Long-term treatment with etanercept significantly reduces the number of proinflammatory cytokine-secreting peripheral blood mononuclear cells in patients with rheumatoid arthritis

H. Schotte, B. Schlüter1, P. Willeke, E. Mickholz, M. A. Schorat, W. Domschke and M. Gaubitz

Medizinische Klinik und Poliklinik B and 1 Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Münster, Germany.

Correspondence to: H. Schotte, Department of Medicine B, Münster University Hospital, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany. E-mail: h.schotte{at}uni-muenster.de


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To determine the influence of etanercept treatment on the number of peripheral blood mononuclear cells (PBMC) secreting immunoregulatory key cytokines and the correlation of these cell counts with treatment response in patients with rheumatoid arthritis (RA).

Methods. Nineteen patients with RA were treated with etanercept as monotherapy. Frequencies of PBMC secreting cytokines were determined by ELISPOT analysis before and after 9 months of therapy and compared with values for healthy controls (HC). The clinical outcome was assessed as defined by the ACR criteria.

Results. Fifteen patients fulfilled the ACR20, seven patients the ACR50 and two patients the ACR70 criteria. Initially elevated numbers of tumour necrosis factor-{alpha}- and interleukin (IL)-1ß-secreting PBMC were reduced to HC levels, and normal or low numbers of IL-6- and interferon-{gamma} (IFN-{gamma})-secreting PBMC were reduced below HC levels. The number of IL-10-secreting PBMC did not differ from that in HC and did not change significantly over time. The pretreatment IFN-{gamma}:IL-10 ratio correlated to reduction in the tender and swollen joint counts.

Conclusions. Long-term treatment with etanercept in patients with RA significantly reduces the numbers of proinflammatory cytokine-secreting PBMC, while the number of IL-10-secreting cells is unaffected. Although the changes described did not affect the safety or efficacy of etanercept therapy, these alterations may account for the long-term systemic effects. The pretreatment IFN-{gamma}:IL-10 ratio may be of prognostic value in predicting the improvement in joint symptoms.

KEY WORDS: Rheumatoid arthritis, Etanercept, Peripheral blood mononuclear cells, ELISPOT, Tumour necrosis factor-{alpha}, Interleukin-1, Interleukin-6, Interleukin-10, Interferon-{gamma}


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Etanercept is a TNF-blocking agent that is effective in the treatment of rheumatoid arthritis (RA) [1]. It reduces the tender and swollen joint counts as well as systemic markers of inflammation. The clinical use of TNF-blocking agents in RA is based on the in vitro finding that TNF-{alpha} is a dominant regulator of the activated inflammatory cascade [2, 3].

Although RA primarily affects the joints, the immunological alterations are by no means restricted to this compartment, but occur systemically. In this respect, elevated levels of pro- and anti-inflammatory cytokines in the peripheral blood of patients with RA have been reported repeatedly [4–8]. Patients with new-onset synovitis exhibited increased numbers of peripheral blood mononuclear cells (PBMC) secreting the Th1 cytokines interleukin (IL)-2 and interferon-{gamma} (IFN-{gamma}) [9]. Otherwise, significantly increased numbers of TNF-{alpha}-secreting PBMC were observed in patients with chronic RA [9]. These data suggest that different cytokine patterns prevail at different disease stages.

The clinical efficacy of etanercept has led to its widespread use in RA today. However, a significant proportion of patients does not respond adequately. Although safety data up to 6 yr do not provide evidence of an increased risk of malignancies, severe infections or autoimmune disorders, this cannot be fully excluded in the long run [10–13]. Thus, the immunomodulatory effects of etanercept in vivo are of interest.

In the present cohort study, we determined frequencies of cytokine-secreting PBMC from RA patients by enzyme-linked immunospot (ELISPOT) analysis before and after 9 months of treatment with etanercept and correlated the results with the clinical outcome. This may improve our understanding of systemic consequences for the immune system and help us to evaluate prognostic parameters for the individual patient's outcome.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The study protocol was approved by the local independent ethics committee (Ethikkommission der Ärztekammer Westfalen-Lippe). After having given written informed consent according to the Declaration of Helsinki, 19 patients with active RA [14] meeting the 1987 American Rheumatism Association (ARA) criteria [15] entered the present study. Twelve patients were women, seven were men, their median age being 50 yr (range 25–66). Median disease duration was 5 yr (range 1–13). The patients were pretreated with one to six (median 3) disease-modifying anti-rheumatic drugs (DMARDs), and had failed at least one of them. DMARDs were discontinued 4 weeks before the start of etanercept treatment. Patients were permitted to continue taking low-dose oral corticosteroids (median 8.75 mg, range 2.5–20, prednisolone equivalent) and/or non-steroidal anti-inflammatory drugs at a stable dosage. As controls, 19 healthy, unrelated, age- and sex-matched volunteers were recruited from the local laboratory staff after exclusion of acute or chronic inflammation.

