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PubMed1997

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis.

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF) T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Arthritis, Rheumatoid↗
PubMed1994

Protein tyrosine kinase activation and protein kinase C translocation are functional components of CD40 signal transduction in resting human B cells.

CD40 is a 47kD glycoprotein expressed on all B cells that plays an important role in B cell development and activation. Previous investigations have focused on signal transduction events in activated B cells and B cell lines, and little information is available regarding CD40 signal transduction in resting, normal B cells. Because CD40 plays a critical role in the regulation and activation of resting B cells, we studied the signaling mechanisms involved in functional responses to CD40 ligation in these cells. Treatment of dense tonsil B cells with either protein tyrosine kinase (PTK) or protein kinase C (PKC) inhibitors, but not with an inhibitor of cyclic nucleotide dependent kinases, resulted in abrogation of CD40-mediated cell adhesion, suggesting a role for both PTK and PKC in CD40-mediated B cell adhesion. Direct evidence for CD40-mediated PTK activation was demonstrated by increased tyrosine phosphorylation as detected by anti-phosphotyrosine Western blots of cell lysates prepared from dense resting tonsil B cells stimulated with biotinylated anti-CD40 monoclonal antibody plus avidin. CD40 engagement also resulted in PKC activation, as detected by translocation of cytosolic PKC activity to the membrane-bound compartment. CD40-mediated PKC translocation was dependent on PTK activation, whereas PTK activity induced by CD40 ligation was independent of PKC activity, suggesting that PTK activation precedes PKC activation in resting B cells stimulated with anti-CD40 mAb. The results of our experiments identify PTK and PKC activation as components of CD40 signal transduction in normal, resting B cells and establish a functional role for these events.

Antigens, CD↗
PubMed2005

TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.

The interaction between CD40 and its ligand, CD154, has been shown to play a role in the onset and maintenance of inflammatory disease. Contributing to this process is the ability of CD40 to signal monocyte and macrophage inflammatory cytokine production. We have shown that this event is dependent on Src family tyrosine kinase activity and the subsequent activation of ERK1/2. To address the role of TNFR-associated factor (TRAF) family members in facilitating this signaling pathway, we transfected a CD40-deficient macrophage cell line with wild-type human CD40, or with CD40 containing disrupted TRAF binding sites. Ligation of either wild-type CD40, or a CD40 mutant unable to bind TRAF2/3/5, resulted in the stimulation of inflammatory cytokine production. However, ligation of a CD40 mutant lacking a functional TRAF6 binding site did not initiate inflammatory cytokine production, and this mutant was found to be defective in CD40-mediated activation of ERK1/2, as well as IkappaB kinase (IKK) and NF-kappaB. Likewise, introduction of a dominant-negative TRAF6 into a wild-type (CD40(+)) macrophage cell line resulted in abrogation of CD40-mediated induction of inflammatory cytokine synthesis. Finally, treatment of monocytes with a cell-permeable peptide corresponding to the TRAF6-binding motif of CD40 inhibited CD40 activation of ERK1/2, IKK, and inflammatory cytokine production. These data demonstrate that TRAF6 acts as a critical adapter of both the Src/ERK1/2 and IKK/NF-kappaB proinflammatory signaling pathways in monocytes and macrophages.

Amino Acid Sequence↗
PubMed2006

Agonistic monoclonal antibody against CD40 receptor decreases lymphocyte apoptosis and improves survival in sepsis.

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8- T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-x(L) was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-x(L), provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.

Animals↗
PubMed1992

CD40 is functionally expressed on human thymic epithelial cells.

