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Efferocytosis impairs pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.

Medeiros AI, Serezani CH, Lee SP, Peters-Golden M - J. Exp. Med. (2009)

Bottom Line: Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo.These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses.They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health Systems, Ann Arbor, MI 48109, USA.

ABSTRACT
The ingestion of apoptotic cells (ACs; termed "efferocytosis") by phagocytes has been shown to trigger the release of molecules such as transforming growth factor beta, interleukin-10 (IL-10), nitric oxide, and prostaglandin E(2) (PGE(2)). Although the antiinflammatory actions of these mediators may contribute to the restoration of homeostasis after tissue injury, their potential impact on antibacterial defense is unknown. The lung is highly susceptible to diverse forms of injury, and secondary bacterial infections after injury are of enormous clinical importance. We show that ACs suppress in vitro phagocytosis and bacterial killing by alveolar macrophages and that this is mediated by a cyclooxygenase-PGE(2)-E prostanoid receptor 2 (EP2)-adenylyl cyclase-cyclic AMP pathway. Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo. These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses. They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

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Efferocytosis inhibits FcR-mediated phagocytosis and bacterial killing by AMs. (A) Jurkat T cells were incubated with 8 μg/ml camptothecin for 5 h and apoptotic cells were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs represent 25.69% of cells. (B) Phagocytosis of IgG RBCs or IgG E. coli was determined after a 90-min pretreatment with ACs at the indicated AC/AM ratios. (C) Phagocytosis of IgG RBCs was determined after pretreatment for the indicated times with ACs added at a ratio of 3:1. (D) Phagocytosis of IgG RBCs was determined after a 90-min pretreatment with viable (VC) or necrotic (NC) Jurkat cells added at a ratio of 3:1. (E) AMs were preincubated with or without ACs (3:1) for 90 min and then infected with K. pneumoniae (50:1). Microbicidal activity was determined and expressed as the percentage survival of ingested bacteria. Results represent the mean ± SEM from three independent experiments, each performed in quintuplicate (B–D) or the mean ± SEM of quintuplicate values from a single experiment representative of three independent experiments (A and E). *, P < 0.05 versus control.
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fig1: Efferocytosis inhibits FcR-mediated phagocytosis and bacterial killing by AMs. (A) Jurkat T cells were incubated with 8 μg/ml camptothecin for 5 h and apoptotic cells were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs represent 25.69% of cells. (B) Phagocytosis of IgG RBCs or IgG E. coli was determined after a 90-min pretreatment with ACs at the indicated AC/AM ratios. (C) Phagocytosis of IgG RBCs was determined after pretreatment for the indicated times with ACs added at a ratio of 3:1. (D) Phagocytosis of IgG RBCs was determined after a 90-min pretreatment with viable (VC) or necrotic (NC) Jurkat cells added at a ratio of 3:1. (E) AMs were preincubated with or without ACs (3:1) for 90 min and then infected with K. pneumoniae (50:1). Microbicidal activity was determined and expressed as the percentage survival of ingested bacteria. Results represent the mean ± SEM from three independent experiments, each performed in quintuplicate (B–D) or the mean ± SEM of quintuplicate values from a single experiment representative of three independent experiments (A and E). *, P < 0.05 versus control.

Mentions: Using a variety of recognition receptors for opsonins or pathogen-associated molecular patterns, AMs ingest and kill pathogens, thereby serving a critical function in immune defense of the delicate gas-exchanging region of the lung. One such receptor that plays an important role in antimicrobial responses (8) and that has been extensively investigated (9) is the Fcγ receptor (FcR) for IgG antibodies. To determine if efferocytosis suppresses in vitro FcR-mediated phagocytosis, rat AMs were preincubated with different ratios of ACs and then challenged with IgG-opsonized erythrocytes (RBCs) or Escherichia coli for 90 min. As a source of ACs, we used Jurkat T cells treated with camptothecin using a protocol resulting in 25.6% of cells in early apoptosis with only 3.1% contamination by late apoptotic or necrotic cells (Fig. 1 A). Because Fadok et al. (3) previously demonstrated that PGE2 production by human macrophages occurred after 90 min of incubation with ACs, we initially used this pretreatment interval. Microscopic visualization (unpublished data) indicated that AMs bound and ingested ACs, as previously reported (10). Preincubation of AMs for 90 min with various ratios of ACs dose-dependently inhibited subsequent FcR-mediated phagocytosis of both RBCs and E. coli (Fig. 1 B), with ≥50% inhibition being observed at AC/AM ratios of 3:1. The inhibition by ACs (3:1) of FcR-mediated ingestion of both targets was also time dependent over a 15–90-min pretreatment interval, and a 16-h pretreatment resulted in near complete suppression (Fig. 1 C). Similar inhibitory effects were obtained when rat thymocytes, rat PMNs, or RLE-6TN rat lung epithelial cells were used as the source of ACs (unpublished data). Preincubation with either viable or necrotic cells had no effect on subsequent FcR-mediated phagocytosis (Fig. 1 D). After their ingestion, macrophages must kill bacteria. Preincubation with ACs (3:1) for 90 min significantly enhanced the intracellular survival of phagocytosed bacteria, reflecting an impairment of AM microbicidal activity against IgG-opsonized Klebsiella pneumoniae (Fig. 1 E). Together, these results demonstrate that preexposure to ACs markedly impairs the ability of AMs to carry out two crucial functions involved in immune defense against bacterial pneumonia: microbial phagocytosis and killing.


Efferocytosis impairs pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.

