We tested the hypothesis that human polymorphonuclear leukocytes (PMN), bearing complement receptors CR1 and CR3, might also synthesize C3, particularly when activated by LPS or cytokines. Northern blot analysis of total RNA, obtained from purified PMN stimulated overnight with LPS or cytokines (IFN-gamma, TNF-alpha, and IL-1) showed the 5.3-kb RNA transcript reported for C3 in hepatocytes and monocytes. No transcripts for C4 and factor B were detected. Time course studies of C3 mRNA expression in PMN treated with LPS or TNF-alpha demonstrated a steady increase with a plateau at 24 h that correlated with secretion of C3, determined by ELISA. In contrast, IFN-gamma and IL-1 induced a transient increase in C3 transcript with a peak around 8 h after stimulation, which was not reflected in an increased rate of C3 secretion. The content of C3 protein in PMN culture media, measured by ELISA, was about 4 ng/ml/10(7) cells after overnight stimulation with LPS or TNF-alpha. A very small amount of C3 (about 0.7 ng/ml/10(7) cells) was detected in supernatants from unstimulated and IFN-gamma- or IL-1-induced PMN. Immunoprecipitation with a polyclonal anti-human C3, followed by SDS-PAGE analysis, from [35S]methionine labeled PMN, revealed the presence in culture supernatants of three major bands at 185, 115 and 70 kDa, corresponding to pro-C3, alpha and beta chains, respectively. Analysis of [14C]methylamine incorporation and of autolytic cleavage showed that the C3 produced in tissue culture by PMN contained an intact thiolester bond. The capacity of PMN to secrete functional C3 in response to LPS and TNF-alpha might be an important mechanism of host defense at sites of inflammation.
Biosynthesis and secretion of complement component C3 by activated human polymorphonuclear leukocytes
LISSANDRINI, Daniele;SORIO, Claudio;
1992-01-01
Abstract
We tested the hypothesis that human polymorphonuclear leukocytes (PMN), bearing complement receptors CR1 and CR3, might also synthesize C3, particularly when activated by LPS or cytokines. Northern blot analysis of total RNA, obtained from purified PMN stimulated overnight with LPS or cytokines (IFN-gamma, TNF-alpha, and IL-1) showed the 5.3-kb RNA transcript reported for C3 in hepatocytes and monocytes. No transcripts for C4 and factor B were detected. Time course studies of C3 mRNA expression in PMN treated with LPS or TNF-alpha demonstrated a steady increase with a plateau at 24 h that correlated with secretion of C3, determined by ELISA. In contrast, IFN-gamma and IL-1 induced a transient increase in C3 transcript with a peak around 8 h after stimulation, which was not reflected in an increased rate of C3 secretion. The content of C3 protein in PMN culture media, measured by ELISA, was about 4 ng/ml/10(7) cells after overnight stimulation with LPS or TNF-alpha. A very small amount of C3 (about 0.7 ng/ml/10(7) cells) was detected in supernatants from unstimulated and IFN-gamma- or IL-1-induced PMN. Immunoprecipitation with a polyclonal anti-human C3, followed by SDS-PAGE analysis, from [35S]methionine labeled PMN, revealed the presence in culture supernatants of three major bands at 185, 115 and 70 kDa, corresponding to pro-C3, alpha and beta chains, respectively. Analysis of [14C]methylamine incorporation and of autolytic cleavage showed that the C3 produced in tissue culture by PMN contained an intact thiolester bond. The capacity of PMN to secrete functional C3 in response to LPS and TNF-alpha might be an important mechanism of host defense at sites of inflammation.
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