CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition

Abstract

The CD300 receptor family members are a group of molecules that modulate a variety of immune cell processes. We show that mouse CD300b (CLM7/LMIR5), expressed on myeloid cells, recognizes outer membrane-exposed phosphatidylserine (PS) and does not, as previously reported, directly recognize TIM1 or TIM4. CD300b accumulates in phagocytic cups along with F-actin at apoptotic cell contacts, thereby facilitating their engulfment. The CD300b-mediated activation signal is conveyed through CD300b association with the adaptor molecule DAP12, and requires a functional DAP12 ITAM motif. Binding of apoptotic cells promotes the activation of the PI3K-Akt kinase pathway in macrophages, while silencing of CD300b expression diminishes PI3K-Akt kinase activation and impairs efferocytosis. Collectively, our data show that CD300b recognizes PS as a ligand, and regulates the phagocytosis of apoptotic cells via the DAP12 signaling pathway.

Figure 1

CD300b binds phosphatidylserine-containing liposomes. (a and b) PS liposomes (DOPC/POPS at 4:1 ratio), PC liposomes (DOPC/POPC at 4:1 ratio) or PE liposomes (DOPC/POPE at 4:1 ratio) were immobilized on a L1 biosensor. Liposome binding was monitored in resonance units (RU) using 10 μg of CD300b-Fc (a) or NITR-Fc chimera proteins (b). Binding was initiated at 60 s and the washout (dissociation phase) begun at 240 s. Data are a representative of three experiments

Figure 2

CD300b recognition of apoptotic thymocytes is dependent on phosphatidylserine. Murine thymocytes were cultured with 10 μM dexamethazone for 6 h to induce apoptosis. (a) Cells were incubated with 0.1 μg CD300b-Fc or NITR-Fc fusion proteins and stained with Annexin V-APC and anti-human IgG Fcγ-FITC antibody. Binding of Fc-chimera proteins to apoptotic thymocytes was analyzed by flow cytometry. (b) Apoptotic thymocytes (0.25 × 10⁶ cells), pretreated with various concentrations of unlabeled Annexin V, were incubated with 0.1 μg CD300b- or NITR-Fc proteins and stained with anti-human IgG Fcγ-FITC antibody. (c) PC-, PE- or PS-coated liposomes (10 μM) were preincubated with 0.1 μg CD300b- or NITR-Fc proteins and then mixed with apoptotic thymocytes. Binding of Fc-chimera proteins to the cell surface was detected using an anti-human IgG Fcγ-FITC antibody. (d) Apoptotic thymocytes were incubated with 0.1 μg CD300b- or NITR-Fc proteins in the presence or absence of 5 mM EDTA or EGTA and stained with anti-human IgG Fcγ-FITC antibody. (b–d) The histograms on the left show examples of binding of Fc-fusion proteins to the apoptotic cells; the graphs on the right shows the mean fluorescence intensity (MFI) values of the bound Fc-fused proteins, from three independent experiments and represent mean±S.E.M.; *P<0.05, ***P<0.001 (Student's t-test)

Figure 3

Indirect binding of TIM1 to CD300b is mediated by phosphatidylserine. (a) EV- or TIM1-transduced Ba/F3 cells were stained with Annexin V-APC, 7-AAD and anti-TIM1-PE antibody. The contour plots show TIM1-PE and Annexin V-APC staining of the 7-AAD^- cell populations. (b) EV- or TIM1-transduced Ba/F3 cells were cocultured with CD300b- or NITR-Fc proteins and stained with 7-AAD, anti-TIM1-PE and anti-human IgG Fcγ-FITC antibodies. Graphs show binding of the Fc-fused proteins and TIM1 to the 7-AAD- cell populations. (c). EV- or TIM1-transduced Ba/F3 cells were preincubated with unlabeled Annexin V (10 μg) and then cells were stained with Annexin V-APC (left panel) or the indicated Fc-fused proteins (right panel), followed by anti-human IgG Fcγ-specific antibody. (d) EV- or TIM1-transduced Ba/F3 cells were incubated with biotin-labeled PC- or PS-coated liposomes, stained with streptavidin-APC and liposome binding was detected by flow cytometry. (e) EV- or TIM1-transduced Ba/F3 cells were cultured with PC- or PS-coated liposomes and incubated with CD300b- or NITR-Fc proteins. Cells were stained with anti-human IgG Fcγ-FITC antibody and analyzed by flow cytometry. The bar graph shows the MFI values+S.E.M. of CD300b-Fc bound to the cell surface; the data are from three independent experiments. (f) Recombinant murine TIM1 protein was immobilized on CM5 biosensor, and binding of CD300b-Fc or NITR-Fc proteins was analyzed by SPR, as in Figure 1. Binding was initiated at time=60 s and the washout (dissociation phase) was begun at t=240 s. (g) Binding of CD300b-Fc or NITR-Fc proteins to TIM1 using a two-step reaction. TIM1 was immobilized on the CM5 sensor chip, and then PC- or PS-coated liposomes were flown over the TIM1 coated chip (first step; 1st). Next, CD300b-Fc or NITR-Fc proteins were injected (second step, 2nd), and the binding to TIM1 was analyzed by SPR. The top panel illustrates a representative result of the SPR analysis, whereas the bottom graph shows the changes in resonance units (RU) following the addition of CD300b-Fc or NITR-Fc (the second step of the reaction). All results are a representative of three independent experiments with a mean+S.E.M.; **P<0.01. ***P<0.001 (Student's t-test)

