We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.
Sarcoidosis is a granulomatous disorder of unknown aetiology. Alveolar macrophages (AM) in sarcoidosis release a variety of mediators important to the pathogenesis of the disease. Complement is essential for the inflammatory response and we investigated whether there were any major defects in the potential for sarcoidosis AM to synthesize complement in vitro. AM from 11 patients with active sarcoidosis and three healthy controls were cultured under serum-free conditions. There was a significant binding of polyclonal (anti-C5, -C6, -C7, -C8) and monoclonal anti-complement antibodies (anti-C3c and anti-C9 neoepitope (aE11] to agarose beads incubated with unstimulated AM for 24, 48, or 72 h. A significant and inhibitable production of soluble C3c, C5, C9, and S-protein was found in the harvested medium as detected by enzyme immunoassays. Activated C3 and C9 were also detected based on neoepitope expression. Presence of co-cultured agarose beads reduced the amount of soluble S-protein due to deposition on the agarose. We argue that the C9 neoepitope is an integral part of the terminal complement complex (TCC), both in the fluid and solid phase when bound to the agarose. In the fluid phase, SC5b-9 was generated, whereas the agarose-bound S-protein is assumed not to be associated with TCC on the beads. The results demonstrate for the first time that AM from sarcoidosis patients synthesize the functional alternative and terminal pathway of complement.
Human monocytes and alveolar and peritoneal macrophages were cultured in serum-free medium with or without endotoxin (ET), agarose beads, or cycloheximide. The cell culture supernatants were collected after various intervals and examined by a monoclonal anti-S-protein antibody in Western blot and in a solid-phase enzyme immunoassay. We found that the phagocytes synthesize and secrete S-protein. ET stimulation or prolonged incubation of the cells did not favour S-protein production, which was inhibited by cycloheximide. Agarose stimulation increased the S-protein level in supernatants from monocyte but not from macrophage cultures.
Alveolar macrophages (AM) from sarcoidosis patients exhibit no detectable defect in their potential to synthesize the functional alternative and terminal pathway of complement. They also synthesize more C9 than AM from healthy controls. Various authors have suggested that sarcoid AM have decreased phagocytic ability. In the present work we studied whether there was any difference in C3 receptor-mediated phagocytosis of serum-treated and native agarose beads by AM recovered from patients with active sarcoidosis compared with controls. AM from seven patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 2, 12, 24, and 48 h. We found a significantly increased CR1 and CR3 receptor-mediated phagocytosis of native agarose beads by AM from the seven patients. CR1 and CR3 were also detected on AM directly recovered from bronchoalveolar lavage fluid using fluorescein-conjugated monoclonal anti-receptor antibodies. The percentage of AM expressing CR appeared to be increased in sarcoidosis. The reason for the enhanced phagocytosis of agarose beads by the sarcoid AM is probably the result of both increased synthesis and receptors of complement. Altered complement production and complement receptors may be important for the pathogenesis of this granulomatous disorder.
The aim of this study was to examine whether soluble neoepitopes of activated C3 (C3b, iC3b, C3c) and C9 are produced by human alveolar macrophages cultured in serum-free medium. There was a significant and inhibitable production of C3 and C9 neoepitopes and C9 by the macrophages from all donors, as detected by enzyme-linked immunosorbent assays based on monoclonal (bH6, aE11) and polyclonal (anti-C9) antibodies. A strong donor-dependent variation in the levels of the C3 neoepitope and C9 (five- to sevenfold) and the C9 neoepitope (twofold) was found. After 1 day (24 h) of incubation, the complement levels were largely unaltered. The presence of an exogenous alternative pathway activator (agarose beads) reduced the amount of soluble complement because of binding to the agarose. However, the relative fraction of C9 neoepitope versus C9 increased (two- to threefold), due to agarose-mediated activation of C9. The results demonstrate activation of the complement system in serum-free alveolar macrophage cultures, irrespective of the presence of a known complement activator.
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