Phylogeny of the plasma regulatory proteins of the complement system.
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Animals↗
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Results for “Complement C4b”
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Regulation of plasma complement C4 and factor b levels in murine systemic lupus erythematosus.
We have investigated plasma concentrations of complement C4 and Factor B in three murine models of systemic lupus erythematosus, MRL-lpr/lpr (H-2k), BXSB (H-2b) and NZB/W (H-2d/z) mice. Levels of C4 and Factor B were also determined in the following control strains: C57BL/6 (H-2b), BALB/c (H-2d) and MRL-+/+ (H-2k). Although complement levels varied independent of disease activity, strain-dependent and intra-strain gender-dependent differences, were detected. Male BXSB mice exhibited significantly lower C4 levels than male C57BL/6 mice from 2.5-5.5 months of age but Factor B levels were not significantly different. Female BXSB, which do not exhibit early disease, had Factor B and C4 levels very similar to C57BL/6 mice. Male and female MRL-lpr/lpr mice, with active disease, had levels of C4 and Factor B which were not significantly different from those in age matched MRL-+/+ mice. While not significantly different from control, several female MRL-lpr/lpr mice with active disease had elevated C4 levels and normal Factor B levels. Male NZB/W, without disease, exhibited an age-dependent decline in plasma C4 but no decline in Factor B levels. In contrast, NZB/W females, with evidence of disease, exhibited age-dependent increases in C4 and Factor B. Interestingly, some animals with elevated C4 had normal Factor B levels and vice-versa. These observations suggest that complement levels do not necessarily reflect disease activity or morbidity patterns in murine lupus and therefore suggest caution in the interpretation of complement levels in the human counterpart of the disease.
Age Factors↗
Absence of induction of IL-1 production in human monocytes by complement fragments.
The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.
Antigen-Antibody Complex↗
Complement receptors and related complement control proteins.
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Carrier Proteins↗
Generation of the classical pathway C3 convertase (EAC4b2a) by proteolytic enzymes.
The formation of EAC 4b2a is a two step reaction: first, the temperature- and time-independent binding of C2 to EAC4b2a resulting in EAC4b2 , secondly, the enzymatically triggered conversion of EAC4b2 to EAC4b2a . In the classical cascade of complement activation, the generation of C3 convertase activity is triggered by the C1 esterase, C1-s, which is part of C-1. Evidence is presented that the enzymes trypsin, chymotrypsin, plasmin, and pronase are also able to activate EAC4b2 to EAC4b2a . Kinetic studies showed that the formation of C3 convertase by these enzymes was dependent on concentration, temperature, and time. The optimal conditions were found as follows: trypsin, 2 micrograms/ml (final conc.) for 8 min at 23 degrees C; chymotrypsin 165 micrograms/ml for 18 min at 23 degrees C; plasmin 0.8 units/ml for 15 min at 23 degrees C; pronase 1.25 microgram/ml for 15 min at 23 degrees C. Even under optimal (tmax) conditions the number of generated EAC4b2a differed from enzyme to enzyme: trypsin (= 100%), pronase (58.3%), chymotrypsin (47.9%), and plasmin (12.9%). The enzymes were also able to generate C3 convertase activity from C2 which was adsorbed to EAC1i4b , a C1 inactivator treated and therefore hemolytically inactive intermediate ( EAC1i4b2 ). These findings underline the biological importance of C1 esterase replacing enzymes.
Animals↗
Inhibition of immune precipitation by complement.
Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.
Animals↗
Cytokines and acute-phase response in acute stroke.
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Acute Disease↗
[Comparison of complement activation between tuberculous and malignant pleuritis].
Complement activation products in pleural effusions were studied to clarify the causes of tuberculous and malignant pleuritis. Pleural SC5b-9, an activation product of complement common pathway, was significantly higher in tuberculous effusions than in malignant ones. In the tuberculous effusions, the levels of SC5b-9 and LDH (a marker of tissue damage) were significantly correlated, but in the malignant effusions these two values were not correlated. In the tuberculous effusions, SC5b-9 and Bb values were significantly correlated, but SC5b-9 and C4d were not, and nor were SC5b-9 and immune complex. In the malignant effusions, SC5b-9 and Bb values were low and were not significantly correlated. These results suggest that complement activation plays a significant role in tuberculous pleuritis, but not in malignant pleuritis.
Antigen-Antibody Complex↗
Construction and characterization of thrombin-resistant variants of recombinant human protein S.
Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Classical and alternative complement pathway activation in paracoccidioidomycosis.
