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Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo

Siobhan Mitchell^*, Graham Thomas, Killeen Harvey^*, David Cottell, Keira Reville^*, Giovanni Berlasconi, Nicos A. Petasis, Lars Erwig^¶, Andrew J. Rees^¶, John Savill, Hugh R. Brady^* and Catherine Godson^*

*Centre for Molecular Inflammation and Vascular Research, Mater Misericordiae Hospital and Department of Medicine and Therapeutics, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, and Dublin Molecular Medicine Centre, Dublin, Ireland; MRC Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom;Department of Chemistry, University of Southern California, Los Angeles, California; Electron Microscopy Laboratory, University College Dublin, Dublin, Ireland;^¶Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom.

Correspondence to Dr. Catherine Godson, Department of Medicine and Therapeutics, University College Dublin, 41 Eccles Street, Dublin 7, Ireland. Phone: 00-353-1-8032188; Fax: 00-353-1-8308404;

	Abstract

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ABSTRACT. Lipoxins (LX) are eicosanoids with antiinflammatory activity in glomerulonephritis (GN) and inflammatory diseases, hypersensitivity, and ischemia reperfusion injury. It has been demonstrated that LXA₄ stimulates non-phlogistic phagocytosis of apoptotic polymorphonuclear neutrophils (PMN) by monocyte-derived macrophages (M) in vitro, suggesting a role for LX as endogenous pro-resolution lipid mediators. It is here reported that LXA₄, LXB₄, the aspirin-triggered LX (ATL) epimer, 15-epi-LXB₄, and a stable synthetic analogue 15(R/S)-methyl-LXA₄ stimulate phagocytosis of exogenously administered excess apoptotic PMN by macrophages (M) in vivo in a classic model of acute inflammation, namely thioglycollate-induced peritonitis. Significant enhancement of phagocytosis in vivo was observed with 15-min exposure to LX and with intraperitoneal doses of LXA₄, LXB₄, 15(R/S)-methyl-LXA₄, and 15-epi-LXB₄ of 2.5 to 10 µg/kg. Non-phlogistic LX-stimulated phagocytosis by M was sensitive to inhibition of PKC and PI 3-kinase and associated with increased production of transforming growth factor–₁ (TGF-₁). LX-stimulated phagocytosis was not inhibited by phosphatidylserine receptor (PSR) antisera and was abolished by prior exposure of M to 1,3-glucan, suggesting a novel M-PMN recognition mechanism. Interestingly, the recently described peptide agonists of the LXA₄ receptor (MYFINITL and LESIFRSLLFRVM) stimulated phagocytosis through a process associated with increased TGF-₁ release. These data provide the first demonstration that LXA₄, LXB₄, ATL, and LX stable analogues rapidly promote M phagocytosis of PMN in vivo and support a role for LX as rapidly acting, pro-resolution signals in inflammation. Engagement of the LXR by LX generated during cell-cell interactions in inflammation and by endogenous LXR peptide agonists released from distressed cells may be an important stimulus for clearance of apoptotic cells and may be amenable to pharmacologic mimicry for therapeutic gain.E-mail: cgodson@mater.ie

	Introduction

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Rapid, efficient and tightly regulated recruitment and clearance of polymorphonuclear neutrophil (PMN) at sites of inflammation are essential components of effective host defense. Evidence from in vitro models and from histopathology suggests that tissue damage mediated by PMN is limited by apoptosis and subsequent phagocytosis of the apoptotic PMN by macrophages (M) and "nonprofessional" phagocytes (1). A direct role for PMN in tissue injury in inflammation and ischemia reperfusion injury of the kidney and other organs is well established (2). Impaired clearance of apoptotic cells by M has been implicated in the pathogenesis of chronic inflammatory conditions, including glomerulonehritis (GN) and systemic lupus erythematosus (SLE) (3). The endogenous signals that promote clearance of apoptotic PMN from an inflammatory focus are still being defined. By dissecting out the mediator systems that regulate this process, it may be possible to design new pro-resolution strategies for inflammatory diseases.

Lipoxins (LX), an acronym for lipoxygenase interaction products, are endogenously produced eicosanoids with anti-inflammatory actions in GN and other diseases (4). LX are formed principally by transcellular metabolism initiated by sequential oxygenation of arachidonic acid by either 15- and 5-lipoxygenases or 5- and 12-lipoxygenases (5). Lipoxin biosynthesis has been demonstrated in a range of inflammatory diseases, including experimental immune complex glomerulonephritis in rats and mice (6,8,9). Platelet-PMN biosynthetic interactions provide the dominant pathway for LX formation in the initial stages of immune complex–mediated glomerulonephritis and are likely superseded by leukocyte interactions with cytokine-activated resident tissue cells as the inflammatory process evolves (9,10). LX were originally proposed to act as "braking signals" in inflammation on the basis of their ability to inhibit neutrophil chemotaxis, adhesion, and transmigration across endothelia and epithelia (reviewed in reference 4). LXA₄ and LXB₄ inhibit LTB₄-induced PMN chemotaxis and PMN-endothelial cell adhesion supported by CD11/CD18 integrins (7). LXA₄ and LXB₄ also attenuate LTC₄- and LTD₄-triggered endothelial hyperadhesiveness for PMN, in part by inhibiting mobilization of P-selectin from Weibel Palade bodies to the endothelial cell surface (6). More recently, the repertoire of antiinflammatory actions of LX has been expanded to include attenuation of cytokine and chemokine release (11,12), synoviocyte metalloproteinases (13), eosinophil trafficking (14), inhibition of mesangial cell proliferation (15), and stimulation of phagocytosis of apoptotic PMN in vitro (16).

