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PubMed2006

[Preparation and identification of soluble trimeric CD40-isoleucine zipper fusion protein].

The interaction of CD40 with its cognate ligand, CD40L (CD154), plays important roles in immune responses. Blockade of CD40-CD40L signal pathway can protect the progression of antibody- and cell-mediated autoimmune diseases, and reduce allograft rejection thus prolonging graft survival, even engendering long-lived antigen-specific tolerance. The present study aims to enhance the binding activity of CD40 by incorporating an isoleucine zipper (IZ) trimeric motif into CD40 ectodomain to promote the formation of soluble CD40 trimers, which would be useful for blocking CD40-CD40L interaction. A prokaryotic expression vector for soluble human CD40 ectodomain fused with an IZ motif and a hexa-histidine (His6) tag at its carboxyl terminus (sCD40IZ) was constructed by multiple round PCR using cloned CD40 cDNA as a template. The recombinant sCD40IZ protein was expressed highly in Escherichia coli (E. coli) with a molecular weight of 27kD, which is consistent with its theoretical value. It mainly existed in inclusion bodies. After refolding from inclusion bodies, soluble sCD40IZ protein was purified by gel filtration. Its molecular weight in solution was about 91kD when determined by gel filtration, suggesting that it most probably existed in the form of trimers. Moreover, this protein could bind to CD40L expressed on Jurkat T cells and its binding activity was significantly higher than that of soluble CD40 without an IZ motif. These results suggest that incorporation of an IZ motif at the carboxyl terminus of soluble CD40 can facilitate the formation of trimers and enhance its binding activity with CD40L. Thus, the trimeric CD40 protein may be used to block CD40-CD40L signal pathway, suggesting that it may have potential application in preventing autoimmune diseases and transplantation rejection.

CD40 Antigens↗

PubMed1997

CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells.

Functional expression of CD40 has recently been described on the surface of HUVEC, and activation of these cells with CD40 ligand (CD40-L) leads to increased adhesion molecule expression. Here, we analyzed the effect of CD40 triggering on cytokine production by HUVEC. CD40-L-transfected fibroblasts, in contrast to their untransfected counterparts, as well as a soluble recombinant human CD40-L/murine CD8alpha chimeric molecule were able to importantly increase (by a mean of fourfold) the production of leukemia inhibitory factor (LIF) by HUVEC. CD40-L displayed an additive effect with IL-4, IL-1alpha, and TNF-alpha on the enhancement of LIF secretion. Cyclosporin A did not affect LIF synthesis by resting or CD40-L-activated HUVEC, whereas dexamethasone diminished the basal level of LIF production and abrogated the CD40-L effect. The secretions of two other proinflammatory cytokines, granulocyte-macrophage CSF and IL-6, were also increased in the presence of CD40-L. However, CD40-L neither affected HUVEC proliferation nor rescued them from IFN-gamma- and TNF-alpha induced apoptosis. Together, these results indicate that endothelial cell activation by CD40-L may play an important role not only in leukocyte recruitment through enhancement of adhesion molecule expression, but also in the maintenance of an inflammatory loop through the increase in proinflammatory cytokine secretion.

Apoptosis↗

PubMed1998

CD40-mediated signal transduction in human airway smooth muscle.

CD40 is a member of the TNF receptor family that was initially described on the surface of B cells. Recently, CD40 has also been described on mesenchymal cells, such as endothelial cells and fibroblasts, where engagement by its ligand CD40 ligand can lead to up-regulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines. Since airway inflammation potentially involves cell-cell interactions of T cells and eosinophils (which express CD40 ligand) with airway smooth muscle (ASM) cells, we postulated that ASM may express CD40 and that engagement of ASM CD40 may modulate smooth muscle cell function. We demonstrate that CD40 is expressed on cultured human ASM and that expression can be increased by treatment with TNF-alpha or IFN-gamma. Cross-linking CD40 on ASM resulted in enhanced IL-6 secretion and an increase in intracellular calcium concentrations, which were dependent on calcium influx. We show that CD40-mediated signaling events include protein tyrosine phosphorylation and activation of NF-kappaB. Pretreatment of ASM with the tyrosine kinase inhibitors genistein or herbimycin inhibited the rapid mobilization of calcium induced via CD40, suggesting that calcium mobilization was coupled to activation of protein tyrosine kinases. In addition, inhibition of calcium influx inhibited both CD40-mediated NF-kappaB activation and enhancement of IL-6 secretion. These results delineate a potentially important CD40-mediated signal-transduction pathway in ASM, involving protein tyrosine kinase-dependent calcium mobilization, NF-kappaB activation, and IL-6 production. Together, these results suggest a mechanism whereby T cell/smooth muscle cell interactions may potentiate airway inflammation.

