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PubMed2004

Requirement for donor and recipient CD40 expression in cardiac allograft rejection: induction of Th1 responses and influence of donor-derived dendritic cells.

Costimulation through the CD40-CD40 ligand (CD40L) pathway is critical to allograft rejection, in that anti-CD40L mAb therapy prolongs allograft survival. However, the majority of studies exploring CD40-CD40L interactions have targeted CD40L. Less is known about the requirement for donor- and/or host-derived CD40 during rejection. This study assessed the relative contributions of donor and recipient CD40 expression to the rejection process. As the effectiveness of costimulatory blockade may be mouse strain dependent, this study explored the requirement for donor and recipient CD40 expression in BALB/c and C57BL/6 mice. Wild-type (WT) and CD40(-/-) BALB/c recipients readily rejected WT and CD40(-/-) C57BL/6 allografts, and rejection was associated with a prominent Th1 response. In contrast, CD40(-/-) C57BL/6 recipients failed to reject WT or CD40(-/-) BALB/c allografts and did not mount Th1 or Th2 responses. However, injection of donor CD40(-/-) dendritic cells induced both Th1 and Th2 responses and allograft rejection in CD40(-/-) C57BL/6 recipients. Finally, WT C57BL/6 mice rejected CD40(-/-) allografts, but this rejection response was associated with muted Th1 responses. These findings demonstrate that 1) CD40 expression by the recipient or the graft may impact on the immune response following transplantation; 2) the requirement for CD40 is influenced by the mouse strain; and 3) the requirement for CD40 in rejection may be bypassed by donor DC. Further, as CD40 is not required for rejection in BALB/c recipients, but anti-CD40L mAb prolongs graft survival in these mice, these results suggest that anti-CD40L therapy functions at a level beyond disruption of CD40-CD40L interactions.

Animals↗
PubMed1996

CD40 is a prognostic marker in primary cutaneous malignant melanoma.

CD40 is a receptor at the surface of B lymphocytes with important functions in the immune response. CD40 has also been found on a variety of carcinoma and melanoma cell lines where it has been suggested to serve as a possible receptor for mitogenic signals. We studied the expression and distribution of CD40 in paraffin sections of 71 uniformly treated malignant melanomas (MMs) with a long clinical follow-up using well known monoclonal antibodies. For comparison, 71 benign nevi were also studied. Common acquired nevi occasionally expressed CD40 in nests or single cells at the dermo-epidermal junction; no immunoreactivity was observed in the dermal part of acquired nevi, and all Spitz' nevi were entirely negative. One-third of large congenital nevi expressed CD40 in small clusters of heavily pigmented, epithelioid cells, corresponding to so-called proliferative nodules. In 41 of 71 MMs, CD40 was expressed in single or clustered neoplastic melanocytes; 9 cases showed CD40 expression only in the radial growth phase, and in 32 cases, the vertical growth phase showed CD40 expression. The same staining pattern was obtained with other anti-CD40 monoclonal antibodies, directed to different epitopes of the CD40 molecule. In 29 of 32 MMs showing CD40 in the vertical growth phase, expression of the CD40 ligand (CD40L) was studied; in 13 of these 29, CD40L was found in the same tumor areas that expressed CD40. Analysis of 28 metastases from 24 MM patients showed in the majority of cases a similar, scattered or nodular staining pattern as observed in the primary tumor. Patients expressing CD40 in the vertical growth phase of their MM did not differ significantly from CD40-negative patients with respect to any of the known prognostic parameters but showed a significantly shorter tumor-free survival. Patients with CD40+ CD40L+ MM tended to have a shorter tumor-free survival than those lacking CD40L. We conclude that CD40 represents a novel prognostic parameter in primary cutaneous MM. The co-localization of CD40 and CD40L suggests an autocrine growth loop in the vertical growth phase of MM.

Biomarkers, Tumor↗
PubMed1990

Identification of the intracytoplasmic region essential for signal transduction through a B cell activation molecule, CD40.