Lipoxin A₄ [(5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid] and its positional isomer lipoxin B₄ [(5S,14R,15S)-trihydroxy-6,8,12-trans-10-cis-eicosatetraenoic acid] are the two principal species formed in mammalian cells (reviewed in reference 4). The rapid metabolic degradation of native LX by dehydrogenation at C15 and possibly -oxidation at C20 has prompted the design and synthesis of stable analogues, which retain many of the properties of the native LX, including inhibition of PMN adhesion and transmigration (17,4). In a cytokine primed milieu, the catalytic activity of aspirin-acetylated cyclooxygenase type 2 (COX-2) is shifted from a cyclooxygenase to 15R lipoxygenase. In this context, 15(R)-HETE is produced and can then be metabolized to 15 epimers of LXA₄ and LXB₄ during cell-cell interactions (18). These aspirin-triggered lipoxins (ATL) also possess antiinflammatory activity (19,20).

Here we demonstrate that LX, ATL, 15-epi-LXB₄, and the stable synthetic LX analogue 15(R/S)-methyl-LXA₄ promote M phagocytosis of apoptotic PMN in vivo. We expand on the signal transduction pathways mediating LX-stimulated phagocytosis and further define the adhesive requirements necessary for efficient phagocytosis. We demonstrate that recently described peptide agonists of the lipoxin receptor (ALXR) can also promote non-phlogistic phagocytosis of apoptotic PMN associated with transforming growth factor–₁ (TGF-₁) release. These data expand on the antiinflammatory repertoire of LX and provide the first evidence of rapid regulation of PMN clearance by endogenous lipid mediators in vivo. This raises the possibility that such a role may be played not only by lipoxygenase-derived lipid mediators but also by necrotactic peptides released by dying cells. The data suggest that it may be possible to actively promote the resolution of inflammatory disease by harnessing the system pharmacologically.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
RPMI 1640 and phosphate-buffered saline (PBS) were obtained from Life Technologies (Grand Island, NY). LXA₄ and LXB₄ were obtained from Cascade Biologicals (Berkshire, UK). 15(R/S)-methyl-LXA_4, 16-phenoxy-LXA₄ and the aspirin-triggered 15-epi-LXB₄ were prepared by total organic synthesis (21). CM-orange (CMO, cell tracker orange) was from Molecular Probes (Oregon, US). Rat cytokines tumor necrosis factor– (TNF-), TGF-, interferon-(IFN-), and TGF-₁ and MCP-1 human enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems (Oxon, UK). The anti-CD36 monoclonal antibody SM (IgM) was provided by Dr. Nancy Hogg (ICRF, London) (22). Anti-_v3 mouse monoclonal antibody (23C6) was purchased from Serotec (Oxford, UK). FITC-conjugated anti-CD36 (FA6–152) and anti-_v3 (anti-CD51/61) (AMF-7) monoclonal antibodies were from Beckman Coulter (Luton, UK). Phosphatidylserine receptor (PSR) antisera were a gift from Dr. Valerie Fadok (National Jewish Medical and Research Center, Denver, CO) (23);isotype-matched control IgM was obtained from Calbiochem (Nottingham, UK). 1,3-glucan was purchased from Accurate Chemicals (Westbury, NY). The MHC-binding peptide (MYFINILTL) (MHC bp) and MMK-1 peptide (LESIFRSLLFRVM) were gifts from Prof. Charles N. Serhan (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.) (19). All other chemicals were obtained from Sigma (Poole, UK).

Human Leukocyte Preparation and Culture
Human monocytes and PMN were isolated from peripheral venous blood drawn from healthy volunteers who had provided informed written consent subsequent to approval from institutional ethics committees. PMN were isolated by density gradient centrifugation and dextran sedimentation and aged for 24 h; apoptosis was verified by flow cytometry as previously documented (16). M were prepared from monocytes collected over Ficoll-Paque as previously reported (16). Adherent monocytes were cultured for 5 to 7 d in RPMI supplemented with 10% autologous serum and 1% penicillin-streptomycin.

M Phagocytosis of Apoptotic PMN In Vitro
M were treated with reagents as indicated and washed with RPMI 1640 before coincubation with aged PMN in 24-well tissue culture plates (4 x 10⁶ PMN/ml of RPMI per well) at 37°C for 30 min. Phagocytosis was assayed by myeloperoxidase (MPO) staining of co-cultures fixed with 2.5% glutaraldehyde as previously reported (16). M were routinely negative for peroxidase staining. For each experiment, the number of M containing one or more PMN in at least five fields (minimum of 400 cells) was expressed as a percentage of the total number of M in duplicate wells. Phagocytosis of apoptotic PMN was confirmed by electron microscopy.

Electron Microscopy
The coincubation cultures was washed with PBS, detached, and centrifuged at 500 xg for 15 min. The cell pellet was fixed in 2.5% glutaraldehyde in PBS for 1 h at room temperature. Glutaraldehyde was removed, and the pellet was washed three times in 6.8% sucrose/PBS. One percent osmium tetraoxide was then added for 1 h at room temperature before a washing step with 6.8% sucrose/PBS. The pellet was then dehydrated in ascending grades of ethanol and embedded in epoxy resin at 60°C. For light microscopy, 1-µm sections were cut with a glass knife and stained with toluene blue. Selected areas were trimmed, and ultrathin sections were cut at 50 nm using a diamond knife; sections were picked up on 100 mesh copper grids and stained with uranyl acetate and lead citrate. These were examined and photographed in a JOEL 2000 EX Temscan at 80KV and using an objective aperture of 100 µm.

Inhibitor and Antibody Studies
M were washed with RPMI 1640 before treatments with the stable cell-permeable cAMP analogue 8-Br-cAMP (2 mM) (24), the protein kinase A inhibitor Rp-cAMP (100 µM) (16), the PI 3-kinase inhibitor LY 294002 (10 µM) (15), the PKC inhibitor GF 109203X (10 µM) (15), the MAP kinase inhibitor PD 98059 (10 µM) (15), or appropriate vehicles for 15 min at 37°C before stimulation with LXA₄ or LXB₄ (1 nM; 15 min; 37°C). The treated cells were washed with RPMI 1640 before coincubation with aged PMN and phagocytosis assayed as described above.

