Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Abstract

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.