Influence of therapy with chimeric monoclonal tumour necrosis factor-
antibodies on intracellular cytokine profiles of T lymphocytes and monocytes in rheumatoid arthritis patients
A. J. Schuerwegh,
J. F. Van Offel,
W. J. Stevens,
C. H. Bridts and
L. S. De Clerck
Department of Immunology, Allergology and Rheumatology, University of Antwerp, Belgium
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Abstract
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Introduction. It has been shown that T lymphocytes and monocytes/macrophages, producing pro-inflammatory cytokines, play a pivotal role in the pathophysiology of rheumatoid arthritis (RA). In recent placebo-controlled double-blind randomized studies, chimeric (human/mouse) tumour necrosis factor-
(TNF
) antibodies (cA2) proved to be very effective in improving clinical disease activity and reducing inflammatory parameters in RA.
Objective. To investigate whether anti-TNF
therapy influences the in vitro intracellular cytokine production in peripheral blood monocytes and T lymphocytes of RA patients after one single (24 h) and multiple intravenous infusions (6 months).
Methods. An intracellular flow cytometric technique was applied to measure interleukin 1ß (IL-1ß), IL-6, TNF
, IL-10 and IL-12 in monocytes and IL-2, IL-4 and interferon-
in T lymphocytes of 15 patients, before, after 24 h and after 6 months of therapy with monoclonal chimeric anti-TNF
antibodies (3 mg/kg, bimonthly i.v.). All patients were on stable therapy with methotrexate (1520 mg/week i.m.). Cytokine content in monocytes was measured directly after blood sampling (basal levels), after 8 h of culture (spontaneous production) and after 8 h of stimulation with lipopolysaccharides (LPS-stimulated production).
Results. Basal levels and production (after 8 h) of IL-1ß, IL-6 and TNF
were significantly decreased 24 h after the first administration of anti-TNF
(for IL-1ß P < 0.01; for IL-6 P < 0.01; for TNF P < 0.003) and after 6 months of therapy (for IL-1ß P < 0.02; for IL-6 P < 0.03; for TNF
P < 0.001). For IL-12, basal levels were significantly decreased 24 h and 6 months after the start of therapy with anti-TNF
antibodies (P=0.0001; P=0.003, respectively). In contrast, IL-10 production increased significantly after 24 h and after 6 months (P=0.02; P=0.01). The TH2/TH1 cytokine ratio in CD4+ T cells was significantly increased after 24 h and after 6 months of anti-TNF
therapy (P=0.003; P=0.0007).
Conclusion. Anti-TNF
therapy might down-regulate the monocytic capacity to produce pro-inflammatory cytokines and induces a shift to a more pronounced anti-inflammatory TH2 cytokine production.
KEY WORDS: Tumour necrosis factor-
, Anti-TNF
therapy, RA, Cytokine.
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Introduction
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Previous investigations have revealed the crucial role of inflammatory cells (T lymphocytes, B cells, monocytes, macrophages) and pro-inflammatory cytokines [interleukin 1ß (IL-1ß), IL-6, tumour necrosis factor-
(TNF
), interferon-
, IL-1
] in the pathogenesis of rheumatoid arthritis (RA) [13]. The rationale for blockade of TNF
was based upon several reports that demonstrated the particular importance of TNF
: high concentrations of TNF
were observed in synovial fluid, synoviocytes and synovial macrophages of patients with RA [4, 5]. Injection of TNF
in animals resulted in synovitis with infiltration of lymphocytes, monocytes and neutrophils in the articular cavity [6]. In addition, a relationship between synovial TNF
expression and disease activity in RA patients was observed [7]. In collagen-induced arthritis in mice, treatment with monoclonal antibodies to TNF
was able to diminish the production of IL-1 and granulocytemacrophage colony-stimulating factor (GM-CSF) by synovial cells in vitro and to ameliorate synovitis and joint destruction [810]. These findings led to the conclusion that TNF
is an important therapeutic target in rheumatoid arthritis: in recent, placebo-controlled, double-blind, randomized studies, anti-TNF
therapy proved to be very effective in improving clinical and laboratory disease activity and reducing inflammatory parameters [1118].
