Effect of interferon-γ on complement gene expression in different cell types

Abstract

We have studied the expression of the complement components C2, C3, factor B, C1 inhibitor (C1-inh), C4-binding protein (C4-bp) and factor H in human peripheral blood monocytes, skin fibroblasts, umbilical vein endothelial cells (HUVEC) and the human hepatoma cell line G2 (Hep G2) in the absence and the presence of interferon-gamma (IFN-gamma). E.l.i.s.a. performed on culture fluids, run-on transcription assays, Northern blot and double-dilution dot-blot techniques confirmed that monocytes expressed all six components, whereas fibroblasts, HUVEC and HepG2 each expressed five of the six components. Fibroblasts and HUVEC did not synthesize C4-bp, and Hep G2 did not produce factor H. In addition to these differences, the synthesis rates of C3, C1-inh and factor H were not the same in all cell types. However, the synthesis rates of C2 and factor B were similar in all four cell types. The half-lives of the mRNAs were shorter in monocytes than in other cell types. Monocyte factor H mRNA had a half-life of 12 min in monocytes, compared with over 3 h in fibroblasts and HUVEC. The instability of factor H mRNA in monocytes may contribute to their low factor H secretion rate. IFN-gamma produced dose-dependent stimulation of C2, factor B, C1-inh, C4-bp and factor H synthesis by all cell types expressing these proteins, but decreased C3 synthesis in all four cell types. Cell-specific differences in the response to IFN-gamma were observed. The increased rates of transcription of the C1-inh and factor H genes in HUVEC were greater than in other cell types, while the increased rate of transcription of the C2, factor B and C1-inh genes in Hep G2 cells was less than in other cell types. IFN-gamma did not affect the stability of C3, factor H or C4 bp mRNAs, but increased the stability of factor B and C1-inh mRNAs and decreased the stability of C2 mRNA. Although these changes occurred in all four cell types studied, the half-life of C1-inh mRNA in monocytes was increased almost 4-fold, whereas the increases in the other cell types were less than 30%. These data show that the constitutive synthesis rates of complement components may vary in the different cell types. They also show that the degree of change in synthesis rates in response to IFN-gamma in each of the cell types often varies due to differences in transcriptional response, sometimes in association with changes in mRNA stability.

Constitutive Expression and Regulation of Rat Complement Factor H in Primary Cultures of Hepatocytes, Kupffer Cells, and Two Hepatoma Cell Lines

“…Although it had not been shown before in these cell types, the IFN-␥-mediated up-regulation that was observed here for HC and KC was not unexpected because this phenomenon had formerly been observed in HUVEC (Brooimans et al, 1989;Dauchel at al, 1990;Lappin et al, 1992;Ripoche et al, 1988b;Schlaf et al, 2001), in primary myoblasts and in two rhabdomyosarcoma cell lines (CRL 1558 and HTB 153) (Legoedec et al, 1995), in primary fibroblasts (Friese et al, 1999;Katz and Strunk, 1988;Lappin et al, 1992;Schwaeble et al, 1991), in fibroblast-like L cells (Munoz-Canoves et al, 1989), in monocytes (Lappin et al, 1992), in the murine liver cell line ϩ/ϩLi (Vik, 1996), and in the human Hep3b hepatoma cell line (Luo and Vik, 1999) and may therefore be a common feature. In the present study, the proinflammatory cytokines TNF-␣, IL-6, and IL-1␤ did not upregulate FH.…”

“…The data concerning the induction of FH in these hepatoma cell lines are also controversial. For the hepatoma cell lines HepG2, HepG3, HepG4, and H4, it has been described that FH-specific mRNA expression cannot be induced by IFN-␥ (Lappin et al, 1992;Schwaeble et al, 1991). In contrast to these findings, Luo and Vik (1999) reported that Hep3b cells that did not express FH constitutively could be stimulated to express FH protein upon treatment with IFN-␥.…”

“…It has been claimed that complement FH also is mainly produced by this organ, although experimental evidence using primary cells of the liver has not yet been provided. Many hepatoma cells of human or murine origin such as Hep3b (Luo and Vik, 1999;Schwaeble et al, 1991), HepG2 (Lappin at al, 1992;Schwaeble et al, 1991), HepG4 (Schwaeble et al, 1991), H4 (Schwaeble et al, 1991), HUH7 (Friese et al, 1999), or ϩ/ϩ Li (Vik, 1996) have been investigated with respect to the expression of FH. Most of the analyzed hepatoma cell lines (HepG2, HepG3, H4) have been found not to express the FH protein constitutively, whereas the human line HUH7 (Friese et al, 1999) and the murine line ϩ/ϩ Li (Vik, 1996) have been found to produce FH in the absence of stimulation.…”

“…These cell types may function as local sources of FH and thereby reduce tissue damage caused by local complement activation. As a model for the investigation of the hepatic synthesis and regulation of FH, various hepatoma cell lines of mouse (ϩ/ϩ Li) (Vik, 1996) and man [Hep3b (Luo and Vik, 1999;Schwaeble et al, 1991), HepG2 (Lappin et al, 1992;Schwaeble et al, 1991), and HepG3, HepG4, H4 (Schwaeble et al, 1991)] have been used in the past. In the present study, we show the first approach to investigate the constitutive expression of FH in primary, cultured liver cells, ie, in hepatocytes (HC) and in Kupffer cells (KC) of rat as well as in two rat hepatomaderived cell lines (FAO and H4IIE).…”

Production of complement components by cells of the immune system

“…The whole C1 complex can be assembled locally, as C1q, C1r and C1s are secreted. Additionally, C2 and C4, which are also part of the CP, have been shown to be secreted by monocytes [26,28,[57][58][59]. Monocytes also secrete various AP components as C3, FB, FD and FP [24,[59][60][61][62][63][64].…”