Differential effects of the tumor necrosis factor alpha-blocker infliximab and etanercept on immunocompetent cells in vitro

International Immunopharmacology 11 (2011) 1724–1731

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Differential effects of the tumor necrosis factor alpha-blocker infliximab and etanercept on immunocompetent cells in vitro Firas Thaher, Sandra Plankenhorn, Reinhild Klein ⁎ Department of Internal Medicine II, University of Tübingen, Otfried-Müller-Str. 10, 72076 Tübingen, Germany

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Article history: Received 25 March 2011 Received in revised form 26 May 2011 Accepted 3 June 2011 Available online 24 June 2011 Keywords: Etanercept Infliximab Immunomodulation Peripheral blood mononuclear cells TNFα-blocker

a b s t r a c t The tumor necrosis factor-alpha (TNFα) antagonists infliximab or etanercept are used in the management of chronic inflammatory disorders but have differences in clinical activity. Here we show that both have different effects on immunocompetent cells in vitro. Peripheral blood mononuclear cells (PBMC) from 20 healthy donors were incubated with infliximab or etanercept alone and in a co-culture with recall-antigens (BCG, tetanus toxoid [TT]). Expression of the activation marker CD69 on different PBMC-subpopulations was determined by flow cytometry, release of Th1-, Th2- and macrophage/monocyte-related cytokines into the supernatants by ELISA. There were strong inter-individual differences in reactivity of PBMC of the 20 donors towards infliximab and etanercept. On the whole group level, both enhanced IL-10 production but had opposite effects on the TNFαand IFNγ-secretion; Th2-cytokine-secretion (IL-13, IL-5) was differentially influenced. IL-13 production was significantly reduced by infliximab but not by etanercept. IL-5 secretion was strongly enhanced in individual subjects but was not significantly influenced on the whole group level. Etanercept but not infliximab significantly decreased the CD69-expression by CD8+ T- and CD56+ natural killer(NK)-cells. Co-culture with recall antigens enhanced most of these reactions. Our data indicate that individual predisposition and immunological reactivity may be an important factor influencing the therapeutic efficacy of anti-TNFα agents. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Tumor necrosis factor-alpha (TNFα) is an important cytokine inducing and maintaining inflammatory processes but being involved also in lipid and glucose metabolism [1]. Therefore, several TNFαneutralizing antibodies and fusion proteins have been constructed for therapeutic purposes such as infliximab, a chimeric monoclonal antiTNFα antibody which consists of three quarters human-constant region immunoglobulin G1 and one quarter mouse variable region Fv, or etanercept, a dimeric fusion protein that joins the human p75 TNFreceptor to the Fc domain of human IgG1. Infliximab is able to bind both, the transmembrane form (mTNFα) and the soluble form of TNFα (sTNF) [2]. Etanercept also neutralizes both, extracellular as well as membrane forms of TNFα, but binding is less effective than that of infliximab; furthermore, etanercept can also bind TNF-β (lymphotoxin) produced by macrophages or T cells [2,3]. Interestingly, TNFα blocker has been meanwhile shown to be ineffective in the treatment of sepsis despite its association with proinflammatory cytokines [4]. But there is no doubt any more about their benefit in ⁎ Corresponding author at: Clinical Immunology, University of Tübingen, Department of Internal Medicine II, Otfried-Müller-Str. 10, 72076 Tübingen, Germany. Tel.: +49 7071 2984479; fax: +49 7071 292760. E-mail address: [email protected] (R. Klein). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.06.005

