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Etanercept decreases synovial expression of tumour necrosis factor-α and lymphotoxin-α in rheumatoid arthritis P Neregård, A Krishnamurthy, S Revu, M Engström, E af Klint, AI Catrina
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Rheumatology Unit, Department of Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden
Objectives: Etanercept is an effective tumour necrosis factor (TNF)-α inhibitor drug with the unique ability to block not only TNF-α but also lymphotoxin (LT)-α, at least in vitro. We aimed to investigate the in vivo effect of etanercept on synovial expression of TNF-α and LT-α. Method: Synovial biopsies from 12 rheumatoid arthritis (RA) patients started on etanercept and 11 RA patients started on infliximab were obtained at baseline and 8 weeks after treatment initiation. Synovial expression of TNF-α and LT-α was evaluated by immunohistochemistry followed by computer-assisted image analysis. Differences between paired samples were analysed by the Wilcoxon test and between groups by the Mann–Whitney test. A p-value < 0.05 was considered statistically significant. Results: Six out of the 12 of the patients started on etanercept achieved an American College of Rheumatology (ACR)50 response. Macroscopic evaluation of the joints during arthroscopy revealed a significant decrease of local inflammation mainly in good ACR50 responders. Synovial expression of both LT-α and TNF-α decreased but the differences did not reach statistical significance at a group level. By contrast, a significant decrease in both LT-α and TNF-α was observed when only good ACR50 responders were analysed. Despite higher levels of baseline synovial TNF-α in the good responders, neither baseline LT-α nor TNF-α could predict clinical response after 8 weeks. A decreasing trend of the synovial levels of LT-α was also observed in good responders to infliximab, but the difference did not reach statistical significance. Conclusions: Etanercept treatment modulates the synovial expression of both TNF-α and LT-α in vivo, a mechanism that might partly explain its clinical efficacy in RA.
Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation with local synovial accumulation of inflammatory cytokines leading to destruction of cartilage and bone. Among these cytokines, tumour necrosis factor (TNF)-α has been proposed as one of the key synovial modulators in RA (1). As a consequence, several TNF-α antagonists have been developed and are currently available for clinical use. Of these drugs, infliximab, adalimumab, certolizumab, and golimumab are monoclonal antibodies against human TNF-α whereas etanercept is a recombinant TNF receptor-Fc fusion protein (2). All of these drugs were originally designed to block TNF-α, but differences in their mechanism of action might result from their different structures and abilities to bind different types of TNF-α molecules (3). In this regard it has long been speculated, for example, that etanercept, the only TNF-α antagonist able to bind
Petra Neregård and Anca Catrina, Rheumatology Unit, Department of Medicine, D2:01, Karolinska University Hospital and Institutet, Stockholm S-17176, Sweden. E-mail: [email protected]
and block both TNF-α and lymphotoxin (LT)-α, might give somewhat different clinical results. One case report suggested that a high level of synovial expression of LT-α might explain why the patient did not respond to initial infliximab therapy but had a good clinical response when etanercept was subsequently used (4). However, when non-responders and responders to infliximab therapy were compared in a follow-up study, no differences in baseline synovial LT-α expression were observed between groups (5). In addition, serum analysis of LT-α failed to identify significant differences in responders compared to non-responders to etanercept in an open-label study (6). However, to date, no studies have addressed the effects of etanercept on synovial expression of LT-α and TNF-α. In the current study we aimed to investigate the effect of etanercept treatment on synovial expression of both TNF-α and LT-α in relation to clinical response. To this end we conducted an open-label arthroscopy study in patients with RA starting with etanercept treatment or after failure of at least one disease-modifying anti-rheumatic drug (DMARD).
Accepted 12 August 2013 © 2014 Informa Healthcare on license from Scandinavian Rheumatology Research Foundation DOI: 10.3109/03009742.2013.834964
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Patients Twelve patients meeting the 1987 American College of Rheumatology (ACR) criteria for RA (7) and having active disease [a mean SE 28-joint Disease Activity Score (DAS28) of 5.6 0.3] were recruited for the study and started on etanercept (nine women and three men with a median age of 47 years, range 40–58). Additionally, 11 patients (eight women and three men with a median age of 58 years, range 25–74) with active RA (a mean SE DAS28 of 5.9 0.4) treated with infliximab were included as controls. Patients were followed up for 8 weeks and were on stable treatment with DMARDs. Non-steroidal anti-inflammatory drugs (NSAIDs) and prednisolone < 10 mg daily were allowed and were stable during the duration of the study. ACR (8) and European League Against Rheumatism (EULAR) response criteria (9) were used to determine the therapeutic response after 8 weeks of treatment. A patient was classified as responder when fulfilling at least ACR50 criteria. The ethics committee at Karolinska University Hospital (Stockholm, Sweden) approved all experiments on human tissue. Informed consent was obtained from all study subjects.
