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J. Biol. Chem., Vol. 282, Issue 8, 5143-5151, February 23, 2007
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5
1 Integrin and Triggers Cell Signaling*
111¶2¶3
From the
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier de l'Université Laval, Québec City, Quebec G1V 4G2, the Laboratoire d'immunologie cellulaire et moléculaire, Centre Hospitalier de l'Université de Montréal, Hôpital Saint-Luc, Montréal, Quebec H2X 1P1, the **Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, Quebec H2W 1T8, the Institut de Recherche en cardiologie, Université de Montréal, Montréal, Quebec H1T 1C8, the Manitoba Centre for Proteomics and Rheumatic Diseases Research Laboratory, Department of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 3P4, and the ¶Département de Médecine, Université de Montréal, Montréal, Québec H3T 3J7, Canada
Received for publication, August 31, 2006 , and in revised form, November 27, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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IIb
3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and IIb3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-51 monoclonal antibody P1D6, and soluble 51. The direct binding of sCD40L to purified 51 was confirmed in a solid phase binding assay. Binding of sCD40L to 51 was modulated by the form of 51 expressed on the cell surface as the activation of 51 by Mn2+ or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of 51 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an 51-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble 51, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, 51, and IIb3) for CD40L. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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It was originally thought that CD40L had only one receptor, CD40, which is a type I transmembrane protein that is a member of the TNF receptor superfamily. CD40 is expressed on the surface of many immune and non-immune cells, including B lymphocytes, monocytes/macrophages, and dendritic cells, as well as platelets, epithelial, and endothelial cells (8). Most biological functions of CD40L have been attributed to its direct interaction with CD40. However, studies using CD40L–/– and CD40–/– mice have suggested that CD40L may also bind to one or more other receptors (9). In support of this hypothesis, it has been elegantly demonstrated that sCD40L interacts with IIb3 (GPIIb/IIIa), an integrin expressed on platelets (10), triggering outside-in signaling and inducing platelet activation and spreading (11). CD40L–/– mice exhibit increased bleeding time (12) and reduced thrombus stability (10), showing that the interaction between sCD40L and the integrin IIb3 is physiologically relevant.
Based on the above observations, we hypothesized that CD40L may bind to another still unknown receptor on human monocytes and induce cellular activation. Our results show that sCD40L binds to CD40-negative human monocytic U937 cells, an interaction that is inhibited by anti-51 integrin (VLA-5) mAb as well as by soluble 51 integrin (s51). The binding of sCD40L to immobilized purified 51 confirms the direct interaction of sCD40L with this integrin. sCD40L induces the translocation of 51 to the Triton X-100-insoluble fraction, the rapid activation of MAPKs ERK1/2 in U937 cells, and IL-8 gene expression, confirming the existence of a third functional receptor for CD40L on 51.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Reagents and Antibodies—Recombinant trimeric soluble CD40L (rsCD40L) (13) was provided by Immunex Corp. (Seattle, OR). Avidin was procured from Sigma. Alexa Fluor-488 labeling of rsCD40L (rsCD40L-A) and avidin (avidin-A) was performed according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Anti-CD40L hybridoma 5C8 (IgG2a) and anti-CD40 hybridoma G28.5 (IgG1) were obtained from the ATCC. The isotype controls anti-TSST-1 mAb 2H8 (IgG1) and anti-SEB mAb 8C12 (IgG2a) were developed in our laboratory. Anti-51 mAb HA5 (IgG2b) and isotype control mouse mAb IgG2b were generously provided by Dr. Bosco Chan (Robarts Research Institute, London, Ontario, Canada). Anti-1 mAb B44 has been previously described (14). The following antibodies were purchased: rabbit anti-5 antibody (Chemicon, Temecula, CA), anti-5 mAb P1D6 (IgG3) (Biomeda, Foster City, CA), and isotype control IgG3 anti-bacterial peptidoglycan MAB983 (Chemicon), goat anti-mouse IgG-fluorescein isothiocyanate antibody (Sigma), rabbit antiphospho-ERK1/2 and anti-ERK1/2 antibodies (Cell Signaling Technology, Inc., Beverly, MA), goat anti-rabbit IgG-HRP antibody and goat anti-mouse IgG-HRP antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-CD40 mAb 82102 (IgG1) (R & D Systems, Minneapolis, MN). Soluble 51 (s51), sV3, and sV5 were produced as previously described (15, 16). Recombinant soluble CD40-Fc was from R & D Systems.
