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• W2079249302 abstract "It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys238 of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling. It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys238 of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling. Primary immune responses are initiated by specific physical interactions between antigen-specific T cells and antigen-presenting cells (APCs), 4The abbreviations used are: APC, antigen-presenting cell; DRM, detergent-resistant membrane; HEK, human embryonic kidney; Ab, antibody; mAb, monoclonal Ab; dl-CD40, disulfide-linked CD40; stCD154, soluble trimeric CD154; MβCD, methyl-β-cyclodextrin; NEM, N-ethylmaleimide; NAC, N-acetyl-l-cysteine; SEE, staphylococcal enterotoxin E superantigen; IL, interleukin; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; WT, wild type; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; TCR, T cell receptor(s); MHC, major histocompatibility complex. 4The abbreviations used are: APC, antigen-presenting cell; DRM, detergent-resistant membrane; HEK, human embryonic kidney; Ab, antibody; mAb, monoclonal Ab; dl-CD40, disulfide-linked CD40; stCD154, soluble trimeric CD154; MβCD, methyl-β-cyclodextrin; NEM, N-ethylmaleimide; NAC, N-acetyl-l-cysteine; SEE, staphylococcal enterotoxin E superantigen; IL, interleukin; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; WT, wild type; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; TCR, T cell receptor(s); MHC, major histocompatibility complex. resulting in bidirectional signal transducing events that modulate cell functions (1Schonbeck U. Libby P. Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). Various cytokine and co-stimulatory receptors provide the input to direct these processes. Elucidating the mechanisms underlying the regulation of cell/cell interactions is thus crucial for further understanding the immunological responses and improving therapeutic strategies aimed at treating cell/cell interaction-mediated human diseases. CD154 and CD40 molecules are pairs of ligand/receptor that belong to the tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamily and that play a pivotal role in cell/cell interactions (1Schonbeck U. Libby P. Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). In B cells, the CD154/CD40 interaction is responsible for clonal expansion, germinal center formation, isotype switching, affinity maturation, and rescue from surface Ig-induced apoptosis. In nonhematopoietic cells, ligation of CD40 with CD154 enhances the secretion of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. The absence or disruption of the CD154/CD40 pathway leads to a severe perturbation of the immune system, for example, in X-linked immunodeficiency with hyper-IgM (1Schonbeck U. Libby P. Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). This observation has been confirmed using CD154 and CD40 knock-out mice (2Xu J. Foy T.M. Laman J.D. Elliott E.A. Dunn J.J. Waldschmidt T.J. Elsemore J. Noelle R.J. Flavell R.A. Immunity. 1994; 1: 423-431Abstract Full Text PDF PubMed Scopus (688) Google Scholar, 3Kawabe T. Naka T. Yoshida K. Tanaka T. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Kikutani H. Immunity. 1994; 1: 167-178Abstract Full Text PDF PubMed Scopus (969) Google Scholar). Like most ligand/receptor pairs, the CD154/CD40 interaction leads to a bidirectional signal that leads to proliferation and IL-2 production (3Kawabe T. Naka T. Yoshida K. Tanaka T. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Kikutani H. Immunity. 1994; 1: 167-178Abstract Full Text PDF PubMed Scopus (969) Google Scholar) as well as various cellular events that modulate T cell functions. Because CD40 has no kinase domain, the transmission of its intracellular signals passes via the recruitment of several adaptor proteins, such as Jak3 and TNFR-associated factor (TRAF) proteins, to specific domains in its cytoplasmic tail. This results in the activation of members of the Src kinase family (such as Lyn and Fyn) and other protein-tyrosine kinases (such as Syk and Btk), phosphatidylinositol 3-kinase, phospholipase Cγ2, and the mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase (ERK) (4Grammer A.C. Lipsky P.E. Adv. Immunol. 