The Mer receptor tyrosine kinase mediates apoptotic cell phagocytosis and modulates macrophage cytokine production. Mer−/− mice have defective clearance of apoptotic debris and develop a systemic lupus erythematosus-like autoimmune syndrome. It was surprising then that B6-Mer−/− recipients of bm12 spleen cells failed to develop anti-dsDNA and anti-chromatin autoantibodies, whereas B6 hosts produced the expected autoimmune chronic graft-vs-host (cGVH) reaction. The lack of autoantibody formation in cGVH was not due to the failure of Mer-deficient hosts to provoke alloreactivity, because Mer−/− spleen cells were recognized by bm12 T cells in MLR. Cell transfer experiments in Rag-knockout mice indicated that the lack of autoantibody production in Mer−/− cGVH disease hosts was due to an intrinsic B cell defect. This defect did not cause a global inability to produce autoantibodies, because in vivo exposure to LPS stimulated production of autoantibodies in both B6 and Mer−/− mice. We further observed that wild-type B6 B cells up-regulated Mer upon activation in cGVH, and that B cells from mice lacking Mer showed a decreased up-regulation of activation-associated cell surface markers. These findings indicate that Mer serves an important role in the activation of self-reactive B cells in systemic autoimmunity.
The injection of spleen cells from bm12 mice into C57BL/6 recipients induces a chronic graft-vs-host (cGVH)3 reaction characterized by systemic autoimmunity similar to that seen in systemic lupus erythematosus (1, 2). The cGVH reaction is driven by MHC class II recognition. In this cGVH model, the recipient strain (B6) differs from the donor B6.C-H2bm12/KhEg (bm12) by only 3 aa in the β-chain of MHC class II molecule I-A (3, 4). The autoreactivity is a result of recognition of recipient B cell MHC class II by donor T cells (5). The disease progresses over a period of weeks to months (4). This model has proven to be a valuable model for elucidating the loss of tolerance that characterizes systemic autoimmunity (6).
The receptor tyrosine kinase Mer (also known as c-Mer, or MerTK) mediates macrophage binding to apoptotic cells and triggers their phagocytosis (7, 8, 9). It belongs to a subfamily of tyrosine kinases, which also includes Axl and Tyro-3. They share the same ligand, growth arrest-specific gene 6, and have similar structure, but a different tissue distribution pattern (7, 10, 11). Mer has been reportedly expressed on the monocytic cell lineage and on platelets, but not on normal lymphocytes or thymocytes (7). Mer-deficient mice gradually develop lupus-like autoimmunity together with reduced efficiency of apoptotic cell clearance (9). These Mer−/− mice are also more susceptible to LPS-induced endotoxic shock (12). Keating et al. (13) overexpressed Mer in lymphocytes and thymocytes under the Vav promoter, and showed that Mer transgenic mice developed T cell acute lymphoblastic leukemia and lymphomas at a significantly higher rate compared with the wild type (WT). Consistent with previous findings (14), the study appears to provide clear evidence that stimulation of Mer leads to activation of transforming and anti-apoptotic pathways. Recently, high expression of Mer was detected on Jurkat cells, and an increased apoptosis rate was shown if Mer expression was suppressed by RNA interference (15).
In the present studies, we examined the role of Mer in cGVH. Surprisingly, mice lacking Mer expression are almost completely protected from the development of cGVH. In addition, we found evidence for Mer expression on normal B cells, and up-regulation of this expression during the cGVH that is transcription independent. These novel findings point to an important role of Mer in B cell autoreactivity, and potentially in the pathogenesis of systemic lupus erythematosus.
Materials and Methods
C57BL/6J (B6, used as Mer+/+ control) and B6.C-H2bm12/KhEg (bm12) mice were originally obtained from The Jackson Laboratory. The C57BL/6J.3H9 (3H9) transgenic mice were obtained from M. Weigert, formerly at Princeton University (Princeton, NJ). Mer−/− mice were generated as described previously (12). The mice used were backcrossed to C57BL/6J for 10 generations. The Mer+/− F1 mice were generated by crossing Mer−/− mice to B6 mice. All mice were subsequently bred and maintained in our mouse colony at the University of Pennsylvania Medical Center. 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.
Induction of cGVH
cGVH disease was induced as previously described (4). Briefly, recipient mice on a C57BL/6 background were injected i.p. with 1 × 108 bm12 splenocytes in single-cell suspensions, prepared by grinding donor spleens against frosted slides in HBSS. Serum samples were prepared from peripheral blood from experimental mice and non-graft-versus-host control animals the day of injection and at weekly intervals thereafter. Mouse sera were stored at −20°C for later analysis.
