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Inhibition of neutrophil migration by the serpin antithrombin III

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Background

Beyond the well-known regulatory role of antithrombin III (ATIII) in haemostasis, anti-inflammatory effects have been described such as the induction of prostacyclin release by endothelial cells or the reduction of tissue accumulation of neutrophils (PMN) in animal models of ischemia-reperfusion and sepsis.

Aim

To determine a role of ATIII beyond its ability to reduce leukocyte-mediated events induced by thrombin, we studied direct effects on the in vitro migration and respiratory burst activity of PMN by the ATIII concentrate Kybernin®P, the monoclonal antibody-purified ATIII thereof and its α and β isoforms.

Methods

PMN of healthy donors were isolated by lymphoprep® followed by hypotonic lysis of contaminating erythrocytes. Chemotactic activity was determined in modified Boyden chambers by the micropore-filter leading front assay in a 48-well system. Respiratory burst activity of PMN was measured fluorimetrically.

Results

Pre-incubation in vitro of human PMN with Kybernin® P, immune-adsorbed ATIII or both isoforms significantly inhibited the migration toward fMet-Leu-Phe (fMLP), interleukin-8 (IL-8), and ATIII itself, in a concentration-dependent manner following a bell-shaped curve. Maximum ATIII effects were seen at a concentration of 1 U/ml, which is the level of ATIII found in normal human plasma. Purified ATIII did not deactivate PMN chemotaxis toward GRO-α, whereas fMLP, IL-8, platelet factor-4 (PF4) and GRO-α itself did. Checkerboard analyses indicate that the inhibition of migration was not due to a general impairment of the chemotactic activityof the cells but most probably involved receptor regulatory processes. Deactivation of chemotaxis toward IL-8 but not GRO-α suggests differential interaction at the CXC receptors 1 and 2. At comparable concentrations purified ATIII and Kybernin® P did not affect resting or stimulated respiratory burst activity of PMN.

Conclusion

Our results suggest that ATIII may be a physiologic regulator of the acute inflammatory response by protecting cells from premature activation.

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Keywords

  • Thrombin
  • Prostacyclin
  • Boyden Chamber
  • Neutrophil Migration
  • Chemotactic Activity