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Factor H (FH) is a key alternative pathway regulator that controls complement activation both in the fluid phase and on specific cell surfaces, thus allowing the innate immune response to discriminate between self and foreign pathogens. However, the interrelationships between FH and a group of closely related molecules, designated the FH-related (FHR) proteins, are currently not well understood. Whereas some studies have suggested that human FHR proteins possess complement regulatory abilities, recent studies have shown that FHR proteins are potent deregulators. Furthermore, the roles of the FHR proteins have not been explored in any in vivo models of inflammatory disease. In this study, we report the cloning and expression of recombinant mouse FH and three FHR proteins (FHR proteins A-C). Results from functional assays show that FHR-A and FHR-B proteins antagonize the protective function of FH in sheep erythrocyte hemolytic assays and increase cell-surface C3b deposition on a mouse kidney proximal tubular cell line (TEC) and a human retinal pigment epithelial cell line (ARPE-19). We also report apparent K D values for the binding interaction of mouse C3d with mouse FH (3.85 μM), FHR-A (136 nM), FHR-B (546 nM), and FHR-C (1.04 μM), which directly correlate with results from functional assays. Collectively, our work suggests that similar to their human counterparts, a subset of mouse FHR proteins have an important modulatory role in complement activation. Further work is warranted to define the in vivo context-dependent roles of these proteins and determine whether FHR proteins are suitable therapeutic targets for the treatment of complement-driven diseases., (Copyright © 2017 by The American Association of Immunologists, Inc.)

A major hurdle for effective stem cell therapy is ongoing inflammation in the target organ. Reconditioning the lesion microenvironment may be an effective way to promote stem cell therapy. In this study, we showed that engineered neural stem cells (NSCs) with complement factor H-related protein 1, a complement inhibitor protein, can attenuate inflammatory infiltration and immune-mediated damage of astrocytes, an important pathogenic progress in patients with neuromyelitis optica spectrum disorders. Furthermore, we demonstrated that transplantation of the complement factor H-related protein 1-modified NSCs effectively blocked the complement activation cascade and inhibited formation of the membrane attack complex, thus contributing to the protection of endogenous and transplanted NSC-differentiated astrocytes. Therefore, manipulation of the lesion microenvironment contributes to a more effective cell replacement therapeutic strategy for autoimmune diseases of the CNS., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Autoantibodies targeting factor H (FH), which is a main alternative complement pathway regulatory protein, have been well characterized in atypical hemolytic uremic syndrome (aHUS) but have been less well described in association with alternative pathway-mediated glomerulopathies (GP). In this study, we studied 17 patients presenting with GP who were positive for anti-FH IgG. Clinical data were collected and biological characteristics were compared with those of patients presenting with anti-FH Ab-associated aHUS. In contrast to the aHUS patients, the GP patients had no circulating FH-containing immune complexes, and their anti-FH IgG had a weaker affinity for FH. Functional studies demonstrated that these Abs induced no perturbations in FH cell surface protection or the binding of FH to its ligand. However, anti-FH IgG samples isolated from three patients were able to affect the factor I cofactor activity of FH. Epitope mapping identified the N-terminal domain of FH as the major binding site for GP patient IgG. No homozygous deletions of the CFHR1 and CFHR3 genes, which are frequently associated with the anti-FH Ab in aHUS patients, were found in the GP patients. Finally, anti-FH Abs were frequently associated with the presence of C3 nephritic factor in child GP patients and with monoclonal gammopathy in adult GP patients, who frequently showed Ig Lchain restriction during reactivity against factor H. These data provide deeper insights into the pathophysiological differences between aHUS and GP, demonstrating heterogeneity of anti-FH IgG., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch's membrane. Bruch's membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch's membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch's membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18-20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.

We identify and characterize two human serum proteins with an apparent molecular mass of 24 and 29 kDa, which are antigenically related to complement factor H. These proteins represent differently glycosylated forms and are encoded by the same mRNA. The corresponding cDNA clone is 1051 bp in size and hybridized to a 1.4-kb mRNA derived from human liver. The predicted translation product represents a protein of 270 amino acids, which displays a hydrophobic leader sequence, indicative of a secreted protein. The secreted part is organized in four short consensus repeats (SCR) and has a single putative N-linked glycosylation site. This predicted sequence is closely related to that of the previously described factor H-related proteins h37 and h42, which are also derived from a 1.4-kb mRNA. Amino acid comparison of these factor H-related proteins showed identical leader sequences, an exchange of three amino acids in SCR1, identical sequences of SCR2, and a lower degree of homology between SCR3-4 (h24 and h29) and SCR4-5 (h37 and h42). In addition, SCR3-4 of h24 and h29 display homology to SCR19-20 of human complement factor H. The relatedness of structural elements of the factor H-related proteins h24, h29, h37, and h42 and of factor H, suggests a function common to these proteins and indicates the existence of a gene family consisting of factor H and at least two factor H-related genes.