CD40 is a 45-kDa glycoprotein expressed on human B lineage cells. Anti-CD40 induces the proliferation of B cells and the extracellular region of CD40 is related to those of a certain kind of growth factor receptors. Therefore, it has been proposed that CD40 might be a receptor for a molecule involved in the growth regulation of B cells. The cDNA coding for CD40 was transfected into the murine B lymphoma cell line M12 and the murine thymoma cell line EL4. The growth of both M12 and EL4 transfectants expressing human CD40 was inhibited by anti-CD40. Phorbol 12-myristate 13-acetate (PMA) augmented the growth inhibitory effects of anti-CD40 on transfectants. The CD40 molecule was constitutively phosphorylated not only in human tonsil B cells but also in transfectants expressing CD40. PMA augmented the phosphorylation of CD40 in these cells. These results indicate that in spite of the growth inhibitory effect of anti-CD40, the augmentative effect of PMA is conserved in CD40+ transfectants and suggest that the transfectant might be useful for the study of signal transduction mechanism through CD40. To investigate which part of the CD40 molecule is important for signal transduction, transfectants expressing mutant CD40 cDNA were established and their growth response to anti-CD40 was evaluated. The mutant molecule, which had an Ala for Thr substitution at position 234, and the deletion mutants lacking Thr234 were inactive in growth signal transduction, indicating that Thr234 itself or the region around Thr234 is essential for signal transduction through CD40.

Animals↗
PubMed2005

Establishment and maintenance of long-term murine gammaherpesvirus 68 latency in B cells in the absence of CD40.

Murine gammaherpesvirus 68 (gammaHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of gammaHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, gammaHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in gammaHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40(-/-)) mice. Here we report that, consistent with previous observations, gammaHV68 efficiently established a latent infection in B cells of CD40(-/-) mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40(-/-) mice 42 days postinfection was observed. In addition, in contrast to gammaHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of gammaHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that gammaHV68 may either directly infect memory B cells present in CD40(-/-) mice or be capable of driving differentiation of naive CD40(-/-) B cells. A possible explanation for the apparent discrepancy between the failure of gammaHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of gammaHV68 B-cell latency in CD40(-/-) mice came from examining virus replication in the lungs of infected CD40(-/-) mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40(-/-) mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.

Animals↗
PubMed2003

CD40 is expressed on ovarian cancer cells and can be utilized for targeting adenoviruses.

PURPOSE: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types in addition to hematopoietic cells. Recent studies show that CD40 expression is related to several carcinomas, although its role in cancer pathobiology is unknown. In this study, we demonstrate the expression of CD40 on several ovarian carcinoma cell lines and the ability of CD40 to mediate targeted adenoviral infection in vitro. EXPERIMENTAL DESIGN: CD40 expression on ovarian cancer cell lines and clinical patient samples was examined by reverse transcription-PCR and flow cytometry. To study the utilization of CD40 for gene delivery, we precomplexed a luciferase coding adenovirus (Ad), Ad5luc1, with a CD40-targeting molecule (CAR/G28). RESULTS: According to our studies, all of the examined ovarian cancer cell lines are expressing CD40. In addition, mRNA for CD40 was detected in every primary tumor sample, suggesting that CD40 is also expressed in vivo. Compared with nontargeted Ad, gene transfer was increased up to 40-fold in CD40+ cells when CD40-targeted Ad was used. Supporting the relation of targeted system to CD40, increasing the amount of targeting fusion protein results in dose response. Furthermore, blockade of CD40 receptors on cell surface decreases the infectability of CD40+ cells with CD40-targeted virus, indicating the specificity of the targeting system for CD40. CONCLUSIONS: These results suggest that CD40 is present in ovarian cancer cells and can be used for targeted gene delivery in a CAR-independent manner, circumventing the problem of the low expression levels of CAR in various cancer cells.

Adenoviridae↗
PubMed2002

Regulation of tumor necrosis factor-alpha expression by CD40 ligation in BV-2 microglial cells.