In the first 3 months the patients received in a randomized, double-blind, placebo-controlled manner either placebo or etanercept at 10, 20, 25 or 50 mg a week subcutaneously. After 3 months, all patients were continued on 25 mg etanercept twice a week for further 6 months. Clinical evaluations were performed by one blinded investigator (HS). Clinical response was assessed as defined by the ACR preliminary criteria for improvement in RA [16], using a 68/66-joint count.

Numbers of cytokine-secreting PBMC were determined by ELISPOT analysis [17, 18]. In brief, isolated PBMC were resuspended in supplemented RPMI 1640. Twofold serial dilutions starting with 100 000 cells/well were incubated for 18 h in nitrocellulose-backed 96-well microtitre plates (Millipore, Bedford, MA, USA). The plates had been precoated overnight with antibodies directed against TNF-{alpha} (2TNF-H34A), IL-1ß (ILB1-H67), IL-6 (5IL6), IL-10 (9D7) and IFN-{gamma} (2G1; Endogen, Cambridge, MA, USA). All assays were performed simultaneously with and without 20 µg/ml of phytohaemagglutinin (PHA) (Sigma, St Louis, MO, USA). As the number of cells spontaneously secreting IFN-{gamma} was negligible, cell counts for this cytokine are presented only under stimulation.

The wells were overlaid with the biotinylated secondary antibodies 2TNF-H33 for TNF-{alpha}, ILB1-H6 for IL-1ß, 7IL6 for IL-6, 12G8 for IL-10 and B133.5 for IFN-{gamma} (Endogen). Alkaline phosphatase-labelled avidin (Sigma) was added and the ELISPOT was developed after addition of 5-bromo-4-chloro-3-indolyl phosphate mixed with nitroblue tetrazolium (Sigma).

Cytokine-secreting cells represented by individual spots on the plates were counted with the Scientific ELISPOT analysis system (Autoimmun Diagnostika, Strassberg, Germany) [19]. Means of four assays for unstimulated cells and six assays for the stimulated cells were calculated. Results are given as cytokine-secreting cells per 100 000 PBMC. The mean intra-assay imprecision (given as the coefficient of variation) for TNF-{alpha} was below 15% and for the other cytokines studied it was below 35%.

Statistical analysis
Data were analysed using the statistical software package SPSS for Windows, release 10.0.5 (SPSS, Chicago, IL, USA). Non-parametric methods were used throughout the report. Differences between unrelated groups were analysed with the Mann–Whitney U-test, whereas analyses for paired samples were performed with Wilcoxon's signed rank test. Bivariate correlations were tested using the Spearman correlation coefficient. P<0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical response
Within 9 months of therapy significant reductions in morning stiffness, tender and swollen joint counts, serum CRP levels and ESR were observed. Fifteen patients improved by 20%, seven by 50% and two by 70%, as defined by the ACR criteria. There were no significant changes in the white blood cell and lymphocyte counts from baseline to month 9 (data not shown).

Frequencies of cytokine-secreting cells
When RA patients before initiation of etanercept treatment were compared with healthy controls, significant differences in the frequencies of cytokine-secreting PBMC became evident. The pretreatment numbers of TNF-{alpha}- and IL-1ß-secreting PBMC were higher in RA patients, while no significant differences were observed with regard to IL-10. In contrast, under stimulation with PHA the frequencies of IL-6- and IFN-{gamma}-secreting PBMC were lower in RA patients, whereas no significant differences in the number of cells spontaneously secreting IL-6 could be detected (Table 1).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Frequencies of cytokine-secreting cells under etanercept treatment

 
After 9 months of treatment, the numbers of cells secreting proinflammatory cytokines were reduced to normal (TNF-{alpha}, IL-1ß) or even subnormal (IL-6 and IFN-{gamma}) levels. In contrast, the number of IL-10-secreting cells was not significantly different from pretreatment levels. Changes in the numbers of cytokine-secreting cells after 9 months did not depend on the etanercept dosage given in the first 3 months (data not shown).

