Departments of Rheumatology, Internal Medicine III and
1 Functional and Experimental Pathology and
2 Special Dermatology, University of Vienna, Vienna, Austria
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Abstract |
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Patients and methods. Peripheral lymphocytes of 39 SSc patients and 47 healthy control persons were studied for apoptosis, Bcl-2 and Bax levels, expression of Fas (CD95) and activation markers (CD25, HLA-DR) as determined by fluorocytometry. Serum Fas and Fas ligand were measured by ELISA.
Results. SSc lymphocytes (mainly CD4+) expressed increased amounts of Bcl-2, while Bax was not elevated. Apoptosis rates of SSc lymphocytes were increased in unsupplemented medium, but returned to normal in the presence of autologous plasma. SSc patients had increased percentages of activated and CD95+ lymphocytes and elevated soluble Fas and soluble FasL levels in serum. Activating anti-CD95 antibodies further increased the apoptosis rate.
Conclusions. Increased in vitro apoptosis, elevated lymphocytic Bcl-2 content and the increased number of Fas-positive T cells are not specific for peripheral blood from SSc patients, but indicate deregulation of lymphocyte homeostasis in this disease.
KEY WORDS: Systemic sclerosis, Lymphocytes, Apoptosis, Cell death, Bcl-2, Bax, Fas.
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Introduction |
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Experimental animals with deficient apoptosis appear unable to eliminate autoreactive cells and therefore exhibit a variety of autoimmune phenomena [814]. For example, Bcl-2 hyperexpression clearly shifts immune cells towards a higher probability of survival [15] and may accordingly lead to lymphoproliferative disorders and/or accelerate autoimmune diseases [11]. In fact, lymphocytes of patients with autoimmune diseases, such as systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), actually demonstrate Bcl-2 overexpression [1619]. Given the animal data, this might lead to the survival of illicit, autoreactive lymphocytes and foster autoimmunity. In addition, such cells might be more resistant to therapy with glucocorticoids or cytotoxic drugs [20].
On the other hand, elevated apoptosis rates have been suggested to play a role in the emergence of autoantibodies. Indeed, in vitro apoptosis rates of SLE lymphocytes were found to be elevated [21], as were the percentages of freshly prepared apoptotic lymphocytes of SLE patients [22]. Moreover, autoantibodies were shown to bind to proteins modified through the process of programmed cell death [23, 24]. It is not clear at the moment how the seemingly counterintuitive findings in SLE and SS are related. On the one hand, the anti-apoptotic protein Bcl-2 is elevated and on the other in vitro apoptosis rates are increased; and autoantibodies react with apoptosis-modified nuclear antigens.
The aetiology and pathogenesis of systemic sclerosis (SSc) are still largely unknown. While the advanced stages of SSc are characterized by the excessive accumulation of collagen [25], perivascular inflammation and endothelial cell apoptosis are already present in the early stages of the disease [25, 26]. Moreover, a variety of humoral and cellular autoimmune phenomena can be observed in SSc [27]. Lymphocytes, and T cells in particular, seem to play an important role in the pathogenesis of SSc [27, 28]: lymphocyte infiltrates are found in SSc skin lesions [29] and SSc alveolitis [30, 31]; SSc T cells are activated [32] and oligoclonally expanded [33, 34]; and changes in the distribution of T-cell subsets have been found [32, 35]. In addition, cytokines, such as interleukin-2 (IL-2) and IL-4, but also IL-6 and tumour necrosis factor (TNF-
) were reported to be elevated in the serum of patients with SSc [3640]. In parallel, elevated serum levels of soluble IL-2 receptor and soluble intercellular adhesion molecule 1 (ICAM-1) indicate the profound activation of the immune system [40, 41]. Finally, high-dose immune-ablating therapy in conjunction with stem-cell or bone-marrow transplantation appears to retard or halt disease progression, at least in some SSc patients [42].