chronic inflammatory diseases such as Crohn's disease (CD), rheumatoid arthritis (RA), ankylosing spondylitis (AS) or psoriasis [5,6]. However, these disorders are not only driven by innate immunity and TNFα but also by T-cells [6]. One could, therefore, postulate that antiTNFα agents not only affect the inflammatory response but probably also other immunological reactions. This is not surprising since TNF receptors are expressed by most nucleated cells with the exception of unstimulated T-cells, and since TNFα is expressed also on the membrane of cytotoxic T cells [2]. Thus, recent studies showed that application of anti-TNFα resulted in an increase of Foxp3+ regulatory T cells and improvement of function [7,8], influenced the expression of IL-2 receptors on T cells, the antibody production by B cells, function of neutrophils, induced lymphocyte/monocyte apoptosis in CD, and influenced gene and mRNA expression of several cytokines in peripheral blood leukocytes [9–12]. However, there are several differences in clinical efficacy between infliximab and etanercept. For instance, infliximab induces clinical remission in Crohn's disease (CD) and rheumatoid arthritis (RA) [13] while etanercept is effective in rheumatoid arthritis but not in CD [13,14]. Also in psoriasis and psoriatic arthritis differences exist in the rapidity of therapeutic improvement [15]. Furthermore, the incidence and spectrum of bacterial or fungal infections varies in patients treated with the two anti-TNFα agents [16,17]; it has been also shown that infliximab induces more frequently autoimmune phenomena

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than etanercept [18]. The scientific basis of these differences in clinical efficacy profiles remains uncertain [2,17,19]. Possible explanations are differences in the abilities of TNF antagonists to achieve adequate serum concentrations, differences to achieve therapeutic concentrations in inflammatory microenvironments or their abilities to bind to TNF and induce therapeutic effects, their specificities in binding capacities including complement activation, differential effects on B-cell or cytotoxic T-cell activity or cytokine production [3,20–23]. We, therefore, wanted to analyse in more detail the effect of antiTNFα agents on different types of peripheral blood mononuclear cells (PBMC) including T helper cell subsets, natural killer cells and macrophages/monocytes. Since those investigations in patients with chronic inflammatory disorders may be hampered by the disease processes themselves or accompanying therapies we chose a ‘neutral’, not-disease-specific, environment, and aimed to elucidate the in vitro influence of infliximab and etanercept on PBMC from healthy donors. Thus, we investigated the effect of both substances on ‘naïve’, i.e. nonstimulated PBMC, and in a second step on PBMC, which had been activated by bacille calmus guérin (BCG) known to activate primarily macrophages and Th1 cells and tetanus toxoid (TT) shown to activate Th2 and regulatory T-cells [24,25]. It will be shown, that TNFα blocker seem to act preferentially on cytotoxic and regulatory T-cells but also NK-cells, and some clear differences in the immunological mode of action of both substances, infliximab and etanercept, will become evident. 2. Materials and methods 2.1. Subjects Twenty healthy individuals (Nos 1–20) were recruited among healthy students and the laboratory staff of the Department of Internal Medicine II, University of Tübingen (7 females, 13 males; age 21–34 years). Exclusion criteria were any acute or chronic diseases including acute allergic reactions and any vaccination within the last 12 months. All volunteers gave written consent to participate in the study. The study was approved by the local ethics committee. 2.2. Antigens and reagents The macrophage/monocyte- and Th1-cell activating antigen BCG (2 ×108 bacteria/50 ml) was obtained from Medac (Wedel, Germany), the Th2/Treg stimulating antigen tetanus-toxoid (TT, 40 IU [international units]) was purchased from Novartis Behring (Marburg, Germany). The monoclonal anti-TNFα-antibody infliximab (REMICADE®) was obtained from Essex Pharma (Munich, Germany), the soluble receptor fusion protein etanercept (Enbrel®) from Wyeth (Münster, Germany). As positive control pokeweed mitogen (PWM; Biochrom AG) was used. 2.3. PBMC cultures PBMC were isolated from 100 ml heparinized blood by centrifugation through Ficoll–Hypaque within 24 h of drawing the blood. This range has been proven in earlier studies to give reliable and reproducible results without significant changes, and cells are still viable after 24 h [24,26–30]. PBMC were collected from the interface, washed twice in Hanks’ salt solution and adjusted to 1 Mio cells/ml in RPMI 1640 medium supplemented with gentamycin and 25% autologous serum as reported in previous studies [24,26,29–31]. Prior to the experiments presented in this study kinetics have been performed to obtain the optimal time points for the following assays. These resulted in incubation of PBMC with the different antigens (see above) for 24 h (flow cytometry) or 7 days (proliferation assay and cytokine production) at 37 °C, 5% CO2 in a humidified atmosphere.

