKP-1 short hairpin RNA enhanced CD40-induced anti-leishmanial functions and significantly protected susceptible BALB/c mice from L. major infection. Similarly, lentivirally overexpressed MKP-3 significantly reduced disease progression and parasite burden in susceptible BALB/c mice. Thus, to our knowledge, our data show for the first time that CD40 reciprocally regulates MKP-1 and MKP-3 expression and activity while the MKPs contribute to the reciprocal CD40 signaling-regulated anti-leishmanial functions. The findings reveal a novel parasite-devised immune evasion strategy and an effective target to redirect CD40-regulated immune responses.