Antibody blockade of cell surface adhesion molecules was affected by exposure of M to anti-CD51/61 mAb (20 µg/ml) and anti-CD36 mAb (SM) ascitic fluid (22) (125 µl/ml) on ice for 20 min. M were then treated with LX (1nM; 15 min; 37°C) or control before coincubation with apoptotic PMN for 30 min, and phagocytosis was assayed as above.

In experiments investigating the role of the phosphatidylserine receptor (PSR), M were treated with PSR antisera (200 µg/ml) and isotype control human IgM on ice for 30 min before treatment with LX and subsequent coincubation with PMN (23). Phagocytosis was assessed by staining the co-cultures for MPO activity as above.

Induction of the PS Recognition System in M
Treatment of M with 1,3-glucan confers PSR dependence on M phagocytosis of apoptotic PMN (25). On day 5, human Mwere treated with 1,3-glucan (25 µg/ml) for 48 h before washing twice with RPMI before treatments with LX assay of phagocytosis.

Rat Bone Marrow–Derived Macrophages: Isolation and Cell Culture
Rat bone marrow–derived M (BMDM) were isolated by standard procedures (26). Briefly, bone marrow cells from the dissected femurs of male Sprague-Dawley rats were flushed with complete medium through a 25-gauge needle to form a single-cell suspension. Cells were cultured in 75-mm tissue culture flasks in Dulbecco’s modified Eagle medium (DMEM) containing 2 mM glutamine, 10% fetal calf serum (FCS), 1% penicillin-streptomycin, and 10% conditioned medium from L929 cells as a source of macrophage-colony stimulating factor (M-CSF). After 7 d, cells were plated into 24-well plates at a concentration of 5 x10⁵ cells/well and rested for 24 h in M-CSF–free medium. Cells were stimulated with murine TNF- (10 ng/ml for 24 h), IFN-/TNF- (10 ng/ml for 4 h and 10 ng/ml for 20 h), or TGF- (5 ng/ml for 24 h) before LXA₄ treatment (1 nM for 15 min). Phagocytosis of aged human PMN was assayed as above.

Assay of TGF-₁ and MCP-1 Release from M
TGF-₁ and MCP-1 were assayed in supernatants from co-cultures of PMN-M by ELISA according to the manufacturer’s instruction (R&D systems).

Assessment of LX-Stimulated M Phagocytosis of Apoptotic PMN In Vivo
Ten-week-old BALB/C mice were housed and treated under UK Home Office-approved conditions in University of Edinburgh, Faculty of Medicine animal facility. An enriched population of inflammatory M was induced in the peritoneal cavity of groups of male BALB/C adult mice by a single intraperitoneal injection of 1 ml of 3% (wt/vol) thioglycollate 5 d before assay (3). On day 4, human PMN were isolated from peripheral blood of healthy volunteers. PMN were labeled with CM-orange (CMO, cell tracker orange) and aged in culture for 24 h to render a portion apoptotic. LXA₄, LXB₄, 15(R/S)-methyl-LXA₄, 16-phenoxy-LXA₄, and 15-epi-LXB₄ were administered intraperitoneally 15 min before instillation of a bolus of labeled, apoptotic PMN (approximate 3:1 ratio of PMN to M) (3). After 30 min, mice were sacrificed and peritoneal cells were harvested by lavage. Cytospin preparations of the lavages were fixed, and the number of Mcontaining ingested CMO-positive, apoptotic PMN was determined by fluorescence microscopy and expressed relative to total number of Mrecovered.

Statistics
Statistical significance was determined by t tests or one-way ANOVA where appropriate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both Lipid and Peptide Agonists for LX Receptors Stimulate Non-Phlogistic Phagocytosis of Apoptotic PMN
Figure 1A shows LX-stimulated M phagocytosis of apoptotic PMN as demonstrated by electron microscopy. As previously reported for LXA₄ in vitro (16), LXB₄ stimulated phagocytosis of apoptotic PMN in this model system, indicating that this pro-resolution effect is shared by the two major LX generated endogenously in mammalian systems. LXA₄ and LXB₄ demonstrated similar dose-response relationships with an EC₅₀ of 0.5 x 10^-9 M and a maximum stimulation observed at 1 nM (Figure 1B). Interestingly, additivity was not observed with simultaneous exposure of M to LXA₄ and LXB₄ (data not shown), indicating the possibility of a common signaling pathway resulting in enhanced phagocytosis. Aspirin acetylation of cyclooxygenase-2 (COX-2) promotes the formation of 15 epi-LX in the context of neutrophil-endothelial cell interactions. The aspirin-triggered epi lipoxins (ATL) are implicated in the antiinflammatory actions of aspirin in vivo. In this study, the ATL 15 epi-LXA₄ also stimulated macrophage phagocytosis (Figure 1B). Recent data indicate that, in addition to LXA₄ and the stable synthetic analogues binding to the ALXR, specific peptides can also bind and elicit responses (19,27). We assessed the effects of such agents on stimulation of M phagocytosis of apoptotic PMN. Treatment of M for 15 min with either MHC bp or MMK-1 at 1 nM significantly enhanced phagocytosis (2-fold and 1.7-fold, respectively) in vitro, and these actions were not additive with LXA₄ (Figure 1C) when used at a suboptimal concentration (0.5 nM)

