RESUMEN
CD1d-mediated lipid antigen presentation activates a subset of innate immune lymphocytes called invariant natural killer T (NKT) cells that, by virtue of their potent cytokine production, bridge the innate and adaptive immune systems. Transforming growth factor (TGF-ß) is a known immune modulator that can activate the mitogen-activated protein kinase p38; we have previously shown that p38 is a negative regulator of CD1d-mediated antigen presentation. Several studies implicate a role for TGF-ß in the activation of p38. Therefore, we hypothesized that TGF-ß would impair antigen presentation by CD1d. Indeed, a dose-dependent decrease in CD1d-mediated antigen presentation and impairment of lipid antigen processing was observed in response to TGF-ß treatment. However, it was found that this inhibition was not through p38 activation. Instead, Smads 2, 3 and 4, downstream elements of the TGF-ß canonical signalling pathway, contributed to the observed effects. In marked contrast to that observed with CD1d, TGF-ß was found to enhance MHC class II-mediated antigen presentation. Overall, these results suggest that the canonical TGF-ß/Smad pathway negatively regulates an important arm of the host's innate immune responses - CD1d-mediated lipid antigen presentation to NKT cells.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD1d/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Presentación de Antígeno/efectos de los fármacos , Línea Celular , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Lípidos/inmunología , Lisosomas/metabolismo , Ratones , Modelos Inmunológicos , Transporte de Proteínas , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.
Asunto(s)
Citoesqueleto de Actina/inmunología , Presentación de Antígeno/inmunología , Antígenos CD1d/inmunología , Quinasas Asociadas a rho/inmunología , Citoesqueleto de Actina/metabolismo , Animales , Antígenos CD1d/metabolismo , Western Blotting , Línea Celular , Humanos , Ratones , Ratones Noqueados , Microscopía Confocal , Quinasas Asociadas a rho/genéticaRESUMEN
We have recently demonstrated that the p38 and ERK1/2 MAP kinases play reciprocal roles in the control of CD1d-mediated antigen presentation. Although the use of specific inhibitors for these pathways clearly had an effect, the effects were not complete, leading to speculations that additional pathways were involved. Here, we show that inhibiting protein kinase C delta (PKCdelta) substantially impairs antigen presentation by murine CD1d1 to NKT cells. This effect was accompanied by marked changes in the intracellular localization of CD1d. Expression of a dominant-negative mutant of PKCdelta in CD1d(+) cells resulted in nearly undetectable endogenous antigen presentation, substantially impaired CD1d recycling, a decrease in MAPK activation, and a decrease in the ability to present low (but not high) concentrations of alpha-galactosylceramide at the cell surface. These data strongly suggest that PKCdelta is a critical regulator of CD1d-mediated antigen presentation and is involved in multiple steps of the process.