Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease.

Atherosclerotic cardiovascular disease (ACVD) is a lipid-driven inflammatory disease and one of the leading causes of death worldwide. Lipid deposits in the arterial wall lead to the formation of plaques that involve lipid oxidation, cellular necrosis, and complement activation, resulting in inflammation and thrombosis. The present study found that homozygous deletion of the CFHR1 gene, which encodes the plasma complement protein factor H-related protein 1 (FHR-1), was protective in two cohorts of patients with ACVD, suggesting that FHR-1 accelerates inflammation and exacerbates the disease. To test this hypothesis, FHR-1 was isolated from human plasma and was found to circulate on extracellular vesicles and to be deposited in atherosclerotic plaques. Surface-bound FHR-1 induced the expression of pro-inflammatory cytokines and tissue factor in both monocytes and neutrophils. Notably, plasma concentrations of FHR-1, but not of factor H, were significantly (p < 0.001) elevated in patients with ACVD, and correlated with the expression of the inflammation markers C-reactive protein, apolipoprotein serum amyloid protein A, and neopterin. FHR-1 expression also significantly correlated with plasma concentrations of low-density lipoprotein (LDL) (p < 0.0001) but not high-density lipoprotein (HDL). Taken together, these findings suggest that FHR-1 is associated with ACVD.

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.

Human complement factor H: molecular cloning and cDNA expression reveals variability in the factor H-related mRNA species of 1.4 kb.

Previously, we have shown that three different mRNA species of 4.3 kb, 1.8 kb and 1.4 kb, related to human complement factor H, are constitutively expressed in the human liver. Probing with our cDNA clone H-46 which represents 920 bp of the 3' end of the 4.3 kb mRNA of factor H on human liver RNA, we always detected the 4.3 kb and the additional, abundantly expressed mRNA species of 1.4 kb in length, indicating that the 1.4 kb transcript is highly homologous to the 3' end of the classical factor H mRNA of 4.3 kb. Using H-46 as a probe, several cDNA clones were isolated from a liver cDNA library and sequenced. The open reading frame of the novel mRNA species encodes a peptide consisting of five internal short consensus repeat motifs (SCR), identifying the translational product to be a member of the SCR family. Sequence comparison with cDNA clones derived from liver RNA of a different donor provided evidence for variability in the factor H related proteins encoded by the 1.4 kb mRNA species. Interestingly, this variability was found to be restricted to the three carboxyterminal SCR domains. Expression data indicate that our variant is not recognized by the monoclonal antibody 3D11.

Interferon-induced transcriptional and post-transcriptional modulation of factor H and C4 binding-protein synthesis in human monocytes.

Cultured human monocytes synthesize factor H (H) and C4 binding protein (C4-bp), as assessed by measuring their presence in culture fluids, and demonstrating the presence of their corresponding mRNAs in total monocyte RNA by Northern blot analysis and by nuclear run-on experiments. H mRNA (4.3 kb and 1.8 kb) could only be detected when cycloheximide (2.5 micrograms/ml) was present in monocyte culture fluid. Recombinant interferon-gamma (IFN-gamma) and lymphoblastoid interferon-alpha (IFN-alpha) produce dose-related increases in the synthesis of H and C4-bp and in the abundance of C4-bp mRNA (2.5 kb). Increased abundance of H mRNA was also seen when cycloheximide (2.5 micrograms/ml) was present in the cultures. Both cytokines increased the transcription rates of the H and C4-bp genes. These changes in transcription were rapid, with significant increases being observed within 30 min of exposure. Following the removal of the cytokines from the cultures the transcription rates of both genes returned to control levels within 4 h. The effects of combining IFN-alpha and IFN-gamma on H and C4-bp transcription rates, mRNA abundances and protein secretion rates were not quantitatively additive. The half-life of H mRNA in monocytes was 15 min, whereas that of C4-bp mRNA was 2 h 45 min. Neither half-life was altered by IFN-gamma.

Prevalence of the 1.8 kb complement factor H mRNA in human lung.

