RESUMEN
Purpose: To determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-â ) in vitro and in a mouse model of Aspergillus fumigatus keratitis. Methods: SREC-â mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-â expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-â small interfering RNA, then the mRNA levels of LOX-1, IL-1ß, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-â mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1ß, and TNF-α mRNA expression levels were tested before and after anti-SREC-â treatment. Results: SREC-â expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-â mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-â small interfering RNA treatment inhibited the expressions of LOX-1, IL-1ß, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1ß, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-â -neutralizing antibody treatment. Conclusions: SREC-â is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-â blockade could be a potential therapeutic approach for FK.
Asunto(s)
Aspergilosis/genética , Aspergillus fumigatus/aislamiento & purificación , Infecciones Fúngicas del Ojo/genética , Regulación de la Expresión Génica/genética , Inmunidad Innata/genética , Queratitis/genética , ARN Mensajero/genética , Receptores Depuradores de Clase F/genética , Adulto , Aspergilosis/inmunología , Aspergilosis/microbiología , Western Blotting , Células Cultivadas , Infecciones Fúngicas del Ojo/inmunología , Infecciones Fúngicas del Ojo/microbiología , Femenino , Humanos , Queratitis/inmunología , Queratitis/microbiología , Masculino , Persona de Mediana Edad , Receptores Depuradores de Clase F/biosíntesisRESUMEN
Ag cross presentation is an important mechanism for CD8(+) T cell activation by APCs. We have investigated mechanisms involved in heat shock protein 90 (Hsp90) chaperone-mediated cross presentation of OVA-derived Ags. Hsp90-OVA peptide complexes bound to scavenger receptor expressed by endothelial cells (SREC-I) on the surface of APCs. SREC-I then mediated internalization of Hsp90-OVA polypeptide complexes through a Cdc42-regulated, dynamin-independent endocytic pathway known as the GPI-anchored protein-enriched early endosomal compartment to recycling endosomes. Peptides that did not require processing could then be loaded directly onto MHC class I in endosomes, whereas longer peptides underwent endosomal and cytosomal processing by aminopeptidases and proteases. Cross presentation of Hsp90-chaperoned peptides through this pathway to CD8(+) T cells was highly efficient compared with processing of free polypeptides. In addition, Hsp90 also activated c-Src kinase associated with SREC-I, an activity that we determined to be required for effective cross presentation. Extracellular Hsp90 can thus convey antigenic peptides through an efficient endocytosis pathway in APCs and facilitate cross presentation in a highly regulated manner.
Asunto(s)
Presentación de Antígeno/inmunología , Reactividad Cruzada/inmunología , Proteínas HSP90 de Choque Térmico/fisiología , Receptores Depuradores de Clase F/fisiología , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células CHO , Cricetinae , Cricetulus , Citosol/inmunología , Citosol/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Glicosilfosfatidilinositoles , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica/inmunología , Receptores Depuradores de Clase F/biosíntesis , Receptores Depuradores de Clase F/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Heat shock protein (HSP) 70 isolated from tumor-dendritic cell (DC) fusions (HSP70.PC-F) induces potent antitumor immunity and prevents growth of such tumors. In the present study, we have examined mechanisms underlying such antitumor activity of the HSP70.PC-F vaccine. The degree of antitumor immunity induced by HSP70.PC-F depended on intact TLR signaling in immunized animals, and mice in which the tlr2 and tlr4 genes were both inactivated did not respond to the vaccine. The reduced responses to HSP70.PC-F vaccine in such tlr knockout mice were restored by immunization of animals with HSP70.PC-F-pulsed wild-type DC, indicating a key role for this cell type in HSP70.PC-F-mediated immunity. Our studies also indicate a role for the scavenger receptor expressed by endothelial cells-1 (SREC-1) in antitumor immunity induced by HSP70.PC-F. These two receptor types appeared functionally interdependent, as indicated by the finding that tlr2 and tlr4 knockout decreases HSP70 binding in double-knockout DC and reduces SREC-1 expression. In addition, TLR-dependent, tumor cell killing was suppressed by SREC-1 knockdown in DC, suggesting a significant role for this receptor in HSP70.PC-F-mediated tumor immunity.