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In a first step, PBMC were incubated only with infliximab or etanercept at concentrations of 1000, 100, 10, 1, and 0.1 μg/ml without any co-stimulation; the concentrations 1–10 μg/ml correspond to the serum concentration of etanercept and the concentration of 10–100 μg/ml to that of infliximab in patients treated with the respective substances [32–36]. In a second step, PBMC were costimulated with TT and BCG at concentrations of 5 TU/ml, and 5 × 10 5 bacteria/ml, respectively. These culture conditions and recall-antigen concentrations had been previously shown to give optimal results in most individuals [24]. 2.4. Proliferation assay For proliferation assays PBMC (1 × 10 6/well) were seeded into 96well cell culture plates and stimulated without antigen (spontaneous proliferation) or with antigens in concentrations as mentioned above. During the last 18 h, the cultures were pulsed with 3H-thymidine (0.74 MBq/ml, 20 μl/well) and then harvested onto glasfibre filters. The incorporated radioactivity was measured by liquid scintillation spectroscopy using a β-counter, and given as counts per minute (cpm). All tests were performed in quadruplicates; mean values were calculated and used for further statistical analysis. 2.5. Cytokine production For cytokine production, 5 × 10 5 PBMC were cultured with the same antigens and antigen concentrations in 24-well culture plates and maintained as indicated above. Culture supernatants were collected at day 7 and kept frozen at −20 °C for up to 30 weeks until quantitative cytokine determination by ELISA as recently described [24,26]. A seven-day stimulation of PBMC for cytokine determination had been shown in previous studies to be optimal for interleukin (IL)-5, IL-13, IL-10, interferon-γ (IFNγ), and tumor necrosis factor (TNF)-β production, while the TNFα-, IL-6 and IL-1 production had already decreased at that time. However, since this decrease occurred in a linear manner and since these cytokines were still present in the supernatants in significant amounts we, nevertheless, used the 7-day supernatant for all cytokines [24] in order to simplify the sample collection. For the detection of cytokines in the supernatants by ELISA wells of 96-well microtiter plates were coated overnight at 4 °C with 1.75 μg/ ml (GM-CSF, IL-6, IL-5, IL-13, IL-10, TNFα, IFNγ, TNFβ) antihuman cytokine monoclonal antibody (Pharmingen, San Diego, CA, USA) in hydrogenbicarbonate buffer, pH 9.6, and blocked for 1 h at room temperature with phosphate buffered saline (PBS 60 mmol/l, pH 7.4) containing 0.5% bovine serum albumin (BSA). Culture supernatants were used undiluted in duplicate and incubated for 2 h at 37 °C; biotinylated anti-human cytokine antibodies (1.25 μg/ml for GM-CSF, IL-6, Il-5, IL-13, IL-10, IFNγ, TNFβ, TNFα; Pharmingen) were added for 2 h at 37 °C. After another incubation for 1 h at room temperature with avidin-peroxidase (2.5 μg/ml), substrate solution (0.5 mg orthophenylene-diamine / ml citrate buffer [pH 5.0] and 0.01% H2O2) was added. The reaction was stopped with 25% sulphuric acid and optical density was measured at 450 nm in a microtiter plate reader. Results were related to a standard curve obtained with the respective recombinant cytokines. Normal values were defined as 100 pg/ml for IL-1, 200 pg/ml for IL-5, 150 pg/ml for IL-13, 200 pg/ml for IL-10, 100 pg/ml for TNFα, 300 pg/ml for IFNγ, and 500 pg/ml for TNFβ [24]. 2.6. Flow cytometry For flow cytometry, again 5 × 10 5 PBMC were cultured in 24 well plates with the same antigens as mentioned above, but in order to measure the early activation marker CD69 the incubation time was only 24 h.

