Induction of caspase- and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment

Abstract

Macrophage recognition and disposal of neutrophils are important steps in the resolution of inflammation. Externalization of phosphatidylserine (PS) on the cell surface serves as a common recognition signal for macrophages and is associated with the apoptosis program in neutrophils. Here, we report that macrophage-differentiated PLB-985 cells induce rapid, caspase-independent PS externalization in human neutrophils. A similar degree of PS externalization was seen when neutrophils were cocultured with gp91(phox)-deficient PLB-985 macrophages, thus demonstrating that macrophage-induced PS externalization was NADPH oxidase-independent. Macrophage-induced PS externalization required cell-to-cell contact and kinase activation and was shown to correlate with neutrophil degranulation. Of note, the degree of engulfment of such PS-positive neutrophils by activated human monocyte-derived macrophages was considerably lower than for neutrophils undergoing constitutive apoptosis, indicating that PS externalization alone is not sufficient for macrophage disposal of neutrophils. However, addition of recombinant milk fat globule epidermal growth factor 8, a PS-binding protein, restored engulfment of the macrophage-cocultured target cells. Finally, neutrophils undergoing spontaneous apoptosis but not macrophage-cocultured neutrophils displayed surface expression and release of annexin I, and the addition of N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe (Boc1), a formyl peptide receptor/lipoxin receptor antagonist, suppressed clearance of apoptotic neutrophils. Conditioned medium from apoptotic neutrophils also promoted the engulfment of macrophage-cocultured neutrophils, and Boc1 blocked this process. Taken together, these studies highlight a novel pathway of PS externalization in primary human neutrophils and also provide evidence for an auxiliary function of annexin I in macrophage clearance of neutrophils.

Fig. 1.

PS externalization on the surface of neutrophils upon cocultivation with PLB-985 macrophages. (A) Neutrophils were cocultured with PLB-985 macrophages or incubated alone at 37°C for the indicated time-points. At the end of incubation, nonadherent cells were harvested, and cells were costained with annexin V-FITC and PI as described in Materials and Methods. (B) Quantification of the percentage of PI-negative, annexin V-positive cells. Data are derived from at least five independent experiments using different donors and are displayed as mean values ± sem. (C) TAMRA-stained neutrophils were cocultured for 1 h with undifferentiated or differentiated PLB-985 or THP-1 cells or incubated alone at 37°C. At the end of incubation, nonadherent cells were harvested, and cells were stained with annexin V-FITC. Only TAMRA-positive cells were included in the analysis by gating on this population. Quantification of the percentage of annexin V-positive cells is shown. Data are derived from at least three independent experiments using different donors and are displayed as mean values ± sem.

Fig. 2.

NADPH oxidase- and iNOS-independent PS externalization in neutrophils cocultivated with PLB-985 macrophages. (A) PLB-985 WT macrophages were pretreated with DPI (10 μM) for 30 min. Neutrophils were then cocultured with PLB-985 WT macrophages for 1 h. Neutrophils cocultivated for 1 h with PLB-985 X-CGD macrophages lacking a functional NADPH oxidase were also included. PS exposure in cocultivated neutrophils was then determined. Data shown are mean values ± sem (n=3). (B) PLB-985 WT macrophages were pretreated with the iNOS inhibitor, 1400W (100 μM), for 1 h, and neutrophils were then added to PLB-985 macrophages. PS externalization in neutrophils was determined following 1 h of coculture. Data are reported as mean values ± sem (n=3).

Fig. 3.

Macrophage-induced PS externalization and degranulation is kinase-dependent. (A) Neutrophils were pretreated with STS (2 μM) for 1 h or with genistein (100 μM), H-7 (6 μM), or rottlerin (30 μM) for 30 min and then added to PLB-985 WT macrophages and cocultured for 2 h. Neutrophils maintained in culture in the absence of PLB-985 macrophages were included as a control. The percentages of annexin V-FITC-positive cells are shown as mean values ± sem (n=5). Spont apop, Spontaneous apoptosis. (B) Neutrophils obtained from cocultivation with PLB-985 macrophages were incubated with FITC-conjugated anti-MPO antibodies or isotype-matched control antibodies for assessment of neutrophil degranulation. Neutrophils treated with the combination of fMLP (10 μM) and CB (10 μg/ml) served as a positive control. Data are displayed as the mean fluorescence intensity (MFI) of three independent experiments (mean±sem).

