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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↗
PubMed1991

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a novel member of the short consensus repeat family related to the carboxy terminal of the classical 150-kDa molecule.

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.

Amino Acid Sequence↗
PubMed1989

Truncated forms of human complement factor H.

By the use of Western-blot analyses with polyclonal anti-(Factor H) antibodies, two low-Mr protein species of Mr 41,000 and 37,000 under non-reducing conditions and 43,000 and 40,000 under reducing conditions are consistently detected together with the well-known 155,000-Mr Factor H in human plasma and serum. These two additional species are also found in plasma, urine and synovial fluids. The 41,000-Mr species but not the 37,00-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the N-terminal part of Factor H. The 37,000-Mr species but not the 41,000-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the C-terminal part of Factor H. The 41,000-Mr and 37,000-Mr species are different from the well-characterized 36,000-Mr N-terminal tryptic fragment of Factor H. They are likely to represent translational products of the short Factor H mRNA species of 1.8 kb and 1.2-1.5 kb occurring in human liver that we have recently described.

Antibodies, Monoclonal↗
PubMed1990

Molecular characterization of human complement factor B subtypes.

While several laboratories have agreed that there are two subtypes of the BF*F alleles, no information has been available until now at the molecular level. The region of the BF gene corresponding to the Ba fragment [1.7 kilobases (kb)] of the BF*S, BF*FA, and BF*FB alleles has been sequenced after specific amplification using the polymerase chain reaction (PCR). A point mutation at codon 7 has been revealed converting a cytosine in the BF*S allele to a thymidine in BF*FB. At the translational level an arginine residue in BF*S is substituted for a tryptophan residue in BF*FB. The amino-terminal sequencing of factor B immunoprecipitated from serum has been carried out from microquantities of protein blotted onto polyvinylidene fluoride (PVDF) membranes. We have shown that the difference between the BF*FA and the BF*FB subtypes in characterized by a glutamine at position 7 in BF*FA and a tryptophan in BF*FB.

Base Sequence↗
PubMed1989

Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products.

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.

Amidohydrolases↗
PubMed1990

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation.

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.

Amino Acid Sequence↗
PubMed1985

Structural polymorphism of murine complement factor H controlled by a locus located between the Hc and the beta 2M locus on the second chromosome of the mouse.

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.

Animals↗
PubMed1984

Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay.

A sensitive solid-phase radioimmunoassay is described which quantitates human D to 1-2 ng/ml. The assay was used to measure the concentration of D in normal and acute-phase sera and sera from individuals with systemic lupus erythematosus. All 3 groups of sera had comparable levels of D with mean values of 1.8, 2.3 and 2.5 micrograms/ml, respectively. Also tested were sera decomplemented in vitro by activators of the classical and alternative pathways. The results indicated that D is not depleted by alternative or classical pathway activation. However, heat inactivation (56 degrees C, 30 min) of serum resulted in almost complete loss of antigenic D.

C-Reactive Protein↗
PubMed1983

Complement factor D concentrations in normal sera: comparison of immunochemical and functional determinations.

The concentration of factor D (D) was determined in the sera of 144 healthy adults and 29 healthy children by enzyme amplified electroimmunoassay (EAE), and by a hemolysis in gel (HIG) assay for D. The 95% geometric confidence areas for D in adults were 75-143% as determined by EAE, and 65-171% by the functional assay. The concentrations were given in per cent of a reference serum pool containing 1.6 mg/l of D as measured by EAE. The correlation coefficient between D values obtained by the two methods was r = 0.52 (p less than 0.001). D levels were lower in children than in adults. In the EAE purified D, and pathological sera with very high D Concentrations, gave lower precipitation peaks with dilutions performed in buffer than with dilutions in D depleted serum or human serum albumin.

Adult↗
PubMed1980

Isolation and characterization of rat complement factor B and its interaction with cell-bound human C3.

Factor B was isolated from fresh rat plasma by sequential chromatography on QAE-A50, Biorex-70, gel filtration on Sephadex G-200 superfine and rechromatography on QAE-A50. That Brat was isolated in its native form was indicated by its migration during immunoelectrophoresis and by its capacity to react with cobra venom factor (CoVF) in the presence of human D to form a C3 convertase capable of cleaving purified rat C3 and human C3. The recovery of Brat was between 8 and 15%; the final material was homogeneous according to SDS-PAGE analysis. Reduction of Brat with DTT in the presence of urea and SDS did not produce detectable peptides of lower molecular weight. Both reduced and unreduced Brat had an apparent molecular weight of 100,000. An antiserum against Brat induced in rabbits recognized only one protein in fresh rat plasma as indicated by immunodiffusion and immunoelectrophoretic analysis. Zymosan treatment of rat serum resulted in the cleavage of Brat into two fragments with alpha and gamma mobility. Native Brat has a beta electrophoretic mobility. The plasma concentration of Brat in Wistar rats was 215 +/- 38 microgram/ml (mean +/- SD).

Animals↗
PubMed1982

Murine complement factor B (BF): sexual dimorphism and H-2-linked polymorphism.

Polymorphism of murine BF is described using agarose gel electrophoresis of EDTA-plasma. The proteins were blotted onto cellulose nitrate sheets and BF was detected by incubation of these sheets with anti-BF serum, anti-IgG serum, and 125I-labeled protein A successively. After autoradiography, four or five main BF bands were found in plasma of male mice. The strain WLL/BrA (H-2bs) carried a more anodal variant than the strains 020/A(H-2pz), B10 (H-2b), B10.A (H-2a), B10.M (H-2f), and OIR (H-2q). In backcross and F2 generations the BF variants always cosegregated with the H-2 haplotypes. In this way linkage to H-2 could be established. When the electrophoretic BF patterns of males and females were compared, a sexual dimorphism was discovered; the females of each strain had only three main BF bands compared with the four or five found in males. However, no difference in level between males and females could be detected, probably because the three BF bands in the females were stronger. These data extend the information on the interspecies homology of the MHC and may open new possibilities for studies of the genetic organization and hormonal regulation of the H-2 complex.

Animals↗
PubMed1990

Regulation of proteolytic activity of complement factor I by pH: C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) have different pH optima for factor I-mediated cleavage of C3b.

C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (approximately 4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.

Antigens, CD↗
PubMed1984

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity.

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.

Amino Acid Sequence↗
PubMed1981

IgG rheumatoid factor, complement and immune complexes in rheumatoid synovitis and vasculitis: comparative and serial studies during cytotoxic therapy.

IgG and IgM rheumatoid factors (IgG-RF and IgM-RF), complement and three assays for immune complexes were measured in 22 patients with rheumatoid arthritis (RA) complicated by either chronic active synovitis or vasculitis. Patients with vasculitis had relatively inactive arthritis but had higher titres of rheumatoid factors, especially IgG-RF, anticomplementary activity (ACA) and lower levels of C4 than those with synovitis. Clq-binding and platelet aggregation (PA) levels were similar in both groups. Serial measurements during cytotoxic therapy showed a close temporal relationship between the clinical features of vasculitis and levels of IgG-RF, ACA and C4 both with remission and with relapse. We suggest that immune complexes containing IgG-RF which activate complement and are detected by ACA are useful markers of rheumatoid vasculitis and may be important in its pathogenesis.

Antigen-Antibody Complex↗