CD226 Expression Deficiency Causes High Sensitivity to Apoptosis in NK T Cells from Patients with Systemic Lupus Erythematosus
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免疫学杂志 2005年第1期
Abstract
Humans and mice with systemic lupus erythematosus (SLE) and related autoimmune diseases have reduced numbers of NK T cells. An association between NK T cell deficiency and autoimmune disease has been identified. However, the mechanisms for reduction of NK T cell number in patients with SLE are unknown. In the present study we report that NK T cells from active SLE patients are highly sensitive to anti-CD95-induced apoptosis compared with those from normal subjects and inactive SLE patients. CD226 expression is deficient on NK T cells from active SLE patients. The expression of one antiapoptotic member protein, survivin, is found to be selectively deficient in freshly isolated NK T cells from active SLE patients. CD226 preactivation significantly up-regulates survivin expression and activation, which can rescue active SLE NK T cells from anti-CD95-induced apoptosis. In transfected COS7 cells, we confirm that anti-CD95-mediated death signals are inhibited by activation of the CD226 pathway through stabilization of caspase-8 and caspase-3 and through activation of survivin. We therefore conclude that deficient expression of CD226 and survivin in NK T cells from active SLE is a molecular base of high sensitivity of the cells to anti-CD95-induced apoptosis. These observations offer a potential explanation for high apoptotic sensitivity of NK T cells from active SLE, and provide a new insight into the mechanism of reduction of NK T cell number in SLE and understanding the association between NK T cell deficiency and autoimmune diseases.
Introduction
Natural killer T cells are a unique T cell subset that coexpresses NK receptors and TCRs (1). Two main subsets of NK T cells have been identified: those positively selected by CD1d (the major subset), and those that are CD1d independent (2). NK T cells play a critical role in modulating the immune responses involved in protection against bacterial or parasitic infections and prevention of autoimmune disease (3, 4, 5). The capacity of NK T cells to activate rapid cytokine expression has been exploited to manipulate the outcomes of autoimmunity and cancer (6). An association between NK T cell deficiency and autoimmune disease has been identified as causal relationship (7, 8, 9). Systemic lupus erythematosus (SLE)4 is a prototypic spontaneous systemic autoimmune disease characterized by autoantibody formation, multiple organ damage, and diverse and variable clinical inflammatory manifestations (10). Recent studies have shown that SLE is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. NK T cells may be part of a regulatory T cell network that inhibits the induction of SLE (11, 12). Humans and mice with lupus and related autoimmune diseases have reduced numbers of CD1d-restricted NK T cells (11, 12), which recover with improvement in disease activity in patients with SLE (12), suggesting a role for these cells in the regulation of SLE (13). An increased rate of apoptosis and decreased clearance of apoptotic cells have been demonstrated in SLE animal models and patients (14, 15), which may play a pathogenic role in the development of the illness. Although the effector mechanisms causing the clinical features of SLE have been studied extensively, the primary defects responsible for the initiation and progression of the disease are still poorly understood, particularly the mechanisms for reduction of NK T cell number in patients with SLE.
Adhesion molecules play fundamental roles in some physiological and pathophysiological processes by mediating leukocyte-endothelial cell adhesion, leukocyte migration, and T cell-APC interactions; regulating several cellular signaling transduction pathways; and even regulating angiogenesis (16). In addition to its role in vascular cell adhesion and signaling, there is growing evidence that certain adhesion molecules, such as CD31, are able to transduce signals to suppress programmed cell death (16). The adhesion molecule CD226, initially named T lineage-specific activation Ag 1, renamed platelet and T cell Ag 1 because of its expression on platelets as well, and also known as DNAX accessory molecule-1 (17, 18, 19), is a transmembrane glycoprotein characterized by two extracellular Ig-like domains and a cytoplasmic portion containing three tyrosine residues (17). CD226 belongs to an Ig superfamily (17, 18, 19) and, with ligands CD112 and CD155 (20), is highly conserved among humans, apes, and monkeys, suggesting very important biological functions (21, 22), such as mediating cytotoxicity and signaling transduction of T cell activation and differentiation (19, 20, 21, 22, 23, 24, 25, 26, 27). CD226 is expressed by virtually all human NK cells, T cells, monocytes, and a small subset of B cells (17, 18, 19, 20). CD226 cooperates with other triggering NK receptors in the induction of NK-mediated cytotoxicity (20). CD226 mediates the intercellular binding between thrombin-activated platelets and HUVEC (25). CD226 and CD18/CD11a molecules are physically and functionally associated in normal cells and are involved in a costimulatory signal for naive T cell differentiation and proliferation (26, 27). CD226/CD112 and CD155 interactions may play a role in the interaction between leukocytes and endothelial cells in the process of leukocyte recirculation (20). To date, there has been no report on the correlation between CD226 signals and regulation of cell apoptosis.
In the present study we report that NK T cells from active SLE patients are more sensitive to anti-CD95-induced apoptosis. CD226 is significantly down-regulated on NK T cells from active SLE patients. Survivin is selectively deficient in NK T cells from active SLE patients. In transfected COS-7 cells and human NK T cells, we confirm that anti-CD95-mediated death signals are inhibited by activation of the CD226 pathway through activation of survivin. A deficiency of CD226 and survivin in NK T cells from active SLE is the molecular basis of the high sensitivity of the cells to anti-CD95-induced apoptosis.
Materials and Methods
Patients, normal subjects, and cell purification
SLE patients who fulfilled at least four of the American Rheumatism Association 1982 revised criteria for SLE (28) were included. The 47 SLE patients (43 women and four men; median age, 32.7 years; range, 20–49 years) had a mean duration of disease of 4.2 years (0.5–11 years). Disease activity was assessed by a modified SLE disease activity index (SLEDAI) score (29). The mean SLEDAI for the active patients was 14.8 (range, 3–30), and that for inactive patients was 0 (29). All patients were informed consent according to institutional guidelines. A total of 20 health volunteers (18 women and two men; median age, 29.5 years; range, 18–46 years) served as controls. NK T cells were purified from mononuclear cells from peripheral blood of patients with SLE or normal subjects as described previously with modification (30). A nylon wood column assay was used for negative selection of CD3+ T cells to avoid preactivation (31), followed by a selection procedure using anti-CD56 mAb-coated Dynabeads (Dynal Biotech) according to the manufacturer’s instructions for CD56+CD3+ cells. The purity of NK T cells ranged from 96–99%, as determined by flow cytometry.
Induction of apoptosis and CD226 activation or blockade of purified NK T cell
For induction of apoptosis of purified NK T cells using immobilized anti-CD95 mAb (ECOS9.1) or anti-CD3 mAb (UCHT1; from BD Pharmingen), the assays were conducted as described previously (32, 33). We generated a mouse anti-human mAb against CD226. For CD226 activation, anti-CD226 mAb was immobilized by adding it to the wells in culture plates (2 μg/ml) at 4°C overnight, followed by washing twice before use. A single cell suspension was added to the plate and cultured for 8 h before subsequent procedures. For CD226 blockade, signal cell suspension was added to soluble mAb against CD226 (5 μg/ml) and kept at room temperature for 4 h before subsequent procedures.
Flow cytometry
For detection of CD226, the cells were incubated with anti-CD226 mAb (5 μg/ml) at 4°C for 60 min, followed by an appropriately labeled secondary Ab. Analysis of V24+V11+ NK T cells was performed on PBMC or purified NK T cell. FITC-labeled anti-human V24 mAb and PE-labeled anti-human V11 mAb were used (Immunotech). For CD95 and CD95L detection, cells were first incubated with CD95 mAb (clone 13) or CD95L mAb (clone 33; BD Biosciences Europe) at 5μg/ml or 5 μg/ml isotype-matched mAb (DakoCytomation) in staining buffer (34). After 20 min, the cells were washed twice with staining buffer and resuspended in 50 μl of CyChrome-conjugated secondary mAb (BD Pharmingen) for 20 min. For detection of apoptosis, cells were stained in staining medium (RPMI 1640, 2% FBS, and 0.1% sodium azide) with 1 μg/ml propidium iodide (PI) for 30 min at 4°C, then stained with FITC-conjugated annexin V with binding buffer (BD Pharmingen) as previously described (35). Coulter XL was used for analyses.
