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First published online 15 January 2003
doi: 10.1242/jcs.00306

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

Department of Pathology, University of Bern, Murtenstr. 31, 3010 Bern, Switzerland

View larger version (100K): [in a new window]   Fig. 1. Morphology of neutrophils exposed to chemotactic peptide or to colchicine without or with preincubation with taxol or the Rho-kinase inhibitor Y-27632. Neutrophils were preincubated for 30 minutes at 37°C followed by a further incubation either in medium for 30 minutes (control) or in medium containing 1 nM fNLPNTL for 1 or 10 minutes or in 10 µM colchicine for 30 minutes. Alternatively, cells were preincubated for 30 minutes in the presence of 10 µM Y-27632 or with 1 µM taxol followed by addition of colchicine (10 µM) and a further incubation for 30 minutes. Cells were fixed with glutaraldehyde and examined using Nomarski optics. Bar, 10 µm. Asterisks indicate typical examples of spherical cells in the control panel and of non-polar cells with ruffles in the panel showing cells stimulated for one minute with fNLPNTL. Arrows indicate the front with protrusions (pseudopods, ruffles), arrowheads the contracted tail (uropod) of typical examples of polarized cells. Note that the cells polarized by colchicine show less ruffling at the front than those stimulated with fNLPNTL

View larger version (19K): [in a new window]   Fig. 2. Stabilization of microtubules with taxol prevents colchicine-induced development of polarity. For controls, neutrophils were either incubated for 60 minutes in medium (open bars) or preincubated for 30 minutes in medium followed by addition of 1 µM (hatched bars) or 20 µM taxol (closed bars) and a further incubation at 37°C. For cells stimulated with colchicine, cells were incubated with DMSO (open bars) or with 1 µM (hatched bars) or 20 µM taxol (closed bars) for 30 minutes at 37°C, followed by the addition of colchicine (10 µM) and a further incubation for 30 minutes. For cells stimulated with the chemotactic peptide fNLPNTL (CP), cells were preincubated for 30 minutes with DMSO (open bars) or with 1 µM (hatched bars) or 20 µM taxol (closed bars) at 37°C followed by addition of 1 nM fNLPNTL and a further incubation for 30 minutes. Cells were fixed with glutaraldehyde and the percentage of polarized cells assessed using Nomarski optics for 100 cells per sample. Mean±s.e.m. of four experiments.

View larger version (16K): [in a new window]   Fig. 3. Concentration-dependent effect of the Rho-kinase inhibitor Y-27632 on the morphology of colchicine-stimulated neutrophils. Neutrophils were preincubated for 30 minutes without or with increasing amounts of Y-27632, as indicated, at 37°C. The incubation was continued for another 30 minutes either in medium or in 10 µM colchicine. Cells were fixed with glutaraldehyde and examined using Nomarski optics. The percentage of spherical cells (circles), cells with front-tail polarity (triangles) and non-polar cells (squares) was determined for 100 cells per sample. In the absence of colchicine and Y-27632, 96±4% of the cells were spherical, 3±3% polarized and 2±1% non-polar. Mean±s.e.m. of five experiments.

View larger version (15K): [in a new window]   Fig. 4. Pertussis toxin selectively inhibits chemotactic-peptide- but not colchicine-induced development of polarity. Neutrophils were treated without or with 450 ng/ml pertussis toxin (Ptx) as described in Materials and Methods, followed by resuspension in Gey's medium and incubation without (control) or with 1 nM fNLPNTL (CP) or with 10 µM colchicine (colch.) for 30 minutes at 37°C. Cells were fixed with glutaraldehyde and the percentage of spherical (open bars) and polarized cells (closed bars) assessed using Nomarski optics for 100 cells per sample. Mean±s.e.m. of three experiments.