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor-alpha (TNF-alpha) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF-alpha in BV-2 microglial cells. There was very little induction of TNF-alpha by ligation of CD40 alone by either cross-linking antibodies against CD40 or recombinant CD40 ligand (CD154). The absence of any increase in TNF-alpha production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF-alpha. However, CD40 ligation markedly induced the production of TNF-alpha as well as the expression of TNF-alpha mRNA in interferon-gamma (IFN-gamma)-stimulated BV-2 glial cells. Ligation of CD40 in CD40-overexpressed cells markedly enhanced the expression of TNF-alpha in the presence of IFN-gamma. To understand the mechanism of CD40 ligation-mediated induction/stimulation of TNF-alpha, we investigated the role of nuclear factor-kappaB (NF-kappaB) and C/EBPbeta. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF-kappaB and not that of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of TNF-alpha and that the activation of C/EBPbeta is also necessary for strong induction of TNF-alpha. Consistently, a dominant-negative mutant of p65 (Delta(p65)) and that of C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of TNF-alpha in BV-2 microglial cells stimulated with the combination of IFN-gamma and CD40 ligand. Taken together, these studies suggest that activation of both NF-kappaB and C/EBPbeta is important for strong induction of TNF-alpha and that CD40 ligation regulates the expression of TNF-alpha by modulating the activation of only NF-kappaB but not that of C/EBPbeta.

Animals↗
PubMed1995

CD40 expression by human fibroblasts.

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.

Adjuvants, Immunologic↗
PubMed2001

Ligation of CD40 stimulates the induction of nitric-oxide synthase in microglial cells.

The present study was undertaken to investigate the role of CD40 ligation in the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 microglial cells and primary microglia. Ligation of CD40 alone by either cross-linking antibodies against CD40 or a recombinant CD40 ligand (CD154) was unable to induce the production of NO in BV-2 microglial cells. The absence of induction of NO production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggests that a signal transduced by the ligation of CD40 alone is not sufficient to induce NO production. However, CD40 ligation markedly stimulated interferon-gamma (IFN-gamma)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-gamma-induced production of NO. This stimulation of NO production was accompanied by stimulation of the iNOS protein and mRNA. In addition to BV-2 glial cells, CD40 ligation also stimulated IFN-gamma-mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mechanism of induction/stimulation of iNOS, we investigated the roles of nuclear factor kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), transcription factors responsible for the induction of iNOS. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone induced the activation of only NF-kappaB but not of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPbeta is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Deltap65) and C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-gamma and CD40 ligand. Stimulation of IFN-gamma-mediated activation of NF-kappaB but not of C/EBPbeta by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-gamma-treated BV-2 microglial cells through the stimulation of NF-kappaB activation. This study illustrates a novel role for CD40 ligation in stimulating the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Animals↗
PubMed2002

CD40 activation in epithelial ovarian carcinoma cells modulates growth, apoptosis, and cytokine secretion.

BACKGROUND/AIMS: CD40, a member of the tumour necrosis factor (TNF) receptor family, is expressed on a variety of haematopoietic cells and is crucial in orchestrating both humoral and cellular immune responses. CD40 is also expressed on some carcinoma cells, where its function remains largely unknown. This study investigated the effects of CD40 ligation on ovarian carcinoma cell growth and apoptosis and on cytokine production, in addition to the role of the NF-kappa B and JNK signalling pathways. METHODS: CD40 expression was measured in epithelial ovarian carcinoma (EOC) biopsies by immunohistochemistry and in EOC cell lines by flow cytometry. To examine the effects of CD40 ligation on cell growth recombinant soluble CD40 ligand was used to stimulate EOC cell lines and growth was measured by MMT assays. Cytokine production was measured by enzyme linked immunosorbent assays interleukin 8 (IL-8) gene transcription was estimated by means of reverse transcription polymerase chain reaction. The integrity of the CD40 signalling pathway in those cell lines that did not produce cytokines in response to CD40 ligation was assessed by the detection of the transcription factor NF-kappa B by an electrophoretic mobility shift assay. To investigate the defect in the NF-kappa B pathway the phosphorylation status of I kappa B alpha was determined by an antibody specific to phosphorylated I kappa B alpha and dissociation of the I kappa B alpha-p65 complex was assessed by co-immunoprecipitation. RESULTS: CD40 is expressed in primary ovarian carcinoma biopsies and EOC cell lines. CD40 ligation resulted in growth inhibition in most of these carcinoma cell lines and was also found to promote apoptosis, with this last effect only being evident in early passage EOC cells. CD40 ligation also induced significant IL-6 and IL-8 production in most of the EOC cell lines examined and it was confirmed for IL-8 that this effect was regulated at the transcriptional level. NF-kappa B activation in response to CD40 ligation was found in three of the EOC cell lines and specific defects in the CD40 induced NF-kappa B pathway were identified in two cell lines. However, CD40 engagement induced JNK activation in all the EOC cell lines. CONCLUSIONS: These data suggest that the CD40 pathway is functional in ovarian carcinoma cells and highlight the need for further studies to provide insight into the role of CD40 in the carcinogenic process and the possible exploitation of this pathway for novel therapeutic approaches.