Real-time quantitative RT-PCR assay
All real-time quantitative RT-PCRs were performed as described previously (36, 37). Briefly, total RNA from purified NK T cells (1 x 105; purity >99%) was prepared using Quick Prep total RNA extraction kit (Pharmacia Biotech) according to the manufacturer’s instructions. RNA was reverse transcribed using oligo(dT)12–18 and Superscript II reverse transcriptase (Invitrogen Life Technologies). The real-time quantitative PCR was performed in special optical tubes in a 96-well microtiter plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems). Using SYBR Green PCR Core Reagents Kit (Applied Biosystems; P/N 4304886), fluorescence signals were generated during each PCR cycle via the 5' to 3' endonuclease activity of AmpliTaq Gold to provide real-time quantitative PCR information. The sequences of the specific primers are listed below: CD226 sense, 5'-TCAAATAGCCACATTGTTTCGGA-3'; CD226 antisense, 5'-AGGGTATATGGTTACGGTTTTACC-3'; survivin sense, 5'-GTGTTTCTTCTGCTTCAAGGAGCT-3'; and survivin antisense, 5'-CAGAGGCCTCAATCCATGGCAGCC-3'. All unknown cDNAs were diluted to contain equal amounts of -actin cDNA. The standards, no template controls, and unknown samples were added in a total volume of 50 μl/reaction. The PCR conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 60 s at 60°C for each amplification. Potential PCR product contamination was digested by uracil-N-glycosylase, because dTTP is substituted by dUTP. Uracil-N-glycosylase and AmpliTaq Gold (PE Applied Biosystems) were applied according to the manufacturer’s instructions.
Blot assays
For mRNA detection (Northern blot), 2 μg of total RNA obtained from each sample were electrophoresed under denaturing conditions, followed by blotting onto Nytran membranes and cross-linking by UV irradiation as previously described (38). CD226 cDNA probe, labeled by [-32P]dCTP, was obtained by PCR amplification of the sequence mentioned above from total RNA from PBMCs from healthy subjects. The membranes were hybridized overnight with 1 x 106 cpm/ml 32P-labeled probe, followed by intensively washing before being autoradiographed. For protein detection (Western blot analysis), the cells were lysed in lysis buffer (0.4% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, 1 mM sodium vanadate, 0.1 mM PMSF, and 2 μg/ml leupeptin and apoprotein, pH 8.0) as previously described (39). Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. The protein concentration was measured using a Bio-Rad protein assay. Protein (40 μg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs (anti-Bcl-2, clone BCL-2/100; anti-caspase-3, clone CPP32–19 (BD Pharmingen); anti-caspase-8, clone H-134; anti-survivin, clone FL-142 (Santa Cruz Biotechnology); anti--actin (Sigma-Aldrich); all at 0.5 μg/ml). Analyses were conducted using ECL detection (Amersham Biosciences).
Cell transient transfection
COS-7 cells were transiently transfected with CMV vector pcDNA3/CD95 and/or CMV vector pcDNA3/CD226 as described previously (20, 40). Briefly, the cells were cultured with DMEM containing 10% FCS, penicillin, and streptomycin. Cells were grown to 70% confluence in 60-mm dishes for 24 h before transfection. The DNA construct of mammalian expression vector pCMV/CD95 and/or pCMV/CD226 (5 μg; or vector only) was mixed with 12 μl of Lipofectamine (Invitrogen Life Technologies) in 2 ml of OptiMEM serum-free medium, added to cells, and incubated for 6 h. The cells were further cultured in 2.5 ml of DMEM containing 10% FCS in 5% CO2. The successful transfection was demonstrated by confocal microscopy as described previously (41). Aspreviously described (42), human NK T cells were transfected CMV vector pcDNA3/CD226 using Amaxa nucleofection technology (Amaxa) with optimization. Briefly, cells were resuspended in solution from nucleofector kit V following the Amaxa guidelines for cell transfection. One hundred microliters of 3 x 106 cell suspension mixed with 0.25 or 2.5 μg of cDNA was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa).
Results
NK T cells from active SLE are more sensitive to apoptosis
Knowing the abnormality of apoptosis of some types of immune cells in patients with SLE and in mice with lupus (7, 8, 9), we examined the death-promoting effect of signals transmitted through CD95-CD95L and CD3-TCR on NK T cells from different subjects. Flow cytometric analysis (Fig. 1) revealed that the numbers of apoptotic and necrotic cells were significantly increased in cultures of NK T cells from active SLE patients (aSLE) in the presence of immobilized anti-CD95 or anti-CD3 mAb (Fig. 1, a and b; p < 0.001) compared with that in cell cultures from normal subjects (i and j). The apoptotic responses in inactive SLE (iSLE) patients were marginally increased (e and f). As a positive control, cells were stimulated with campotothecin, which induces identical apoptosis in all three types of subjects (c, g, and k), whereas isotype Abs for anti-CD95 and anti-CD3 did not induce apoptosis in the cells (data not shown). The cell cultures for distinct subject groups in the absence of stimuli showed the unstimulated basic lines of cell apoptosis (d, h, and l). The summary of data (mean ± SD) for apoptotic cells (PI– annexin V+) of six experiments performed in each subject group is shown in Fig. 1B, documenting the sensitivity to apoptosis of NK T cells from aSLE patients. As shown in Fig. 1C, the total fractions of dead cells (including apoptotic and necrotic) in NK T cells from normal subjects and iSLE patients were significantly lower than that in aSLE patients in anti-CD95- and anti-CD3-induced apoptotic assays, but identical levels were observed in campotothecin-induced apoptosis in all three types of subjects. These results could confirm that NK T cells from active SLE were highly sensitive to apoptotic induction by the CD95-CD95L pathway or the CD3-TCR pathway. To explore the reasons for the phenomena observed above, we examined the levels of CD95 and CD95L expression on NK T cells from various subjects. Flow cytometry revealed that CD95 was expressed in identical levels on NK T cells from different subjects (Fig. 2), and that the expression of CD95L on NK T cells from aSLE patients was slightly higher than those in normal subjects and iSLE patients, strongly suggesting that the greater sensitivity to apoptosis of NK T cells from aSLE patients could not be explained by the levels of CD95 and CD95L expression on the cells. We also examined the sensitivity of total CD3+ T cells, B cells, and monocytes to anti-CD95-induced apoptosis. The results showed that aSLE CD3+ T cells were slightly, but significantly, more sensitive to anti-CD95-induced apoptosis than cells from iSLE and normal subjects; otherwise, there were no significant differences in sensitivity to anti-CD95-induced apoptosis in distinct types of cells among aSLE, iSLE, and normal subjects (data not shown).
CD226 expression is deficiently expressed on NK T cells from aSLE patients
We examined the expressions of some adhesion molecules (L-selectin, CD18/CD11a, CD15, CD28, CD29, CD31, CD34, CD44, CD49a-e, CD51, CD54/ICAM-1, CD62E, and VCAM-1/VLA-4) on NK T cells from three types of subjects, and either the results were in agreement with previous reports or no differences were found among the three types of subjects (data not shown). To search for the mechanism of the high apoptotic sensitivity of NK T cells from aSLE patients, we examined the expression of CD226 on NK T cells from aSLE patients, iSLE patients, and normal subjects. The results from flow cytometric analyses shown in Fig. 3 documented that there were different numbers of NK T cells in the three groups of subjects. There were only 1.5% of V24+V11+ NK T cells of the total T cell population in aSLE patients and 21% of CD226-positive cells (Fig. 3A). There were 8% V24+V11+ NK T cells of the total T cell population in normal subjects and 88% of CD226-positive cells (Fig. 3C), whereas there were 4% V24+V11+ NK T cells of the total T cell population in inactive SLE patients and 47% of CD226-positive cells (Fig. 3B). To confirm the observation mentioned above, we performed a real-time quantitative RT-PCR assay to detect the different expressions of CD226 at the mRNA level. The results (Fig. 3D) showed that CD226 mRNA was expressed at high levels in freshly isolated NK T cells from normal subjects (1.3 x 104 copies). CD226 mRNA expressions was significantly decreased in NK T cells from aSLE patients (1.2 x 103 and 0.9 x 103 copies in two separate cases) compared with that in normal subjects, whereas there was an increase in NK T cells from the iSLE patients (6 x 103 copies). A linear relationship between cycle threshold and log starting quantity of standard DNA template or target cDNAs was detected (data not shown). In all experiments, the correlation coefficients were 0.95–0.99. The same pattern of CD226 mRNA and protein expressions in NK T cells from aSLE patients, iSLE patients, and normal subjects was confirmed by Northern and Western blots (Fig. 3, E and F). We also examined the CD226 expression levels in total CD3+ T cells, B cells, and monocytes. There were no significant differences in CD226 expression levels in total CD3+ T cells, B cells, and monocytes from aSLE, iSLE, and normal subjects, determined by real-time RT-PCR assay and confirmed by Northern and Western blot analyses (data not shown).