View larger version (28K): [in a new window]   Fig. 5. PI 3-kinase is involved in chemotactic-peptide- but not in colchicine-induced development of polarity. (A) Effect of wortmannin on chemotactic-peptide- and colchicine-induced polarity. Neutrophils were preincubated for 10 minutes with DMSO or with 25, 50 or 100 nM wortmannin (WT) at 37°C. The incubation was continued for another 30 minutes in medium (control) or with 10 µM colchicine (colch.) or with 1 nM fNLPNTL (CP). Cells were fixed with glutaraldehyde and the percentage of polarized cells assessed using Nomarski optics for 100 cells per sample. Mean±s.e.m. of five experiments. (B) Akt phosphorylation in response to chemotactic peptide and colchicine. Stimulation with chemotactic peptide: neutrophils were preincubated for 10 minutes with DMSO or with 25, 50 or 100 nM wortmannin at 37°C. The incubation was continued for another 5 minutes in the absence or presence of 1 nM fNLPNTL. Stimulation with colchicine: neutrophils were either incubated for 40 minutes at 37°C in medium or were exposed, after preincubation in medium for 10-35 minutes, for the times indicated to 10 µM colchicine (total incubation time: 40 minutes). Cells were precipitated with TCA and subjected to immunoblotting in order to visualize phosphorylated Akt (phospho-Akt). The blot was then stripped and reprobed with an antibody that reacts with Akt independently of its activation state. Immunoblots representative of three experiments are shown.

View larger version (17K): [in a new window]   Fig. 6. Treatment with colchicine does not induce phosphorylation of p42/44 MAPK in neutrophils. Neutrophils were either incubated for 40 minutes at 37°C in medium or were exposed, after preincubation in medium for 10-35 minutes, for the times indicated to 1 nM fNLPNTL or to 10 µM colchicine (total incubation time: 40 minutes). Cells were precipitated with TCA and subjected to immunoblotting in order to visualize phosphorylated p42/44 MAPK. An immunoblot representative of four experiments is shown. Note, for the control and the samples incubated with colchicine, 1.8x105 cells were applied per lane; for the sample incubated with fNLPNTL, 0.2x106 cells were applied per lane.

View larger version (17K): [in a new window]   Fig. 7. Translocation of Rok from cytosol to the membrane in neutrophils treated with colchicine or chemotactic peptide. (A,B) Neutrophils were preincubated for 15 minutes at 37°C without or with 10 µM taxol. The incubation was continued for another 5 minutes in medium (control) or with 10 µM colchicine (colch.) or with 10 µM lumicolchicine (lumicolch.). For the incubation with chemotactic peptides, cells were preincubated for 5 minutes at 37°C, followed by addition of 1 nM fNLPNTL (CP) and a further incubation for 15 minutes. Membrane and cytosolic fractions were subsequently prepared and analyzed by immunoblotting for the presence of Rok (arrow). (A) Immunoblot of a typical experiment; (B) Quantitative evaluation of A (mean±s.e.m. of three experiments; cells with colchicine: n=7). (C,D) Neutrophils were preincubated for 15 minutes at 37°C, without or with 10 µM taxol. The incubation was continued for another 15 minutes either in medium (control) or with 1 nM fNLPNTL (CP) and a further incubation for 15 minutes. The amount of Rok present in the membrane fraction was then analyzed by immunoblotting. (C) Immunoblot of a typical experiment (duplicates of each assay are shown); (D) quantitative evaluation of C (mean±s.e.m. of six experiments).

View larger version (27K): [in a new window]   Fig. 8. Colchicine treatment increases myosin light chain phosphorylation in neutrophils in a Y-27632-sensitive manner. Neutrophils were either incubated for 50 minutes at 37°C in medium or in 10 µM Y-27632 or were preincubated in the absence or presence of 10 µM Y-27632 for 30 minutes followed by a further incubation with 1 nM fNLPNTL for 5 minutes or with 10 µM colchicine for 20 minutes as indicated. Cells were precipitated with TCA and subjected to immunoblotting. (A) Immunoblot of a representative experiment (for cells exposed to colchicine, duplicates are shown). (B) Quantitative evaluation of A (mean±s.e.m. of five to six experiments; control with Y-27632: n=2).

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