Flow Cytometric Analysis—For the rsCD40L-A binding assay, cells were incubated in binding assay medium (RPMI 1640, HEPES 10 mM, BSA 1%) containing 200 ng of rsCD40L-A or avidin-A (2 x 105 cells/100 µl) for 1 h at 37 °C in a humidified incubator and a 5% CO2 atmosphere. For competition binding with mAbs directed against cell surface molecules (CD40 and 51), cells (preincubated with 10 µg of human IgG/106 cells in staining medium for 15 min at 37 °C) were incubated with mAb for 30 min at 37 °C prior to labeling with rsCD40L-A. For competition binding with s51 or mAbs (10-fold molar excess) directed against soluble molecules, rsCD40L-A and avidin-A were incubated with s51 or mAbs for 1 h at 37 °C prior to the addition of the cells. Cell surface analyses with mAbs were performed as previously described (17). Washed cells were analyzed on a FACSort (BD Biosciences, Mountain View, CA).
Integrin Activation—Cells (106/ml HBSS) were incubated with Mn2+ (1 mM in HBSS) or DTT (10) mM in HBSS) for 30 min at room temperature. The DTT-treated cells then were washed twice in HBSS and resuspended in HBSS. The Mn2+-stimulated cells were used without washing.
Cell Binding Assay—The wells of microtiter plates (Nunc Maxisorp, VWR International Ltd., Mississauga, Ontario, Canada) were coated with gelatin (30 mg/ml in PBS) for 2 h at 37 °C. Unbound gelatin was removed, and the wells were air-dried for 1 h at 37 °C. Fibronectin (5 µg/ml in PBS, Chemicon) was added to the wells, and the plates were incubated overnight at 4 °C. The wells were washed with PBS and blocked with 1% BSA in PBS for 1 h at room temperature. Control wells were coated with gelatin and BSA. Cells (5 x 104 cells/well in PBS) were added to the wells, and the plates were incubated for 1 h at 37 °C. Unbound cells were removed, and the wells were washed three times with PBS under mild agitation. Bound cells were analyzed under a microscope (Zeiss Axiovert 100, Carl Zeiss, Inc., Thornwood, NY) and photographed with a 3-CCD Color video camera, model DXC-390P (Sony Electronics Inc., Park Ridge, NJ). The images were analyzed with Northern Eclipse 6.0 software (Empix Imaging Inc., Mississauga, Ontario, Canada). The adherent cells were then fixed with 1% paraformaldehyde in PBS for 30 min at room temperature and stained with 0.5% crystal violet in 20% methanol. After thoroughly washing the wells with tap water, the cells were lysed with 1% SDS in water, and the absorbance at 595 nm (Thermomax microplate reader, Molecular Devices, Sunnyvale, CA) was determined.
Solid Phase Binding Assay—The wells of microtiter plates (Nunc Maxisorb) were coated with 4 µg/ml of purified s51, V3, V5, or soluble recombinant CD40-Fc in PBS (pH 7.5, 50 µl/well) overnight at room temperature. After three washes with PBS containing 0.05% Tween 20 (PBS-T), the wells were blocked with 0.5% BSA in PBS for 2 h at room temperature. After three washes with PBS-T, rsCD40L was added to the wells at the indicated concentration, and the plate was incubated for 3 h at room temperature. The wells were washed three times with PBS-T, and bound rsCD40L was detected using goat anti-CD40L-biotin (R & D Systems, 2 h, at room temperature) and streptavidin-HRP (Sigma, 2 h, at room temperature), and revealed with 3,3',5,5'-tetramethylbenzidine substrate (Sigma).
Cell Stimulation—U937 and BJAB cells were incubated in serum-free medium for 4 h at 37°C and stimulated with rsCD40L (250 ng/5 x 105 cells) for 5 and 15 min at 37 °C. The stimulation was stopped by the addition of hot 2x SDS sample buffer containing 10% 2-mercaptoethanol, protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Sigma). After boiling for 7 min, cell lysates were separated by SDS-PAGE for Western blot analysis.
Receptor Translocation to the Cytoskeleton—U937 and BJAB cells were stimulated with rsCD40L (250 ng/5 x 105 cells) for 30 min at 37 °C in binding assay medium and washed three times in PBS. Cells were lysed in Triton X-100 buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1x complete protease inhibitor mixture (Roche Applied Science), and 1% Triton X-100 (Fisher Scientific) for 30 min on ice. The cell lysates were centrifuged at 16,000 x g for 15 min at 4 °C. Soluble and insoluble fractions were separated by SDS-PAGE under non-reducing conditions and analyzed by immunoblotting.
