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Mouse macrophages can be stimulated by interferon (IFN)-␥ and bacterial lipopolysaccharide (LPS) to produce nitric oxide (NO) as the result of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate ␥-interferonactivated site (GAS). In transfection studies reported here, it was demonstrated that a luciferase reportergene construct, containing four synthetic copies of the iNOS GAS, was inducible when transfected macrophages were stimulated with either IFN-␥, LPS, or a combination of the two. Consistent with this finding were other transfection analyses, which showed that responsiveness of the intact iNOS promoter to these same agents was significantly reduced when two conserved nucleotide positions within the GAS were mutated. Oligonucleotide probes, which mimicked the iNOS GAS, formed a complex with proteins that appeared in the nuclei of IFN-␥ or IFN-␥ ؉ LPS-treated macrophages within 30 min of stimulation, as shown by electrophoretic mobility shift assay. LPS alone also caused the the appearance of a nuclear protein capable of binding the iNOS GAS-containing oligonucleotide; however, in contrast to binding induced by IFN-␥, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1␣, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1␣ to the iNOS promoter's GAS is required for optimal induction of the iNOS gene by IFN-␥ and LPS.

In this chapter, current concepts about the mechanisms of action of endotoxin are reviewed. Particular attention is focused upon endotoxin-induced production of soluble mediators from macrophages and mononuclear cells and on the potential contribution of these mediators to endotoxin shock. In many cases, the interrelationships between these mediators as primary and/or secondary consequences of endotoxin stimulation of mononuclear phagocytes are discussed. Final comments address the relevance of these mediators to the therapy of endotoxin shock.

SummaryPreculture of thioglycollate-elicited C3HeB/FeJ mouse peritoneal macrophages in vitro with subthreshold stimulatory concentrations of lipopolysaccharide (LPS) can induce hyporesponsiveness (desensitization) to both tumor necrosis factor ot (TNF-cr and nitric oxide (NO) production when these cells are subsequently stimulated with 100 ng/ml of LPS. We have established, however, that the primary dose of LPS required for inducing downreguhtion of NO production is significantly lower than that required for inducing downregulation of TNF-o~ production. Further, when LPS-pretreated macrophages become refractory to subsequent LPS stimulation for NO production, the secondary LPS-stimulated TNF-o~ production is markedly enhanced, and vice versa. These results indicate that LPS-induced TNF-c~ and NO production by macrophages are differentially regulated, and that the observed desensitization process may not reflect a state in which macrophages are totally refractory to subsequent LPS stimulation. Rather, our data suggest that LPS-pretreated macrophages become selectively primed for differential responses to LPS. The LPS-induced selective priming effects are not restricted to LPS stimulation, but extend as well to stimuli such as zymosan, Staphylococcus aureus, and heat-killed Listeria monocytogenes.

The experiments by Sultzer and Nilsson (1), and later by Watson and Riblet (2), established that spleen cells from the C3H/HeJ strain of mouse were refractory to the mitogenic effects of bacterial lipopolysaccharides (LPS). More recently, however, experiments from our laboratory (3) demonstrated that spleen cells from C3H/HeJ mice were in fact responsive to some preparations of LPS but not to others, and that the method of extraction played a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol had potent mitogenic activity. Our data showed that the mitogenic activity of such positive preparations of LPS coisolated with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsC1. In addition, lipid A isolated from positive preparations of LPS was also capable of stimulating C3H/HeJ spleen cells. Taken together, these experiments provided rather convincing data that it was the LPS (in particular the lipid A) itself, or some contaminant very tightly bound to the lipid A, which was responsible for its biological activity.We further demonstrated that treatment of positive preparations of LPS with hot phenol rendered such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains was only moderately decreased. These experiments would suggest either that the phenol treatment chemically alters the lipid A region of the LPS molecule or that such treatment removes the putative tightly bound contaminant responsible for C3H/HeJ mitogenesis.In the experiments reported here, we have explored in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells. For these experiments we have utilized our earlier observations that the antibiotic polymyxin B forms a highly stable molecular complex with the lipid A region of LPS (4), and that such polymyxin B-LPS complexes are unable to mitogenically stimulate B lymphocytes (5). In addition, we have attempted to distinguish between the two potential modes of action of phenol on LPS, namely, the chemical alteration of * This is publication number 906 from the

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