CD40 Antigens↗

PubMed1996

Human epidermal keratinocytes are induced to secrete interleukin-6 and co-stimulate T lymphocyte proliferation by a CD40-dependent mechanism.

Although the expression and function of CD40 on B lymphocytes has been well studied, the significance of CD40 on non-lymphoid cells such as keratinocytes (KC) is not as well characterized. We demonstrate in this report that CD40 is expressed by virtually all human KC, and that it functions as an important signaling molecule. Flow cytometry of undifferentiated and terminally differentiated KC indicated that both cell types expressed CD40, as determined by binding to monoclonal antibodies and a recombinant CD40 ligand fusion protein; interferon-gamma (IFN-gamma) treatment of KC increased CD40 expression. Cultured KC also expressed 1.5-kb CD40 transcripts. Activation of KC cell surface CD40 using the monoclonal antibody G28.5 resulted in the rapid generation of a 50-kDa tyrosine phosphorylated polypeptide, as well as a dose-dependent increase in the secretion of interleukin-6, a cytokine that has been linked to KC proliferation. KC also co-stimulated a significant T lymphocyte proliferative response to the mitogen phytohemagglutinin that was CD40 dependent. These data indicate that KC constitutively express a low level of functional CD40 and regulate their expression in response to IFN-gamma. These data support the concept that KC, via their expression of CD40, have the capacity to amplify inflammation in the skin by interacting with CD40 ligand-bearing T cells.

CD40 Antigens↗

PubMed1996

Activation of mitogen-activated protein kinases via CD40 is distinct from that stimulated by surface IgM on B cells.

CD40 plays critical roles in B cell proliferation and differentiation in response to T cell-dependent antigenic stimulation. It has been suggested that CD40-mediated biological activities are transduced by a CD40 receptor-associated factor, CRAF1 and probably by protein tyrosine kinase Lyn and its substrates, phospholipase C gamma (PLC gamma) and phosphatidylinositol-3 kinase (PI-3 kinase). Here, we describe the novel finding that a mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (ERK) cascade is involved in CD40 signaling in mouse B cells. Analysis of ERK activities in the B cell lymphoma cell line WEHI 231, which shows an increase in DNA synthesis or arrest of the cell cycle by cross-linking of CD40 or surface IgM (sIgM) cross-linking, respectively, indicated that one of the ERK isoforms, ERK2, was preferentially and rapidly activated after CD40 cross-linking. The CD40-mediated ERK2 activation was comparable to that after sIgM stimulation, although the activity was reduced toward the basal level within several minutes after stimulation. In contrast, ERK1 and ERK2 were activated to a similar extent by sIgM cross-linking, and the activities remained stable for at least 10 min. Furthermore, similar features of differential activation of ERK isoforms were observed in normal resting B cells in CD40 and sIgM signaling. These results suggest divergent regulatory pathways for ERK1 and ERK2 activation, and they support the notion that CD40 signaling may utilize a limited set of elements in the ERK cascade. Co-stimulation of WEHI 231 cells with anti-CD40 mAb rescues the cells from anti-IgM-mediated apoptosis, whereas this co-stimulation resulted in activation of ERK isoforms comparable to that in sIgM stimulation, without a synergistic effect. This result indicates the dominance of ERK activation in sIgM signaling over that of CD40, and it suggests that ERK activation may not be linked to the biological effect that CD40 stimulation in this cell line.

Animals↗

PubMed1995

Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation.

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF-gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation.

Animals↗

PubMed1994

Antibodies to murine CD40 protect normal and malignant B cells from induced growth arrest.