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5 antibody overnight at 4 °C followed by goat anti-rabbit IgG-HRP antibody or with mouse anti-CD40 antibody overnight at 4 °C followed by goat anti-mouse IgG-HRP antibody. The phosphorylation of ERK1/2 was assessed by immunoblotting using phospho-specific Abs according to the manufacturer's instructions. Membranes were stripped (62 mM Tris-HCl, pH 6.8, 2% SDS/100 mM, 2-mercaptoethanol, 30 min, 50 °C) and reprobed with antibody recognizing total ERK1/2. Antigen-antibody complexes were revealed with ECL (GE Healthcare, Mississauga, Ontario, Canada).
Analysis of IL-8 mRNA Expression—U937 cells (2 x 106 cells/100 µl) were treated with 100 ng of rsCD40L in RPMI 5% fetal bovine serum at 37 °C for the indicated time points. Reactions were stopped by adding ice-cold RPMI, and cells were isolated by spinning at 14,000 rpm at 4 °C. Total RNA was prepared from each sample using the RNeasy total RNA isolation Kit (Qiagen Inc., Mississauga, Ontario, Canada). Single strand cDNA for a PCR template was synthesized from 1 µg of total RNA using a primer, oligo(dT)12–18 (Invitrogen), and superscript III reverse transcriptase (Invitrogen) under the conditions indicated by the manufacturer. Reverse transcription was inactivated at 95 °C for 5 min, and the products were kept on ice until needed for the PCR. Specific primers were designed from cDNA sequence for IL-8 and -actin. Each cDNA was amplified by PCR using TaqDNA polymerase (Invitrogen). The sequences of the primers were as follows: IL-8F (5'-GCCAAGGAGTGCTAAAGAAC-3'), IL-8R (5'-CACTGGCATCTTCACTGATTCTTG-3'), -actin F (5'-AATCTGGCACCACACCTTCT-3'), and -actin R (5'-TAATGTCACGCACGATTTCC-3'). Conditions for PCR were 35 cycles of 94 °C for 45 s, 55 °C for 45 s, and the 72 °C for 1 min. Additional 10 min of 72 °C was performed at the end of the PCR reaction. The products were analyzed on a 1% agarose gel containing ethidium bromide. The expected sizes of the PCR products for IL-8 and -actin were 280 and 400 bp, respectively. We did not detect any band when we performed PCR without adding the cDNA template in this study. Genomic DNA contaminants were examined by performing PCR reaction on 1 µg of total RNA at similar conditions, and no contaminants were detected. Densitometric analyses were performed on each detected band using a Molecular Imager Gel Doc System and Quantity One analysis software from Bio-Rad. Results shown are normalized for two conditions, first based on -actin levels at each time points and thereafter, based on the level of expression of each gene of samples indicated as time 0 (non-treated samples). The ratio was then blotted as -fold increase of IL-8 mRNA after rsCD40L treatment versus time of the treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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rsCD40L Bound to 51—Based on the above results, and because sCD40L also binds to IIb3 (10, 11), which is selectively expressed on platelets (18), hematopoietic progenitors (19), and mast cells (20) but not on U937 cells (data not shown), we hypothesized that binding of rsCD40L to U937 cells may also be mediated by other members of the integrin superfamily. We first looked for integrin superfamily members that are expressed on U937, K562, and HEK 293 cells but not on BJAB cells. Among others, we found that 51 was expressed constitutively on U937 cells (Fig. 2A), K562 cells (data not shown) (21), and HEK 293 cells (data not shown) (22) but not on BJAB cells (Fig. 2A). To determine whether rsCD40L-A could bind to 51, we performed a competitive binding assay using soluble 51 (s51) as bait for rsCD40L-A. Fig. 2B shows that preincubation of rsCD40L-A with s51 substantially inhibited the binding of rsCD40L-A to U937 cells. Preincubation of rsCD40L-A with s51 did not affect rsCD40L-A binding to CD40 on B cells, suggesting that sCD40L could bind concomitantly to both CD40 and 51.
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51 is involved in the binding of rsCD40L to U937 cells, cells were preincubated with anti-5 mAb P1D6 or isotype control mAb and then incubated with rsCD40L-A. Fig. 2C shows that mAb P1D6 inhibited the interaction of rsCD40L-A to U937 cells but, as expected, did not affect the binding of rsCD40L-A to 51-negative BJAB cells. Thus, the results presented here support our contention that 51 is the third receptor for sCD40L.
