Glucocorticoids relieve collectin-driven suppression of apoptotic cell uptake in murine alveolar macrophages through downregulation of SIRPα

Abstract

The lung environment actively inhibits apoptotic cell (AC) uptake by alveolar macrophages (AMøs) via lung collectin signaling through signal regulatory protein α (SIRPα). Even brief glucocorticoid (GC) treatment during maturation of human blood monocyte-derived or murine bone marrow-derived macrophages (Møs) increases their AC uptake. Whether GCs similarly impact differentiated tissue Møs and the mechanisms for this rapid response are unknown and important to define, given the widespread therapeutic use of inhaled GCs. We found that the GC fluticasone rapidly and dose-dependently increased AC uptake by murine AMøs without a requirement for protein synthesis. Fluticasone rapidly suppressed AMø expression of SIRPα mRNA and surface protein, and also activated a more delayed, translation-dependent upregulation of AC recognition receptors that was not required for the early increase in AC uptake. Consistent with a role for SIRPα suppression in rapid GC action, murine peritoneal Møs that had not been exposed to lung collectins showed delayed, but not rapid, increase in AC uptake. However, pretreatment of peritoneal Møs with the lung collectin surfactant protein D inhibited AC uptake, and fluticasone treatment rapidly reversed this inhibition. Thus, GCs act not only by upregulating AC recognition receptors during Mø maturation but also via a novel rapid downregulation of SIRPα expression by differentiated tissue Møs. Release of AMøs from inhibition of AC uptake by lung collectins may, in part, explain the beneficial role of inhaled GCs in inflammatory lung diseases, especially emphysema, in which there is both increased lung parenchymal cell apoptosis and defective AC uptake by AMøs.

Figures

Figure 1

Fluticasone rapidly and specifically increases uptake and binding of AC by murine AMø. (A-E) AC uptake. Adherence-purified AMø from normal C57 BL/6 mice were treated in chamber slides with fluticasone (2 nM unless indicated) for 0-6 h, then AC were added at a 10:1 ratio for 2 h. Slides were washed and stained using H&E, then ingested AC were counted at 100X magnification under oil. A. Graphic timeline of a phagocytosis assay. B, C. Kinetics of GC-augmented AC uptake. D, E. Dose-response of GC-augmented AC uptake. (F-J) AC binding. Adherence-purified AMø from normal C57 BL/6 mice were treated in chamber slides with fluticasone (2 nM unless indicated) for 0-6 h, then AC were added at a 100:1 ratio for 20 min. Slides were washed and stained using H&E, then surface bound AC were counted at 100X magnification under oil. F. Graphic timeline of a binding assay. G, H. Kinetics of GC-augmented AC binding. I, J. Dose-response of GC-augmented AC binding. Data are mean ± SE of 5-8 mice assayed individually in at least two independent experiments per condition. **, statistically significant for untreated, p

Figure 2

Fluticasone rapidly downregulates SIRPα and increases efferocytosis without a requirement for new protein synthesis. A, Murine AMø were treated with 2 nM fluticasone for 0, 1, 3 or 6 h. RNA was collected at each time point and analyzed by real-time RT-PCR with GAPDH as the housekeeping gene; results are displayed as fold increase from untreated. B, Murine AMø were pre-treated with 5 μM cycloheximide for 1 h followed by 2 μM fluticasone for 5 h, then AC were added at a 10:1 ratio for 2 h. Slides were washed and stained using H&E, then ingested AC were counted at 100X magnification under oil. C, D. Surface SIRPα protein. Murine AMø treated with 2μM fluticasone for 6 or 24 h, then analyzed by flow cytometry for surface expression of SIRPα. Cells shown are gated CD45+CD19-TCRβ-. C. Representative dot plot. D. Average percent of CD11c+SIRPα- cells within gated CD11c+ population. Data are mean ± SE of 5-7 individual mice assayed individually in at least two independent experiments per condition. *, statistically significant from untreated, p<0.05 and **, statistically significant from untreated, p<0.01 by one-way ANOVA with Bonferroni post-hoc testing.

Figure 3

Azithromycin but not simvastatin has additive effects on efferocytosis by murine AMø. (A-D) Affect of multi-agent treatment on efferocytosis. Murine AMø were treated with Murine AMø were treated with 500 ng/mL azithromycin, 10 μM simvastatin or media alone. After 18 h, 2 μM fluticasone was added for a further 6 h, then AC were added at a 10:1 ratio for 2 h. Slides were washed and stained using H&E, then ingested AC were counted at 100X magnification under oil. A, B. Simvastatin and Fluticasone. C, D. Azithromycin and Fluticasone. Data are presented as the mean ± SE of seven mice assayed individually in three independent experiments. **, statistically significant than fluticasone alone, p

Figure 4

Simvastatin downregulates SIRPα expression while azithromycin does not. A, B. Surface SIRPα protein. Murine AMø treated with 10 μM simvastatin or 500 ng/mL azithromycin for 24 h, then analyzed by flow cytometry for surface expression of SIRPα. Cells shown are gated CD45+CD19-TCRβ-. A. Representative dot plot. B. Average percent of CD11c+SIRPα- cells within gated CD11c+ population. *, statistically significant from other conditions, p<0.05 by one-way ANOVA with Bonferroni post-hoc testing. C. Murine AMø were pre-treated with 5 μM cycloheximide (CHX) for 1 h followed by 10 μM simvastatin or 500 ng/mL azithromycin for 24 h, then AC were added at a 10:1 ratio for 2 h. Slides were washed and stained using H&E, then ingested AC were counted at 100X magnification under oil. Data in B, C are mean ± SE of 5-7 mice assayed individually in at least two independent experiments. **, statistically significant from no cyclohexamide, p<0.01 by one-way ANOVA with Bonferroni post-hoc testing.

Figure 5

SP-D activates SIRPα pathway in PMø and makes PMø sensitive to fluticasone-driven increase in AC clearance. (A-C) Surface SIRPα protein. Murine PMø were treated with 2 μM fluticasone for 6 or 24 h, then analyzed by flow cytometry for surface expression of SIRPα. Cells shown are gated CD45+CD19-TCRβ-. A. Representative dot plot. B. Average percent of CD11b+SIRPα- cells within gated CD11b+ population. C, Average MFI of SIRPα on gated CD11b+ cells. D, Fluticasone rescues SP-D inhibition of AC uptake. Murine PMø were treated with 25 μg/mL SP-D for 4 h, followed by control media or 2 μM fluticasone for 5 h, then AC were added at a 10:1 ratio for 2 h. Slides were washed and stained using H&E, then ingested AC were counted at 100X magnification under oil. Data are mean ± SE of 5-8 mice assayed individually in at least two independent experiments per condition. **, statistically significant, p<0.01 by one-way ANOVA with Bonferroni post-hoc testing.

Figure 6

Model of GC regulation of SIRPα-mediated control of murine AMø efferocytosis. A. In untreated AMø, which express high amounts of SIRPα, lung collectins SP-D and SP-A (not shown) signal constitutively through SIRPα, activating SHP-1 and leading to downstream activation of RhoA. By inhibiting Rac-dependent mobilization of actin, the lung collectins tonically impede efficient uptake of AC by AMø, even though SP-A and SP-D can also bind AC. B. Treatment with fluticasone (triangles) reduces SIRPα surface expression, in part via transrepression of SIRPα by ligand-occupied GRα homodimers (brackets). The consequent decreased activation of SHP-1 relieves inhibition of Rac, permitting efficient AC uptake. Based on data in the current study, plus previously published data (17, 36, 37, 50).