Mx000

Boyden chambers of 3 µm pore size (Corning Incorporated, Corning, NY, USA) was used to assess chemotaxis for neutrophils. MLE-12 cells (mouse lung epithelial cell lines, ATCC CRL-2110; American Type Culture Collection, Manassas, VA, USA) were plated in 24-well plates (4×10⁵ per well) and incubated with the three groups of lung CD4⁺ T cells (blank control group, negative control group and SOCS3 overexpressing group) for 48 h. The culture supernatant was harvested. Then, the treated supernatant was added to the lower chambers, while neutrophils were added to the top chambers for incubation for another 90 min at 37°C in a humidified atmosphere with 5% CO₂. The filters were fixed with ethanol and stained with DAPI to visualize the nuclei. The number of cells that had migrated through the entire thickness of the filter was evaluated, and the results were expressed as the chemotactic index, being the number of cells that migrated towards the sample divided by the number of cells that migrated towards the control medium. Triplicate chambers were used in each experiment and five fields were examined in each filter. CXCL1 and CXCL5 were measured in the culture supernatant using commercially available ELISA kits (catalog nos. MKC00B and MX000; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.