Lastly, to confirm that sCD40L binds directly to 51, a solid phase binding assay was developed using immobilized soluble CD40-Fc as a control receptor (Fig. 3A). The results presented in Fig. 3B clearly demonstrate that rsCD40L binds directly to purified 51 in a dose-dependent manner. In contrast, rsCD40L did not bind to other purified integrins, V3 (Fig. 3C) and V5 (Fig. 3D), confirming the specificity of the rsCD40L-51 interaction. Thus, 51, like CD40 and IIb3, is a receptor for sCD40L.
Chemical Agents That Increase the Affinity of 51 for Fibronectin Negatively Affected the Binding of rsCD40L to U937 Cells—51 is constitutively expressed on the cell surface in an inactive form that cannot bind fibronectin (reviewed in Ref. 23). Conformational changes triggered by outside-in or inside-out signaling result in the activation of the integrin (23) allowing it to bind to its natural ligand. Chemical agents such as Mn2+ and DTT can promote such changes (21). We wondered whether the activation of 51 integrin could also modulate the binding of rsCD40L to U937 cells. First, we confirmed that U937 cells did not constitutively bind to fibronectin and that Mn2+ and DTT strongly promoted their adhesion to fibronectin as evaluated by microscopy (Fig. 4A) and a colorimetric assay (Fig. 4B). In contrast, similar treatments of 51-negative BJAB cells did not promote their attachment to fibronectin (Fig. 4). Second, conformational changes induced by these chemical agents expose a 1 epitope, the mAb B44 epitope (21). Indeed, the results presented in Fig. 5A show that treatments with Mn2+ or DTT induced the expression of the B44 epitope on U937 cells but not on BJAB cells. We then assessed the binding of rsCD40L-A to U937 cells treated with Mn2+ or DTT. Interestingly, the treatment of U937 cells with Mn2+ reduced the binding of rsCD40L-A while the treatment with DTT almost completely inhibited the binding of rsCD40L-A to U937 cells (Fig. 5B). In contrast, similar treatments of BJAB cells had no effect on the binding of rsCD40L-A to CD40 (Fig. 5B). Thus, changes in the conformation of 51 that promote its binding to fibronectin prevent its interaction with sCD40L.
rsCD40L Induced 51 Recruitment to the Cytoskeleton in CD40-negative U937 Cells—One consequence of the binding of 1 integrins with their ligands is their association with the cell cytoskeleton (24). We thus looked at whether the interaction of rsCD40L with 51 would also result in its association with the cytoskeleton. To assess the recruitment of 51 to the cytoskeleton, cells were incubated with rsCD40L for 30 min at 37 °C and solubilized in Triton X-100 buffer. The soluble (Sol) and insoluble (Ins) fractions were separated by centrifugation and analyzed by immunoblotting with a rabbit polyclonal anti-5 Ab (Fig. 6A). 51 was found exclusively in the Triton X-100-soluble fraction of unstimulated U937 cells, whereas a significant amount of 51 translocated into the Triton-X-100-insoluble fraction of rsCD40L-stimulated U937 cells. As expected, rsCD40L induced the translocation of CD40 to the detergentinsoluble fraction of BJAB B cells (25) and the formation of CD40 homodimers (26) (Fig. 6B). Thus, like its natural ligand, fibronectin, the interaction of rsCD40L with 51 triggered its association with the cytoskeleton.
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51 by rsCD40L. The stimulation of monocytes with membrane CD40L or sCD40L induces the activation of the MAPK pathways ERK1 and -2 (27–29). The binding of ligand to 51 also induces the activation of MAPK pathways (30). We thus investigated the phosphorylation of ERK1/2 in U937 cells following stimulation with rsCD40L and found, as shown in Fig. 7A, that ERK1/2 was rapidly phosphorylated in rsCD40L-activated U937 cells. As expected (31), rsCD40L induced the activation of ERK1/2 in BJAB cells (Fig. 7A). These results indicated that, like its classic ligand fibronectin, the interaction of sCD40L with 51 triggers the activation of signaling pathways, confirming the functional status of this interaction.
