Carbon dioxide suppresses macrophage superoxide anion production independent of extracellular pH and mitochondrial activity

Background

Superoxide anions released by activated macrophages during surgery are considered to be responsible for local cellular damage. Application of CO₂ pneumoperitoneum during laparoscopy affects superoxide anion release, but the underlying mechanism remains unclear and the data reported are conflicting. We investigated the direct and pH-mediated impact of CO₂ and air on macrophage superoxide anion production.

Methods

Cells of the NR 8383 rat macrophage cell line were incubated for 2 hours in 5% CO₂, 100% CO₂, and room air or pH 7.4, pH 6.5, and pH 5.5. The extracellular pH was monitored during incubation. At 0, 2, and 6 hours after incubation, the release of superoxide anions was determined fluorometrically. The mitochondrial activity was determined via the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] during and after incubation.

Results

Extracellular pH decreased to 6.4 during incubation in a CO₂ atmosphere. The release of superoxide anions was significantly reduced immediately after CO₂ incubation. It was restored at all other time-points. Decreasing the extracellular pH to 6.5 had no effect on superoxide anion release, whereas acidification of the extracellular milieu to pH 5.5 significantly suppressed subsequent superoxide release. Mitochondrial activity was significantly decreased by CO₂ up to 2 hours and by acidic milieu up to 6 hours. Incubation in room air had no effect.

Conclusions

Incubation in CO₂ can directly suppress macrophage superoxide anion production. This effect is of short duration, fully reversible, and not correlated to changes in extracellular pH or mitochondrial activity. Air contamination does not affect macrophage superoxide anion release. We speculate that CO₂ pneumoperitoneum could attenuate the intraoperative free radical production by directly inhibiting superoxide anion release of macrophages without long-lasting suppression of macrophages and their capacity to release superoxide anions postoperatively.