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Lipoxins (LX), aspirin-triggered LX (ATL), and peptide agonists promote non-phlogistic macrophage (M) phagocytosis of apoptotic polymorphonuclear neutrophils (PMN). (A) M were treated with LXA4 (1nM for 15 mins) before coincubation with aged PMN for 30 min. Electron microscopy was performed as described in Materials and Methods. (B) LXB4 and the aspirin-triggered 15-epi-LXB4 stimulate Mphagocytosis. M were treated with LXB4 (1 nM) or 15-epi-LXB4 (1 x10-11 M) for 15 min before coincubation with aged PMN for 30 min. Data are fold induction ± SEM (n = 4);*P < 0.05 versus control. (C) Peptide agonists of LXA4 receptor (ALXR) stimulate Mphagocytosis. M were treated with MHC bp or MMK-1 synthetic peptide (1 nM for 15 min) alone or in combination with LXA4 (1 nM) for 15 min before coincubation with aged PMN for 30 min. Data are mean ± SEM (n = 4);*P < 0.05 versus control. (D) LXA4 and peptide agonists stimulate the release of transforming growth factor–1 (TGF-1). M were treated with LXA4, MHC bp, and MMK-1 (1 nM) for 15 min at 37°C before coincubation with apoptotic PMN. After 30 min, supernatants were collected and TGF-1 production was measured by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean TGF-1 production (pg/ml) ± SD. (E) LXA4 and peptide agonists do not provoke release of MCP-1. M were treated with LXA4, MHC bp, and MMK-1 (1 nM) for 15 min at 37°C before coincubation with apoptotic PMN. After 30 min, supernatants were collected and MCP-1 production was measured by ELISA. Data are expressed as mean MCP-1 production (pg/ml) ±SD.

LX-Stimulated Phagocytosis: The Role of cAMP, PKC, PI 3-kinase, and MAP Kinase
The signal transduction pathways mediating Mphagocytosis of apoptotic PMN are poorly understood. A role for cyclicAMP in this process has been demonstrated. Consistent with previous findings for LXA₄ (16), LXB₄-stimulated phagocytosis was inhibited by the cell-permeable analogue 8-Br-cAMP and mimicked by the PKA inhibitor Rp-cAMP (vehicle, 34.5 ± 5.35; LXB₄, 64.5 ±7.5;* 8-Br-cAMP, 21.6 ± 3.2; 8-Br-cAMP + LXB₄, 17.63 ± 2.7; Rp-cAMP, 50.0 ± 2.98;* Rp-cAMP +LXB₄, 68.0 ± 5.3;* data are % phagocytosis ±SEM; n = 4 *P < 0.05 versus control).

Given the important role of PI 3-kinase, PKC and erk as modulators of M cytoskeletal and/or adhesive events critical to phagocytic activity (1), we explored their role in LX-stimulated phagocytosis of apoptotic PMN. Previous exposure of M to the PKC inhibitor GF 109203X blunted LX-triggered phagocytosis (Table 1). The PI 3-kinase inhibitor LY 294002 also reduced LX-stimulated phagocytosis. In contrast, the MAP kinase inhibitor PD 98059 was without effect on LX-stimulated phagocytosis (control, 11.5 ± 3.4; LXB₄, 21.5 ± 5.3; PD, 11.3 ± 3.6; PD +LXB₄, 22.1 ± 4.6; % phagocytosis ± SEM). Interestingly, the serine-threonine phosphatase inhibitor okadaic acid inhibited LX-stimulated phagocytosis, suggesting a modulatory role for phosphatases in the resolution process (Table 1).

Enhancement of M Phagocytosis of Apoptotic PMN In Vitro: A PSR-Independent Mechanism

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. LX-stimulated phagocytosis is phosphatidylserine receptor–independent. (A) Phosphatidylserine receptor (PSR) antisera did not block LX-stimulated phagocytosis. M were treated with PSR antisera (200 µg/ml) or isotype control Ab on ice for 30 min before treatment with LXA4 (1 nM for 15 mins at 37°C) and subsequent coincubation with apoptotic PMN. Data are mean % phagocytosis ± SEM (n = 3);*P < 0.05. (B) 1,3-glucan treatment abolishes sensitivity of M to LX-stimulated phagocytosis. M were treated with 25 µg/ml 1,3-glucan for 48 h before treatment with LXA4 and LXB4 (1 nM for 15 mins at 37°C) and coincubation with apoptotic PMN for 30 min. Data are expressed as mean phagocytosis ± SEM (n = 4);*P < 0.05 versus control. (C) PSR Ab and 1,3-glucan treatment of M elicits TGF-1 release. TGF-1 release from M treated with control, PSR Ab (200 µg/ml), isotype control Ab, and 1,3-glucan (25 µg/ml) for 18 h at 37°C was determined by ELISA. Data are expressed as mean cytokine production ±SD.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. LX stimulate phagocytosis of bone marrow–derived M (BMDM) under basal conditions and after cytokine programming. (A) BMDM were treated with LXA4 (1 nM for 24 h), 15(R/S)-methyl-LXA4, and 16-phenoxy-LXA4 (1 x10-11 M for 24 h). BMDM were treated with (B) tumor necrosis factor– (TNF-) (10 ng/ml for 24 h) or (C) interferon- (IFN-)/TNF- (10 ng/ml for 4 h and 10 ng/ml for 20 h) before treatment with LXA4 (1 nM for 15 mins), 15(R/S)-methyl-LXA4 or 16-phenoxy-LXA4 (1 x10-11 M for 15 mins). After 30-min coincubations with aged PMN, phagocytosis was assessed by staining the cocultures for MPO activity. A minimum of 400 cells were counted to determine the number of M that had ingested one or more PMN. Results are expressed as fold/basal phagocytosis ± SEM (n = 4);*P < 0.05 versus control.

