Microtubule-disruption-induced and chemotactic-peptide-induced migration of human neutrophils: implications for differential sets of signalling pathways

Abstract

Neutrophil granulocytes rely on a functional actin network for directed migration. Microtubule disassembly does not impair receptor-linked chemotaxis,instead it induces development of polarity and chemokinesis in neutrophils concomitant with polarized distribution of α-actinin and F-actin. Cells stimulated with colchicine, which disassembles microtubules, migrate with a speed comparable to cells exposed to chemotactic peptide. We investigated signalling pathways involved in colchicine-induced neutrophil polarization and migration. Colchicine-induced development of polarity was insensitive to treatment with pertussis toxin, in contrast to chemotactic-peptide-induced shape changes, which were completely abolished by this treatment. Thus,colchicine does not appear to act via activating heterotrimeric Giproteins. Colchicine does also not seem to act via phosphatidylinositol 3-kinase, as it failed to induce phosphorylation of its downstream target Akt and the potent phosphatidylinositol 3-kinase inhibitor wortmannin failed to inhibit colchicine-induced shape changes. By contrast, wortmannin significantly reduced chemotactic-peptide-induced shape changes. However, the Rho-kinase inhibitor Y-27632 (10 μM) inhibited colchicine-induced development of polarity by 95±3% (n=5) and chemokinesis by 76±9% (n=3), which suggests that the Rho-Rho-kinase pathway has a crucial role in polarity and migration. Indeed, treatment of cells with colchicine induced a significant increase in membrane-bound Rho-kinase II,which is indicative of activation of this protein. This membrane translocation could be prevented by taxol, which stabilizes microtubules. Colchicine also induced a marked increase in myosin light chain phosphorylation, which could be suppressed by Y-27632 and by taxol. In summary, we provide evidence that microtubule disassembly induces in neutrophils a selective activation of Rho-kinase, bypassing activation of heterotrimeric Gi proteins and phosphatidylinositol 3-kinase. This process is sufficient for induction of chemokinesis and mediates increased phosphorylation of myosin light chain and accumulation of F-actin and α-actinin in the leading edge.

Neutrophil microtubules suppress polarity and enhance directional migration

“…In the absence of added chemoattractant, nocodazole induced polarity in Ϸ80% of neutrophils from human peripheral blood (19). Nocodazole induced similar polarity in dHL-60 cells, although in a smaller proportion of cells (21%; Table 1, which is published as supporting information on the PNAS web site).…”

“…Microtubules of a polarized neutrophil radiate toward its leading and trailing edges from an MTOC located just behind, just in front of, or between lobes of the nucleus (15)(16)(17). Microtubule-depolymerizing drugs like colchicine and nocodazole induce neutrophils to polarize and crawl in random directions, in the absence of chemoattractant (18,19); in contrast, the drugs weaken or abolish polarity and migration induced by these drugs in most vertebrate cells. The unusual response to microtubule disruption is accompanied by elevated activities of a small GTPase, Rho, and Rho-dependent phosphorylation of myosin light chains (18,19), which probably account for the induction of polarity and migration, because both are substantially inhibited by exposure to Y-27632, a pharmacologic inhibitor of a Rhodependent kinase, p160-ROCK (19).…”

“…Microtubule-depolymerizing drugs like colchicine and nocodazole induce neutrophils to polarize and crawl in random directions, in the absence of chemoattractant (18,19); in contrast, the drugs weaken or abolish polarity and migration induced by these drugs in most vertebrate cells. The unusual response to microtubule disruption is accompanied by elevated activities of a small GTPase, Rho, and Rho-dependent phosphorylation of myosin light chains (18,19), which probably account for the induction of polarity and migration, because both are substantially inhibited by exposure to Y-27632, a pharmacologic inhibitor of a Rhodependent kinase, p160-ROCK (19). Finally, neutrophils whose microtubules were disrupted by colchicine oriented correctly in a shallow gradient of attractant (9) and reoriented correctly toward an attractant from a micropipette placed one cell length away from the cell (8); in a more stringent test of directionfinding ability, microtubule disruption caused neutrophils to migrate toward spores of Candida albicans in abnormally contorted migration paths with wide turns (20).…”

γ-Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion

“…Proteins that play a role in the regulation of the microtubule were identified in the GABA B R-Akt signaling module and GAB-A B R activation resulted in disassembly of the tubulin network in a PI3K-dependent manner. In neutrophils, disassembly of the microtubule network has been shown to induce development of polarity, a key feature required for chemokinesis and chemotaxis (24). Recent studies have suggested a role for the GABA B R in directed migration of embryonic cortical neuron, and motility of spinal neuroblasts, thus supporting the concept that these receptors can guide directed migration or growth (25).…”

“…Microtubule disassembly induces receptor-linked neutrophil chemotaxis (24). Additionally, GABA receptors have been shown to induce directed migration of embryonic cortical cells (25) hence, we hypothesized that GABA B R activation may induce neutrophil chemotaxis.…”