Fig. 4.

Phagocytosis of primary human PLB-985-cocultivated neutrophils by activated HMDM. (A) Comparison of PS externalization in neutrophils following cocultivation with (co-culture) or without (control) PLB-985 WT macrophages for 2 h versus neutrophils undergoing spontaneous apoptosis. Data are shown as mean values ± sem (n=3). (B) Phagocytosis of neutrophils maintained in culture as indicated above. Neutrophils were added to M-CSF-activated HMDM and cocultured for 1 h at 37°C prior to assessment of the degree of phagocytosis. Data are shown as mean values ± sem (n=4). (C) HMDM engulfment of control neutrophils or neutrophils undergoing spontaneous apoptosis was monitored in the presence or absence of PLS (1 mM). Data are reported as mean values ± sem (n=3).

Fig. 5.

MFG-E8 enhances phagocytosis of neutrophils with PLB-985 macrophage-induced PS externalization. (A) HMDM were pretreated with MFG-E8 (WT) or D89E MFG-E8 mutant protein (1 μg/ml) for 30 min, and the recombinant proteins were added again at the time of cocultivation. Neutrophils were cocultured with HMDM for 1 h, and the percentages of phagocytosis-positive macrophages were determined. Neutrophils undergoing spontaneous apoptosis and neutrophils maintained in culture for 2 h in the absence of PLB-985 macrophages were included for comparison. Data are derived from four independent experiments and are reported as mean values ± sem. (B) Cocultivation of neutrophils and HMDM was performed as above, and the percentages of macrophages that carried one or greater than or equal to two target cells in the presence or absence of MFG-E8 (1 μg/ml) were determined. Data shown are mean values ± sem (n=3). (C) Representative images showing HMDM engulfment of TAMRA-labeled target neutrophils (red) in the presence or absence of MFG-E8 (1 μg/ml). Hoechst 33342 (blue) was used for visualization of cell nuclei. Arrows indicate macrophages that have ingested more than one target cell. Original magnification, ×40.

Fig. 6.

Annexin I is involved in the phagocytosis of neutrophils with PLB-985 macrophage-induced PS externalization. (A) Annexin I externalization in neutrophils undergoing spontaneous apoptosis versus neutrophils cocultured with PLB-9865 macrophages or cultured alone (control) was analyzed by flow cytometry using specific antiannexin I antibodies. The percentages of annexin I-positive cells with background subtracted (isotype-matched control antibodies) are shown. Data are reported as mean values ± sem (n=3). (B) Secretion of annexin I into the culture medium was analyzed by Western blotting. Supernatants from control neutrophils cultured alone for 2 h or neutrophils maintained in culture for 22 h (spontaneous apoptosis) and supernatants from cocultures of PLB-985 macrophages and neutrophils (“co-culture 2 h”) or neutrophils cocultured with PLB-985 macrophages for 2 h and then incubated alone in fresh medium for 2 h (“co-culture 2h + 2h”) were analyzed using specific antiannexin I antibodies. A comparison of the latter two samples reveals that cocultured neutrophils fail to secrete annexin I, whereas PLB-985 macrophages may release annexin I following interaction with neutrophils. For comparison, immunoblotting of total cell lysates of the corresponding samples is also shown. Results are representative of several independent experiments. (C) Phagocytosis of apoptotic neutrophil target cells (in vitro culture for 22 h) by HMDM in the presence or absence of the formyl peptide receptor/lipoxin receptor antagonist, Boc1 (50 μM). (D) Phagocytosis of neutrophils cocultured with PLB-985 macrophages by HMDM pretreated with conditioned medium from apoptotic neutrophils in the presence or absence of Boc1 (50 μM). Data shown in Panels C and D are mean values ± sem (n=4).