LX Stimulate M Phagocytosis of Exogenously Administered Apoptotic PMN In Vivo
To determine if LX stimulate M phagocytosis of apoptotic PMN in vivo, we employed the mouse (BALB/C) thioglycollate-elicited peritonitis model (3). An enriched population of inflammatory M were elicited with thioglycollate, and mice were then treated with a bolus intraperitoneal injection of LXA₄ (1 or 2 µg for 15 min) before instillation of CMO-labeled apoptotic human PMN. It was necessary to use this approach given the difficulties in assessing endogenous generation and processing of apoptotic cells during acute inflammation. Thiry minutes after the instillation of apoptotic PMN intraperitoneally, lavaged cells were assayed by fluorescence microscopy. Figure 4A shows CMO-labeled apoptotic PMN, autofluorescence lavaged macrophages, and lavaged macrophages from LX-treated mice. LXA₄ (1 µg and 2 µg) stimulated a 1.8-fold and a 2.2-fold increase in phagocytosis, respectively, as demonstrated in Figure 4B. Significant enhancement of phagocytosis was also seen in LXA₄-treated mice when lavages were performed 15 min and 30 min after the instillation of apoptotic PMN (Figure 4C). The ability of LXB₄, the 15 epimeric ATL, and stable synthetic LXA₄ analogue to influence clearance of apoptotic PMN at an inflammatory site was also investigated. As seen with LXA₄, LXB₄ (1 µg) promoted phagocytosis in vivo an action shared with the aspirin-triggered epimer 15-epi-LXB₄ (0.5 µg) and 15(R/S)-methyl-LXA₄ (0.5 µg), which secured a twofold increase in phagocytosis (Figure 5). In aggregate, these data provide the first demonstration that native LX, ATL, and LX stable analogues promote clearance of apoptotic PMN in vivo and support their role as rapidly acting endogenous pro-resolution agents in inflammation.

View larger version (43K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. LX rapidly stimulate M phagocytosis of apoptotic PMN in vivo. BALB/C adult mice were treated with thioglycolate (3%) for 5 d before intraperitoneal administration of LX or vehicle (15 min) before instillation of aged human CMO-labeled PMN. Cytospin preparations of peritoneal lavages were prepared, and ingested PMN was quantified by fluorescent microscopy. In each experiment, a minimum of 400 cells was counted and the number of M containing one or more apoptotic PMN was expressed as a percentage of the total number of M. (A) Upper panel, CMO-labeled aged PMN;middle panel, auto-fluorescent lavaged macrophages; lower panel, LXA4-stimulated M phagocytosis of apoptotic PMN in vivo; the thin arrows point to ingested PMN. (B) LXA4 stimulates phagocytosis in vivo. A 250-µl bolus of vehicle or LXA4 (1 µg and 2 µg) was administered intraperitoneally for 15 min before a 30-min PMN coincubation. Phagocytosis was assessed as described above. Data are mean % phagocytosis ± SEM (n = 3); *P < 0.005 versus control. (C) LXA4 significantly stimulates phagocytosis after 15-min and 30-min coincubation with excess apoptotic PMN. Mice were treated with 1 µg LXA4 for 15 min before either a 15-min or a 30-min coincubation with apoptotic PMN in vivo. Phagocytosis was assayed as described above. Data are expressed as mean % phagocytosis ± SEM (n = 3);*P < 0.005;**P < 0.0005 versus control.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. LX-stimulated Mphagocytosis of apoptotic PMN in vivo. An action shared with the ATL and the LX stable analogue. A 250-µl bolus of control, LXB4 (1 µg,), 15(R/S)-methyl-LXA4 (0.5 µg), and 15-epi-LXB4 (0.5 µg) was given intraperitoneally for 15 min before a 30-min PMN coincubation. Phagocytosis was determined as described in Figure 4. Data are expressed as mean % phagocytosis ± SEM (n = 3 to 5);*P < 0.05;***P < 0.0005 versus control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

v3

To investigate the mechanism whereby LX-stimulated Mphagocytosis of apoptotic PMN, we have used in vitro models: LX-stimulated human M and cytokine-primed rat BMDM. In preliminary experiments, we investigated whether LX might stimulate phagocytosis of targets other than apoptotic PMN, namely latex beads and apoptotic lymphocytes. However, in our experiments the level of phagocytosis observed basally with such materials is extremely high, and it was not possible to observe an incremental increase in response to LX.

BMDM are essentially uncommitted phagocytes that can develop phenotypically distinct properties after cytokine programming, resulting in the development of inflammatory (TGF-), cytotoxic (IFN-/TNF-), and reparative (TNF-) phenotypes (30). Exposure of TNF-–programmed M to LX further enhances their ability to phagocytose apoptotic PMN, whereas LX rescues the compromised phagocytic activity of TNF-– and IFN-–primed M. These data suggest that LX may stimulate PMN clearance by M within the complex multi-mediator milieu that typifies inflammation in vivo.

To identify the signaling molecules involved in LX-stimulated phagocytosis, we have employed modulators of pathways implicated in LX signaling (15,33) and/or phagocytosis (reviewed in reference 34). We demonstrate a role for PKC and PI 3-kinase in LX-stimulated phagocytosis. It is likely that LXA₄ and LXB₄ stimulate phagocytosis through novel PKC isoforms (35) on the basis of the contrasting actions of the two LX on calcium mobilization (36). The 3'-phosphorylated substrates of PI 3-kinase are important regulators of several downstream components of phagocytosis, including Rho activation, a consequence of LX stimulation of M(37).

A modulatory role for cAMP is suggested by our observations that both LXA₄- (16) and LXB₄-stimulated phagocytosis was inhibited by cell-permeable 8 Br-cAMP and mimicked by a PKA inhibitor. In this context, it is noteworthy that phosphorylation of CD36 by PKA may reduce its ability to bind apoptotic cells (16,38);furthermore, phagocytosis is dependent on cytoskeletal rearrangements, which are regulated by monomeric GTPases, including Rho (39), and Rho activation is negatively regulated by PKA (40).

