Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects many tissues and organs. The major immunopathological findings of SLE include defective immune regulation with the breakdown of immune tolerance, autoantibody formation followed by immune complex deposition, cytokine imbalance, and inflammation [1]. Failure of phagocytes to remove apoptotic cells has been suggested to allow excessive release of autoantigens and to lead to the induction of autoimmunity, although the underlying mechanisms remain unclear [2‐6]. In addition, CD4⁺CD25^highFoxP3⁺ regulatory T cells (Tregs), which are pivotal in the maintenance of T-cell homeostasis and are critical regulators of immune tolerance [7, 8], exhibit quantitative and/or qualitative deficiencies in SLE that may contribute to the development of lupus pathogenesis [9, 10].

CD200 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily, and is expressed by a variety of cells, including B cells, activated T cells, follicular dendritic cells (DCs), and neurons [11‐13]. CD200 consists of extracellular, transmembrane, and intracellular domains, although its intracellular region lacks a signaling motif [11‐13]. CD200 receptors include CD200R1 to CD200R4, of which CD200R1 has the highest binding affinity [14]. The distribution of CD200 receptors is mainly on myeloid-derived cells, such as DCs, macrophages, and also activated T cells [15, 16]. The known immunoregulatory roles of the CD200/CD200R1 pathway include suppression of the degranulation of mast cells and basophils [17] and negative regulation of macrophage function [18].

Hoek and colleagues found that CD200-deficient mice had increased endogenous activation of macrophages/myeloid cells in the central nervous system, with enhanced susceptibility to experimental allergic encephalomyelitis and collagen-induced arthritis [18]. Administration of CD200R-Ig to disrupt CD200-CD200R interaction also increased the susceptibility of mice to collagen-induced arthritis. Furthermore, Broderick and colleagues reported that blockade of CD200 resulted in the early onset of experimental autoimmune uveoretinitis in mice [19]. In addition, Rosenblum and colleagues studied CD200-knockout mice in a model of UV-mediated induction of tolerance to hapten, and suggested that the expression of CD200 in skin cells plays a role in autoimmune congenital alopecia [20]. Finally, Gorczynski and colleagues showed that tumor growth in vivo can be monitored by levels of soluble CD200 in serum of tumor-bearing animals [21], whereas Moreaux and colleagues found significant overexpression of CD200 in a variety of cancers compared with normal cells or tissues and suggested that CD200 might be a potential therapeutic target and prognostic factor for a large array of malignancies [22].

While available evidence highlighted an important role of CD200/CD200R1 in experimental autoimmune diseases, the role of CD200/CD200R1 in human autoimmune diseases such as SLE remains unknown. We therefore explored the expression and function of CD200/CD200R1 in subjects with SLE.

Despite the data in animal models - including collagen-induced arthritis and experimental allergic encephalomyelitis - suggesting that CD200/CD200R1 may play a role in prevention of autoimmune diseases, information on the role of the CD200/CD200R axis in human diseases - especially in SLE - is very limited. Our study demonstrated that the percentage of CD200⁺ cells in CD4⁺ T cells, plasmacytoid DCs and myeloid DCs of SLE patients was significantly higher than that for HCs. In addition, serum levels of CD200 in SLE patients were also significantly higher than those for HCs.

As CD200 lacks an intracellular signaling motif, most - if not all - of its immunological function relates to its capacity to engage and signal via its receptors, of which CD200R1 seems to be most prominent. Functional studies confirmed this by showing that CD200Fc induced phosphorylation of DOK2 in CD4⁺ T cells. Notably, we found that CD200R1 expression in SLE patients was significantly lower than HCs in CD4⁺ T cells and DCs. The dysregulated expression of CD200/CD200R1 in SLE had functional consequences since CD4⁺ T-cell proliferation was increased by blocking CD200R1 with specific antibody, whereas DC migration and Th17 cell differentiation were decreased and Treg generation was enhanced by engaging CD200R with CD200Fc. These results are all consistent with the conclusion that the deranged expression of both CD200 and CD200R1 in SLE contributes to the functional abnormalities characteristic of this autoimmune disease. Notably, most of the activity of CD200-CD200R1 engagement is usually believed to relate to inhibiting the activity of myeloid cell function [35]. However, we found that CD200R1 expression was also decreased on CD4⁺ T cells and at least the activity in regulating Tregs appeared to involve a direct effect on T cells. These findings suggest a broader spectrum of activity of CD200R1 signaling than has previously been appreciated.

Overproduction of autoantibodies in SLE is believed to be caused by insufficient removal of apoptotic cells and material by macrophages and DCs. Our study demonstrated that SLE patients had a higher proportion of spontaneous early apoptotic lymphocytes compared with HCs. The amount of apoptotic material in SLE patients may exceed the capacity of macrophages to remove it, allowing DCs to become involved in the process of apoptotic cell clearance. Under these circumstances, DCs can become either tolerogenic or stimulatory, depending on the nature of the receptors employed and the available cytokines. As CD200 expression on early apoptotic lymphocytes was increased in SLE patients, we examined whether the increased expression of CD200 on early apoptotic lymphocytes might have had an effect on their binding and uptake by DCs. We demonstrated that early apoptotic cells were more likely to be bound and engulfed by DCs than living cells. The explanation for this could be that although early apoptotic cells remain morphologically intact, specific signals - such as expression of lysophosphatidylcholine - were upregulated on the cell surface, which mediated recognition by DCs and macrophages [36, 37]. Our study also revealed that the binding and phagocytosis of early apoptotic cells that were CD200-positive were lower than those that did not express CD200, suggesting that CD200 expression in SLE could provide a signal to DCs - presumably by binding CD200R, which limits their capacity to bind and ingest apoptotic material. Aberrant expression of CD200 may therefore contribute to the decreased clearance of apoptotic material found in SLE.