Human complement factor H, described as a 155,000 molecular weight (MW) component, is a key factor in the control of the alternative pathway of complement activation. Using two human factor H cDNA clones, designated R2a (a clone derived from the 3' end of the factor H coding sequence) and B38-1 (a clone derived from the 5' end of the factor H coding sequence), as probes, three factor H-specific transcripts of 4.3 kb, 1.8 kb and 1.5 kb are usually detected in human liver, in equal abundance. Using these two factor H cDNAs to probe human lung RNA, there was evidence of a singular distribution of the factor H mRNA species in human lung compared to liver, in equal prevalence of the 1.8 kb factor H mRNA over the 4.3 kb factor H mRNA (a three- to fourfold difference). No significant expression of the 1.5 kb mRNA was detected. The prevalence of the 1.8 kb complement factor H mRNA leads to the speculation that the predominant factor H form biosynthesized in lung tissue is a truncated form of the factor H molecule.

Enhanced synthesis of factor B induced by tumor necrosis factor in human skin fibroblasts is decreased by IL-4.

IL-4 is a T cell-derived lymphokine that has multiple biologic functions, affecting B cells, T cells, mast cells, monocytes, macrophages, and hematopoietic progenitor cells. We report that IL-4 also affects human skin fibroblasts. These effects were primarily in modulating the effects of other mediators of inflammation on these cells, with IL-4 producing little or no effects by itself. Synthesis of C proteins in human skin fibroblasts is modulated by TNF and other mediators of inflammation. TNF increased synthesis of factor B by 18.6-fold; IL-4 reduced the TNF effect on factor B synthesis by 83%. This effect started with concentrations of IL-4 as low as 0.1 ng/ml. The effect of IL-4 on the TNF-induced increase in synthesis of factor B occurred after 2 h incubation, the time when the effects of TNF alone were first observed. IL-4 also abrogated the effects of IL-1 and LPS on factor B synthesis, but did not affect the increase in synthesis of factor B induced by IFN-gamma. In contrast to the pattern for effect of IL-4 on factor B synthesis, the TNF-induced rate of synthesis of C3 was augmented by IL-4. The specificity of the IL-4 effect was also apparent when comparing the effect of IL-4 on the TNF-induced synthesis of factor B to the effects on total protein synthesis (increased 1.5-fold) and the TNF-induced increases in synthesis in C1r, C1s, C1 inhibitor, C2, and factor H (no changes). The IL-4-induced decrease in synthesis of factor B was mediated at a pretranslational level and was dependent on synthesis of a new protein. This interaction between IL-4 and mediators of inflammation can potentially modulate the inflammatory response in the setting of activation of Th cells.

Differential regulation of complement factor H and C3 production in human umbilical vein endothelial cells by IFN-gamma and IL-1.

Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C3. Addition of T cell growth factor to HUVEC enhanced factor H production, and caused a profound increase in C3 production. In the present study, investigations were initiated to characterize the effects of purified rIL-1 and rIFN-gamma on the production of factor H and C3 at the protein and mRNA level. IFN-gamma enhanced factor H production in a dose-dependent fashion and a two-fold increase was observed with an optimal dose of 200 U/ml, whereas IFN-gamma had no effect on C3 production. IL-1 inhibited factor H secretion, but the production of C3 was increased 10-fold at an optimal dose of 500 pg/ml of IL-1. Kinetic experiments demonstrated that addition of IL-1 to HUVEC resulted in an induction of C3 production after more than 24 h, whereas IFN-gamma already had a significant effect on factor H production after 8 h of culture. IL-1 in combination with IFN-gamma had a synergistic effect on C3 production. The effects of IL-1 and IFN-gamma on factor H and C3 production by HUVEC could be blocked by using neutralizing amounts of antibodies specific for these cytokines. Northern blot analysis showed that factor H mRNA expression was enhanced in IFN-gamma-treated HUVEC and C3 mRNA was induced in IL-1-treated HUVEC, indicating that the observed increase of factor H and C3 probably is controlled by enhancement of transcription or stability of the transcript.

Biosynthesis of complement factor H by human umbilical vein endothelial cells. Regulation by T cell growth factor and IFN-gamma.

The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.

Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1.

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.

Synthesis and regulation of complement protein factor H in human skin fibroblasts.