To further investigate the correlation between the sensitivity to apoptosis and the level of CD226 expression on the cells mentioned above, we conducted anti-CD95-induced apoptosis in NK T cells from normal, iSLE, and aSLE subjects by preactivating and blocking the function of CD226. We used immobilized or soluble anti-CD226 mAb to activate or block CD226 on the cells, respectively. As shown in Fig. 4A, preactivation of CD226 could rescue NK T cells from aSLE patients from anti-CD95-induced apoptosis, although CD226 expression was low in freshly isolated cells (Fig. 3). Compared with the results shown in Fig. 1Aa (55% of dead cells), there were only 14% of dead NK T cells from active SLE, in which the cells were preactivated by anti-CD226 (Fig. 4Aa). The blockade function of CD226 reduced the resistance of anti-CD95-induced apoptosis in NK T cells from normal subjects, although CD226 expression was high in freshly isolated cells. The percentage of total dead cells reached 58% (Fig. 4A). Isotype Ab had no such effects (Fig. 4A, c, f, and i). As shown in Fig. 4B, the total fractions of dead cells (including apoptotic and necrotic) in NK T cells from different subjects showed patterns similar to that in Fig. 4A, confirming that activation of CD226 could rescue NK T cells from anti-CD95-induced apoptosis, whereas blockade of CD226 function could significantly reduce the resistance of NK T cells from aSLE patients to anti-CD95-induced apoptosis. Of note, the explanation for the finding that CD226 activation still could rescue low expressed CD226 aSLE NK T cells from apoptosis could be that abundant binding sites of CD226 in experimental system (anti-CD226 mAb at 2 μg/ml was immobilized) were sufficient to activate aSLE NK T cells through limited CD226 receptors. We also observed that preactivation of CD226 could rescue NK T cells from active SLE patients from anti-CD3-induced apoptosis (data not shown), confirming the rescuing function of CD226 activation from apoptosis.
Mechanisms of CD226 involved in high sensitivity to anti-CD95-induced apoptosis in NK T cells from aSLE patients
To examine the mechanisms of CD226 expression and activation involved in high sensitivity to anti-CD95-induced apoptosis in NK T cells from active SLE patients, we first examined the expression levels of some important substrates of the apoptosis pathways in the cells. Western blots revealed that key apoptotic executioner molecules, procaspase-8 and procaspase-3, were expressed at identical levels in freshly isolated NK T cells from different subjects (Fig. 5A). Western blot showed that the protein levels of one antiapoptotic member, Bcl-2, in freshly isolated NK T cells from different subjects were also identical (Fig. 5B). Interestingly, one antiapoptotic member protein, survivin, was selectively expressed at very low levels or was absent in freshly isolated NK T cells from active SLE patients, whereas it was expressed at high levels in freshly isolated NK T cells from normal subjects and iSLE patients (Fig. 5B). To confirm deficient expression of survivin in NK T cells from aSLE patients, we examined the expression of survivin mRNA in NK T cells from aSLE patients, iSLE patients, and normal subjects. We conducted a real-time quantitative RT-PCR assay to detect the different expressions of survivin at the mRNA level. The results (Fig. 5C) showed that survivin mRNA was expressed at a very low level in freshly isolated NK T cells from active SLE patients (0.8 x 103 copies). Survivin mRNA expression was significantly increased in NK T cells from the normal subjects (5.0 x 103 copies) compared with that from aSLE patients, whereas there was also an increase in NK T cells from the iSLE patients (3.5 x 103 copies). The same pattern of survivin mRNA expression in NK T cells from aSLE patients, iSLE patients, and normal subjects were confirmed by Northern blot (Fig. 5D). Thus, it might be important to maintain a certain level of expression of survivin to balance between proapoptotic and antiapoptotic pathways in cells. We also examined the survivin expression levels in total CD3+ T cells, B cells, and monocytes. The results showed that there were no significant differences in survivin expression levels in total CD3+ T cells, B cells, and monocytes from aSLE, iSLE, and normal subjects, determined by real-time RT-PCR assay and confirmed by Northern and Western blot analyses (data not shown).
To determine the mechanism of CD226 activation to rescue NK T cells from programmed death, we conducted a series of experiments to investigate the activities of a number of proapoptotic and antiapoptotic members during the process of anti-CD95-induced apoptosis in cells from aSLE patients under different conditions of CD226 activation, as indicated in Fig. 6. In the antiapoptotic pathway systems, Bcl-2 activity under different conditions of CD226 activation was seemly identical in aSLE NK T cells. CD226 activation significantly up-regulated survivin activity, which rescued the cells from apoptosis (Fig. 6A). In the proapoptotic pathway systems, active forms of caspase-8 and caspase-3 were significantly reduced after activation of CD226, suggesting that anti-CD95-mediated death signals were inhibited by activation of the CD226 pathway (Fig. 6, B and C). Thus, abnormal expression of survivin and deficient expression of CD226 were the molecular bases of the high sensitivity of NK T cells from aSLE patients to anti-CD95-induced apoptosis.
CD226 activation protects CD95- and CD226-transfected COS-7 cells from apoptosis
To confirm the observation of a protective effect of CD226 from apoptosis of NK T cells from active SLE patients, we chose a cell line, COS-7, that was able to express CD95 and CD226 after transfection and in which apoptosis was inducible by anti-CD95 mAb (20, 40). We had a successful double transfection of CD95 and CD226 (data not shown). Flow cytometric analysis revealed that the numbers of apoptotic and necrotic cells were significantly increased in culture of CD95- or/and CD226-transfected COS-7 cells in the presence of immobilized anti-CD95 mAb compared with that of vector-only-transfected cells or CD226-only-transfected cells (data not shown). We used immobilized or soluble anti-CD226 mAb to preactivate or block CD226 function in double-transfected COS-7 cells, respectively (data not shown). Preactivation of CD226 mAb could rescue COS-7 cells from anti-CD95-induced apoptosis (data not shown). As expected, the blockade of CD226 restored the sensitivity of double-transfected COS-7 cells to anti-CD95-induced apoptosis (data not shown). Isotype Ab for anti-CD226 had no such effect (data not shown). To confirm the mechanisms of the rescuing function of CD226 activation in COS-7 cells from anti-CD95-induced apoptosis, we first examined the expression levels of some important substrates of apoptosis pathways in the cells during the apoptotic process (data not shown). Western blots revealed that key apoptotic executioner molecules, procaspase-8 and procaspase-3, were stabilized by preactivation of CD226, where COS-7 cells were rescued from anti-CD95-induced apoptosis (data not shown). Western blot also showed that the protein levels of one antiapoptotic member, Bcl-2, in COS-7 cells under different conditions were identical (data not shown). Interestingly, the antiapoptotic protein, survivin, was selectively activated under the condition of CD226 preactivation, where COS-7 cells were rescued from anti-CD95-induced apoptosis (data not shown). Thus, we confirmed that in the transfected COS-7 cell system, anti-CD95-mediated death signals were inhibited by activation of the CD226 pathway through stabilization of caspase-8 and caspase-3 and through activation of survivin.
CD226 transfection rescues aSLE NK T cells from anti-CD95-induced apoptosis
To confirm the finding of a correlation between CD226 expression and resistance to anti-CD95-induced apoptosis in the COS-7 cell system, we applied Amaxa nucleofection technology to transfect cDNA encoding CD226 into human NK T cells from aSLE patients, iSLE patients, and normal subjects to investigate the effect of CD226 expression on resistance to anti-CD95-induced apoptosis. The data in Fig. 7, A and B, demonstrate that successful transfection of CD226 took place; particularly NK T cells from aSLE patients were well transfected with cDNA encoding CD226, as documented by Western blot (Fig. 7A) and flow cytometry (Fig. 7B). The results (Fig. 7, C and D) from flow cytometric analysis revealed that the number of apoptotic cells was significantly decreased in the culture of sufficient CD226-transfected (2.5 μg) NK T cell from active SLE patients in the presence of immobilized anti-CD95 (Fig. 7, Cd and D; p < 0.001) compared with that of untransfected cells and vector only (Fig. 7C, a and b). Low dose CD226 transfection (0.25 μg) was insufficient to rescue aSLE NK T cell from anti-CD95-induced apoptosis (Fig. 7Cc). A sufficient CD226 transfection (25 μg) could also rescue iSLE NK T cell from anti-CD95-induced apoptosis (Fig. 7, Ch and D; p < 0.01); even the apoptotic responses in inactive SLE patients were marginally increased (Fig. 7C, e–g). CD226 transfection in normal NK T cells had little effect on anti-CD95-induced apoptosis (Fig. 7, C, i–l, and D), because normal NK T cells already expressed abundant CD226 (Fig. 3). However, experiments of CD226 transfection in normal NK T cells showed that the transfection procedure itself did not affect the sensitivity of cells to apoptosis (Fig. 7C, a, e, and i). Thus, the results confirmed the correlation between CD226 expression and resistance to anti-CD95-induced apoptosis in the human NK T cell system.