Frequencies of cytokine-secreting cells and clinical characteristics
The number of IL-10-secreting PBMC before the start of therapy correlated inversely with the initial swollen joint count of the patients (r = –0.53; P<0.05, data not shown). No correlation of cytokine-secreting cells with other clinical characteristics was observed throughout the study period.

Pretreatment frequencies of cytokine-secreting cells and treatment response
No correlation of the initial frequencies of cytokine-secreting cells either with the course of individual clinical characteristics or with the response according to the ACR criteria was observed. However, a significant correlation of the pretreatment ratio of IFN-{gamma}:IL-10 secreting PBMC with the reduction from month 0 to month 9 in the tender joint count (r = 0.49; P<0.05) and swollen joint count (r = 0.52; P<0.05) became evident (Fig. 1). The pretreatment ratio of IFN-{gamma}:IL-10-secreting PBMC was significantly lower in RA patients (median 0.8, interquartile range 0.4–1.9) than in healthy controls (median 5.0, interquartile range 2.9–9.5; P<0.001) and did not change significantly over time.



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 1. Ratio of IFN-{gamma}- to IL-10-secreting PBMC and reduction in tender and swollen joints. The scatter plots show the correlation of the pretreatment ratio of IFN-{gamma}- to IL-10-secreting cells in RA patients with the reduction in tender and swollen joints from baseline to month 9 under etanercept treatment (r = 0.49 and r = 0.52 respectively; P<0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Etanercept therapy is effective in most patients with RA, but a significant proportion do not respond adequately. Though safety data for etanercept treatment up to 6 yr do not provide evidence of an increased risk of malignancies, autoimmune disorders or severe infections, long-term effects cannot be definitively excluded. Thus, a more detailed understanding of the immunomodulatory effects of etanercept in vivo is needed. For this purpose we investigated the frequencies of cytokine-secreting PBMC under treatment with etanercept.

Before the initiation of etanercept treatment, in patients with chronic, active RA we observed increased frequencies of spontaneously TNF-{alpha}- and IL-1ß-secreting PBMC compared with healthy controls. Our results corroborate and extend previous findings in chronic RA patients [9, 20]. Because TNF-{alpha} and IL-1ß are key proinflammatory cytokines, our data indicate systemic activation of the inflammatory cascade in RA patients. At first glance, our finding of an unaltered or even low frequency of IL-6-secreting PBMC seems to be contradictory to the well-documented elevation of IL-6 serum levels which has been shown to be related to disease activity [21]. However, the elevated IL-6 serum levels in RA may predominantly derive from other cellular sources, e.g. the inflamed synovium. The observation of decreased IFN-{gamma} production has previously been attributed to specific T-cell defects in RA [22].

In our study, long-term treatment with etanercept for 9 months led to a substantial clinical improvement in the RA patients. This effect was paralleled by a significant reduction in PBMC-secreting proinflammatory cytokines to healthy control levels or even below them, while the number of IL-10-secreting cells did not change significantly. Thus, our data provide evidence for profound systemic immunomodulation under long-term etanercept treatment in vivo. In contrast to our results, short-term treatment with etanercept was not associated with any alterations [20].

The systemic effects of etanercept may be the consequence of the crucial role of TNF-{alpha} in the complex cytokine network regulating immune and inflammatory responses. From in vitro studies it is known that TNF-{alpha} induces the secretion of IL-1 and IL-6 [23, 24], which are mediators of erosive cartilage and bone damage [25, 26]. Moreover, via IL-12, TNF-{alpha} induces the secretion of IFN-{gamma} [27], which is primarily produced by Th1 cells and augments both transcription and mRNA stability of proinflammatory cytokines in monocytes and macrophages [28]. IL-1, IL-6 and IFN-{gamma} in turn stimulate the release of TNF-{alpha}, thus perpetuating a vicious circle of chronic inflammation [29]. In contrast, IL-10, which is also induced by TNF-{alpha}, acts as a negative regulator of TNF-{alpha}, IL-1, IL-6 and IFN-{gamma} production [30]. Thus, the blockade of TNF-{alpha} by etanercept interrupts a positive feedback loop of TNF-{alpha} production, while a negative regulatory loop via IL-10 remains unaltered. This down-regulation of systemic inflammatory activity has important biological consequences. On the one hand, the dysregulated immune response in RA is suppressed, resulting in a substantial clinical improvement. On the other hand, these proinflammatory cytokines share important antiproliferative properties and are required for normal immune responses. Thus, withdrawal and down-regulation of these cytokines may be associated with tumour development, infection or autoimmune processes.