Synthesis of complement proteins and their regulation in resident cells of the central nervous system are important pathophysiologic factors that can affect the outcome of inflammatory central nervous system diseases. Primary cultures of rat astrocytes constitutively express C3 mRNA and produce C3 protein; both of them were enhanced by LPS or by a live as well as inactivated Newcastle disease virus, a neurotropic paramixovirus. TNF, IL-1 beta, and IL-8 also increased the levels of C3 mRNA and protein whereas IL-1 alpha and IL-6 had no effect, although all of these cytokines are inducible by LPS. LPS stimulation in the presence of cycloheximide decreased the LPS-mediated C3 mRNA induction by 60%. These data suggest that LPS effect on C3 regulation is mediated directly by LPS as well as by LPS-induced cytokines. Interestingly, C3 mRNA induced by Newcastle disease virus or inactivated Newcastle disease virus was inhibited by protein kinase inhibitors, H-7 and staurosporine, whereas these inhibitors had no effect on C3 induction mediated by LPS or cytokines, indicating the existence of different signal transduction pathways.

Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.

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.

Three allotypes of murine factor H have been identified serologically in the previous study (denoted H.1, H.2, and H.3). A cDNA clone coding for the entire length of murine factor H was isolated from a library constructed from the livers of STR/N mice which have H.2 allotype and was fully sequenced. The insert of this clone (STR309) contained 4184 nucleotides and consisted of a 47-bp 5' noncoding region, a 54-bp coding for leader peptide, a 3648 bp for the mature factor H protein, and a 435-bp 3' noncoding region. Compared with the previously reported sequence of the cDNA clone (MH8) isolated from B10.WR mice that have H.1 allotype, the size of the protein coding region was exactly the same, but 21 nucleotide substitutions resulting in 15 amino acid replacements were observed. The amino acid replacement/nucleotide substitution ratio (0.71) is far higher than those observed in the allotypic variations of other proteins. Four 15-base oligonucleotide probes specific for either STR309 or MH8 were synthesized and used in Northern blot analysis. The probes specific for STR309 hybridized with mRNA isolated from the livers of STR/N mice but not with mRNA from the livers of BALB/c mice that have H.1 allotype, whereas the reverse pattern was observed with the oligonucleotide probes specific for MH8. These results strongly suggest that the nucleotide sequence of STR309 represents H.2 allotype of factor H protein, providing an example of an unusual allotype with high ratio of amino acid replacements to nucleotide substitutions.

We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.

Human Factor H (beta 1H) was found to be polymorphic after neuraminidase treatment and isoelectric focusing (IEF) under completely denaturing conditions. Three variants, FH 1, FH 2, and FH 3, were identified in a sample population of 81 unrelated caucasoid individuals. Family studies demonstrated correct mendelian segregation of FH 1, FH 2, and FH 3. Our data indicate that these genetic variants of human Factor H are encoded by three codominant alleles, FH*1, FH*2, and FH*3, at a single autosomal locus FH. In the sample analyzed, the gene frequencies of FH*1, FH*2, and FH*3 were, respectively, 0.691, 0.302, and 0.006.

Complementary DNA probes corresponding to the factor H and C5 polypeptides have been used to determine the chromosomal localizations of these two complement components. Both probes revealed complex and polymorphic arrays of DNA fragments in Southern blot analysis of mouse genomic DNA. Following the distribution of these bands in panels of somatic cell hybrids carrying various combinations of mouse chromosomes on a constant rat or Chinese hamster background allowed the localization of the C5-associated fragments to proximal chromosome 2 and the localization of the factor H-associated fragments to chromosome 1 or chromosome 3. Following the inheritance of DNA restriction fragment-length polymorphisms revealed by the probes in recombinant inbred mouse strains allowed the factor H-associated fragments to be mapped to Sas-1 on chromosome 1, and the C5-associated fragments to be mapped to Hc. Analysis of three-point crosses, in turn, placed the latter locus 19 cM distal to Sd on chromosome 2. We have designated the two loci Cfh and C5, respectively. This genetic analysis raises the possibility that C5 and factor H are both encoded by complex loci composed of distinct structural and regulatory genes.

Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.