To further investigate the possible mechanisms responsible for the clinical efficacy of anti-TNF
therapy, we studied the short-term (24 h) and long-term (6 months) effects of anti-TNF
therapy on in vitro intracellular cytokine production in T lymphocytes and monocytes.
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Methods and patients
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Study population
Fifteen patients with active rheumatoid arthritis (RA), fulfilling the diagnostic criteria of the American College of Rheumatology for the classification of rheumatoid arthritis [19], were evaluated before, after 24 h and after 6 months (just before administering the sixth infusion) of therapy with monoclonal chimeric anti-TNF
antibodies (3 mg/kg i.v. at week 0, week 2, week 6, week 14, week 22, week 30) in combination with stable doses of methotrexate (12.520 mg/week i.m.) and stable low-dose corticosteroids (510 mg/day) or non-steroidal anti-inflammatory drugs (NSAIDs) (Table 1
). The disease duration of the RA patients was 47 (23279) months [median (range)]. Active disease was considered if the following criteria were present: morning stiffness lasting for more than 20 min, presence of at least six painful joints and four swollen joints and an erythrocyte sedimentation rate (ESR) of more than 28 mm/h or a C-reactive protein (CRP) of at least 1 mg/dl. The clinical response at month 6 was performed by a blinded assessor and defined according to the American College of Rheumatology definitions of ACR 20, ACR 50 and ACR 70 [20]. A placebo-controlled, double-blind study was ethically not acceptable since the efficacy of anti-TNF
therapy is already well established in placebo-controlled, double-blind studies [11, 14]. All patients gave informed consent for the study.
Analysis of lymphocyte subsets
An aliquot of 100 µl of peripheral blood was incubated for 15 min in the dark at 4°C with 10 µl of multi-colour monoclonal antibody panels (CD3-PerCP+CD4-FITC+CD8-PE; CD45-PerCP+CD3-FITC+CD19-PE; BD Biosciences, Erembodegem, Belgium). Subsequently, the remaining red blood cells were lysed with Facslysis (BD Biosciences) for 10 min at room temperature. Cells were fixed and pelleted at 400 g for 10 min. Afterwards, the cells were resuspended in phosphate-buffered saline (PBS) and analysed by FACScan flow cytometry (BD) within 24 h.
Intracellular cytokine analysis in lymphocytes
Cytokine-producing T lymphocytes were determined as previously described [21]. Briefly, peripheral whole blood cultures were incubated for 6 h at 37°C and 5% CO2 humidified atmosphere in the presence of 50 ng/ml phorbol-12-mirystate-13-acetate (PMA, Sigma, St Louis, MO, USA), 1 µ/ml ionomycin (Sigma) and 1 µg/ml brefeldin A (Sigma) or brefeldin A alone. After stimulation, 100 µl of cells were stained with CD8-FITC (BD) and CD3-PerCP (BD). Cells were lysed with FacsLysis (BD) and permeabilized in 0.3% saponin in PBS. Cells were stained for 30 min with monoclonal anti-cytokine antibodies labelled with phycoerythrin (PE) (anti-Hu IL-2-PE, anti-Hu IL-4-PE, anti-interferon-PE; BD). Twenty thousand cells were measured on a FACScan (BD) flow cytometer and analysed with WinMDI 2.8 software. Analysis gates were set on lymphocytes according to forward and sideward scatter properties. CD4+ T lymphocytes were defined and gated out as CD3+CD8- and CD8+ T cells as CD3+CD8+ using a compensated fluorescence plot. Within this gate, intracellular cytokine-producing cells were determined in histogram mode (PE emission). Markers were set on the 99th percentile using isotype-matched irrelevant antibodies (mouse IgG1 RPE, Serotec Ltd, Oxford, UK) as a reference. Results were expressed as the percentage of cytokine-producing cells. T helper (TH2/TH1) and T cytotoxic (TC2/TC1) ratios were calculated in the following manner: values for IL-4 (representing a TH2/TC2 cytokine) were divided by values for interferon-
or IL-2 (representing a TH1/TC1 cytokine)-positive CD4+ or CD8+ T lymphocytes.