Corticosteroid medication affects CD226 and survivin expression as well as sensitivity to apoptosis in NK T cells from aSLE patients
To examine the effects of corticosteroid medication on CD226 and survivin expression as well as sensitivity to apoptosis in NK T cells from aSLE patients, as shown in Table I, six patients with active SLE were recruited and were administrated prednisone as a major therapy. At the time of treatment, blood samples were taken. The data showed that CD226 expression in NK T cells at the mRNA and protein levels was significantly increased after successful treatment with prednisone. It began at wk 4 of treatment. Survivin expression in NK T cells was also significantly increased. It began at wk 6 of treatment. The anti-CD95-induced apoptosis in NK T cells also appeared to significantly decrease at wk 6 of treatment as the SLEDAI score decreased.
Together these data show that NK T cells from active SLE patients were highly sensitive to anti-CD95-induced apoptosis compared with that in normal subjects and iSLE patients. CD226 expression was deficiently expressed on NK T cells from aSLE patients. Survivin was found to be selectively expressed at a very low level in freshly isolated NK T cells from active SLE patients. CD226 preactivation significantly up-regulated survivin activation, which could rescue NK T cells from aSLE patients from anti-CD95-induced apoptosis. The observations were confirmed in transfected COS-7 cells and human NK T cells in terms of the correlation between activation of CD226 and stabilization of death signals.
Discussion
Development of SLE is associated with the emergence of autoreactive Th cells (43) and is accompanied by a reduction in regulatory T cells (44, 45). The nature and specificity of regulatory T cells that inhibit autoantibody production and development of lupus remain largely undefined. Some regulatory T cells are T cells or VH peptide-reactive CD8+ T cells that inhibit autoantibody production by ablating autoreactive B cells (44). Recent reports that humans and mice with lupus have reduced numbers of NK T cells (10, 11) suggest that NK T cells may be part of a regulatory T cell network to inhibit the induction of SLE. To understand the mechanism of reduction of NK T cell number in SLE, we focused our study on the sensitivity of NK T cells to induction of apoptosis during active and inactive disease processes and on determining its mechanisms. In the present study we have demonstrated that CD226 is significantly down-regulated on NK T cells from aSLE patients, which may be one of the reasons why NK T cells from aSLE patients are more sensitive to apoptosis. Abnormal and deficient expression of CD226 and survivin are the molecular bases of the high sensitivity of NK T cells from aSLE patients to anti-CD95-induced apoptosis. To our knowledge this is the first report of CD226-deficient expression on NK T cells from aSLE patients, providing direct evidence that the abnormality in CD226-induced high sensitivity to apoptosis is responsible for the reduction of NK T cell number in patients with active SLE.
In humans, the function of NK/NK T cells is regulated by a series of surface receptors that either inhibit or enhance NK/NK T-mediated cytolytic activity. When NK cells encounter potential target cells, killer inhibitory Ig-like receptors and/or the CD94/NKG2A heterodimer sense the expression of HLA class I molecules at the cell surface (46, 47). When appropriate inhibitory receptor/HLA class I ligand interactions occur, the receptors deliver negative signals that inhibit NK/NK T-mediated killing. In the absence of such interactions, as may occur in tumor or virus-infected target cells, non-HLA class I-specific activating receptors are allowed to deliver triggering signals to NK/NK T cells, resulting in target cell killing. Several non-HLA class I-specific activating receptors have been identified, including NKp46, NKp30, NKp44, and NKG2D (48, 49). The leukocyte adhesion molecule CD226, expressed by virtually all human NK cells, T cells, and monocytes and by a small subset of B cells, has been shown to participate in the induction phase of NK cell activation (19, 20, 26). CD226 function is dependent on the surface expression of CD18/CD11a (26, 27). Upon mAb-mediated cross-linking, CD226 transduces activating signals, resulting in tyrosine phosphorylation of CD226 itself and enhancement of cytotoxicity and cytokine production in both T and NK cells (19, 20, 26); it is also involved in the costimulatory signal for naive T cell differentiation and proliferation (20, 26, 27). In the present study at the protein and mRNA levels we have demonstrated that CD226 is significantly down-regulated on V24+V11+ NK T cells from aSLE patients (Figs. 3 and 4). Meanwhile, there are only 1.5% V24+V11+ NK T cells of the total T cell population in aSLE patients. We used immobilized or soluble anti-CD226 mAb to preactivate or block CD226 on the cells, respectively. Preactivation of CD226 can rescue NK T cells from active SLE patients from anti-CD95-induced apoptosis. Compared with the results shown in Fig. 1 (55% of dead cells), there are only 14% dead NK T cells from active SLE, where the cells were preactivated by anti-CD226 (Fig. 5). The findings that signals by CD226 pre activation in aSLE NK T cells increase the resistance to induction of apoptosis strongly suggest that CD226 signals can cross-talk with cell death pathways by either stabilization of the death executioner system or activation of the cell apoptosis inhibitor system, resulting in resistance of cells to apoptotic signals.
To explore the molecular basis of CD226 rescuing functions on NK T cells from active SLE patients, we examined the activity of a number of adaptor proteins during the process. Two major cell death pathways, termed the extrinsic and intrinsic pathways of apoptosis, exist in mammalian cells (50). The extrinsic pathway is initiated by engagement and aggregation of TNF family death receptors (such as CD95) through a series of death domain-containing adaptor molecules that recruit and directly activate cytosolic caspase 8, which, in turn, converts procaspase 3 to caspase 3, the central executioner of the apoptotic process (51, 52). The intrinsic pathway is intimately tied to the integrity of mitochondria and other intracellular organelles. The normal and healthy cells are maintained by a carefully regulated balance of proapoptotic and antiapoptotic members of the Bcl-2 family of proteins. In mitochondria of normal and healthy cells, resident antiapoptotic proteins, such as Bcl-2 and Bcl-xL, effectively bind up and neutralize proapoptotic proteins (53) and prevent perturbation of the outer mitochondrial membrane. A series of endogenous caspase inhibitors, known as inhibitors of apoptosis, normally interact with and inhibit the enzymatic activity of the initiator caspase 9 and the effectors caspases 3 and 7 (54). Both the location and the activation state of proapoptotic and antiapoptotic components of the cell death pathway are crucial for controlling cellular decisions about life and death. Survivin, a member of the inhibitors of apoptosis family, is potentially involved in both the inhibition of apoptosis and the control of cell division (55, 56, 57). Being essential for embryonic development (55), survivin expression in adult human tissues is usually reported in the context of its relationship to cancer. It is prominently expressed in the majority of human cancers, including those of colon, breast, lung, and high grade non-Hodgkin’s lymphoma (58). There are reports that survivin is also expressed in some normal human adult tissues, including bone marrow, colonic mucosa, endometrium, thymus, and keratinocytes (59, 60). We have observed that survivin is selectively expressed at very low levels in NK T cells from aSLE patients compared with the level in freshly isolated NK T cells from normal subjects (Fig. 6). Furthermore, CD226 activation up-regulate significantly the expression and activation of survivin, which may be one of the reasons to rescue the cells (Fig. 7).
What is the essential correlation between the defective expression of CD226 and the signaling abnormalities identified to date in aSLE NK T cells? There is a mounting body of evidence indicating increased intracellular calcium in T cells from SLE patients due to defective expression of the TCR -chain (61, 62, 63, 64, 65). Several previous studies of CD226 (20, 21, 26, 27) indicate that this molecule is a signaling element that triggers cytotoxicity by engagement of extracellular ligand or cellular counter-receptor (CD155 and CD112) and postulate that CD226 is induced upon T cell activation, and the expression persists unless cells are exposed to signal that elevates intracellular free calcium (21). Hence, the increased intracellular free calcium concentration in SLE T cells may lead to down-regulation of CD226 expression and, thus, increased apoptosis of NK T cells.
Generally, deficient expression of survivin and CD226 on aSLE NK T cells establishes a molecular basis of sensitivity of the cells to anti-CD95-induced apoptosis. These observations offer a potential explanation for the high apoptotic sensitivity of aSLE NK T cells and provide a new insight into the mechanism of reduction of NK T cell number in SLE and understanding the association between NK T cell deficiency and autoimmune diseases.
Footnotes
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.
1 This work was supported by the National Science Foundation of China (Grants 39870674 and 30030130); a special grant from the Personnel Department of Wuhan University, China; the Science Foundation of Anhui Province, China (Grant 98436630); the Education and Research Foundation of Anhui Province, China (Grant 98JL063); the Research Foundation of the Health Department of Hubei Provincial Government, China (Grant 301140344); and the National Key Basic Research Program of China (2001CB510004).
2 D.T. and L.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Tan Jinquan, Department of Immunology, Wuhan University School of Medicine, Dong Hu Road 115, Wuchang 430071, Wuhan, Peoples Republic of China. E-mail address: jinquan_tan@hotmail.com; or Dr. Jin Boquan, Department of Immunology, Fourth Military Medical University, Xian, Peoples Republic of China. E-mail address: immu_jin@fmmu.edu.cn
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; aSLE, active SLE; iSLE, inactive SLE; PI, propidium iodide; SLEDAI, SLE disease activity index.