Interestingly, apoptosis and apoptosis-regulating intracellular proteins have not been a major focus of interest in SSc in spite of several indications of a role of autoimmunity in SSc pathogenesis. Therefore, in the present study a possible increase in lymphocytic Bcl-2 in SSc was analysed. In parallel, we measured the in vitro lymphocyte apoptosis rates in SSc and attempted to shed light on the question of whether this in vitro rate actually represents in vivo effects. It will be demonstrated that Bcl-2, but not Bax, is overexpressed, while apoptosis is enhanced in vitro. Interestingly, the findings in patients with graft-vs-host disease (GVHD) were similar to those in patients with scleroderma.
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Patients and methods |
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Preparation and labelling of peripheral blood cells
Peripheral blood mononuclear cells (PBMC) were separated immediately after venipuncture on a FicollPaque gradient (Pharmacia Biotech, Uppsala, Sweden). Apoptosis was investigated as described below. Surface stainings were performed according to standard procedures using antibodies against CD3, CD4, CD19, CD28, CD69, HLA-DR (Becton Dickinson, San Jose, CA, USA), CD8 (Dako, Glostrup, Denmark), CD25 (Serotec, Kidlington, UK), CD45 RO and CD95 (Immunotech, Marseilles, France) directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyll protein (PerCP). Intracellular staining was performed as described [16] employing the FIX+PERM kit (An der Grub, Vienna, Austria) and a monoclonal FITC-conjugated anti-Bcl-2 antibody (Dako) or a polyclonal rabbit-anti-Bax antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by a PE-conjugated donkey anti-rabbit IgG F(ab')2 fragment (Accurate, Westbury, NY, USA), respectively. Unrelated isotype-matched FITC- or PE-conjugated mouse antibodies (mIgG1 FITC antibody from Dako and mIgG1 PE antibody from Becton Dickinson) or unconjugated polyclonal antibodies (rabbit immunoglobulin; Accurate) served as negative controls. Bcl-2 fluorescence was recorded using linear signal amplification. The mean fluorescence intensity (mfi) was calculated after subtracting the negative control signals.
Detection of apoptosis
Cells were processed either directly after the FicollPaque separation or after 24 or 48 h of culture in RPMI medium (37°C, 5% CO2). PBMC (2x106) were stained by nick- and end-labelling employing terminal deoxynucleotidyl transferase and deoxyuridine triphosphate (TUNEL technique) directly conjugated to FITC (Boehringer Mannheim, Mannheim, Germany) [44]. In addition, similar numbers of PBMC were stained with FITC-conjugated Annexin V and propidium iodide (Annexin V/FITC-Kit; Bender, Vienna, Austria) [45] and dead lymphocytes, i.e. propidium iodide-positive cells, were excluded in FACS (fluorescence-activated cell sorter) analysis. Whereas the TUNEL assays detect DNA fragmentation, a later step in programmed cell death [46], Annexin V detects phosphatidyl serine on the outer cell membrane and is therefore indicative of early apoptosis.
Cell culture
Ten millilitres of whole heparinized venous blood freshly drawn from nine SSc patients was diluted 1:2 with Dulbecco's modified Eagle medium (DMEM). Two hundred microlitres per staining was triple-stained immediately and after 24 and 48 h, using FITC-, PE- and PerCP-conjugated irrelevant control antibodies or monoclonal antibodies against CD3, CD4 and CD8 (all from Becton Dickinson). Red blood cells were then lysed with Simultest IMK lymphocyte lysing solution (Becton Dickinson) and the CD4/CD8 ratio was determined by fluorocytometry gating for CD3+ lymphocytes.
In a series of further experiments, freshly obtained PBMC were divided into two portions. One portion of PBMC was incubated in RPMI medium supplemented with 50% autologous plasma and the second portion in RPMI alone. After 24 h of incubation at 37°C, the in vitro apoptosis rates, as measured with Annexin V (to investigate early apoptosis), were compared.
In another series of experiments, cells were incubated for 24 h in 5 ml of unsupplemented RPMI medium with or without the addition of 150 ng/ml of the apoptosis-blocking anti-CD95 antibody ZB4 (Immunotech) or the apoptosis-inducing antibody CH11 (Immunotech), and the percentage of apoptotic cells was measured by the TUNEL assay, determining the percentage of cells in the definitive process of cell death.