Intracellular cytokine analysis in monocytes
Intracellular cytokine production by monocytes was determined as previously described [22]. Cytokine content in monocytes was measured directly after blood sampling (basal level), after 8 h of culture (spontaneous production) and after 8 h of stimulation with lipopolysaccharides (LPS-stimulated production). Peripheral blood cultures were stimulated for 8 h with 1 µg/ml lipopolysaccharide Escherichia coli (LPS, Serotype 026:B6, Sigma) and 1 µg/ml brefeldin A (LPS-stimulated production), or brefeldin A alone (spontaneous production). After culture, or immediately after blood sampling (to evaluate basal cytokine levels in monocytes), 100 µl of whole blood was stained with CD14-FITC (BD), lysed with FacsLysis buffer (BD), fixed and made permeable. Cells were incubated with PE-labelled anti-cytokine antibodies against IL-1ß, IL-6 and TNF
(BD).
Forty thousand events were measured on a FACScan flow cytometer and analysed with WinMDI software. Analysis gates were set on CD14+ cells according to fluorescein isothiocyanate (FITC) emission and side scatter. Within this gate, intracellular cytokine production was evaluated. Measurements were standardized using microspheres with different fluorescence intensities (DAKO FluoroSpheres, Code No K 0110 Glostrup, Denmark). Results were expressed as molecules of equivalent soluble fluorescein (MESF) units [23]. Isotype-matched irrelevant antibodies (mouse IgG1 RPE, Serotec) were used as controls.
Statistics
Differences in numbers of T-cell subsets and cytokine production before and after therapy were calculated with Friedman's test and Wilcoxon's rank test. Correlations were assessed with Spearman's rank correlation test.
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Results
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Disease activity
After 6 months, 14 patients showed reduced symptoms and signs of RA (Table 2
), as judged by ACR 20 (20% of improvement); nine of 15 patients achieved ACR 50.
White blood cell counts
Therapy with anti-TNF
resulted in an increase in lymphocyte counts in the peripheral blood, 24 h after the first infusion (Table 3
). The elevation of lymphocyte numbers was mainly due to an absolute increase of CD3+ T cells (P=0.0001) (Table 3
). The absolute number of CD4+ T cells increased drastically after 24 h (P=0.0003), with no differences after 6 months (Table 3
). Similarly, the number of CD8+ T cells increased after 24 h (P=0.0006). There was no change in numbers of peripheral blood B lymphocytes (CD19+). In addition, a rapid decrease of absolute numbers of monocytes (CD14+) was found (P=0.01) (Table 3
).
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TABLE 3. Monocytes and lymphocyte subsets in RA patients before (d0), after 24 h (d1) and after 6 months of therapy (m6)
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Lymphocytic cytokines
Twenty-four hours after treatment with anti-TNF
, the percentages and absolute numbers of IL-4, interferon-
and IL-2-positive CD4+ T cells were significantly increased (P=0.05, P=0.004, P=0.03, respectively) (Table 4
). The rise in IL-4-positive CD4+ T cells was more pronounced than the rise in interferon-
and IL-2-positive CD4+ T cells, resulting in a significant increase of the TH2/TH1 cytokine ratio in the peripheral blood (for IL-4/IL-2 P=0.0006 and for IL-4/interferon-
P=0.0007, respectively) (Fig. 1
). After 6 months, there was still an augmented TH2/TH1 cytokine ratio (for IL-4/IL-2 P=0.01 and for IL-4/interferon-
P=0.003, respectively) (Fig. 1
).
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TABLE 4. Percentage and absolute numbers of IL-2-, IL-4- and interferon- -positive T lymphocytes in RA patients before (d0), after 24 h (d1) and after 6 months of therapy (m6)
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For CD8+ T cells, the percentages and absolute numbers of interferon-
-, IL-2- and IL-4-positive cells were also higher after 24 h (P=0.0001, P=0.01) (Table 4
). In contrast, the absolute numbers of both interferon-
-, IL-2- and IL-4-positive CD8+ T cells did not alter after 6 months of therapy, compared with baseline values (Table 4
). The TC2/TC1 cytokine ratio, represented by IL-4/IL-2 and IL-4/interferon-
, did not change for CD8+ T cells.