To function, CD200 needs to bind to CD200R on cell surfaces. Our data confirmed that T cells expressed CD200R1. Since a previous animal study suggested that CD200/CD200R signaling had an effect on cytokine balance [27], we investigated whether CD200/CD200R1 could affect the balance of T-cell subsets in SLE. We found that CD200Fc reduced Th17 cell differentiation in SLE, but not in HCs. These results suggest that Th17 cell differentiation in SLE may be regulated by engagement of CD200R, such that signaling through this pathway limited Th17 cell differentiation. It is possible that the downregulation of CD200R in SLE resulted in less regulation of Th17 cell differentiation, which could be corrected by the increased availability of CD200. On the contrary, anti-CD3/CD28-stimulated T-cell proliferation was promoted by antagonistic anti-CD200R1 in a concentration-dependent manner in SLE patients but not HCs, suggesting that anti-CD200R1 may block the endogenous signal provided by increased expression of CD200 and, thereby, permit increased CD4⁺ T-cell proliferation. In summary, these results indicate that the CD200-CD200R1 pathway exerts a number of regulatory influences on T-cell function, either directly or through the action of DCs, and that the dysregulation of surface expression of these molecules may contribute to some of the immunoregulatory abnormalities characteristic of SLE.

In untreated active SLE patients, the proportion of CD4⁺CD25^highFoxP3⁺ Tregs was significantly lower than in HCs (see Additional file 7). Pallasch and colleagues demonstrated that antagonistic anti-CD200 antibody could promote chronic lymphocytic leukemia cell-induced proliferation of antigen-specific T cells and reduce the proportion of CD4⁺CD25^highFoxP3⁺ cells [38]. Gorczynski and colleagues showed that, in BL/6 bone-marrow cells, anti-CD200R2/3 mAb (not CD200R1) could promote the development of tolerogenic DCs through a TGF-β-dependent (but not IL-10) and CTLA-4-dependent pathway, induce more CD4⁺CD25^highFoxP3⁺ Tregs, and inhibit the mixed lymphocyte reaction in a MHC-restricted and antigen-specific manner [39]. These results all suggested that the activation of the CD200/CD200R axis could exert an immunosuppressive function via promoting Treg proliferation and inhibiting effector T-cell function. Our study found that TGF-β induced generation of CD4⁺CD25^highFoxP3⁺ T cells in HCs, whereas this was not seen in SLE patients. This finding is consistent with a previous study that demonstrated defective expression of TGF-β signal transduction molecules in most SLE patients [40]. Interestingly, we found that the addition of CD200Fc rescued the defective generation of CD4⁺CD25^highFoxP3⁺ T cells in SLE patients, indicating that CD200 could intervene in the TGF-β signaling pathway and promote Treg generation. This effect appeared to be directly mediated by T cell-T cell interaction since these studies were carried out with sorted T cells. Specific signals and cytokines mediate the differentiation of Tregs and Th17 cells [41, 42]. The current data imply that signaling through CD200R1 may be one important influence on these pathways of T-cell differentiation. Increased signaling through CD200R1 may bias toward Tregs and away from Th17 cells, and thus may be beneficial in SLE.

Downregulation of CD200R1 in SLE may contribute to impaired generation of regulatory signals, and increased production of CD200 in vivo could bind to other receptors such as CD200R2 to CDR200R4 [14], thereby transmitting stimulatory signals leading to the enhanced differentiation of Th17 cells, as has been reported [43]. Moreover, it has been reported that CD200 engagement of CD200R1 could induce tolerogenic DCs, which in turn could promote differentiation of Tregs [39, 44, 45].

In our study, however, experiments were carried out with purified T cells, making this a less probable explanation for the findings. CD200R1 expression by DCs was also downregulated in SLE, however, suggesting that reduced generation of tolerogenic DCs in the context of decreased Tregs could contribute to unregulated development of Th17 cells.

Taking the results together, we have demonstrated in SLE patients that the number of CD200⁺ cells as well as the serum level of CD200 were significantly higher than in HCs, whereas CD200R1 expression was significantly lower than in HCs, especially in CD4⁺ T cells and DCs. In addition, in SLE patients, exogenous CD200Fc reduced the proportion of Th17 cells and rescued the defective generation of CD4⁺CD25^highFoxP3⁺ T cells, whereas anti-CD200R1 antibody promoted anti-CD3/CD28-induced CD4⁺ T-cell proliferation. Moreover, we found that CD200 on early apoptotic cells was increased in SLE patients and may serve to limit their binding and phagocytosis by DCs. These data collectively indicate that CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in this autoimmune disease.