Detection of autoantibodies in sera by ELISA
Autoantibodies were determined by ELISA, as previously described (4). Briefly, polyvinyl microtiter plates (Dynatech Laboratories) were coated with optimal concentrations of either dsDNA (2.5 μg/ml, prepared from calf thymus; Sigma-Aldrich) or chromatin (3 μg/ml, purified from chicken erythrocyte nuclei). For the anti-dsDNA ELISA, plates were first coated with poly(l-lysine) (1 μg/ml; Sigma-Aldrich), before incubation with autoantigens. Ags were diluted in borate-buffered saline (BBS), added to plates, and incubated overnight at 4°C. The plates were then washed with BBS and blocked with BBT (BBS, 0.4% Tween 80, and 0.5% BSA) for 2 h at 4°C. Serum samples, diluted 1/250 in BBT, were added in duplicate and incubated overnight at 4°C. Alkaline phosphatase-conjugated goat anti-mouse IgG (Fcγ fragment specific; Jackson ImmunoResearch Laboratories) was added as secondary Ab. Serum from an older MRL/lpr mouse with high-titer anti-DNA provided a positive standard curve at serial 2-fold dilutions from 1/250 to 1/128,000. The plates were washed and incubated with substrate paranitrophenyl phosphate (Sigma-Aldrich) for 30 min at 37°C. The plates were read at various time points with an automated ELISA reader (Molecular Devices). For rheumatoid factor (RF) ELISA, plates were coated with mouse IgG1 (2 μg/ml; BD Pharmingen) and detected with goat anti-mouse IgM-alkaline phosphatase (Jackson ImmunoResearch Laboratories).
The following conjugated Abs were purchased from BD Pharmingen: allophycocyanin anti-CD19, FITC anti-CD86, FITC anti-CD80, PE anti-I-Ab, FITC anti-CD21, FITC anti-CD22, PE anti-CD23, PE anti-CD24, PE anti-Fas, and streptavidin-PE. Biotin anti-Mer was purchased from R&D Systems. Cell surface staining was routinely performed, as previously described (16). Cells were blocked with 2.4G2, followed by direct incubation with labeled Abs for 40 min, and washed. An additional 30-min incubation with streptavidin-PE was performed to detect biotinylated Abs. Cells were fixed in PBS containing 1% paraformaldehyde and analyzed on a BD Biosciences FACScan. Relative fluorescence intensity was plotted on a logarithmic scale using CellQuest software.
B cell purification with magnetic beads
B cells used in real-time PCR were purified with an anti-CD43-negative selection kit purchased from Miltenyi Biotec. Briefly, splenic cell suspensions were incubated with anti-CD43 magnetic beads at 4°C for 15 min at a concentration of 10 μl beads/107 cells in 90 μl of MACS buffer (PBS with 0.5% BSA, 2 mM EDTA). After washing, cells were resuspended in the MACS buffer for AutoMACS separation (negative selection program was used to avoid any cell activation). The purity of cell separation was checked by flow cytometry. B cells were also purified through sorting of CD19+ cells for the same purpose, which gave a purity of 98%.
Mixed leukocyte reaction
Splenocytes isolated from B6, Mer−/−, and bm12 mice were used as responders and stimulators. Stimulators were irradiated at 1000 rad and cocultured with responder cells (ratio 1:1, and final concentration: 2 M cells/ml) in a 37°C, 5% CO2 incubator in triplicate wells in 96-well round-bottom plates. After 4 days, the cells were pulsed with 1 μCi/well [3H]thymidine for 18 h, and proliferation was determined as the mean 3H incorporation in triplicate cultures. PMA/Ionomycin (500 ng/ml; Sigma-Aldrich) was used as positive control in the culture.
Adoptive B cell transfer
B cells were purified through magnetic sorting (AutoMacs, CD43 MicroBeads kit, negative selection, purity 95%; Miltenyi Biotec). Single-cell suspensions (1 × 106 cells in 200 μl of HBSS buffer) from B6 mice or Mer−/− mice were transferred i.v. into Rag-knockout (KO) mice at day −1, and followed by induction of cGVH, as described previously, at day 0. Serum samples were collected at day 0 and weekly afterward. Anti-dsDNA and anti-chromatin Abs were analyzed by ELISA.