Figure 4

CD300b-mediated engulfment of phosphatidylserine liposome-coated beads requires the co-expression of DAP12. (a) L929 cells transduced with the indicated constructs were incubated with PC- or PS-coated liposome beads for 20 min at 37 °C. The cells were fixed, and imaged by microscopy. The DIC images show cells and liposome-coated beads; the scale bar is 20 μm. The inserts show a close-up of a single cell. (b) PKH67-labeled L929 cells transduced with the indicated constructs were incubated with PC- or PS-coated liposome beads for 30 min. After homogenization of the cells, the beads were harvested, and their phagocytosis was determined by analysis of the percentage of PKH67⁺-beads using flow cytometry. The beads that became fluorescent were regarded as the engulfed beads, as they acquired the fluorescence due to their encapsulation within PKH67⁺-cell membranes (phagosomes). The contour plot indicates the gating strategy, and illustrates a representative result (upper panel). The bar graph (lower panel) shows the quantification of the percentage of engulfed liposome-coated beads. Data represent mean+S.E.M.; ***P<0.001 (ANOVA)

Figure 5

CD300b promotes efferocytosis in a DAP12-dependent manner. (a) EV-, DAP12-, CD300b- and CD300b-DAP12-expressing L929 cells were labeled with CFSE, mixed with pHrodo-labeled apoptotic cells (AC) and the percentage of engulfment of (pHrodo⁺) cells was analyzed by confocal microscopy (a) and flow cytometry (b). (a) CFSE-labeled L929 cells are shown green, while engulfed pHrodo⁺-labeled apoptotic cells are displayed in red (lower panel); the DIC images are shown in the upper panel. Scale bars, 20 μm. (b) The contour plots (upper panel) illustrate the gating strategy whereas the quantification of the percentage of pHrodo⁺ cells is summarized in the bar graph (bottom panel; error bars represent S.E.M.). (c) L929 cells expressing Myc-tagged CD300b were cultured in glass-bottom dishes, and incubated with TFL4-labeled apoptotic cells (AC, blue). The cells were then fixed, washed and stained using anti-cMyc antibody, followed by Alexa647-conjugated secondary antibody (green). F-actin was stained using phalloidin-Alexa568 (red). Scale bars, 5 μm. (d) EV-, DAP12- and CD300b-DAP12-expressing L929 cells were cultured in the presence or absence of PC-, PE- or PS-coated liposomes (10 μM) and then mixed at a 1:4 ratio with pHrodo-labeled apoptotic cells (AC) for 60 min. Percentage of cells containing apoptotic thymocytes (pHrodo⁺-cells) was analyzed by flow cytometry, as described in b. (e) pHrodo-labeled apoptotic cells (AC) were cultured in the presence or absence of 50 μg/ml Annexin V, CD300f-Fc, CD300b-Fc or NITR-Fc proteins and then incubated with EV-, DAP12- and CD300b-DAP12-expressing L929 cells at a 4:1 ratio for 60 min. Percentage of pHrodo⁺-cells was analyzed by flow cytometry, as in b. (f) EV-, DAP12-, DAP12m-, CD300b-DAP12-, CD300b-DAP12m-expressing L929 cells were mixed with pHrodo-labeled apoptotic cells (AC) at a 1:4 ratio, and the percentage of pHrodo⁺-cells was analyzed by flow cytometry, as in b. All data shown are derived from three independent experiments, and the bar graphs shown in b and d–f represent mean+S.E.M.; **P<0.01 (Student's t-test)

Figure 6

CD300b regulates macrophage-mediated phagocytosis of apoptotic cells. (a) J774.1 cells transduced with short hairpin scramble control RNA (shControl), or shRNA targeting CD300b (shCD300b) were stained with Alexa488-labeled anti-CD300b antibody. The efficiency of silencing was assessed by monitoring CD300b cell surface expression via flow cytometry. (b) Control or CD300b-silenced J774.1 cells were mixed with pHrodo-labeled apoptotic cells (AC; 1:4 ratio), and the percentage of J774.1-pHrodo⁺ cells was analyzed by flow cytometry. The contour plots (upper panel) illustrate the gating strategy, whereas the quantification of the percentage of J774.1-pHrodo⁺-cells from three independent experiments is summarized in the bar graph (bottom panel; error bars represent S.E.M.); *P<0.05, **P<0.01 (Student's t-test). (c) Control or CD300b-silenced J774.1 cells were incubated with or without apoptotic cells (AC) for the indicated length of time, and the activation of DAP12-dependent signaling pathways was assessed by western blot analysis with anti-pSyk, anti-pAkt, anti-pErk, anti-pPI3K antibodies. Immunoblot using anti-Syk, anti-Erk, anti-Akt and anti-PI3K-antibodies served as loading controls