CD40 is a prominent B cell Ag also found on certain epithelial cells and on carcinomas. In this report, we analyzed CD40 distribution in the human thymus. CD40 was not found on the majority of CD45-positive thymocytes, but was present in a CD45-negative stromal cell population. Immunohistology showed CD40 expression on cortical thymic epithelial cells (TEC), medullary TEC, thymic interdigitating cells, and thymic B cells. CD40 was not found on thymocytes, endothelial cells, or on the fibroblasts of the septae. Expression of CD40 was specifically maintained on cultured TEC and not found on cultured thymic fibroblasts. IL-1 alpha, TNF-alpha, IFN-gamma, but not IL-4, significantly up-regulated the membrane expression of CD40 on cultured TEC. The regulation of CD40 was similar to that of ICAM-1, and contrasted with that of MHC class II Ag, which could only be induced by IFN-gamma but not by IL-1, TNF, or IL-4. Northern blot analysis showed the presence of a 1.4-kb mRNA transcript for CD40 in TEC, which was up-regulated by IL-1 and IFN-gamma. Cross-linking of CD40 at the surface of human TEC in the absence of IL-1 stimulation failed to induce cytokine secretion. Triggering of TEC with anti-CD40 mAb in conjunction with IFN-gamma and IL-1 stimulation increased granulocyte-macrophage CSF secretion in a dose-dependent manner. The effect was visible as early as 24 h after triggering, occurred in the absence of cellular proliferation, and was specific for CD40 since triggering of other TEC membrane Ag such as ICAM-1 or MHC class I molecules had no effect to increase cytokine production in TEC. These data demonstrate that CD40 is expressed and is a functional molecule at the surface of the epithelial cells of the thymus.

Antigens, CD↗
PubMed1993

Immunoregulatory role of CD40 in human B cell differentiation.

CD40 is a member of a family of surface molecules with homology to the nerve growth factor receptor and is expressed on B cells. The role of CD40 in regulating human B cell differentiation and the production of specific Ig isotypes was examined. In the absence of other stimuli, anti-CD40 and IL-4 induced the secretion of IgM, IgG, and IgE by purified human peripheral blood B cells. However, addition of formalinized Staphylococcus aureus (SA) enhanced the response. In the presence of SA, anti-CD40 and IL-4 induced the secretion of IgM, IgG1, IgG2, IgG3, and IgG4 in addition to IgE. No consistent effect on IgA secretion was demonstrated. The combination of SA, anti-CD40, and IL-4 induced one tenth of the total Ig production that was stimulated by SA and IL-2, with a different distribution of Ig H chain isotypes. B cells stimulated with SA, anti-CD40, and IL-4 secreted a greater percentage of IgG4 and IgE and a decreased percentage of IgG1 and IgA, compared with that secreted in response to SA and IL-2. Anti-CD40 did not induce Ig production in combination with IL-2 and did not alter the Ig secreted in response to SA and IL-2. Stimulation with SA, anti-CD40, and IL-4 caused Ig H chain isotype switch, inasmuch as IgG and IgE secretion could be induced from preimmune cord blood B cells and IgD+ naive adult B cells and IgE secretion could be induced from IgE- adult B cells. Ig secretion by purified B cells stimulated with SA, IL-4, and anti-CD40, but not with SA and IL-2, was significantly enhanced by anti-CD18 or anti-ICAM-1 mAb, suggesting that homotypic aggregation induced by anti-CD40 might limit Ig production. Finally, anti-CD40 was found to inhibit Ig production by B cells cultured with anti-CD3-activated T cells. These results suggest that engagement of the CD40 molecule on B cells provides signals that permit IL-4 to induce Ig isotype switching. This response is limited by LFA-1-mediated homotypic interactions of B cells. Engagement of CD40 by the ligand expressed by activated T cells appears to be important for the activation of B cells during T cell-B cell collaboration and the production of all isotypes of Ig.

Antibody Formation↗
PubMed1996

CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma.

The CD40 antigen is a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily and is involved in cell proliferation, differentiation, and survival. Using different monoclonal antibodies, we found CD40 expression by immunohistochemistry on CD31- and CD34-positive Kaposi's sarcoma spindle cells in all tumors of 18 HIV-1 seropositive and 4 HIV-1 seronegative patients. Western blot analysis of tumor lysates detected a 48- to 50-kd glycoprotein corresponding to the CD40 antigen expressed by B lymphocytes. CD40 expression was also detectable in one of four cultures of spindle cells derived from Kaposi sarcoma tissue. Treatment of the CD40-positive spindle cells but not of the CD40-negative ones with interferon-gamma up-regulated CD40 surface expression. Besides on Kaposi sarcoma tumor cells, CD40 was distinctly present on vascular endothelial cells in areas within and adjacent to the tumors and in benign inflammatory lesions such as granulation tissue of HIV-1-negative patients. In contrast, CD34-negative endothelia of thin walled vessels, most likely lymphatics, were predominantly CD40 negative. Only faint or no CD40 expression was found on endothelial cells in normal skin. We conclude from our data that expression of the CD40 antigen by endothelial cells is up-regulated during tissue inflammation. As signaling through CD40 is able to increase cell survival, expression of CD40 by Kaposi sarcoma tumor cells might play an important role in the pathogenesis of this neoplasm.