2000; 76: 61-178Crossref PubMed Google Scholar). Biochemical studies have shown that ligated CD40, along with other signaling molecules, translocate and cluster into sphingolipid- and cholesterol-enriched microdomains and engage specific intracellular signaling pathways (5Grassme H. Bock J. Kun J. Gulbins E. J. Biol. Chem. 2002; 277: 30289-30299Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 6Grassme H. Jendrossek V. Bock J. Riehle A. Gulbins E. J. Immunol. 2002; 168: 298-307Crossref PubMed Scopus (217) Google Scholar). These discrete detergent-resistant membrane (DRM) microdomains are very dynamic areas of the cell membrane that serve as platforms for cell signaling (7Zeyda M. Stulnig T.M. Prog. Lipid Res. 2006; 45: 187-202Crossref PubMed Scopus (67) Google Scholar). Although it is well established that CD154 is mainly present in a trimeric form at the cell surface and/or following release from the cell membrane (8van Kooten C. Banchereau J. J. Leukocyte Biol. 2000; 67: 2-17Crossref PubMed Scopus (1160) Google Scholar), little research has been conducted on CD40 homodimers (9Braesch-Andersen S. Paulie S. Koho H. Nika H. Aspenstrom P. Perlmann P. J. Immunol. 1989; 142: 562-567PubMed Google Scholar) and their functional importance during T cell/APC interactions. We have previously reported that ligation of CD40 with soluble leucine zipper trimeric CD154 (stCD514) leads to the formation of the disulfide-linked CD40/CD40 (dl-CD40) homodimer (10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar). A high degree of CD40 clustering and subsequent dl-CD40 homodimer formation are selectively required for certain CD40-triggered responses such as phosphatidylinositol 3-kinase-dependent expression of B7-2 on human B cells and IL-8 production (10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar). We show here that the translocation of CD40 to DRM microdomains is a prerequisite for dl-CD40 homodimer formation and that a cysteine residue at position 238 is involved in dl-CD40 homodimer formation. We also provide evidence showing that CD154/CD40 bidirectional signaling triggered during cognate T cell/APC interaction is critically dependent on CD40 homodimer formation. Cell Cultures, Antibodies, and Reagents—All of the cells used in this study were obtained from the ATCC (Manassas, VA). BJAB and Jurkat D1.1 cells were maintained in RPMI 1640 containing 5% fetal bovine serum, l-glutamine, penicillin, and streptomycin (Wisent, St-Bruno, Canada). HEK 293 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, l-glutamine, penicillin, and streptomycin (Wisent). The hybridomas producing the mouse mAbs directed against human CD40 (G28-5; IgG1) were obtained from ATCC. The IgG1 isotype controls were produced in our laboratory. The horseradish peroxidase-conjugated anti-mouse IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Trimeric CD154 was a generous gift from Immunex Corp. (Seattle, WA). Cell culture agents, electrophoresis grade chemicals, methyl-β-cyclodextrin (MβCD), 5,5′-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide (NEM), and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich. Induction of CD40/CD40 Homodimers—CD40 ligations were performed by incubating B cells (20 × 106 cells/ml) with medium alone or medium containing 5 μg/ml of soluble trimeric CD154 (stCD154; Immunex) at 37 °C for 30 min. Alternatively, B cells were stimulated with 10 μg/ml of anti-CD40 mAb G28-5 (ATCC) on ice for 15 min, washed twice, resuspended in RPMI containing 5% fetal bovine serum and 2 μg/ml of goat anti-mouse IgG (Jackson ImmunoResearch, Cedarlane Laboratories Ltd., Burlington, Canada), and incubated for a further 10 min at 37 °C. Detergent-soluble and -insoluble Fractionation—CD40 stimulation and sucrose gradient density flotation were performed as previously described (10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 12Bouillon M. El Fakhry Y. Girouard J. Khalil H. Thibodeau J. Mourad W. J. Biol. Chem. 2003; 278: 7099-7107Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, the cells were lysed in ice-cold TNE buffer (10 mm Tris, pH 7.5, 150 mm NaCl, and 5 mm EDTA) containing 1% Triton X-100, 2 mm orthovanadate, and a mixture of protease inhibitors (Roche Applied Science) for 30 min on ice. The cell lysates were diluted in an equal volume of 85% sucrose, overlaid with 2.4 ml of 35% sucrose and 1 ml of 5% sucrose, and centrifuged at 35,000 rpm for 16 h at 4 °C. Eleven 380-μl fractions were harvested from the tube, starting at the top. The pellet was washed and resuspended in 380 μl of TNE