Gordon D. Ross

Analysis of the different types of leukocyte membrane complement receptors and their interaction with the complement system.

The specificity, distribution, and structure of 8 different types of leukocytes membrane complement (C) receptors (CR1, CR2, CR3, and receptors for C1q, beta 1H, C3e, C3a, and C5a) are discussed. Recent data are reviewed on the synthesis of C components by macrophages and B lymphocytes, and how these components may function in the activation of these two cell type by the C system. Commonly used C receptor assay procedures are evaluated in terms of both specificity and sensitivity. Specific assay procedures are recommended for measuring CR1 (C4b-C3b receptor), CR2 (C3d receptor), CR3 (C3bi receptor), and the beta 1H receptor. Assays include both rosette and fluorescence procedures for detection of C receptors on either mouse or human leukocytes. Primary systems have been selected for optimal sensitivity and specificity, and where possible, acceptable alternative systems that are less sensitive or specific are suggested for laboratories lacking facilities for C purification.

Opsonization and Membrane Complement Receptors

Publisher Summary This chapter discusses opsonization and membrane complement receptors. Opsonization is the important process in host defense by which particles or complexes are made readily ingestible for uptake by phagocytic cells. Specific serum proteins, known as opsonins, coat particles and cause the particles to bind avidly to phagocytes and trigger ingestion. The complement (C) system plays a major role in opsonization by coating particles such as bacteria with fixed C3 and C4. This leads to binding of the bacteria to phagocyte C3 receptors and the clearance of the bacteria. Viruses, soluble immune complexes, and tumor cells are opsonized and removed by a similar mechanism. Other non-C serum proteins may also opsonize bacteria, particularly IgG antibacteria antibody and fibronectin. For each type of opsonin, there is an opsonin-specific membrane receptor on phagocytes responsible for binding particles coated with that opsonin. In the blood, C-coated particles and immune complexes are bound first to erythrocytes by way of C3 receptors and then, the erythrocytes serve as a vehicle that transports the bound complexes to macrophages in the liver that strip the complexes from the red cell surface and return the red cells to the circulation. At tissue sites of infection, C activation generates C5a, which attracts phagocytic cells via specific C5a receptors.

Identification of a spontaneously shed fragment of B cell complement receptor type two (CR2) containing the C3d-binding site.

CR2 is a 140-kilodalton glycoprotein expressed on B lymphocytes which binds to both C3d and Epstein-Barr virus (EBV). The present study identified a 72-kilodalton C3d-binding protein (gp72) in the spent culture media of Raji B lymphoblastoid cells as a spontaneously shed fragment of the 140-kilodalton CR2 molecule. Both polyclonal and monoclonal antibodies (AB) were used in several assay systems to detect antigenic determinants shared between the gp72 fragment and CR2. Rabbit antibodies to either intact CR2 or gp72 blocked C3d receptor activity, and this inhibitory activity was removed by absorption of anti-CR2 with purified gp72.OKB7, a monoclonal anti-CR2 AB that blocks both C3d and EBV binding to CR2, reacted specifically with both CR2 and gp72, whereas both anti-B2 and HB-5 monoclonal anti-CR2 AB, that block neither of these receptor sites, were unreactive with gp72. The data suggested that the gp72 fragment was not present as such in intact cells, but rather was a product of cells generated by proteolysis of CR2. Thus, intrinsically labeled gp72 was isolated from Raji cell media by affinity chromatography on OKB7-Sepharose, but only intact CR2 was isolated from the Raji cell fraction solubilized in the presence of protease inhibitors. Several lines of evidence suggested that gp72 was not a second type of C3d receptor that was distinct from CR2. First, Raji cells expressed nearly identical amounts of OKB7 and HB-5 epitopes when analyzed by flow cytometry or radioimmune assay, excluding the possibility that B cells expressed OKB7 antigens in both CR2 and a distinct HB-5-negative C3d receptor. Second, all Raji cell surface C3d receptor activity was associated with HB-5-reactive CR2 molecules. We conclude that gp72 represents a spontaneously shed proteolytic fragment of CR2 that contains the C3d-binding site and the closely associated OKB7 epitope.

Complement receptor structure and function

Membrane complement receptors (CRs) are now recognized as important cell-bound components of the complement system and research into the structure and function of complement receptors is one of the main thrusts of current complement research.

Synthesis of complement component C5 by human B and T lymphoblastoid cell lines.

Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lymphoblastoid cells with both distributions of C5 were shown to synthesis a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A)+ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by immunoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but an α subunit of slightly smaller relative mass.

Regulation of Macrophage Functions by Complement, Complement Receptors, and IgG-Fc Receptors

Membrane receptors provide the essential recognition function required for control of macrophage responses to changes in their cellular environment. Considering the complexity and number of different functions performed by macrophages, it is likely that only a small proportion of the total number of different types of receptors are presently known. Among these various receptors there are at least six distinct types of complement (C) receptors and three different types of IgG-Fc receptors (Table 1). Of these nine types of receptors, only four have been characterized structurally. Although a function has been described for six of these receptors, it is possible that each of these receptors may control several other additional functions that are presently unknown.