The cell surface molecules involved in M-apoptoticyte recognition include lectins, integrins, such as M_v3 that co-operate with CD36, PSR, scavenger receptors, and CD14 and specific bridging molecules, including thrombospondin and C1q (1). The ligand for Mreceptors on apoptotic cells are less well documented; activation of a phospholipid scramblase in apoptotic cells results in exposure of phosphatidylserine (41), a ligand for the recently described M PSR (23). PS-dependent phagocytosis of apoptotic leukocytes by mouse thioglycollate-elicited peritoneal M has previously been shown, whereas quiescent M were reported to utilize an _v3-dependent mechanism (1). Recent data indicate that phagocytosis by either activated or quiescent M is PSR-dependent (23,29). Here we report that in monocyte-derived M LX-stimulated phagocytosis required the _v3-CD36 complex but was independent of the PSR. This conclusion is based on sensitivity to anti-_v3 and CD36 antisera and insensitivity to PSR antisera. Further evidence of PSR independence are provided from 1,3-glucan–treated M. This maneuver subverts the _v3-CD36 system to a PS-dependent mechanism (25). LX-dependent phagocytosis was not observed in 1,3-glucan–treated M (importantly, the previously documented effects of stimulation of M with PSR antisera and with 1,3-glucan on stimulating TGF-release (25,28,42) were observed). Therefore, LX-stimulated phagocytosis of apoptotic cells is not necessarily dependent on the PSR. An analogous situation has recently been described by Cocco and Ucker (43) and for dexamethasone-augmented phagocytosis (44). A role for PS in LX-stimulated phagocytosis is not excluded as PS may bind to alternative receptors, e.g., a ligand for the Axl/Tyro3 Mer family of receptor tyrosine kinase growth arrest specific gene 6 (GAS 6) mediates PS binding to M(45), and PS facilitates CD36-TSP-_v3–mediated uptake of apoptotic cells (25).

The LXA₄ receptor (ALXR) (also known as the FPRL-1 receptor) is a G-protein–coupled receptor (46) expressed in diverse cell types (4). LXB₄ does not compete with [³H]LXA₄ for binding at the ALXR in PMN (47). In addition to binding LXA₄ and its analogues, the ALXR has recently been shown to bind specific peptides (19,27), the acute phase protein serum amyloid A (SAA) (48), prion protein (49), and amyloid (50). More recently, an additional receptor for peptide agonists has been described, namely FPRL-2. FPRL-2 is expressed on monocyte/macrophages but not PMN (51). LX and its analogues act via the ALXR to inhibit PMN chemotaxis. In contrast to the inhibitory effects of LX on PMN function, LX have been reported to activate monocyte chemotaxis, and this effect is shared with the ALXR peptide ligands (18). This contrasting data obtained from peptide and LX ligands in monocytes and PMN suggests involvement of the FPRL-2 receptor (19,27,51). It was of interest to investigate whether peptide agonists of ALXR could mimic the effects of LX on phagocytosis of apoptotic PMN. The peptides investigated were an MHC bp derived from mitochondrial NADPH dehydrogenase and a synthetic rogue peptide (MMK-1) (19,27). Exposure of M to either peptide stimulated phagocytosis to levels comparable to those seen with LX. Interestingly, additivity was not observed on addition of suboptimal concentrations of LX and peptides together, suggesting a common mechanism. The release of N-formylated peptides from mitochondria of damaged cells is a signal for PMN chemotaxis. On the basis of our data, we propose that MHC bp and MMK-1 peptides might also act to stimulate macrophage phagocytosis of apoptotic cells via the ALXR or the more recently described FPRL-2 (51). We have verified the potential for peptide-activated phagocytic activity in the resolution of inflammation, showing that it is coupled to TGF-₁ release.

Our data demonstrating LX-stimulated phagocytosis of apoptotic PMN in vitro suggest a role for this process in promoting the resolution of inflammation in vivo. Consistent with this hypothesis are the data that demonstrate that LXA₄ and LXB₄ stimulated phagocytosis of human apoptotic PMN in the murine BALB/C model of thioglycollate-induced peritonitis, an established model of inflammation (3,52). Furthermore, the aspirin-triggered 15-epi-LXB₄ and the stable LX analogue 15(R/S)-methyl-LXA₄ (19) also stimulated phagocytosis. The enhanced efficacy and stability of LX analogues in modulation of leukocyte trafficking in models of inflammation and second organ injury after local and systemic administration has led to the proposal that these compounds might have therapeutic potential (17–20). Consistent with this are our data that highlight the attractiveness of LX stable analogues, which are resistant to metabolic inactivation by monocytes/macrophages yet retain the activity of the native LX (17,19,21). Comparable stimulation of phagocytosis was observed with 0.5-µg doses of 15(R/S)-methyl-LXA₄ and 15-epi-LXB₄ to that seen with 2-µg doses of the native LXA₄. However, it was not feasible to investigate detailed dose-response relationships; therefore, conclusions about relative potency and efficacy in this model cannot be made. In previous reports (19), 10 µg of intravenously administered ATL were found to be effective in modulating leukocyte trafficking; it is obviously difficult to make a direct comparison between the availability via the different routes of administration. As described previously (see above), aspirin acetylation of COX-2 promotes the biosynthesis of epimeric LX (18), which can act locally to downregulate leukocyte activity (18–20) and, as suggested by our data, promote the clearance of apoptotic PMN from an inflammatory site. This may be particularly relevant in inflammation, where COX-2 expression is upregulated (4) and may have clinical implications for the use of COX-2 inhibitors, which leave the 15 lipoxygenase activity of the enzyme intact.

	Acknowledgments

 
We are grateful to Valerie Fadok for providing PSR antisera. We thank Paola Maderna for helpful comments during the preparation of this manuscript. Work in the authors’ labs are supported by the Wellcome Trust (047273 [JS]; 058601 [HRB & CG]), the Health Research Board Ireland (CG, HRB), Enterprise Ireland (CG, HRB), the Punchestown Renal Research Fund (HRB), the National Kidney Research Fund (R22/1/99 [JS]), the EU (JS,CG,HRB), and the NIH (P01DE13499 [NP]).

