Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

Failure of the synthetic androgen, danazol, to increase protein S levels and prevent thrombosis in a patient with severe protein S deficiency.

A 34-year-old man with recurrent deep and superficial thromboses was found to have severe protein S deficiency. Treatment with both warfarin and adjusted-dose subcutaneous heparin failed to completely prevent thrombosis. Based on reports of increases in the endogenous anticoagulants (protein C, protein S, antithrombin III and plasminogen) with synthetic androgen therapy, the patient was treated with danazol for 8 weeks. Although the levels of antithrombin III, protein C and plasminogen increased, no change in the levels of total or free protein S or C4b binding protein was observed. Treatment was discontinued at 8 weeks when the patient developed a recurrence of superficial thrombophlebitis. The role of synthetic androgens in the treatment of patients with inherited thrombotic disorders is reviewed and potential reasons for treatment failure in this patient are discussed.

Human anti-endotoxin antibody HA-1A mediates complement-dependent binding of Escherichia coli J5 lipopolysaccharide to complement receptor type 1 of human erythrocytes and neutrophils.

HA-1A has been shown clinically to decrease mortality in septic patients with gram-negative bacteremia. In this study, the ability of HA-1A to augment the serum complement-dependent immune adherence of 125I-labeled Escherichia coli J5 lipopolysaccharide (LPS) to human erythrocytes (RBC) and polymorphonuclear leukocytes (PMNL) was evaluated. In vitro studies indicated three things: HA-1A mediates immune adherence of 125I-J5 LPS to human RBC and PMNL in a dose-dependent manner; under these conditions, high concentrations of LPS (400 ng/mL) could be specifically bound. Immune adherence occurs via the classical complement pathway as demonstrated by its calcium dependence; HA-1A-J5 LPS-C' immune complexes bound to CR1 on human RBC and PMNL. PMNL binding and internalization of immune complexes was demonstrated by trypsin stripping of externally bound immune complexes. These studies support the proposal that HA-1A can lower the bioavailability of endotoxin by mediating binding and potential clearance of LPS via human RBC through the reticuloendothelial system or via direct internalization by peripheral blood PMNL.

Effect of thiol compounds on human complement component C4.

Thiol compounds have been investigated as inhibitors of the covalent binding reaction of human complement protein C4 using Sepharose-C1s as a combined activating and binding surface. o- and p-substituted aminothiophenols are equally effective inhibitors, whereas the m-substituted compound is a less potent inhibitor. The anti-hypertensive drug captopril is also shown to inhibit the covalent binding reaction. A comparison of the effects of these compounds on the covalent binding reaction of isolated C4A and C4B has been made. Results suggest that a Pro-to-Leu substitution in C4B is likely to account for the differences in inhibitory potency of C4B compared with C4A observed with the aromatic inhibitors.

Immunoblotting human C4 bound to human erythrocytes in vivo and in vitro.

A study of C4 bound to human erythrocytes in vitro and in vivo has been made by immunoblotting with mouse monoclonal anti-C4c and anti-C4d and human polyclonal anti-C4d (Rodgers and Chido) following SDS-PAGE. Multi-banded patterns differentiated between C4A and C4B isotypes. Treatment of EC4b with trypsin eliminated immunoblotting but not agglutination reactions. Serum inactivation (factor I) of EC4b resulted in banding patterns similar to those obtained from patients' EC4d. Treatment of EC4b membranes with NH2OH affected many of the bands, two were lost, one was markedly reduced and others had altered SDS-PAGE mobility. Interpretation of the bands has been made in terms of C4-acceptor complexes and inactivation fragments of C4. A distinct difference in the banding of C4A and C4B isotypes has been detected.