Serine/threonine phosphorylation in cellular signaling for alveolar macrophage phagocytic response to endotoxin

Protein serine/threonine (ser/thr) phosphorylation is an early signaling ev ent in macrophage activation. We investigated the changes in stress-activat ed protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats cha llenged with intratracheal endotoxin. Animals were sacrificed 90 min post i ntratracheal lipopolysaccharide (LPS, 100 mu g/rat) challenge. AMs were inc ubated with or without phosphatase inhibitors at 37 degrees C for 30 min. P hagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs we re determined. LPS challenge activated SAPK/JNK activity and enhanced phago cytosis of AMs without altering phosphatase activity in these cells. Inhibi tion of phosphatase 1 and 2A activity with okadaic acid and calyculin A exe rted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concen tration of 1 mu M increased the mean channel fluorescence intensity (MCF) a nd the percentage of cells engaged in phagocytosis (percent phagocytosis) i n AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 mu M enhanced both the MCF and percent phagocytosis of AMs from LPS-chal lenged rats. Calyculin A at a concentration of 10 nM increased the MCF phag ocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagoc ytosis of AMs in both saline and LPS groups. AM CD18 expression was not alt ered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (caly culin A) of AM CD18 expression. These results suggest that ser/thr phosphor ylation and dephosphorylation participate in mediating the phagocytic respo nse of AMs to LPS.