Fluorocytometry
Immediately after the staining procedure, cells were analysed by fluorocytometry with a Becton Dickinson FACScan. In all experiments, gates were carefully set for lymphocytes, excluding the monocyte population [16]. Cells brighter than the respective isotype control or the appropriate negative controls for the TUNEL (TdT omitted) and Annexin (no Annexin added) assays were defined as positive in surface immunofluorescence and apoptosis stainings, respectively. Intracellular mean fluorescence intensity was measured on a linear scale as described [16]; only those channels having more than 99% of the cells stained with a negative control antibody were included in the measurement. In order to avoid technical artefacts of cytofluorimetry, instrument settings were kept constant during the period of the experiments; alignment constancy was carefully monitored to constant channels by means of fluorescent beads (Immunocheck; Coulter, Hialeah, FL, USA) and samples of healthy controls were tested in parallel with patient samples. Forward- and side-scatter comparison of the lymphocytes of SSc patients and healthy controls did not reveal differences with regard to cell size and granularity.
Serum levels of soluble Fas and soluble Fas ligand
We evaluated levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) in serum samples of SSc and GVHD patients and of 20 healthy controls employing commercially available ELISA kits (Medical and Biological Laboratories, Nagoya, Japan).
Statistics
Numerical data are expressed as mean±S.D. Student's t-tests and paired t-tests were employed for the comparison of groups and paired samples, respectively. Pearson correlation coefficients were calculated to investigate possible associations between variables. P values<0.05 were considered significant.
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Results |
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Bcl-2 is increased in SSc T cells
The mfi of Bcl-2 in 47 healthy control individuals was 299±50. This value was independent of sex (male 303±42, female 292±56) and age [<40 yr (mean 30), 296±54; >40 yr (mean 59); 303±27]. Compared with these values, Bcl-2 was clearly elevated in patients suffering from SSc (369±88 mfi, P<0.0001). Lymphocyte Bcl-2 levels were similar in patients with the diffuse and limited forms of SSc (375±93 and 364±85, respectively) and in patients with GVHD (386±21) (Fig 1). There were no significant differences between healthy controls and any of the patient groups with regard to the mfi of the negative control samples or the scatter parameters (not shown).
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Bax expression is similar for patients and controls
Like Bcl-2, Bax was expressed in essentially all peripheral lymphocytes of both patients and healthy controls. Unlike Bcl-2, however, there was no difference in Bax expression between lymphocytes of SSc patients and those of healthy individuals (271±73 vs 269±99, not significant; Fig 3). Bax expression was independent of sex (male 290±94, female 258±81) and age (<40 yr 272±101, >40 yr 268±74). As with Bcl-2, there were no significant differences between the mean fluorescence intensities of the negative control samples and scatter parameters of patients and controls (not shown).
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SSc lymphocyte apoptosis is not increased ex vivo
As expected [48], apoptotic cells as measured with the TUNEL technique were virtually undetectable ex vivo both among SSc and healthy lymphocytes (calculated values 0.5±0.3 vs 0.3±0.3%, not significant; Fig 4, 0 h). Since the lack of TUNEL-positive cells directly after cell preparation could be due to the removal of dead cells by the Ficoll procedure, we also used Annexin V to test freshly purified PBMC for early apoptotic cells. Indeed, we found remarkably high percentages of Annexin-positive cells among both SSc and healthy lymphocytes, but the percentages were similar (18.5±9.6 vs 21.1±6.4%, not significant). This finding does not support a higher percentage of apoptotic cells in the peripheral blood of SSc patients. However, it cannot rule out an increased in vivo apoptosis rate, since dying cells are typically cleared rapidly, as soon as they exhibit surface changes.
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SSc lymphocytes show increased in vitro cell death rates
We therefore next tested the apoptosis rate of SSc lymphocytes in vitro in plain medium. After 24 h of in vitro culture, the percentage of TUNEL-positive cells was increased in the SSc cell population compared with healthy control cells (17.2±11.1 vs 10.4±3.4%, P=0.02; Fig 4). After a second day of culture this difference was even more pronounced (35.4±15.1 vs 17.7±5.6%, P=0.01; Fig. 4
). Again, there were no significant differences between males and females or between younger and older control persons (not shown). Thus, PBMC from patients with SSc show enhanced in vitro cell death rates, presumably due to increased in vitro apoptosis.
