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The Mer receptor tyrosine kinase is both an important mediator of apoptotic cell phagocytosis and a regulator of macrophage and DC cytokine production. Since phenotypically distinguishable macrophages are known to have different functions, we have examined Mer expression of murine splenic macrophages. We also used serum deficient in the Mer ligand, growth arrest-specific protein 6 (Gas6) to define better the role of this Mer ligand in macrophage function. By immunofluorescence staining, we found Mer to be strongly expressed in splenic red pulp, largely on platelets. We also found Mer expression on marginal zone macrophages. Strikingly, all tingible body macrophages bore Mer. In functional phagocytosis assays of apoptotic cells, Gas6 appeared to be the sole ligand for Mer, and this system accounted for about 30% of splenic macrophage phagocytosis of apoptotic cells. Taken together, the expression pattern of mer on macrophage subpopulations in the spleen and its Gas6-dependent role in macrophage phagocytosis suggest an important role for Mer in the modulation of immune responses.
Clearance of apoptotic cells through phagocytosis plays an important role during embroygenesis and normal tissue homeostasis. The Mer receptor tyrosine kinase (RTK) is an important receptor for apoptotic cells and is essential for their normal clearance [1; 2]. Mer belongs to the Tyro-3 subfamily of RTK, with a conserved KW(I/L)A(I/L)ES signature sequence in the kinase domain. Its expression is limited to certain hematopoietic lineages, namely monocytes/macrophages, dendritic cells (DC), NK cells, megakaryocytes, platelets, and, as we have shown recently, certain activated B cells [3; 4; 5]. The expression pattern of Mer in the immune system and its function in modulating inflammatory responses have received much attention lately [2; 6; 7; 8], but its expression in intact organs of the immune system is not well understood.
The principal ligand for Mer is believed to be growth arrest-specific protein 6 (Gas6), a member of the vitamin K-dependent protein family. Gas6 binds and activates all three receptors in the Tyro-3 subfamily [9; 10; 11; 12; 13], but has a remarkably lower affinity for Mer (3–10-fold lower) [10; 14]. Gas6 shares ~40% amino acid identity and has a similar domain organization with protein S, a negative regulator of blood coagulation . Gas6 has been extensively studied and repeatedly reported to bind and activate Mer , yet the role of protein S as an additional ligand for Mer remains unclear [1; 5]. Human protein S was initially reported to activate murine Tyro-3 [9; 17]. More recently, Uehara et al found that human protein S oligomerization is required for human Mer autophosphorylation in a culture system using fetal serum .
To understand better the function of Mer in the regulation of immune system in lymphoid organs, we investigated the expression pattern of Mer in the mouse spleen and the contribution of Gas6 in mediating Mer-mediated macrophage phagocytosis. We found that Mer was present on all CD68+ macrophages and on a fraction of MZ (marginal zone) macrophages, but not on metallophilic macrophages. Interestingly, Mer mediated macrophage phagocytosis was mediated mainly through the soluble bridge ligand Gas6.
Biotin conjugated anti-mouse Mer antibody and recombinant mouse Gas6 (rmGas6) were purchased from R&D Systems (Minneapolis, MN). Anti-mouse FITC-CD11b, PE-CD11b, FITC-B220, PE-CD4, FITC-CD61, and FITC-CD11c were from BD Biosciences (San Jose, CA). Macrophage specific antibodies, PE-MARCO, FITC-MOMA-1, and PE-CD68 were obtained from Serotec Inc. (Raleigh, NC).
C57BL/6J (B6) mice (Jackson Laboratories, Bar Harbor, ME) were used as WT controls. Mer-KO mice were generated as described previously  and were 10 generations backcrossed to B6 mice. The Gas6-KO mice were generated by Dr. Carmeliet  and were subsequently bred and maintained in our mouse colony at the University of Pennsylvania or at Temple University. Recipient and donor mice were sex and age matched within each independent experiment. All of the experimental procedures performed on these animals were conducted according to the guidelines of the Institutional Animal Care and Use Committee.
Mouse spleen was snap-frozen in liquid nitrogen. Sections (5 μm) were air-dried for 10 minutes, followed by 30 minutes incubation with 1% BSA in PBS. Sample sections were then blocked with 3% BSA in PBS. Mer expression was detected by overnight incubation with biotin-labeled anti-Mer at 4°C and visualized with PE-streptavidin or FITC-streptavidin. All other antibodies were incubated with tissue sections for 4–6 hrs at 4°C. Slides were mounted in Anti-fading Aqueous Mounting Medium (Biomeda Corp., Foster City, CA), and images were acquired using an Olympus BX60 fluorescence microscope equipped with camera (Center Valley, PA).