buffer. Alternatively, the cells were lysed in ice-cold TNE buffer for 30 min on ice. The Triton-soluble fraction was separated from the Triton-insoluble fraction by centrifugation at 14,000 rpm for 30 min at 4 °C. The Triton-insoluble pellets were washed, resuspended in 380 μl of TNE buffer, and homogenized by sonication. To disrupt sphingolipid- and cholesterol-enriched microdomains, BJAB cells were pretreated with 10 mm MβCD for 10 min at 37 °C. For thiol alkylation and the antioxidant treatments, the cells were treated with cell-permeable NEM thiol alkylating agent or NAC antioxidant for 60 min at 37 °C. The concentrations were maintained throughout the CD40 stimulation treatments. Immunoblotting—To study homodimer formation by intermolecular disulfide bond formation, the samples were diluted 1:5 in 5× Laemmli sample buffer under nonreducing conditions (without β-mercaptoethanol). The samples were boiled for 5 min and resolved by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk and 0.1% Tween 20 in phosphate-buffered saline, the CD40 molecules were probed with anti-CD40 (G28-5; 1:2,000) for 1 h at 37 °C followed by horseradish peroxidase-conjugated anti-mouse IgG Ab (1:10,000) for 1 h at room temperature and were then visualized using an enhanced chemiluminescence detection kit (GEHealthcare Bioscience Inc.). Flow Cytometry Analyses—CD40 and HLA-DR expression were examined using anti-CD40 (G28-5) or anti-HLA-DR mAbs (L243), followed by Alexa fluor 488-conjugated anti-mouse IgG. A total of 104 cells gated by light scatter on live cells were analyzed by FACScan (Becton Dickinson). Plasmid Constructs and Stable Transfection—The expression vectors CD40-C6Q, CD40-C17A, CD40-C238A, and CD40-ΔCyto were generated by site-directed mutagenesis using the pCEP4-CD40 wild type (CD40-WT) as a template (10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). HEK 293 cells were transfected by DNA-calcium phosphate precipitation (Clontech, Mountain View, CA) and selected using 400 μg/ml hygromycin (Roche Applied Science). HEK 293 cells co-expressing CD40 and HLA-DR were generated by transfecting cells expressing CD40-WT or CD40-C238A or cells mock transfected with the pBud-DR1-α/β expression vector (a generous gift from Dr. Jacques Thibodeau, Université de Montréal) and selected in 160 μg/ml and 200 μg/ml of Zeocin (InvivoGen, Cederlane Laboratories, Burlington, Canada) and hygromycin, respectively. Detection of IL-8 and IL-2—For the detection of IL-8 protein, HEK 293 transfectants (5 × 104/well; in triplicate) were allowed to adhere in 96-well plates for 24 h before being stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate or anti-CD40 mAbs plus secondary Abs as described above. IL-8 release was detected by ELISA as previously described (13Akoum A. Lawson C. McColl S. Villeneuve M. Mol. Hum. Reprod. 2001; 7: 859-866Crossref PubMed Scopus (98) Google Scholar). For IL-2, HEK 293 transfectants (5 × 104/well; in triplicate) were plated in 96-well plates and co-cultured with the same number of Jurkat D1.1 T cells expressing Vβ8, which is recognized by staphylococcal enterotoxin E (SEE) superantigen (a generous from Dr. Sekaly, Université de Montréal). The cells were incubated for 48 h in the absence or presence of 100 ng/ml of SEE, and IL-2 production was monitored using an ELISA kit (Mabtech Inc.). The ELISA results were analyzed using Statview 4.5 (Abacus Concept, Inc., Berkeley, CA). All of the values are expressed as the means of triplicates ± standard error of the mean. Induction of dl-CD40 Homodimers in DRM Microdomains—There is a growing body of evidence to indicate that oligomerization of CD40 leads to a rapid increase in dl-CD40 homodimer formation (10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar) and that some CD40 signaling requires DRMs integrity (14Vidalain P.O. Azocar O. Servet-Delprat C. Rabourdin-Combe C. Gerlier D. Manie S. EMBO J. 2000; 19: 3304-3313Crossref PubMed Scopus (165) Google Scholar). These observations prompted us to investigate the role of DRM microdomains in dl-CD40 homodimer formation using cold 1% Triton X-100 solubilization over sucrose density gradient. To this end, BJAB B cells were left untreated or were treated with stCD154. They were then lysed, fractionated, and analyzed by Western blot under nonreducing conditions. As shown in Fig. 1A, we observed three major phenomena in stCD154-treated cells compared with untreated cells: (a) there was a significant translocation of CD40 to DRM microdomains, (b) CD40 homodimer formation was mainly detected in DRMs, and (c) there was no detectable CD40 in the pellets (fraction 12). Triton-insoluble fractions (DRMs) were found exclusively in the low density fractions (3 and 4), in which raft-associated molecule ganglioside GM1 was detected, whereas the Triton-soluble proteins were present in the high density fractions (9Braesch-Andersen S. Paulie S. Koho H. Nika H. Aspenstrom P. Perlmann P. J. Immunol. 