Medeiros AI, Serezani CH, Lee SP, Peters-Golden M - J. Exp. Med. (2009)

Efferocytosis inhibits FcR-mediated phagocytosis and bacterial killing by AMs. (A) Jurkat T cells were incubated with 8 μg/ml camptothecin for 5 h and apoptotic cells were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs represent 25.69% of cells. (B) Phagocytosis of IgG RBCs or IgG E. coli was determined after a 90-min pretreatment with ACs at the indicated AC/AM ratios. (C) Phagocytosis of IgG RBCs was determined after pretreatment for the indicated times with ACs added at a ratio of 3:1. (D) Phagocytosis of IgG RBCs was determined after a 90-min pretreatment with viable (VC) or necrotic (NC) Jurkat cells added at a ratio of 3:1. (E) AMs were preincubated with or without ACs (3:1) for 90 min and then infected with K. pneumoniae (50:1). Microbicidal activity was determined and expressed as the percentage survival of ingested bacteria. Results represent the mean ± SEM from three independent experiments, each performed in quintuplicate (B–D) or the mean ± SEM of quintuplicate values from a single experiment representative of three independent experiments (A and E). *, P < 0.05 versus control.
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Related In: Results  -  Collection

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fig1: Efferocytosis inhibits FcR-mediated phagocytosis and bacterial killing by AMs. (A) Jurkat T cells were incubated with 8 μg/ml camptothecin for 5 h and apoptotic cells were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs represent 25.69% of cells. (B) Phagocytosis of IgG RBCs or IgG E. coli was determined after a 90-min pretreatment with ACs at the indicated AC/AM ratios. (C) Phagocytosis of IgG RBCs was determined after pretreatment for the indicated times with ACs added at a ratio of 3:1. (D) Phagocytosis of IgG RBCs was determined after a 90-min pretreatment with viable (VC) or necrotic (NC) Jurkat cells added at a ratio of 3:1. (E) AMs were preincubated with or without ACs (3:1) for 90 min and then infected with K. pneumoniae (50:1). Microbicidal activity was determined and expressed as the percentage survival of ingested bacteria. Results represent the mean ± SEM from three independent experiments, each performed in quintuplicate (B–D) or the mean ± SEM of quintuplicate values from a single experiment representative of three independent experiments (A and E). *, P < 0.05 versus control.
Mentions: Using a variety of recognition receptors for opsonins or pathogen-associated molecular patterns, AMs ingest and kill pathogens, thereby serving a critical function in immune defense of the delicate gas-exchanging region of the lung. One such receptor that plays an important role in antimicrobial responses (8) and that has been extensively investigated (9) is the Fcγ receptor (FcR) for IgG antibodies. To determine if efferocytosis suppresses in vitro FcR-mediated phagocytosis, rat AMs were preincubated with different ratios of ACs and then challenged with IgG-opsonized erythrocytes (RBCs) or Escherichia coli for 90 min. As a source of ACs, we used Jurkat T cells treated with camptothecin using a protocol resulting in 25.6% of cells in early apoptosis with only 3.1% contamination by late apoptotic or necrotic cells (Fig. 1 A). Because Fadok et al. (3) previously demonstrated that PGE2 production by human macrophages occurred after 90 min of incubation with ACs, we initially used this pretreatment interval. Microscopic visualization (unpublished data) indicated that AMs bound and ingested ACs, as previously reported (10). Preincubation of AMs for 90 min with various ratios of ACs dose-dependently inhibited subsequent FcR-mediated phagocytosis of both RBCs and E. coli (Fig. 1 B), with ≥50% inhibition being observed at AC/AM ratios of 3:1. The inhibition by ACs (3:1) of FcR-mediated ingestion of both targets was also time dependent over a 15–90-min pretreatment interval, and a 16-h pretreatment resulted in near complete suppression (Fig. 1 C). Similar inhibitory effects were obtained when rat thymocytes, rat PMNs, or RLE-6TN rat lung epithelial cells were used as the source of ACs (unpublished data). Preincubation with either viable or necrotic cells had no effect on subsequent FcR-mediated phagocytosis (Fig. 1 D). After their ingestion, macrophages must kill bacteria. Preincubation with ACs (3:1) for 90 min significantly enhanced the intracellular survival of phagocytosed bacteria, reflecting an impairment of AM microbicidal activity against IgG-opsonized Klebsiella pneumoniae (Fig. 1 E). Together, these results demonstrate that preexposure to ACs markedly impairs the ability of AMs to carry out two crucial functions involved in immune defense against bacterial pneumonia: microbial phagocytosis and killing.

Bottom Line: Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo.These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses.They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health Systems, Ann Arbor, MI 48109, USA.

ABSTRACT
The ingestion of apoptotic cells (ACs; termed "efferocytosis") by phagocytes has been shown to trigger the release of molecules such as transforming growth factor beta, interleukin-10 (IL-10), nitric oxide, and prostaglandin E(2) (PGE(2)). Although the antiinflammatory actions of these mediators may contribute to the restoration of homeostasis after tissue injury, their potential impact on antibacterial defense is unknown. The lung is highly susceptible to diverse forms of injury, and secondary bacterial infections after injury are of enormous clinical importance. We show that ACs suppress in vitro phagocytosis and bacterial killing by alveolar macrophages and that this is mediated by a cyclooxygenase-PGE(2)-E prostanoid receptor 2 (EP2)-adenylyl cyclase-cyclic AMP pathway. Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo. These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses. They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

Show MeSH
Related in: MedlinePlus