Department of Internal Medicine II (Hematology, Oncology, Immunology and Rheumatology), 1 Department of Medical Biometry, 2 Department of Ophthalmology, University Hospital, Tübingen, 3 Department of Ophthalmology, University Hospital, Essen, 4 Department of Internal Medicine, Division of Clinical Immunology and Rheumatology, 5 Department of Ophthalmology, University Hospital, Freiburg, Germany.
Correspondence to: I. Kötter, Department of Internal Medicine II (Hematology/Oncology/Immunology/Rheumatology), University Hospital, Otfried-Müller Strasse 10, D-72076 Tübingen, Germany. E-mail: ina.koetter{at}med.uni-tuebingen.de
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Abstract |
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Methods. Fourteen patients with ocular BD were evaluated before and at weeks 4 and 24 of IFN- treatment and compared with 10 healthy controls. PBMC were stained with monoclonal antibodies and measured by flow cytometry.
Results. Compared with the controls there is a significant elevation of monocytes (CD14+), CD8+/ T cells, CD3+/
T cells, natural killer (NK) cells (CD56+/CD16+) and activated/regulatory T cells (CD4+/CD25+ and CD8+/CD25+) in patients with active BD before treatment with IFN-
2a. Numbers of naïve T cells (CD8+/CD45+RA+/RO, CD4+/CD45+RA+/RO) were significantly lower. Under therapy, NK cells, CD8+/
T cells and CD3+/
T cells decreased significantly, whereas B cells increased. The previously reduced expression of HLA class I on monocytes in HLA-B51-positive patients rose to levels comparable to HLA-B51-negative patients.
Conclusion. These results implicate the participation of NK cells and T cells, especially CD8+/
T cells, in the pathogenesis of BD and may explain one mechanism by which IFN-
2a exerts therapeutic effects. Alternatively, they may result indirectly from remission induction by IFN-
2a. The reduced expression of HLA class I on monocytes in HLA-B*51-positive patients might reflect an impaired expression of and antigen presentation by HLA-B*51.
KEY WORDS: IFN-, Behçet's disease, Lymphocyte subpopulations
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Introduction |
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Recent studies have revealed the central role of T-cell mediated immune response in the pathogenesis of BD. Although there are conflicting reports on the number of CD4+ and CD8+ T cells in the peripheral blood of BD patients, an increase in the proportions of T cells and especially CD8+
T cells has been consistently found [26]. Additionally, CD56+ natural killer (NK) cells and CD45RA+
T cells were shown to be increased in BD patients [4, 7, 8]. CD4+/CD25+ T cells were significantly elevated in patients with active uveoretinitis [9]. Cytokine research hinted at a T helper 1 (Th1) profile in BD [10].
Interferon-2a (IFN-
2a) has been found to be clinically effective in several patient groups with BD [11]. Recently, our group could show efficacy in 50 patients with active retinal vasculitis [12]. From an immunological point of view this is quite surprising, as IFN-
is known to divert the T-cell response in the direction of Th1 which is predominant in BD [13].
The aim of this study was to investigate the effects exerted by IFN-2a on lymphocyte subpopulations and monocytes in BD.
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Materials and methods |
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The clinical study (treatment of ocular BD with IFN-2a) as well as the blood samples and studies of intracellular cytokines in the Behçet's patients, healthy controls and patients with spondyloarthropathy were approved by the local ethics committee.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated from 20 ml heparinized blood by Ficoll-Paque 1077 gradient (Biochrom KG, Berlin, Germany) centrifugation (30 min, 400g). The PBMC at the interface were collected, washed twice in phosphate-buffered saline (PBS, Dulbecco's, Invitrogen-Lifetechnologies, Karlsruhe, Germany), containing 0.5% bovine serum albumin (BSA, Sigma, Steinheim, Germany), resuspended in RPMI-1640 culture medium (Invitrogen-Lifetechnologies) supplemented with 10% heat-inactivated fetal calf serum (FCS, CC-Pro, Neustadt, Germany) and gentamicin 100 mg/l (Merck, Darmstadt, Germany) (complete RPMI) and stored at 180°C for further analysis.
