RESUMO
The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.
Assuntos
Variação Genética , Receptores CXCR3/genética , Linfócitos T/metabolismo , Acetamidas/farmacologia , Western Blotting , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/farmacologia , Quimiocina CXCL11/genética , Quimiocina CXCL11/farmacologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/farmacologia , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Fator Plaquetário 4/genética , Fator Plaquetário 4/farmacologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Receptores CXCR3/agonistas , Receptores CXCR3/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Members of the chemokine (Chemotactic cytokines) superfamily and their receptors play a major role in trafficking of immune cells under homeostatic and inflammatory conditions. The chemokine receptor CXCR3 is expressed mainly on activated T lymphocytes and binds three pro-inflammatory, interferon-gamma-inducible chemokines: monokine induced by IFN-gamma (Mig/CXCL9), IFN-gamma-induced protein-10 (IP-10/CXCL10) and IFN-gamma-inducible T-cell alpha-chemoattractant (I-TAC/CXCL11). CXCR3 and its agonists are involved in a variety of inflammatory pathologies, making this receptor an attractive target for the design of new anti-inflammatory drugs. Interestingly, a growing body of evidence suggests the existence of at least two novel variants of CXCR3, namely CXCR3-B and CXCR3-alt, which present challenges in the design of new anti-inflammatory drugs targeting CXCR3. In this chapter, we describe the collection, isolation and activation of human peripheral blood-derived T lymphocytes and methods to examine the expression of CXCR3 and its atypical variants at both mRNA and protein levels, as well as protocols for exploring the biochemical and functional responses of T lymphocytes to all known CXCR3 agonists.