| NKG2D is a key homodimeric activation receptor expressed on the cell surface of almost all NK cells,γδcells, some cytolytic CD8+ T cells and NKT cells, and normally absent on CD4+ T cells but can be induced by TNF-αand IL-15. Several ligands that bind to NKG2D are members of the MHC class I chain-related family, MICA and MICB. Normally, MICA/B have a restricted tissue distribution in intestinal epithelium, and can be stress-induced molecules by viral and bacterial infections, malignant transformation, and proliferation acting as danger signals to alert NK cells and CD8+ T lymphocytes through engagement of the NKG2D activating receptor. In addition, the MIC/NKG2D interaction may lower the threshold of TCR activation by specific antigen and thus trigger or exacerbate an autoimmune response. Data from the literature strongly support the idea that the NKG2D-MIC activation pathway participates in the development of immune mediated disorders. Recently there had been some reports about a novel subset of NKG2D+ CD4+ T cells in some autoimmune disease such as rheumatoid arthritis, HTLV-1-associated neurologic disease, and Crohn's diseases. Upregulation of MICA/B ligands and high level of IL-15/ TNF-αactivate the effector CD4+ T cells and induce the expression of NKG2D. This subset T cells show the deficiency of the surface marker of CD28 and can secret perforin and granzyme B acting as cytolytic and autoreactive T cells. Later, some groups have been reported that anomalous NKG2D expression on circulating CD4+ T cells in patients with Wegener's granulomatosis .Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the deposition of immune complexes with an inflammatory/necrotic phenomenon in different tissues, mainly kidney, skin, blood vessels and central nervous system. Multiple immune abnormalities are characteristic of this condition, including the synthesis of different autoantibodies, enhanced synthesis of IFN-γand defective immunoregulatory function. IFN-γis secreted by lymphoctyes in vivo and plays an essential role in immunologic defence even in immunoregulation. We and others previous study demonstrated that IFN-γparticipate in the crosstalk between NK cells and monocytes by inducing or enhancing the expression of MIC and mIL-15 on monocytes. Considering that IL-15 can induce NKG2D expression on CD4+ T cells, we wonder whether monocytes in patients with SLE aberrantly displayed enhanced expression of MIC and mIL-15 on the surface due to the increased concentration of serum IFN-γ. If this is true, we want to know whether there is a subpopulation of CD4+ T cells expressing NKG2D existing in SLE patients, which maybe induced by mIL-15 on CD14+ monocytes. It is of great interest to further dissect the mechanisms of NKG2D/MIC interaction during the crosstalk of CD4+ T cells and CD14+ monocytes.Thus, in this study, we investigate the NKG2D receptor expression on CD4+ T cells as well as MIC and mIL-15 expression on CD14+ monocytes in SLE patients. The methods is as follows:1. We detected and analyze the frequency of CD4+NKG2D+ T cells and MIC+ monocytes and the expression of mIL-15 between SLE patients and normal control. Also, we analyzed whether there are correlation between the frequency of MIC+ monocytes and SLEDAI.2. We detected the concentration of IFN-γand IFN-αin sera of SLE patients and normal controls.Then we investigated the effects of IFN-γ, IFN-αon MIC expression of primary monocytes and monocytic cell lines, for example, THP-1 and U937. We also nalyzed whether there are correlation between the concentration of IFN-γin patients sera and MIC+ monocytes.3. With cocultue system, we investigated the effect of monocytes derived from SLE patients and normal controls on the induction of NKG2D receptor expression on CD4+ T cells. Furthermore, we decteted this effect of IFN-γ-treated monocytes on NKG2D expression in different time point and different cell ratio. In some expriments, anti-IL-15 antibodies were adding to the coculture system.4. We detected the activatory marker of CD25 and CD69 and IFN-γproduction of the cocultured CD4+ T cells. In some expriments, anti-IL-15 antibodies,anti-MICs antibodies and transwell were adding to the coculture system.5.We investigated the effect of IL-15 and NKG2D/MIC on the cocultured CD4+ T cells. We detected activatory marker of CD25 and CD69 , IFN-γlevel either using ELISA and intracellular cytokines staining , and the production of perforin and granzyme B in the absence or presence of transwell, anti-IL-15 Abs and anti-MIC Abs. In addition, we observed the proliferatin of cocultred CD4+ T cells by using Cell Counting Kit-8 ( CCK-8)The results are as follows:1. In normal controls, the NKG2D expression was nearly not observed on CD4+ T PBLs (1.2-8.3%, mean 3.4±1.5%) compared with 8-33% (mean 20.1±7.3%) in SLE. The expression of MIC on monocytes is (5–12%, mean7.2±2.4%). In normal controls compared with (11–76%, mean 37±18.1%) in patients. When analyzing the expression of membrane bound IL-15 (mIL-15) on monocytes, we found that mIL-15 expression of CD14+ monocytes in SLE patients (25-48%, mean 36±7.3%) was significantly higher than those in normal controls (5-13%, mean 7.6±2. 5%) .The expression of MIC on monocytes is significantly correlated with SLEDAI. 2. Elevated serum IFN-γhas been found in SLE patients. Our results showed that the concentration of IFN-γin SLE patients (150.3-669.5 pg/mL, mean 429.7±145 pg/mL) was remarkablely higher than those in normal controls (18.8-88.2 pg/mL, mean 73.8±18.9pg/mL),while IFN-α(198.3-489.2pg/mL, mean319±86.1pg/mL) in patients compared with (58.8-322.2 pg/mL, mean151.9±78.5pg/mL) in normal controls. .The concentration of IFN-γin patients is significantly correlated with expression of MIC on monocytes. IFN-γcan upregulated the expression of MIC on primary monocytes and monocytic cell lines. 3. IFN-γpretreated monocytes or freshly isolated monocytes induced NKG2D expression on CD4+ T cells, and can activate CD4+ T Cells via NKG2D/MIC requiring IL-15. 4. Monocytes expressing MIC and mIL-15 promote NKG2D+CD4+ T cells activation via NKG2D/MIC interaction in a IFN-γ-dependent manner.5. The activated CD4+ T cells produced IFN-γand upregulated the MIC and mIL-15 on monocytes. Hence in a feedback , CD4+ T cells and monocytes can crosstalk in IFN-γand mIL-15 dependent manner via NKG2D/MIC interation and activate reciprocally. This crosstalk maybe contribute the exacerbation of the pathogenesis of SLE. |
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