Acquired Immunodeficiency Syndrome↗
PubMed1997

IL-4 and IL-10 modulation of CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis.

Previous studies have demonstrated that the interaction of CD40 on monocytes with CD40 ligand, present on activated CD4+ T cells, induces monocyte inflammatory cytokine synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the maintenance and/or exacerbation of nonseptic (e.g., autoimmune) inflammatory responses. In the present study the effects of the modulatory cytokines IL-4 and IL-10 on CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis were examined. Both IL-4 and IL-10 decreased CD40-dependent IL-1beta synthesis in a dose-dependent manner individually and synergized in this effect when used concurrently, with minimal effect on CD40 surface expression. CD40 signaling of IL-1beta synthesis was shown to be dependent on the induction of protein tyrosine kinase (PTK) activity, and both IL-4 and IL-10 diminished CD40-mediated tyrosine phosphorylation of monocyte cellular proteins. However, IL-4, but not IL-10, blocked CD40-mediated rescue from apoptosis, an event that we have demonstrated previously to be dependent on PTK activity as well. Together these results suggest that in monocytes 1) both IL-4 and IL-10 target CD40-induced PTK activity in the down-regulation of IL-1beta synthesis; and 2) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway, in that these cytokines are synergistic with respect to their abilities to inhibit CD40-mediated IL-1beta synthesis and differ in their abilities to block CD40-mediated rescue from apoptosis.

Apoptosis↗
PubMed1999

Membrane-bound CD154, but not CD40-specific antibody, mediates NF-kappaB-independent IL-6 production in B cells.

CD40, a member of the tumor necrosis factor receptor (TNF-R) superfamily, is expressed on the surface of B cells, where its engagement results in IL-6 secretion. In this study, we characterize the specific molecular requirements for CD40-mediated IL-6 production. Engagement of CD40 on either a B cell line or normal mouse splenic B cells with a membrane-bound form of CD154 (also known as CD40L or gp39) induced IL-6 secretion as well as up-regulation of IL-6 mRNA, but cross-linking CD40 with agonistic anti-CD40 mAb did not, although these mAb induce many other CD40 activation events, including the nuclear translocation of the transcription factor NF-kappaB. Using a mouse B cell line stably transfected with various human CD40 (hCD40) cytoplasmic truncation and point mutants, we show that the region from amino acids 202 to 225 in the cytoplasmic domain of CD40 is necessary for IL-6 secretion. However, the carboxy-terminal 32 amino acids are not, although these residues are required for CD40-mediated NF-kappaB activation. In addition, CD40 mutants previously shown to lack binding to TRAF2 and -3 are fully capable of inducing IL-6 production. Thus, CD40-mediated IL-6 induction is independent of NF-kappaB activation and the binding of TRAF2 and -3, but CD40 must be engaged by trimeric CD154 on cell membranes to activate production of IL-6.

Animals↗
PubMed2000

Macrophages, smooth muscle cells, endothelial cells, and T-cells express CD40 and CD40L in fatty streaks and more advanced human atherosclerotic lesions. Colocalization with epitopes of oxidized low-density lipoprotein, scavenger receptor, and CD16 (Fc gammaRIII).

