Involvement Of CD226~+ NK Cells In Immunopathogenesis Of Systemic Lupus Erythematosus

Nature killer (NK) cells are defined as large granular lymphocytes that belong to the innate immune system. They do not express T cell antigen receptors (TCR) or B cell antigen receptors (BCR) but they express the non-antigenic specificity receptors, including inhibitory as well as some activating members. They were named "natural killers" because of the initial notion that they do not require pre-activation in order to kill certain tumor cells. NK cells play important roles in the early protection against viruses and kill cancer cells in vivo. In addition, they can regulate immune response by intercellular contact or by producing a variety of cytokines and chemokines including IFN-γ, TNF-α/β, IL-2, IL-10, IL-13, CCL2 (MCP-1), CCL3 (MIP1-a), CCL4 (MIP1-b), CCL5 (RANTES), XCL1 (lymphotactin) and CXCL8 (IL-8). Now the role of NK cells in autoimmunity is attracting increased attention, such as Diabetes, Encephalomyelitis, Multiple Sclerosis and Systemic Lupus Erythematosus. However, they are revealing both the disease-promoting as well as disease-protective roles in the different disease models.Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease that can affect any issue or organ of the body. The disease occurs nine times more often in women than in men, especially in women in child-bearing years ages 15 to 35. Although the pathogenesis of SLE has been extensively investigated and we have known much about the relationship between disease and adaptive immune system, the contribution of innate immune system especially NK cells in the development of SLE pathology is still unclear. Autoimmune diseases develop in steps, including a priming phase in secondary lymphoid tissues followed by homing to the target organ and tissue destruction. Innate immune system play a role early in the disease may therefore be ignored. Furthermore, the new knowledge on NK-cell biology implicates NK cells as possible players in all these phases.Base on these considerations, our study is to investigate how NK cells participate in the pathogenesis of SLE and the reason why the proportion and absolute number of circulating NK cells are significantly lower in SLE patients. Meanwhile, we compare the different functions of NK cells between peripheral circulation and local tissue. Through these studies we hope to find better methods for SLE clinical diagnosis and treatment. Patients were divided into two groups based on disease activity, which was estimated by the SLEDAI (active SLE patients: SLEDAI≥4, patients in remission: SLEDAI﹤4). Peripheral blood samples obtained from healthy donors were served as controls. The cell phenotype and proportion were determined by flow cytometry, and the absolute numbers of different cell subtypes were calculated. The expression of inhibitory and activating NK cell receptors was analyzed by flow cytometry. NK cell populations were enriched from whole blood by negative selection using the MACS NK Cell Isolation Kit. CD226~+ NK cells and CD226- NK cells were also separated by magnetic-bead purification with MACS kits. Cells were cultured with cytokines or human plasma, and flow cytometry were used to test the change of cells that cultured with different stimuli. Annexin V and PI staining were used to detect the apoptosis of cell subtypes. ELISA and intracellular cytokine staining were used to examine the secretion of different cytokines. The expression of genes that predominantly induced by type I IFN was tested by Semiquantitative real-time RT-PCR. Tissue injury was assessed by H&E staining. The major findings and conclusions of our study are shown as follows:1. Reduced proportion of circulating CD226~+ NK cells in SLE patients with active diseaseAs the immune state of SLE is a poorly understood, involving abnormality of both the innate and adaptive arms of the immune system, the proportions of several lymphocyte subsets, such as T (CD4+ or CD8+), B, NK, NKT, MDC and PDC cells, in the peripheral blood were determined by flow cytometry. We find that the absolute number as well as the proportion of CD3-CD56+ NK cells was significantly lower in active SLE patients than in healthy controls and patients in remission. The multiple functions of NK cells can be regulated by interactions between inhibitory and activating families of NK receptors and their respective ligands. So we compared the expression of a number of representative inhibitory and activating NK cell receptors. Nonetheless, we did not observe any differences between the patient groups and the healthy controls in the expression of NKG2A, CD94, CD85j, CD69, NKp46 and NKG2D by NK cells. However, the