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Cell staining was performed using cocktails for the demonstration of activated CD4+ T-cells (FastImmune™CD4/CD69/CD3), CD8+ T-cells (FastImmune™CD8/CD69/CD3), CD19+ B-cells (FastImmuneTMCD19/ CD69/CD45), and CD56+ NK-cells (FastImmune™ CD56/CD69/CD45) (all obtained from Becton–Dickinson [San Jose, CA]). The PBMC were incubated with the respective antibodies or IgG isotype control antibodies (BD Biosciences Pharmingen). A minimum of 10,000 lymphocytes were counted. Quadrants were set based upon the isotype controls for each antibody. Results were expressed as the percentage of CD69 expressing cells of the respective cell types (CD4+/CD3+;CD8+/ CD3+; CD19+/CD45+; CD56+/CD45+). Furthermore, the percentage of CD4 +CD3+ and CD8+ CD3+ T-cells, CD16 + CD45+ B- and CD56 + CD45+ NK-cells was determined. 2.7. Investigation of the viability of PBMC In order to proof the viability of cells, for all experiments PBMC were incubated with the mitogen poke weed mitogen (PWM; 10 μg/ ml) in parallel to the other antigens. When we did not obtain positive reactions with respect to proliferation, cytokine production or CD69 expression with this mitogen, we concluded that the PBMC were

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either not viable or that there were other technical problems. The experiments were excluded from the analysis and repeated. 2.8. Statistics Statistical analysis was performed with SPSS (version 15.0) applying the Wilcoxon signed rank test for paired parameters. Differences were considered significant for p b 0.05. 3. Results 3.1. Influence of infliximab and etanercept on ‘naïve’ PBMC 3.1.1. Effect on cytokine production In a first step we analysed the influence of infliximab and etanercept on the presence of TNFα, their major target, in the supernatants of PBMC from 20 healthy individuals. As expected, after incubation with infliximab TNFα could be hardly detected in the PBMC supernatants (Fig. 1a). In contrast, incubation with etanercept at a low concentration (0.1 μg/ml) lead to a significant increase of TNFα-concentration in the supernatants which was not any more observed after increasing the dose.

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Fig. 1. Influence of infliximab and etanercept on the spontaneous TNFα- (a), and IFNγ- (b), IL-5 (c) and IL-10-production (d) by PBMC from 20 healthy volunteers. Values of individual probands (Nos 1–20) are given. There are strong inter-individual differences in cytokine response towards both substances. Thus, in proband No 7 there was a significant reduction of the production of TNFα, IFNg, IL-5, and IL10 after incubation of PBMC with low doses of infliximab or etanercept. In other probands there was a strong enhancement of cytokine production (for instance in probands no. 3, 12, 18 there was an induction of TNFα-production by etanercept, in probands no. 12, 13 there was an increase of IFNγ release, in probands no. 6, 8, 12, 18, 19 the IL-10 production was induced) Significances as compared to the cytokine production without infliximab or etanercept (0 μg/ml) on the whole group level are given. * p b 0.05, n.s. = not significant. - = mean.

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On the production of the Th1 cytokine IFNγ both substances had just the opposite effect; thus, infliximab increased in individual volunteers the IFNγ-production – although at the whole group levels the differences were not significant – while incubation of the PBMC with etanercept significantly reduced its secretion (Fig. 1b). Infliximab slightly reduced the spontaneous production of the Th2 cytokine IL-13 at the whole group level (Fig. 1c), while etanercept had no significant effect. On IL-5 production both substances had no significant effect at the whole group level, although it was strongly induced in individual patients (data not shown). Similar data were obtained for the production of IL-10, a cytokine produced by Th2 cells but also regulatory T cells (Fig. 1d). The production of the B-cell activating cytokine IL-6 was stimulated by both substances, infliximab and etanercept, by PBMC from several probands but not at the whole group level (not shown). For all cytokines there were strong inter-individual differences. PBMC from one subject (no. 7) produced already spontaneously several

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cytokines, although there was no evidence for an infectious, allergic or other inflammatory process in this individual.

3.1.2. Effect on the expression of the activation marker CD69 In another step we wanted to see whether infliximab or etanercept affect the state of activation of PBMC from the 20 subjects as indicated by the expression of the activation marker CD69. At the group level infliximab had no significant effect on CD69 expression by CD4+ (not shown) or CD8+ T cells (Fig. 2a), B-cells (not shown) or NK-cells (Fig. 2b). Also etanercept did not influence the CD69 expression by CD4 + CD3+ T-cells and B-cells (not shown), but it significantly reduced the expression of CD69 by CD8 + CD3+ cytotoxic T-cells (Fig. 2a) and CD56 + CD45+ NK-cells (Fig. 2b). However, as already shown for the cytokine production, there were great individual variations; thus CD69 was induced on CD8, CD19 and CD56 cells in some individuals (for instance No. 5, 9, 10,12, 18, 19) by at least one of the two TNFα-blockers (not shown).