CD40-CD40L receptor-ligand interaction plays a central role in antigen presentation, immunological reactions, and in T-cell and macrophage activation. Since all these mechanisms are important for the pathogenesis of atherosclerosis, we studied the expression profile of CD40-CD40L in different types of human atherosclerotic lesions using double immunostaining techniques with cell type-specific antibodies. Normal human intima did not contain CD40 or CD40L immunoreactivity. From type-II lesions (fatty streaks) to advanced type-VI lesions (complicated plaques), colocalization of CD40 and CD40L was observed in T cells (CD3+ cells), macrophages (CD68+ cells), and smooth muscle cells (HHF35+ cells). No correlation was found between the lesion type and CD40-CD40L expression. Positive lesions had dense infiltrations of macrophages and macrophage-derived foam cells together with T cells. The most intensive immunoreactivity for the CD40 receptor and its ligand CD40L was found in macrophage- and T-cell-rich pockets, where both cell types were in close contact with each other. The majority of macrophages, and especially those of macrophage-derived foam cells, were positive for both CD40 and CD40L. A small subset of the lesion macrophage population (10-20%) consisted of cells positive only for either CD40 or CD40L, suggesting the presence of a subpopulation of macrophages more active in inflammatory processes than in lipid uptake. Intimal smooth muscle cells in and around the macrophage-rich areas as well as some of the medial smooth muscle cells near the lesions stained positive for CD40 and CD40L. Moderate to faint expression of these proteins was also found in endothelium. In addition, CD40-CD40L immunoreactivity colocalized with epitopes characteristic of oxidized low-density lipoprotein, scavenger receptor class A, and CD16 (Fc gammaRIII), thus suggesting the involvement of CD40-CD40L and these pathogenetic mediators in foam cell formation, progression of atherosclerotic lesions, and differentiation of immunologically active subsets of macrophages. These results support the hypothesis that CD40-CD40L interaction is involved in atherogenesis and that it might provide a target for future therapeutic interventions.

Adult↗
PubMed2003

Selective regulation of CD40 expression in murine dendritic cells by thiol antioxidants.

Interaction of CD40 on dendritic cells (DC) with CD40 ligand induces interleukin-12 (IL-12) production by these DC during the antigen presentation. Thus, the level of CD40 expression appears to influence the capability of DC to induce a T helper 1 (Th1) response. However, it is not fully understood how CD40 expression on DC is regulated. In the present study, we examined the effects of the reducing agents, N-acetyl-l-cysteine (NAC) and reduced glutathione (GSH), on tumour necrosis factor-alpha (TNF-alpha)-induced phenotypic changes in murine DC. TNF-alpha markedly increased the expression on DC of major histocompatibility complex (MHC) and the costimulatory molecules, CD40, CD80 and CD86. Both NAC and GSH completely abolished the TNF-alpha-induced enhancement of CD40 expression, but had no considerable effect on the expression of CD80, CD86 and MHC. The marked decrease of CD40 protein with NAC was also detected by Western blotting, but was not associated with the expression level of CD40 mRNA in DC. Thus, NAC appears to reduce CD40 expression on DC by regulating a post-transcriptional pathway. The inhibitory effect of NAC or GSH on TNF-alpha-induced CD40 expression was released by simply removing these agents from the culture. In contrast, culture of TNF-alpha-treated DC with NAC or GSH markedly decreased the expression of CD40 within 12 hr. These results demonstrate that reducing agents selectively, rapidly and reversibly regulate CD40 expression on DC, which may eventually affect the capability of DC for Th1/Th2 polarization.

Acetylcysteine↗
PubMed2003

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis.

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.

Androstadienes↗
PubMed1995

Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals.

CD40 is expressed on a variety of cells, including B cells, monocytes, dendritic cells, and fibroblasts. CD40 interacts with CD40L, a 30-33-kD activation-induced CD4+ T cell surface molecule. CD40L-CD40 interactions are known to play key roles in B cell activation and differentiation in vitro and in vivo. We now report that normal human endothelial cells also express CD40 in situ, and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections from normal spleen, thyroid, skin, muscle, kidney, lung, or umbilical cord were studied for CD40 expression by immunohistochemistry. Endothelial cells from all tissues studied express CD40 in situ. Moreover, human umbilical vein endothelial cells (HUVEC) express CD40 in vitro, and recombinant interferon gamma induces HUVEC CD40 upregulation. CD40 expression on HUVEC is functionally significant because CD40L+ Jurkat T cells or CD40L+ 293 kidney cell transfectants, but not control cells, upregulate HUVEC CD54 (intercellular adhesion molecule-1), CD62E (E-selectin), and CD106 (vascular cell adhesion molecule-1) expression in vitro. Moreover, the kinetics of CD40L-, interleukin 1-, or tumor necrosis factor alpha-induced CD54, CD62E, and CD106 upregulation on HUVEC are similar. Finally, CD40L-CD40 interactions do not induce CD80, CD86, or major histocompatibility complex class II expression on HUVEC in vitro. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vitro. Moreover, they suggest a mechanism by which activated CD4+ T cells may augment inflammatory responses in vivo by upregulating the expression of endothelial cell surface adhesion molecules.

