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PubMed1992

Surface epithelium related activation of complement differs in Crohn's disease and ulcerative colitis.

IgG1 and activated complement are colocalised on the colonic epithelial brush border in active ulcerative colitis. To investigate whether such deposition is specific for ulcerative colitis, we examined ethanol fixed mucosal specimens from 18 patients with Crohn's colitis and 14 with terminal ileitis by indirect two colour immunofluorescence staining. Monoclonal antibodies to the IgG subclasses and to neoepitopes of activated complement C3b and the terminal complement complex were used in combination with rabbit antiserum to C1q, C4c or cytokeratin. Granular deposition of C3b and terminal complement complex were observed at the luminal face of the surface epithelium in 10 of 18 patients with Crohn's colitis. Specimens from eight of 14 patients with ileal involvement were intensely stained for activated complement (primarily C3b) within the surface mucus layer. No epithelial IgG, C1q or C4c deposition was observed. The results suggest that early and late phase complement activation takes place at the luminal face of the epithelium in Crohn's disease. The absence of colocalised IgG and complement components involved in the classical activation pathway (C1q and C4c), however, suggest that other immunopathological mechanisms (the alternative pathway?) are primarily involved in Crohn's disease in contrast with ulcerative colitis.

Adolescent↗
PubMed1988

Clinical application of new technique that measures C4d for assessment of activation of classical complement pathway.

A new laser nephelometric technique that measures C4d for the assessment of the activation of the classical complement pathway was developed. C4d was isolated from other larger C4 related molecules at a final concentration of polyethylene glycol of 12% and then quantitated by laser nephelometry using a commercially available antiserum, which reacts with C4d determinants. C4d standard (100%) was produced by exhaustive activation of the classical pathway in pooled normal human serum using heat aggregated human immunoglobulin. Serial dilutions of the standard provided a reference curve against which clinical samples were read. Patients with rheumatoid arthritis showed significantly higher C4d values (mean 53.8%) than controls (21.7%; p less than 0.001). The technique proved accurate, rapid, and suitable for the routine laboratory evaluation of complement activation through the classical pathway, and it may be useful in the management of those conditions in which complement activation has a pathogenic role.

Adolescent↗
PubMed1992

Classical complement activation induced by pregnancy: implications for management of connective tissue diseases.

AIMS: To determine the effect of pregnancy on C4d concentrations and to assess whether C4d remains a useful disease activity marker in the management of connective tissue diseases during pregnancy. METHODS: Plasma C3, C4, and C4d concentrations were measured in 83 women at various stages of normal pregnancy and compared with those in 80 non-pregnant controls. RESULTS: C3 concentrations in the pregnant women were significantly raised (p = 0.0001) and the C4 concentrations were reduced (p = 0.0007), and accompanied by a significant increase in C4d (p = 0.0001). The C4d:C4 ratio was higher in the pregnant women (p = 0.0001). CONCLUSIONS: Pregnancy induces activation of the classical complement pathway. C4d concentrations cannot be used to monitor disease activity in patients with connective tissue diseases during pregnancy.

Adolescent↗
PubMed1991

In vitro formation of complement activation products by lipopolysaccharide chemotypes of Salmonella minnesota.

We have applied immunoassays for complement activation products C4d, fragment Bb and the protein S-C5b-9 neoantigen (S-MAC) to assess activation of classical, alternative and terminal pathways, respectively, by lipopolysaccharides (LPS) from the smooth strain (SS) of Salmonella minnesota and the shallow rough (core) mutants R60, R345, R5 and R7. Incubations of sera (n = 6) with LPS generated small and insignificant quantities of Bb and S-MAC in the case of Rb, Rc and Rd chemotypes and slightly greater quantities with Ra. SS-LPS brought about significant (p = 0.01) increases in the formation of both Bb and S-MAC. No significant changes were observed in the concentration of C4d. Polymyxin B enhanced Bb and S-MAC production by SS-LPS, optimally at the lowest concentration of polymyxin B studied, 10 ng/ml. These data confirm and extend observations about complement activation by LPS and suggest that immunoassay may be useful in studying mechanisms of complement activation.

Adult↗
PubMed1990

Activation of the alternative pathway of complement in psoriatic lesional skin.

The complement system may play an important role in the inflammatory reaction of psoriasis. While the classical pathway of the complement has been shown to be activated in psoriasis, there have been few reports on the involvement of the alternative pathway in the inflammatory reactions in psoriasis. Complement fragments, C4d and Bb, are released at the time of the classical and alternative pathway activation, respectively. The presence of the C4d or Bb fragment, therefore, denotes a preceding complement activation through the classical and/or alternative pathway. In the present study, we have measured the levels of C4d and Bb in psoriatic scale extracts using enzyme immunoassay techniques. The scales of these dermatoses contained higher levels of C4d and Bb detectable by enzyme immunoassay than those in the stratum corneum of noninflammatory skin. These results suggest that the alternative as well as the classical pathway of complement are activated in psoriatic lesional skin.

Complement Activation↗
PubMed1992

Pleural SC5b-9 in differential diagnosis of tuberculous, malignant, and other effusions.

