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PubMed1989

Membrane-associated complement factor H on lymphoblastoid cell lines Raji expresses a co-factor activity for the factor I-mediated cleavage of C3b.

Evidence of factor H was found on the surface of Raji cells derived from two different lines; a 150,000 MW species identical to 150,000 MW plasma factor H was detected. Rosette experiments performed at physiological ionic strength using EAC3b or EC3b were negative. In contrast, Raji cells expressed a co-factor activity in the factor I-mediated cleavage of fluid-phase C3b. This activity was cell number dependent and was inhibited by anti-factor H but not by anti-CR1. Raji cells did not secrete factor H in the extracellular medium.

B-Lymphocytes↗
PubMed1988

Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents.

The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [35S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.

Adjuvants, Immunologic↗
PubMed1990

Hereditary subtypic patterns detected in the Ba fragment of complement factor B: occurrence of four common alleles in Japanese.

A procedure which can detect subtype-specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan-mediated fragmentation of BF in serum via alternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2-4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one-to-one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA-FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged sialic acid.

Alleles↗
PubMed1988

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Animals↗
PubMed1992

cis and trans elements differ among mouse strains with high and low extrahepatic complement factor B gene expression.

Factor B (Bf), an enzyme of the alternative pathway of complement activation, is one of four major histocompatibility complex (MHC) class III genes. To ascertain the genetic mechanism for tissue-specific constitutive and regulated expression of Bf, we sequenced the regulatory regions 5' of the gene from mice of different H-2 MHC haplotypes and assessed trans-acting factors, specific DNA binding nucleoproteins, in liver and kidney. Striking tissue-specific differences in constitutive expression of Bf were demonstrated in mice of H-2f or H-2z haplotypes when compared with H-2d or H-2u (kidney and intestinal Bf in H-2d or H-2u much greater than H-2f or H-2z). These differences correlated with a point nucleotide substitution 3 bp downstream of the upstream Bf initiation site that affects interaction with a DNA binding protein. This and additional cis differences localize the sequence substitutions responsible for previously identified restriction fragment length polymorphisms among inbred mouse strains and also reveal two previously unrecognized polymorphisms generated by SmaI and HinfI digestion. Evidence for differences in trans was found in a comparison of DNA binding nucleoproteins from kidney, but not liver, of B10.PL when compared with B10.M. These data, together with the high degree of sequence homology between human and mouse Bf 5' flanking regions, should prompt a search for polymorphic restriction sites and cis binding elements in the Bf promoter that could serve as markers of human MHC-associated renal pathology and variants in local MHC class III gene expression.

Animals↗
PubMed1987

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.

B-Lymphocytes↗
PubMed1985

A quantitative method for assessing the third complement factor (C3) attached to the surface of opsonized Pseudomonas aeruginosa: interrelationship between C3 fixation, phagocytosis and complement consumption.

A direct enzyme-linked immunoassay is described for the determination of C3 that becomes attached to the surface of bacteria upon incubation in serum. The assay uses horseradish peroxidase-conjugated rabbit antiserum specific for human C3. In this study various Pseudomonas aeruginosa strains, Escherichia coli and Staphylococcus aureus were opsonized under different conditions and the amount of C3 fixed to the bacterial surface was measured directly. The extent of C3 fixation was compared with the percentage of phagocytosis by human polymorphonuclear leukocytes and with the complement consumption in the fluid phase. It was demonstrated that C3 fixation closely correlated with the percentage of phagocytosis but not with complement consumption. The method can be performed with relatively simple equipment and could be used in routine laboratories in order to determine the extent of opsonization of Pseudomonas aeruginosa and other bacteria and to detect opsonic defects in the sera of patients.

Complement Activation↗
PubMed1991

Effect of complement-protein-C3b density on the binding of complement factor H to surface-bound C3b.

Various amounts of the activation fragment C3b of the complement (C) protein C3 were coupled to Sepharose 4B by catalysis with the C3 convertase of the alternative pathway of C. The binding of radioactively labelled C proteins B and H (= factor H) to the C3b-carrying particles was assayed. It was found that the relative binding of H, but not of B, fell rapidly with decreasing densities of solid-phase C3b, suggesting a sigmoidal relationship between C3b density and binding of H. To study the phenomenon in more detail, preformed C3b was coupled to activated thiopropyl-Sepharose 6B at various densities. By using this model system, it was shown that the binding of H/unit amount of C3b was positively correlated to C3b density up to a C3b concentration of about 0.5 mg/ml of gel, whereas binding of B was independent of C3b density. The results show that accumulation of high densities of C3b on a surface creates high-affinity binding sites for H. Because H has recently been shown to form dimers in solution, the interaction of dimeric H with neighbouring C3b molecules is a likely explanation for the phenomenon. The C3b density effect may be a regulatory mechanism keeping the activation of the alternative pathway of C on activating surface within reasonable limits.

Complement C3-C5 Convertases↗
PubMed1986

A family with complement factor I deficiency.

A family with inherited factor I deficiency is described. The proband was a 19-year-old Caucasian female with one episode of meningococcal meningitis and one episode of suspected septicaemia of unknown cause. Two obligate and two probable heterozygotes with factor I levels below the lower limit of the reference range were identified. None of these exhibited increased susceptibility to infectious diseases. The inheritance was autosomal codominant. In addition, molecular heterogeneity of factor H in plasma from the proband but not from any other family members was demonstrated by crossed immunoelectrophoresis. The migration of factor H component of fast electrophoretic mobility was retarded by antibodies to C3c and C3d, suggesting the presence of a fluid-phase complex between factor H and excess C3b generated by the uncontrolled activity of the amplification loop.

