Effect of pH on MMP-9 release by LPS-stimulated neutrophils

Introduction Acidosis and alkalosis are complications that may be causes of poor prognosis in critically ill patients. In particular, the mortality rate of patients that go under alkalosis conditions is greater than that in acidosis. Certain neutrophil responses can be affected by changes in the external pH: extracellular acidic conditions enhance neutrophil proinflammatory responses triggered by conventional agonists (1). Neutrophils have different kind of granules: primary granules are Myeloperoxidase (MPO) positive and MMP-9 negative; secondary granules contain lactoferrin, a little amount of MMP-9 and other molecules; tertiary granules contain the larger amount of MMP-9. The aim of this study is to verify if MMP-9 released by stimulated neutrophils may be higher in extracellular alkalosis respect acidosis conditions. This may have implications in severity of alkalosis and acidosis. Materials and Methods Neutrophils were isolated from buffycoats of healthy subjects by dextran sedimentation and Ficoll-Hypaque gradient centrifugation. Contaminating RBC were removed by hypotonic lysis. Cell pellets were resuspended in NaCl 0.9% and than counted. 2x106 cells were placed in bicarbonate-buffered RPMI 1640 medium at various pH (7.0 to 7.8) with 1% heat-inactivated FBS and LPS 10 mg/ml and placed at 37 °C for 30 minutes; conditioned mediums were collected and stored at –80 °C until MMP-9 assay. Total MMP-9 was measured using commercially Activity Assay System. Results Extracellular acidosis and alkalosis enhance neutrophil proinflammatory response. We have studied the release of MPO and MMP-9 “in vitro” in the pH range from 7.0 to 7.8. After LPS-stimulation, MPO released doesn’t show significative difference in the pH range studied. Instead, the amount of MMP-9 released increases both under acidic and basic conditions (P<0.001) compared to physiological pH (7.4). Furthermore, the amount of MMP-9 released at basic pH is greater than that released at acidic pH (P<0.001).We have also analysed the pH dependence of MMP-9 proteolytic activity. We have observed that in the pH range from 7.0 to 7.8 MMP-9 shows an higher activity when the pH is over 7.4 (physiological pH). Conclusions Neutrophil response to a proinfammatory stimulus is differentially affected by the environmental pH. While the release of primary granules seems to be insensitive to the analysed pH, the release of tertiary granules is sensitive to small pH changes and in particular when the pH shifts to slightly basic conditions. The increased release of MMP-9 at higher pH together with its proteolytic activity dependence from the pH suggest that MMP-9 could be one of the adverse factors in the prognosis of alkalosis and acidosis conditions. (1) Trevani et al., Extracellular acidification induces human neutrophil activation. Journal of Immunology 62: 4849-4857; 1999