MR-positive macrophages associate with SP-D in the healthy and injured lung, but MR is not involved in SP-D uptake. (A) Flow cytometry strategy for analyzing lung macrophages for the association with SP-D and their expression of MR. Single-cell suspensions were stained for CD45, F4/80, CD11b, SiglecF, and CD11c. CD45⁺F4/80⁺CD11b⁺ cells were separated into SiglecF⁺ cells (alveolar macrophages), SiglecF⁻CD11c⁺ cells (interstitial macrophages), and SiglecF⁻CD11c⁻ cells (monocyte-derived macrophages; Misharin et al., 2013). (B) Quantification of the number of macrophages from each subpopulation associating with SP-D in unchallenged lungs and lungs of bleomycin-treated mice. Experimental conditions were as described in Fig. 4 E. (C) Level of SP-D (Alexa Fluor 647 SP-D MFI) associated with SP-D⁺ subpopulations of macrophages. n = 4. One-way ANOVA was used to test for statistical significance. (D) Fraction of each subpopulation of macrophages positive for MR expression. n = 5. (E and F) Endogenous SP-D was determined in BAL collected from unchallenged (E) and LPS-treated (F) WT mice and MR-deficient littermates by sandwich ELISA. BAL from LPS-challenged mice was collected 48 h after challenge. n = 8 (E) and 7 (F). (G) Internalization of radiolabeled SP-D and collagen type I by alveolar macrophages isolated from lungs of WT mice and MR-deficient littermates. CPM, counts per minute. n = 3. A two-tailed Student’s t test was used for test of significance (E–G).