Received for publication January 16, 2004. Accepted for publication November 12, 2004.
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Humans and mice with systemic lupus erythematosus (SLE) and related autoimmune diseases have reduced numbers of NK T cells. An association between NK T cell deficiency and autoimmune disease has been identified. However, the mechanisms for reduction of NK T cell number in patients with SLE are unknown. In the present study we report that NK T cells from active SLE patients are highly sensitive to anti-CD95-induced apoptosis compared with those from normal subjects and inactive SLE patients. CD226 expression is deficient on NK T cells from active SLE patients. The expression of one antiapoptotic member protein, survivin, is found to be selectively deficient in freshly isolated NK T cells from active SLE patients. CD226 preactivation significantly up-regulates survivin expression and activation, which can rescue active SLE NK T cells from anti-CD95-induced apoptosis. In transfected COS7 cells, we confirm that anti-CD95-mediated death signals are inhibited by activation of the CD226 pathway through stabilization of caspase-8 and caspase-3 and through activation of survivin. We therefore conclude that deficient expression of CD226 and survivin in NK T cells from active SLE is a molecular base of high sensitivity of the cells to anti-CD95-induced apoptosis. These observations offer a potential explanation for high apoptotic sensitivity of NK T cells from active SLE, and provide a new insight into the mechanism of reduction of NK T cell number in SLE and understanding the association between NK T cell deficiency and autoimmune diseases.
Introduction
Natural killer T cells are a unique T cell subset that coexpresses NK receptors and TCRs (1). Two main subsets of NK T cells have been identified: those positively selected by CD1d (the major subset), and those that are CD1d independent (2). NK T cells play a critical role in modulating the immune responses involved in protection against bacterial or parasitic infections and prevention of autoimmune disease (3, 4, 5). The capacity of NK T cells to activate rapid cytokine expression has been exploited to manipulate the outcomes of autoimmunity and cancer (6). An association between NK T cell deficiency and autoimmune disease has been identified as causal relationship (7, 8, 9). Systemic lupus erythematosus (SLE)4 is a prototypic spontaneous systemic autoimmune disease characterized by autoantibody formation, multiple organ damage, and diverse and variable clinical inflammatory manifestations (10). Recent studies have shown that SLE is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. NK T cells may be part of a regulatory T cell network that inhibits the induction of SLE (11, 12). Humans and mice with lupus and related autoimmune diseases have reduced numbers of CD1d-restricted NK T cells (11, 12), which recover with improvement in disease activity in patients with SLE (12), suggesting a role for these cells in the regulation of SLE (13). An increased rate of apoptosis and decreased clearance of apoptotic cells have been demonstrated in SLE animal models and patients (14, 15), which may play a pathogenic role in the development of the illness. Although the effector mechanisms causing the clinical features of SLE have been studied extensively, the primary defects responsible for the initiation and progression of the disease are still poorly understood, particularly the mechanisms for reduction of NK T cell number in patients with SLE.
Adhesion molecules play fundamental roles in some physiological and pathophysiological processes by mediating leukocyte-endothelial cell adhesion, leukocyte migration, and T cell-APC interactions; regulating several cellular signaling transduction pathways; and even regulating angiogenesis (16). In addition to its role in vascular cell adhesion and signaling, there is growing evidence that certain adhesion molecules, such as CD31, are able to transduce signals to suppress programmed cell death (16). The adhesion molecule CD226, initially named T lineage-specific activation Ag 1, renamed platelet and T cell Ag 1 because of its expression on platelets as well, and also known as DNAX accessory molecule-1 (17, 18, 19), is a transmembrane glycoprotein characterized by two extracellular Ig-like domains and a cytoplasmic portion containing three tyrosine residues (17). CD226 belongs to an Ig superfamily (17, 18, 19) and, with ligands CD112 and CD155 (20), is highly conserved among humans, apes, and monkeys, suggesting very important biological functions (21, 22), such as mediating cytotoxicity and signaling transduction of T cell activation and differentiation (19, 20, 21, 22, 23, 24, 25, 26, 27). CD226 is expressed by virtually all human NK cells, T cells, monocytes, and a small subset of B cells (17, 18, 19, 20). CD226 cooperates with other triggering NK receptors in the induction of NK-mediated cytotoxicity (20). CD226 mediates the intercellular binding between thrombin-activated platelets and HUVEC (25). CD226 and CD18/CD11a molecules are physically and functionally associated in normal cells and are involved in a costimulatory signal for naive T cell differentiation and proliferation (26, 27). CD226/CD112 and CD155 interactions may play a role in the interaction between leukocytes and endothelial cells in the process of leukocyte recirculation (20). To date, there has been no report on the correlation between CD226 signals and regulation of cell apoptosis.
In the present study we report that NK T cells from active SLE patients are more sensitive to anti-CD95-induced apoptosis. CD226 is significantly down-regulated on NK T cells from active SLE patients. Survivin is selectively deficient in NK T cells from active SLE patients. In transfected COS-7 cells and human NK T cells, we confirm that anti-CD95-mediated death signals are inhibited by activation of the CD226 pathway through activation of survivin. A deficiency of CD226 and survivin in NK T cells from active SLE is the molecular basis of the high sensitivity of the cells to anti-CD95-induced apoptosis.
Materials and Methods
Patients, normal subjects, and cell purification
SLE patients who fulfilled at least four of the American Rheumatism Association 1982 revised criteria for SLE (28) were included. The 47 SLE patients (43 women and four men; median age, 32.7 years; range, 20–49 years) had a mean duration of disease of 4.2 years (0.5–11 years). Disease activity was assessed by a modified SLE disease activity index (SLEDAI) score (29). The mean SLEDAI for the active patients was 14.8 (range, 3–30), and that for inactive patients was 0 (29). All patients were informed consent according to institutional guidelines. A total of 20 health volunteers (18 women and two men; median age, 29.5 years; range, 18–46 years) served as controls. NK T cells were purified from mononuclear cells from peripheral blood of patients with SLE or normal subjects as described previously with modification (30). A nylon wood column assay was used for negative selection of CD3+ T cells to avoid preactivation (31), followed by a selection procedure using anti-CD56 mAb-coated Dynabeads (Dynal Biotech) according to the manufacturer’s instructions for CD56+CD3+ cells. The purity of NK T cells ranged from 96–99%, as determined by flow cytometry.
Induction of apoptosis and CD226 activation or blockade of purified NK T cell
For induction of apoptosis of purified NK T cells using immobilized anti-CD95 mAb (ECOS9.1) or anti-CD3 mAb (UCHT1; from BD Pharmingen), the assays were conducted as described previously (32, 33). We generated a mouse anti-human mAb against CD226. For CD226 activation, anti-CD226 mAb was immobilized by adding it to the wells in culture plates (2 μg/ml) at 4°C overnight, followed by washing twice before use. A single cell suspension was added to the plate and cultured for 8 h before subsequent procedures. For CD226 blockade, signal cell suspension was added to soluble mAb against CD226 (5 μg/ml) and kept at room temperature for 4 h before subsequent procedures.
Flow cytometry
For detection of CD226, the cells were incubated with anti-CD226 mAb (5 μg/ml) at 4°C for 60 min, followed by an appropriately labeled secondary Ab. Analysis of V24+V11+ NK T cells was performed on PBMC or purified NK T cell. FITC-labeled anti-human V24 mAb and PE-labeled anti-human V11 mAb were used (Immunotech). For CD95 and CD95L detection, cells were first incubated with CD95 mAb (clone 13) or CD95L mAb (clone 33; BD Biosciences Europe) at 5μg/ml or 5 μg/ml isotype-matched mAb (DakoCytomation) in staining buffer (34). After 20 min, the cells were washed twice with staining buffer and resuspended in 50 μl of CyChrome-conjugated secondary mAb (BD Pharmingen) for 20 min. For detection of apoptosis, cells were stained in staining medium (RPMI 1640, 2% FBS, and 0.1% sodium azide) with 1 μg/ml propidium iodide (PI) for 30 min at 4°C, then stained with FITC-conjugated annexin V with binding buffer (BD Pharmingen) as previously described (35). Coulter XL was used for analyses.
