A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation - PubMed (original) (raw)

A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation

Susan Sather et al. Blood. 2007.

Abstract

Membrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of the extracellular domain or alternative mRNA splicing yielding a secreted protein. Mertk (Mer) is in a receptor tyrosine kinase family with Axl and Tyro-3, and all 3 receptors share the Gas6 ligand. Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo. We have found that the membrane-bound Mer protein is cleaved in the extracellular domain via a metalloproteinase. The cleavage results in the production of a soluble Mer protein released in a constitutive manner from cultured cells. Significant amounts of the soluble Mer protein were also detected in human plasma, suggesting its physiologic relevance. Cleavage of Mer was enhanced by treatment with LPS and PMA and was specifically inhibited by a tumor necrosis factor alpha-converting enzyme metalloproteinase inhibitor. As a decoy receptor for Gas6, soluble Mer prevented Gas6-mediated stimulation of membrane-bound Mer. The inhibition of Gas6 activity by soluble Mer led to defective macrophage-mediated engulfment of apoptotic cells. Furthermore, soluble Mer decreased platelet aggregation in vitro and prevented fatal collagen/epinephrine-induced thromboembolism in mice, suggesting a potential therapeutic use for soluble Mer in the treatment of clotting disorders.

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Figures

Figure 1

Figure 1

Mer extracellular domain is released into the medium of cultured cell lines. (A) J774 (mouse) or (B) U937 (human) monocytic cells were grown overnight in serum-free medium. CM was collected and concentrated 10-fold, cells were lysed, and a sample of each CM was deglycosylated with PNGase F. (C) Concentrated CM from overnight cultures of human cell lines. (D) Concentrated CM from HSB-2 and U937 cells untreated and digested with PNGase F. Cell lysates (cells), CM, and PNGase-digested medium (CM + PNG) were analyzed by SDS-PAGE and immunoblotting with antibodies against mouse (A) or human (B-D) Mer extracellular domain. Untreated sMer (sMer) and deglycosylated sMer (degly sMer) are indicated in panel D.

Figure 2

Figure 2

sMer is shed from primary cells in culture and is detected in mouse and human blood. (A) Elicited peritoneal macrophages and dissociated splenocytes from wild-type mice were cultured in serum-free DMEM for 7 hours. Cell lysates and conditioned medium from the macrophages and splenocytes, and 5 μL serum from wild-type mice or mice with the Mer gene disrupted (KD) were analyzed by SDS-PAGE and immunoblotting. (B) Human monocyte-derived macrophages (Μφ), MVECs, platelets, and plasma samples were examined for the presence of Mer by Western blotting. Cultured monocyte-derived macrophages and MVECs express 205- and 185-kDa Mer glycoform, respectively, and sMer was present in CM from these cells. A 165-kDa Mer protein was detected in pelleted platelets, and sMer extracellular domain proteins of 110 to 140 kDa were abundant in pooled plasma from healthy donors (George King Bio-Medical, Overland Park, KS) and in plasma from free-flowing blood (plasma), and subsequently after clotting, in serum from the same donor (serum).

Figure 3

Figure 3

LPS or PMA stimulates release of Mer ectodomain. Surface expression of Mer on J774 cells treated with 50 ng/mL LPS for 4 hours (A) or treated with 50 nM PMA for 45 minutes (B) was evaluated by flow cytometry. Expression on untreated cells at each time point is shown for comparison. (C) J774 cells were incubated in serum-free medium with or without 50 ng/mL LPS or (D) treated with 50 nM PMA for the indicated times. Mer and actin present in cell lysates and sMer released into the medium at each time point were analyzed by immunoblotting. (E) The carboxyl-terminal portion of cleaved Mer remains cell-associated. Elicited peritoneal macrophages from MerKD or wild-type mice were cultured overnight, then placed in serum-free medium and treated with 100 ng/mL LPS for the indicated times. Cells were lysed, and proteins from equal numbers of cells were separated by SDS-PAGE and immunoblotted with an antibody against the C-terminus of Mer. Membrane-bound Mer receptor (200 kDa), which is absent from MerKD macrophages, decreases with time of LPS treatment. The C-terminal 60-kDa fragment of Mer that accumulates with LPS treatment is indicated by an asterisk.