Immunofluorescent staining
The frozen cell preparation was rapidly thawed in a 37°C water bath. After washing with complete RPMI, PBMC (1 x 106 per well) were incubated for 30 min at 4°C in the dark with 10 ml fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridium chlorophyll protein (PerCP) or allophycocyanin (APC) conjugated monoclonal antibodies (mAbs) against the following surface markers: CD3 (clone SK7, PerCP labelled), CD4 (clone SK3, FITC, PE and APC labelled), CD8 (clone SK1, FITC, PE and PerCP labelled), CD14 (clone M5E2, APC labelled), CD16 (clone NKP15, FITC labelled), CD19 (clone 4G7, PerCP labelled), CD20 (clone L27, PE labelled), CD25 (clone 2A3, PE labelled), CD45RA (clone HI100, PE labelled), CD45RO (clone UCHL-1, APC labelled), CD56 (clone MY31, PE labelled), the T cell ß receptor (clone WT31, FITC labelled), T cell receptor (clone 11F2, FITC labelled), HLA class I (clone G46-2.6, FITC labelled) and HLA class II (clone L243, PE labelled). All mAbs were purchased from BD Bioscience, San Jose, California, USA. Isotype matched control mAbs labelled with FITC, PE, PerCP and APC were used as controls (BD Bioscience, San Jose, California, USA). After incubation, the cells were washed in PBS with 0.5% BSA, centrifuged and the supernatant was removed. The cells were fixed with 5% formaldehyde in PBS for 15 min. Following a final washing step with complete RPMI, the cells were resuspended in 200 ml PBS, 0.5% BSA and kept at 4°C in the dark until analysis.
Flow cytometric analysis
Four-colour analysis was performed using a FACSCalibur (BD Bioscience, San Jose, California, USA) and Cellquest software (BD Bioscience, San Jose, California, USA). Lymphocyte subpopulations and monocytes were selected using a forward and side scatter gate for lymphocytes and a different gate for monocytes. For each analysis 20 000 events for surface phenotype expression were acquired. Non-specific staining and autofluorescence were determined using isotype matched controls.
Statistical analysis
Cell count percentages were transformed to logits to ensure homoscedasticity. Analyses of variance with factors patient and week were used to test for longitudinal treatment effects. The geometric means of healthy controls and patients before treatment were compared by Welch's test (t-test for unequal variances). The largest effects are shown as boxplots with whiskers extending to the outermost data point that lies less than 1.5 times the interquartile range (IQR) away from the box that contains half the observations and a line at the empirical median (M). Medians and their 95% confidence intervals (CI) computed from the logits and transformed back to percentages were tabulated. Assumptions of normality were checked by normal quantile plot of homoscedasticity, independence by residual-by-predicted plot and absence of outliers by both.
To test ratios of median HLA expression for systematic longitudinal fluctuation over weeks 0, 4 and 24 and the differences between cell types and HLA types and the interaction of these two, we included these factors and the factor patient nested under HLA type in an ANOVA model explaining the logarithms of median HLA expression. There was no need for MANOVA, as the Mauchly criterion was not significant at the 5% level of significance which was assumed for all tests locally and multiply when the BonferroniHolm procedure was applied to the analyses of cell count percentages. Computations were carried out with JMP 5.0.1.2. (SAS Institute Inc., Cary, NC 2003).
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Results |
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Baseline distribution of PBMC of patients with active BD versus healthy controls
The analysis of lymphocyte subpopulations and monocytes before therapy with IFN-2a in 14 patients and in healthy controls is summarized in Table 3.
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Monocytes
The percentage of monocytes (CD14+) from patients with active BD before IFN-2a treatment was significantly higher than in healthy controls [P = 0.006; M patients (IQR) 25.1% (12.7%); M controls (IQR) 14.5% (5.7%)].
Changes in PBMC subpopulations after 4 and 24 weeks of IFN-2a treatment in patients with BD
To determine whether differences in the distribution of defined lymphocyte subpopulations or monocytes might associate with IFN-2a efficacy in BD, the percentages of the different subgroups of
ß T cells,
T cells, B cells, NK cells and monocytes before and at weeks 4 and 24 of IFN-
2a treatment were analysed (Table 3).
