Increase of peripheral CXCR3 positive T lymphocytes upon treatment of RA patients with TNF-{alpha} inhibitors

D. Aeberli1, M. Seitz1, P. Jüni1,2,3 and P. M. Villiger1

1 Department of Rheumatology and Clinical Immunology/Allergology, University Hospital, 2 Division of Epidemiology and Biostatistics, Department of Social and Preventive Medicine, University of Berne, Berne, Switzerland and 3 MRC Health Services Research Collaboration, Department of Social Medicine, University of Bristol, Bristol, UK.

Correspondence to: P. M. Villiger, Department of Rheumatology and Clinical Immunology/Allergology; University Hospital, CH-3010 Bern, Switzerland. E-mail: peter.villiger{at}insel.ch


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To explore the regulation of factors involved in lymphocyte trafficking in patients with rheumatoid arthritis (RA) undergoing treatment with tumour necrosis factor {alpha} (TNF-{alpha}) inhibitors.

Methods. We examined 14 consecutive patients with RA according to ACR criteria prior to and during treatment with TNF-{alpha} inhibitors (seven etanercept, seven infliximab) and determined disease activity using the Disease Activity Score (DAS-28). Peripheral blood mononuclear cells were isolated before and after 6 and 14 weeks of treatment and analysed immediately for CD3, CD4 and CD8, expression of chemokine receptors CXCR3 and CCR4, CD45RO phenotype and for expression of interferon {gamma} (IFN-{gamma}) and interleukin 4 (IL-4) using four-colour flow cytometry.

Results. We found significant increases in CD4 and CD8 T lymphocytes expressing CXCR3 after 6 and 14 weeks. The overall proportion of T lymphocytes expressing CCR4 appeared unchanged. More than half of peripheral CD4 T lymphocytes showed a memory phenotype (CD45RO), with a non-significant increase under TNF-{alpha} inhibition. Upon activation, up to 30% of CXCR3+/CD4 T cells expressed IFN-{gamma}, while IL-4-expressing cells were rare. There was a robust negative correlation between CXCR3+/CD4 T lymphocytes and DAS-28.

Conclusions. TNF-{alpha} inhibition with infliximab and etanercept results in sustained accumulation of CXCR3 positive T lymphocytes in the peripheral blood of RA patients. This suggests altered lymphocyte trafficking during TNF-{alpha} inhibition.

KEY WORDS: TNF, Etanercept, Infliximab, CXCR3, Th1, T lymphocyte, Disease activity score, Inhibition


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Characteristic features of rheumatoid arthritis (RA) are hyperplasia of the synovial lining and an infiltration by inflammatory cells, including monocytes and B and T lymphocytes. The mechanisms that regulate the migration and accumulation of these cells in inflamed joints are not clearly understood, but it is generally accepted that chemokines and their receptors are crucial for the recruitment and positioning of the cells in the RA synovium [1].

There is marked expression of the chemokine receptors CXCR3 and CCR5 on memory CD4 T lymphocytes [1, 2]. The fact that these cells express interferon-{gamma} (IFN-{gamma}) but not IL-4 suggests that CXCR3 may be used to identify Th1 T cells. Other receptors, such as CCR4, are predominantly present on Th2 type T lymphocytes [3].

The vast majority of T lymphocytes in inflamed joints from RA patients are CD45RO memory cells that are CXCR3-positive and express IFN-{gamma} [4, 5]. Inhibition of tumour necrosis factor {alpha} (TNF-{alpha}) leads to a reduction of these cells in the joints [6]. It is unclear, however, whether this local decrease in inflammatory cells results from an increased rate of apoptosis or from altered cell trafficking and peripheral cell pooling [7]. Infliximab was reported to be associated with a peripheral increase in the Th1:Th2 ratio [8] and peripheral accumulation of T cells expressing CCR5 [9]. Conversely, it induced apoptosis in peripheral blood monocytes from patients with Crohn's disease in one study [10]. However, a subsequent report found no increased rate of apoptosis in response to infliximab in synovial biopsies from RA patients [11].

