Treatment with an inhibitory monoclonal antibody to mouse factor B protects mice from induction of apoptosis and renal ischemia/reperfusion injury.

Complement activation in the kidney after ischemia/reperfusion (I/R) seems to occur primarily via the alternative complement pathway. The ability of an inhibitory mAb to mouse factor B, a necessary component of the alternative pathway, to protect mice from ischemic acute renal failure was tested. Treatment with the mAb prevented the deposition of C3b on the tubular epithelium and the generation of systemic C3a after renal I/R. Treated mice had significantly lower increases in serum urea nitrogen and developed significantly less morphologic injury of the kidney after I/R. For gaining insight into potential mechanisms of protection, the activity of caspases within the kidney also was measured, and it was found that caspases-2, -3, and -9 increased in a complement-dependent manner after renal I/R. Apoptotic cells were detected by terminal deoxynucleotidyl transferase catalyzed labeling of DNA fragments, and mice in which the alternative pathway was inhibited demonstrated significantly less apoptosis than control mice. Thus, use of an inhibitory mAb to mouse factor B effectively prevented activation of complement in the kidney after I/R and protected the mice from necrotic and apoptotic injury of the tubules.

Modulation of complement component (C3 and factor B) biosynthesis by a histone deacetylase inhibitor in human intestinal epithelial cells.

Sodium butyrate enhances TNF-alpha-induced complement C3 secretion but suppresses TNF-alpha-induced factor B secretion in intestinal epithelial cells. To further evaluate the mechanism underlying these responses, we assessed the effects of trichostatin A, a compound structurally unrelated to butyrate and a potent inhibitor of histone deacetylase. The C3 and factor B secretion was evaluated by enzyme-linked immunosorbent assay (ELISA) and Northern blot, and the activation of transcription factor was assessed by an electrophoretic gel mobility shift assay (EMSA). Like sodium butyrate, trichostatin A enhanced TNF-alpha-induced C3 secretion, but suppressed TNF-alpha-induced factor B secretion. These effects were also observed at the level of mRNA. EMSAs indicated that trichostatin A weakly suppressed TNF-alpha-induced NF-kappaB and NF-IL6 activation. These observations differ from previous reports that sodium butyrate potently suppressed NF-kappaB activation but enhanced NF-IL6 activation. Trichostatin A modulated TNF-alpha-induced C3 and factor B secretion in a manner similar to that induced by sodium butyrate, suggesting that both sodium butyrate and trichostatin A exert certain counter-regulatory effects associated with histone hyperacetylation. However, it remains to be determined which factors other than histone acetylation are responsible for the counter-regulation of TNF-alpha-induced C3 and factor B gene expression.

Methionine modification impairs the C5-cleavage function of cobra venom factor-dependent C3/C5 convertase.

The complement-mediated lysis of guinea pig erythrocytes by cobra venom factor (CVF) decreased by 50-60% within 2 min of treatment with 5 mM sodium periodate at 0 degree C. This loss of activity paralleled modification of 3-4 Met; other amino acids and sugar residues of the oligosaccharide chains were not affected. Treatment with N-chlorosuccinimide or chloramine-T under conditions that specifically modified 3-4 readily-oxidizable Met also caused 50-60% loss of CVF activity. The secondary structure of CVF was not altered by these modifications. Methionine-modified CVF (MetCVF) supported the cleavage of factor B by factor D with equal efficiency as that of untreated CVF to form C3/C5 convertase (MetCVF,Bb) of the alternative pathway. MetCVF,Bb and CVF,Bb were indistinguishable with respect to C3 cleavage. However, the C5-cleavage ability of MetCVF,Bb was significantly lower than that of CVF,Bb. These results suggest the involvement of Met in CVF binding of C5.

Suppression of alternative complement pathway activity by radiographic contrast media.

The authors examined the effect of four different kinds of contrast media (ionic/non-ionic, monomer/dimer) on the activation of the complement (C) system (haemolytic activity and anaphylatoxin generation) in vitro. In addition, the authors compared the effect of contrast media on inulin-mediated generation of the anaphylatoxin derivative C3a des Arg in sera from urticarial reactors and their non-reacting controls. It was observed that the incubation of commercial iohexol, ioxaglate, iodixanol and meglumin amidotriz solutions in normal human serum (NHS) resulted in a dose-dependent decrease in the haemolytic activity of the alternative C pathway. Contrary to expectations the contrast media did not activate C in NHS. Instead, inulin-induced generation of C3a des Arg was inhibited by all the four contrast media. The strongest inhibitor was ioxaglate, an ionic dimer. No significant difference between the urticarial reactors and non-reactors in the inhibition of C3a des Arg generation was observed. In analyzing the mechanism of C inhibition we found that the contrast media solutions, particularly the ionic ones, prevented formation of the alternative pathway C3 convertase, C3bBb, by inhibiting the binding of factor B to surface-associated C3b molecules. The results suggest that the previously observed decrease in haemolytic C titres by contrast media is due to direct suppression of C activity rather than activation-induced consumption.