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Savill J, Fadok VA: Corpse clearance defines the meaning of cell death. Nature 407: 784–787, 2000[CrossRef][Medline]
2. Johnson R, Klebanoff SJ, Couser WG: Cellular mediators of immune injury: Neutrophils, In: Immunologic Renal Diseases,edited by Neilson EG, Couser WG: Philadelphia, Lippincott-Raven, 1997, pp 547–560
3. Taylor PR, Carugati A, Fadok VA, Cook HT, Andrews M, Carroll MC, Savill JS, Henson PM, Botto M, Walport MJ: A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo. J Exp Med 192: 3359–3366, 2000
4. McMahon B, Mitchell S, Brady HR, Godson C: Lipoxins: Revelations on resolution. TiPS 22: 391–395, 2001
5. Serhan CN, Hamberg M, Samuellson B: Lipoxins: Novel series of biologically active compounds formed from arachidonic acid in human leukocytes. Proc Natl Acad Sci USA 81: 5335–4339, 1984[Abstract/Free Full Text]
6. Papayianni A, Serhan CN, Philips ML, Rennke HG, Brady HR: Transcellular biosynthesis of Lipoxin A₄ during adhesion of platelets and neutrophils in experimental immune complex glomerulonephritis. Kidney Int 47: 1295–1302, 1994
7. Papayianni A Serhan, CN and Brady HR Lipoxin A₄ and B₄ inhibit leukotriene-stimulated interactions of human neutrophils and endothelial cells. J Immunol 156: 2264–72, 1996[Abstract]
8. Mayadas TN, Mendrick DL, Brady HR, Tang T, Papayianni A, Assmann K: Acute passive anti-glomerular basement membrane nephritis in P-selectin deficient mice. Kidney Int 49: 1342–1349, 1996[Medline]
9. Munger KA, Montero A, Fukunaga M, Uda S, Yura T, Imai E, Kaneda Y, Valdivielso JM, Badr KF: Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis. Proc Natl Acad Sci USA 96: 13375–13380, 1999[Abstract/Free Full Text]
10. Katoh T, Lakkis FG, Makita N, Badr KF: Co-regulated expression of glomerular 12/15-lipoxygenase and interleukin-4 mRNAs in rat nephrotoxic nephritis. Kidney Int 46: 341–349, 1994[Medline]
11. Hachicha M, Pouliot M, Petasis NA, Serhan CN: Lipoxin [LX] A₄ and aspirin-triggered 15-epi-LXA₄ inhibit tumor necrosis factor alpha-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J Exp Med 189: 1923–1929, 1999[Abstract/Free Full Text]
12. Gronert K, Gewirtz A, Madara JL, Serhan CN: Identification of a human enterocyte lipoxin A₄ receptor that is regulated by interleukin[IL]-13 and interferon- and inhibits tumor necrosis factor -induced IL-8 release. J Exp Med 187: 1285–1294, 1998[Abstract/Free Full Text]
13. Sodin-Semrl S, Taddeo B, Tseng D, Varga J, Fiore S: Lipoxin A₄ inhibits IL-1 -induced IL-6, IL-8 and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases. J Immunol 164: 2660–2666, 2000[Abstract/Free Full Text]
14. Bandeira-Melo C, Bozza PT, Diaz BL, Cordeiro RS, Jose PJ, Martins MA, Serhan CN: Cutting Edge: Lipoxin (LX) A₄ and aspirin-triggered 15-epi-LXA₄ block allergen induced eosinophil trafficking. J Immunol 164: 2267–2271, 2000[Abstract/Free Full Text]
15. McMahon B, Stenson C, McPhilips F, Fanning A, Brady H, Godson C: Lipoxin A₄ antagonizes the mitogenic effects of leukotriene D₄ in human renal mesangial cells. J Biol Chem 275: 27566–27575, 2000[Abstract/Free Full Text]
16. Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR: Cutting edge: Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 164: 1663–1667, 2000[Abstract/Free Full Text]
17. Maddox JF, Colgan SP, Clish CB, Petasis NA, Folkin VV, Serhan CN: LXB₄ regulates human monocyte/neutrophil adherence and motility: design of stable LXB₄ analoges with increased biological activity. FASEB J 12: 487–494, 1998[Abstract/Free Full Text]
18. Claria J, Serhan CN: Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc Natl Acad Sci USA 92: 9475–9479, 1995[Abstract/Free Full Text]
19. Chiang N, Fierro IM, Gronert K, Serhan CN: Activation of lipoxin A₄ receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation. J Exp Med 191: 1197–1207, 2000[Abstract/Free Full Text]
20. Clish CB, O’Brien JA, Gronert K, Stahl GL, Petasis NA, Serhan CN: Local and systemic delivery of a stable-aspirin-triggered lipoxin prevents neutrophil recruitment in vivo. Proc Natl Acad Sci USA 96: 8247–8252, 1999[Abstract/Free Full Text]
21. Serhan CN, Maddox JF, Petasis NA, Akritopoulou-ZanzeI, Papayianni A, Brady HR, Colgan SP, Madara JL: Design of Lipoxin A4 stable analogues that block transmigration and adhesion of human neutrophils. Biochemistry 34: 14609–14615, 1995[CrossRef][Medline]
22. Savill JS, Hogg N, Ren Y, Haslett C: Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 90: 1513–15, 1992
23. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekwitz RAB, Henson PM: A receptor for phosphatidylserine specific clearance of apoptotic cells. Nature 405: 85–90, 2000[CrossRef][Medline]
24. Rossi AG, McCutcheon J, Roy N, Chilvers ER, Haslett C, Dransfield I: Regulation of macrophage phagocytosis of apoptotic cells by cAMP. J Immunol 160: 3562–3568, 1998[Abstract/Free Full Text]
25. Fadok VA, Warner ML, Bratton DL, Henson PM: CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor [_{v: 3}]. J Immunol 161: 6250–6257, 1998[Abstract/Free Full Text]
26. Lake FR, Nobel PW, Henson PM, Richies DW: Functional switching of macrophage responses to tumor necrosis factor- (TNF-) by IFNs: Implications for the pleiotropic activities of TNF-. J Clin Invest 93: 1661–16, 1994
