Il 1β il 1f2

EL4.NOB-1 cells (2 × 10⁵/170 µL) were plated in 96-well plates and incubated for 3 h. The preincubated EL4.NOB-1 cells were stimulated with IL-1β/IL-1F2 (200 pg/10 µL: R&D Systems) and various concentrations (37.25–250 ng/20 µL) of purified rmIL-1Ra, commercially available mIL-1Ra (R&D Systems), or commercially available human IL-1Ra (hIL-1Ra) (R&D Systems) for 24 h. After incubation, the supernatants of the EL4.NOB-1 cells were collected, and the concentration of IL-2 was assessed using the mouse IL-2 ELISA (R&D Systems).

The levels of NO, PGE₂, and cytokines (TNF-α, IL-1β, IL-6) in the culture supernatant of RAW264.7 cells cocultured with KBL346 were measured. The amount of NO produced by macrophages was analyzed using the Griess reaction. The Griess reagent was prepared by mixing Griess reagent A (1% sulfanilamide in 5% phosphoric acid) (sulfanilamide, Sigma-Aldrich Co., St. Louis, MO, USA, 33626-100G) (phosphoric acid, Sigma-Aldrich Co., 49685-100ML) and Griess reagent B (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in DW) (Sigma-Aldrich Co., 33461-25G) at a 1:1 ratio. Absorbance was measured at 540 nm after mixing equal amounts of the sample and Griess reagent And incubating in the dark for 20 min. The concentration of NO was calculated from a standard calibration curve obtained using sodium nitrite (NaNO₂) (Sigma-Aldrich Co., 7632-00-0). The levels of PGE₂ and cytokines in the cocultured cell supernatant were measured using ELISA kits: PGE₂ (Cayman CHEMICAL, Ann Arbor, Michigan, USA, 514010), TNF-α (558534), IL-6 (555240) (BD Biosciences, San Jose, CA, USA), and IL-1β/IL-1F2 (R&D SYSTEMS, Minneapolis, MN, USA, DY401).

Plasma samples from 22 ZZ AATD and 21 non-AATD controls were analyzed for CX3CL1/Fractalkine using Duoset kit (R&D Systems, Minneapolis, MN, assay sensitivity 0.072 ng/ml, detection range 0.2–10 ng/ml). Cell-free culture supernatants were analyzed directly or stored at −80°C. ELISA Duoset kits for TNF-α (assay detection range 15.6–1000 pg/ml), IL-1β/IL-1F2 (assay detection range 3.91–250 pg/ml), and IL-6 (assay detection range 9.38–600 pg/ml) were purchased from R&D Systems (Minneapolis, MN) and were used according to the manufacturer’s instructions. Plates were measured on Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). Measurements were carried out in duplicates.

For the quantification of cytokines, the ileum were weighed and homogenized in PBS containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA), phenylmethylsulfonyl fluoride 0.1 mM (Sigma- Aldrich, St. Louis, MO, USA), benzethonium chloride 0.1 mM (Sigma-Aldrich, St. Louis, MO, USA), EDTA 10 mM (Synth, São Paulo, São Paulo, Brazil), and aprotinin A 20 KIU (Sigma-Aldrich, St. Louis, MO, USA). Afterwards, this material was homogenized, centrifuged at 3,000 g for 10 min and the supernatants collected for cytokine assay. Plates were coated with purified monoclonal antibodies reactive with cytokines IL- 10, IL-12 p70 and IL-1β/IL-1F2 (R&D Systems, Inc, USA), overnight at 4°C. Then, plate wells were washed, supernatants were added, and plates were again incubated overnight at 4°C. On the third day, biotinylated monoclonal antibodies against cytokines (R&D Systems, Inc, USA) were added on the plates and incubated for 2 h, at room temperature. Colour was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml) and 0.04% (v/v) H₂O₂ substrate in sodium citrate buffer. The reaction was stopped by the addition of 20 μl/well of 2N H₂SO₄. The absorbance was measured at 492 nm using a Microplate Reader Model 680 (BIO-RAD).