B6 and Mer-KO mice were injected i.p. with 1 ml of 3% thioglycolate (in 1 × PBS, 3 months aged in the dark). Peritoneal macrophages were collected at day 5 with wash buffer (RPMI1640 with 2% FBS and 0.2 mM EDTA). 500μl cell suspensions were placed in 24-well plates at a concentration of 2×106 cells/ml in complete medium (RPMI with 10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 0.1% 2-ME). After 2hrs, nonadherent cells were washed off with PBS and the remaining cells used for phagocytosis assay.
Mouse blood samples from Gas6-KO and WT mice were collected by intracardiac puncture terminal bleeding under anesthesia. Mouse sera were collected after overnight clotting at 4°C. Sera were then stored at −20°C until further assay. Heat-inactivation was performed by incubating serum samples at 56°C for 30 minutes.
Single-cell thymocyte suspensions were made by pushing thymi from 4–6 weeks old B6 mice through cell strainers (50μm). After washing twice with PBS, cells were labeled with CFSE (Invitrogen, Carlsbad, CA), and then rendered apoptotic by exposure to 500 Rads of γ-irradiation followed by 4 hrs culture in complete RPMI medium. Apoptosis was verified using an Annexin-V/7-AAD Apoptosis detection kit (BD Pharmingen, San Diego, CA) . Apoptotic thymocytes were co-cultured for 4 hrs with phagocytes at various ratios as indicated. The phagocytes were extensively washed with PBS containing EDTA to remove physically bound but not internalized apoptotic cells. Percentages of macrophages that had ingested labeled thymocytes were determined by FACS analysis, with gating on CD11b-PE and CFSE.
Cell surface staining was routinely performed as previously described . Cells were blocked with 2.4G2 (anti-Fc receptor monoclonal), followed by direct incubation with labeled Abs for 40-min and washing. An additional 30-min incubation with streptavidin-PE was performed to detect biotinylated Abs. Cells were fixed in PBS containing 1% paraformaldehyde and were then analyzed on a BD Biosciences FACscan (Mountain View, CA). Relative fluorescence intensity was plotted on a logarithmic scale using CellQuest software.
Data are presented as means ± SD. Statistical significance was determined using Student’s t test.
We have previously reported the expression pattern of Mer in the hematopoietic lineages . We confirmed that Mer-KO mice (with the tyrosine kinase domain replaced by a neo cassette) fully disrupted surface expression of Mer on activated peritoneal macrophages  (Figure 1). We, therefore, used Mer-KO mice as negative controls to study the expression pattern of Mer in the spleen. As shown in Figure 2, the most intense expression of Mer was found in red pulp. This expression was colocalized to platelets (Figure 2C). Consistent with previous findings [3; 4], Mer was not expressed on T- or B- cells (Figure 2A and B). Dendritic cells (DC) and macrophages (M) expressed Mer on their surfaces (Figure 2D and E).
Within the hematopoietic lineage, the highest expression of Mer was shown on macrophages . Macrophages play an essential role in apoptotic cell clearance and antigen capture and presentation. We identified different subpopulations of macrophages using unique markers, namely MARCO for MZ macrophages, MOMA for metallophilic macrophages, and CD68 for tingible body macrophages. Strikingly, all CD68+ macrophages also expressed Mer (Figure 3A). We found a small portion of MZ macrophages expressed Mer (Figure 3B). No overlap was shown on MOMA positive macrophages (Figure 3C).
Because mouse serum is generally not used to evaluate the efficiency of phagocytosis of mouse macrophages, it was necessary to test the phagocytosis efficiency using different concentrations of mouse serum. We found that 10% mouse serum in the assay was needed for efficient macrophage phagocytosis, as in other assay systems  (Figure 4A). Therefore, we use 10% mouse serum for the rest of assays. This concentration also makes our results comparable to published data. On the other hand, heat inactivation led to a ~20% reduction in overall macrophage phagocytosis (Figure 4B left), presumably due to denaturation of complement components in the fresh serum. The relative decrease in phagocytosis that resulted from heat-inactivation of serum was not significantly different for WT and Mer-KO macrophages. FBS-mediated macrophage phagocytosis was unchanged when heat-inactivated serum was used (Figure 4B right), probably reflecting the absence of active complement in the stored FBS.