Antibodies, Monoclonal↗
PubMed2002

CD40-mediated p38 mitogen-activated protein kinase activation is required for immunoglobulin class switch recombination to IgE.

BACKGROUND: Signaling through CD40 activates multiple kinases and signal pathways that drive diverse CD40-mediated biologic functions. The specific pathways activated by CD40 signaling involving CD40-dependent Ig class switch recombination (CSR) have not been defined. OBJECTIVE: We sought to dissect CD40-activated signaling required for CD40-mediated Ig CSR by using the specific signal pathway inhibitors, with the emphasis on CD40-activated p38 mitogen-activated protein kinase (p38 MAPK) signaling in CD40-mediated CSR to IgE. METHODS: Human B cells were costimulated with IL-4 plus anti-CD40 in the presence or absence of specific signal pathway inhibitors. Ig production, kinase phosphorylation, IgH epsilon germline transcripts and Smu-Sepsilon recombination were examined, and their relationships were analyzed. RESULTS: CD40-dependent IgE induction was inhibited by the specific p38 MAPK inhibitor SB203580 but not by the extracellular signal-regulated protein kinase-specific inhibitor PD98059 or the phosphatidylinositol 3-kinase-specific inhibitor LY294002. CD40 activation of p38 MAPK correlated with CD40-dependent IgE production, and IgE suppression by SB203580 correlated with the inhibition of CD40-activated p38 MAPK phosphorylation. Suppression of IgE production by SB203580 was not due to inhibition of cell proliferation because SB203580 did not suppress IL-4 plus alpha-CD40-induced cell proliferation. SB203580, but neither PD98059 nor LY294002, inhibited CD40-dependent Smu-Sepsilon recombination, as determined by using a digestion circularization PCR assay. The inhibitory effects of SB203580 on IgE production and Smu-Sepsilon recombination were directly related to its ability to suppress production of Ig epsilon germline transcripts. CONCLUSION: These results demonstrate that p38 MAPK is required for CD40-mediated class switching to IgE.

B-Lymphocytes↗
PubMed1993

Regulation of expression of the ligand for CD40 on T helper lymphocytes.

Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell-delivered contact signals to B cells and may contribute to the high degree of specificity in B cell responses.

Animals↗
PubMed1997

Characterization of CD40 signaling determinants regulating nuclear factor-kappa B activation in B lymphocytes.

CD40 signaling to B cells is important for generating an effective humoral immune response. CD40 ligation leads to B cell activation events such as proliferation, Ig secretion, isotype switching, and up-regulation of cell surface molecules, as well as the generation of memory B cells. Many of these events are dependent upon the ability of CD40 to activate the transcription factor NF-kappa B (NF-kappa B). To define the CD40 signaling components upstream of NF-kappa B activation and the functional consequences downstream of NF-kappa B activation, we examined mouse B cell transfectants expressing wild-type or mutant human CD40. Analysis of CD40 cytoplasmic domain truncation and point mutants defined a 10-amino acid CD40 cytoplasmic signaling determinant required for NF-kappa B activation. A threonine residue at position 234, previously shown to be important for CD40 association with TNF receptor-associated factor 2 (TRAF2), TRAF3, and TRAF5, was not required for NF-kappa B activation. This suggests that in B cells, CD40-induced NF-kappa B activation can occur independently of TRAF2 and TRAF5 association. NF-kappa B activation was independent of the transmembrane domain of CD40, suggesting that it is independent of p23, a molecule that associates with CD40 in a region other than the cytoplasmic domain. Proteasome-dependent inhibitory kappa B alpha (I kappa B alpha) and I kappa B beta degradation occurred downstream of CD40 ligation and preceded CD40-mediated NF-kappa B nuclear translocation. CD40- or pervanadate-mediated I kappa B tyrosine phosphorylation was not detected. NF-kappa B activation correlated with the ability of CD40 to induce Ab secretion and the up-regulation of ICAM-1 and LFA-1. However, NF-kappa B activation was insufficient for CD40-mediated up-regulation of B7-1, Fas, and CD23.

