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Lipopolysaccharide impairs macrophage cytoplasmic pH regulation under conditions simulating the inflammatory
C J Swallow1, S Grinstein, O D Rotstein
1Department of Surgery, Toronto General Hospital, Ontario, Canada.
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.
Insights
Lipopolysaccharide (LPS) impairs macrophage pH regulation when L-arginine is present at low concentrations found in vivo. This finding suggests a mechanism that could compromise macrophage function in inflammatory environments.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophages (m phi s) must maintain cytoplasmic pH (pHi) in acidic inflammatory environments.
- L-arginine metabolism at high concentrations impairs m phi pHi regulation.
- In vivo L-arginine concentrations are significantly lower than typical in vitro conditions.
Purpose of the Study:
- To investigate the in vivo relevance of L-arginine's effect on m phi pHi regulation.
- To examine m phi pHi regulation under low L-arginine concentrations mimicking inflammatory conditions.
- To assess the impact of lipopolysaccharide (LPS) on this process.
Main Methods:
- Murine peritoneal macrophages were used.
- pHi regulation was assessed by measuring recovery from an acid load using a pH-sensitive fluorescent probe.
- Cells were incubated with varying concentrations of L-arginine and LPS.
Main Results:
- At low L-arginine concentrations (12.5 microM) mimicking in vivo conditions, LPS markedly inhibited m phi pHi recovery.
- LPS alone did not inhibit pHi recovery; inhibition was L-arginine-dependent.
- Inhibition occurred rapidly (within 60 min) upon exposure to LPS and L-arginine.
Conclusions:
- LPS-stimulated L-arginine metabolism impairs macrophage pHi regulation under conditions mimicking the in vivo inflammatory microenvironment.
- This pH modulation may compromise macrophage function in acidic inflammatory sites.
- The findings highlight a novel mechanism affecting immune cell function in inflammation.