Figure 8. Protease web affects validation in vivo.

(A) Tris-Tricine 15% SDS-PAGE and MALDI-TOF mass spectrometry analyses of LIX cleavage following incubation with wild-type (WT) or MMP8-deficient (KO) murine polymorphonuclear leukocytes (PMNs) for up to 3 h after PMA stimulation to release PMN proteases to the culture medium. (B) Sequence of the N- and C-terminal regions of LIX with cleavage sites by PMN MMP8 and the unknown protease (?) annotated. (C) Tris-Tricine SDS-PAGE analysis of LIX cleavage by PMNs after addition of protease inhibitors: AEBSF, 2-aminoethyl benzenesulfonyl fluoride hydrochloride; α1-PI, α1-proteinase inhibitor; SLPI, secreted leukocyte proteinase inhibitor. (D) LIX cleavage by murine (m) MMP8 and murine neutrophil elastase (mNE) analyzed by 15% Tris-Tricine SDS-PAGE analysis and MALDI-ToF mass spectrometry. E:S, enzyme-to-substrate ratio; “Marker,” molecular weight markers as indicated. (E) Network effects on LIX cleavage. Proteases are green, inhibitors red, and other substrate proteins are grey. Edges are cleavages (green, with arrow head) or inhibitions (red, with “T” head). (F) MMP8 cleavage of α1-proteinase inhibitor (α1-PI). The serine protease inhibitor α1-PI was incubated with MMP8 for 16 h at 37°C in 50 mM Tris, 200 mM NaCl, 5 mM CaCl₂, pH 7.4 containing 1 mM APMA. The enzyme-to-substrate (E:S) ratio ranged from 1∶5 to 1∶5,000 (w:w). Reactions were visualized on a 10% SDS-PAGE (silver stained). Below, a time course of MMP8 cleavage of α1-PI at 1∶50 (w:w) E:S ratio. (G) Bronchioalveolar lavage of mice stimulated with LPS. WT, wild-type mouse; KO, MMP8 knockout mouse. LPS (2 µg) was instilled in the lungs of female mice, and after 48 h, the mice were sacrificed and the lungs lavaged with PBS. Cell-free bronchioalveolar lavage from three mice was pooled and concentrated by acetone precipitation. α1-PI detection was with Alexa-conjugated antibodies (Molecular Probes) on the LiCOR Odyssey. (H) Numbers of PMNs in the bronchioalveolar lavage after LPS stimulation with (n = 3) and without (n = 3) instillation of GW311616 (GW), a specific neutrophil elastase inhibitor.