Interaction between s51 and rsCD40L Did Not Interfere with the CD40L-induced Activation of ERK1/2 via CD40—We showed above by flow cytometry analysis that purified s51 prevented the interaction of rsCD40L with U937 cells. As expected, the interaction of rsCD40L with s51 also completely prevented the activation of the ERK1/2 in U937 cells (Fig. 7B). An interesting outcome of the binding experiments was the observation that rsCD40L may interact concomitantly with CD40 and 51 (Fig. 2B). This suggested that sCD40L bound to 51 can trigger signaling in CD40-positive cells. Indeed, our data in Fig. 7B show that rsCD40L bound to s51 induced the activation of ERK1/2 in BJAB cells. Thus, sCD40L may serve as a molecular bridge between CD40 and 51 expressed on two different cells and trigger signal transduction in both cells.
rsCD40L Induced IL-8 Gene Expression in U937 Cells—The interaction of monocytes with fibronectin leads to inflammatory cytokines expression (30, 32), cellular responses that could also be induced in these cells by sCD40L-triggered signaling. To assess the expression of biological mediators, we analyzed IL-8 gene expression in U937 cells stimulated with 100 ng of rsCD40L for 15 min to 4 h. In U937 cells, rsCD40L induced a weak but significant IL-8 gene expression (Fig. 8A), reaching more than a 2.5-fold increase at 2 h (Fig. 8B). Thus, the interaction of sCD40L with cells in a CD40-independent manner also promotes the expression of inflammatory mediators.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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IIb3, and, if such binding occurs, to identify the new CD40L receptor(s). The main findings of this study are that 1) sCD40L bound to 51, a widely distributed cell surface receptor, 2) the interaction of sCD40L with 51 was prevented by conformational changes of 51 that result in its activation and its binding to fibronectin, 3) binding of sCD40L to 51 induced signaling, translocation of 51 to the Triton X-100-insoluble fraction, and chemokine gene expression, and 4) sCD40L may simultaneously bind to CD40 and 51 on the cell surface. Thus, 51 is a functional receptor for sCD40L, the third described so far, two being members of the integrin family.
Extensive research over the last decade has documented the central role of CD40L in immune responses (reviewed in Refs. 33 and 34) and in the development of autoimmunity (reviewed in Refs. 35 and 36) and inflammatory disorders (reviewed in Refs. 2 and 37). It became evident that CD40L is not only expressed by activated T cells but also by other cell types following activation, especially in chronic inflammatory diseases and autoimmunity, and that it plays a broader role than initially thought. Indeed, the expression of CD40L by immune cells is augmented and prolonged in many disease conditions, including systemic lupus erythematosus (38), rheumatoid arthritis (39, 40), inflammatory bowel disease (37), and cardiovascular disease (41), and is induced in non-immune cells such as fibroblasts (42) and endothelial cells (43). CD40L is also released as a soluble, trimeric, biologically functional molecule, and an increased level of circulating sCD40L is also a feature of many chronic inflammatory and autoimmune conditions (41, 44–46). The importance of CD40L in the development and maintenance of these inflammatory disorders has been demonstrated in mouse models and in a few human studies where an anti-CD40L treatment has been shown to be beneficial (35, 36, 47). CD40, the classic ligand for CD40L, is also expressed on many immune and non-immune cell types in inflammatory and autoimmune diseases (2, 36, 37), which supports the contention that the CD154-CD40 axis plays a crucial role in autoimmunity and inflammatory disorders. However, the scope of the role of CD40L in cellular immunity and inflammation is not limited just to its interaction with CD40. André et al. (10, 11) have shown that sCD40L can bind to IIb3, triggering platelet activation, and is involved in thrombus stabilization (10, 11). We show here that 51 is also a functional receptor for sCD40L.
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51 is a cell surface receptor that binds to the extracellular matrix and, in doing so, provides cells with adhesive properties and a transmembrane link between the extracellular environment and the intracellular cytoskeleton. Ligand binding to integrins triggers signaling pathways such as MAPKs, leading to cytoskeleton organization and regulating cell fate and behavior (48–50). Like other integrins, 51 is not constitutively active and, in that form, cannot bind to fibronectin, its ligand (23). sCD40L, however, binds to inactive 51, and the activation of 51 by chemical agents that induce conformational changes in a manner similar to that observed in the ligand-occupied integrin (21) prevents the binding of sCD40L. Thus, although 51 acts as receptor for fibronectin and sCD40L, these two ligands cannot bind to 51 simultaneously.
The interaction of 51 with the ECM or ligation by anti-5 antibodies induces the activation of ERK pathways in various cell types, including B cell progenitor cell line (51), fibroblasts (52), chondrocytes (53), and, as shown here, monocytic U937 cells (54). In many cell types such as chondrocytes (55), synovial cells (56), and monocytes (30, 54), signals transduced via 51 result in cell survival, proliferation, and cytokine and chemokine expression. We show herein that the engagement of 51 by sCD40L on U937 cells is also functionally relevant, because it induces the activation of ERK signaling pathways and the association of 51 with the Triton X-100-insoluble fraction. The trimeric nature of rsCD40L (13) and native sCD40L (6, 57) may facilitate the formation of clusters of 51 that promotes the activation of signaling pathways and the association of 51 with the cytoskeleton in a manner similar to the ECM (24, 48).