Adult↗
PubMed1984

The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.

Animals↗
PubMed1990

Genetic polymorphism of complement factor H in cattle.

Bovine factor H was found to be polymorphic by the combined techniques of SDS-polyacrylamide electrophoresis of bovine plasma and immunoblotting. Three phenotypes (S, SF, F) were identified in a sample population of 149 cattle. Variant S and F differed by an apparent molecular weight of 5000 daltons. Family studies demonstrated Mendelian segregation of variants S and F. The data indicate that these genetic variants of bovine factor H are encoded by two codominant alleles at a single autosomal locus.

Alleles↗
PubMed1985

A DNA restriction fragment length polymorphism in the complement region of the human MHC shows an absolute correlation with polymorphism of complement factor B(Bf) defined by isoelectric focusing.

The gene coding for properdin factor Bf is located in the human major histocompatibility complex and is closely linked to the genes coding for the complement components C2 and C4. Recently, by Southern blotting techniques, a restriction fragment length polymorphism was identified using the endonuclease Taq I, which subdivides haplotypes carrying the F allele of factor Bf. The F allotype has also been subdivided at the protein level by isoelectric focusing into two subtypes Fa and Fb. We have investigated the DNA of 41 healthy unrelated individuals with known BfF subtypes using the 2.3 kb factor Bf cDNA probe to determine if there is any correlation between the Taq I polymorphism and F subtype. We have found that in 23 individuals who carried the Fb subtype a 6.6 kb Taq I fragment was present. The remaining 18 individuals carried the Fa subtype and showed only the 4.5 kb Taq I fragment on Southern blotting (P = 10(-12). This striking correlation (r = 1) between the Fb protein and DNA polymorphism is surprising especially as the 4.5 kb and 6.6 kb Taq I fragments overlap the Bf and C2 genes and the polymorphic Taq I site is located within the C2 gene.

Alleles↗
PubMedDate not supplied

[Behavior of complement factors and plasma proteins in burns].

The behaviour of three complement components (C3, C4 and C3 activator), alpha 1, alpha 2, IgA, IgG, IgM, total proteins, and gamma globulins was followed 114 patients at the C.T.O. Turin, Burns Centre with burns covering from 15% to over 70% of the body surface. Forty-two died within a relatively short time. In the remaining 72, the parameters were studied almost daily for over 30 days, or even until a final cure was obtained. The complement data and, more particularly, the distinct increased in alpha-globulins made it clear that burns can be acute inflammations. In addition, initial immune depression is followed by a prompt response of the complement system (responsible for aspecific immunity). That this change is vital in the progress of the disease may be deduced from the fact that complement component levels were constantly below normal in the patients who died.

Alpha-Globulins↗
PubMed1982

Isolation, characterization and N-terminal sequences of the CNBr-cleavage peptides from human complement Factor B. Localization of a free thiol group and a sequence defining the site cleaved by factor D.

Nine CNBr-cleavage peptides from Factor B (a component of the alternative pathway of complement) were isolated. Each was characterized by amino acid analysis and automated Edman degradation. One peptide contained a methionyl bond resistant to cleavage by CNBr. The number of CNBr-cleavage peptides is in agreement with the results of amino acid analysis of Factor B and the fragments Ba and Bb. A total of 358 unique residues were identified from the N-terminal sequences of the CNBr-cleavage peptides. These represent approx. 50% and 60% of the total residues of Factor B and fragment Bb respectively. Alignment of two CNBr-cleavage peptides (CB-VIc and CB-IV) provided a continuous segment of 140 residues. This sequence contained the site cleaved by Factor D to generate the Ba and Bb fragments during the activation of complement. Peptide CB-IV contained a free thiol group at a position corresponding to residue 33 of fragment Bb. Amino sugar analyses of Factor B and of fragments Bb and Ba indicated that all the carbohydrate structures of factor B are N-linked to asparagine through N-acetylglucosamine. The two carbohydrate-attachment sites of the Bb fragment were identified.

Amino Acid Sequence↗
PubMed1990

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.

Blotting, Northern↗
PubMed1989

Tissue-specific initiation of murine complement factor B mRNA transcription.

Factor B (Bf) gene expression is regulated independently in hepatic and extrahepatic tissues. In studying murine factor B expression, we found two mature mRNA species for Bf in kidney and intestine (2.7 and 2.4 kb) but in other tissues, including liver, lung, spleen, heart, brain, and skeletal muscle, only a single Bf transcript (2.4 kb) was observed. These two Bf transcripts in kidney or intestine are identical in structure except for a 300 bp 5' extension found in the 2.7-kb mRNA. The long Bf transcript originates from a tissue-specific alternative site of transcriptional initiation upstream of the initiation site for the 2.4-kb mRNA, as determined by primer extension and nuclease protection assays. The upstream initiation site lacks a typical promoter motif and is only 80 bp downstream from the terminus of the murine C2 gene. After stimulation in vivo with endotoxin, only the 2.4-kb mRNA increases in kidney indicating independent regulation of the two Bf transcripts. The differences in 5' untranslated region generated in these two Bf mRNA species may affect local concentrations of factor B protein in kidney and intestine by mechanisms depending on differential translation rates or stability of mRNA.

Acute-Phase Reaction↗