We hypothesized that TNF-{alpha} inhibition is associated with a reduced trafficking of Th1 type lymphocytes into inflamed joints, resulting in a peripheral pooling of these cells that is reflected by a pronounced peripheral increase of CXCR3 expression. The objectives of our study were therefore to comparatively explore the behaviour of T lymphocyte populations at the systemic level in response to TNF-{alpha} inhibition with etanercept or infliximab, and to determine the correlation between laboratory findings and clinical response in RA patients, with a focus on the patterns of CXCR3 and CCR4 expression.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Seven consecutive patients (five females) about to undergo anti-TNF-{alpha} therapy with etanercept (25 mg subcutaneously, twice per week) and seven consecutive patients (six females) about to receive infliximab (3 mg/kg body weight intravenously, every 2–6 weeks) were recruited for this prospective study. All patients satisfied the American College of Rheumatology criteria for RA, had erosive radiographic features of RA and had failed to respond to conventional disease-modifying anti-rheumatic drugs. Their median age was 61 yr (range 36–73) and their median disease duration was 11 yr (1–25). Patient characteristics at baseline were similar in etanercept and infliximab groups. The co-medication remained unchanged until the end of the study. One physician who was unaware of the type of anti-TNF-{alpha} therapy performed all clinical and laboratory evaluations. All 14 patients were examined at baseline and 6 weeks. For logistical reasons, only five out of seven patients in each group could be examined at 14 weeks. The study protocol was approved by the relevant research ethics committee. All patients gave written informed consent.

We determined disease activity at baseline and after 6 weeks using the recently developed and validated Disease Activity Score based on 28 joints (DAS-28) [12]. Peripheral blood was obtained in EDTA before and during anti-TNF-{alpha} treatment. Fresh mononuclear cells were isolated by centrifugation on Ficoll–Paque. The cells were washed with phosphate-buffered saline (PBS) and immediately stained for flow cytometry or resuspended in RPMI with 10% fetal calf serum (FCS) for the determination of cytokine expression. A total of 0.2 x 106 cells were stained in PBS buffer containing 1 mg/ml of human immunoglobulin G and 0.02% NaN3 with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin-conjugated anti-CD8, peridinin chlorophyll protein-conjugated anti-CD3, and allophycocyanin (APC)-conjugated anti-CXCR3 or biotinylated anti-CCR4 followed by streptavidin–APC or APC-conjugated anti-CD45RO. For negative controls, cells were stained with the same concentration of isotype-matched control antibodies. All antibodies and secondary reagents were from BD Pharmingen (San Jose, CA, USA). Cell-associated fluorescence was analysed by four-colour flow cytometry (FACScalibur; Becton Dickinson, Mountain View, CA, USA).

Peripheral blood mononuclear cells were prestained with Cy-Chrome-conjugated anti-CD4 and APC-conjugated anti-CXCR3 and activated with 10 ng/ml phorbol-12-myristate-13-acetate and 1 µg/ml ionomycin in the presence of 10 µg/ml brefeldin A for 4 h, the staining pattern of CD4 and CXCR3 expression being preserved. The activated cells were fixed with 2% paraformaldehyde, permeabilized with 0.5% saponin/2% FCS in PBS and stained with FITC-conjugated anti-IFN-{gamma} and phycoerythrin-conjugated anti-IL-4 as well as the isotype-matched control antibodies. The samples were then analysed with a FACScalibur (Becton Dickinson).

We used the Wilcoxon rank-sum test to compare decreases in DAS-28 and chemokine receptor expression between etanercept and infliximab groups, and the Wilcoxon matched-pairs signed-ranks test to compare chemokine receptor expression before and during anti-TNF-{alpha} treatment. To determine the association between the proportion of receptor-positive T cells and DAS-28, we calculated Spearman's {rho} and used exploratory linear regression models with the proportion of receptor-positive T cells as the independent variable. Analyses were performed in STATA 8.2 (Stata, College Station, TX, USA). No adjustments were made for multiple comparisons, P values are two-sided.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
There was a decrease in disease activity, reflected by a drop in the median DAS-28 from 6.6 to 4.7 (P = 0.001) and the median CRP from 23 mg/l to 8 mg/l (P = 0.007) 6 weeks after treatment beginning. The median decrease was similar in the etanercept and infliximab groups for DAS-28 (–2.55 vs –2.45, P = 0.48), and for CRP (–6 mg/l in both groups, P = 0.90).

