Role of intracellular chloride in the reversible activation of neutrophil beta(2) integrins: A lesson from TNF stimulation

The process of beta(2) integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-d ependent 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 o f PMN, stimulated this study, which investigates the role of alterations of Cl-i(-) in the activation of beta(2) integrins triggered by TNF. Here we s how that TNF enhances the expression of activation-specific neoepitopes of beta(2) integrins, namely, epitope 24, a unique epitope present on all thre e leukocyte integrin alpha subunits, and epitope CBRM1/5, localized to the I domain on the alpha-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitope s are dependent on a drop in Cl-i(-) because 1) inhibition of Cl-i(-) decre ase is invariably accompanied by inhibition of beta(2) integrin activation, 2) Cl-i(-) decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl--free buffers, activates beta(2) integrins, and 3) re storation of the original Cl-i(-) levels is accompanied by deactivation of beta(2) integrins, We also show that Cl-i(-) decrease is required for TNF-i nduced cytoplasmic alkalinization, but such a rise in pH(i) does not seem t o be relevant for beta(2) integrin activation. The results of our study emp hasize the role of Cl- as a new PMN "second messenger.".