Protein S was reported as a ligand for Mer mediated signaling . We set out to test whether mouse protein S might also facilitate macrophage phagocytosis through Mer. We collected peritoneal macrophages 5-days after i.p. thioglycolate. Macrophages (CD11b+) that ingested apoptotic cells were analyzed by FACS after 4-hr co-culture with apoptotic cells. As shown in Figure 5A, macrophage phagocytosis was reduced to a similar degree (compared to wild type cells in normal serum), when either Gas6 was absent or when cells lacked the receptor Mer. The reduction of macrophage phagocytosis was apparent at all ratios tested of macrophages to thymocytes. We further confirmed our findings using the combination of Mer-KO macrophages and Gas6-KO serum in the assay with the 4M of apoptotic cells (Figure 5B). Data are thus consistent with the notion that Gas6 is the major ligand for Mer-mediated phagocytosis.
We tested the blocking function of goat anti-mouse polyclonal antibody (R&D system) in mouse serum dependent phagocytosis, in order to explore the potential therapeutic application of anti-Mer antibody in Mer-mediated mouse disease model. Figure 6 shows this antibody could block phagocytosis by WT macrophages down to the level seen with Mer-KO macrophages.
Identifying the expression of Mer among macrophage subpopulations in lymphatic organs will aid in understanding the role of Mer in the clearance of apoptotic cells, including lymphocytes. We have shown that Mer-KO mice develop a lupus-like autoimmune syndrome and splenomegaly. In addition, excessive apoptotic cells and cell debris were observed in Mer-KO mice spleens after exogenous infusion of apoptotic cells [8; 20]. Therefore, the anatomical localization and expression pattern of Mer in spleen are of particular interest. Mer seems to be universally expressed among CD68-bearing macrophages — so-called tingible body macrophages. These cells have been reported to be of critical importance in engulfing apoptotic debris, and, in lymph nodes from SLE patients, these cells have a marked deficiency in uptake of apoptotic cells . CD68+ tingible body macrophages are found in splenic germinal centers, where they engulf newly generated apoptotic B cells. Our data suggest that Mer may be of special importance in the elimination of apoptotic B cells generated in the processes of negative selection and affinity maturation. In the absence of Mer, the build-up of these apoptotic B cells, which express nuclear antigens and other self proteins, may spur the development of autoimmunity by providing an antigenic stimulus for nearby autoreactive B cells. Together with the overactivated macrophages found in Mer-deficient mice, the potentially immunogenic apoptotic B cells may stimulate the spontaneous development of autoimmunity. Another important receptor/ligand system that facilitates uptake of apoptotic debris, milk fat globule–EGF factor 8 (MFG-E8)/lactadherin, is also centered on these tingible macrophages , reinforcing the notion that they are central in apoptotic debris clearance.
The splenic marginal zone is the first area of the spleen encountered by circulating antigens and is believed to be of central importance in capturing antigens. Further, MZ B cells have been reported to contribute disproportionately to the production of autoantibodies, supporting a crucial role for the MZ in the genesis of autoimmunity. Given the importance of Mer in phagocytosis of apoptotic debris and in regulating macrophages activation, we were surprised to observe that Mer was not present on the metallophillic macrophages that are so important in the MZ. Instead. Mer was expressed on a small portion of MZ macrophages, which may represent a previously unrecognized MZ macrophage population. A proportion of these Mer+ MZ macrophages expresses MARCO, and may be functionally distinguishable from other MZ macrophages. The exact function of MARCO is unknown, but it has been suggested that it facilitates cell adhesion and shape change . Recently, a role for MARCO in the clearance of apoptotic cell was reported . Co-expression of Mer with MARCO might be necessary for the engulfment and antigen presentation of specific pathogens.
We addressed another important issue concerning how Mer functions in macrophages in vivo. This concerned the physiological ligand for Mer. While Gas6 was first identified as a ligand for Axl and subsequently a common ligand for all three members (Tyro-3, Axl, and Mer) within the same subfamily , it is unclear whether this molecule is the only ligand and a strong case has been made for human protein S as an alternative ligand . A difficulty has been that all studies were done in a culture system involving bovine serum. Considering the cross-species activation of receptor by protein S , care must be taken when interpreting data. Our data, using murine serum from wild type and from Gas6-KO mice, indicate that Gas6 is indeed the major ligand for Mer-mediated phagocytosis. Gas6-KO mice do not develop autoimmune disease (our unpublished data), despite their apparent inability to phagocytose apoptotic cells through Mer. This may indicate that Mer signaling for regulation of macrophage cytokine production may proceed through alternative pathways, viz., protein S, while phagocytosis of apoptotic cells through Mer largely depends on Gas6. These studies will require confirmation in other systems, but underline the complexity of the signaling through Mer and other related molecules in regulation of innate and adaptive immunity.
We thank Dr. Peter Carmeliet at Katholieke University Leuven, Belgium, for providing Gas6 knockout mice. This work was supported by grants from the NIDCR and the US Department of Veterans Affairs.
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