Recombinant human C5a induces transcription but not translation of interleukin 1 beta mRNA in human monocytes.
The effect of recombinant human C5a (rhC5a) on the synthesis of interleukin 1 beta (IL-1 beta) was investigated in human monocytes, isolated by leukapheresis and countercurrent elutriation. rhC5a induced IL-1 beta mRNA synthesis in a dose- and time-dependent manner. Maximal induction was achieved at 3 h with rhC5a concentrations of 200 to 500 ng/ml. The maximal rhC5a-stimulated mRNA induction was about 75% of that observed using LPS as the stimulus (300 ng/ml). The inducing activity of rhC5a could be neutralized by preincubation with a polyclonal anti-C5a antibody. On the other hand rhC5a in optimal concentrations only weakly stimulated IL-1 beta protein synthesis, as measured by a two-site directed enzyme-linked immunoassay. When compared on Northern blots, a slightly reduced mobility of C5a-stimulated IL-1 beta mRNA was observed relative to LPS-stimulated RNA. To exclude the possibility that structural defects in the IL-1 beta mRNA are responsible for the weak translational efficiency after C5a stimulation a primer extension experiment was performed. No difference in the length of the extended fragment was detected between LPS and C5a-stimulated RNA, suggesting that the 5'-regions of the RNAs are identical. When LPS- or C5a-stimulated RNA was used to program IL-1 beta synthesis in an in vitro translation system from rabbit reticulocytes, no difference in translational efficiency was observed. Our results indicate that in human monocytes two signals for IL-1 beta gene expression are necessary, a signal for transcriptional activation and another signal to induce translation of the mRNA.
Geiger
T
T
Ciba-Geigy Ltd., Research Department, Basel, Switzerland.
Rordorf
C
C
Galakatos
N
N
Seligmann
B
B
Henn
R
R
Lazdins
J
J
Erard
F
F
Vosbeck
K
K
D015152:Blotting, Northern; D015936:Complement C5a / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides; D009000:Monocytes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D014158:Transcription, Genetic
D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015936:Complement C5a