Human keratinocytes produce the complement inhibitor factor I: Synthesis is regulated by interferon-gamma

Extrahepatic complement synthesis is believed to play an important role in host defense and inflammation at tissue and organ level. In the epidermis the most abundant cell type, keratinocytes have been shown to produce C3, factor B and factor H. In the present study, we investigated the synthesis of factor I by human keratinocytes. We also studied whether proinflammatory cytokines IL-1alpha, IL-6, TGF-beta1, TNF-alpha and IFN-gamma regulate factor I synthesis in keratinocytes. Human keratinocytes constitutively expressed factor I mRNA and produced factor I protein. Amongst the above-mentioned cytokines, only IFN-gamma regulated the synthesis of factor I, and this effect occurred predominantly at pre-translational level. Factor I produced by keratinocytes was functionally active in cleaving C3b. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor I, and that this synthesis is regulated by IFN-gamma.     

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H

Factor H is an abundant plasma glycoprotein that plays a critical role in the regulation of the complement system in plasma and in the protection of host cells and tissues from damage by complement activation. Several recent studies have described the association of genetic variations of the complement factor H gene (CFH) with atypical haemolytic uraemic syndrome (aHUS), age-related macular degeneration (AMD) and membranoproliferative glomerulonephritis (MPGN). This review summarizes our current knowledge of CFH genetics and examines the CFH genotype-phenotype correlations that are helping to understand the molecular basis underlying these renal and ocular pathologies.     

Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.     

Synthesis of complement proteins in the human chorion is differentially regulated by cytokines

The aim of the current paper was to determine the chorion's contribution to complement synthesis in the placenta and its regulation by cytokines. Biosynthetic labeling followed by immunoprecipitation with polyclonal antibodies was performed in chorionic tissue and chorion-derived cells. Eight complement proteins, factor B, C3, C1r, C1s, C1 inhibitor, factor H, C4 and C2 were detected in chorionic tissue and were secreted extracellularly. In chorion-derived cells, IL-1beta stimulated factor B synthesis but had no effect on C1r, C1 inhibitor, C1s, factor H and C4. TNFalpha had no stimulative effect on any of the complement proteins tested. In contrast, both IL-1beta and TNFalpha highly induced IL-6 secretion in chorion-derived cells, demonstrating the overall responsiveness of these cells to these stimuli. Interestingly, IFN-gamma increased the synthesis of C1s, C1r, C1 inhibitor, C4 and factor H in chorion-derived cells. The fact that the latter two complement proteins have opposing effects on immune activation of the complement cascade demonstrates the complex balance required to both maintain an ability to ward off infections but simultaneously suppress the immune response to enable tolerance of the allograft fetus.     

The C-terminus of complement factor H is essential for host cell protection

Complement is a powerful self-amplifying system of innate immune defense with the capacity to eliminate microbes directly. Factor H is a central regulator in plasma which protects host tissue from complement mediated damage. Here we characterize the relevance of surface attached factor H, and study the regulatory activity of factor H on endothelial cells. Although these cells expressed membrane bound regulators, cell bound factor H contributed substantially to complement regulatory activity at the cell surface. Blockade of the C-terminus of factor H with monoclonal antibodies inhibited cell binding of this soluble regulator and resulted in enhanced complement activation on the cells. In the absence of factor H, increased deposition and slower inactivation of C3b resulted in higher amount of membrane attack complexes on the cell surface. When the membrane regulators CD55 and CD59 were removed by enzymatic treatment, complement mediated cell lysis was enhanced in the absence of factor H. Importantly, inhibition of the C-terminus did not compromise the regulatory function of factor H in fluid phase. Altogether these data point to a highly relevant, yet so far underestimated role of factor H for complement control at cellular surfaces, and reveal a decisive role of the factor H C-terminus in host cell recognition and protection.     

Translational mini-review series on complement factor H: renal diseases associated with complement factor H: novel insights from humans and animals

Factor H is the major regulatory protein of the alternative pathway of complement activation. Abnormalities in factor H have been associated with renal disease, namely glomerulonephritis with C3 deposition including membranoproliferative glomerulonephritis (MPGN) and the atypical haemolytic uraemic syndrome (aHUS). Furthermore, a common factor H polymorphism has been identified as a risk factor for the development of age-related macular degeneration. These associations suggest that alternative pathway dysregulation is a common feature in the pathogenesis of these conditions. However, with respect to factor H-associated renal disease, it is now clear that distinct molecular defects in the protein underlie the pathogenesis of glomerulonephritis and HUS. In this paper we review the associations between human factor H dysfunction and renal disease and explore how observations in both spontaneous and engineered animal models of factor H dysfunction have contributed to our understanding of the pathogenesis of factor H-related renal disease.     

Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3

E2A, HEB, E2-2, and daughterless are basic helix-loop-helix (bHLH) proteins that play key roles in multiple developmental pathways. The DNA binding activity of E2A, HEB, and E2-2 is regulated by a distinct class of inhibitor HLH proteins, the Id gene products. Here, we show that Id3 is required for major histocompatability (MHC) class I- and class II-restricted thymocyte positive selection. Additionally, H-Y TCR-mediated negative selection is severely perturbed in Id3 null mutant mice. Finally, we show that E2A and Id3 interact genetically to regulate thymocyte development. These observations identify the HLH inhibitory protein Id3 as an essential component required for proper thymocyte maturation.     

Regulation of C3 and factor H synthesis of human glomerular mesangial cells by IL-1 and interferon-gamma.

Previous reports have shown production of complement components C4, C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL-2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time- and dose-dependent manner. To investigate whether certain defined cytokines from TCGF were responsible for the observed effect, we tested various cytokines for their effect on complement production by MC. It is shown that IL-1 induces C3 synthesis whereas factor H production is up-regulated by IFN-gamma, in both a dose- and time-dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL-1 could be blocked with antibodies specific for IL-1, and also that TCGF and IFN-gamma enhanced factor H synthesis could both be blocked with antibodies specific for IFN-gamma. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA-polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCGF and IL-1. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up-regulated by TCGF and IFN-gamma. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.     

Translational mini-review series on complement factor H: structural and functional correlations for factor H

The 155-kDa glycoprotein, complement factor H (CFH), is a regulator of complement activation that is abundant in human plasma. Three-dimensional structures of over half the 20 complement control protein (CCP) modules in CFH have been solved in the context of single-, double- and triple-module segments. Proven binding sites for C3b occupy the N and C termini of this elongated molecule and may be brought together by a bend in CFH mediated by its central CCP modules. The C-terminal CCP 20 is key to the ability of the molecule to adhere to polyanionic markers on self-surfaces where CFH acts to regulate amplification of the alternative pathway of complement. The surface patch on CCP 20 that binds to model glycosaminoglycans has been mapped using nuclear magnetic resonance (NMR), as has a second glycosaminoglycan-binding patch on CCP 7. These patches include many of the residue positions at which sequence variations have been linked to three complement-mediated disorders: dense deposit disease, age-related macular degeneration and atypical haemolytic uraemic syndrome. In one plausible model, CCP 20 anchors CFH to self-surfaces via a C3b/polyanion composite binding site, CCP 7 acts as a 'proof-reader' to help discriminate self- from non-self patterns of sulphation, and CCPs 1-4 disrupt C3/C5 convertase formation and stability.     

DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferon-gamma

Recombinant human tumor necrosis factor (rhuTNF) induced DNA fragmentation in sensitive cell lines. This fragmentation was similar to that caused by lymphotoxin-containing cytotoxic T cell culture supernatants. The percentage of DNA cleaved correlated with the degree of cell growth inhibition shown by individual cell lines. DNA fragmentation was first seen after 12 h treatment, and increased slowly with time. The presence of 100 microM ZnSO4 inhibited the rhuTNF-induced DNA cleavage in MCF-7 cells. Recombinant interferon-gamma (rhuIFN-gamma) did not induce DNA cleavage, although it reduced the growth of all the cell lines used in this study. However, it interacted with rhuTNF to produce a doubling in the percentage of DNA fragmentation, and increased cytotoxicity in rhuTNF-sensitive cell lines. Pretreatment with rhuIFN-gamma for 1 h prior to rhuTNF treatment also enhanced DNA fragmentation and cell killing.     

Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells

Thymosin beta 4 (Tβ4) is the major G-actin sequestering molecule and is abundant in lymphoid tissues. However, it is not clear what regulates Tβ4 expression and what its function is on natural killer (NK) cells. We investigated whether interleukin-18 (IL-18) has a role in Tβ4 expression and if enhanced Tβ4 influences IL-18-mediated interferon-gamma (IFN-γ) secretion. In this study, recombinant human IL-18 (rhIL-18) enhanced the endogenous level of Tβ4 through p38MAPK and JNK signaling pathway in the human NK cell line, NK-92MI. Overexpression of endogeneous Tβ4 stimulated IFN-γ expression and secretion. Additionally, pretreatment with an inhibitor for Tβ4 decreased IL-18-enhanced IFN-γ secretion, and transfection with Tβ4-specific short hairpin RNA resulted in reduction of IFN-γ production in primary NK cells as well as in the human NK cell line. Taken together, these data indicated that Tβ4 is regulated by IL-18 and is involved in IL-18-enhanced IFN-γ secretion in NK cells.