RESUMEN
Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X(L) (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X(L) expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.
Asunto(s)
Apoptosis , Complemento C5a/metabolismo , Proteínas de la Membrana , Sepsis/metabolismo , Timo/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Proteínas Portadoras/biosíntesis , Caspasa 3 , Caspasa 6 , Caspasa 9 , Caspasas/metabolismo , Complemento C5a/inmunología , Grupo Citocromo c/metabolismo , Activación Enzimática , Masculino , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Tamaño de los Órganos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Conejos , Ratas , Ratas Long-Evans , Timo/citología , Proteína X Asociada a bcl-2 , Proteína Letal Asociada a bcl , Proteína bcl-XRESUMEN
We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M >> anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M >> anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.