Chymase

To generate BMMCs for both in vitro experiments and in vivo reconstitution studies, bone marrow was flushed from mouse femurs and cultured in RPMI medium containing 10% FBS, penicillin and streptomycin, HEPES, 1% supernatant from Cho-KL cells containing stem cell factor (produced in-house), and recombinant IL-3 (5 ng/mL, R&D Systems, Minneapolis, MN; #203-IL-010). MCs were verified to be >95% pure by toluidine blue staining (Sigma-Aldrich, St. Louis, MO) prior to use. Peripheral reconstitution of Sash mice was performed by injecting 1 × 10⁷ BMMCs i.v., followed by a 6-week period to allow complete engraftment, as previously described^{42 (link),64 (link)}. CNS reconstitution of Sash mice was performed by injecting 1 × 10⁶ MCs i.c.v., followed by a 16-week period for engraftment in the brain^{43 (link)}. For depletion of CNS-resident MCs in the MCPT5-CRE; iDTR^fl/fl mice, DT (dose 0.5 µg/kg) was injected i.c.v. into the brain at days 0 and 4, with experiments performed on day 6. This dose was found to be optimal because higher doses of DT were observed to be toxic. For administration of chymase, 30 ng of purified chymase (Sigma-Aldrich, St. Louis, MO; #97501-92-3) is injected i.p. at days 1 and 3 post-infection of Sash mice.

Primary lung fibroblasts from healthy individuals or patients with IPF in monoculture or in co-culture with LAD2 in 6-well plates were cultured for 72 h, followed by 24 h of starvation in DMEM containing 0.4% FCIII before addition of fresh DMEM containing 0.4% FCIII with or without chymase (1 ng/mL), tryptase (75 ng/mL) or a combination of chymase (1 ng/mL) and tryptase (75 ng/mL) (both from Sigma-Aldrich, St Louis, MO, USA). The chosen concentrations of chymase and tryptase had previously been evaluated [19 (link)]. All incubations were made at 37 °C with 5% CO₂. Cell supernatants were collected after 72 h and stored at −20 °C.

Proteases used were Chymase (Sigma C8118), Cathepsin D (Sigma SRP6415), Cathepsin G (RP-77525), Cathepsin E (Biovision 7842), Pepsin (Roche 10108057001), Trypsin porcine pancreas (Sigma T0303-1G), Lysozyme from chicken egg white (Sigma L6876), Napsin A (RND 8489-NA). Digestion experiments were carried out with purified human hemoglobin (Sigma H7379) and recombinant or purified proteases. 100 μg hemoglobin (ca. 1.56 nmol) were digested with Chymase (in Tris-HCl 0.05 M, pH 8.0/0.26 M NaCl), Cathepsin D, G and E (in 0.2 M citrate buffer, pH 5.0), Pepsin (in 20 mM sodium acetate buffer, pH 3.5), Trypsin (in 0.1 M Tris–HCl, with 10 mM CaCl2, pH 8), Lysozyme (in 10 mM Tris-HCl, pH 8) or Napsin A (in 0.2 M NaCl, 0.1 M sodium acetate, pH 3.6). All proteases were used at a 1:100 molar ratio (15 pmol) and reactions were incubated at 37°C for 2 h.