A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester - PubMed (original) (raw)

A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester

Michael Miksa et al. J Immunol Methods. 2009.

Abstract

Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8(-/-) macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells.

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Figures

Figure 1

Figure 1

pHrodo-SE labeling of apoptotic cells and responsiveness to changes in pH. (a) Annexin V staining of non-treated (non-apoptotic) and dexamethasone-treated (apoptotic, 0.1 μM for 16h) rat thymocytes, as analyzed by flow cytometry. (b) Dose-dependent pHrodo-SE staining of apoptotic cells. 107 apoptotic thymocytes were stained with 0.01-1 μg/ml pHrodo-SE, as analyzed by flow cytometry. (c) Responsiveness of pHrodo-SE-labeled apoptotic thymocytes to changes in pH. pHrodo-SE-labeled cells (0.01 μg/ml) were exposed to a pH of 7.4, 4.0 and 3.0 by adding HCl to the cell suspension, as analyzed by flow cytometry. Mean fluorescent intensity (MFI) is shown next to FACS plot.

Figure 2

Figure 2

Fluorescent intensity of pHrodo-SE-labeled apoptotic thymocytes increases after engulfment by macrophages. Splenic macrophages were labeled with FITC-anti-CD11b/c (OX42) and thymocytes with pHrodo-SE (0.02 μg/ml). Cells were coincubated for 60 min, collected and fixed with 1%PFA prior to fluorescent microscopy. Top three panels show lymphocytes in red (pHrodo-SE+), macrophages in green (CD11b/c) and a merged image, of the same area to the right. Boxes in the merged image indicate the 10× magnification shown below. Arrowheads indicate free floating and attached apoptotic thymocytes and arrows indicate engulfed apoptotic cells. Magnification: 400×.

Figure 3

Figure 3

Effect of pHrodo-SE on phagocytosis. Thymocytes were collected from normal rats and incubated with (apoptotic) or without (non-apoptotic) 0.1μM of dexamethasone for 16h. Cells were labeled with pHrodo-SE, cocultured with peritoneal macrophages for 60min, then stained with FITC-anti-CD11b/c and analyzed by flow cytometry immediately. Representative phagocytosis result shown of CD11b/c and pHrodo-SE (gated on CD11b/c+) of (a) non-apoptotic thymocytes, and (b) apoptotic thymocytes. (c) Average percent phagocytosis index ± SEM of CD11b/c+pHrodo+ cells. (d) Average mean fluorescent intensity (MFI) ± SEM of CD11b/c+pHrodo+ cells. *P<0.05 vs. non-apoptotic, Student's _t_-test, _n_=3.

Figure 4

Figure 4

Verification of deficient phagocytosis of apoptotic cells by MFGE8-deficient splenic macrophages. (a) Representative FACS histograms. MFGE8-deficient splenic CD11b+ macrophages were incubated with autologous pHrodo-labeled thymocytes (0.02 μg/ml*106 cells) for 5 to 180 minutes and analyzed by flow cytometry in 3days. (b) Line graph showing means ± SEM (percent phagocytosing macrophages) of triplicates of a representative experiment. *P<0.05 vs. Control, two-way ANOVA and Student Newman Keuls' test, _n_=3. (c) Average mean fluorescent intensity (MFI) and SEM of CD11b+pHrodo-SE+ cells in one representative experiment performed in triplicate. This represents an estimate for the amount of phagocytized cells by the macrophages. *P<0.05 vs. Control, two-way ANOVA and Student Newman Keuls' test, _n_=3.

Figure 5

Figure 5

MFGE8-deficient resident peritoneal macrophages compensate for a delay in apoptotic cell clearance. MFGE8-deficient peritoneal CD11b+ macrophages were incubated with autologous pHrodo-SE-labeled thymocytes (0.02 μg/ml*106 cells) for 5 to 180 minutes and analyzed by flow cytometry. Means ± SEM (percent phagocytosing macrophages) of triplicates of a representative experiment. *P<0.05 vs. Control, two-way ANOVA and Student Newman Keuls' test, _n_=3.

Figure 6

Figure 6

Flow chart of pHrodo-SE-staining and phagocytosis assay protocol.

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