Role of Intracellular Chloride in the Reversible Activation of Neutrophil β2 Integrins: A Lesson from TNF Stimulation

The process of β2 integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes’ (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl− content (Cl−i) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl−i in the activation of β2 integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of β2 integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin α subunits, and epitope CBRM1/5, localized to the I domain on the α-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl−i because 1) inhibition of Cl−i decrease is invariably accompanied by inhibition of β2 integrin activation, 2) Cl−i decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl−-free buffers, activates β2 integrins, and 3) restoration of the original Cl−i levels is accompanied by deactivation of β2 integrins. We also show that Cl−i decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pHi does not seem to be relevant for β2 integrin activation. The results of our study emphasize the role of Cl− as a new PMN “second messenger.”