Antigens, CD↗
PubMed1995

CD40 cross-linking inhibits specific antibody production by human B cells.

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation, antibody secretion, heavy chain switching and rescue from apoptosis after somatic mutation in the germinal centre. The importance of these manifold responses to CD40 activation for humoral immunity is exemplified by the inability of boys with X-linked hyper IgM syndrome to make IgG, IgE or IgA due to a mutation in in the gene coding for CD40 ligand (CD40L). In the present study, we have investigated the effect of CD40 ligation on specific antibody production by human B cells to influenza virus. The antibody response was T cell dependent and specific for the strain of influenza virus used as antigen. Addition of either CD40 mAb or recombinant trimeric CD40L profoundly inhibited specific antibody production. Antibody production by unseparated tonsillar mononuclear cells and by T-depleted B cells stimulated with antigen in the presence of T cell replacing factor were equally inhibited with CD40 antibody showing that the effect was due to ligation of CD40 on B cells rather than blocking of T cell help. The specific antibody detected in these experiments was mostly IgG with little or no IgM and was obtained from surface IgM B cells consistent with activation of a secondary (memory) response. Co-stimulation of tonsillar B cells with CD40 antibody and anti-IgG induced proliferation of IgG+ B cells. These results suggest that CD40 ligation can inhibit specific antibody responses and stimulate proliferation in the same IgG+ (memory) B cell subpopulation. Addition of CD40 antibody during the first 24-48 h of the response was required for inhibition, suggesting that the effect was on early B cell activation and/or proliferation required for antibody production. There was no correlation, however, between the ability of CD40 mAb to stimulate proliferation and inhibit antibody production. We suggest that early activation of CD40 in the specific antibody response inhibits the formation of plasma cells and promotes instead the generation of memory cells.

Antibodies, Monoclonal↗
PubMed1997

Thymic overexpression of CD40 ligand disrupts normal thymic epithelial organization.

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.

Animals↗
PubMed2005

CD40: a mediator of pro- and anti-inflammatory signals in renal tubular epithelial cells.

Infiltration of immune cells into the renal interstitium is characteristic of chronic inflammatory kidney diseases. CD4+ T cells and platelets express CD40 ligand (CD40L) and are reported to mediate proinflammatory events in renal proximal tubular epithelial cells (RPTEC) via interaction with CD40. In other cell types, CD40 signals can also induce protective genes. Here, human RPTEC were treated with sCD40L to ligate CD40, and a significant increase in the generation of proinflammatory reactive oxygen species was found; however, CD40-activated cells did not undergo apoptosis. This suggests that CD40 signals may simultaneously induce antiapoptotic genes for cytoprotection of RPTEC. Heme oxygenase-1 (HO-1) expressed in RPTEC serves as a protective gene, but it is not known whether it is regulated by CD40. Next, RPTEC were transiently transfected with a full-length HO-1 promoter-luciferase construct and were treated with sCD40L. CD40 ligation was found to significantly increase HO-1 promoter activity. By electrophoretic mobility shift assay, it was confirmed that CD40 signaling induced the transcriptional activation of HO-1 through the binding of NF-kappaB to its promoter. By Western blot analysis, a marked increase in HO-1 protein expression following CD40 ligation was also found. These observations are of clinical significance because it was found that CD40 and HO-1 are induced in expression in vivo in inflamed rejecting kidney biopsies and co-expressed in renal tubules. Therefore, ligation of CD40 in RPTEC promotes both inflammatory and anti-inflammatory processes. Regulating the balance between these two events may be of importance in the prevention of tubular injury associated with renal disease.

Apoptosis↗
PubMed2001

Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.

