Involvement of Antilipoarabinomannan Antibodies in Classical Complement Activation in Tuberculosis

We examined alternative and classical complement activation induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. After exposure to BCG, there were higher levels of the terminal complement complex in sera from Indian tuberculosis patients than in sera from healthy controls. The addition of BCG with or without EGTA to these sera indicated that approximately 70 to 85% of the total levels of the terminal complement complex was formed by classical activation. Sera from Indian tuberculosis patients contained more antibody to lipoarabinomannan (LAM) than sera from healthy Indians. Levels of anti-LAM immunoglobulin G2 (IgG2), but not anti-LAM IgM, correlated positively with classical activation induced by BCG in the sera. By flow cytometry, deposition of C3 and terminal complement complex on bacilli incubated with normal human serum was demonstrated. The anticomplement staining was significantly reduced in the presence of EGTA and EDTA. Flow cytometry also revealed the binding of complement to BCG incubated with rabbit anti-LAM and then with factor B-depleted serum. This indicates that classical activation plays a major role in complement activation induced by mycobacteria and that anti-LAM IgG on the bacilli can mediate this response. Classical complement activation may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes, which may influence the course after infection.Mycobacterium tuberculosis is a facultative intracellular parasite, and several studies have focused upon the mechanisms by which mycobacteria enter mononuclear phagocytes. The complement system plays a major role in opsonizing mycobacteria for cellular uptake. It has been shown that monocyte complement receptors (CR) mediate the phagocytosis of M. tuberculosis and Mycobacterium bovis BCG coated with C3 by alternative complement activation (17, 26). Phenolic glycolipid 1, which is found in abundance on Mycobacterium leprae, fixes C3 via serum antibody (Ab) binding and classical-pathway complement activation and mediates phagocytosis by monocytes (27). The authors have also shown that serum from healthy adults contains Ab to lipomannan, lipoarabinomannan (LAM), and arabinogalactan (28), and it is well established that anti-M. tuberculosis Ab occur in both tuberculous and nontuberculous individuals (1). Production of such Ab in the latter group may be influenced by BCG vaccination widely used against tuberculosis to induce cell-mediated protection against the disease or by exposure to epitopes shared by avirulent environmental mycobacteria and M. tuberculosis. Moreover, the presence of complement-activating Ab to avirulent mycobacteria that cross-react with M. tuberculosis may be decisive for the development of localized instead of disseminated tuberculosis (6).Complement activation culminates in the formation of the terminal complement complex (TCC). The presence of TCC containing C5b-9 with or without vitronectin (24) on the bacterial surface may explain the reported uptake of bacilli via monocyte vitronectin receptors (25). The soluble terminal complement activation product C5a is a potent chemotaxin and stimulator and may recruit activated host monocytes that can be invaded. Recently, the binding of M. tuberculosis to CR3 expressed in Chinese hamster ovary cells was reported to be predominantly nonopsonic (7). Previously, we have shown that antigen (Ag) 85C of M. bovis BCG and M. tuberculosis promotes monocyte CR3-mediated uptake of beads coated with mycobacterial products (13). Interestingly, 85C could be a ligand for the non-iC3b-binding epitope in CR3 found to bind M. tuberculosis to macrophages (29). In addition, several other ligands and receptors, unrelated to complement, are known to participate in the uptake of mycobacteria in mononuclear phagocytes (2, 12, 25, 30).We wanted to study complement activation induced by BCG and M. tuberculosis Ag in sera from nontuberculous and tuberculous subjects. Especially, we wished to investigate classical complement activation and its relationship to the specificity of anti-M. tuberculosis Ab. Therefore, sera from healthy subjects and tuberculosis patients were exposed to mycobacteria and differences in soluble complement activation products in the sera were examined by an enzyme-linked immunosorbent assay (ELISA) specific for neoepitopes in the activation products (11, 21). Ab to mycobacteria in the populations were identified, and the levels were determined by ELISA. In addition, deposition of complement on BCG exposed to different sera and Ab was studied by flow cytometry.(This work was presented at the Sixth European Meeting on Complement in Human Disease, Innsbruck, Austria 12 to 15 March, 1997.)