At baseline, CXCR3 was expressed on a higher percentage of blood CD8 T cells than on CD4 T cells (median 43% vs 17%) (Fig. 1, top). TNF-{alpha} inhibition markedly changed the frequency of receptor-expressing cells, with an increase in the median proportion of CXCR3+ CD4 and CD8 T cells after 6 and 14 weeks. Concomitant with the increase in the percentage of cells expressing CXCR3, the cells also expressed a higher density of this receptor. We found no consistent evidence for a difference between groups, with median increases of 7% vs 1% after 6 weeks (P = 0.14) and 14% vs 20% after 14 weeks (P = 0.91). The overall frequency of CD4 and CD8 T cells expressing CCR4 appeared to be unaffected by the anti-TNF-{alpha} therapy. CCR4 was expressed by a median of 23% CD4 T cells and 7% of CD8 T cells (Fig. 1, middle).



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FIG. 1. Box and whisker plots presenting the percentages of CD4 (left) and CD8 T cells (right) expressing CXCR3 (top), CCR4 (middle) and CD45RO (bottom) before and after 6 and 14 weeks of treatment. The box represents ranges from the 25th to the 75th quartile; the horizontal line in the box represents the median; the whiskers indicate the range. P values are for differences between baseline and follow-up examinations from the Wilcoxon matched-pairs signed-ranks test. Data from the etanercept and infliximab groups are combined. Note that only five out of seven patients could be examined in each group at 14 weeks for logistical reasons.

 
The frequency of CD4 (50%) and CD8 (15%) T cells in the total lymphocyte fraction remained constant during anti-TNF-{alpha} therapy. The majority of CD4 CXCR3 T cells expressed CD45RO (97%). Figure 1 (bottom) indicates that there was some evidence for an increase in the median proportion of CD45RO memory cells among the CD4 T-cell subset (58% at baseline vs 68% at 14 weeks, P = 0.14), but not among CD8 T cells (38% vs 45%, P = 1.00). Up to 30% of the CXCR3+ CD4 T cells expressed IFN-{gamma}, but IL-4 could only be detected in a small percentage of cells upon stimulation for 4 h.

Because of similar increases in CXCR3+ CD4 T cells upon treatment with etanercept and infliximab, we combined data from both groups when determining the association between disease activity and the percentage of CXCR3+ CD4 T cells. There was a negative correlation between DAS-28 and the percentage of CXCR3+ CD4 T lymphocytes at 6 weeks (Spearman's {rho} = –0.59, P = 0.026). Univariable linear regression analysis indicated a decrease in DAS-28 of 0.57 points associated with every 10% increase in CXCR3+ CD4 T cells [95% confidence interval (CI) –1.20 to 0.06, P = 0.071]. Conversely, the association between DAS-28 and CXCR3+ CD8 T lymphocytes was less pronounced (Spearman's {rho} = –0.32, P = 0.27) and non-significant in any of our correlation and regression analyses. CRP at 6 weeks was significantly correlated with DAS-28 (Spearman's {rho} = 0.58, P = 0.03), but not with CXCR3+ CD4 T cells at 6 weeks (Spearman's {rho} = 0.00, P = 0.99).

In exploratory sensitivity analyses, we adjusted for DAS-28 at baseline in the linear regression model and found the association between DAS-28 and CXCR3+ CD4 T lymphocytes similar, but statistically significant (–0.59 DAS-28 points per 10% increase in CXCR3+ CD4 T cells, 95% CI –1.14 to –0.03, P = 0.039). Additional inclusion of CRP at 6 weeks in the model yielded a significant association between DAS-28 and CXCR3+ CD4 T lymphocytes (–0.55 points, 95% CI –1.10 to –0.01, P = 0.048), but not between DAS-28 and CRP (+0.29 points per 10 mg/l increase in CRP, 95% CI –0.20 to 0.79, P = 0.22). Finally, the association between DAS-28 and CXCR3+ CD4 T lymphocytes appeared to be more pronounced, but non-significant after exclusion of the two individuals with the highest percentage of CXCR3+ CD4 T cells at 6 weeks (–1.04 points per 10% increase in CXCR3+ CD4 T cells, 95% CI –2.69 to 0.60, P = 0.19).

