Extracellular pH Controls Chemotaxis of Neutrophil Granulocytes by Regulating Leukotriene B4 Production and Cdc42 Signaling.

Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed, acidic tissue. The aim of this study was to elucidate the role of the microenvironmental pH in neutrophil chemotaxis. To this end, we combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pH_i) in varied extracellular pH (pH_e). Observational studies were complemented by biochemical analyses of leukotriene B4 production and activation of the Cdc42 Rho GTPase. Our data show that pH_i of neutrophils dose-dependently adapts to a given pH of the extracellular milieu. Neutrophil chemotaxis towards C5a has an optimum at pH_i~7.1 and its pH_i dependency is almost parallel to that of LTB4 production. Consequently, a shallow pH_e gradient, resembling that encountered by neutrophils during extravasation from a blood vessel (pH~7.4) into the interstitium (pH~7.2), favors chemotaxis of stimulated neutrophils. Lowering pH_e below pH6.8, predominantly affects neutrophil chemotaxis, while the velocity is largely maintained. Inhibition of the Na⁺/H⁺-exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil chemotaxis at the optimal pH_i irrespective of the high LTB4 production. Neutrophil migration/chemotaxis is almost completely abrogated by inhibiting LTB4 production or blocking its receptor (BLT1). The abundance of the active GTP-bound form of Cdc42 is strongly reduced by NHE1 inhibition or pH_e6.5. In conclusion, we propose that the pH dependence of neutrophil chemotaxis towards C5a is caused by a pH_i-dependent production of LTB4 and activation of Cdc42. Moreover, it requires the activity of the Na⁺/H⁺ exchanger NHE1.