A monoclonal antibody against soluble phase-terminal complement complex (SC5b-9) was used to try to differentiate pleural effusions of tuberculous vs malignant and other origin. Effusions of tuberculous origin showed a significantly higher SC5b-9 level than did plasma, suggesting activation of complement in the pleural space. All 26 patients with tuberculous effusions showed SC5b-9 levels in pleural fluid exceeding 2.0 mg/L, while 20 with malignant effusions had levels less than 2.0 mg/L. However, rheumatoid, some parapneumonic, and treated malignant effusions showed SC5b-9 levels above 2.0 mg/L. Considering a value exceeding 2.0 mg/L, the specificity and sensitivity of the SC5b-9 estimation in tuberculosis were 0.74 and 1.0, respectively. The mean values for C4d and Bb fragments of complement were significantly (p < 0.05) higher in the tuberculous than in the malignant effusions. However, the values for Bb in 16 (62 percent) of the 26 patients with tuberculous or malignant effusions were in the same range. The activity of adenosine deaminase (ADA) was higher in the tuberculous than in the malignant effusions. While 18 of 26 patients with tuberculous effusions showed an ADA value exceeding 50 mU/ml, the estimated cutoff point (sensitivity = 0.69), 35 of the 36 nontuberculous effusions showed a true negative value (specificity = 0.97). A correlation between ADA and SC5b-9 values was observed in pleural effusions. These observations suggest that the estimation of SC5b-9 in pleural fluid presents a new approach to differentiating tuberculous vs malignant effusions.

Adenosine Deaminase↗
PubMed1991

Complement fragment C4d and Bb levels in inflammatory skin diseases (e.g. SLE, atopic dermatitis, erythroderma and pustulosis palmaris et plantaris) for assessment of complement activation.

The complement is one of the major effector system in the process of inflammation. Complement activation has been shown to occur in inflammatory dermatoses such as systemic lupus erythematosus, atopic dermatitis, erythroderma of unknown origin, and pustulosis palmaris et plantaris by the elevated blood levels of complement fragments. To clarify the complement activation, especially the alternative pathway involvement, we have measured the concentrations of classical pathway-specific C4d and alternative pathway-derived Bb in the plasma of patients with these inflammatory disorders at a mild to exacerbated stage. Only the SLE plasma showed significantly elevated Bb levels. These results suggest that assessments of plasma C4d and Bb levels may be of value in monitoring the involvement of the complement system in patients with inflammatory dermatoses with significant complement activation.

Adult↗
PubMed1991

[The relationship between complement C3 receptors (CR1, CR3) on polymorphonuclear leukocytes and complement fragments during hemodialysis].

The expression of complement receptor type 1 (CR1) and type 3 (CR3) on polymorphonuclear leukocytes (PMNs) and generation of complement fragments, C3a, C5a, C4d, iC3b and Bb, were studied in patients during hemodialysis using cuprammonium rayon (Cu) membranes. Furthermore, the relation between the expression of CR1 and CR3 on PMNs from healthy donors and complement fragments was investigated. The expression of CR1 and CR3 on PMNs increased during hemodialysis. Plasma C3a, C5a and iC3b levels increased in the first 15 minutes and then decreased at 120 minutes of dialysis. But plasma Bb level remained high until the end of hemodialysis. Purified Bb had no effect on the expression of CR1 and CR3 on PMNs, but C5a augmented those expression in vitro. Nafamostat mesilate, an artificial proteinase inhibitor, inhibited augmentation of complement receptors on PMNs in concentration dependent fashion. C5a generated through the activation of complement was thought to take an important role in the increased expression of CR1 and CR3 on PMNs.

Aged↗
PubMed1991

[Circulating immune complexes and complement breakdown products in childhood IgA nephropathy].

Circulating immune complexes (CIC), mainly IgA-CIC have been frequently detected in IgA nephropathy and recently increased levels of C3 fragments which indicate C3 activation have been reported. However, little is known about the relationship between CIC and complement activation. We determined CIC by the solid-phase anti-C3 Facb enzyme immunoassay in 37 children with IgA nephropathy to investigate the relationship between CIC and clinical and/or histological findings, and also determined C3 fragments whether CIC correlate with complement activation. IgA-CIC were detected in 78% (27/37) with a mean level of 11.9 +/- 3.9 micrograms/ml, which was significantly higher than other glomerular diseases (P less than 0.05). IgA-CIC levels were also found significantly higher in 27 cases with proteinuria than in 10 cases without proteinuria (P less than 0.05). IgG-CIC were detected in 67% (12/18) with a mean level of 4.1 +/- 2.6 micrograms/ml, which was not significantly different from other glomerular diseases. No striking correlation was noted to exist between CIC levels at renal biopsy and the histological severity, because CIC are often present intermittently. C3d was quantitated by the rocket immunoelectrophoresis and C3 by the single radial immunodiffusion to determine the C3d/C3 ratio. The mean value of C3d/C3 was 0.63 +/- 0.19 which was significantly higher than a corresponding value for 15 healthy controls of 0.27 +/- 0.06 (P less than 0.05). Levels of IgA-CIC were found to have a significant positive correlation between C3d/C3 determined simultaneously in 33 cases (r = 0.43, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1991

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↗
PubMed1990

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain.

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.

Antibodies, Monoclonal↗