Figure 6
Figure 6. Inhibitory role of CD300a on the engulfment of dead cells. (A) Expression of CD300a on macrophages. Human monocyte derived macrophages were stained with anti-CD14 PE-Cy7 and anti-CD300a PE and analyzed by flow cytometry. The quadrants in the dot plot were determined according to the binding of isotype negative control antibodies (not shown). (B) Effect of CD300a-Ig on the phagocytic function of macrophages. PKH67 labeled dexamethasone treated DT40 cells were incubated with purified proteins, that is, CD300a-Ig, LAIR-1-Ig, and annexin V, and then they were subjected to a phagocytosis assay as described in “Methods.” Macrophages, electronically gated by the expression of CD14, that have ingested DT40 cells are positive for PKH67. A representative experiment is shown. Numbers in the dot plots represent the percentage of macrophages that have engulfed dead DT40 cells. Bar graph represents the average ± SEM. Results shown are from 5 independent experiments. (C) Histogram representation of CD300a expression on macrophages after siRNA knockdown. The broken line histograms shown CD300a expression on nontransfected cells; the gray line corresponds to the CD300a expression on cells transfected with nontarget siRNA (NT-siRNA); and the black line histogram shows CD300a expression after transfection with siRNA specific for CD300a (CD300a-siRNA). Data shown is a representative of 6 independent donors. (D) Knockdown of CD300a increases the phagocytic activity of macrophages. PKH26 labeled dexamethasone treated thymocytes were subjected to a phagocytosis assay with macrophages transfected with NT-siRNA or with CD300a-siRNA. Macrophages, electronically gated by the FSC and SSC parameters that have ingested thymocytes are positive for PKH26. A representative experiment is shown (left panel). Numbers in the contour plots represent the percentage of macrophages that have engulfed dead thymocytes. The graph represents the percentage of macrophages that have engulfed (% of phagocytosis) dead cells (right panel). Each pair of symbols corresponds to a different donor. (E) L929 cells were stably transfected with empty or CD300a vector. The histograms (top left panel) show the staining with anti-CD300a Ab of empty vector transfected cells (dotted black line) and CD300a transfected cells (black line). The gray histogram represents unstained cells. L929 cells were mixed with PKH26 labeled dexamethasone treated thymocytes for 60 minutes, treated with trypsin/EDTA to detach the cells, and analyzed by flow cytometry. L929 cells, electronically gated by the FSC and SSC parameters that have ingested dead thymocytes, are positive for PKH26. A representative experiment is shown (bottom panel). Numbers in the dot plots represents the percentage of L929 cells that have engulfed dead thymocytes. The graph represents the percentage of L929 cells that have engulfed (% of phagocytosis) dead cells (top right panel). Each pair of symbols corresponds to an independent experiment. (F) Functional recognition of PE by CD300a. Levels of β-galactosidase activity generated by culture of BWZ.36, BWZ.36 LAIR-1/CD3ζ (BWZ.36 LAIR-1), and BWZ.36 CD300a/CD3ζ (BWZ.36 CD300a) cells on uncoated plates or plates coated with isotype-control antibody, anti-CD300a antibody, PC, PS, or PE. The lipids (PC, PS, and PE) were air-dried and the isotype controls and anti-CD300a antibodies were immobilized on a 24-well plate. Data shown is representative of 2 independent experiments.

Inhibitory role of CD300a on the engulfment of dead cells. (A) Expression of CD300a on macrophages. Human monocyte derived macrophages were stained with anti-CD14 PE-Cy7 and anti-CD300a PE and analyzed by flow cytometry. The quadrants in the dot plot were determined according to the binding of isotype negative control antibodies (not shown). (B) Effect of CD300a-Ig on the phagocytic function of macrophages. PKH67 labeled dexamethasone treated DT40 cells were incubated with purified proteins, that is, CD300a-Ig, LAIR-1-Ig, and annexin V, and then they were subjected to a phagocytosis assay as described in “Methods.” Macrophages, electronically gated by the expression of CD14, that have ingested DT40 cells are positive for PKH67. A representative experiment is shown. Numbers in the dot plots represent the percentage of macrophages that have engulfed dead DT40 cells. Bar graph represents the average ± SEM. Results shown are from 5 independent experiments. (C) Histogram representation of CD300a expression on macrophages after siRNA knockdown. The broken line histograms shown CD300a expression on nontransfected cells; the gray line corresponds to the CD300a expression on cells transfected with nontarget siRNA (NT-siRNA); and the black line histogram shows CD300a expression after transfection with siRNA specific for CD300a (CD300a-siRNA). Data shown is a representative of 6 independent donors. (D) Knockdown of CD300a increases the phagocytic activity of macrophages. PKH26 labeled dexamethasone treated thymocytes were subjected to a phagocytosis assay with macrophages transfected with NT-siRNA or with CD300a-siRNA. Macrophages, electronically gated by the FSC and SSC parameters that have ingested thymocytes are positive for PKH26. A representative experiment is shown (left panel). Numbers in the contour plots represent the percentage of macrophages that have engulfed dead thymocytes. The graph represents the percentage of macrophages that have engulfed (% of phagocytosis) dead cells (right panel). Each pair of symbols corresponds to a different donor. (E) L929 cells were stably transfected with empty or CD300a vector. The histograms (top left panel) show the staining with anti-CD300a Ab of empty vector transfected cells (dotted black line) and CD300a transfected cells (black line). The gray histogram represents unstained cells. L929 cells were mixed with PKH26 labeled dexamethasone treated thymocytes for 60 minutes, treated with trypsin/EDTA to detach the cells, and analyzed by flow cytometry. L929 cells, electronically gated by the FSC and SSC parameters that have ingested dead thymocytes, are positive for PKH26. A representative experiment is shown (bottom panel). Numbers in the dot plots represents the percentage of L929 cells that have engulfed dead thymocytes. The graph represents the percentage of L929 cells that have engulfed (% of phagocytosis) dead cells (top right panel). Each pair of symbols corresponds to an independent experiment. (F) Functional recognition of PE by CD300a. Levels of β-galactosidase activity generated by culture of BWZ.36, BWZ.36 LAIR-1/CD3ζ (BWZ.36 LAIR-1), and BWZ.36 CD300a/CD3ζ (BWZ.36 CD300a) cells on uncoated plates or plates coated with isotype-control antibody, anti-CD300a antibody, PC, PS, or PE. The lipids (PC, PS, and PE) were air-dried and the isotype controls and anti-CD300a antibodies were immobilized on a 24-well plate. Data shown is representative of 2 independent experiments.

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