Differential Involvement of Ca2+ and Actin Filament in Leukocyte Shape Change

Abstract

At the sites of inflammation, leukocytes are confronted with mediators which induce different cellular responses like chemotaxis, degranulation and respiratory burst. Morphologically, these responses are accompanied by changes in the cells’ shape. In this study, we investigated the involvement of the actin cytoskeleton and Ca²⁺ in the shape change responses of human eosinophils and neutrophils to chemoattractants and correlated the obtained findings to degranulation and respiratory burst using flow cytometry. Shape change was recorded as an increase in forward scatter. Degranulation was measured as the cell surface upregulation of the granule-associated marker CD63. Respiratory burst was determined fluorimetrically as the oxidation of dihydrorhodamine 123. The involvement of actin filaments and phospholipase C (PLC) was investigated with the actin inhibitor cytochalasin B and the selective PLC inhibitor U-73122, respectively. The data that we obtained demonstrated that granulocytes exhibit 2 distinct types of shape change responses when stimulated with chemoattractants: (i) one type is induced by chemokines like eotaxin and interleukin 8, which are poor degranulators, and also by classical chemoattractants, C5a and formyl-methionyl-leucyl-phenylalanine; this shape change depends on the activation of PLC and functional actin filaments, but does not require Ca²⁺ influx from outside; (ii) the second type of shape change is not stimulated by chemokines, but can be seen with classical chemoattractants which are also potent inducers of degranulation and respiratory burst. This type of shape change does not require any functional actin filaments, but appears to be a consequence of degranulation and depends essentially on the activation of PLC and Ca²⁺ influx from the extracellular space.