The role of cytoplasmic microtubules in polymorphonuclear leukocyte chemotaxis: Evidence for the release hypothesis by means of time-lapse analysis of PMN movement relative to dot-like attractants

Abstract

The present experiments were designed to elucidate the role of cytoplasmic microtubules in the chemotaxis of human polymorphonuclear leukocytes (PMNs) by means of the Boyden chamber technique and by means of analysis of PMN locomotion around a dot-like attractant. Casein induced positive chemotaxis in a small and variable fraction of the PMNs in the Boyden chamber. The movements of individual PMNs in coverslip preparations of clotted autoplasma were analysed as regards velocity of locomotion, locomotive index and net radial dislocation relative to the cell centre, with or without a yeast-phagocytosing leukocyte as a dot-like attractant. PMNs without obvious attractants tended to leave the visual field, i.e. they had a negative net radial dislocation relative to the centre of the visual field. Their locomotive indices suggested that their disappearance from the visual field was due to random movement. In contrast, the locomotive indices of PMNs influenced by attractants suggested the presence of both positive and negative chemotaxis in the population of moving PMNs. Yeast-phagocytosing leukocytes attracted wandering PMNs isolated by the Isopaque-Ficoll method (IF-PMNs) with a force which approximately balanced the basic tendency of the IF-PMNs to leave the visual field. Selective pretreatment of the moving IF-PMNs with podophyllic acid ethylhydrazide (SPI), 0.5 μg/ml (1.05 × 10 −6 M), did not inhibit their attraction towards the central yeast phagocyte. The attraction of wandering IF-PMNs towards the central yeast phagocyte was inhibited by selective pretreatment of the phagocytes with SPI, 0.5 μg/ml. These observations indicate that cytoplasmic microtubules have an essential role in the release of chemotactic substances from phagocytosing leukocytes but not in the direction-finding of attractant-approaching PMNs. From the present observations by means of SPI, it is suggested that antitubulin inhibition of the release of chemotactic substances from phagocytosing leukocytes is the mechanism of inhibition of PMN chemotaxis by sub-antimitotic antitubulin concentrations in vitro. The latter phenomenon is thought to reflect the cellular basis of the anti-inflammatory action of the antitubulins.