Monocytic cytokines
Basal levels and spontaneous production (after 8 h of culture) of IL-1ß, IL-6 and TNF
were significantly decreased 24 h after the first administration of anti-TNF
and after 6 months of therapy (P < 0.05) (Fig. 2
). Stimulated production (after 8 h of culture with LPS) of IL-1ß, IL-6 and TNF
showed a similar rapid decline after 24 h and 6 months of treatment (P < 0.05) (Fig. 2
).
Basal IL-12 levels were significantly decreased after 24 h and after 6 months, compared with baseline: 1240 (5871908) MESF (molecules of equivalent soluble fluorescein) [median (range)] after 24 h and 1120 (7361763) MESF after 6 months, compared with baseline 1509 (12512419) MESF (P=0.0001, P=0.003, respectively). In contrast, LPS-stimulated levels of IL-10 rose significantly after 24 h and after 6 months: 1402 (10104509) MESF after 24 h and 1170 (8003180) MESF after 6 months, compared with baseline 970 (6073323) MESF (P=0.02, P=0.01, respectively).
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Discussion
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It has been shown that therapy with chimeric monoclonal TNF
antibodies leads to a rapid improvement of clinical and biological signs of inflammation in RA [1114]. There is considerable evidence for two major mechanisms of action: first, there is a reduced cell influx of inflammatory cells into the joints, due to down-regulation of the synovial endothelial adhesiveness and the chemotactic gradient [2427]; second, there is an inhibition of the pro-inflammatory cytokine cascade. It seems that inflammation in RA, characterized by initial TNF
production, which subsequently enhances the production of other pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12 and GM-CSF, can be inhibited by blocking TNF
[2631].
To our knowledge, this is the first study that investigated the influence of both the short-term (24 h) and long-term (6 months) effect of anti-TNF
therapy on intracellular cytokine profiles in T lymphocytes and monocytes. In contrast, previous studies only evaluated the cytokine profile after a single infusion of anti-TNF
[2933]. In the present study, we observed a clear reduction of capacity to produce IL-1ß, IL-6, TNF
and IL-12 in monocytes, immediately after the first infusion of anti-TNF
therapy, which persisted after 6 months of therapy. A flow cytometric intracellular technique was applied in this study to evaluate the altered capacity of monocytes to produce pro-inflammatory cytokines. By blocking produced cytokines intracellularly in the Golgi apparatus, we could avoid complexation of cytokines with their inhibitors, which could be an explanation for some discrepancies in other studies, detecting cytokines in serum with ELISAs: Oshima et al. [31] detected no substantial serum IL-1ß, Lorenz et al. [30] observed a decrease in IL-1ß production, whereas Charles et al. [32] found no difference in serum IL-1ß levels after a single infusion with anti-TNF
antibodies.
Our findings regarding IL-6 production on a short- and longer-term basis are completely in line with previous data that reported decreased serum IL-6 production [2932]. The decrease in IL-6 could explain the rapid and sustained decrease in acute-phase CRP, also observed in other trials with anti-TNF
therapy [11, 1618, 32].
On the basis of in vitro experiments, which showed that IL-12 production in RA was partly inhibited by anti-TNF
[34], it was expected that TNF
neutralization would lead to down-regulation of IL-12 production, which was observed in our study.
By serum studies using ELISA assays, not distinguishing immunoreactive from bioactive cytokines, it has not been possible to test equivocally the prediction that TNF blockade will inhibit TNF
production in vivo [31, 32]. In our study, basal, spontaneous and LPS-stimulated TNF
production significantly decreased after 24 h and after 6 months of therapy. In accordance with our findings in peripheral blood monocytes, analysis of synovial tissue before and after treatment showed that TNF blockade was able to reduce TNF
synthesis in the joints [28].
Taken together, this study provides evidence that anti-TNF
blockade can deactivate the pro-inflammatory cytokine cascade, as demonstrated by reductions in serum CRP, IL-1, IL-6, TNF
and IL-12 production.
