MMP-12 cleaves mCXCL5 and impairs PMN recruitment to LPS in Mmp12^−/− mice. (A) E coli was used to express 2 batches of active human MMP-12 (hMMP-12; lanes 1 and 2) and one batch of murine MMP-12 (mMMP-12; lane 3). Protease integrity and purity was verified on silver-stained 10% SDS-PAGE gels and Western blotted using antibodies directed to the human and murine MMP-12 catalytic domain as appropriate. Arrow indicates active MMP-12. Molecular mass markers (× 10⁻³ Da) are shown. (B) Analysis of MMP-12 proteolysis of murine CXCL5 incubated with or without the MMP inhibitor 1 μmol/L prinomastat (MMPI) as a control on a silver-stained 15% Tris-Tricine SDS-PAGE gel with arrow indicating cleaved mCXCL5. (C) Identification of human and murine MMP-12 cleavage products by MALDI-TOF MS. Deconvolution of mass spectrometry data revealed that MMP-12 cleaved mCXCL5 (LIX) in the same position as MMP-8.¹⁶  Arrow indicates the cleavage site of human and murine MMP-12. (D) PMN responsiveness to LPS (n = 8) or PBS vehicle control (n = 6) injected into dorsal air pouches of Mmp12^−/− or wild-type mice was measured 8 hours after injection by myeloperoxidase activity in the lavage fluids. Standard error bars are shown. P values calculated with Mann-Whitney nonparametric test.