A sensitive method to detect synthesis of the functional classical, alternative and terminal pathway of complement by cells cultured in vitro

Abstract

A new method used to study in vitro synthesis by human monocytes and alveolar macrophages of the essential complement components for the functional classical, alternative and terminal pathway is presented. The method is based on accumulation of major complement components on activators of the alternative (agarose beads) and classical (lgM-sensitized sheep erythrocytes; ElgM) pathway during co-culture with the phagocytes. There was a time-dependent increase in binding of labelled protein to the co-cultured activators, demonstrating de novo protein synthesis by the phagocytes. Moreover, there was a significant binding to the co-cultured agarose beads and ElgM of monoclonal anti-C3c, anti-C3g, polyclonal anti-C5-C9 and of two monoclonal antibodies (poly C9-MA and MCaEll) to a neoantigen of polymerized C9 present in the terminal complement complex (TCC). In addition, we found a significant binding of polyclonal anti-C4 antibodies to co-cultured ElgM. Incubation of the activators in human serum, subsequently revealed the same pattern of antibody binding. There was no binding of anti-S protein antibodies to the activators after incubation with serum or with the phagocytes. We thus conclude that mononuclear phagocyte-produced complement in the form of C3b, iC3b, and the TCC (C5b-9) was deposited on both activators, whereas C4b was detected on the ElgM. It is our hope that this method can be applied when studying complement biosynthesis by cells other than mononuclear phagocytes.

Key Words:

• cells
• functional complement system
• in vitro
• synthesis