27. Klein C, Paul JI, Sauve K, Schmidt MM, Arcangeli L, Ransom J, Trueheart J, Manfredi JP, Broach JR, Murphy AJ: Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nature Biotechnol 16: 1334–1337, 1998[CrossRef][Medline]
28. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM: Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF, PGE₂, and PAF. J Clin Invest 101: 890–898, 1997
29. Fadok V, deCathelineau A, Daleke DL, Henson PM, Bratton DL: Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J Biol Chem 276: 1071–1077, 2001[Abstract/Free Full Text]
30. Erwig LP, Kluth DC, Walshe GM, Rees AJ: Initial cytokine exposure determines function of macrophages and renders them unresponsive to other cytokines. J Immunol 161: 1983–1988, 1998[Abstract/Free Full Text]
31. Savill J, Smith J, Sarraf C, Ren Y, Abbott F, Rees A: Glomerular mesangial cells and inflammatory macrophages ingest neutrophils undergoing apoptosis. Kidney Int 42: 924–936, 1992[Medline]
32. Botto M, Dell’Agnola C, Bygrave AE, Thompson EA, Cook HT, Petry F, Loos M, Pandolfi PP, Walport MJ: Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nature Genetics 19: 56–59, 1998[CrossRef][Medline]
33. Maderna P, Godson C, Hannify G, Murphy M, Brady HR: Influence of lipoxin A₄ and other lipoxygenase-derived eicosanoids on tissue factor expression. Am J Physiol Cell 279: 945–953, 2000
34. Kwiatkowska K, Sobota A: Signalling pathways in phagocytosis. Bioessays 21: 422–431, 1999[CrossRef][Medline]
35. Larsen EC, DiGennaro J, Saito N, Mehta S, Loegering DJ, Mazurkiewicz JE, Lennartz MR: Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells. J Immunol 165: 2809–2817, 2000[Abstract/Free Full Text]
36. Romano M, Maddox J, Serhan CN: Activation of human monocytes and the acute monocytic leukemia cell line (THP-1) by lipoxins involves unique signaling pathways for LXA₄ versus LXB₄. J Immunol 157: 2149–2154, 1996[Abstract]
37. Maderna P Cottell, D. Berlasconi G Petasis, NA Brady HR, Godson C: Lipoxins induce actin rearrangement in monocytes and macrophages but not in neutrophils. Am J Pathol 160: 2275–2283, 2002[Abstract/Free Full Text]
38. Ren Y, Silverstein RL, Allen J, Savill JS: CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis. J Exp Med 181: 1857–1862, 1995[Abstract/Free Full Text]
39. Hall A, Aderem A: Mechanisms of phagocytosis. Curr Opin Immunol 8: 36–40, 1996[CrossRef][Medline]
40. Lang P, Gerbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M, Bertoglio J: Protein kinase A phosphorylation of Rho A mediates the morphological and functional effects of cAMP in cytotoxic lymphocytes. EMBO J 15: 510–519, 1996[Medline]
41. Frasch SC, Henson PM, Kailey JM, Janes DM, Fadox VA, Bratton DL: Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase C delta. J Biol Chem 275: 23065–23073, 2000[Abstract/Free Full Text]
42. McDonald PP, Fadok VA, Bratton D, Henson PM: Transcriptional and translational regulation of inflammatory mediated production by endogenous TGF- in macrophages that have ingested apoptotic cells. J Immunol 163: 6164–6171, 1999[Abstract/Free Full Text]
43. Cocco RE, Ucker DS: Distinct modes of macrophages recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure. Mol Biol Cell 12: 919–930, 2001[Abstract/Free Full Text]
44. Giles KM, Ross K, Rossi AG, Hotchin NA, Haslett C, Dransfield I: Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/pyk2 phosphorylation, and high levels of active Rac. J Immunol 167: 976–986, 2001[Abstract/Free Full Text]
45. Scott R, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Shelton Earp H, Matsushima GK: Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411: 207–210, 2001[CrossRef][Medline]
46. Fiore S, Maddox J, Perez HD, Serhan CN: Identification of a human cDNA encoding a functional high affinity lipoxin A₄ receptor. J Exp Med 180: 253–260, 1994[Abstract/Free Full Text]
47. Fiore S, Ryeom SW, Weller PF, Serhan CN: Lipoxin recognition sites. Specific binding of labeled Lipoxin _A4 with human neutrophils. J Biol Chem 267: 16168–16179, 1992[Abstract/Free Full Text]
48. Su SB, Gong W, Gao JL, Shen W, Dunlop NM, Murphy PM, Oppenheim JJ, Wang JM: A seven transmembrane, G- protein coupled receptor, FPRL1 mediates the chemotactic activity of serum amyloid A for human phagocytic cells. J Exp Med 189: 395–402, 1999[Abstract/Free Full Text]
49. Yingying L, Yazawa H, Gong W, Yu Z, Ferrans VJ, Murphy PM, MingJ: Cutting Edge: The neurotoxic prion peptide fragment PrP_106–126 is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1. J Immunol 166: 1448–1451, 2001[Abstract/Free Full Text]
50. Le Y, Gong W, Tiffany HL, Tumanov A, Nedospasov S, Shen W, Dunlop NM, Gao JL, Murphy PM, Oppenheim JJ, Wang JM: Amyloid (beta)42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1. J Neurosci 2, RC123, 2001
51. Christophe T, Karlsonn A, Dugave C, Rabiet MJ, Boulay F, Dahlgren C: The synthetic peptide Trp-Lys-Try-Met-Val-Met-NH₂ specifically activates PMN through FPRL1/LXA₄R and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2. J Biol Chem 276: 21585–21593, 2001[Abstract/Free Full Text]
52. Huynh, M-L, Fadok, VA Henson, P Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-1 secretion and the resolution of inflammation. J Clin Invest 109: 41–50, 2002[CrossRef][Medline]

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