In addition to the decreased capacity of monocytes to produce pro-inflammatory cytokines, a decrease in absolute monocyte counts was observed, which has been described previously [25, 2931]. One of the underlying mechanisms could be complement lysis of membraneous TNF
-expressing monocytes that bind the anti-TNF
antibody [35]; an alternative explanation would be antibody-dependent cellular cytotoxicity of membraneous TNF
-expressing monocytes [35]. Recently, a third mechanism for the decrease in monocyte counts is reported: the capacity of anti-TNF
antibodies to induce apoptosis of monocytes in patients with Crohn's disease [36].
In addition, we demonstrated an increased capacity of monocytes to produce IL-10, which is in accordance with previous findings of increased serum levels of IL-10 after one infusion of anti-TNF
therapy [31]. This up-regulated IL-10 production can contribute to the correction of the disequilibrium between pro- and anti-inflammatory cytokines in monocytes of RA patients. In vitro experiments reported enhanced pro-inflammatory cytokine (IL-1, IL-6, IL-8, TNF
, GM-CSF) production by blocking IL-10 or a reduction by addition of exogenous IL-10 [37], demonstrating an important role for IL-10 as an anti-inflammatory and immunoregulatory cytokine in monocytes.
In our study, an increased absolute number of lymphocytes was observed: several reports describe an increase of peripheral blood TH1 cells in patients treated with anti-TNF
, associated with the down-regulation of adhesion molecules in the synovium [25, 26, 31, 33], which could be an important factor for inhibiting TH1 cells from migrating into the synovium and inhibiting the release of their TH1 cytokines in the synovium.
We investigated whether TNF
therapy alters the amount of type 1 (represented by IL-2 and interferon-
) and type 2 (represented by IL-4) CD4+ and CD8+ T lymphocytes within the peripheral blood of RA patients. We could confirm the data presented by Maurice et al. [33] and Lorenz et al. [29], who showed a rise for IL-4-, IL-2- and interferon-
-positive CD4+ T cells shortly after a single infusion. Moreover, we demonstrated that the increased number of IL-4-, interferon-
- and IL-2-positive CD4+ T cells was still present after 6 months of anti-TNF
therapy. This increase of TH1 and TH2 cytokine production, at the same time as the up-regulation of IL-10 production and down-regulation of IL-12, is a very interesting and unexpected finding since IL-10 was originally discovered as a TH1-inhibiting cytokine [3840], whereas IL-12 is known to play an important role in the development of a TH1 response and subsequent interferon-
production in RA [41].
According to Cope et al. [42], increased IL-2, IL-10 and interferon-
production in T lymphocytes and improved proliferative responses were reported to occur after prolonged in vitro and in vivo TNF
blockade. Further investigation of patients will allow us to define how TNF
therapy enhances T-cell responsiveness and type 1/type 2 cytokine production. The ratio of TH2/TH1 was increased in comparison with baseline in our study, suggesting a normalization of the initial TH1 cytokine predominance in RA patients towards a more pronounced anti-inflammatory TH2 cytokine balance, in contrast to the study of Maurice. This discrepancy may be partially due to methodological differences (optimal incubation and use of brefeldin A or monensin to block cytokine secretion) [22, 4346].
In conclusion, repeated administration of anti-TNF
therapy might down-regulate the monocytic capacity to produce pro-inflammatory cytokines and induce a shift to a more pronounced anti-inflammatory TH2 cytokine balance. Further investigation on cytokine profiles in doseresponse studies with anti-TNF
therapy has to be performed in order to elucidate the complex mechanisms of anti-TNF
therapy, responsible for the clinical efficacy in rheumatoid arthritis.
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Acknowledgments
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We would like to thank Centocor/Schering-plough for providing infliximab. We thank also the staff of the polyclinic of ImmunologyAllergologyRheumatology of the University Hospital of Antwerp for their support. We are especially grateful to P. Claus for her expert administrative and technical assistance.
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Notes
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Correspondence to: W. J. Stevens, University of Antwerp UIA, Department of Immunology, Allergology and Rheumatology, Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail: wim.stevens{at}ua.ac.be 
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Submitted 18 December 2001;
Accepted 18 October 2002