A further goal of the present investigation was to study the association of systemic cytokine secretion with the clinical characteristics of RA patients. Our finding of an inverse correlation of the frequency of IL-10 secreting PBMC with the swollen joint count suggests a potential protective role of constitutive high IL-10 secretion against joint swelling in RA. This hypothesis is supported by data of Verhoef et al. [31], which show a strong negative correlation between IL-10 production and radiographic joint damage as well as its progression.

Prognostic parameters for the outcome under etanercept treatment are of special interest. In patients with new-onset synovitis, a low Th1:Th2 cytokine ratio in the peripheral blood has been shown to be associated with a favourable DMARD response [32]. In contrast, we found that the pretreatment ratio of IFN-{gamma}:IL-10-secreting PBMC correlated positively with the reduction in tender and swollen joint counts. A possible explanation for this discrepancy are the different patterns of cytokine secretion in new-onset synovitis with a high IFN-{gamma}:IL-10 ratio, which inverts in chronic RA [9]. Our data concerning the IFN-{gamma}: IL-10 ratio suggest that patients with a predominant Th1 cytokine pattern experience the greatest benefit from etanercept treatment.

In summary, our results provide evidence that long-term treatment with etanercept substantially down-regulates the activated systemic proinflammatory cytokine cascade in RA. The ratio of IFN-{gamma}- to IL-10-secreting PBMC may in the future serve as a prognostic parameter for the clinical response to etanercept treatment.