The alternative pathway of C activation is Ag-independent and forms a first line of defense against infection before immune response. The C3 convertase, C3bBb, formed during activation of the alternative pathway is tightly regulated, with destabilization produced by factor H. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrated that human skin fibroblasts synthesized and secreted factor H protein. Two forms of the protein were identified, the approximately 160-kDa form seen more prominently in serum and a 45-kDa form that has also been identified in serum. The cells contained two forms of factor H mRNA, 4.4 and 1.8 kb. IFN-gamma increased factor H protein synthesis and mRNA content. No effect was observed with LPS. Neither HepG2 cells or human peripheral blood monocytes synthesized factor H protein or contained factor H mRNA.

Expression of complement factor H on the cell surface of the human monocytic cell line U937.

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.

Biosynthesis and postsynthetic processing of human C3b/C4b inactivator (factor I) in three hepatoma cell lines.

Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.

Partial H (beta 1H) deficiency and glomerulonephritis in two families.

H (beta 1H) controls the C3b amplification loop by its ability to displace Bb from the alternative pathway convertase, C3b,Bb, and acts as a cofactor with I (C3b inactivator) to produce inactive C3b. Serum C3 levels are dependent to a large extent on the levels of H and I. Partial H deficiency was found in two families. The index case in Family 1 had vasculitis, thrombocytopenia, proteinuria, and depressed serum H and C3 levels. The index case in Family 2 had depressed serum H and B (Factor B) levels and IgA nephropathy which progressed to renal failure. His sister also had IgA nephropathy and depressed serum H and C3 levels. The depressed serum C3 level, B level, and H level could be responsible for the development of the immune diseases found in some members of these families.

Synthesis of two components of human complement, beta 1H and C3bINA, during fetal life.

The levels of beta 1H and C3bINA were estimated in sera from human fetuses, cord and maternal samples. Both components of complement were detected in fetuses more than 12 weeks old. The serum concentrations tended to increase with the gestational age. The mean levels of beta 1H and C3bINA in cord sera were near 54 and 61% of the mean values in sera from normal adults. Elevated levels of beta 1H were observed in maternal sera at the end of the gestational period. When the levels of beta 1H and C3bINA were compared with those of two other components of complement, it was confirmed that the mean levels of C9 were low in fetal and newborn sera, while the serum concentrations of both C9 and Factor B were elevated in maternal samples. Newly synthesised beta 1H was detected in the culture fluids of fetal liver and peritoneal cells, as judged by the incorporation of labelled aminoacids and the autoradiography of he specific immunoprecipitates in agar gel.

Increased biosynthesis of complement components by cultured monocytes, synovial fluid macrophages and skynovial membrane cells from patients with rheumatoid arthritis.

Monocytes, synovial fluid (SF) and synovial membrane (SM) macrophages from patients with rheumatoid arthritis (RA) were maintained in short-term tissue culture for up to 10 days, and the synthesis of C4, C2, C3, C5, factor B(B), D, properdin (P), C3b inactivator (C3bINA) and beta 1H globulin studied. Functionally active C2, B, D, P, C3bINA and beta 1H were synthesized by the cells in each type of culture. C4, C3 and C5 could be detected, but were functionally inactive. RA monocytes synthesized more C2 than monocytes from patients with degenerative joint disease (DJD) (P < 0.001). Similar studies revealed that SF macrophages synthesized more C3 than SM macrophages (P < 0.001) which in turn produced more C2 than monocytes (P < 0.001). Other experiments showed that SF macrophages synthesized more of each component than the other cell types. SM macrophages made more C2 than B than RA and DJD monocytes, but synthesized only small quantities of P, D and beta 1H. RA monocytes synthesized more of each component than DJD monocytes. The results of these studies show that (1) in RA, complement components can be synthesized locally in the inflamed joints, and (2) local factors in the joints probably stimulate complement synthesis.

Biosynthesis of the complement components and the regulatory proteins of the alternative complement pathway by human peripheral blood monocytes.

Short-term cultures of human peripheral blood monocytes were shown to synthesize the alternative pathway complement components C3, factors B (B) and D (D), and properdin, the regulatory proteins C3b inactivator (C3bINA) and beta 1H, in addition to C2, C4, and C5. B, D, properdin, C3bINA, and C2 were detected by functional assays, whereas beta 1H, C4, C3, and C5 could only be detected using immunochemical procedures. Immunoperoxidase localization studies showed that all the cells in each culture contained each component, so it is possible that all monocytes synthesize each component. It is concluded that cells of the monocyte-macrophage series form a mobile source of complement components and regulatory proteins which can be concentrated at sites of inflammation.