We have previously described the production of polyclonal anti-murine CD40 antibodies that specifically bind recombinant murine CD40 expressed on L cells and induce vigorous proliferation of normal murine B lymphocytes. The current study utilizes these antibodies to explore the distribution and function of CD40 in murine B cell development. Murine CD40 is expressed at high levels by normal splenic B cells and all Ig-positive B cell lymphomas tested to date. It is not expressed by the 70Z/3 pre-B cell line, BaF3 pre-B cell line, or by numerous T cell and myeloid cell lines. 70Z/3 pre-B cells can be induced to express CD40 by LPS stimulation of the cells. Stimulation of purified splenic B cells with anti-CD40 antibodies causes upregulation of class II MHC antigens, CD23, and ICAM-1 and results in extensive aggregation of the cells. Antibodies to murine CD40 are extremely effective at rescuing malignant and normal B cells from induced growth arrest. Anti-CD40 antibodies protect WEHI-231 and CH31 B lymphoma cells from growth arrest induced by soluble anti-IgM antibodies, TGF beta, or a combination of both stimulants. Similarly, anti-IgM preactivated normal splenic B cells which normally die rapidly from growth arrest after 1 or 2 days culture produce a vigorous proliferative response to subsequent stimulation with anti-CD40 antibodies plus IL-4. Interestingly, anti-CD40 antibodies provide little to no protection against B lymphoma growth arrest induced by immobilized anti-IgM antibodies. These data confirm and extend functional properties assigned previously to human CD40 and identify numerous defined murine model systems to explore the molecular basis of CD40-mediated protection from induced B cell growth arrest.

Animals↗

PubMed2004

CD40 expression on human pancreatic duct cells: role in nuclear factor-kappa B activation and production of pro-inflammatory cytokines.

AIMS/HYPOTHESIS: Human pancreatic duct cells are closely associated with islet beta cells, and contaminate islet suspensions transplanted in Type 1 diabetes mellitus patients. Activated duct cells produce cytotoxic mediators and possibly contribute to the pathogenesis of Type 1 diabetes mellitus or islet graft rejection. As CD40 transduces activation signals involved in inflammatory and immune disorders, we investigated CD40 expression on duct cells and their response to CD40 engagement. METHODS: CD40 expression on human pancreatic duct cells was analysed by flow cytometry and immunohistochemical analyses. To assess the function of CD40 expression on duct cells, activation of the transcription factor nuclear factor-kappa B was determined using electrophoretic mobility shift assay and ELISA. Cytokine mRNA levels were quantified by real-time RT-PCR, and protein levels by Luminex technology. RESULTS: Isolated human pancreatic duct cells and Capan-2 cell lines were found to express constitutively CD40. The expression of CD40 on duct cells was confirmed in vivo on human normal and pathological pancreatic specimens. CD40 ligation on Capan-2 cells induced rapid nuclear factor-kappa B activation, and supershift assays demonstrated that p50/p65 heterodimers and p50/p50 homodimers were present in the activated complexes in the nucleus. This activation was accompanied by tumour necrosis factor-a and interleukin-1beta mRNA accumulation. Tumour necrosis factor-alpha protein secretion was confirmed in CD40-activated Capan-2 cells and in isolated human pancreatic duct cells. CONCLUSIONS/INTERPRETATION: Interaction between activated T lymphocytes expressing CD40 ligand and duct cells expressing CD40 may contribute to the immune responses involved in Type 1 diabetes mellitus and islet graft rejection. Interfering with CD40-mediated duct cell activation could alleviate beta cell damage of immune origin.

CD40 Antigens↗

PubMed2005

A functional CD40 receptor is expressed in pancreatic beta cells.

AIMS/HYPOTHESIS: Despite differences in function and embryonic origin, pancreatic islet cells and neurons express proteins belonging to the tumour necrosis factor receptor superfamily. While neurons express the CD40 receptor, it is unknown whether islet cells also express it. We investigated CD40 expression in human and mouse pancreatic islets as well as in NIT-1 insulinoma cells. METHODS: CD40 expression was studied by reverse transcriptase polymerase chain reaction, flow cytometry, immunohistochemistry and western blot. Responses mediated by CD40 were assessed by a luciferase gene reporter assay following stimulation with a CD40 agonist antibody. RESULTS: We found that CD40 is expressed in mouse and human pancreatic islet cells. CD40 is expressed by beta cells, and its expression is upregulated by proinflammatory cytokines (IL-1beta, IFN-gamma and TNF-alpha). CD40 signalling in NIT-1 insulinoma cells activates nuclear factor kappa-B, demonstrating that CD40 is functional. CONCLUSIONS/INTERPRETATION: We present evidence that, in addition to immune cell types, mouse and human pancreatic beta cells express CD40. Its expression is upregulated by proinflammatory stimuli, and signalling through this receptor activates NF-kappaB. We suggest that the effects of inflammatory stimuli that affect beta cell function and survival may be also mediated by signalling through the CD40 receptor. Thus, CD40 may have a role in processes associated with islet autoimmunity and transplantation.