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Fig. 2. Influence of infliximab and etanercept on the spontaneous expression of the activation marker CD69 by CD8 + CD3+ T-cells (a) and CD56 + CD45+ NK-cells (b). Values of individual probands (Nos 1–20) are given. In general, there was a reduced expression of CD69 by CD8 + CD3+ T-cells, although in individual probands a significant enhancement was observed (proband no. 5, 9, 16, 19). Etanercept signmificantly decreased the CD69 expression by CD56 + CD45+ NK cells, while infliximab had no effect. Significances as compared to the CD69-expression without infliximab or etanercept (0 μg/ml) on the whole group level are given. n.s. = not significant; * p b 0.05, ** p b 0.01, *** p b 0.001. - = mean.

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Fig. 3. Influence of infliximab and etanercept on the BCG-induced TNFα (a) and IL-6 production (b) by PBMC from 20 healthy volunteers. Values of individual probands (Nos 1–20) are given. Incubation of BCG-activated PBMC with infliximab completely suppressed the TNFa-production in all individuals while etanercept lead to a significant increase. BCG-induced IL-6 production was significantly increased by infliximab while etanercept had no effect. Significances as compared to the BCG-induced TNFα- and IL-6 production without infliximab or etanercept on the whole group level are given. n.s. = not significant; * p b 0.05, ** p b 0.01, *** p b 0.001. - = mean.

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3.2.1. Effect on cytokine production In order to mimic an ‘inflammatory environment’ in vitro, PBMC were incubated with recall antigens known to activate predominantly Th1 cells and macrophages (BCG) or Th2- and Treg cells (Tetanus toxoid). On the production of macrophage/monocyte cytokines by BCGstimulated PBMC infliximab and etanercept had different effect; thus, infliximab lead to a significant decrease of TNFα – as expected – while etanercept unexpectedly significantly enhanced the TNFα-production (Fig. 3a). The secretion of IL-6 was influenced in an opposing way; infliximab induced its release while it was hardly influenced by etanercept (Fig. 3b). Both, infliximab and etanercept, significantly decreased the BCGinduced production of IFNγ (Fig. 4a); interestingly, both also significantly reduced the production of the TT-induced production of the Th2 cytokines IL-13 (Fig. 4b) and IL-5 (not shown). However, there was one proband (no 7), in whom TT-induced IL-5 production was strongly enhanced (not shown). In contrast, the TT-induced production of IL-10 was strongly induced by incubation with infliximab and etanercept reaching statistical significance at the whole group level for etanercept (Fig. 4c).

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3.2.2. Effect on the expression of the activation marker CD69 Incubation of the PBMC with BCG lead to a significant increase of the expression of the activation marker CD69 by CD4+- and CD8+ T-cells as well as by CD19+ B- and CD56+ NK-cells. This activation of PBMC-subtypes was neither influenced by infliximab nor by etanercept (not shown). TT induced only on CD56+ NK-cells the expression of CD69, and this was significantly decreased by etanercept but not infliximab (Fig. 5).

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3.3. Influence of infliximab and etanercept on the proliferative response of ‘naïve’ and recall-antigen activated PBMC Furthermore, the proliferative response of PBMC towards infliximab and etanercept was investigated by 3H-thymidine incorporation

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Fig. 4. Influence of infliximab and etanercept on the BCG-induced IFNγ-production (a) as well as the TT-induced IL-13 (b) and IL-10 production (c) by PBMC from 20 healthy volunteers. Individual values are given. On the whole group level, both substances significantly decreased the BCG-induced IFNγ-production although it was strongly induced in some individual probands (Nos 7, 14, 11). Also TT-induced IL-13 production was significantly reduced by inflilximab and etanercept. In contrast, etanercept (but not infliximab) significantly enhanced the TT-induced IL-10 production. Significances as compared to the BCG-induced IFNγ and TT-induced IL-13 or IL-10 production without infliximab or etanercept on the whole group level are given. n.s. = not significant; * p b 0.05, ** p b 0.01, *** p b 0.001. - = mean.

