Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Use of antisera to the isolated alpha and beta subunits of C3 as probes to study functional sites present on particle-bound C3b but absent on native soluble forms of C3.

The effect of antisera to the isolated alpha and beta chains of C3 on certain C3b-dependent reactions has been studied. C5-mediated haemolysis of EAC1423b was inhibited preferentially by antiserum to the alpha chain, whereas antiserum to the beta chain inhibited the formation of C3bBb. The anti-beta chain antiserum also stabilised C3bBbP, and rendered the enzyme relatively resistant to accelerated decay in the presence of factor H. These and previous findings that anti-alpha and anti-beta IgG bind to restricted subsets of antigenic determinants on C3/C3b suggest that these antisera affect C3b function through the binding of antibodies to active binding sites exclusively exposed by bound C3b. The anti-alpha and anti-beta antibody probes are currently being further developed to verify this interpretation.

Characterisation of a new murine B cell lymphoma.

The characterisation of a new murine B cell lymphoma, A31, is described. Histopathological examination of passaged tumour indicates that initial infiltration occurs in the spleen, lymph nodes, Peyer's patches and liver, while in the terminal phase the bone marrow, gonads and occasionally the central nervous system become involved. The terminal spread is coincidental with the leukaemic phase in the tumour. The tumour cells show typical B cell characteristics in vitro. These include surface immunoglobulin (Ig) of mu, kappa isotype, surface Ia, Thy-1 negativity and an increased uptake of tritiated thymidine following incubation with lipopolysaccharide. A31 cells secrete low levels of IgM into the tissue culture fluid. Short-term culture produced only 100 ng IgM per 10(7) cells over 8 h and no tumour-associated monoclonal band could be detected in the serum of tumour-bearing mice. Chromosomal karyotypes of A31 cells gave model numbers 2n=40 normal, and 2n=41, with partial trisomy of chromosome 2, and trisomy of 17. There was loss of a chromosome 6 and the Y chromosome, together with the translocation of part of an 11 to one of the two unidentified marker chromosomes. The responses of lymphoma-bearing mice to therapeutic levels of cyclophosphamide and vincristine sulphate and also to whole body X-radiation are illustrated. This tumour may help in unravelling the complex biology of B cell lymphoma and because of its low level of Ig secretion, be of particular value in experimental immunotherapy.

Enhancement of hamster alveolar macrophage phagocytic activity by lymphokines.

Modulation of phagocytic activity of resident hamster pulmonary alveolar macrophages was accomplished by incubation of the cells in lymphokines prepared by stimulation of hamster splenocytes with concanavalin A or alloantigens in mixed lymphocyte cultures. Alveolar macrophages preincubated in either of these lymphokine preparations possessed significantly greater ability to ingest IgG or IgM plus complement-coated sheep erythrocytes, via their Fc or complement receptors, respectively, than macrophages exposed to control preparations. Ingestion of yeast particles also was enhanced with macrophages incubated in supernatants from cultures of stimulated splenocytes. Supernatant fluids from either mitogen- or alloantigen-stimulated splenocytes possessed migration inhibitory activity with characteristics similar to MIF from other animals; the phagocytosis-enhancing activity shared some of these characteristics.

Fc- and complement receptor-dependent degradation of soluble immune complexes and stable immunoglobulin aggregates by guinea pig monocytes, peritoneal macrophages, and Kupffer cells.

The degradation of soluble immune complexes (ICx) and stable soluble immunoglobulin aggregates was studied in vitro. To obtain insight into the capacity of phagocytes from different organs to degrade soluble ICx we studied monocytes, peritoneal macrophages, and Kupffer cells. Peritoneal macrophages and Kupffer cells degrade similar amounts of aggregated guinea pig IgG2 (AIgG) and ICx per cell. Monocytes were at least ten times less effective than peritoneal macrophages and Kupffer cells. The presence of normal guinea pig serum as a source of complement enhanced the degradation of ICx and AIgG by peritoneal macrophages and monocytes. There was, however, a diminished degradation of ICx and AIgG by freshly isolated Kupffer cells in the presence of complement. After culturing of the Kupffer cells for 40 hours, there was an increase in the density of C3b receptors and a concomitant reversal of inhibition of AIgG degradation in the presence of complement. It can be concluded from the present experiments that the capacities of peritoneal macrophages and Kupffer cells to degrade soluble ICx and AIgG are comparable and that monocytes are much less active than peritoneal macrophages and Kupffer cells.

Fibronectin promotes binding but not ingestion of agarose beads by mouse macrophages adn human monocytes.

We have examined to what extent human fibronectin associated with agarose beads with a 5- to 10-micron diameter mediates binding and uptake of the beads by mouse macrophages and human monocytes. Native agarose beads preincubated with 125I-fibronectin were neither associated with nor taken up by mouse macrophages after 30 min of incubation under serum-free conditions. When fibronectin was cross-linked to cyanogen bromide-activated agarose beads or incubated with gelatinized beads, this resulted in a significant increase in particle binding by macrophages and monocytes as compared with gelatinized beads, whereas the fraction of cells with ingested particles remained unaltered. Native agarose beads activated by cyanogen bromide and treated with ethanolamine were to a greater extent associated with and taken up by phagocytes than fibronectin- or gelatin-coated beads. Our results indicate that fibronectin acts as an adhesive glycoprotein and not as an opsonin. Since agarose beads are activators of the alternative pathway of complement, and fibronectin is reported to bind to factor C3, we speculate that cell-derived C3b is bound to the beads and fibronectin-coated beads are ingested by the phagocytes via complement C3b receptors on the cells.

Phagocytosis inhibits the production of C2 by human monocytes.

Production of the second complement component by cultured human monocytes was suppressed by the addition of a variety of phagocytosable particles. The degree of suppression was related to the concentration of particles, but was unrelated to particle size, the presence of surface sialic acid, antibody or C3b. The failure to reverse suppression by prostaglandin synthetase inhibitors indicates that it is not mediated by prostaglandins.