Animals↗

PubMed1994

Enhancement of IgE production by anti-CD40 antibody in atopic dermatitis.

It has been recently recognized that the obligate requirement for T cells in the development of IgE responses can be substituted for by anti-CD40 antibody. In this study of patients with atopic dermatitis and high IgE levels, we have analyzed the role of the CD40 molecule in IgE production. Costimulation of peripheral blood mononuclear cells (PBMCs) from normal donors with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody resulted in a selective increase in IgE production; either reagent alone, however, was ineffective. In contrast, addition of anti-CD40 monoclonal antibody alone to PBMCs or B cells from patients with atopic dermatitis markedly increased IgE production, even in the absence of exogenous IL-4. With the use of an ELISA spot assay, this increase in IgE production was attributed to an expansion of IgE-secreting B cells. In anti-IgM-stimulated lymphocyte cultures from patients with atopic dermatitis the costimulation with anti-CD40 induced strong lymphocyte proliferation. Similar results were observed with anti-IgM plus IL-4. The augmentation induced by anti-CD40 was inhibited by addition of anti-IL4 to anti-CD40-treated atopic dermatitis cells. In normal subjects the effects of anti-CD40 alone on IgE production could be observed after pretreatment of normal PBMCs with IL-4 for 3 days. The effects of anti-CD40 in atopic dermatitis may be explained in part by differences in CD40 expression. In freshly isolated PBMCs from patients with atopic dermatitis, the mean fluorescence intensity of CD40 expression on B cells was increased when compared with PBMCs from nonatopic donors, ans stimulation of normal or atopic dermatitis PBMCs with IL-4 increased the intensity of CD40 staining of cells.

Adolescent↗

PubMed1996

The functional CD40 antigen of fibroblasts may contribute to the proliferation of rheumatoid synovium.

This paper demonstrates that CD40 is expressed on rheumatoid synovial pannus and primary fibroblast cell lines established from rheumatoid and osteoarthritic synovium as well as normal skin. Among various tested cytokines, interferon-gamma (IFN-gamma) and to a lower extent, tumour necrosis factor-alpha (TNF-alpha) were found to upregulate CD40 expression on fibroblasts. Synovial and skin fibroblasts cultured over CD40 Ligand transfected L cells (L-CD40 L) demonstrate a CD40 specific increase of DNA synthesis as measured by tritiated thymidine incorporation. Cell-cycle analysis and enumeration of viable cells further show that CD40 induced fibroblast proliferation. Costimulation with L-CD40 L and IFN-gamma resulted in maximal proliferation. Engagement of fibroblasts CD40 increased the IL-1-induced production of granulocyte macrophage-colony stimulating factor and macrophage inflammatory protein-1 alpha MIP-1 alpha. CD40 L activated fibroblasts showed decreased levels of CD40, but only marginal alterations of other cell-surface antigens. Taken together, the present results indicate that fibroblasts express functional CD40 and suggest a possible role of CD40 L expressing cells, such as activated T cells and mast cells, in the development of synovium hyperplasia.

Animals↗

PubMed2001

Antibodies to CD40 induce a lethal cytokine cascade after syngeneic bone marrow transplantation.