The percentage of Annexin V-positive, propidium iodide-excluding cells after 24 h of culture was consistent with such an elevated in vitro apoptosis rate. Again, the percentage of apoptotic cells was significantly higher for SSc than for healthy PBMC (30.4±13.6 vs 17.0±5.4, P<0.0003; Fig 5). Taken together, these findings provide evidence for an increased in vitro cell death rate in SSc, which appears to be due to enhanced in vitro apoptosis.
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The in vitro apoptosis rate is almost normal in autologous plasma
Since the in vitro apoptosis rate in unsupplemented medium was increased and since several cytokines are elevated in the serum of scleroderma patients, we hypothesized that the higher in vitro apoptosis rate of SSc lymphocytes could be due to acute serum and cytokine deprivation. In a series of further experiments, we therefore divided fresh PBMC into two portions and incubated one in unsupplemented RPMI and the other in RPMI supplemented with 50% autologous plasma. We found a significant decrease in early apoptosis (as measured by Annexin V) in SSc patients, from 28.3±8.7% (cells cultured without plasma) to 19.4±7.4% (cells cultured in autologous plasma; P=0.004). The findings were similar for PBMC of healthy individuals (decrease from 21.0±3.8 to 15.3±4.3%, P=0.003). Whereas for cells cultured in unsupplemented RPMI the differences between patients and controls were significant, the addition of serum reduced the scleroderma apoptosis rate to a virtually normal level in the vast majority of the patients (Fig 6).
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CD95 (Fas) expression is elevated on SSc lymphocytes
In vivo-activated lymphocytes are more dependent on life-sustaining cytokines [49] which are present in the serum and, therefore, are very likely to undergo in vitro apoptosis. The activation of lymphocytes, however, often enhances the expression of CD95 (Fas). As CD95 is known to transduce apoptosis signals, we also studied the surface expression of Fas on lymphocytes.
Lymphocyte surface-staining demonstrated an increased percentage of CD95+ lymphocytes in SSc patients compared with healthy controls (57.3±16.0 vs 41.5±13.5%, P<0.0001). This elevated expression of Fas was similar for diffuse (55.8±19.6%, P=0.002 vs healthy controls) and limited SSc (58.5±13.0%, P<0.0001 vs healthy controls) and was also found in GVHD patients (64.1±11.6%, P=0.0003 vs healthy controls) (Fig 7). Since CD95 expression has been reported to be higher in older persons [50], we also compared the SSc population with an age-matched (56±18 yr) subgroup consisting of those healthy controls who were aged over 40 yr. In spite of the smaller size of this control group and its tendency towards slightly higher CD95 percentages, the difference was still significant (57.3±16.0 vs 44.7±12.6%, P=0.03).
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Elevated serum levels of sFas and sFasL
Like levels of cellular CD95, levels of sFas were clearly elevated in SSc patients compared with healthy controls (1.46±0.52 vs 0.95±0.36 ng/ml, P<0.005). Interestingly, we obtained similar findings in GVHD patients (1.69±1.31 ng/ml). Serum levels of sFasL were higher in SSc than in controls (130±70 vs 86±40 pg/ml, P=0.02), but not in GVHD patients (69±30 pg/ml).
Effects on CD4/CD8 ratio
The combination of increased amounts of (anti-apoptotic) Bcl-2 among the CD4+ lymphocyte subset and higher percentages of Fas receptor-positive cells among the CD8+ lymphocytes could lead to the increased CD4/CD8 ratio found in SSc [32]. We tested this hypothesis by following the CD4/CD8 ratio of SSc lymphocytes in whole blood cultures supplemented with equal volumes of DMEM. In line with our hypothesis, the CD4/CD8 ratio was 2.5±1.3 on the day of blood-drawing and then increased to 2.8±1.4 after 24 h (paired t-test P<0.05) and to 3.1±1.6 after 48 h (paired t-test P<0.05 for the intervals from 0 to 48 h and from 24 to 48 h, respectively).
Clinical correlations
As described above, neither Bcl-2, nor Fas expression discriminated between patients with limited and diffuse SSc. Similarly, there was no correlation between individual clinical or laboratory variables and the expression of Bcl-2, Bax or Fas. Moreover, no correlation to any therapy could be discerned (data not shown).