Amino Acid Sequence↗
PubMed2006

Transfection and ligation of CD40 in human oral keratinocytes affect proliferation, adhesion and migration but not apoptosis in vitro.

AIMS: CD40 expression is restricted to Keratinocytes of normal epidermis or stratified squamous epithelium of oral mucosa. Ligation of CD40 inhibits keratinocyte proliferation and apoptosis. The aim of this study was to investigate the functional significance of CD40 in the proliferation, apoptosis, adhesion and migration of human oral keratinocytes in vitro. METHODS: The CD40-negative oral keratinocyte line OSC19, its CD40-positive transfected derivative (OSC19T-CD40) and null transfectants (OSC19T-control), with and without stimulation by soluble protein CD40 ligand (sCD40L) or anti-CD40 antibodies were used. RESULTS: OSC19T-CD40 showed significantly (P < 0.001) slower growth than the null transfectants and parent cells. OSC19T-CD40 proliferation was inhibited by ligation with sCD40L and blocking by two anti-CD40 antibodies, but stimulated by a third. Binding of CD40 with ligand or antibody had no effect on keratinocyte apoptosis in any cell line. The capacity of OSC19T-CD40 cells to adhere to CD40L-coated wells was significantly greater (P < 0.001) than that of parent OSC19 and OSC19T-control cells, and the migration rate of OSC19T-CD40 cells was significantly higher than parent OSC19 (P = 0.038 on fibronectin, P = 0.004 on Matrigel) or OSC19T-control (P =0.017 on fibronectin, P = 0.013 on Matrigel) cells. CONCLUSIONS: CD40 is an important molecule in keratinocyte homeostasis, and has more than one ligand. The ligand that is bound may be critical in oral epithelial homeostasis, the development of malignancy and the behaviour of the subsequent tumour.

Apoptosis↗
PubMedDate not supplied

CD40 activation as potential tool in malignant neoplasms.

BACKGROUND: CD40, a cell surface molecule, is expressed on B-cell malignancies and many different solid tumors. It is capable of mediating diverse biological phenomena such as the induction of apoptosis in tumors and stimulation of the immune response. It has thus been studied as a possible target for antitumor therapy. The general aim of this review is to focus the attention of clinical oncologists on the involvement of CD40 in tumors and the rationale of CD40-activation-based therapies in new, biologically oriented antitumor protocols. METHODS: A Medline review of published papers about the role of CD40 activation in cancer therapy. RESULTS: Many authors have shown that CD40 activation promotes apoptotic death of tumor cells and that the presence of the molecule on the surface of carcinoma lines is an important factor in the generation of tumor-specific T-cell responses that contribute to tumor cell elimination. The CD40 ligand (CD40L) is the natural ligand for CD40; it is expressed primarily on the surface of activated T lymphocytes. Preclinical studies suggest that CD40-CD40L interaction could be useful for cytotoxicity against CD40-expressing tumors and for immune stimulation. Tumor inhibition was observed when tumor cells were treated with agonistic anti-CD40 monoclonal antibodies or with the soluble form of CD40L. The results of the first phase I clinical trial to treat cancer patients with subcutaneous injection of recombinant human CD40L have been recently reported. Immunohistochemical studies have revealed that detection of CD40 in primary cutaneous malignant melanoma and lung cancer may have a negative prognostic value. Interestingly, up-regulation of CD40 was observed in the tumor vessels of renal carcinomas and Kaposi's sarcoma, suggesting possible involvement of CD40 in tumor angiogenesis. Recently, it has also been shown that CD40 engagement on endothelial cells induces in vitro tubule formation and expression of matrix metalloproteinases, two processes involved in the neovascularization and progression of tumors. CONCLUSIONS: CD40 activation represents an exciting target for hematological malignancies and solid tumors expressing the molecule, but its functional role in cancer development still remains unclear and controversial.

Animals↗
PubMed2004

Signaling and protein associations of a cell permeable CD40 complex in B cells.

