RESUMO
In addition to the membrane-bound form, CD154 also exists as a soluble molecule originating from an intracellular and membrane cleavage. We have previously shown that CD154 cleavage from T cell surface is mediated by CD40 and involves the action of ADAM10/ADAM17 enzymes. In the aim of defining the importance of CD154 maintained on cell surface, we generated a CD154 mutated at the cleavage site. Our data show that the double mutation of E112 and M113 residues of CD154 abolishes its spontaneous release and the CD40-mediated cleavage from cell surface but does not affect its binding to CD40. We also demonstrated that both the release of CD154 from the intracellular milieu and its CD40-mediated cleavage from cell surface are highly dependent on ADAM10/ADAM17 enzymes. The CD154-EM mutant was shown capable of inducing a more prominent apoptotic response in susceptible B cell lines than the wild-type (WT) form of the molecule. In addition, human B cells cultured in the presence of the CD154-EM mutant exhibited upregulated proliferative responses compared with the CD154-WT. The CD154-EM mutant was also shown to trigger differentiation of human B cells, reflected by an increased Ig production, more significantly than CD154-WT. Thus, our data strongly suggest that cleavage-resistant CD154 is a more prominent stimulant than the cleavable form of the molecule. Therefore, a maintained expression of CD154 on cell membrane and a disturbed cleavage of the molecule could be a mechanism by which CD154 is involved in some pathological conditions and should be revisited.
Assuntos
Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Membrana Celular/metabolismo , Espaço Intracelular/metabolismo , Linfócitos T/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Apoptose , Ligante de CD40/genética , Diferenciação Celular , Células HEK293 , Humanos , Imunoglobulinas/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Proteólise , Transdução de SinaisRESUMO
In addition to its classical receptor, CD40, it is now well established that CD154 also binds αIIbß3, α5ß1, and αMß2 integrins. Although these integrins are all members of the same family, they bind CD154 differently. The current investigation aims to analyze the interaction of CD154 with α5ß1 and αMß2 and investigate its role in bidirectional signals in various human cell lines. Results obtained herein indicate that the CD154 residues involved in the interaction with α5ß1 are N151 and Q166, whereas those involved in αMß2 binding are common to residues required for CD40, namely Y145 and R203. Soluble CD40/CD154 or αMß2/CD154 complexes do not interfere with the binding of CD154 to α5ß1-positive cells, but inhibit the binding of CD154 to CD40- or αMß2-positive cells, respectively. Ligation of CD154 on CD154-positive cells with soluble CD40, αIIbß3, α5ß1, or αMß2 stimulates intracellular signaling, including MAPK phosphorylation. Given that CD154 exists as a trimer, our data strongly suggest that CD154 may bind concomitantly to two receptors of the same or different family, and biologically activate cells expressing both receptors. The characterization of CD154/receptor interactions helps the identification of new therapeutic targets for the prevention and/or treatment of CD154-associated autoimmune and inflammatory diseases.
Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Integrina alfa5beta1/metabolismo , Antígeno de Macrófago 1/metabolismo , Animais , Antígenos CD40/genética , Antígenos CD40/imunologia , Ligante de CD40/genética , Ligante de CD40/imunologia , Linhagem Celular Tumoral , Drosophila melanogaster , Expressão Gênica , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/imunologia , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
CD20 is an attractive therapeutic target given the success of its monoclonal antibody, Rituximab, in the treatment of B-cell malignancies and B-cell-mediated autoimmune diseases. Treatment with Rituximab causes a rapid depletion of B cells and a decrease in disease symptoms. Despite the clinical efficiency of Rituximab, its mechanism of action is not completely understood. In this study, we aimed at further investigating the Rituximab-induced cell death and the factors affecting such responses. Our results indicate that Rituximab-induced cell death depends on the nature of the cells and levels of CD20 expression on the cell surface. Coexpression of CD20 with CD40, a member of the TNF receptor family that is known to be physically associated with CD20 on the cell surface, enhances the apoptotic response induced by Rituximab. Inhibiting the formation of CD40 disulfide-bound-homodimers, a process required for some CD40 signaling, further enhances Rituximab-induced cell death. Cell death induced by anti-CD40 mAb is also upregulated by the presence of CD20, suggesting a bidirectional influence of the CD20/CD40 association. Moreover, treating cells with both anti-CD20 and anti-CD40 antibodies improves the cell death response induced by a single-agent treatment. These results highlight the role of the CD20/CD40 association in triggering B-cell depletion and may pave the way for an alternative more efficient therapeutic strategy in treating B-cell-mediated disorders.
Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Antígenos CD20/metabolismo , Antineoplásicos/metabolismo , Antígenos CD40/metabolismo , Membrana Celular/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Antígenos CD20/genética , Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/química , Antígenos CD40/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Expressão Gênica , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Mutação , Ligação Proteica , Multimerização Proteica , Receptores Fc/metabolismo , RituximabRESUMO
CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5ß1 or αMß2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Proteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Antígenos CD40/química , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Proteína Quinase C/metabolismo , Proteólise , Transdução de Sinais , Solubilidade , Linfócitos T/citologiaRESUMO
In addition to the membrane-bound molecule, soluble CD154 (sCD154) is also detected at high levels in the medium of activated T cells and platelets and in the serum of patients suffering from different inflammatory diseases. This sCD154 is the result of cleavage of the full-length molecule between the glutamic acid residue at position 112 (E112) and methionine at position 113 (M113) and can be derived from the intracellular milieu and from cleavage of cell surface molecules. We have recently reported that substitution of both E112 and M113 by alanine inhibits intracellular and CD40-induced membrane cleavage of CD154 and procures to CD154 an increased biological function as compared with cleavable CD154. Thus, in this study, and in the aim of developing tools inhibiting cleavage of CD154 from the cell surface, we generated a panel of anti-human CD154 mAbs. One of the derived mAbs that did not alter the binding of sCD154 to CD40, named in this study Clone 8 mAb, totally lost its binding activity against cells expressing CD154 mutated at its E112 and M113 residues. Treatment with Clone 8 mAb was shown to completely abolish CD40-dependent and -independent cleavage of CD154 from the cell surface. Our study is highlighting the development and characterization of an innovative therapeutic tool capable of inhibiting the release/cleavage of CD154 from cells and thus maintaining its availability on the cell surface and the high probably of increasing its potency as an activator of CD40-induced responses.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Ligante de CD40/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Células HEK293 , Humanos , Células Jurkat , CamundongosRESUMO
CD40 has an important role in T cell-B cell interaction which rescues B lymphocytes from undergoing apoptosis. However, various studies have demonstrated that CD40 can also play a direct role in the induction of specific cell death and thus in the inhibition of tumour cell proliferation. Our previous studies showed that CD40-mediated cell death was independent of caspases and required no de novo protein synthesis. Knowing that CD40 signaling is mediated by its association with several intracellular effectors, including members of TNFR-associated factors (TRAFs) family, the goal of the present study is to investigate the mechanisms involved in the induction of cell death by CD40. Our data reveals that CD40-mediated cell death required lysosomal membrane permeabilization and the subsequent cathepsin B release. In addition, CD40 homodimer formation, a phenomenon known to be necessary for some CD40-mediated signals, was shown to negatively regulate cell death induced by CD40. Moreover, using HEK293 cells ectopically expressing CD40 deficient in TRAF binding, we showed that CD40-mediated apoptosis occurred in the absence of TRAF2 and TRAF3 association, but was significantly reduced when CD40 was deficient in its TRAF6 binding. Therefore, by outlining the role of lysosomal pathways and intracellular effectors, namely TRAF6 in CD40-mediated cell death, our study identifies new targets for anti-cancer therapy.