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Discussion |
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Expression of the cell death receptor Fas (CD95) was increased among SSc CD8+ but not CD4+ cells compared with those of healthy controls, and the increase in Fas was mainly seen on activated, HLA-DR+ T lymphocytes. The elevated percentage of Fas+ lymphocytes was accompanied by elevated levels of sFas and FasL in SSc serum [52]. Taken together, these results indicate that the increase in Fas expression most probably reflects a physiological response to the ongoing cell activation in SSc. Additional experiments with an activating anti-Fas antibody led to a further increase in apoptosis, suggesting that the expressed Fas receptors are able to mediate apoptosis signals. Thus, SSc patients have more activated T cells than controls and these activated T cells express more Fas receptors, indicating that their programmed cell death is primed.
The data on apoptosis are consistent with such an assumption. Whereas increased percentages of early apoptotic lymphocytes have been seen in freshly prepared PBMC of SLE patients [22], we could not detect such a difference when comparing similarly prepared PBMC of SSc patients and healthy individuals. However, after 24 h of culture in serum-free medium, and to an even greater extent after 48 h of culture, higher percentages of apoptotic cells were detected among SSc than among control cells. Moreover, addition of plasma reverted this increased level of apoptosis to normal levels (Fig 6). Interestingly, high levels of cytokines, such as IL-2, IL-4, IL-6, IL-10 and IL-13, have been found in SSc sera [37, 38]. Since activated lymphocytes are more dependent on such life-sustaining cytokines, the increased in vitro apoptosis of SSc cells probably reflects the fate of higher frequencies of activated lymphocytes.
On the other hand, it is noteworthy that large proportions of the patients lymphocytes were still viable after 24 h in serum-free culture, indicating that only a minority of cells would undergo apoptosis under in vivo conditions and supporting the notion that the observed increase in Bcl-2 may have functional consequences.
The increased expression of Bcl-2 among CD4+ T cells in conjunction with the anti-apoptotic effects of Bcl-2 suggests that some T-cell populations could expand more easily. Moreover, the lack of increased expression of CD95 on CD4+ cells indicates that the propensity of these cells to undergo apoptosis is not increased. Since CD4+ cells have been attributed with pathogenetic importance [29], the findings presented here support this assumption. The combination of increased Bcl-2 with unaltered CD95 expression could foster the expansion of illicit, pathogenically important T-cell subpopulations.
The changes observed did not correlate with clinical or laboratory variables of the disease. They were also found equally among patients with the limited and diffuse forms of SSc. This could thus represent common features of scleroderma patients other than clinical subgroup or stage of the disease.
Interestingly, findings similar to those in SSc were obtained in patients with GVHD in all respects: Bcl-2 and Fas expression as well as sFas serum levels were increased. Some of these observations have also been made recently by others [53]. This may simply mean that the increases in Bcl-2 and Fas are not specific for scleroderma. In fact, both have been found in other systemic autoimmune diseases, such as SLE and SS [16, 18, 19, 54]. One could therefore argue that GVHD simply represents another of these conditions and is similarly associated with T-cell involvement. From our previous studies it is also clear, however, that this finding is not simply a consequence of inflammation, since patients with active rheumatoid arthritis did not have increased Bcl-2 in their lymphocytes [48]. As an alternative explanation, therefore, the findings may also fit the idea of microchimaerism as a potential cause of scleroderma, an idea that has recently been fostered by clinical similarities, statistical data, and the detection of nucleated fetal cells in SSc skin lesions [5557].
Fas and Bcl-2 regulate different pathways of apoptosis that may serve distinct functions in lymphocyte homeostasis and in the maintenance of T-cell tolerance. In summary, accelerated in vitro apoptosis as well as increased Fas and Bcl-2 protein expression have been observed in patients with SSc; since such changes, albeit with differences in fine specificity, have been described in other connective tissue diseases, they reflect the hyperactivation of T lymphocytes from patients with these diseases and may be related to the deregulation of the lymphocytic control of apoptosis, which in itself is likely to be functional in the generation and/or perpetuation of autoimmune disease.
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Acknowledgments |
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Notes |
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References |
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