CD40 binding produces multifaceted growth signals in normal and malignant B cells, whereas its physiological role is less well characterized in epithelial cancers. We examined the growth outcome of CD40 ligation in human breast cancer cells, using CD40+ (T47D and BT-20) and CD40-negative (MCF-7, ZR-75-1) cell lines as defined by flow cytometric analysis, immunohistochemistry, and reverse transcription-PCR. Treatment with the soluble recombinant CD40 ligand (CD40L) molecules gp39 or CD40L-trimer significantly reduced [3H]thymidine uptake in BT-20 and T47D cells by up to 40%, but did not affect the growth of CD40-negative MCF-7 or ZR-75-1 cells. Similarly, significant growth inhibition was observed after co-incubation with CD40L-transfected murine L cells (55.0 +/- 8.9%, P < 0.001) that express membrane CD40L constitutively, or with paraformaldehyde-fixed, CD3+ CD40L+ PBLs from three different HLA-mismatched donors (39.7 +/- 3.7%, P < 0.01). Untransfected L cells and non-CD40L-expressing lymphocytes did not produce significant growth inhibition. The in vivo antitumorigenic effects of CD40L were examined using a s.c. severe combined immunodeficient-hu xenograft model. Pretreatment with two different soluble recombinant CD40L constructs (CD40L and gp39) produced similar xenograft growth-inhibitory effects [67 +/- 24% (n = 4), and 65 +/- 14% (n = 8) inhibition, respectively], which were reversed by co-treatment with the CD40L-neutralizing antibody LL48. In vitro analysis indicated that CD40L-induced growth inhibition was accompanied by apoptotic events including cell shrinkage, rounding, and detachment from the adherent T47D culture monolayer. Thirty-one and 27% of gp39-treated T47D and BT-20 cells underwent apoptosis, respectively, as compared with 56 and 65% from the same cell lines after treatment with the Fas agonistic antibody CH-11. An up-regulation of the proapoptotic protein Bax in T47D and BT-20 cells was observed, which indicated that this Bcl-2 family member may contribute to this growth-inhibitory effect. To explore the clinical relevance of CD40L-CD40 interaction, retrospective immunohistochemical analysis was carried to characterize in situ CD40- and CD40L-expression in breast cancer patient biopsies. All of the infiltrating ductal (5 of 5 cases tested) and lobular (4 of 4 cases) breast carcinomas, carcinomas in situ (6 of 6 cases), and mucinous carcinoma tested (1 case) expressed CD40. Varying proportions of tumor cells also expressed CD40L in the majority of infiltrating ductal (3 of 5 cases) and lobular (3 of 4 cases) carcinomas, and carcinomas in situ (4 of 6 cases), as determined by immunohistochemistry and validated by RT-PCR detection of the CD40L message in only CD40L positive-staining cases. Tumor infiltrating mononuclear cells from infiltrating carcinomas and carcinomas in situ expressed CD40 (10 of 10 cases), but less commonly CD40L (1 case of infiltrating lobular carcinoma, 2 cases of carcinoma in situ). Our findings indicate that the CD40 signaling pathway is active in human breast carcinoma cells. However, tumor-infiltrating lymphocytes from primary tumor tissues may be limited in their capacity to directly modulate tumor growth through the CD40L-CD40 loop.

Animals↗
PubMed2003

Stimulation of CD40 inhibits Fas- or chemotherapy-mediated apoptosis and increases cell motility in human gastric carcinoma cells.

The widespread expression of CD40, a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily, is likely to account for the central role of CD40 in the regulation of humoral immunity and host defense. Interestingly, the expression of the CD40 in various types of carcinoma cells was often observed and conveys signals regulating diverse cellular responses, ranging from proliferation to growth suppression. Thus, the biologic role of the CD40-CD40L interaction in solid tumors is still controversial. In this study, we investigated the expression and function of the CD40 in gastric carcinoma cells. In 3-4,5 dimethylthiozol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and Annexin V/propidium iodide staining, CD40 stimulation using a soluble form of CD40 ligand did not affect cell viability, but significantly inhibited Fas-mediated or chemotherapy-mediated apoptosis in three CD40-positive gastric cancer cell lines. Moreover, in migration assay, CD40 stimulation induced an elevation of cell motility in CD40-positive gastric carcinoma cells. Our results show that the CD40 expression on gastric carcinoma makes cells less vulnerable to apoptosis induced by Fas or chemotherapy. These results suggest that the CD40 expression on gastric carcinoma may be associated with cell survival and elevation of cell motility.

Antineoplastic Agents, Phytogenic↗