Fig. 4. DEL-1 enhances efferocytic activity through interaction with phosphatidylserine and α_vβ₃ integrin.

(a, b) Saturation binding of (a) full-length DEL-1, or (b) DEL-1 protein that lacks the discoidin I-like domains expressed as an Fc fusion protein (DEL-1[E1-E3]-Fc) to liposomes composed of PC/Chol. (PC) or PC/Chol./PS (PS) (n = 3 preparations in (a) and n = 2 preparations in (b)). (c, d) Primary BMDMs from wild-type mice (n = 3 separate cell isolations) (c), or human monocyte-derived macrophages (MDMs) (n = 5 separate cell isolations) (d) were co-cultured for 30 min with apoptotic neutrophils (mouse or human, respectively) that were pre-stained with BCECF-AM ((2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) in the presence of Fc control, full-length DEL-1-Fc or DEL-1 protein lacking the discoidin I-like domains (DEL-1[E1-E3]-Fc) (each 500 ng/ml). Relative efferocytosis index is shown and was calculated as the ratio of macrophages that have phagocytosed apoptotic material to the total number of macrophages. Data are expressed relative to the Fc control group set as 1. (e) Wild-type mice were injected i.p. with thioglycollate and 72 h thereafter they received a second i.p. injection of 5 × 10⁶ pre-stained apoptotic neutrophils together with 5 μg of either Fc control or DEL-1-Fc; 3 h after the latter injection, mice were sacrificed and the number of phagocytic macrophages was analyzed (n = 11 mice for Fc control and 10 mice for Del-1-Fc). (f) In vitro phagocytosis assay was performed as described in (c). Relative efferocytosis index of BMDMs from mice deficient in β3 integrin (Itgb3^KO) and of BMDMs from respective control mice (WT) in the presence of Fc control or DEL-1-Fc is shown (n = 3 separate cell isolations per genotype). *P < 0.05, **P < 0.01, ***P < 0.001, NS, non-significant (One-way ANOVA with Dunnett’s (c, d) or Tukey’s (f) multiple comparisons test; two-tailed Student’s t-test (e)). Data are means ± SEM and are derived from three experiments (a), from two experiments (b, e), from one experiment (c, f), or are pooled from five experiments (d).