IL-13 results in differential regulation of the complement proteins C3 and factor B in tumour necrosis factor (TNF)-stimulated fibroblasts.

IL-13, like IL-4, a product of activated T cells, has multiple biological actions, primarily on B cells and monocytes. The purpose of the present study was to compare the effects of IL-13 with those of IL-4 on the synthesis of complement proteins in fibroblasts. Dermal fibroblasts were developed from skin biopsies. Confluent monolayers were stimulated with the relevant cytokine or combinations of cytokines and biosynthetically labelled with 35S-methionine. The specific proteins were analysed using immunoprecipitation and SDS-PAGE. Addition of IL-13 to fibroblast cultures treated with TNF-alpha resulted in a dose-dependent increase in C3 protein biosynthesis and a concomitant down-regulation of factor B protein biosynthesis. In TNF-stimulated fibroblasts, the addition of IL-13, 100 ng/ml, induced a 2.45-fold increase in the synthesis of C3, while in the same cells under identical conditions the synthesis of factor B was only 42% of the level without IL-13. Similar effects of IL-13 were noted on IL-1-treated fibroblasts. These effects were specific for C3 and factor B, and no alteration of the constitutive or TNF-induced synthesis of C1s or C1 inhibitor proteins was observed. IL-13 altered the synthesis of C3 and factor B proteins also in fibroblasts stimulated with interferon-gamma (IFN-gamma) in addition to TNF, in the same direction as it did in cells stimulated with TNF alone. IL-13 has similar effects to those of IL-4 on the synthesis of C and factor B in TNF- and IL-1-stimulated fibroblasts. The observed effects of IL-13 are IL-4-independent, as anti-IL-4 antibody abrogates IL-4-induced effects, but has no effect on IL-13-induced responses. This interaction between different cytokines on the synthesis of proinflammatory and immunoregulatory proteins may have significance, particularly at local sites of inflammation, and may affect the synthesis of complement proteins in inflamed joint as in rheumatoid arthritis.

Cytokine-regulated production of the major histocompatibility complex class-III-encoded complement proteins factor B and C4 by human glomerular mesangial cells.

Local production of complement within normal or diseased kidneys could be of importance during local inflammatory reactions. In the present study, we demonstrate that human MCs are able to synthesize the MHC class-III-encoded complement proteins factor B and C4 in vitro. This synthesis is strongly upregulated following stimulation with cytokine-containing supernatants of activated peripheral blood mononuclear cells. All primary cell lines tested so far are able to synthesize factor B and C4 after stimulation. To determine more specifically whether defined cytokines are able to enhance factor B and C4 complement production, MCs were stimulated with IL-1 alpha, IFN-gamma, and TNF-alpha. Factor B synthesis was increased in a dose-dependent fashion by IL-1 alpha, TNF-alpha, and IFN-gamma, whereas C4 synthesis was only upregulated by IFN-gamma. Furthermore, factor B synthesis was upregulated after stimulation with IFN-alpha, -beta, and -gamma and C4 synthesis only by IFN-gamma. The synthesis of factor B and C4 was inhibited by cycloheximide, suggesting de novo protein synthesis. The cytoplasmic localization of both components was shown by immunofluorescence studies. Northern and dot blot analysis revealed induction of factor B and C4 mRNA after stimulation with cytokines.

Purification from Vipera lebetina (desert adder) venom of a protein that depletes human complement.

A rapid and efficient procedure for purification from Vipera lebetina venom of a low molecular weight anticomplement protein is described. The procedure used gel filtration on Superose 12, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrated on SDS-PAGE as a single band of about 25,000 Da under nonreducing conditions and as a band of 16,000 Da under reducing conditions. Its isoelectric point was estimated to be 7.6 +/- 0.1. The isolated Vipera lebetina protein was found to decrease the hemolytic activity in human serum measured by assays for classical pathway and alternative pathway activation. The loss of the complement activity could be ascribed, at least in part, to a proteolytic cleavage of the alpha chains of C3 and C4. This protein was also found to be without action on human blood coagulation and on purified fibrinogen and Factor B.