title: Fas-mediated neutrophil apoptosis and associated A1 protein expression during systemic inflammation are regulated independently of both tumor necrosis factor receptors.
abstract: TNFR-1 (p55) and Fas share a death domain which is critical for apoptosis signaling whereas TNFR-p55 and TNFR-2 (p75) can activate NF-kappaB leading to anti-apoptotic proteins expression such as A1. The purpose of this study was to elucidate the role(s) of TNFR-p55 and TNFR-p75 in Fas-mediated neutrophil apoptosis and A1 expression in a mouse model of endotoxemia. Gene knockout (KO) (p55-/-, p75-/-, p55(-/-)/p75(-/-)) or wild type (WT) mice were injected i.p. with saline or LPS (4 microg/g) followed by collecting peripheral blood after 24 h. Neutrophil apoptosis was assessed by propidium iodide staining using two-color flow cytometry with granulocyte-specific Gr1-FITC after 6-h whole blood culture with or without Fas agonist Jo2 (300 ng/ml) in the presence or absence of cycloheximide (CHX, 30 microg/ml). Membrane-associated receptors (Fas, TNFR-p55 and TNFR-p75) and cytoplasmic A1 expression of freshly isolated neutrophils were assessed by one-color flow cytometry and western blotting respectively. Compared with the group-WT/Sal, Jo2 induced apoptosis only in the presence of CHX (J+C). J+C-induced apoptosis was significantly lower in the group-p55(-/-)/Sal and p55(-/-)/p75(-/-)/Sal but not in the group-p75(-/-)/Sal. J+C-induced apoptosis was inhibited similarly in all the LPS-injected WT and KO mice. Strong A1 expression was also induced similarly in all the LPS-injected WT and KO mice. Fas and TNFR-p55 expression was normal and TNFR-p75 was significantly increased in all the LPS-injected WT and KO mice although absence of the appropriate surface receptors was confirmed in the KO mice. We conclude that p55 normally plays a proapoptotic role, but p75 appears to play a minimal role in Fas-mediated neutrophil apoptosis. During endotoxin-induced systemic inflammation, both TNFR-p55 and TNFR-p75 appear to be of minimal importance for modulation of Fas-mediated apoptosis and associated A1 protein expression despite normal Fas/TNFR-p55 and increased TNFR-p75 expression in neutrophils.
authors.affiliation: Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903,USA.
authors.forename: Joji
authors.initials: J
authors.lastname: Kotani
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Avallone
authors.forename: Edward
authors.initials: E
authors.lastname: Lin
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goshima
authors.forename: Kishor
authors.initials: K
authors.lastname: Gandhi
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Lowry
authors.forename: Steve E
authors.initials: SE
authors.lastname: Calvano
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000097:blood / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015415:Biomarkers / Q000097:blood; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000097:blood / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D007249:Inflammation / Q000097:blood / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018124:Receptors, Tumor Necrosis Factor / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D051818:Replication Protein C; D019014:fas Receptor / Q000097:blood*
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D019014:fas Receptor; D051818:Replication Protein C