The following study presents evidence of complement activation in paracoccidioidomycosis (PCM). Twenty-eight untreated patients were studied from endemic areas of Parana in southern Brazil. The activation of the classical pathway, evaluated by the C4d/C4 ratio, was significantly elevated in the patients compared to the control population (p < 0.005). Six patients were examined prospectively with the C4d/C4 assay during treatment and they showed a decrease in this ratio associated with clinical improvement. The activation of the alternative pathway was determined by rocket immunoelectrophoresis of fragment Ba. These levels were also significantly higher in the patient group in comparison to the controls (p < 0.0005). The prospective study also showed a significant variation in the Ba levels associated with clinical improvement (p < 0.01). Furthermore, the levels of C3, C4, CH50 and anti-Paracoccidioides brasiliensis IgG were determined in all patients. The anti-P. brasiliensis IgG levels showed a weak positive correlation with the C4d/C4 ratio (r[S] = 0.45; p < 0.03). The C3, C4 and CH50 levels did not show significant variations from the normal ranges. Our results suggest the involvement of both complement pathways, classical and alternative, in PCM and their association with disease activity.
Adolescent↗
Complement activation in platelet concentrates is surface-dependent and modulated by the platelets.
Activation of platelets during storage may contribute to the loss of function and viability known as the platelet storage lesion. We investigated the role of complement activation as a possible mediator of the platelet storage lesion. We studied platelet bags stored up to 5 days under standard blood bank conditions and monitored the generation of several complement activation fragments from both the classical and alternative pathways. To assess the role of noncellular artificial surfaces in the activation of complement, parallel bags were prepared containing platelet-poor plasma. The levels of C4d and C3a increased steadily over time in storage, as did the level of the inactivated membrane attack complex SC5b-9. Generation of C4d, C5a, and SC5b-9 was greater in the absence of platelets than when platelets were present in the container. C5a levels in both groups were low and remained so during storage, suggesting that the C5a generated became surface associated. Using flow cytometry we detected C3 and C3a, but not C9, on the platelet surface. The percentage of C3-positive platelets peaked at the third day of storage; by day five platelet-associated C3 had declined. Decay accelerating factor expression on the platelet surface increased with time in storage and in parallel with CD63 expression. Based on the C4d levels, complement activation proceeded via the classical pathway; minimal generation of the alternative pathway activation fragment. Bb was seen in either the presence or absence of platelets. As an indirect measure of the activation of C1, the functional level of C1 esterase inhibitor (C1INH) was determined. C1INH levels declined over time in storage in bags containing only plasma; however, in the presence of platelets, the levels remained constant presumably because of release of C1INH from the alpha-granules of activated platelets.
Blood Platelets↗
Classical pathway of complement activation in mammalian kidneys.
Two monoclonal antibodies (mAb M4d2 and M4d3) specific for the alpha 2-fragment (C4d), and one antibody (mAb M4c3) specific for the gamma-chain of human complement protein C4, have been tested for cross-reactivity against mammalian complement. These mAb have also been found to react with C4 from guinea-pig (mAb M4d2 and M4c3) as well as from cattle, baboon and rhesus monkey (mAb M4d3 and M4c3) in an activation ELISA. Since reactivity of mAb M4d2 and M4c3 included guinea-pig complement, the specific recognition of mammalian C4 could be confirmed with sera from C4-deficient (def') guinea-pigs. mAb M4d2 or M4d3, but not mAb M4c3, stained glomerular deposits within renal tissue sections from pig, cattle and guinea-pig. In the case of mAb M4d2, specificity of that staining could also be demonstrated in kidney specimens from C4-def' guinea-pigs. It can be concluded that, as in humans, the C4d fragment is also present in mammalian glomeruli. Compared with normal guinea-pigs, the C4-def' and C2-def' animals showed markedly increased glomerular deposits of IgM. It appears that glomerular deposition of complement C4d in mammals: (1) indicates activation via the classical pathway; (2) represents a general phenomenon of renal homeostasis; and (3) seems to be involved in the physiological clearance of immune complexes.
Animals↗
Decreased protein S activity in sickle cell disease.
In sickle cell disease (SCD), vaso-occlusion is a complex process involving cellular, vascular and humoral factors and possibly thrombotic events. We studied three physiological inhibitors of the coagulation system, antithrombin III (AT III), protein C (PC) and protein S (PS), in three groups of subjects: 27 homozygous patients observed either in crisis or in a steady state, 23 heterozygous patients and 30 healthy subjects. PS study included the measurement of total and free PS antigen, PS activity and C4bBP antigen. In heterozygous subjects the results were similar to those of controls, but in homozygous subjects abnormalities of PS and to a lesser extent PC were observed. Values of PC were extremely variable with 10 cases lower than the normal range (2 SD of the mean) and 17 others within this range. In all cases total PS antigen was slightly reduced (77 +/- 18%, M +/- SD) with a more marked decrease of free antigen (59 +/- 17%) and normal values of C4bBP. Levels of PS activity were greatly reduced and lower than those of free antigen with a mean ratio of PS activity to free antigen of 0.6. These abnormalities were associated with significantly high concentrations of fibrinogen D-dimers. PS deficiency in SCD may be at least partly due to adsorption of free PS to aminophospholipids abnormally expressed on sickle cells membranes, microvesicles and activated platelets, while the discrepancy between PS activity and free antigen could reflect proteolytic inactivation of PS by traces of thrombin.