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Fig. 5. Influence of infliximab and etanercept on the TT-induced expression of the activation marker CD69 by CD56 + CD45+ NK-cells. Values of individual probands (Nos 1–20) are given. The expression of CD69 by NK-cells preactivated with TT was significantly reduced by etanercept while infliximab had no significant effect. Significances as compared to the TT-induced CD69 expression without infliximab or etanercept on the whole group level are given. n.s. = not significant; * p b 0.05, ** p b 0.01, *** p b 0.001. - = mean.

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assay. Comparing the values at the group levels for the two substances at the different concentrations, there were no significant differences. However, in a few individuals a strong dose dependent increase of proliferation was obtained (Fig. 6a). The BCG- and TT-induced proliferation of PBMC was significantly inhibited by increasing concentrations of etanercept and infliximab (Fig. 6b, c). 4. Discussion

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Fig. 6. a) Influence of Infliximab and etanercept on the proliferative response of PBMC from 20 healthy individuals. Values of individual probands (Nos 1–20) are given. There was a strong increase of the spontaneous proliferation of PBMC by infliximab and to a less extent by etanercept in some individuals (no. 3, 18) but on the whole group levels values were not significant. b, c) Influence of infliximab and etanercept on the BCG- (b) and TT-induced (c) proliferative response of PBMC from 20 healthy volunteers. Values of individual probands (Nos 1–20) are given. Proliferative response of PBMC to BCG or TT was significantly reduced by infliximab and etanercept. Significances as compared to the spontaneous, BCG- or TT-induced proliferation without infliximab or etanercept on the whole group level are given. Cpm = counts per minute; n.s. = not significant; * p b 0.05, ** p b 0.01, *** p b 0.001. - = mean.

From the presented data obtained with PBMC from healthy donors several major conclusions can be drawn: 1.) there is a rather heterogeneous inter-individual response of PBMC from different healthy individuals towards both TNFα-blockers infliximab and etanercept with respect to proliferation and cytokine production; 2.) infliximab and etanercept have opposite effects on the release of TNFα and IL-6 by PBMC by non-stimulated and BCG-activated PBMC; 3.) both substances reduce the production of IFNγ; 4.) the effect of the two antiTNFα agents on the release of the Th2 cytokines IL-5 and IL-13 depends upon the state of activation of PBMC; 5.) both agents enhance the release of IL-10; 6.) etanercept, but not infliximab, decreases the expression of the activation marker CD69 on NK-cells and to a less extend on CD8+ T-cells; 7.) most immunological reactions were observed already with low concentrations of infliximab or etanercept (0.1 or 1 μg/ml). All these data were obtained with PBMC incubated only with etanercept or infliximab alone but became more significant when the lymphocytes were co-stimulated with recall-antigens such as BCG activating macrophages and Th1 cells or TT activating Th2-, regulatory T-cells and NK cells [24,37]. We did not use a mitogen such as phytohemagglutinin or poke weed mitogen as applied by other authors [11] because in our experience their application in PBMC is too strong and pluripotent to differentiate specific effects. In the present study we used PWM only as a positive control to evaluate the responsiveness of PBMC, individuals, whose PBMC did not react with this mitogen were excluded from the study. At the group level, most immunological reactions were reduced by both substances. A decrease of T cell responsiveness has been observed also in vivo in patients with AS treated with TNFα antagonists [38], and it has been shown that TNF blockade suppresses IFNγ synthesis by T cells and expression of type 1 genes like IFNγ and IL-12 receptor beta, which might explain cases of severe mycobacterial infection and/or reactivation of tuberculosis undergoing treatment with infliximab [10,11,39]. Interestingly, both anti-TNFα agents strongly enhanced the release of the Th2-cytokine IL-5 by PBMC from individual probands, while production of IL-13 was rather reduced. For both substances a significant reduction of IL-5 and IL-13 production was observed when PBMC were co-cultured with the Th2-inducing antigen TT. This might explain why autoimmune phenomena and even autoimmune diseases can be induced by TNFα blockade in some patients in vivo[18], but also why some patients with systemic lupus erythematosus, which has a strong Th2-component [40], seem to benefit from this kind of therapy (although experience is still rather limited) [41]. A downregulation of the type 2 genes IL-4, -5, and -9 has been also described for etanercept while infliximab had no effect in this respect [10]. Inter-individual variations in reactivity towards TNFα blocker have been already observed by Appel et al [38] and Coury et al., [42], and it has been reported that in practice of rheumatology approximately one-third of patients demonstrate no clinical improvement in response to TNFα blocker, while another third demonstrate a partial response, and one-third an excellent and sustained response [43]. Their modes of action as well as their side effects may, therefore, depend to a large extend upon the individual immunoreactivity determined by genetic or environmental factors. Moreover, we found that infliximab but not etanercept induced the B-cell activating factor IL-6 which may activate autoreactive B-cells [44]. Furthermore, etanercept and to a less extent infliximab enhanced the IL-10 production. In another study using Jurkat cells, Mitoma et al