CD40 stimulation, by either antibody or ligand, has been shown to inhibit the growth of a variety of neoplastic cells, both in vivo and in vitro. In this study, we assessed the effects of CD40 stimulation using a murine agonistic CD40 monoclonal antibody (MoAb) (FGK115) or a soluble recombinant murine CD40 ligand (srmCD40L) in both lethally irradiated and nonirradiated BALB/c mice. Toxicity after CD40 stimulation was not observed in nonirradiated animals receiving up to 100 microg of the agonist anti-CD40 MoAb. However, as little as 10 microg of the agonistic anti-CD40 MoAb induced acute toxicity resulting in 100% morbidity of lethally irradiated animals by 4 days after irradiation. Histological evaluation of animals receiving anti-CD40 MoAb revealed severe intestinal lesions with disruption of the villi, goblet cell depletion, and crypt hyperplasia of the small intestine, colon, and cecum. Delaying the administration of anti-CD40 MoAb or reducing the amount of irradiation given resulted in increased survival and less severe lesions. Analysis of serum cytokine levels in lethally irradiated mice receiving agonistic anti-CD40 showed a marked increase of interferon (IFN)-gamma. Lethally irradiated IFN-gamma knockout mice given the agonistic anti-CD40 MoAb demonstrated significant increases in survival and minimal gut lesions compared with wild-type mice receiving the same regimen, suggesting that IFN-gamma plays a major role in this toxic reaction. These results indicate that CD40 stimulation using agonistic antibodies following lethal irradiation leads to a fatal, cytokine-induced disease affecting the intestine.

Animals↗

PubMed2002

CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-kappa B, AND CBF1.

CD40-induced activation of cytokine gene expression in dendritic cells (DC) is an important process in the initiation of primary immune responses. We have determined the intracellular signaling events that lead to CD40 ligation-induced activation of interleukin-6 (IL-6) gene transcription in a murine DC line, FSDC, that is phenotypically representative of bone marrow-derived DC. IL-6 reverse transcriptase-PCR and promoter assays established the responsiveness of FSDC to anti-CD40 ligation. Further promoter assays showed that the transcription factors NF-kappaB and AP-1 are downstream transcriptional mediators of CD40-induced IL-6 gene expression. Anti-CD40 treatment of FSDC stimulated increased expression of specific NF-kappaB (p50:p65) and AP-1 (c-Jun, JunB, JunD, and c-Fos) DNA-protein complexes. Overexpression of an IkappaB-alpha super-repressor or a dominant negative JunD resulted in a strong inhibition of CD40-inducible IL-6 promoter activity supporting a role for both transcription factors. Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDC followed by stimulation with an anti-human CD40 antibody. These experiments revealed that anti-CD40 stimulation of NF-kappaB and IL-6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2. Induction of IL-6 mRNA by anti-CD40 treatment was found to be a transient event (24 h) and was followed by a diminution of IL-6 transcript to levels below those found in unstimulated cells. This loss of IL-6 expression was associated with reduced p50:p65 NF-kappaB DNA binding and elevated binding of CBF1 to a site overlapping the NF-kappaB site. Overexpression of CBF1 resulted in a profound inhibition of basal and anti-CD40-induced IL-6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of the IL-6 response. The physiological relevance of these molecular events to DC function is discussed.

Amino Acid Motifs↗

PubMed2002

CD40-mediated activation of NF-kappa B in airway epithelial cells.

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.

Antigens, CD↗

PubMed2003

Mouse endothelial CD40 expression does not play a role during the development of transplant arteriosclerosis.

Endothelial cells (ECs) are of major interest in allograft rejection. The authors and others have recently shown in different mouse allograft models that, during the development of transplant arteriosclerosis, donor ECs are replaced by recipient ECs. It was the aim of this study to characterize the phenotype of ECs during the development of transplant arteriosclerosis further, with particular interest in their capability to express CD40. Abdominal aortic allografts were transplanted across a full major histocompatibility complex (MHC) barrier using BALB/c (H-2d) mice as donors and C57BL/6 (H-2b) mice as transplant recipients. Aortic allografts were harvested on days 7 and 30 after transplantation and analyzed by double-immunohistochemistry for expression of CD40 and CD31. As the authors have previously shown that ECs in the aortic allograft model are of donor origin on day 7 after transplantation, whereas by day 30 donor ECs have been replaced by ECs of recipient origin, these two time points were chosen for analysis of CD40 expression. Double-immunohistochemistry for CD40 and CD31 revealed no endothelial expression of CD40 at either time point. These initial results obtained with the anti-CD40 monoclonal antibody (mAb) (clone 3/23) were confirmed by two further experiments using two different anti-CD40 mAbs (clone HM40-3 and clone 1/10) that bind to epitopes of the CD40 receptor distinct to that of 3/23. Neither mAb revealed endothelial expression of CD40. Further analysis of the composition of the cellular infiltrate demonstrated that the majority of macrophages were CD40 positive. The data indicate that in this mouse model of aortic transplantation, neither recipient- nor donor-derived ECs express CD40 during the development of transplant arteriosclerosis. Therefore, in contrast to rat or human ECs, CD40 expression by mouse ECs does not seem to play a major role in this form of allograft rejection.