Real-time quantitative RT-PCR assay
All real-time quantitative RT-PCRs were performed as described previously (36, 37). Briefly, total RNA from purified NK T cells (1 x 105; purity >99%) was prepared using Quick Prep total RNA extraction kit (Pharmacia Biotech) according to the manufacturer’s instructions. RNA was reverse transcribed using oligo(dT)12–18 and Superscript II reverse transcriptase (Invitrogen Life Technologies). The real-time quantitative PCR was performed in special optical tubes in a 96-well microtiter plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems). Using SYBR Green PCR Core Reagents Kit (Applied Biosystems; P/N 4304886), fluorescence signals were generated during each PCR cycle via the 5' to 3' endonuclease activity of AmpliTaq Gold to provide real-time quantitative PCR information. The sequences of the specific primers are listed below: CD226 sense, 5'-TCAAATAGCCACATTGTTTCGGA-3'; CD226 antisense, 5'-AGGGTATATGGTTACGGTTTTACC-3'; survivin sense, 5'-GTGTTTCTTCTGCTTCAAGGAGCT-3'; and survivin antisense, 5'-CAGAGGCCTCAATCCATGGCAGCC-3'. All unknown cDNAs were diluted to contain equal amounts of -actin cDNA. The standards, no template controls, and unknown samples were added in a total volume of 50 μl/reaction. The PCR conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 60 s at 60°C for each amplification. Potential PCR product contamination was digested by uracil-N-glycosylase, because dTTP is substituted by dUTP. Uracil-N-glycosylase and AmpliTaq Gold (PE Applied Biosystems) were applied according to the manufacturer’s instructions.
Blot assays
For mRNA detection (Northern blot), 2 μg of total RNA obtained from each sample were electrophoresed under denaturing conditions, followed by blotting onto Nytran membranes and cross-linking by UV irradiation as previously described (38). CD226 cDNA probe, labeled by [-32P]dCTP, was obtained by PCR amplification of the sequence mentioned above from total RNA from PBMCs from healthy subjects. The membranes were hybridized overnight with 1 x 106 cpm/ml 32P-labeled probe, followed by intensively washing before being autoradiographed. For protein detection (Western blot analysis), the cells were lysed in lysis buffer (0.4% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, 1 mM sodium vanadate, 0.1 mM PMSF, and 2 μg/ml leupeptin and apoprotein, pH 8.0) as previously described (39). Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. The protein concentration was measured using a Bio-Rad protein assay. Protein (40 μg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs (anti-Bcl-2, clone BCL-2/100; anti-caspase-3, clone CPP32–19 (BD Pharmingen); anti-caspase-8, clone H-134; anti-survivin, clone FL-142 (Santa Cruz Biotechnology); anti--actin (Sigma-Aldrich); all at 0.5 μg/ml). Analyses were conducted using ECL detection (Amersham Biosciences).
Cell transient transfection
COS-7 cells were transiently transfected with CMV vector pcDNA3/CD95 and/or CMV vector pcDNA3/CD226 as described previously (20, 40). Briefly, the cells were cultured with DMEM containing 10% FCS, penicillin, and streptomycin. Cells were grown to 70% confluence in 60-mm dishes for 24 h before transfection. The DNA construct of mammalian expression vector pCMV/CD95 and/or pCMV/CD226 (5 μg; or vector only) was mixed with 12 μl of Lipofectamine (Invitrogen Life Technologies) in 2 ml of OptiMEM serum-free medium, added to cells, and incubated for 6 h. The cells were further cultured in 2.5 ml of DMEM containing 10% FCS in 5% CO2. The successful transfection was demonstrated by confocal microscopy as described previously (41). Aspreviously described (42), human NK T cells were transfected CMV vector pcDNA3/CD226 using Amaxa nucleofection technology (Amaxa) with optimization. Briefly, cells were resuspended in solution from nucleofector kit V following the Amaxa guidelines for cell transfection. One hundred microliters of 3 x 106 cell suspension mixed with 0.25 or 2.5 μg of cDNA was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa).
Results
NK T cells from active SLE are more sensitive to apoptosis
Knowing the abnormality of apoptosis of some types of immune cells in patients with SLE and in mice with lupus (7, 8, 9), we examined the death-promoting effect of signals transmitted through CD95-CD95L and CD3-TCR on NK T cells from different subjects. Flow cytometric analysis (Fig. 1) revealed that the numbers of apoptotic and necrotic cells were significantly increased in cultures of NK T cells from active SLE patients (aSLE) in the presence of immobilized anti-CD95 or anti-CD3 mAb (Fig. 1, a and b; p < 0.001) compared with that in cell cultures from normal subjects (i and j). The apoptotic responses in inactive SLE (iSLE) patients were marginally increased (e and f). As a positive control, cells were stimulated with campotothecin, which induces identical apoptosis in all three types of subjects (c, g, and k), whereas isotype Abs for anti-CD95 and anti-CD3 did not induce apoptosis in the cells (data not shown). The cell cultures for distinct subject groups in the absence of stimuli showed the unstimulated basic lines of cell apoptosis (d, h, and l). The summary of data (mean ± SD) for apoptotic cells (PI– annexin V+) of six experiments performed in each subject group is shown in Fig. 1B, documenting the sensitivity to apoptosis of NK T cells from aSLE patients. As shown in Fig. 1C, the total fractions of dead cells (including apoptotic and necrotic) in NK T cells from normal subjects and iSLE patients were significantly lower than that in aSLE patients in anti-CD95- and anti-CD3-induced apoptotic assays, but identical levels were observed in campotothecin-induced apoptosis in all three types of subjects. These results could confirm that NK T cells from active SLE were highly sensitive to apoptotic induction by the CD95-CD95L pathway or the CD3-TCR pathway. To explore the reasons for the phenomena observed above, we examined the levels of CD95 and CD95L expression on NK T cells from various subjects. Flow cytometry revealed that CD95 was expressed in identical levels on NK T cells from different subjects (Fig. 2), and that the expression of CD95L on NK T cells from aSLE patients was slightly higher than those in normal subjects and iSLE patients, strongly suggesting that the greater sensitivity to apoptosis of NK T cells from aSLE patients could not be explained by the levels of CD95 and CD95L expression on the cells. We also examined the sensitivity of total CD3+ T cells, B cells, and monocytes to anti-CD95-induced apoptosis. The results showed that aSLE CD3+ T cells were slightly, but significantly, more sensitive to anti-CD95-induced apoptosis than cells from iSLE and normal subjects; otherwise, there were no significant differences in sensitivity to anti-CD95-induced apoptosis in distinct types of cells among aSLE, iSLE, and normal subjects (data not shown).
CD226 expression is deficiently expressed on NK T cells from aSLE patients
We examined the expressions of some adhesion molecules (L-selectin, CD18/CD11a, CD15, CD28, CD29, CD31, CD34, CD44, CD49a-e, CD51, CD54/ICAM-1, CD62E, and VCAM-1/VLA-4) on NK T cells from three types of subjects, and either the results were in agreement with previous reports or no differences were found among the three types of subjects (data not shown). To search for the mechanism of the high apoptotic sensitivity of NK T cells from aSLE patients, we examined the expression of CD226 on NK T cells from aSLE patients, iSLE patients, and normal subjects. The results from flow cytometric analyses shown in Fig. 3 documented that there were different numbers of NK T cells in the three groups of subjects. There were only 1.5% of V24+V11+ NK T cells of the total T cell population in aSLE patients and 21% of CD226-positive cells (Fig. 3A). There were 8% V24+V11+ NK T cells of the total T cell population in normal subjects and 88% of CD226-positive cells (Fig. 3C), whereas there were 4% V24+V11+ NK T cells of the total T cell population in inactive SLE patients and 47% of CD226-positive cells (Fig. 3B). To confirm the observation mentioned above, we performed a real-time quantitative RT-PCR assay to detect the different expressions of CD226 at the mRNA level. The results (Fig. 3D) showed that CD226 mRNA was expressed at high levels in freshly isolated NK T cells from normal subjects (1.3 x 104 copies). CD226 mRNA expressions was significantly decreased in NK T cells from aSLE patients (1.2 x 103 and 0.9 x 103 copies in two separate cases) compared with that in normal subjects, whereas there was an increase in NK T cells from the iSLE patients (6 x 103 copies). A linear relationship between cycle threshold and log starting quantity of standard DNA template or target cDNAs was detected (data not shown). In all experiments, the correlation coefficients were 0.95–0.99. The same pattern of CD226 mRNA and protein expressions in NK T cells from aSLE patients, iSLE patients, and normal subjects was confirmed by Northern and Western blots (Fig. 3, E and F). We also examined the CD226 expression levels in total CD3+ T cells, B cells, and monocytes. There were no significant differences in CD226 expression levels in total CD3+ T cells, B cells, and monocytes from aSLE, iSLE, and normal subjects, determined by real-time RT-PCR assay and confirmed by Northern and Western blot analyses (data not shown).
