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. 2021 Mar 22;6(6):e147282. doi: 10.1172/jci.insight.147282

Figure 4. IL-1β is necessary for donor alveolar macrophages (AM) to produce CCL2.

Figure 4

(A) AM were isolated from WT or Il1r–/– mice and incubated in vitro with PBS or 1 ng/μL of mouse recombinant IL-1β. Ccl2 mRNA expression was measured by qPCR 24 hours after incubation (n = 6–8). (B) AM isolated from WT mice were incubated for 30 minutes in vitro with DMSO (control), 50 μM LY294002 (LY), 10 μM Bisindolylmaleimide-I (Bis), or 1 μM BAY11-7082 before adding 1 ng/μL of mouse recombinant IL-1β. Ccl2 mRNA expression was measured by qPCR 24 hours after incubation (n = 3–7). (C) Donor AM were isolated from WT or Il1r–/– allografts 24 hours after transplant, and Ccl2 mRNA expression was measured by qPCR (n = 4–5). (D) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) from allografts harvested at the indicated times from experiments in which donor lungs were treated with PBS or anakinra, or in which recipients were treated with isotype control and anti–IL-1β antibody (n = 3 per group). Graphs show means ± SD. Graphs in A–C were analyzed by unpaired Student’s t test. Graph in D was analyzed by 2-way ANOVA, followed by Sidak’s post hoc test. *PBS versus anakinra; #isotype versus anti–IL-1β antibody. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ###P < 0.001; ####P < 0.0001.