lipid-a and 3-4-dehydroproline

Studies were designed to further define the modulatory role of complement subcomponent C1q in macrophage activation by Lipid A to mediate production of TNF and cytotoxic nitric oxide (NO). Pretreatment of macrophages for 24 h with 2.5 mM 3,4,dehydro-D,L-proline (DHP), an inhibitor of C1q secretion, suppressed their response to Lipid A activation for cytotoxicity of P815 tumor targets which correlated with a corresponding decrease in NO production. In contrast, DHP-pretreated macrophages displayed an increase in the release of TNF in response to Lipid A as compared to untreated controls. Time kinetic studies indicated that DHP-pretreated macrophages produced higher sustained levels of TNF activity during 1 to 24 h culture with Lipid A than did untreated control macrophages. This was confirmed by increased TNF mRNA expression in response to Lipid A by DHP-treated cells. DHP-pretreated macrophages had reduced levels of cell surface C1q as determined by cytofluorometric analysis of the binding of FITC-labeled anti-C1q, F(ab')2. Macrophages were also found to have reduced binding capacity for phycoerythrin-labeled rTNF (PE-TNF) by cytofluorometric analysis following DHP treatment. Exposure of DHP-pretreated macrophage to soluble C1q at 4 degrees C restored their reduced binding of PE-TNF. C1q was confirmed to bind to macrophages at 4 degrees C as detected by FITC anti-C1q, F(ab')2 and such C1q binding promoted a corresponding increased binding of PE-TNF. Macrophages which were plated over immobilized C1q were also markedly enhanced in their binding of PE-TNF probe. Our results indicate that the inhibition of macrophage secretion of C1q by DHP pretreatment, was accompanied by an increased TNF mRNA expression and release with a decrease in NO generation following Lipid A activation. Since TNF binding to DHP-treated macrophages was reconstituted by the binding of exogenous C1q to the cells, it appears that C1q may be involved in the modulation of autocrine binding of TNF for subsequent generation of cytotoxic NO.

Topics: Animals; Complement C1q; Cytotoxicity, Immunologic; Flow Cytometry; Humans; Lipid A; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred C3H; Nitric Oxide; Proline; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The inhibitors of C1q biosynthesis and secretion, 3,4-dehydro-DL-proline (DHP) and 2,2'-dipyridyl, were previously shown to suppress murine macrophage FcR-dependent phagocytosis and cytolysis of IgG-opsonized RBC targets. Inasmuch as non-antibody macrophage activators also bind C1q to initiate C1 activation, we determined the effects of these same inhibitors of C1q biosynthesis on activation of macrophages for antibody-independent, nonspecific tumor cytotoxicity by lipid A and a variety of other non-antibody activators. Preexposure of mouse inflammatory peritoneal macrophages to either DHP (0.5 to 2.5 mM) or 2,2'-dipyridyl (0.1 to 0.3 mM) for 24 h produced a dose-related suppression of their response to activation by lipid A to mediate tumor cytotoxicity of L1210 mouse leukemia targets. Inhibition of C1q secretion by DHP-treated macrophages was confirmed both by a complement hemolytic assay and by autoradiographic analysis of [35S]methionine-labeled culture supernatants. DHP-treated macrophages were inhibited in their response to direct activation and triggering of IFN-gamma-primed macrophages by lipid A, Poly I:C, and cobra venom factor for tumor cytotoxicity. DHP inhibited macrophage activation for antibody-dependent cellular cytotoxicity of L1210 tumor targets mediated by antitumor target IgG. The addition of exogenous purified C1q (2 micrograms/ml) to macrophages after DHP treatment, reconstituted their response to activation for both antibody-independent and antibody-dependent tumor cytotoxicity. Our results indicate that C1q synthesis and secretion by effector macrophages is a prerequisite for the initiation of their activation by both immune complex and by non-antibody agents that also bind C1q. It now appears that macrophage-derived C1q may act as an auxiliary amplification signal for autocrine-like modulation of the initiation of macrophage activation by both the antibody-dependent and independent pathways.

Topics: 2,2'-Dipyridyl; Animals; Antibody-Dependent Cell Cytotoxicity; Complement C1q; Cytotoxicity, Immunologic; In Vitro Techniques; Interferon-gamma; Lipid A; Macrophage Activation; Macrophages; Mice; Neoplasms, Experimental; Proline; Pyridines; Secretory Rate