1989; 142: 562-567PubMed Google Scholar, 10Reyes-Moreno C. Girouard J. Lapointe R. Darveau A. Mourad W. J. Biol. Chem. 2004; 279: 7799-7806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar), in which membrane phosphatase CD45 was detected (data not shown). We previously demonstrated that the ligation of CD40 with anti-CD40 Abs is not efficient in inducing significant dl-CD40 homodimer formation. However, efficient homodimer formation occurs when the anti-CD40 Abs are cross-linked with a secondary antibody (11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar). We thus set out to assess the distribution of CD40 in cells treated with anti-CD40 Abs alone or with cross-linked anti-CD40 Abs. As can be seen in Fig. 1B, the anti-CD40 Abs treatment did not promote dl-CD40 homodimer formation. This failure to trigger dl-CD40 homodimer formation can be explained by the failure of anti-CD40 Abs to induce CD40 translocation to DRMs (Fig. 1B). In contrast, significant CD40 translocation to DRMs and homodimer formation in DRMs (Fig. 1B, fractions 3 and 4) were induced in cells incubated with cross-linked anti-CD40 Abs. CD40 translocation to and homodimer formation in DRM microdomains also occurred in Ramos, LG2, and primary human tonsillar B cells, indicating that these microdomains play a role in CD40 dimer formation (data not shown). Together these results showed that a high affinity multivalent ligand/receptor interaction is required for CD40 to translocate to DRMs and that the relocalization of CD40 within these microdomains may be necessary to form CD40/CD40 homodimers. Prerequisite Requirement of DRM Integrity for dl-CD40 Homodimer Formation—To assess the role of DRM integrity in dl-CD40 homodimer formation, we treated cells with MβCD, a drug that disrupts the integrity of these microdomains by extracting cholesterol, prior to CD40 oligomerization. Because fractionation of cell lysates on a sucrose density gradient revealed no monomeric or dimeric CD40 in the pellets (Fig. 1, A and B, fraction 12), we performed straightforward ice-cold Triton-soluble (cytosolic) and Triton-insoluble (DRM and cytoskeleton) fractionation. Using this method, we found that treating cells with cross-linked anti-CD40 antibody resulted in the translocation of CD40 monomer from the Triton-soluble fraction to the Triton-insoluble fraction (DRM) and dl-CD40 homodimer formation (Fig. 1C). Disrupting DRMs prior to CD40 stimulation totally impaired CD40 translocation to the Triton-insoluble fraction as well as dl-CD40 formation (Fig. 1C). This inhibition was not due to lower surface CD40 expression, because MβCD had no effect on CD40 expression (Fig. 1D) or cell viability (data not shown), indicating that the integrity of DRMs is critical for dl-CD40 homodimer formation. Involvement of Extracellular Cysteines 6 and 17 in CD40 Localization—While seeking to determine the potential residue(s) involved in CD40-mediated disulfide bound formation, we previously demonstrated that a point mutation (C6Q) in the extracellular domain of the CD40 abolishes dl-CD40 homodimer (11Girouard J. Reyes-Moreno C. Darveau A. Akoum A. Mourad W. Mol. Immunol. 2005; 42: 773-780Crossref PubMed Scopus (15) Google Scholar). However, the basis of this inhibition was not investigated. As shown in Fig. 2, treatment of HEK 293 cells stably expressing the CD40-C6Q mutant at levels comparable with that of CD40-WT with cross-linked anti-CD40 antibody resulted in impaired CD40 homodimer formation. However, this inhibition was also accompanied by an almost complete inhibition of CD40 translocation to DRMs (Fig. 2B). Because the integrity of these microdomains and CD40 translocation are required for inducing CD40 homodimer formation, these results suggest that it was the impairment of CD40 translocation to the DRMs that inhibited CD40-C6Q dimerization. Based on the disulfide structural model of members of the TNFR superfamily (15Jones M.D. Hunt J. Liu J.L. Patterson S.D. Kohno T. Lu H.S. Biochemistry. 