    Acknowledgments
 
We wish to thank all patients who participated in the present study. Etanercept was kindly provided by Wyeth-Ayerst Research, Münster, Germany.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Moreland LW, Baumgartner SW, Schiff MH et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N Engl J Med 1997;337:141–7.[Abstract/Free Full Text]
  2. Butler DM, Maini RN, Feldmann M, Brennan FM. Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures. Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist. Eur Cytokine Netw 1995;6:225–30.[ISI][Medline]
  3. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440.[CrossRef][ISI][Medline]
  4. al Janadi M, al Balla S, al Dalaan A, Raziuddin S. Cytokine production by helper T cell populations from the synovial fluid and blood in patients with rheumatoid arthritis. J Rheumatol 1993;20:1647–53.[ISI][Medline]
  5. Tanabe M, Ochi T, Tomita T et al. Remarkable elevation of interleukin 6 and interleukin 8 levels in the bone marrow serum of patients with rheumatoid arthritis. J Rheumatol 1994;21:830–5.[ISI][Medline]
  6. Llorente L, Richaud-Patin Y, Fior R et al. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus. A potential mechanism of B lymphocyte hyperactivity and autoimmunity. Arthritis Rheum 1994;37:1647–55.[ISI][Medline]
  7. Schulze-Koops H, Lipsky PE, Kavanaugh AF, Davis LS. Elevated Th1- or Th0-like cytokine mRNA in peripheral circulation of patients with rheumatoid arthritis. Modulation by treatment with anti-ICAM-1 correlates with clinical benefit. J Immunol 1995;155:5029–37.[Abstract]
  8. Vazquez-Del Mercado M, Delgado-Rizo V, Munoz-Valle JF, Orozco-Alcala J, Volk HD, Armendariz-Borunda J. Expression of interleukin-1 beta, tumor necrosis factor alpha, interleukins-6, -10 and -4, and metalloproteases by freshly isolated mononuclear cells from early never-treated and non-acute treated rheumatoid arthritis patients. Clin Exp Rheumatol 1999;17:575–83.[ISI][Medline]
  9. Kanik KS, Hagiwara E, Yarboro CH, Schumacher HR, Wilder RL, Klinman DM. Distinct patterns of cytokine secretion characterize new onset synovitis versus chronic rheumatoid arthritis. J Rheumatol 1998;25:16–22.[ISI][Medline]
  10. Moreland LW, Cohen SB, Baumgartner SW et al. Long-term safety and efficacy of etanercept in patients with rheumatoid arthritis. J Rheumatol 2001;28:1238–44.[ISI][Medline]
  11. Robinson WH, Genovese MC, Moreland LW. Demyelinating and neurologic events reported in association with tumor necrosis factor alpha antagonism: by what mechanisms could tumor necrosis factor alpha antagonists improve rheumatoid arthritis but exacerbate multiple sclerosis? Arthritis Rheum 2001;44:1977–83.[CrossRef][ISI][Medline]
  12. Shakoor N, Michalska M, Harris CA, Block JA. Drug-induced systemic lupus erythematosus associated with etanercept therapy. Lancet 2002;359:579–80.[CrossRef][ISI][Medline]
  13. Brown SL, Greene MH, Gershon SK, Edwards ET, Braun MM. Tumor necrosis factor antagonist therapy and lymphoma development: twenty-six cases reported to the Food and Drug Administration. Arthritis Rheum 2002;46:3151–8.[CrossRef][ISI][Medline]
  14. Felson DT, Anderson JJ, Boers M et al. The American College of Rheumatology preliminary core set of disease activity measures for rheumatoid arthritis clinical trials. The Committee on Outcome Measures in Rheumatoid Arthritis Clinical Trials. Arthritis Rheum 1993;36:729–40.[ISI][Medline]
  15. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  16. Felson DT, Anderson JJ, Boers M et al. American College of Rheumatology. Preliminary definition of improvement in rheumatoid arthritis. Arthritis Rheum 1995;38:727–35.[ISI][Medline]
  17. Czerkinsky C, Andersson G, Ekre HP, Nilsson LA, Klareskog L, Ouchterlony O. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 1988;110:29–36.[CrossRef][ISI][Medline]
  18. Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 1983;65:109–21.[CrossRef][ISI][Medline]
  19. Vaquerano JE, Peng M, Chang JW, Zhou YM, Leong SP. Digital quantification of the enzyme-linked immunospot (ELISPOT). Biotechniques 1998;25:830–4, 836.[ISI][Medline]
  20. Berg L, Lampa J, Rogberg S, van Vollenhoven R, Klareskog L. Increased peripheral T cell reactivity to microbial antigens and collagen type II in rheumatoid arthritis after treatment with soluble TNFalpha receptors. Ann Rheum Dis 2001;60:133–9.[Abstract/Free Full Text]
  21. Houssiau FA, Devogelaer JP, Van Damme J, de Deuxchaisnes CN, Van Snick J. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1988;31:784–8.[ISI][Medline]
  22. Franchimont P, Reuter A, Vrindts-Gevaert Y et al. Production of tumour necrosis factor-alpha, interferon-gamma and interleukin-2 by peripheral blood mononuclear cells of subjects suffering from rheumatoid arthritis. Scand J Rheumatol 1988;17:203–12.[ISI][Medline]
  23. Dinarello CA, Cannon JG, Wolff SM et al. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med 1986;163:1433–50.[Abstract]
  24. Smith JW, Urba WJ, Curti BD et al. The toxic and hematologic effects of interleukin-1 alpha administered in a phase I trial to patients with advanced malignancies. J Clin Oncol 1992;10:1141–52.[Abstract]
  25. Saklatvala J, Sarsfield SJ, Townsend Y. Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever. J Exp Med 1985;162:1208–22.[Abstract]
  26. Ohshima S, Saeki Y, Mima T et al. Interleukin 6 plays a key role in the development of antigen-induced arthritis. Proc Natl Acad Sci USA 1998;95:8222–6.[Abstract/Free Full Text]
  27. Kitagawa M, Mitsui H, Nakamura H et al. Differential regulation of rheumatoid synovial cell interleukin-12 production by tumor necrosis factor alpha and CD40 signals. Arthritis Rheum 1999;42:1917–26.[CrossRef][ISI][Medline]
  28. Lee JY, Sullivan KE. Gamma interferon and lipopolysaccharide interact at the level of transcription to induce tumor necrosis factor alpha expression. Infect Immun 2001;69:2847–52.[Abstract/Free Full Text]
  29. Philip R, Epstein LB. Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, gamma-interferon and interleukin-1. Nature 1986;323:86–9.[ISI][Medline]
  30. Chernoff AE, Granowitz EV, Shapiro L et al. A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses. J Immunol 1995;154:5492–9.[Abstract/Free Full Text]
  31. Verhoef CM, van Roon JA, Vianen ME, Bijlsma JW, Lafeber FP. Interleukin 10 (IL-10), not IL-4 or interferon-gamma production, correlates with progression of joint destruction in rheumatoid arthritis. J Rheumatol 2001;28:1960–6.[ISI][Medline]
  32. van der Graaff WL, Prins AP, Dijkmans BA, van Lier RA. Prognostic value of Th1/Th2 ratio in rheumatoid arthritis. Lancet 1998;351:1931.[ISI][Medline]
Submitted 9 November 2003; revised version accepted 12 February 2004.