Fig. 4

MARCO⁺ AMs produced CCL6 and were regulated by UGRP1 in aged lungs. The lung tissues of aged mice (20–24 months old) were compared with the young mice (10–16 weeks old). A Production of CCL6 in MARCO⁺ AMs and MARCO⁻ AMs. Lung MNCs were prepared and then analyzed by flow cytometry analysis. MARCO⁺AMs (CD45⁺ F4/80⁺ CD11c⁺) or MARCO⁻ AMs (CD45⁺ F4/80⁺ CD11c⁺) were gated to show the expression of CCL6. Histograms are shown respectively. B Frequency and absolute number of MARCO⁺ CCL6⁺ AMs in the aged lungs compared with the young lungs. C The levels of CCL6 in the serum, lung tissues (n = 6 for each group) and BALF (1.0 mL/mouse, n = 5 for each group) were detected by ELISA. Clodronate liposomes (50 μL/mouse) were administrated i.n. twice every 72 h to deplete AMs in the lungs. D Co-staining of MARCO and CCL6 in the lung tissues detected by immunofluorescence. The arrows indicate the positive cells in the lung tissues. Scale bar, 25 μm. MARCO⁺ CCL6⁺ cell numbers were counted and analyzed. Each symbol represents the average of 10 fields of vision (63 ×) from an individual sample. There were 6 mice in each group. Data are shown as the mean ± SEM. Comparisons by unpaired two-tailed Student’s t-test. **** p < 0.0001. (E) Purified AMs (CD45⁺ F4/80⁺ CD11c⁺) (1 × 10⁵ cells/well) were stimulated with UGRP1 (300 ng/mL) for 48 h in the DMEM containing 10% FBS, and then the levels of CCL6 in the culture supernatants were detected by ELISA. Anti-MARCO (20 μg/mL) was used to block the ligand-receptor interaction. There were 6 samples in each group. Data are shown as the mean ± SEM. Comparisons by two-way analysis of variance (ANOVA) followed by Tukey’s test. ns, not significant (p > 0.05), **** p < 0.0001