Adolescent↗
Complement activation and immune complexes in juvenile rheumatoid arthritis.
We investigated the potential role of the complement system in juvenile rheumatoid arthritis (JRA) by examining plasma for levels of complement activation fragments C4d and Bb. We correlated findings with both disease activity and laboratory abnormalities, including immune complex (IC) levels. While there was a strong correlation between active disease and both C4d and Bb plasma levels in JRA, no strong correlations could be made between IC levels and complement activation products; weak associations were seen between complement activation fragments and erythrocyte sedimentation rate. Our data suggest that plasma complement activation is a concomitant of active disease in JRA; however, its role in the pathophysiology of JRA remains uncertain.
Antigen-Antibody Complex↗
Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein.
The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.
Amino Acid Sequence↗
Human complement proteins C3b, C4b, factor H and properdin react with specific sites in gp120 and gp41, the envelope proteins of HIV-1.
Recently we reported the basic phenomenon of an interaction between the envelope glycoproteins of HIV-1 gp120 and gp41 and components of the human complement system, i.e. activated C4 (C4b) and activated C3 (C3b) and the complement regulator proteins factor H and properdin. In this study we analyze these interactions in detail. Using 46 overlapping peptides of gp120 attached to microtiter plates, binding of activated human C3 to 6 regions in gp120 was found (aa 100-129, 161-190, 231-250, 301-328, 410-449, 470-499). In competition assays with soluble peptides, representatives of four of these regions were capable to partially inhibit C3b binding to immobilized gp120. Activated human C4 interacted only with peptides covering aa 410-449, but both in direct binding assays and fluid phase inhibition studies. The multi-reactivity of gp120 with C3b was also supported by the fact that gp120 agglutinated erythrocytes coated with C3b. Guided by partial aa sequence homology of gp120 and human C4b binding protein (C4bp) as well as human properdin we detected binding of anti-properdin to aa 100-129 in gp120 and of anti-C4bp to aa 410-449 in gp120. This cross-reactivity was also confirmed by a monoclonal antibody directed against aa 416-443 of gp120, which could be shown to bind C4bp. Interestingly, aa 310-328, part of the V3-loop, were found to show an aa sequence similarity to human complement receptor type 3 (alpha-chain). Consequently, of the 4 (or possibly 6) interaction sites of gp120 with activated human C3, 3 may bind due to imitation of either properdin, CR3 or C4bp. In addition to C4b and C3b, we detected interaction of factor H with gp120; it selectively bound to aa 102-129. Using 14 overlapping peptides of gp41 attached to plates, we identified 4 areas in gp-41 (aa 561-585, 587-605, 615-635, 651-675) which bound human factor H. All of them except the first region partially inhibited factor H binding to gp41 in competition assays with soluble peptides. Properdin bound only to 2 regions (aa 584-614, 651-675). The first 3 sites in gp41 were already shown by us to share homology to sites in human C3. The region around aa 651-675 now also turned out to be similar to human C3. These data demonstrate that the interaction of both, gp120 and gp41, with the complement system is polyvalent and complex.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
C4b-binding protein, a regulatory protein of complement.
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Alleles↗
Glomerular deposition of the complement C4 isotypes C4A and C4B in glomeruonephritis.
BACKGROUND: Complement C4 is a component of the classical complement pathway, which is a major mediator of inflammation in many forms of glomerulonephritis. The two isoforms of C4-C4A and C4B-differ in their physicochemical and functional properties. METHODS: The glomerular deposition of C4A and C4B was investigated in 39 cases of glomerulonephritis with classical pathway activation using isotype-specific monoclonal antibodies 99H7 (C4A) and 1288 (C4B) and indirect immunofluorescence. Complement C4 phenotypes of all patients were determined by agarose gel electrophoresis and immunoprecipitation. RESULTS: Three biopsies contained only the isotype C4B. C4 phenotyping revealed complete C4A deficiency in these three patients. Both isotypes C4A and C4B were detected in 36 biopsies. In 19 (53%) thereof staining for both isotypes was identical. In the remaining 17 (47%), staining intensity of C4A predominated over C4B. The distribution of these two staining patterns did not differ between membranous glomerulonephritis and lupus nephritis. They were also independent of C4A and C4B allotypes including the presence or absence of null alleles at either gene locus. In no case was C4B staining stronger than C4A staining. Serum creatinine and proteinuria did not differ between patients with identical and C4A-dominant C4 deposition. CONCLUSIONS: The most likely but still hypothetical explanation for predominance in glomerular deposition of C4A over C4B in many cases of immune complex-mediated glomerulonephritis is the greater affinity of C4A to protein-containing immune complexes as compared to C4B.
Adolescent↗