Signaling through the CD40 receptor activates diverse molecular pathways in a variety of immune cell types. To study CD40 signaling complexes in B cells, we produced soluble CD40 cytoplasmic domain multimers that translocate across cell membranes and engage intracellular CD40 signaling pathways. As visualized by fluorescence microscopy, rapid transduction of recombinant Antennapedia-isoleucine zipper (Izip)-CD40 cytoplasmic domain fusion protein (Antp-CD40) occurred in both the DND39 B cell line and human tonsillar B cells. Upon cellular entry, Antp-CD40 activated NF-kappaB-dependent transcription, induced proteolytic processing of p100 to the p52/NF-kappaB2 subunit, and increased expression of CD80 and CD54 on the surface of B cells. Antp-CD40 transduction of B cells did not, however, activate detectable levels of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase and did not up-regulate CD95 expression. Analysis of Antp-CD40 complexes recovered from transduced B cells revealed that Antp-CD40 associated with endogenous TRAF3 and Ku proteins. Multimerization of Antp-CD40, or extensive clustering of transmembrane CD40, diminished the disruptive effect of the T254A mutation in the TRAF2/3 binding site of the CD40 cytoplasmic domain. Taken together, these results indicate that Antp-CD40 mimics some of the natural CD40 signaling pathways in B cells by assembling partially functional signaling intermediates that do not require plasma membrane localization. We present a novel approach for delivering pre-activated, soluble receptor cytoplasmic domains into cells and recovering intact signaling complexes for molecular analysis.

Amino Acid Sequence↗
PubMed2004

Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.

We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells. To determine the potential residue(s) involved in disulfide bonds formation and subsequent CD40-induced IL-8 expression, we generated a CD40 mutant in which the extracellular cysteine 6 was replaced by a glutamine (CD40-C6Q). CD40-induced IL-8 mRNA expression and protein synthesis were studied in stably transfected HEK 293 cells that were sorted out along with similar levels of expression of wild type (CD40-WT) and CD40-C6Q molecules. In contrast to cells expressing CD40-WT protein, disulfide-linked CD40/CD40 dimer formation was completely abolished in HEK 293 cells expressing CD40-C6Q proteins. Abolishment of disulfide-linked CD40/CD40 dimers in these transfected cells was sufficient to inhibit CD40-induced mRNA expression and secretion of IL-8. This study identifies the extracellular cysteine 6 of CD40 molecules as a potential molecular target to disrupt the expression of CD40-induced pro-inflammatory cytokines by epithelial cells.

B-Lymphocytes↗
PubMed1999

CD40 expression by human bronchial epithelial cells.

CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.

Bronchi↗
PubMed2005

Differential expression and regulation of murine CD40 in regional vascular beds.

There is emerging evidence for a role of the CD40/CD40 ligand (CD40L) dyad as a signaling mechanism in different inflammatory conditions. The aims of this study were to 1) quantify the constitutive and induced expression of CD40 in different regional vascular beds of the mouse and 2) assess the role of CD40L as a modulator of vascular endothelial CD40 expression. The dual radiolabeled monoclonal antibody technique was used to quantify the expression of endothelial CD40 in control and LPS-challenged wild-type (WT) mice. Significant constitutive CD40 expression was detected in several vascular beds of WT mice with lung, kidney, and small intestine exhibiting the highest expression, whereas the liver and stomach showed no detectable baseline expression. LPS administration elicited two- to sevenfold increases in CD40 expression in several tissues (heart, kidney, and intestine) within 4 h, whereas other organs (brain) required up to 48 h to exhibit CD40 upregulation. CD40 expression was not detected in unstimulated or LPS-challenged CD40-/- mice. Constitutive expression of CD40 was profoundly reduced in unstimulated CD40L-/- mice, but the LPS-induced CD40 upregulation did not differ between CD40L-/- and WT mice. Depletion of platelets or T lymphocytes, the major CD40L-expressing cells in blood, also resulted in a profound reduction in basal CD40 expression. These findings demonstrate significant endothelial expression of CD40 under basal conditions in different vascular beds and increased CD40 expression after endothelial cell activation with LPS. Platelet- and T-lymphocyte-associated CD40L appears to play a major role in regulating the density of CD40 expression on vascular endothelial cells in vivo.

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