Figure 8. Ccl2 mRNA expression by Sftpc^I73T AT2 cells precedes BALF CCL2 and recruitment of Ly6C^hiCD64^– monocytes in lung tissue.

(A) Flow cytometry analysis for EpCAM (CD326) and CD45 expression in AT2 cells isolated from I^ER-SP-C^I73T/I73TFlp^–/– and I^ER-SP-C^I73T/I73TFlp^+/– mice. Dot plot showing EpCAM⁺CD45^– (epithelial), EpCAM^–CD45⁺ (immune), and double-negative populations as a percentage of total cells from each preparation (n = 22 animals). (B) qRT-PCR of AT2 RNA for Ccl2 expression (18S-normalized) for I^ER-SP-C^I73T/I73TFlp^+/– group expressed as fold change (black dots) over I^ER-SP-C^I73T/I73TFlp^–/– controls pooled from both time points (gray dots). *P < 0.05 versus using 1-way ANOVA followed by Tukey’s post hoc test. (C) CCL2 (MCP-1) ELISA of BALF from I^ER-SP-C^I73T/I73TFlp^–/– and I^ER-SP-C^I73T/I73TFlp^+/– cohorts. Dot plots with mean ± SEM are shown. Controls were pooled from I^ER-SP-C^I73T/I73TFlp^–/– at all 3 time points (all less than detectable limit). *P < 0.05 versus control group using 1-way ANOVA followed by Tukey’s post hoc test. (D) Representative flow cytometry plot of lung cell suspensions for CD45⁺Ly6C^hiCD64^– populations in I^ER-SP-C^I73T/I73TFlp^–/– and I^ER-SP-C^I73T/I73TFlp^+/– mice on day 3. Dot plot (3 mice pooled per data point) with mean ± SEM showing relative percentage of Ly6C^hiCD11c^–CD64^–CD11b⁺CD24^–Ly6G^– monocytes. Multiple comparisons were made by 1-way ANOVA followed by Tukey’s post hoc test. For Ly6C^hi populations, *P < 0.05 for I^ER-SP-C^I73T/I73TFlp^+/– versus day 3 I^ER-SP-C^I73T/I73TFlp^–/–; **P < 0.05 for 3 day I^ER-SP-C^I73T/I73TFlp^+/– group versus I^ER-SP-C^I73T/I73TFlp^+/– at 7 or 14 days.