Animals↗

PubMed2001

Marrow-derived CD40-positive cells are required for mice to clear Cryptosporidium parvum infection.

To clear a Cryptosporidium parvum infection, mice need CD4+ T cells, major histocompatibility complex class II, and an intact CD40-CD154 signaling pathway. CD40 is constitutively expressed on marrow-derived cells such as dendritic cells and B lymphocytes and is induced by gamma interferon (IFN-gamma) on most somatic cells. To determine whether the CD40 needed to clear a C. parvum infection has to be on marrow-derived mononuclear cells or on the epithelial cells that normally harbor the parasite, we transplanted CD40-/- mice with CD40+/- bone marrow and then infected them with C. parvum. These chimeras cleared the C. parvum infection, while CD40+/- controls transplanted with CD40-/- marrow cells remained infected. CD40 expression on marrow-derived cells therefore suffices for a C. parvum infection to be cleared, while CD40 expression on intestinal epithelial cells is not sufficient. There was no difference between the acquisition of CD69 and CD154 by mesenteric lymph node T cells of C. parvum-infected animals with intact or disrupted CD40-CD154 pathways. CD4 T cells entered the intestinal laminae propriae of C. parvum-infected animals whether or not the CD40 genes of these recipients were intact. These results suggest that, for a C. parvum infection to be cleared, CD40 is not necessary for T-cell activation but may instead contribute to an effector pathway of marrow-derived cells.

Adoptive Transfer↗

PubMed2005

TRAF6 is required for TRAF2-dependent CD40 signal transduction in nonhemopoietic cells.

The emerging role of CD40, a tumor necrosis factor (TNF) receptor family member, in immune regulation, disease pathogenesis, and cancer therapy necessitates the analysis of CD40 signal transduction in a wide range of tissue types. In this study we present evidence that the CD40-interacting proteins TRAF2 and TRAF6 play an important physiological role in CD40 signaling in nonhemopoietic cells. Using mutational analysis of the CD40 cytoplasmic tail, we demonstrate that the specific binding of TRAF2 to CD40 is required for efficient signaling on the NF-kappaB, Jun N-terminal protein kinase (JNK), and p38 axis. In fibroblasts lacking TRAF2 or in carcinoma cells in which TRAF2 has been depleted by RNA interference, the CD40-mediated activation of NF-kappaB and JNK is significantly reduced, and the activation of p38 and Akt is severely impaired. Interestingly, whereas the TRAF6-interacting membrane-proximal domain of CD40 has a minor role in signal transduction, studies utilizing TRAF6 knockout fibroblasts and RNA interference in epithelial cells reveal that the CD40-induced activation of NF-kappaB, JNK, p38, and Akt requires the integrity of TRAF6. Furthermore, we provide evidence that TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2. These observations provide novel insight into the mechanisms of CD40 signaling and the multiple roles played by TRAF6 in signal transduction.

CD40 Antigens↗

PubMed2006

Anthocyanin prevents CD40-activated proinflammatory signaling in endothelial cells by regulating cholesterol distribution.

OBJECTIVE: Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a key element in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin on CD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism. METHODS AND RESULTS: Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatory status, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-induced nuclear factor-kappaB (NF-kappaB) activation. TRAF-2 played pivotal role in CD40-NF-kappaB pathway as TRAF-2 small interference RNA (siRNA) diminished CD40-induced NF-kappaB activation and inflammation. TRAF-2 overexpression increased CD40-mediated NF-kappaB activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulation by CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-kappaB activation. Exposure to anthocyanin not only interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipid rafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor. CONCLUSIONS: Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling by preventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin.

Analysis of Variance↗