Arthroscopy and synovial biopsy Synovial biopsy samples were obtained by arthroscopy from all patients before and after 8 weeks of treatment with either etanercept or infliximab. The biopsies were taken from an area adjacent to the cartilage–pannus junction, snap frozen in liquid nitrogen, and maintained at –70 C until sectioned. Macroscopic evaluation of joint inflammation was performed by analysis of recorded arthroscopy photographs as described previously (10). In brief, hypertrophy was scored on a four-point scale as follows: 0 ¼ a thin and transparent synovial mass (SM), 1 ¼ minor thickening, 2 ¼ granulations, and 3 ¼ villi; vascularity was scored on a four-point scale: 0 ¼ thin and scattered vessels, 1 ¼ low vessel density, 2 ¼ intermediated vessel density, and 3 ¼ densely packed vessel; synovitis was scored on a four-point scale grading every visible aspect of synovitis (hypertrophy, vascularity, and hyperaemia) as assessed globally by the observer: 0 ¼ normal synovial membrane and white inactive fibrotic tissue, 1 ¼ low active synovitis, 2 ¼ intermediate active synovitis, and 3 ¼ highly active synovitis.
Tissue preparation and immunohistochemical analysis Cryostat sections (7 μm) were fixed in 2% formaldehyde and stored at –70 C until analysed. Immunohistochemistry was used to detect synovial LT-α and TNF-α using a mouse monoclonal anti-LT-α antibody (clone 359-81-11, ab21506, Abcam, USA) and a mouse monoclonal anti-human TNF-α (2C8, Nordic Biosite AB, Sweden). Isotype controls using mouse immunoglobulin (Ig)G1 (X 0931 from DAKO Cytomation, Denmark) were included.
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P Neregård et al
Stained synovial biopsy sections were evaluated semiquantitatively in random order by two independent observers (SR and PN) using a four-point scale (0 ¼ no positive cells, 1 ¼ occasional positive cells, 2 ¼ scattered positive cells and/or focal positive cells, and 3 ¼ extensive positive cells). The observers were unaware of patient identity. The synovial expression of each marker was evaluated by computer-assisted image analysis by a single observer (PN) unaware of the identity of each section. The results were expressed as the percentage of positive stained area relative to the total tissue area. Statistical analysis Differences between paired samples were analysed by the Wilcoxon test and differences between groups of patients by the Mann–Whitney test. A p-value < 0.05 was considered statistically significant. Results Clinical findings Following 8 weeks of treatment, 10 out of 12 patients treated with etanercept were EULAR responders, either moderate (5/10) or good (5/10) responders. Ten out of 12 etanercepttreated patients reached ACR20 response criteria, with only six out of 12 patients reaching at least ACR50 response. In the infliximab-treated group, nine out of 11 patients were EULAR responders, either moderate (6/9) or good (3/9) responders. Eight out of 11 infliximab-treated patients reached ACR20 criteria, with only five out of 11 patients reaching at least ACR50 response.
Arthroscopy findings Macroscopic evaluation of the joints during arthroscopy and scoring of local inflammation on video pictures revealed a significant decrease of local inflammation mainly in responders. Scoring according to our previously published protocol revealed a decrease in the hypertrophy score from a median of 2.75 (range 1–3) to a median of 1.75 (range 1–3), a decrease in the vascularity score from a median of 1.5 (range 1–2.5) to a median of 1 (range 0–3), a decrease in the synovitis score from a median of 2 (range 1–3) to 1.25 (range 0–3). (10).
Characterization of the synovial expression of LT-α and TNF-α LT-α was present in a majority of the biopsies obtained from the etanercept-treated patients (11 out of 12 biopsies obtained at baseline and eight out of 12 biopsies obtained after 8 weeks of treatment) with large variations between different patients (median 1.5%, range 0.02–57.9%,). LT-α was mainly expressed in macrophages of both lining and sublining layer and vessels (Figure 1). TNF-α expression
Etanercept decreases synovial LT-α and TNF-α
A
87
C
B
Figure 1. Brown diaminobenzidine immunoperoxidase staining showing expression of LT-α (A) in the lining layer, and (B) in endothelial and (C) mononuclear cells of the sublining layer. Original magnification 250. Inserts show close-up pictures.
