Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

Abstract

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.

Keywords: cysteine protease; inflammation; neutrophil; protease; protease inhibitor; serine protease.

FIGURE 1.

CatC in PLB-985 and HL-60 cells. A, structures of proCatC (left) and mature CatC (right). Arrows indicate the recognition domains of antiCatC antibodies (Ab1, Ab2). B, Western blot analysis of 30-fold concentrated PLB-985 and HL-60 cell supernatants (50 μg of protein/lane) using anti-CatC antibodies Ab1 (left) and Ab2 (right). The cells were cultured for 24 h or 48 h. C, Western blot analysis of PLB-985 cell lysates (50 and 200 μg of protein/lane) and HL-60 cell lysates (50 and 200 μg of protein/lane) using anti-CatC antibodies Ab1 (left) and Ab2 (right). Similar results were obtained in three independent experiments. Recombinant CatC, proCatC, and neutrophil lysates (50 and 20 μg of protein/lane) were used as controls. PMNs, polymorphonuclear neutrophils.

FIGURE 2.

Intracellular localization and secretion of mature CatC by activated cells. A, sideward (SS) versus forward (FS) scatter plot of undifferentiated (left panel) and differentiated PLB-985 cells (right). B, confocal microscopy of undifferentiated (left) and differentiated (right) PLB-985 cells immunostained with anti-CatC (Ab1) antibodies (green) and anti-golgin-84 antibodies (red) showing the initial localization of CatC in the Golgi of immature cells and its distribution throughout the cell after differentiation. C, CatC activity in cell-free supernatants of undifferentiated and differentiated PLB-985 cells after treatment with the calcium ionophore A23187. FU, fluorescence unit. D, immunoblot analysis of 20- or 80-fold concentrated supernatants of neutrophils (S1 to S7) after their activation with A23187 using the anti-CatC antibody Ab1. Similar results were found in three independent experiments.

FIGURE 3.

Active PR3 in PLB-985. A, immunodetection of purified PR3, PR3 in PLB-985, HL-60, and neutrophil lysates using Bt-[PEG]₆₆-PYDA^P(O-C₆H₄-4-Cl)₂, a selective biotinylated probe of proteolytically active PR3. PMNs, polymorphonuclear neutrophils. B, RP-HPLC fractionation of cleavage products on a C18 cartridge after incubating the PR3 substrate Abz-VADnVADYQ-EDDnp with a PLB-985 cell lysate. Peak 1 is Abz-VADnV, and 2 is ADYQ-EDDnp. Similar results were observed in three independent experiments.

FIGURE 4.

In vitro processing of proCatC by CatS. A, human recombinant proCatC-His₆ (1 μm) was incubated with recombinant CatS (0.2 μm) for different incubation times at 37 °C, and its fragmentation was investigated by Western blotting using anti-CatC antibodies Ab1 (upper panel) and Ab2 (lower panel). B, ProCatC-His₆ (5 μm) was incubated at 37 °C with recombinant CatS (0.2 μm), and its fragmentation was analyzed by Western blotting using an anti-His₆-tag antibody. C, ProCatC-His₆ (1 μm) was incubated at 37 °C with recombinant CatS (0.2 μm) in the presence or absence of the CatC inhibitor I_CatC (2 μm). CatC was analyzed by Western blotting using anti-CatC antibody (Ab1). Similar results were observed in three independent experiments.

FIGURE 5.

Structure of reversible CatS and CatC nitrile inhibitors used in this study. A, compound 1, (S)-2-amino-N-((1R, 2R)-1-cyano-2-(4′-(4-methylpiperazin-1-ylsulfonyl) biphenyl-4-yl)cyclopropyl)butanamide. B, compound 2, N-(1-cyanocyclopropyl)-3-((2,3-difluorobenzyl)sulfonyl)-2-((2,2,2-trifluoro-1-(4-fluorophenyl)ethyl)amino)propanamide. C, compound 3, β-(2-thienyl)-l-alanyl-d-phenylalanine nitrile.

FIGURE 6.

Processing of proCatC in PLB-985 cells cultured in the presence or absence of synthetic inhibitors. A, cell surface CD11b expression was analyzed by flow cytometry. Undifferentiated PLB-985 were cultured for 1 week in the presence of I_CatS (10 μm), I_CatC (2 μm), or after adding the DMSO/DMF containing buffer alone. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentage of CatC, PR3, and CatG activity was determined using the respective selective substrates for each protease and is given in the box. B, PLB-985 cells were differentiated into neutrophil-like in the presence of DMF and with or without I_CatS (10 μm) or I_CatC (2 μm) or DMSO/DMF. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The % of CatC, PR3, and CatG activity measured using their respective selective substrates were shown in the box. Cell surface CD11b expression was analyzed by flow cytometry as in A. C, undifferentiated PLB-985 cells cultured in the presence of E64c (100 μm), E64d (100 μm), or DMSO alone for 48 h were lysed. Cell lysates and supernatants of PLB-985 cells were analyzed by Western blotting using anti-CatC antibody Ab1 (left and right panels, respectively). Similar results were observed in five independent experiments.