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[45] reported that infliximab but not etanercept induced an increased production of IL-10. The immunological effects of the TNFα blocker may, therefore, be greatly influenced by the in vitro conditions. Regulatory T cells (Treg) are an important source of IL-10 production [46], i.e. this cell type might have been activated in vitro by etanercept. We, therefore, also analysed the effect of TNFα-blocker on the expression of Foxp3, a marker of regulatory T cells [47] and, indeed, found a strong enhancement (own unpublished observation). A restoration of function of Treg by infliximab in RA and CD has been described [48,49], and IL-10 administration improves psoriasis [50]. Since Treg suppress Th1 as well as Th2 cells [46], their enhancement in vivo and in vitro by TNFα-blockers may be another therapeutic mechanism of these substances. Another source of IL-10 are Th17 cells which have been found to play an important role in several autoimmune disorders including RA and CD [51] but could not be investigated in the present study. As expected, we found a significant reduction of TNFα in the PBMC supernatants by infliximab; surprisingly, however, incubation with etanercept on the contrary strongly enhanced the TNFα levels. These data are somewhat in contrast to other reports, which described also in vitro a reduction of TNFα by etanercept; these discrepancies to our results could be explained by different in vitro conditions as concentrations of TNFα-blockers or cell lines used by the authors [52,53]. Furthermore, the action of etanercept on membrane as well as soluble TNFα is ‘reversible’, whereas the binding of monoclonal anti-TNFα antibodies is more effective [2,3,12], i.e. dissociation of etanercept and TNFα could have lead to increased TNFα levels in the cell culture supernatants. However, whether differences in association and dissociation rates may play a role is controversially discussed [12,21]. On the other hand, infections, especially tuberculosis, are more often observed in patients treated with infliximab than with etanercept [54], and there are reports showing that in vivo during therapy of patients with AS or Sjögren's disease with etanercept the TNFα production is increased while treatment with infliximab decreased its production [55,56]. This was attributed to a counter-regulatory action due to peripheral TNFα blockade by etanercept. Finally, reverse signalling by transmembrane TNFα in response to anti-TNFα antibodies but not the soluble receptors has been proposed as an explanation for the differences in therapeutic response [23,45]. In accordance with the findings by Haider [10] we did not detect any significant effects of TNFα-inhibitors on total CD3+ T cells, B- cells or NK cells in vitro (data not shown). However, it was of interest that etanercept but not infliximab suppressed the expression of the activation marker CD69 on CD8 + CD3+ T-cells which might correlate to the decreased proliferative response and IFNγ production shown in the present study and the decrease of CD8+ T cell mediated antimicrobial activity against Mycobacterium tuberculosis reported in patients with RA and AS [57]. Even more pronounced was the reduction of CD69+ by anti-TNFα agents on NK-cells either in ‘naïve’ PBMC cultures or in PBMC cultures co-stimulated with BCG or TT known to activate NK-cells either directly or via T cell cytokines [37,58]. An increase of NK-cells has been observed in inflamed joints and synovial fluid of RA patients as well as in psoriatic skin lesions indicating that these cells may be involved in the pathogenetic or inflammatory response [59,60]. NK cells constitutively express membrane TNFα [61] and could be, therefore, another therapeutic target for anti-TNFα agents. On the other hand, the decrease of activated NK-cells may be responsible for the enhancement of haematological tumors in patients treated with TNFα-inhibitors [62,63]. Of course, these preliminary observations data on the effect of TNFα-blockers on NK-cell activity have still to be backed up by further investigations, but they are in accordance with the previous observation that key effector molecules of T cells and/or NK cells (IL-1β, IL-8, granzyme B) are down regulated by infliximab [10]. Performing those in vitro studies, the question always arises whether the concentration of the drugs/agents used in vitro correspond to the

