Permeability was assessed in MLVEC cultures treated with (A) IL-1β at indicated concentrations and (B) after adding IL-1R–neutralizing (2.5 μg/ml) or control antibody for 24 hours. For A, a representative experiment of 3 biological replicates is shown. Data represent mean ± SD of 3 technical replicates. For B, data are presented as mean ± SD of 4 biological replicates. One statistical outlier was excluded in the group receiving IL-1β treatment only. **P < 0.01; ***P < 0.001; ****P < 0.001, 1-way ANOVA with post hoc Holm-Šídák test (A and B). RFU, relative fluorescence unit. (C) MLVECs were treated with IL-1β (0, 0.5, and 1 ng/ml) for 24 hours, fixed, and stained with ZO-2. Nuclei were counterstained with DAPI. Images are representative of 3 independent experiments. (D) MLVECs were treated with IL-1R–neutralizing or control antibody for 30 minutes prior to the addition of IL-1β (1 ng/ml). Cell lysates were analyzed for ZO-2 by Western blotting. The same blot was reprobed for GAPDH. Blots are representative of 3 independent experiments. (E) Frozen tissues of B6 CD45.1⁺ lung grafts, collected 24 hours after transplantation into splenectomized B6 CD45.2⁺ hosts that received B6 WT or B6 IL-1β–deficient monocytes and (F) human donor grafts at the conclusion of cold ischemia and 2 hours after reperfusion were stained with antibodies for ZO-2 (red) and CD31 (green). Nuclei were counterstained with DAPI. Arrowhead indicates colocalization of CD31 and ZO-2. Scale bar: 20 μm. Images were taken with a ×40 objective lens. The relative mean fluorescence intensity (MFI) of ZO-2 staining was quantified and is represented graphically for E and F. For E, data are expressed as median with interquartile range. n = 5 per group. *P < 0.05, Mann-Whitney U test. For F, n = 8 patients per group. *P < 0.05, paired t test.