T cells
NK cells
The elevated levels of CD56+/CD16+ NK cells significantly decreased (P = 0.025) and reached those of the healthy controls [M (IQR) for patients at week 0, 13% (8.4%); M (IQR) for patients at week 24, 5.8% (6.3%); M (IQR) for healthy controls, 4.4% (7.8%)] (Fig. 4a).
B cells
Although there were no significant differences in the percentage of CD19+/CD20+ B cells between week 0 and week 4 and week 4 and 24, a substantial increase occurred in week 24. Overall, this was highly significant [P = 0.004; week 0, M (IQR) 9.7% (5.5%); week 24, M (IQR) 11.7% (16.6%)] (Fig. 4b).
Monocytes
Within the first month of treatment the previously elevated monocyte percentages (CD14+) further increased, although this did not reach significance [M (IQR) for patients at week 0, 25.1% (12.7%); at week 4, 20.2% (23.1%); M (IQR) for healthy controls, 14.5% (5.7%)].
HLA class I expression on lymphocytes and monocytes before and under IFN-2a treatment
HLA class I expression at baseline was higher on monocytes than on lymphocytes. There was a tendency towards lower expression of HLA class I on monocytes in HLA-B*51-positive patients in comparison to HLA-B*51-negative patients. The expression of HLA class I increased significantly on both cell types under IFN-2a (P<0.001) (Fig. 5).
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Discussion |
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Consistent with these results, an increase in the proportions of CD4/8 double-negative and of CD8+ T cells has been repeatedly found in patients with BD by other groups, irrespective of whether the patients were of British, Japanese or Middle Eastern origin [26].
T cells account for 2 to 5% of all CD3+ T cells in peripheral blood. Although the double-negative
T cells (CD4/CD8) are the most common
receptor-positive cells, there was only a trend towards elevation of this subpopulation compared with the healthy controls. Thus, the CD8+
T cells are mainly responsible for the overall significant elevation of
T cells. As the other groups who previously described the elevation of
T cells in patients with active BD did not discriminate between double-negative and CD4+ or CD8+
T cells, we cannot determine whether this is consistent with the findings in other BD populations examined thus far. Only one report discriminated between the subgroups of
T cells. In that study, proliferative responses of CD8+
+ T cells were significantly increased in patients with BD, whereas the responses of the other subgroups of
T cells were similar to those of the healthy controls [15]. One could therefore argue that the CD8+
T cells play a major role in the pathogenesis of BD, for example by recognizing MICA-binding peptides, as suggested by Nishida et al. [15].
Some investigators have reported increased percentages of NK cells in patients with active BD [7, 8]. Suzuki and colleagues showed increased percentages of both NK (CD16+/CD56+) cells and CD56+ T cells [8]. On the other hand, Eksioglu-Demiralp et al. [16] found increased CD4+/CD16+ and CD4+/CD56+ T-cell subsets in BD, but could not demonstrate elevated levels of CD16+/CD56+ NK cells in their series. CD4+/CD25+ activated T cells were described as elevated in patients with BD and active uveoretinitis [9].
An elevation of CD14+ monocytes was described by Sahin et al. [17]. In parallel, the monocyte activation marker soluble CD14 (sCD14) was also found to be raised in the serum of BD patients [17].
We found a reduction in CD45+/RA+RO mostly naïve T cells in the peripheral blood which had not previously been reported for patients with BD. Although one earlier publication mentioned a predominant infiltration by CD4+/CD45+/RO+ memory T cells expressing HLA-DR in skin with a pathergy reaction [18], we did not observe any differences in the memory subpopulations of CD4+ or CD8+ T cells in the peripheral blood of patients and healthy controls. This possible discrepancy could, for example, be due to homing of this subpopulation to the inflamed tissues.
IFN-2a is a recombinant interferon with antiviral, anti-proliferative and wide-ranging immunomodulatory properties. With respect to the present results, recent studies on the immunological effects of IFN-
have proposed differential influences on lymphocyte subpopulations, monocytes and dendritic cells in vitro, in healthy volunteers and in several disease states, either virally induced such as hepatitis C or haematological such as chronic myelogenous leukaemia (CML).