serum levels reached by therapy in vivo. In previous studies for etanercept plasma concentrations in the range of 2–5 μg/ml and for infliximab in the range of 10–100 μg/ml have been calculated [32–34,36], which are comparable to the concentrations used in our study resulting in immunological responses (0.1–10 μg/ml). In conclusion we have shown that TNFα-blocker may exert their therapeutic function not only by their binding of TNFα and modification of cell death as reported by other authors [3,20–22,45] but also by their direct interaction with immunocompetent cells, especially cytotoxic Th1 cells, regulatory T cells or NK cells probably via membrane bound TNFα, as already suggested by Haider et al [10]. This would also explain some of the differential effects of infliximab and etanercept in vivo and in vitro. In order to avoid for the systematic analysis of the in vitro effect of TNFα blocker the complex immunological alterations in patients with chronic inflammatory disorders, we selected for the present study healthy individuals. Of course, it will be now important to analyse the observed effects also with PBMC of patients with RA or CD before and during the course of treatment with TNFα-blocker. It will be also of interest to see whether distinct immunological reactions correlate with response to treatment. However, one has also to be aware, that a detailed analysis of antiinflammatory mechanisms of TNFα inhibitors is always hampered by the fact that the array of genes regulated by TNF in leukocytes is largely unknown [10]. Conflict of interest The authors declare no competing financial or commercial interests. Acknowledgements F.T. was supported by the Deutsche Forschungsgemeinschaft, Bonn Bad Godesberg (graduate school GRK 794). References [1] Popa C, Netea MG, van Riel PLCM, van der Meer JWM, Stalenhoef AFH. The role of TNF-α in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 2007;48:751–62. [2] Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther 2008;117:244–79. [3] Dinarello CA. Differences between anti-tumor necrosis factor-a monoclonal antibodies and soluble TNF receptors in host defense impairment. J Rheumatol 2005;32:40–7. [4] Reinhart K, Karzai W. Anti-tumor necrosis factor therapy in sepsis: update on clinical trials and lessons learned. Crit Care Med 2001;29(Suppl 7):121–5. [5] Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu Rev Immunol 2001;19:163–96. [6] Lew W, Bowcock AM, Krueger JG. Psoriasis vulgaris: cutaneous lymphoid tissue supports T-cell activation and 'Type 1' inflammatory gene expression. Trends Immunol 2004;25:295–305. [7] Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4 + CD25hi T-regulatory cells. Blood 2006;108:253–61. [8] Ehrenstein MR, Evans JG, Singh A, Moore S, Warnes G, Isenberg DA, et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFα therapy. J Exp Med 2004;200:277–85. [9] Tilg H, Moschen A, Kaser A. Mode of function of biological anti-TNF agents in the treatment of inflammatory bowel diseases. Expert Opin Biol Ther 2007;7:1051–9. [10] Haider AS, Cohen J, Fei J, Zaba LC, Cardinale I, Toyoko K, et al. Insights into gene modulation by therapeutic TNF and IFNg antibodies: TNF regulates IFNg production by T cells and TNF-regulated genes linked to psoriasis transcriptome. J Invest Dermatol 2008;128:655–66. [11] Moriconi F, Raddatz D, Ho NAH, Yeruva S, Didas J, Ramadori G. Quantitative gene expression of cytokines in peripheral blood leukocytes stimulated in vitro: modulation by the anti-tumor necrosis factor-alpha antibody infliximab and comparison with the mucosal cytokine expression in patients with ulcerative colitis. Transl Res 2007;150:223–32. [12] Scallon B, Cai A, Solowski N, Rosenberg A, Song X-Y, Shealy D, et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther 2002;301:418–26.

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