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G-protein- and phosphodiesterase-dependent regulation of neutrophil migration by antithrombin III involving CXC-receptor 1

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Background

Antithrombin III (ATIII) is a serpin with a newly uncovered regulatory role in neutrophil (PMN) chemotaxis. ATIII and platelet factor-4 (PF4) differentially inhibited migration of PMN toward the CXC-chemokines, interleukin-8 (IL-8) and GRO-α, and the classical attractants, fMet-Leu-Phe (fMLP) and C5a.

Aim

To determine signalling events and involvement of CXC-receptors (CXCR) 1 and 2 in ATIII-induced inhibition of directed PMN migration, we studied the in vitro effects of ATIII in the presence of specific CXCR1 and CXCR2 antibodies (mAb), pertussis toxin or various blockers of signalling enzymes of PMN.

Methods

As in the absence of other attractants, highly purified ATIII has been shown to itself induce PMN migration, effects were determined in modified Boyden chemotaxis chambers by the micropore-filter leading front assay in a 48-well system, after pretreatment of PMN with antibodies, pertussis toxin or enzymes blockers.

Results

Preincubation of PMN with effective concentrations of pertussis toxin, staurosporine or 3-isobutyl-1-methylxantine completely blocked ATIII-induced migration whereas treatment with bisindolylmaleimide (GFX), a selective protein kinase C blocker, wortmannin or tyrphostin-23, had no effect. In assays of IL-8-induced migration, PMN responses to the enzyme blockers were comparable, whereas PF4-induced responses differed as they were also sensitive to GFX. Migration of PMN toward ATIII was significantly reduced by pre-treatment of cells with CXCR1 but not CXCR2 mAb, whereas migration toward IL-8 was antagonised by both CXCR1 and CXCR2 mAb. The mAbs had no effect on fMPL-induced PMN migration. A mAb to IL-1R, which was used as a control, was inactive as well.

Conclusion

Effects of ATIII on PMN migration appear to involve specific signalling pathways including CXCR1, G-proteins and phosphodiesterase and staurosporine-sensitive enzymes other than protein kinase C (i.e. protein kinase A or phospholipase D).

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Keywords

  • Staurosporine
  • Wortmannin
  • Pertussis Toxin
  • Neutrophil Migration
  • Specific Signalling Pathway