LT-α before treatment LT-α after treatment TNF-α before treatment TNF-α after treatment
SQ score
IA score
Correlation coefficient
1 (1–2) 1 (1–2) 2 (1–3) 1 (1–2)
1.5 (0.3–9.9) 0.3 (0.2–7.3) 11.7 (2.7–19) 5.6 (1.5–10.8)
0.8 0.9 0.9 0.8
was generally higher than that of LT-α (median 11.7%, range 0.2–86.8%). The same pattern of expression was observed in the infliximab-treated group, with LT-α being present in a majority of the biopsies (10 out of 11 biopsies obtained at baseline and six out of 11 obtained after 8 weeks of treatment) and with large inter-individual variations.
Etanercept down-regulates synovial LT-α and TNF-α expression in good clinical responders Semi-quantitative and quantitative computer-assisted image analysis revealed a general decrease in the levels of both LT-α and TNF-α at a group level, without
reaching statistical significance (Table 1 and Figure 2). However, when patients were divided according to their clinical response into good responders (at least ACR50 response) and non-responders, we observed a significant decrease in the levels of both LT-α and TNF-α in the responders (Figure 3) but no changes in the nonresponders (Figure 4). Baseline TNF-α expression tended to be higher in the responders (median 18.9%, range 0.6–86.8) than the non-responders (median 9.9, range 0.2–16), without reaching statistical significance. No difference was observed for LT-α (median of 1.5%, range 0.3–14.7 in responders; median of 2.5%, range 0.2–11.1 in non-responders). Levels of expression of neither LT-α nor TNF-α at baseline could predict clinical response after
Synovial LT-α expression
Synovial TNF-α expression
A
B
% of positive stained area
60 % of positive stained area
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Table 1. Semi-quantitative (SQ) and computer-assisted image analysis (IA) results.
25 20 15 10 5 0 Before etanercept
After etanercept
90 85 80 75 25 20 15 10 5 0 Before etanercept
After etanercept
Figure 2. Graphs showing results from computer-assisted image analysis of synovial expression of (A) LT-α and (B) TNF-α in etanercept-treated RA patients.
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P Neregård et al A
B
C % of positive stained area
*
LT-a
Before etanercept
4 2 0
After etanercept
Before etanercept
E
F % of positive stained area
D
TNF-α
Before etanercept
After etanercept *
90 80 20 10 0
After etanercept
Before etanercept
After etanercept
Figure 3. Brown diaminobenzidine immunoperoxidase staining showing a significant decrease in synovial expression of LT-α (A) before treatment and (B) after treatment and of TNF-α (D) before treatment and (E) after treatment in a responder to etanercept therapy. Graphs show results from computerassisted image analysis of synovial (C) LT-α and (F) TNF-α expression in responders to etanercept therapy.
B
C % of positive stained area
A
LT-α
Before etanercept D
60 50 40 20 10 0
After etanercept
Before etanercept
After etanercept
Before etanercept
After etanercept
E F % of positive stained area
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16 14 12 10
TNF-α
Before etanercept
After etanercept
90 80 70 20 10 0
Figure 4. Brown diaminobenzidine immunoperoxidase staining showing no significant changes in synovial expression of LT-α (A) before treatment and (B) after treatment and of TNF-α (D) before treatment and (E) after treatment in a non-responder to etanercept therapy. Graphs show results from computer-assisted image analysis of synovial expression of (C) LT-α and (F) TNF-α in non-responders to etanercept therapy.
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Etanercept decreases synovial LT-α and TNF-α
A
B
89
Goodresponders % of positive stained area
C
LT-α
Before infliximab
40 20 0
After infliximab E
D
60
Before infliximab
After infliximab
Before infliximab
After infliximab
Non-responders % of positive stained area
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F
LT-α
Before Infliximab
After infliximab
80 60 40 20 0
Figure 5. Brown diaminobenzidine immunoperoxidase staining showing synovial expression of LT-α in a responder (A before treatment and B after treatment) and a non-responder (D before treatment and E after treatment) to infliximab treatment. Graphs show results from computer-assisted image analysis of synovial LT-α infliximab responders (C) and non-responders (F).
