Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

Abstract

Orientation of nucleus, centriole, microtubules and microfilaments within human neutrophils in a gradient of chemoattractant (5% Escherichia coli endotoxin-activated serum) was evaluated by EM. Purified neutrophils were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 .mu.m) micropore filters permitted pseudopod penetration but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e., cellular asymmetry with linear alignment of nucleus, centriole, microtubule array and pseudopods. Activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil population. Cytochalasin B (3.0 .mu.g/ml) prevented migration and decreased the microfilaments seen but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicine, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10-8 to 10-7 M. These colchicine effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, formation of narrow pseudopods within a substrate and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicine, but not lumicolchine, minimally decreased activated random migration and markedly inhibited directed migration but had no effect on random migration. Although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are apparently essential for normal pseudopod formation and orientation and maximal unidirectional migration during chemotaxis.