Taqman-based real-time PCR was used to amplify a 59-bp sequence of the Mer open reading frame on the kinase domain, which is absent in the Mer−/− mice. The sequences used in this assay were as follows: forward primer, 5′-GCCGCATTGCCAAAATG-3′; reverse primer, 5′-TCCGCCAGGCTCTCGAT-3′; minor groove binder-labeled probe, 5′-TGTGAAGTGGATCGC-3′. Tests were done in triplicate, and the mean was used as the final result. The same volume of RNA extract (1 μg) was used in all PCR. The reaction was run on a 7500 Real Time PCR System (Applied Biosystems).
Mice were injected i.p. with 200 μg of LPS (from Escherichia coli 055:B5; Sigma-Aldrich). Sera samples were collected, and anti-dsDNA Ab was measured as described previously. B cell activation markers were analyzed by FACS 16 h after LPS injection.
Data are presented as the mean ± SD. Statistical significance was determined using Student's t test.
Mer-KO mice are protected from the development of cGVH
cGVH disease was established in unirradiated recipient mice by i.p. injection of a single dose of 1 × 108 age/sex-matched bm12 donor spleen cells. Mice were followed for serum autoantibody levels. As expected, the Mer+/+-positive control mice showed clear elevations of autoantibody titers of anti-dsDNA, anti-chromatin, and RF. The pattern of a gradual falloff in titer over time was similar for anti-dsDNA and RF, with a later peak for anti-chromatin. Surprisingly, the Mer−/− mice showed no increase in autoantibody titers (Fig. 1, A and B), except for a very modest, transient elevation of RF at week 2 (Fig. 1,C). Mer−/− mice also failed to develop the splenomegaly that is characteristic of this model (Fig. 1 D), nor did they develop the mild ascites that was seen in the WT recipients (data not shown). These findings indicate an unexpected role of Mer in the development of cGVH.
To ask whether Mer deficiency might protect against autoantibody production in mice with a genetic predisposition to generate anti-DNA autoantibodies, we generated Mer−/− anti-dsDNA Ab transgenic mice using the 3H9 H chain knock-in mice. In these 3H9 transgenic mice, a rearranged V-D-J construct has been inserted into the J region, downstream of the D regions (17). The 3H9 H chain produces an anti-dsDNA Ab when paired with ∼1/3 of L chains. The C57BL/6.3H9 (3H9) mice are more susceptible to cGVH, yet they do not spontaneously show anti-dsDNA Abs in their sera (18). Fig. 2 shows that neither Mer−/− nor Mer−/−/3H9 mice developed detectable anti-dsDNA or anti-chromatin autoantibodies after bm12 cell transfer, whereas B6 control and 3H9 mice developed substantial titers of anti-dsDNA and anti-chromatin (Fig. 2). These data emphasize the profound resistance of Mer−/− mice to response in the cGVH.
Splenocytes from bm12 mice proliferated equally in response to Mer+/+ and Mer−/− spleen cells
One potential explanation for the failure of Mer−/− mice to respond to the cGVH would be the lack of alloreactive stimulation of bm12 T cells by class II+ cells in the Mer−/− recipient mice. However, an in vitro MLR showed no difference between Mer+/+ and Mer−/− irradiated stimulator cells (Fig. 3). Conversely, there was similar [3H]thymidine incorporation when Mer+/+ and Mer−/− spleen cells were cultured with irradiated bm12 target cells. Thus, it was unlikely that the deficient cGVH in Mer−/− mice was due to a failure to stimulate the alloreactive donor T cells.
Failure to develop cGVH in Mer−/− mice is not due to dominant genes linked to Mer
If the resistance to cGVH in Mer−/− mice was indeed due to Mer deficiency, it should be corrected in Mer+/− heterozygotes. In fact, F1 mice (B6 × Mer−/−) showed no significant differences compared with B6 in the cGVH production of anti-dsDNA and anti-chromatin autoantibodies (Fig. 4, B and C). It was of interest that there was an intermediate level of Mer expression on splenic macrophages from Mer+/− F1 mice in comparison with Mer+/+ and Mer−/− mice (Fig. 4 A). Thus, these results exclude the involvement of other dominant gene(s) (perhaps linked to Mer), such as a minor histocompatibility mismatch. They also suggest that there is no gene dosage effect of Mer on cGVH.