Figure 4

Figure 4

LPS-induced production of sMer is independent of protein synthesis and is blocked by metalloproteinase inhibitors. (A) A 6-cm plate of J774 cells was cultured in the presence of 10 μg/mL cycloheximide for 30 minutes, rinsed with serum-free medium, and then incubated in serum-free medium containing 10 μg/mL cycloheximide and 50 ng/mL LPS for 1 hour. Two additional untreated plates were incubated in serum-free medium with or without 50 ng/mL LPS for 1 hour. sMer released into the medium and Mer and p53 in cell lysates were monitored by Western blot. (B) J774 cells were treated with 50 ng/mL LPS to stimulate the cleavage of the Mer extracellular domain. EDTA (5 mM), 200 μM TAPI-0, or DMSO (vehicle used to dissolve TAPI) was added to cell cultures as indicated for 2 hours. Mer remaining in cells and sMer released into the medium after each treatment were detected by Western blot.

Figure 5

Figure 5

The soluble ectodomain of Mer binds to Gas6 and inhibits Gas6 signaling. (A) Gas6/Mer complexes in wild-type C57Bl/6 serum were detected by immunoprecipitating with a mouse anti-Gas6 antibody and by performing a Western blotting with an anti–mouse Mer antibody. The Gas6/Mer complexes were not observed in the control serum from C57Bl/6 MerKD mice. (B) A chimeric recombinant protein consisting of the mouse Mer extracellular domain fused to the Fc region of human IgG was incubated with recombinant mouse Gas6 (rmGas6). As a control for nonspecific binding, a parallel experiment was performed with a Ret receptor ectodomain/Fc chimera and mGas6. Complexes pulled down with protein G Sepharose beads were run on SDS-PAGE gels, and bound Gas6 was detected by immunoblotting with anti–mouse Gas6 antibody. Also shown is the input rmGas6. (C) J774 cells were starved in serum-free medium and then treated for 10 minutes with 200 nM mGas6 and 200 nM Mer/Fc or Ret/Fc as indicated. Cell lysates were analyzed for phospho-Mer and total Mer content. (D) J774 cells incubated with or without mGas6 and Mer/Fc as in panel C and phospho-AKT and total AKT were monitored.

Figure 6

Figure 6

Mer/Fc inhibits apoptotic cell engulfment. (A) Fluorescence analysis of phalloidin-stained J774 cells (green) engulfing Cy3-labeled apoptotic Jurkat T cells (red) in the absence (top) or presence (bottom) of 50 nM Mer/Fc. DNA is stained with Hoechst and appears blue. Bar represents 10 μm. (B) In vitro engulfment assay using J774 macrophages incubated with apoptotic Jurkat T cells in the absence or presence of increasing doses of Mer/Fc protein (n = 3) or (C) Mer/Fc plus 10 nM rhGas6 (n = 2). (D) Engulfment by cells incubated with Mer/Fc or Ret/Fc control. Results are expressed as percent of untreated control and reflect the phagocytic index of the samples assayed. The phagocytic indices of untreated controls never fell below 25.5. *P < .001.

Figure 7

Figure 7

Mer extracellular domain inhibits platelet aggregation induced by ADP and collagen and protects mice against collagen-epinephrine–induced thrombosis. (A-D) In vitro platelet aggregation was performed using human PRP and was analyzed on a BioData aggregometer. (A-B) Aggregation response of platelets in response to 2 μM ADP (A) or 4 μM ADP (B) following preincubation with different concentrations of Mer/Fc. (C) Platelet aggregation induced by 4 μM ADP after pretreatment with Ret/Fc. (D) Aggregation of platelets in response to 10 μg/mL collagen with and without preincubation with Mer/Fc. Squares on the x-axis represent 15-second intervals. Data shown are representative of 3 independent experiments. (E) Survival of mice pretreated with Mer/Fc (n = 10) or saline (PBS control, n = 8) after collagen-epinephrine injection. Protection from fatal thromboembolism by Mer/Fc was significant (P < .022, log-rank test). (F-G) Hematoxylin and eosin staining of lungs from control mouse (F) or Mer/Fc-pretreated mouse (G) after collagen-epinephrine injection. Extensive platelet thromboembolism is seen in control mice (arrows) compared to the Mer/Fc-pretreated mice. Scale bar represents 100 μm.

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