COMPARISON OF THE EFFECTS OF SALMONELLA-MINNESOTA RE595 LIPOPOLYSACCHARIDE, LIPID-A AND MONOPHOSPHORYL LIPID-A ON NITRIC-OXIDE, TNF-ALPHA, AND IL-6 INDUCTION FROM RAW-264.7 MACROPHAGES

Lipopolysaccharide (LPS) exhibits a wide variety of bioactivities. Alt hough it was generally proposed that the lipid A component represented the active center responsible for most of the bioactivities of LPS, a variety of lipid A partial structures and analogues were reported to have different properties. Lipopolysaccharide of the Re595 mutant of S almonella minnesota is lack of O and part of the core polysaccharide ( 2 keto-3-deoxyoctanate (KDO) left on lipid A). Re595 lipid A (LA) and Re595 monophosphoryl lipid A (MPLA) differ in structure from Re595 LPS by lacking KDO and KDO plus phosphoryl group respectively. Whether th ese lipid A-common Re595 LPS preparations differed in activities, we i nvestigated their effects on nitric oxide (NO), TNF-alpha, IL-6, and I L-12 induction from murine macrophage cell line RAW 264.7. RAW 264.7 c ells (2 x 10(5) cells ml(-1)) were stimulated with these LPS preparati ons at 1 mu g ml(-1) for 48 h. Re595 LPS, Re595 LA and Re595 MPLA sign ificantly induced NO, TNF-alpha and IL-6 production; NO, TNF-alpha and IL-6 inducing capacities were in the order of LPS = LA > MPLA, LPS = LA = MPLA, and LPS = LA > MPLA respectively. However, these preparatio ns did not induce IL-12 production from RAW cells even when stimulated in combination with IFN-gamma (20 U ml(-1)). IFN-gamma itself also in duced NO, TNF-alpha and IL-6 production from RAW 264.7 cells. When RAW 264.7 cells were stimulated with IFN-gamma plus any of these preparat ions, effects were additive and synergistic for NO and 1L-6 responses respectively. But TNF-alpha responses of RAW cells against these prepa rations were almost equal when cultured alone or in combination with I FN-gamma. Pre-treatment of RAW cells either with LPS, LA or MPLA at lo w concentration (0.1 mu g ml(-1)) for 60 min before pulsing with IFN-g amma (20 IU ml(-1)) plus LPS (1 mu g ml(-1)) for an additional 48 h, s ignificantly (P < 0.01) decreased NO response. Although to a lesser ex tent, TNF-alpha and IL-6 responses were also decreased. Complete inhib ition of NO inducing effect of these LPS preparations was achieved wit h polymyxin B at 40 mu g ml(-1). But the concentration of polymyxin B to get a significant (P < 0.05) inhibitory effect on LPS was four time s higher than that for LA or MPLA. Unexpectedly, polymyxin B also inhi bited INF-gamma-induced NO production from RAW cells in a dose-depende nt fashion. These findings suggested that effect of LPS was dependent, at least in part, on both the LPS polysaccharide chain length and the hydrophilic portion of LPS. In addition, not only LPS but also LA and MPLA exert either enhancing or suppressive effects, depending on thei r concentrations and the timing of their addition with respect to cost imulators. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.