B cells from Mer−/− mice do not have an activated phenotype upon cGVH induction
Our previous studies have shown that host B cells from mice undergoing cGVH show phenotypic changes indicative of diffuse activation (19). We questioned whether Mer−/− host B cells, although unable to produce autoantibodies under these conditions, might nevertheless show evidence of activation. Mature splenic B cells in Mer−/− mice showed no apparent phenotypic differences compared with age/sex-matched Mer+/+ mice, nor did transitional B cells gated on CD93+ (data not shown). Two weeks after the induction of cGVH, splenic B cells from Mer−/− mice displayed an intermediate activation phenotype, as compared with B6 controls (Fig. 5). Mer−/− B cells up-regulated MHC II and CD86, and down-regulated CD21, although to a much lesser extent. Thus, the allogeneic stimulus had some effect on Mer−/− B cells, even if they produced very little autoantibodies.
B cells in Mer−/− mice can be autoreactive under polyclonal stimulation
Studies have shown that in vivo challenge with LPS in B6 mice results in lupus-like features (20). We asked whether the inability of Mer−/− B cells to produce autoantibodies in the cGVH was due to a failure to develop an autoreactive Ig repertoire. We stimulated B cells diffusely by injecting a single dose of 200 μg of LPS into Mer−/− mice and sex/age-matched Mer+/+ controls. Fig. 6,A shows that B cells from Mer−/− responded as well as Mer+/+ mice. Both Mer−/− mice and Mer+/+ mice produced comparable amounts of anti-dsDNA Ab (Fig. 6,A). Activation and developmental markers were also analyzed by flow cytometry. Sixteen hours after LPS administration, B cells generated a similar activation profile in Mer−/− mice as in Mer+/+ mice (Fig. 6 B). B cells in Mer−/− mice thus have autoreactive potential under conditions other than the cGVH.
Mer−/− B cells are intrinsically unresponsive to alloreactive stimulation
The failure of Mer−/− B cells to undergo activation during cGVH could be due to intrinsic B cell unresponsiveness or could be due to the lack of Mer-expressing cells (such as macrophages or dendritic cells) in the B cell environment. To address this issue, we adoptively transferred 10 million purified B cells from Mer−/− mice into Rag-KO recipient mice (deficient in B and T cells, but with intact Mer expression). These chimeric mice were challenged with 1 × 108 bm12 spleen cells i.p. on the following day. A control group received B cells from Mer+/+ mice. Strikingly, the B cells from Mer−/− mice failed to produce either anti-dsDNA or anti-chromatin Ab, whereas the Mer+/+ B cells responded as expected (Fig. 7). These results indicated that the defect in cGVH autoantibody production in Mer−/− mice was intrinsic to their B cells.
B cells up-regulate surface expression of Mer after cGVH
The cell transfer results are compatible with the possibility that Mer expression on B cells is required for them to respond to allogeneic help, although it is possible that their intrinsic defect is acquired from their Mer-deficient environment. The latter mechanism would be parallel to what we have found in CD4−/− mice, in which the development of B cells in the absence of the CD4 T cell subset results in their inability to respond in the cGVH (19, 21). In addition, several groups have reported a failure to detect Mer on lymphocytes (7, 10). Similarly, we also failed to detect Mer on resting B cells (Fig. 8,A, left panel). Two weeks after the induction of cGVH, however, B cells from Mer+/+ mice showed a clear up-regulation of surface Mer, which was not seen in Mer−/− mice (Fig. 8,A, left panel). This up-regulation of Mer was not seen on B cells activated by LPS injection (Fig. 8,A, right panel). Real-time PCR indicated the presence of Mer mRNA in B cells from Mer+/+ mice, but not from Mer−/− mice (Fig. 8,B). Similar levels of message were found in WT B cells purified by two different protocols (right and left panels of Fig. 8 B). Strikingly, the amount of message did not increase with cGVH, which suggests that the rise in surface expression is controlled by posttranscriptional regulation.
The mouse model of cGVH provides important insights into B cell loss of tolerance in autoimmunity. Recipient B cells, driven by allogeneic T cells, are responsible for the autoantibody production. Donor B cells disappear shortly after transfer and are not stimulated by the host T cells. Recipient B cells undergo polyclonal activation, accompanied by B cell phenotypic changes and increases in total serum levels of Igs (IgM and IgG), followed by the appearance of specific autoantibodies (19). We had predicted that Mer−/− mice would develop more rapid or robust cGVH, given their susceptibility to the spontaneous loss of tolerance presumably because of defective clearance of apoptotic cells (9). It was surprising then that Mer-deficient mice were highly protected from the development of cGVH, and their B cells showed little evidence of activation by alloreactive T cells.
