Figure 3. FcgRIIA shedding requires... | Rockefeller University Press

Figure 3.

$Figure 3. FcgRIIA shedding requires serine proteases. (A) Cell surface levels of FcgRIIA (IV.3) was analyzed by flow cytometry upon R848 activation in the presence of a pan protease inhibitor or inhibitors of matrix metalloproteases (GM6001, 10 µM), cysteine proteases (E-64, 1 µM), serine proteases (AEBSF, 100 µM), neutrophil elastase (Elastase inhibitor IV, 25 µM), cathepsin G (chymostatin, 10 µg/ml), or furin (chloromethylketone (CMK, 25 µM). The experiment was repeated three (E-64), four (Pan Prot.inh., P < 0.0001; AEBSF, P = 0.0004; chymostatin; and CMK, P = 0.0038), five (GM6001), and seven (NEi) times; combined results are shown and compared using paired Student’s t test. (B) Neutrophils were incubated with furin (100 ng/ml) or CMK 30 min before addition of R848. BAFF cell surface expression was analyzed by flow cytometry. The experiment was repeated seven times; combined results are shown and compared using paired Student’s t test (R848, P = 0.0095; R848+Furin, P = 0.0027; R848+CMK, P = 0.002). (C) Neutrophils were incubated with furin (100 ng/ml) in presence or absence of R848 and analyzed for FcgRIIA levels by flow cytometry. The experiment was repeated nine times; combined results are shown. (D and E) Supernatant from activated neutrophils was fractionated and analyzed for capacity to induce shedding of monocyte FcgRIIA (D) without or (E) with prior boiling of the fractions. In E, the 30-kD pool was used. In panel D, the experiment was repeated four (30kD pool), six (10kD and 100 kD) or seven (30 kD fractions) times; combined results are shown and compared using paired Student’s t test (>30 kD, P = 0.0003; <30 kD, P = 0.0015; 30 kD pool, P = 0.016 and P = 0.018 as compared with supernatant and <30 kD fraction, respectively; >10 kD, P < 0.0001; <10 kD, P = 0.0002; >100k D, P = 0.0001). In E, the experiment was repeated three (30 kD fraction and pool) or six (boiled supernatant) times; combined results are shown and compared using paired Student’s t test (boiled supernatant, P = 0.0035; Boiled >30 kD, P = 0.017; Boiled <30 kD, P = 0.011). *, P < 0.05; **, P < 0.01; ***, P < 0.001.$

FcgRIIA shedding requires serine proteases. (A) Cell surface levels of FcgRIIA (IV.3) was analyzed by flow cytometry upon R848 activation in the presence of a pan protease inhibitor or inhibitors of matrix metalloproteases (GM6001, 10 µM), cysteine proteases (E-64, 1 µM), serine proteases (AEBSF, 100 µM), neutrophil elastase (Elastase inhibitor IV, 25 µM), cathepsin G (chymostatin, 10 µg/ml), or furin (chloromethylketone (CMK, 25 µM). The experiment was repeated three (E-64), four (Pan Prot.inh., P < 0.0001; AEBSF, P = 0.0004; chymostatin; and CMK, P = 0.0038), five (GM6001), and seven (NEi) times; combined results are shown and compared using paired Student’s t test. (B) Neutrophils were incubated with furin (100 ng/ml) or CMK 30 min before addition of R848. BAFF cell surface expression was analyzed by flow cytometry. The experiment was repeated seven times; combined results are shown and compared using paired Student’s t test (R848, P = 0.0095; R848+Furin, P = 0.0027; R848+CMK, P = 0.002). (C) Neutrophils were incubated with furin (100 ng/ml) in presence or absence of R848 and analyzed for FcgRIIA levels by flow cytometry. The experiment was repeated nine times; combined results are shown. (D and E) Supernatant from activated neutrophils was fractionated and analyzed for capacity to induce shedding of monocyte FcgRIIA (D) without or (E) with prior boiling of the fractions. In E, the 30-kD pool was used. In panel D, the experiment was repeated four (30kD pool), six (10kD and 100 kD) or seven (30 kD fractions) times; combined results are shown and compared using paired Student’s t test (>30 kD, P = 0.0003; <30 kD, P = 0.0015; 30 kD pool, P = 0.016 and P = 0.018 as compared with supernatant and <30 kD fraction, respectively; >10 kD, P < 0.0001; <10 kD, P = 0.0002; >100k D, P = 0.0001). In E, the experiment was repeated three (30 kD fraction and pool) or six (boiled supernatant) times; combined results are shown and compared using paired Student’s t test (boiled supernatant, P = 0.0035; Boiled >30 kD, P = 0.017; Boiled <30 kD, P = 0.011). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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