ENDOTOXIN TOLERANCE IS ASSOCIATED WITH DECREASED PROSTAGLANDIN-H SYNTHASE-1 AND SYNTHASE-2

Lipopolysaccharide (LPS) induces macrophage protein and eicosanoid syn thesis. Previous studies have shown that LPS induction of eicosanoid m etabolism is transcription dependent and that prostaglandin (PG) H syn thase is the committed step in the conversion of arachidonic acid (AA) to thromboxane (Tx) B-2. LPS tolerance induced by sublethal in vivo i njections of LPS renders rats resistant to LPS in vivo and macrophages refractory to LPS-stimulated eicosanoid synthesis in vitro. This stud y examined potential activity and content changes in constitutive and mitogen inducible PGH synthase in LPS-stimulated control and tolerant macrophages. TxB(2) levels were measured to evaluate basal PGH synthas e activity and stimulation by Salmonella enteritidis LPS (50 mu g/ml), calcium ionophore A-23187, and AA. All tolerant macrophage groups dem onstrated decreased TxB(2) synthesis. TxB(2) synthesis stimulated by A A in tolerant cells was decreased by 70% (P < 0.05) compared with cont rol macrophages. In subsequent studies changes in PGH synthase content were examined in rat peritoneal macrophages from tolerant and control rats incubated with and without LPS. Immunoblot analysis of PGH synth ase-1 (constitutive) demonstrated no increase in cells stimulated with LPS compared with basal but was diminished by 62 +/- 9% (n = 4, P < 0 .05) in tolerant macrophages compared with control cells. Immunoblot a nalysis of PGH synthase-2 (mitogen inducible) demonstrated induction i n response to LPS that was maximal between 12 and 24 h. PGH synthase-2 was also induced in tolerant macrophages in response to LPS but was l ess than in control cells. The results demonstrate that endotoxin tole rance is associated with reduced activity and content of PGH synthase- 1 and a decreased LPS induction of PGH synthase-2. This downregulation of both isozymes makes tolerance a unique phenomenon to examine the m echanisms for regulation of the expression of PGH synthases-1 and -2.