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. 2022 Feb 8;7(3):e149870. doi: 10.1172/jci.insight.149870

Figure 4. ADAM8 protease is expressed in human leukocytes and detected in lung fluids of patients with ARDS.

Figure 4

(A) Workflow for protease profiling of PMNs at baseline and during activation with TNF-α (20 ng/mL) using 7 FRET-based peptide substrates (PEPDab 005, 008, 010, 011, 013, 014, and 052). To deduce a profile of specific MMP and ADAM activities, peptide cleavage patterns from 3 independent donors were calculated using a nonlinear kinetic model; proteolytic signatures were compared to a reference matrix of catalytic efficiencies to deconvolute protease identities. (B) Proteolytic profiling infers increased activity of known neutrophil MMPs and significant levels of active ADAM8; *, P < 0.05; ****, P < 0.0001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. (C) Representative images of ADAM8-positive leukocytes (arrowheads, brown color) in lung tissue of patients with ARDS and healthy controls. Scale bar, 50 μm. (See enlarged sections in Supplemental Figure 12.) (D) Representative time-lapse fluorimetry of healthy control BAL (gray) and BAL from patients with ARDS from pneumonia (black; ARDS survivor, red; ARDS nonsurvivor) using the most ADAM8-specific FRET reporter, PEPDab013 (mean ± SD of 3 technical replicates); right, box-and-whisker plots of inferred ADAM8 activity in BAL samples of control patients (gray, n = 4), ARDS survivors (black, n = 5), and ARDS nonsurvivors (red, n = 6). *, P < 0.05; ***, P < 0.001; 1-way ANOVA followed by Tukey’s multiple-comparison test. (E) Representative time-lapse fluorimetry of BAL of patients with mild (cyan) or severe PGD (blue) using PEPDab 013 (mean ± SD of 3 technical replicates); right, box-and-whisker plots of inferred ADAM8 activity in BAL samples of mild PGD (cyan, n = 16) and severe PGD (blue, n = 16). **, P < 0.005, Student’s t test with Welch’s correction. nd, not detectable.