To further investigate the correlation between the sensitivity to apoptosis and the level of CD226 expression on the cells mentioned above, we conducted anti-CD95-induced apoptosis in NK T cells from normal, iSLE, and aSLE subjects by preactivating and blocking the function of CD226. We used immobilized or soluble anti-CD226 mAb to activate or block CD226 on the cells, respectively. As shown in Fig. 4A, preactivation of CD226 could rescue NK T cells from aSLE patients from anti-CD95-induced apoptosis, although CD226 expression was low in freshly isolated cells (Fig. 3). Compared with the results shown in Fig. 1Aa (55% of dead cells), there were only 14% of dead NK T cells from active SLE, in which the cells were preactivated by anti-CD226 (Fig. 4Aa). The blockade function of CD226 reduced the resistance of anti-CD95-induced apoptosis in NK T cells from normal subjects, although CD226 expression was high in freshly isolated cells. The percentage of total dead cells reached 58% (Fig. 4A). Isotype Ab had no such effects (Fig. 4A, c, f, and i). As shown in Fig. 4B, the total fractions of dead cells (including apoptotic and necrotic) in NK T cells from different subjects showed patterns similar to that in Fig. 4A, confirming that activation of CD226 could rescue NK T cells from anti-CD95-induced apoptosis, whereas blockade of CD226 function could significantly reduce the resistance of NK T cells from aSLE patients to anti-CD95-induced apoptosis. Of note, the explanation for the finding that CD226 activation still could rescue low expressed CD226 aSLE NK T cells from apoptosis could be that abundant binding sites of CD226 in experimental system (anti-CD226 mAb at 2 μg/ml was immobilized) were sufficient to activate aSLE NK T cells through limited CD226 receptors. We also observed that preactivation of CD226 could rescue NK T cells from active SLE patients from anti-CD3-induced apoptosis (data not shown), confirming the rescuing function of CD226 activation from apoptosis.
Mechanisms of CD226 involved in high sensitivity to anti-CD95-induced apoptosis in NK T cells from aSLE patients
To examine the mechanisms of CD226 expression and activation involved in high sensitivity to anti-CD95-induced apoptosis in NK T cells from active SLE patients, we first examined the expression levels of some important substrates of the apoptosis pathways in the cells. Western blots revealed that key apoptotic executioner molecules, procaspase-8 and procaspase-3, were expressed at identical levels in freshly isolated NK T cells from different subjects (Fig. 5A). Western blot showed that the protein levels of one antiapoptotic member, Bcl-2, in freshly isolated NK T cells from different subjects were also identical (Fig. 5B). Interestingly, one antiapoptotic member protein, survivin, was selectively expressed at very low levels or was absent in freshly isolated NK T cells from active SLE patients, whereas it was expressed at high levels in freshly isolated NK T cells from normal subjects and iSLE patients (Fig. 5B). To confirm deficient expression of survivin in NK T cells from aSLE patients, we examined the expression of survivin mRNA in NK T cells from aSLE patients, iSLE patients, and normal subjects. We conducted a real-time quantitative RT-PCR assay to detect the different expressions of survivin at the mRNA level. The results (Fig. 5C) showed that survivin mRNA was expressed at a very low level in freshly isolated NK T cells from active SLE patients (0.8 x 103 copies). Survivin mRNA expression was significantly increased in NK T cells from the normal subjects (5.0 x 103 copies) compared with that from aSLE patients, whereas there was also an increase in NK T cells from the iSLE patients (3.5 x 103 copies). The same pattern of survivin mRNA expression in NK T cells from aSLE patients, iSLE patients, and normal subjects were confirmed by Northern blot (Fig. 5D). Thus, it might be important to maintain a certain level of expression of survivin to balance between proapoptotic and antiapoptotic pathways in cells. We also examined the survivin expression levels in total CD3+ T cells, B cells, and monocytes. The results showed that there were no significant differences in survivin expression levels in total CD3+ T cells, B cells, and monocytes from aSLE, iSLE, and normal subjects, determined by real-time RT-PCR assay and confirmed by Northern and Western blot analyses (data not shown).
To determine the mechanism of CD226 activation to rescue NK T cells from programmed death, we conducted a series of experiments to investigate the activities of a number of proapoptotic and antiapoptotic members during the process of anti-CD95-induced apoptosis in cells from aSLE patients under different conditions of CD226 activation, as indicated in Fig. 6. In the antiapoptotic pathway systems, Bcl-2 activity under different conditions of CD226 activation was seemly identical in aSLE NK T cells. CD226 activation significantly up-regulated survivin activity, which rescued the cells from apoptosis (Fig. 6A). In the proapoptotic pathway systems, active forms of caspase-8 and caspase-3 were significantly reduced after activation of CD226, suggesting that anti-CD95-mediated death signals were inhibited by activation of the CD226 pathway (Fig. 6, B and C). Thus, abnormal expression of survivin and deficient expression of CD226 were the molecular bases of the high sensitivity of NK T cells from aSLE patients to anti-CD95-induced apoptosis.
CD226 activation protects CD95- and CD226-transfected COS-7 cells from apoptosis
To confirm the observation of a protective effect of CD226 from apoptosis of NK T cells from active SLE patients, we chose a cell line, COS-7, that was able to express CD95 and CD226 after transfection and in which apoptosis was inducible by anti-CD95 mAb (20, 40). We had a successful double transfection of CD95 and CD226 (data not shown). Flow cytometric analysis revealed that the numbers of apoptotic and necrotic cells were significantly increased in culture of CD95- or/and CD226-transfected COS-7 cells in the presence of immobilized anti-CD95 mAb compared with that of vector-only-transfected cells or CD226-only-transfected cells (data not shown). We used immobilized or soluble anti-CD226 mAb to preactivate or block CD226 function in double-transfected COS-7 cells, respectively (data not shown). Preactivation of CD226 mAb could rescue COS-7 cells from anti-CD95-induced apoptosis (data not shown). As expected, the blockade of CD226 restored the sensitivity of double-transfected COS-7 cells to anti-CD95-induced apoptosis (data not shown). Isotype Ab for anti-CD226 had no such effect (data not shown). To confirm the mechanisms of the rescuing function of CD226 activation in COS-7 cells from anti-CD95-induced apoptosis, we first examined the expression levels of some important substrates of apoptosis pathways in the cells during the apoptotic process (data not shown). Western blots revealed that key apoptotic executioner molecules, procaspase-8 and procaspase-3, were stabilized by preactivation of CD226, where COS-7 cells were rescued from anti-CD95-induced apoptosis (data not shown). Western blot also showed that the protein levels of one antiapoptotic member, Bcl-2, in COS-7 cells under different conditions were identical (data not shown). Interestingly, the antiapoptotic protein, survivin, was selectively activated under the condition of CD226 preactivation, where COS-7 cells were rescued from anti-CD95-induced apoptosis (data not shown). Thus, we confirmed that in the transfected COS-7 cell system, anti-CD95-mediated death signals were inhibited by activation of the CD226 pathway through stabilization of caspase-8 and caspase-3 and through activation of survivin.
CD226 transfection rescues aSLE NK T cells from anti-CD95-induced apoptosis
To confirm the finding of a correlation between CD226 expression and resistance to anti-CD95-induced apoptosis in the COS-7 cell system, we applied Amaxa nucleofection technology to transfect cDNA encoding CD226 into human NK T cells from aSLE patients, iSLE patients, and normal subjects to investigate the effect of CD226 expression on resistance to anti-CD95-induced apoptosis. The data in Fig. 7, A and B, demonstrate that successful transfection of CD226 took place; particularly NK T cells from aSLE patients were well transfected with cDNA encoding CD226, as documented by Western blot (Fig. 7A) and flow cytometry (Fig. 7B). The results (Fig. 7, C and D) from flow cytometric analysis revealed that the number of apoptotic cells was significantly decreased in the culture of sufficient CD226-transfected (2.5 μg) NK T cell from active SLE patients in the presence of immobilized anti-CD95 (Fig. 7, Cd and D; p < 0.001) compared with that of untransfected cells and vector only (Fig. 7C, a and b). Low dose CD226 transfection (0.25 μg) was insufficient to rescue aSLE NK T cell from anti-CD95-induced apoptosis (Fig. 7Cc). A sufficient CD226 transfection (25 μg) could also rescue iSLE NK T cell from anti-CD95-induced apoptosis (Fig. 7, Ch and D; p < 0.01); even the apoptotic responses in inactive SLE patients were marginally increased (Fig. 7C, e–g). CD226 transfection in normal NK T cells had little effect on anti-CD95-induced apoptosis (Fig. 7, C, i–l, and D), because normal NK T cells already expressed abundant CD226 (Fig. 3). However, experiments of CD226 transfection in normal NK T cells showed that the transfection procedure itself did not affect the sensitivity of cells to apoptosis (Fig. 7C, a, e, and i). Thus, the results confirmed the correlation between CD226 expression and resistance to anti-CD95-induced apoptosis in the human NK T cell system.
Corticosteroid medication affects CD226 and survivin expression as well as sensitivity to apoptosis in NK T cells from aSLE patients
To examine the effects of corticosteroid medication on CD226 and survivin expression as well as sensitivity to apoptosis in NK T cells from aSLE patients, as shown in Table I, six patients with active SLE were recruited and were administrated prednisone as a major therapy. At the time of treatment, blood samples were taken. The data showed that CD226 expression in NK T cells at the mRNA and protein levels was significantly increased after successful treatment with prednisone. It began at wk 4 of treatment. Survivin expression in NK T cells was also significantly increased. It began at wk 6 of treatment. The anti-CD95-induced apoptosis in NK T cells also appeared to significantly decrease at wk 6 of treatment as the SLEDAI score decreased.