A specific influence of IFN- on
T cells has up to now only been reported for a patient with large granular lymphocyte leukaemia, where the leukaemic
+ blasts could be reduced in vivo by treatment with IFN-
[19]. The proportion of CD8+
T cells, which comprises about 25% of all
+ T cells, was reported to be reduced by IFN-
treatment in patients with hepatitis C [20]. The same authors also reported a significant reduction of the primarily elevated NK cells in patients with chronic hepatitis C. The NK cell count was also reported to be markedly reduced after IFN-
treatment in vivo in patients with myasthenia gravis [21] although NK cell activity was nonetheless found to be induced. This phenomenon was consistently observed by many other groups under different circumstances under in vitro conditions, and in vivo in different disease states [2224]. The number of monocytes and neopterin serum levels (a marker for monocyte activation) were observed to be augmented by IFN-
in healthy volunteers [25]. Furthermore, IFN-
induces the expression of CD14 on monocytes [26]
B-cell numbers increased under IFN- treatment for chronic hepatitis C [20]. In vitro examinations by Su and David proved that this could be explained by inhibition of B-cell receptor-mediated apoptosis [27].
In the present study, we found that under treatment with IFN-2a, NK cells, CD8+/
T cells and CD3+/
T cells decreased significantly, whereas B cells increased. The previously reduced expression of HLA class I on monocytes in HLA-B*51-positive patients rose to levels comparable to HLA-B*51-negative patients. There was a tendency towards decreased expression of HLA class I molecules on CD14+ monocytes from HLA-B*51-positive versus HLA-B*51-negative active BD patients, but this did not reach significance, probably because of small sample size (only four HLA-B*51-negative patients). This finding stands in contrast to comparable data on HLA-B27, the expression of which was shown to be increased on lymphocytes [28]. HLA class I expression is also known to be induced by IFN-
[29], especially on CD14+ monocytes, as described by Piazzola et al. [30]. HLA-B*51 is possibly only expressed at low levels and may be efficiently induced by IFN-
. The relevance of the finding of reduced expression of HLA class I antigens on PBMC of HLA-B*51-positive patients with BD is not clear, as no monoclonal antibody with specificity for HLA-B*51 was available. Therefore, further studies with antibodies specific for HLA-B*51 are required to clarify whether this reduction is truly due to lowered expression of HLA-B*51.
The other primarily increased populations in BD patients blood remained elevated irrespective of IFN- treatment. The increase in B cells and monocytes under therapy may explain at least some of the side-effects of IFN-
such as autoantibody production, autoimmune phenomena and fever.
Eight of our patients were on immunosuppressive agents (prednisolone, and in one case CSA) immediately prior to initiation of IFN-2a treatment. All these patients had active ocular disease (and 13 of them also had other manifestations of BD) irrespective of these immunosuppressants. Glucocorticosteroids are known to reduce all lymphocyte subpopulations [31] whereas CSA does not influence the number of NK or
T cells but reduces cytokine secretion by T cells and NK cell function [32, 33]. Thus, if anything, the numbers of NK and
T cells in the patients at week 0 would have been increased even more without recent immunosuppressive treatment. The reductions in NK and
T cells observed during IFN-
2a treatment are either caused by IFN-
2a itself or generally indicate remission of BD. The effects of IFN-
may either be direct (by inducing apoptosis of the
T cells or NK cells) or indirect via the influence it exerts on the cytokine network [34], for example by a decrease in IL-18 or IL-21, which are known to augment the number and activity of NK cells, or by inhibition of the release of IL-12, preventing the development of antiviral CD8+ T cells [35]. It has been described previously that patients with BD in remission do not exhibit increased percentages of NK and
T cells [4, 36]. Thus, the reduction of NK and
T cells could merely indicate remission of BD, and would also be expected to occur under the influence of other remitting agents such as CSA, azathioprine or high-dose glucocorticosteroids. Further studies comparing the effects of IFN-
to those of other disease-remitting agents are required.
It is unclear whether the significant increases in T cells and NK cells in patients with active BD are specific for BD or also occur in other vasculitides or HLA class I-associated diseases such as spondyloarthropathies. However, there are some reports hinting at a participation of
T cells in reactive arthritis [37], and it is well-known that
T cells are also increased in viral and bacterial infections [38] and in response to heat shock proteins [39] and even to non-protein antigens [40]. This raises the question of the disease-specificity of the present findings, but on the other hand may hint at the participation of infectious agents or heat shock proteins in the pathogenesis of BD.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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Notes |
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References |
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