To explore the cellular response to TNF-{alpha} inhibition in other autoimmune diseases, we examined two patients with Crohn's disease and two patients with ankylosing spondylitis before and 4 weeks after treatment with 5 mg/kg of infliximab. The increases in CXCR3+ T cells were similar to those observed in our RA patients.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Examining 14 consecutive RA patients before and during treatment with the TNF-{alpha} inhibitors etanercept and infliximab, we found a marked increase in CXCR3+ Th1-type T lymphocytes in peripheral blood associated with TNF-{alpha} inhibition. Exploratory data from patients with Crohn's disease and ankylosing spondylitis suggest that these effects may occur irrespectively of the targeted condition.

The marked effect of TNF-{alpha} on adhesion molecule and chemokine expression observed in our study suggests that TNF-{alpha} blockade alters cell traffic. Previous studies have provided direct and indirect evidence for this hypothesis [2, 6]. The fate of local immune competent cells upon treatment with TNF-{alpha} inhibitors is unclear, however. A recent study suggested complement-mediated cell lysis of TNF-expressing target cells, as well as apoptosis in blood monocytes and in lamina propria T lymphocytes of patients with Crohn's disease [10, 13]. In contrast, no increase in the rate of apoptosis was found in biopsy specimens from RA patients [11]. The discrepancies between outcomes in Crohn's disease and RA could be due to chance, differences in dosages of TNF-{alpha} inhibitors or differences in cellular characteristics.

Alternatively, cells are pooled in the circulation. In line with this hypothesis, an increase in circulating Th1-type CD4+ lymphocytes in response to treatment with infliximab was reported [8]. A recent study described an accumulation of CCR5-expressing T lymphocytes in RA patients upon TNF-{alpha} blockade in peripheral blood [9]. However, studies in ankylosing spondylitis produced conflicting results. Whereas infliximab down-regulated the non-specific and specific T-cell cytokine response [14], treatment with etanercept resulted in an increase in IFN-{gamma} production by activated lymphocytes [15]. Our study provides evidence for a marked increase in peripheral CXCR3+ CD4 T lymphocytes upon TNF-{alpha} blockade, which is likely to reflect a pooling of immune competent cells at the systemic level.

Using Spearman's rank correlation and linear regression models, we found a robust association between the proportion of CXCR3+ CD4 T lymphocytes and the disease activity as determined by DAS-28 after 6 weeks, which appeared to be independent of CRP levels. The proportion of CXCR3+ CD4 T lymphocytes could therefore represent an additional dimension of treatment response in patients undergoing TNF-{alpha} inhibition. Larger studies are needed to confirm this observation and to determine its clinical implications.

Arguably, our findings may be unrelated to local inflammation at the level of joints. However, the phenotype of detected cells corresponded to the phenotype of the majority of T lymphocytes in RA joints [16, 17]. There was an increase in CXCR3+ but not in CCR4+ T lymphocytes displaying the memory phenotype CD45RO. Activation of these cells led to expression of IFN-{gamma}; in contrast, IL-4 could rarely be detected.

We conclude that TNF-{alpha} inhibition is likely to result in a pooling of Th1-type lymphocytes in peripheral blood, which is reflected by a peripheral increase in CXCR3+ expression. The fact that these immune-competent cells appear not to be purged, but merely pooled in the peripheral blood, may contribute to the understanding of autoimmune phenomena associated with TNF-{alpha} inhibitors [18, 19].


    Acknowledgments
 
We thank Pius Loetscher of the Theodor Kocher Institute, Christoph Müller of the Department of Pathology and Hans-Uwe Simon and Ines Schmid of the Department of Pharmacology, University of Bern, Switzerland for helpful comments and technical support. PJ is a Senior Research Fellow funded by the Swiss National Science Foundation (PROSPER grants 3233-066377.01 and 3200-066378.01).

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 5 June 2004; revised version accepted 10 September 2004.



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