proportion of CD226~+ NK cells was significantly lower in active disease than in healthy controls and patients in remission. We also find that the proportion of CD226~+ NK cells was lower in diseased MRL/lpr mice compared with pre-disease MRL/lpr mice and MRL/mp mice. These data indicate that both the proportion and absolute number of circulating NK cells is reduced in SLE patients with active disease, but is gradually restored to near normal levels in remission, and most of the NK cells lost in active SLE and diseased MRL/lpr mice might be CD226 positive.2. Dynamic changes in CD226~+ NK cell populations in SLE patients are associated with disease activityTo further assess the status of CD226 expression on NK cells in SLE patients, dynamic observations were made on fifteen SLE patients during therapy. The proportion of NK cells was markedly lower in six patients (four received prednisolone, two were treated with both prednisolone and hydroxychloroquine) during the exacerbation stage. Further analysis identified a sustained decrease in CD226~+ NK cells in these patients during therapy. By contrast, the other nine patients whose conditions were improved after medication (they all received prednisolone as monotherapy), and the NK cell populations were not significantly reduced. Moreover, the proportion of CD226~+ NK cells was raised in the nine patients during therapy. Taken together, these data suggest that the decreased expression of CD226 on NK cells is correlated with disease activity in SLE patients and therapy possibly restores the frequency of CD226~+ NK cells, at least in some patients.3. Expression of the CD226 ligands CD112 and CD155 on DCs in SLE patientsWe evaluated the expression of two CD226-specific ligands, CD112 and CD155, on circulating PBMCs in SLE patients. Interestingly, they were mostly expressed on PDCs in both SLE patients and healthy controls. CD112 expression levels on PDCs were slightly increased in SLE patients compared with healthy controls.Considering that CD226 is highly expressed on NK cells, whereas CD226-specific ligands CD112 and CD155 are substantially expressed on PDCs in SLE patients, it is possible that increased CD226 ligands–CD226 pathway is involved in the pathogenesis of SLE.4. Activation and apoptosis of CD226~+ NK cells are induced by IFN-αfrom active SLE plasmaIn this study, several proinflammatory and immunoregulatory cytokines were measured by ELISA in plasma obtained from SLE patents and healthy controls. We find that the levels of IFN-αin plasma samples from active SLE patients were higher than that in healthy controlsand patients in remission. Moreover, levels of IFN-αwere high in three patients with active disease but declined to normal levels after therapy. To further test the IFN-αactivity in plasma from SLE patients, PBMCs from healthy donor were stimulated with different plasma samples, and the relative expression of 4 IFIGs (PRKR, IFIT1, MX1and IFI44) in PBMCs was tested after 24h incubation. IFIGs were induced by plasma from active patients, and the neutralizing antibodies to IFN-αcould inhibit the active SLE plasma activity. The results from flow cytometry shown that IFN-αwas detectable from PDCs in SLE patients, but not in healthy controls. Furthermore, we find that in vitro cell culture experiments, where PBMCs isolated from healthy controls were stimulated with either IFN-αor active SLE plasma, the activation of NK cells occurred almost simultaneously with down-regulation of CD226 expression. Meanwhile, active SLE plasma could induce up-regulation of Annexin V expression on CD226~+ rather than CD226- NK cells.Taken together, these experiments confirm that IFN-αstimulation causes NK cells to undergo activation-induced cell death. Interestingly, it is likely that IFN-αpredominately down-regulates the proportion of CD226~+ NK cells. Therefore, the reduced frequency of circulating CD226~+ NK cells in active SLE patients is mirrored in vitro using proper concentrations of IFN-αor active SLE plasma to stimulate the PBMCs.5. Activated CD226~+ NK cells mediate kidney injury in MRL/lpr miceWe chose a mouse model that mimics human SLE. Our results indicate that NK cells was recruited from the periphery into kidneys in pre-disease MRL/lpr mice and most of the infiltrating NK cells were CD226~+ subset. Furthermore, kidney-infiltrating NK cells in MRL/lpr mice displayed an activated phenotype including up-regulated expression of CD69 and high production of IFN-γ, granzyme B and perforin whichmay contribute to inflammation and eventual kidney injury.Taken together, these data suggest that activated CD226~+ NK cells are responsible, at least to some extent, for the kidney injury observed in SLE.