8 weeks. In contrast to the etanercept-treated patients, the decrease in synovial levels of LT-α (from a median of 13.2%, range 6.1–52.4 to a median of 5.7%, range 0.9–23.5) observed in the good responders to infliximab did not reach statistical significance (Figure 5).
Discussion This is the first study to investigate the effect of etanercept on synovial cytokine expression in relation to clinical response to therapy. We have demonstrated that etanercept decreases synovial expression of both TNF-α and LT-α and that this effect is restricted to clinical responders. Clinical response to etanercept could not be predicted by the differential synovial expression of these cytokines at baseline. Etanercept is the only TNF-α antagonist acting as a soluble receptor and able to block not only TNF-α but also LT-α, at least in vitro. High levels of expression of synovial LT-α have therefore been proposed as a possible mechanism to explain why certain patients might respond to etanercept while being resistant to treatment with TNF-α antagonists that block TNF-α exclusively, such as infliximab (4). However, this original speculation was dismissed in a subsequent study that demonstrated no significant differences in synovial baseline expression of
LT-α in responders and compared to non-responders to infliximab. We further confirm this by demonstrating similar baseline expression of synovial LT-α in both responders and non-responders to etanercept treatment. LT-α is as effective as TNF-α in promoting inflammatory changes in synovial-derived fibroblasts, and has been proposed as an important modulator of synovial inflammation in RA (11). In the current study, LT-α was found to be present in both lining and sublining layers in a majority of RA patients, with high interindividual variations, similar to what is described for other cytokines (12). Etanercept treatment resulted in significant down-regulation of both LT-α and TNF-α in good responders to therapy. A similar trend was also observed in the good responders to infliximab treatment, but without reaching statistical significance. Larger studies are needed to determine whether the observed reduction in LT-α levels is a drug-specific effect. Along with other studies, we previously demonstrated that treatment with both etanercept and infliximab decreases synovial cellularity in general, and the number of CD68positive macrophages in particular, through decreased cell recruitment and apoptosis induction (13–15). It is therefore possible that the decrease in the levels of synovial TNF-α and LT-α found in the current study might reflect this cell number reduction. However, normalization of the synovial TNF-α and LT-α levels to the total cell surface area (an indirect measurement of the cell numbers) in the computer-
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assisted image analysis also suggests a direct reduction in the synovial cytokine levels. Previously it was suggested that baseline levels of synovial TNF-α might predict a good response to TNF-α antagonists (16, 17), a finding that was not confirmed in another study (18). In our study we observed a tendency towards higher baseline levels of synovial TNF-α expression in responders compared to nonresponders to etanercept, but the difference was not statistically significant. In conclusion, we have demonstrated that etanercept treatment modulates in vivo synovial expression of both TNF-α and LT-α, an additional mechanism that might explain the clinical efficacy observed with this drug in clinical practice.
Acknowledgements This research has received funding from the Initial Training Networks 7th Framework Programme Osteoimmune (289150), the European 7th Framework Programme (FP7/2007-2013) Euro-TEAM (305549), the Innovative Medicines Initiative ‘Be The Cure’ (115142-2), and the Swedish Research Council.
References 1. Feldmann M, Brennan FM, Foxwell BM, Maini RN. The role of TNF alpha and IL-1 in rheumatoid arthritis. Curr Dir Autoimmun 2001;3:188–99. 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. Makrygiannakis D, Catrina AI. Apoptosis as a mechanism of action of tumor necrosis factor antagonists in rheumatoid arthritis. J Rheumatol 2012;39:679–85. 4. Buch MH, Conaghan PG, Quinn MA, Bingham SJ, Veale D, Emery P. True infliximab resistance in rheumatoid arthritis: a role for lymphotoxin alpha? Ann Rheum Dis 2004;63:1344–6. 5. Buch MH, Bingham SJ, Bejarano V, Bryer D, White J, Emery P, et al. Therapy of patients with rheumatoid arthritis: outcome of infliximab failures switched to etanercept. Arthritis Care Res 2007;57:448–53. 6. Bingham CO 3rd, Ince A, Haraoui B, Keystone EC, Chon Y, Baumgartner S. Effectiveness and safety of etanercept in subjects with RA who have failed infliximab therapy: 16-week, open-label, observational study. Curr Med Res Opin 2009;25:1131–42.