1997; 36: 14914-14923Crossref PubMed Scopus (30) Google Scholar) and the CD40 structural model (16Bajorath J. Aruffo A. Proteins. 1997; 27: 59-70Crossref PubMed Scopus (27) Google Scholar), Cys6 forms a disulfide intraloop with Cys17 on the extracellular domain of CD40. We thus hypothesized that a Cys17 mutation should also destabilize the intramolecular disulfide bond and prevent CD40 translocation to DRM and CD40 dimerization. Indeed, similar to CD40-C6Q, treatment of CD40-C17A-expressing cells (Fig. 2A) resulted in total inhibition of CD40 translocation to and homodimer formation in DRMs (Fig. 2B). These results strongly suggest that the intramolecular disulfide bond between these two cysteines (Cys6 and Cys17) might play an important role in the localization of CD40 molecules in these microdomains following CD40 stimulation. Involvement of Cytoplasmic Cysteine 238 in dl-CD40 Homodimer Formation—Having demonstrated that DRM microdomains integrity was required for CD40 homodimer formation and the indirect involvement of Cys6 and Cys17,we then generated HEK 293 cells expressing truncated CD40 (CD40-ΔCyto) (Fig. 3A) to study the possible requirement of intracellular signaling to induce dl-CD40 homodimer and to determine which residues mediate this phenomenon. We found that deleting the cytoplasmic domain of CD40 did not alter the extraction of CD40 from the Triton-insoluble fraction, but it did completely inhibit dl-CD40 homodimer formation (Fig. 3B). These results strongly imply that the formation of the CD40 homodimer requires a specific cytoplasmic-mediated intramolecular disulfide bond. We thus investigated the role of the sole cytoplasmic cysteine (Cys238) in the induction of CD40 homodimer. To this end, we generated a CD40-C238A mutant in which Cys238 was replaced by alanine. Stimulating CD40-C238A-expressing cells (Fig. 3A) revealed that there was a significant translocation of CD40 from the Triton-soluble to the Triton-insoluble fraction in the absence of dl-CD40 homodimer formation (Fig. 3B), strongly suggesting the crucial role for Cys238 in the induction of CD40 homodimers. Role of Reactive Oxygen Species in dl-CD40 Homodimer Formation—It is well established that thiol disulfide exchange reactions are catalyzed by proteins with disulfide isomerase activity (17Turano C. Coppari S. Altieri F. Ferraro A. J. Cell. Physiol. 2002; 193: 154-163Crossref PubMed Scopus (396) Google Scholar). The evidence gathered from the above experiments suggested that an intracellular disulfide isomerase activity is involved in the induction of intramolecular disulfide bond formation with Cys238. To confirm this hypothesis, we utilized a chemical approach to further illustrate this phenomenon. To this end, BJAB cells were preincubated with cell-permeable NEM thiol alkylating agent. The NEM concentrations used were selected based on preliminary experiments in which the treatment had no effect on cell viability (less than 5%) or CD40 surface expression levels (data not shown). As shown in Fig. 4A, cells preincubated with NEM showed a significant, dose-dependent reduction in dl-CD40 dimer formation compared with untreated BJAB cells. The cysteine residues of proteins are sensitive to oxidation by reactive oxygen species (ROS) (18Chevion M. Berenshtein E. Stadtman E.R. Free Radic. Res. 2000; 33: S99-S108PubMed Google Scholar). Thiols maintain the intracellular redox homeostasis, and the functioning of a number of proteins that contain free thiols can thus be reproducibly modified by the formation of disulfide bonds induced by ROS. ROS has been reported to be transiently increased in primary B cells and WEHI 231 B cell line by cross-linking the CD40 receptor, which may serve as second messengers linking CD40 engagement on B cells to improve downstream activation events (19Lee J.R. Mol. Cell Biochem. 