Inhibitor of C1q secretion suppresses the macrophage response to lipid A for nitric oxide but not for TNF production: evidence for a role of C1q in autocrine binding of TNF.
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.
Leu
R W
RW
Oklahoma Medical Research Foundation, Noble Center for Biomedical Research, Oklahoma City.
Stewart
C A
CA
Herriott
M J
MJ
Fast
D J
DJ
Rummage
J A
JA
D000818:Animals; D015922:Complement C1q / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D006801:Humans; D008050:Lipid A / Q000494:pharmacology*; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000096:biosynthesis*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism
D008050:Lipid A; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C002195:3,4-dehydroproline; D015922:Complement C1q; D011392:Proline