Endotoxin fails to stimulate inositol triphosphate production in macrophages.

Unlabelled: Inositol Triphosphate (IP3) production is an early cell signaling event which leads to mobilization of intracellular calcium (Ca++). We examined whether bacterial endotoxin (lipopolysaccharide, LPS) stimulates IP3 production in macrophages pretreated with LPS (tolerant) or not.

Methods: RAW 264.7 macrophages were cultured at 5 x 10(6) cells in RPMI supplemented with 10% FCS. LPS tolerance was induced by pretreating macrophages (Tol) for 19 h with 10 ng/ml of LPS. Non-tolerant (Non-Tol) macrophages received no LPS pretreatment. Macrophages were next washed, repleted with fresh media, and stimulated with 100 ng/ml LPS. Paired cultures were stimulated with 1 microM platelet activating factor (PAF), a known stimulant of IP3 production. Following 1, 10, and 15-min stimulation intervals, IP3 was extracted with trichloroacetic acid and measured by receptor displacement assay.

Results: LPS did not stimulate IP3 production in either Non-Tol or Tol macrophages. In contrast, PAF stimulated significant increases in IP3 levels within 1 min in both Non-Tol (9.5 +/- 3.0 pmol/ml) and Tol (9.5 +/- 2.4 pmol/ml) macrophages. Non-Tol IP3 levels returned to baseline by 10 min, while Tol IP3 levels remained significantly elevated (8.2 +/- 1.7 pmol/ml).

Conclusions: Unlike PAF, bacterial LPS fails to stimulate IP3 production in macrophages. Furthermore, IP3 production could not be elicited in cultured macrophages repetitively stimulated with LPS.