Further FACS analysis revealed an unexpected up-regulation of Mer on the surface of normal B cells upon cGVH activation, because mouse Mer expression had not previously been reported on lymphocytes (7). Real-time PCR showed the presence of Mer mRNA in WT B cells. Because two preparations of B cells purified by very different protocols gave very similar levels of message, we feel it is unlikely that contamination of the B cells is responsible for these results. The lack of any change in the level of message with cGVH suggested the possibility that posttranscriptional regulation of Mer may permit rapid B cell responses to alloreactive stimulation. The lack of Mer did not appear to play a significant role in naive B cell maturation and/or activation, because transitional B cell (data not shown) and mature B cell phenotypes in naive Mer−/− mice were no different from those seen in normal mice. Whether acquired Mer expression plays a role in the B cell activation and/or autoreactivity remains unknown. The finding of an intrinsic role of B cells in the adoptive transfer experiments is consistent with our observation that normal B cells acquire Mer during cGVH. Thus, expression of Mer on B cells might be required for them to respond to allogeneic help. Alternatively, it is possible that a lasting B cell defect is actually a result of their maturation in a Mer-deficient environment. With gene-manipulated mice, we can construct animals in which B cell maturation and/or activation occur in different microenvironments. To facilitate these studies, a Mer/Rag double-KO mice strain is currently under development in our facility. Together with our Rag-KO mice, this strain will be a valuable tool in studying the important role of Mer in the development of cGVH and allow us to take further steps into the mechanism of cGVH. In vitro B cell activation, such as BCR, anti-CD40, with or without combination with IL-4 may also provide insight into the direct role of Mer on B cell activation. Mer−/− mice retained the ability to produce anti-dsDNA and to undergo activation under the influence of LPS, indicating that autoreactive B cells were capable of Mer-independent activation. This is understandable also in light of the autoimmunity that spontaneously develops in aging Mer−/− mice (9). Further work is needed to define the precise role of Mer in B cell activation and the development of autoantibodies.
The fact that Mer−/− mice develop spontaneous autoimmunity (9) raises the question whether there are distinct roles of B cells in spontaneous autoimmunity vs induced autoimmunity. Several arguments favor this perspective. First, spontaneous autoimmunity occurs only in old mice with 30–50% frequency, whereas the nearly complete resistance to induced cGVH was demonstrated at 6- to 8-wk mice. Second, autoantibodies in these two models are different. Mer−/− mice mainly develop anti-chromatin, with sporadic anti-ssDNA and rheumatoid factor. In cGVH, Mer−/− mice showed no detectable levels of anti-dsDNA and anti-chromatin autoantibodies, with slightly increased rheumatoid factor. Third, the spontaneous development of autoantibodies is due to a gradual increase of apoptotic debris caused by insufficient clearance of Mer−/− macrophages. A direct T→B cell interaction and stimulation are required in the model of cGVH, although the exact mechanism is still unknown.
These data support the view that there are distinct pathways of activation of autoreactive B cells, in particular that activation via class II MHC may require an intact Mer molecule, whereas activation by LPS or via the accumulation of apoptotic cells is independent of Mer. In addition to its important role in triggering apoptotic ingestion (22, 23), Mer may mediate an anti-apoptotic signal that may permit persistence of autoreactive B cells in certain circumstances. Both transforming and anti-apoptotic abilities of Mer have been observed experimentally through its intracellular region (14, 24). Studies have also shown overexpression of Mer on human cancers, such as mantle cell lymphoma, E2A-PBX1 B cell leukemia, and alveolar rhabdomyosarcoma (25, 26, 27). Considering the anti-apoptotic effects of Mer, B cells might simply die after allostimulation without Mer. Against this notion are observations that autoreactive B cell death does not correlate with the failure in general B cell activation during the early stage of cGVH (Fig. 5).
Taken together, our data emphasize the importance of Mer in regulation of autoreactivity and indicate that its role may be more complex than previously thought. Future investigations will clarify the pathways whereby Mer influences B cell activation in autoimmunity.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by grants from the Alliance for Lupus Research, the Lupus Research Institute, the Lupus Foundation of South Jersey, the U.S. Department of Veterans Affairs, and the National Institutes of Health (DE017590, R01AR34156, and AI063626). P.L.C. is a scholar of the Mary Kirkland Lupus Foundation, which provided support for W-H.S.
Abbreviations used in this paper: cGVH, chronic graft-versus-host; BBS, borate-buffered saline; KO, knockout; RF, rheumatoid factor; WT, wild type.