Pivotal Role for α1-Antichymotrypsin in Skin Repair

Daniel C Hoffmann,Christine Textoris,Felix Oehme,Tobias Klaassen,Andreas Goppelt,Axel Römer,Burkhard Fugmann,Jeffrey M Davidson,Sabine Werner,Thomas Krieg,Sabine A Eming

doi:10.1074/jbc.m111.249979

Abstract

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

Highlights

␣1-Antichymotrypsin (␣1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair
By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of ␣1-ACT and the mouse homologue Spi-2, respectively
By examining human and murine skin wounds, we showed that the physiological repair response is associated with increased ␣1-ACT/Spi-2 gene expression at the wound site

Summary

EXPERIMENTAL PROCEDURES

Animals—C57BLKS/J-mϩ/ϩLeprdb/wt mice (The Jackson Laboratory, Bar Harbor, ME) were mated; mice homogenous for the leptin receptor mutation developed diabetes (db/db), and mice lacking the mutation (WT) were used as control animals. ␣1-ACT staining on mouse wound tissue was performed on paraffin sections using the Dako Envision anti-rabbit HRP kit (DakoCytomation, Hamburg, Germany) with 3-amino-9-ethylcarbazole as substrate. To assess complex formation of r␣1-ACT with its target proteases, r␣1-ACT was diluted to a concentration of 1.5 mg/ml in 25 mM Tris buffer, pH 7.5, and cathepsin G (AppliChem) was diluted to a concentration of 1 mg/ml in 25 mM NaCH2COOH, pH 5.5, containing 400 mM NaCl. The two components were mixed in different ratios as indicated to give a final volume of 50 ␮l and were incubated for 5 min at 37 °C to allow formation of the complex. For the stabilized exudate samples, phosphoric or formic acid was added prior to r␣1-ACT, so that the processing order was exudate ϩ acid ϩ r␣1-ACT 3 incubation 3 internal standard ϩ Tris buffer. Significance was analyzed using unpaired Student’s t test for Gaussian distribution or the Mann-Whitney test

RESULTS

Findings

DISCUSSION