FIGURE 7.

Processing of proCatC in HL-60 cells cultured with or without synthetic inhibitors. A, HL-60 cells were cultured for 1 week in medium containing I_CatS (10 μm), I_CatC (2 μm), I_CatS/I_CatC (10 μm/2 μm), DMF, or DMSO/DMF, and then total cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentages of residual CatC, PR3, and CatG activity toward their respective selective substrates were given in the box. B, HL-60 cells were cultured for 1 week in medium containing I_CatS (10 μm), I_CatC (2 μm), and I_CatS/I_CatC inhibitors (10 μm/2 μm), and their lysates were analyzed by Western blotting using anti-CatC antibody (Ab2). C, HL-60 cells were cultured for 48 h in medium containing I_CatS (10 μm), E64c (100 μm), or E64d (100 μm), and then total cell extracts were analyzed by Western blotting using anti-CatC antibody (Ab1). Similar results were observed in five independent experiments. D, diagram summary showing the processing of proCatC in HL-60 cells cultured in the presence of I_CatS or E64d. Arrows indicate the position of proteolytic cleavages in proCatC. CP, cysteine protease. E, partial sequence of the CatC propeptide (residues 1–65) showing the N-terminal proteolysis-sensitive extension (residues 1–24) and the structurally conserved sequence found in CatL-like cysteine proteases (residues 24–65). Model structure was obtained using cysteine peptidase C (PDB code 4HWY; Ref. 37) as a template.

FIGURE 8.

The effect of CatS and CatC inhibitors on the levels and activity of PR3 in HL-60 cells. A, HL-60 cells exposed to I_CatS (10 μm), I_CatC (2 μm), I_CatS/I_CatC (10 μm/2 μm), or DMSO/DMF for 1 week were lysed, and immune-reactive PR3 was analyzed by Western blotting using an antiPR3 antibody (Ab). GAPDH was used as the loading control. B, the same lysates were used to label proteolytically active PR3 using a selective biotinylated PR3 activity based-probe Bt-[PEG]₆₆-PYDA^P(O-C₆H₆-4-Cl)₂. Lysates incubated with or without the biotinylated PR3 activity-based probe was examined by Western blotting after SDS-PAGE using extravidin peroxidase. Similar results were observed in three independent experiments.

FIGURE 9.

CatC in BALF from healthy subjects and in BALF cell lysates, alveolar macrophages, and BEAS-2B bronchial epithelial cells. A, anti-CatC (Ab1) immunoblots of 30-fold concentrated BALF (H1 to H4) from healthy subjects. Shown are immunoblots with anti-CatC antibodies Ab1 and Ab2 of BALF cell lysates (inset, immunostaining of healthy human lung tissue with Anti-CatC (Ab1) showing the CatC in the cytoplasm of alveolar macrophages) (B), cultured alveolar macrophage lysates and supernatants (C), and cultured BEAS-2B cell lysates and supernatants (D). PMNs, polymorphonuclear neutrophils. Similar results were observed in three independent experiments.

FIGURE 10.

CatC in lung secretions from patients with chronic inflammatory lung diseases dominated either by macrophages or by neutrophils. A, immunoblots of 30-fold concentrates of BALF from patients with histiocytosis X (HX), desquamative interstitial pneumonia (DIP), or sarcoidosis (SC) using anti-CatC antibody Ab1. B, immunoblots of 20-fold concentrates of sputum supernatants from patients with cystic fibrosis (CF7 to CF9) and from patients with neutrophilic asthma (A1 and A2). The S4 control was a supernatant of ionophore-activated neutrophils.

FIGURE 11.

CatC in the BALF of rodents and macaques. A, initial rates of hydrolysis of the CatC substrate Gly-Phe-AMC (50 μm) by BALF from mice, rats, and macaques before and after incubation with the CatC diazomethyl ketone inhibitor Gly-Phe-CHN₂ (25 μm). CatC activity was detected in the control BALF of rodents but not in that of macaques (n = 5) before LPS treatment. FU, fluorescence unit. B, immunoblotting of BALF from rodents and macaques showing the active CatC heavy chain in the BALF of rodents and that of LPS-treated macaques. Similar results were observed in three independent experiments.

FIGURE 12.

Summary of the activation of neutrophil serine proteases in neutrophilic precursors showing that almost complete inhibition of CatC is required to prevent the production of active NSPs at inflammatory sites. CP, cysteine protease.