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The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF-alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.

A simple method is described for the identification of mononuclear cells bearing the complement receptor. Gram-negative bacteria are directly fluoresceinated and then incubated in non-immune human serum, which leads to complement fixation via activation of the alternative complement pathway. Complement receptor-bearing mononuclear cells (CRMC) will form easily readable rosettes with these bacteria. The method requires no antibody preparation or purification, uses human serum rather than purified complement components or mouse serum, and gives results that correlate closely (r = 0.9015) with the EAC method of identifying CRMC. Furthermore, these indicator cells cannot bind to mononuclear cells via the E or IgG Fc receptors, eliminating this source of possible error. This method may be used for the identification of CRMC in suspension or frozen tissue sections.

Human erythrocytes have a surface receptor for the third component of complement (C3b). This receptor mediates the immune adherence (IA) reaction. In this study, erythrocytes from over 100 normal donors and from donors with rare blood group types were examine for IA reactivity. All donors had red cells with high titer IA activity, with the exception of one Rh null individual whose erythrocytes had barely detectable IA activity. Five other Rh null donors had normal IA titers. Because of the apparent defect in the expression of the red cell C3b receptor in this one individual, the mononuclear cell C3b receptors of this donor were studied and found to be normal. These findings suggest that expression of the erythrocyte C3b receptor may be controlled by factors which differ from those determining expression of the mononuclear cell C3b receptor.

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.