Together these data show that NK T cells from active SLE patients were highly sensitive to anti-CD95-induced apoptosis compared with that in normal subjects and iSLE patients. CD226 expression was deficiently expressed on NK T cells from aSLE patients. Survivin was found to be selectively expressed at a very low level in freshly isolated NK T cells from active SLE patients. CD226 preactivation significantly up-regulated survivin activation, which could rescue NK T cells from aSLE patients from anti-CD95-induced apoptosis. The observations were confirmed in transfected COS-7 cells and human NK T cells in terms of the correlation between activation of CD226 and stabilization of death signals.
Discussion
Development of SLE is associated with the emergence of autoreactive Th cells (43) and is accompanied by a reduction in regulatory T cells (44, 45). The nature and specificity of regulatory T cells that inhibit autoantibody production and development of lupus remain largely undefined. Some regulatory T cells are T cells or VH peptide-reactive CD8+ T cells that inhibit autoantibody production by ablating autoreactive B cells (44). Recent reports that humans and mice with lupus have reduced numbers of NK T cells (10, 11) suggest that NK T cells may be part of a regulatory T cell network to inhibit the induction of SLE. To understand the mechanism of reduction of NK T cell number in SLE, we focused our study on the sensitivity of NK T cells to induction of apoptosis during active and inactive disease processes and on determining its mechanisms. In the present study we have demonstrated that CD226 is significantly down-regulated on NK T cells from aSLE patients, which may be one of the reasons why NK T cells from aSLE patients are more sensitive to apoptosis. Abnormal and deficient expression of CD226 and survivin are the molecular bases of the high sensitivity of NK T cells from aSLE patients to anti-CD95-induced apoptosis. To our knowledge this is the first report of CD226-deficient expression on NK T cells from aSLE patients, providing direct evidence that the abnormality in CD226-induced high sensitivity to apoptosis is responsible for the reduction of NK T cell number in patients with active SLE.
In humans, the function of NK/NK T cells is regulated by a series of surface receptors that either inhibit or enhance NK/NK T-mediated cytolytic activity. When NK cells encounter potential target cells, killer inhibitory Ig-like receptors and/or the CD94/NKG2A heterodimer sense the expression of HLA class I molecules at the cell surface (46, 47). When appropriate inhibitory receptor/HLA class I ligand interactions occur, the receptors deliver negative signals that inhibit NK/NK T-mediated killing. In the absence of such interactions, as may occur in tumor or virus-infected target cells, non-HLA class I-specific activating receptors are allowed to deliver triggering signals to NK/NK T cells, resulting in target cell killing. Several non-HLA class I-specific activating receptors have been identified, including NKp46, NKp30, NKp44, and NKG2D (48, 49). The leukocyte adhesion molecule CD226, expressed by virtually all human NK cells, T cells, and monocytes and by a small subset of B cells, has been shown to participate in the induction phase of NK cell activation (19, 20, 26). CD226 function is dependent on the surface expression of CD18/CD11a (26, 27). Upon mAb-mediated cross-linking, CD226 transduces activating signals, resulting in tyrosine phosphorylation of CD226 itself and enhancement of cytotoxicity and cytokine production in both T and NK cells (19, 20, 26); it is also involved in the costimulatory signal for naive T cell differentiation and proliferation (20, 26, 27). In the present study at the protein and mRNA levels we have demonstrated that CD226 is significantly down-regulated on V24+V11+ NK T cells from aSLE patients (Figs. 3 and 4). Meanwhile, there are only 1.5% V24+V11+ NK T cells of the total T cell population in aSLE patients. We used immobilized or soluble anti-CD226 mAb to preactivate or block CD226 on the cells, respectively. Preactivation of CD226 can rescue NK T cells from active SLE patients from anti-CD95-induced apoptosis. Compared with the results shown in Fig. 1 (55% of dead cells), there are only 14% dead NK T cells from active SLE, where the cells were preactivated by anti-CD226 (Fig. 5). The findings that signals by CD226 pre activation in aSLE NK T cells increase the resistance to induction of apoptosis strongly suggest that CD226 signals can cross-talk with cell death pathways by either stabilization of the death executioner system or activation of the cell apoptosis inhibitor system, resulting in resistance of cells to apoptotic signals.
To explore the molecular basis of CD226 rescuing functions on NK T cells from active SLE patients, we examined the activity of a number of adaptor proteins during the process. Two major cell death pathways, termed the extrinsic and intrinsic pathways of apoptosis, exist in mammalian cells (50). The extrinsic pathway is initiated by engagement and aggregation of TNF family death receptors (such as CD95) through a series of death domain-containing adaptor molecules that recruit and directly activate cytosolic caspase 8, which, in turn, converts procaspase 3 to caspase 3, the central executioner of the apoptotic process (51, 52). The intrinsic pathway is intimately tied to the integrity of mitochondria and other intracellular organelles. The normal and healthy cells are maintained by a carefully regulated balance of proapoptotic and antiapoptotic members of the Bcl-2 family of proteins. In mitochondria of normal and healthy cells, resident antiapoptotic proteins, such as Bcl-2 and Bcl-xL, effectively bind up and neutralize proapoptotic proteins (53) and prevent perturbation of the outer mitochondrial membrane. A series of endogenous caspase inhibitors, known as inhibitors of apoptosis, normally interact with and inhibit the enzymatic activity of the initiator caspase 9 and the effectors caspases 3 and 7 (54). Both the location and the activation state of proapoptotic and antiapoptotic components of the cell death pathway are crucial for controlling cellular decisions about life and death. Survivin, a member of the inhibitors of apoptosis family, is potentially involved in both the inhibition of apoptosis and the control of cell division (55, 56, 57). Being essential for embryonic development (55), survivin expression in adult human tissues is usually reported in the context of its relationship to cancer. It is prominently expressed in the majority of human cancers, including those of colon, breast, lung, and high grade non-Hodgkin’s lymphoma (58). There are reports that survivin is also expressed in some normal human adult tissues, including bone marrow, colonic mucosa, endometrium, thymus, and keratinocytes (59, 60). We have observed that survivin is selectively expressed at very low levels in NK T cells from aSLE patients compared with the level in freshly isolated NK T cells from normal subjects (Fig. 6). Furthermore, CD226 activation up-regulate significantly the expression and activation of survivin, which may be one of the reasons to rescue the cells (Fig. 7).
What is the essential correlation between the defective expression of CD226 and the signaling abnormalities identified to date in aSLE NK T cells? There is a mounting body of evidence indicating increased intracellular calcium in T cells from SLE patients due to defective expression of the TCR -chain (61, 62, 63, 64, 65). Several previous studies of CD226 (20, 21, 26, 27) indicate that this molecule is a signaling element that triggers cytotoxicity by engagement of extracellular ligand or cellular counter-receptor (CD155 and CD112) and postulate that CD226 is induced upon T cell activation, and the expression persists unless cells are exposed to signal that elevates intracellular free calcium (21). Hence, the increased intracellular free calcium concentration in SLE T cells may lead to down-regulation of CD226 expression and, thus, increased apoptosis of NK T cells.
Generally, deficient expression of survivin and CD226 on aSLE NK T cells establishes a molecular basis of sensitivity of the cells to anti-CD95-induced apoptosis. These observations offer a potential explanation for the high apoptotic sensitivity of aSLE NK T cells and provide a new insight into the mechanism of reduction of NK T cell number in SLE and understanding the association between NK T cell deficiency and autoimmune diseases.
Footnotes
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.
1 This work was supported by the National Science Foundation of China (Grants 39870674 and 30030130); a special grant from the Personnel Department of Wuhan University, China; the Science Foundation of Anhui Province, China (Grant 98436630); the Education and Research Foundation of Anhui Province, China (Grant 98JL063); the Research Foundation of the Health Department of Hubei Provincial Government, China (Grant 301140344); and the National Key Basic Research Program of China (2001CB510004).
2 D.T. and L.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Tan Jinquan, Department of Immunology, Wuhan University School of Medicine, Dong Hu Road 115, Wuchang 430071, Wuhan, Peoples Republic of China. E-mail address: jinquan_tan@hotmail.com; or Dr. Jin Boquan, Department of Immunology, Fourth Military Medical University, Xian, Peoples Republic of China. E-mail address: immu_jin@fmmu.edu.cn
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; aSLE, active SLE; iSLE, inactive SLE; PI, propidium iodide; SLEDAI, SLE disease activity index.
Received for publication January 16, 2004. Accepted for publication November 12, 2004.
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