Prostaglandin E(2) (PGE(2)) can have pro- or anti-inflammatory effects, depending on engagement of different PGE(2) receptor (EP) subtypes. The role of EPs in regulating autoimmune inflammation was studied in the murine arthritis/lupus model induced by pristane. Peritoneal macrophages were isolated (biomagnetic beads) from BALB/c, DBA/1, or C57BL/6 mice treated with pristane (intraperitoneally, 3 months earlier) or thioglycolate (3 days earlier) or with untreated controls. EPs, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells were cultured unstimulated or stimulated with lipopolysaccharide (LPS) or LPS + interferon-gamma in combination with EP subtype-specific agonists. Tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 production was tested by enzyme-linked immunosorbent assay (culture supernatant) and flow cytometry. TNF-alpha mRNA levels also were examined. High levels of EPs (EP4/2>EP1>EP3), iNOS, and COX-2 mRNA were expressed in peritoneal macrophages from pristane-treated but not untreated or thioglycolate-treated mice (RT-PCR). TNF-alpha production was inhibited 50-70% at 2-24 h by EP4/2 agonists, whereas IL-6 was enhanced up to approximately 220%. TNF-alpha inhibition is mediated partly via the protein kinase A pathway and partly via IL-6. Intracellular TNF-alpha staining was inhibited 20% by EP4/2 agonists. TNF-alpha mRNA levels were inhibited 50-70% at 2-24 h, indicating that TNF-alpha inhibition was partly at the level of transcription. EP1/3 agonists had little effect. Synovial cells from mice with pristane-induced arthritis (DBA/1) also expressed EP2/4, and the EP2/4 agonist inhibited TNF-alpha production. PGE(2) can modulate inflammatory reactions via the EP2/4 receptor through its regulation of TNF-alpha and IL-6. Modification of EP signaling may be a new therapeutic strategy in inflammatory/autoimmune diseases.