2003; 252: 1-7Crossref PubMed Scopus (19) Google Scholar). To investigate the role of ROS in the formation of the dl-CD40 homodimer formation after CD40 cross-linking, BJAB cells were pretreated with the NAC, which is a source of sulfhydryl groups and acts as an antioxidant-scavenging ROS. The NAC concentrations used were selected based on preliminary experiments in which the treatment had no effect on cell viability (less than 5%) or CD40 surface expression levels (data not shown). As shown in Fig. 4B, dl-CD40 homodimer formation was significantly reduced in the presence of NAC (50%) in a dose-dependent fashion (Fig. 4B), suggesting for the first time that ROS play a role in the de novo oxidation of the thiol disulfide equilibrium required for the formation of these homodimers in DRM microdomains. Functional and Bidirectional Role of dl-CD40 Homodimer Signaling—Because the replacement of Cys238 in the cytoplasmic tail by alanine prevented CD40 dimer formation but not DRMs association, we next investigated the effect of the DRM/CD40 association and/or CD40 dimerization on CD40-induced IL-8 secretion. Cells expressing comparable levels of CD40-WT or CD40-C238A (Fig. 3A) were treated with cross-linked anti-CD40 antibody. As can be seen in Fig. 5A, CD40 cross-linking on CD40-WT-expressing cells induced IL-8 secretion significantly, whereas it had no effect on CD40-C238A-expressing cells. In contrast, stimulation with phorbol 12-myristate 13-acetate resulted in a significant increase in the secretion of IL-8 protein by all transfectants. These results point to the crucial role of dl-CD40 homodimer formation but not DRM association in CD40-mediated IL-8 secretion. Efficient T cell activation requires signals from T cell receptors (TCR) by nominal antigens presented in the context of self-MHC class II and other co-stimulatory molecules. The binding of CD154 to CD40 is also required for T cell activation, as indicated by the absence of germinal center formation in CD40 receptor knock-out mice (20van Essen D. Kikutani H. Gray D. Nature. 1995; 378: 620-623Crossref PubMed Scopus (377) Google Scholar) and the triggering of cell signaling pathways in T cells by anti-CD154 mAbs (21Brenner B. Koppenhoefer U. Grassme H. Kun J. Lang F. Gulbins E. FEBS Lett. 1997; 417: 301-306Crossref PubMed Scopus (52) Google Scholar). During T cell/APC contact, the CD154/CD40 interaction leads to bidirectional signaling that mediates the responses of both cell types (21Brenner B. Koppenhoefer U. Grassme H. Kun J. Lang F. Gulbins E. FEBS Lett. 1997; 417: 301-306Crossref PubMed Scopus (52) Google Scholar). We thus hypothesized that dl-CD40 homodimer formation might also be important for T cell activation. To test this hypothesis, we used an in vitro superantigen-mediated T cell activation model. Jurkat D1.1 T cells that bear Vβ8, which is recognized by SEE (22Yellin M.J. Lee J.J. Chess L. Lederman S. J. Immunol. 1991; 147: 3389-3395PubMed Google Scholar) and that express high levels of CD154 were co-cultured with HEK 293 mock transfected cells or stable transfectants expressing CD40-WT, HLA-DR, CD40-WT/HLA-DR, or CD40-C238A/HLA-DR in the presence or absence of SEE. All transfectants with comparable levels of surface HLA-DR or CD40 expression were sorted out (Fig. 5C). As shown in Fig. 5B, IL-2 production was induced when SEE was presented by cells expressing HLA-DR alone (150 ± 20 pg/ml). Co-expression of CD40-WT with HLA-DR resulted in a significant enhancement of IL-2 production by D1.1 Jurkat cells (600 ± 115 pg/ml). Conversely, when SEE was presented by cells co-expressing the CD40-C238A mutant with HLA-DR, the level of IL-2 production decreased to levels very similar to that detected in Jurkat cells co-cultured with cells expressing HLA-DR alone (195 ± 30 pg/ml) (Fig. 5B). Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. However, the mechanisms by which a cell uses its functional membrane to organize receptor oligomerization are poorly understood. Receptor clustering in DRM microdomains, where some signaling molecules reside and others are recruited where they may interact with the clustered receptors, has recently been identified as a central event in the initiation of signal transduction of many immune receptors, notably in signaling through B cell receptors (23Pierce S.K. Nat. Rev. Immunol. 2002; 2: 96-105Crossref PubMed Scopus (270) Google Scholar, 24Cheng P.C. Brown B.K. Song W. Pierce S.K. J. Immunol. 2001; 166: 3693-3701Crossref PubMed Scopus (122) Google Scholar), MHC class II (12Bouillon M. El Fakhry Y. Girouard J." @default.
• W2079249302 created "2016-06-24" @default.
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• W2079249302 date "2007-07-01" @default.
• W2079249302 modified "2023-10-18" @default.
• W2079249302 title "Requirement of Oxidation-dependent CD40 Homodimers for CD154/CD40 Bidirectional Signaling" @default.
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