Lipopolysaccharide impairs macrophage cytoplasmic pH regulation under conditions simulating the inflammatory microenvironment

Abstract

Within the acidic inflammatory milieu, macrophages (m phi s) must maintain their cytoplasmic pH (pHi) within a range conducive to optimal function. It was previously shown that metabolism of L-arginine at concentrations present in vitro in RPMI medium (1.14 mM) impairs the ability of m phi s to regulate pHi. However, concentrations of L-arginine in vivo reportedly range from approximately 100 microM in serum to less than or equal to 50 microM in wounds. To investigate the potential in vivo relevance of this inhibition, m phi pHi regulation was examined following incubation with low concentrations of L-arginine that mimic the inflammatory microenvironment, in the presence or absence of lipopolysaccharide (LPS). pHi regulation was evaluated as the ability of thioglycolate-elicited murine peritoneal m phi s to recover from an imposed cytoplasmic acid load. The m phi pHi was measured using a pH-sensitive fluorescent probe. Following incubation for 2 h in the absence of LPS, the pHi recovery rate was equivalent in cells incubated with and without L-arginine. Coincubation with LPS, however, resulted in marked inhibition of pHi recovery at L-arginine concentrations as low as 12.5 microM. The inhibition was not due to LPS alone, since LPS without L-arginine was not inhibitory. Inhibition of pHi recovery was observed at LPS concentrations ranging from 10 ng/ml to 10 micrograms/ml. The L-arginine-dependent inhibition was apparent within 60 min of exposure to LPS, in both freshly harvested cells and cells preincubated for 2 h in the absence of L-arginine and then exposed to both L-arginine and LPS. Under conditions mimicking the in vivo setting, LPS-stimulated L-arginine metabolism impairs m phi pHi regulation. Modulation of pHi by this mechanism may compromise m phi function within the acidic microenvironment of inflammation.