A number of cell types such as monocytes, macrophages, and dendritic cells express both CD40 and 51. CD40L is important for macrophage activation as shown by the fact that CD40L knock-out mice have defective T cell-dependent macrophage-driven functions (58). To date, signaling and subsequent cellular responses induced by CD40L in CD40-positive cells have been attributed solely to its interaction with CD40, although in most cases the identity of the CD40L receptor has not been confirmed. The results presented herein point to the need for a reassessment of the contribution of CD40 to these cellular responses. Indeed, stimulation of CD40-positive/51-positive monocytes and macrophages with sCD40L or membrane-bound CD40L induces the activation of ERK (27, 28) and the synthesis of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF- (27, 28, 59). These cellular responses are also induced in monocytes by fibronectin (30), mainly via its interaction with 51 (32). The signals induce in U937 cells by sCD40L lead to IL-8 gene expression, which suggests that the CD40L-51 interaction in addition to the CD40L-CD40 interaction contributes to the pathogenesis of inflammatory conditions where cells such as neutrophils play a crucial role. Interestingly, vitamin D3-differentated U937 cells have been reported to produce tissue factor in a CD40L-dependent but in a CD40-independent manner when incubated with CD40L+ T cells (60). It is thus tempting to hypothesize that tissue factor is induced in these cells by 51-mediated signals.
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51 and CD40 simultaneously, as shown by cell binding and cell activation experiments in BJAB cells. The CD40 binding site has been mapped to the interface between adjacent CD40L monomers (61). The simultaneous binding of 51 and CD40 to sCD40L suggests that sCD40L interacts with 51 outside the CD40 binding site. These results raise the possibility that sCD40L trimers may serve as a molecular bridge between CD40 and 51 expressed on various cells types, triggering signal transduction in such cells, or may cross-link the two receptors expressed on the same cell, triggering signaling-related events via both the receptors.
Thus, the responses of monocytes as well as other CD40-positive and 51-positive cell types to sCD40L may be induced through both CD40 and 51 individually or in combination. The signaling pathways and subsequent cellular responses triggered by sCD40L would depend on the availability of the various receptors for interactions with sCD40L. For example, platelets express all three receptors for sCD40L that have been identified to date (CD40, 51, and IIb3). Platelets are also the major source of circulating sCD40L (1) and, through their cell surface expression and release of CD40L, can play an important role in the immune response (62) and in inflammation (63). The roles played by each of three sCD40L receptors in platelet activation remain to be determined. Moreover, the expression of IIb3 is not restricted to platelets and their progenitors as was recently shown with hematopoietic progenitors (64) and mast cells (20), which also expressed 51 (65). Like platelets, activated mast cells express both integrins 51 and IIb3, and CD40L (66), and could be activated via 51 and IIb3 following their interaction with CD40L. 51, through its interaction with sCD40L, may play an important role in immune and inflammatory responses, because this integrin is broadly expressed (67). This possibility is currently under investigation in our laboratory. Given the important role of CD40L in inflammatory and autoimmune diseases, the identification of 51 as a new sCD40L receptor may turn out to be crucial in understanding the development of these diseases and may lead to the development of potential new strategies to counter them.
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FOOTNOTES |
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1 These authors contributed equally to this work.
2 Supported by a postdoctoral fellowship from the Fonds de la Recherche en Santé du Québec.
3 To whom correspondence should be addressed: Centre Hospitalier de l'Université de Montréal, Campus St. Luc, Pavillon Edouard-Asselin, 264 boulevard René Lévesque Est, Montréal, Quebec H2X 1P1, Canada. Tel.: 514-890-8000 Ext. 35287; Fax: 514-412-7314; E-mail: MW.Mourad{at}umontreal.ca.
4 The abbreviations used are: CD40L, CD40 ligand; rsCD40L, recombinant soluble CD40L; rsCD40L-A, Alexa Fluor 488-labeled rsCD40L; avidin-A, Alexa Fluor 488-labeled avidin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; s51, soluble 51; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL-8, interleukin-8; HRP, horseradish peroxidase; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; DTT, dithiothreitol; PBS, phosphate-buffered saline.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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