PGE2 has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE2 production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE2 than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE2 produced, macrophage-derived IL-12 and T cell-derived IFN-γ production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A2 mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE2 by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE2 synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE2 by macrophages, which is regulated by secretory phospholipase A2 and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.

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PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-γ production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-γ production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-γ production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-γ production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-γ and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.

We demonstrated that interleukin 1 (IL-1), a potent peptide mediator in immune and inflammatory responses, stimulates the synthesis of cAMP in a variety of IL-i-responsive cell targets. We also showed that cAMP analogs and cAMP-inducing agents can replace IL-1 in the induction of interleukin 2 receptors on lymphocytes as well as in phytohemagglutinin-induced murine thymocyte proliferation. By use of IL-1 and the cAMP-inducer, forskolin, a direct correlation between the induced level of cAMP and the degree of lymphocyte interleukin 2 receptor expression or thymocyte proliferation was established. Our results indicate that cAMP may be an important intracellular second messenger for IL-1.Interleukin 1 (IL-1), a peptide hormone produced by macrophages as well as several other cell types, stimulates the growth and differentiation of numerous cell types including lymphocytes, neutrophils, fibroblasts, synovial cells, osteoclasts, hepatocytes, and adipocytes (1-4) as well as being a potent fever inducer (5). In addition, IL-1 has been suggested to play an important role in the pathophysiology of a variety of chronic inflammatory diseases (6, 7).Within the last few years, tremendous progress has been made in our understanding of the biological and biochemical properties of IL-1. Two forms of IL-1, termed a and ,B, were cloned, sequenced, and expressed (8-12), and the receptor for IL-1 was characterized (13-15). However, the events that occur after the interaction of IL-1 with its receptor have not been elucidated. Several studies have shown that IL-1 can induce the expression of interleukin 2 receptors (IL-2R) on lymphocytes (16,17). Recently, we reported that YT cells, a human natural killer-like cell line, possess IL-1 receptors and also increase their expression of IL-2R in response to (18).cAMP Assay. Cells were incubated in the presence or absence of IL-1 and drugs at 37°C for varying periods. The incubations were stopped by the addition of ice-cold C13CCOOH (final concentration, 6%). The cells were then sonicated in the C13CCOOH, and the particulate material was removed by centrifugation. The Cl3CCOOH-soluble fraction was extracted once with ether before measurement of cAMP content using an RIA kit (Amersham/Searle). Cell membranes were prepared from the various cell types by hypotonic lysis in 10 mM Tris-HCl, pH 7.5, containing aprotinin (10 ,g/ml), pepstatin (1 ,ug/ml), leupeptin (1 ,ug/ml), and Na-p-tosyl-L-lysine chloromethyl ketone (50 ,ug/ml) at 4°C (30 min on ice followed by Dounce homogenization). The homogenate was centrifuged at 500 x g for 5 min at 4°C. The supernatant was recovered and recentrifuged. The nucleifree supernatant was then layered over 0.25 M sucrose and centrifuged at 15,000 x g for 30 min at 4°C. The membranecontaining pellet was resuspended in 20 mM Hepes, pH 7.2, and washed several times before use. The washed membranes were suspended in a buffer containing 20 mM Hepes, pH 7.2/50 ,uM ATP/0.3% bovine serum albumin/10 mM MgCl2/pyruvate kinase (5 ,g/ml)/4.5 mM phosphoenolpyruvat...