RESUMO
Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were >or=10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities.
Assuntos
Adjuvantes Imunológicos/farmacologia , Oncorhynchus mykiss/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Anticorpos/imunologia , Northern Blotting , Movimento Celular/efeitos dos fármacos , Inflamação/genética , Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Oncorhynchus mykiss/genética , Fagocitose/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/farmacologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaAssuntos
Citocinas/imunologia , Peixes/imunologia , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Peixes/genética , Peixes/fisiologia , Humanos , Interleucina-1/genética , Interleucina-1/imunologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Filogenia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The predicted rainbow trout mature interleukin-1 beta (IL-1 beta) peptide has been produced as a recombinant protein in E. coli. The bioactivity of this molecule has been studied using trout head kidney cell preparations and a trout macrophage cell line (RTS11). Trout rIL-1 beta was shown to increase the expression level of IL-1 beta, cyclooxygenase (COX2) and MHC class II beta chain transcription, as determined by Northern blot analysis. Stimulatory doses of rIL-1 beta were typically > or =10 ng/ml. Induction of IL-1 beta expression occurred within 1h post-stimulation with trout rIL-1 beta and was maximal 3-6h post-stimulation. Trout rIL-1 beta was also able to increase murine D10.G4.1 cell proliferation and trout head kidney leukocyte phagocytic activity, in a dose-dependent manner. However, equivalent D10.G4.1 cell proliferation was induced with approximately 1000-fold lower doses of human rIL-1 beta. That LPS contamination did not contribute to the effects seen was confirmed by determining its concentration in the trout rIL-1 beta preparation, and demonstrating that the rIL-1 beta activity was inhibited by heating or pre-incubation with a polyclonal anti-trout rIL-1 beta antibody.
Assuntos
Interleucina-1/biossíntese , Oncorhynchus mykiss/imunologia , Proteínas Recombinantes/biossíntese , Animais , Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/genética , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologiaRESUMO
The present work provides the first information concerning the chemoattractant activity of trout recombinant IL-1beta and its derived peptides, referred to as P1, P2 and P3. The predicted rainbow trout mature interleukin-1beta peptide was produced as a recombinant protein in Escherichia coli. The first peptide, P1, corresponded to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and had an MW of 137 kDa. It was equivalent to a region known to be part of the receptor binding domain from the mammalian crystal structure of IL-1beta complexed to its receptor. P2 was used as control peptide, consisting of the same 12 amino acids as P1, but arranged in a random sequence (VVEEYIRAPPTT). P3 was synthesised to complex with an adjacent region of the IL-1 receptor, and corresponded to fragment 207-216 (YRRNTGVDIS) of the trout sequence, with an MW of 1.18 kDa. Migration was stimulated when leucocytes were exposed to concentrations of > or = 10 ng ml(-1) rIL-1beta. Peptide P3 also induced leucocyte migration, with an optimal dose of 0.25 mM being recorded. While P1 had no effect on cell migration when used alone, synergism was evident as a consequence of combining P1 with a suboptimal dose (0.01 mM) of P3. No synergism occurred when cells were exposed to a combination of P3 and the control peptide P2.