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P Neregård et al 7. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 8. Felson DT, Anderson JJ, Boers M, Bombardier C, Furst D, Goldsmith C, et al. American College of Rheumatology. Preliminary definition of improvement in rheumatoid arthritis. Arthritis Rheum 1995;38:727–35. 9. van Gestel AM, Prevoo ML, van ‘t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL. Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis. Comparison with the preliminary American College of Rheumatology and the World Health Organization/International League Against Rheumatism Criteria. Arthritis Rheum 1996;39: 34–40. 10. af Klint E, Catrina AI, Matt P, Neregrad P, Lampa J, Ulfgren AK, et al. Evaluation of arthroscopy and macroscopic scoring. Arthritis Res Ther 2009;11:R81. 11. Calmon-Hamaty F, Combe B, Hahne M, Morel J. Lymphotoxin α revisited: general features and implications in rheumatoid arthritis. Arthritis Res Ther 2011;13:232. 12. Ulfgren AK, Grondal L, Lindblad S, Khademi M, Johnell O, Klareskog L, et al. Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment. Ann Rheum Dis 2000;59:439–47. 13. Smeets TJ, Kraan MC, van Loon ME, Tak PP. Tumor necrosis factor alpha blockade reduces the synovial cell infiltrate early after initiation of treatment, but apparently not by induction of apoptosis in synovial tissue. Arthritis Rheum 2003;48:2155–62. 14. Catrina AI, Trollmo C, af Klint E, Engstrom M, Lampa J, Hermansson Y, et al. Evidence that anti-tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages, but not lymphocytes, in rheumatoid arthritis joints: extended report. Arthritis Rheum 2005;52:61–72. 15. Suzuki Y, Inoue K, Chiba J, Inoue Y, Kanbe K. Histological analysis of synovium by treatment of etanercept for rheumatoid arthritis. Int J Rheum Dis 2009;12:7-13. 16. Wijbrandts CA, Dijkgraaf MG, Kraan MC, Vinkenoog M, Smeets TJ, Dinant H, et al. The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium. Ann Rheum Dis 2008;67:1139–44. 17. Ulfgren AK, Andersson U, Engstrom M, Klareskog L, Maini RN, Taylor PC. Systemic anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha synthesis. Arthritis Rheum 2000;43:2391–6. 18. Buch MH, Reece RJ, Quinn MA, English A, Cunnane G, Henshaw K, et al. The value of synovial cytokine expression in predicting the clinical response to TNF antagonist therapy (infliximab). Rheumatology (Oxford) 2008;47:1469–75.
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Etanercept decreases synovial expression of tumour necrosis factor-α and lymphotoxin-α in rheumatoid arthritis P Neregård, A Krishnamurthy, S Revu, M Engström, E af Klint, AI Catrina
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Rheumatology Unit, Department of Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden
Objectives: Etanercept is an effective tumour necrosis factor (TNF)-α inhibitor drug with the unique ability to block not only TNF-α but also lymphotoxin (LT)-α, at least in vitro. We aimed to investigate the in vivo effect of etanercept on synovial expression of TNF-α and LT-α. Method: Synovial biopsies from 12 rheumatoid arthritis (RA) patients started on etanercept and 11 RA patients started on infliximab were obtained at baseline and 8 weeks after treatment initiation. Synovial expression of TNF-α and LT-α was evaluated by immunohistochemistry followed by computer-assisted image analysis. Differences between paired samples were analysed by the Wilcoxon test and between groups by the Mann–Whitney test. A p-value < 0.05 was considered statistically significant. Results: Six out of the 12 of the patients started on etanercept achieved an American College of Rheumatology (ACR)50 response. Macroscopic evaluation of the joints during arthroscopy revealed a significant decrease of local inflammation mainly in good ACR50 responders. Synovial expression of both LT-α and TNF-α decreased but the differences did not reach statistical significance at a group level. By contrast, a significant decrease in both LT-α and TNF-α was observed when only good ACR50 responders were analysed. Despite higher levels of baseline synovial TNF-α in the good responders, neither baseline LT-α nor TNF-α could predict clinical response after 8 weeks. A decreasing trend of the synovial levels of LT-α was also observed in good responders to infliximab, but the difference did not reach statistical significance. Conclusions: Etanercept treatment modulates the synovial expression of both TNF-α and LT-α in vivo, a mechanism that might partly explain its clinical efficacy in RA.
Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation with local synovial accumulation of inflammatory cytokines leading to destruction of cartilage and bone. Among these cytokines, tumour necrosis factor (TNF)-α has been proposed as one of the key synovial modulators in RA (1). As a consequence, several TNF-α antagonists have been developed and are currently available for clinical use. Of these drugs, infliximab, adalimumab, certolizumab, and golimumab are monoclonal antibodies against human TNF-α whereas etanercept is a recombinant TNF receptor-Fc fusion protein (2). All of these drugs were originally designed to block TNF-α, but differences in their mechanism of action might result from their different structures and abilities to bind different types of TNF-α molecules (3). In this regard it has long been speculated, for example, that etanercept, the only TNF-α antagonist able to bind
Petra Neregård and Anca Catrina, Rheumatology Unit, Department of Medicine, D2:01, Karolinska University Hospital and Institutet, Stockholm S-17176, Sweden. E-mail: [email protected]
and block both TNF-α and lymphotoxin (LT)-α, might give somewhat different clinical results. One case report suggested that a high level of synovial expression of LT-α might explain why the patient did not respond to initial infliximab therapy but had a good clinical response when etanercept was subsequently used (4). However, when non-responders and responders to infliximab therapy were compared in a follow-up study, no differences in baseline synovial LT-α expression were observed between groups (5). In addition, serum analysis of LT-α failed to identify significant differences in responders compared to non-responders to etanercept in an open-label study (6). However, to date, no studies have addressed the effects of etanercept on synovial expression of LT-α and TNF-α. In the current study we aimed to investigate the effect of etanercept treatment on synovial expression of both TNF-α and LT-α in relation to clinical response. To this end we conducted an open-label arthroscopy study in patients with RA starting with etanercept treatment or after failure of at least one disease-modifying anti-rheumatic drug (DMARD).
Accepted 12 August 2013 © 2014 Informa Healthcare on license from Scandinavian Rheumatology Research Foundation DOI: 10.3109/03009742.2013.834964
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Method
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Patients Twelve patients meeting the 1987 American College of Rheumatology (ACR) criteria for RA (7) and having active disease [a mean SE 28-joint Disease Activity Score (DAS28) of 5.6 0.3] were recruited for the study and started on etanercept (nine women and three men with a median age of 47 years, range 40–58). Additionally, 11 patients (eight women and three men with a median age of 58 years, range 25–74) with active RA (a mean SE DAS28 of 5.9 0.4) treated with infliximab were included as controls. Patients were followed up for 8 weeks and were on stable treatment with DMARDs. Non-steroidal anti-inflammatory drugs (NSAIDs) and prednisolone < 10 mg daily were allowed and were stable during the duration of the study. ACR (8) and European League Against Rheumatism (EULAR) response criteria (9) were used to determine the therapeutic response after 8 weeks of treatment. A patient was classified as responder when fulfilling at least ACR50 criteria. The ethics committee at Karolinska University Hospital (Stockholm, Sweden) approved all experiments on human tissue. Informed consent was obtained from all study subjects.
Arthroscopy and synovial biopsy Synovial biopsy samples were obtained by arthroscopy from all patients before and after 8 weeks of treatment with either etanercept or infliximab. The biopsies were taken from an area adjacent to the cartilage–pannus junction, snap frozen in liquid nitrogen, and maintained at –70 C until sectioned. Macroscopic evaluation of joint inflammation was performed by analysis of recorded arthroscopy photographs as described previously (10). In brief, hypertrophy was scored on a four-point scale as follows: 0 ¼ a thin and transparent synovial mass (SM), 1 ¼ minor thickening, 2 ¼ granulations, and 3 ¼ villi; vascularity was scored on a four-point scale: 0 ¼ thin and scattered vessels, 1 ¼ low vessel density, 2 ¼ intermediated vessel density, and 3 ¼ densely packed vessel; synovitis was scored on a four-point scale grading every visible aspect of synovitis (hypertrophy, vascularity, and hyperaemia) as assessed globally by the observer: 0 ¼ normal synovial membrane and white inactive fibrotic tissue, 1 ¼ low active synovitis, 2 ¼ intermediate active synovitis, and 3 ¼ highly active synovitis.
Tissue preparation and immunohistochemical analysis Cryostat sections (7 μm) were fixed in 2% formaldehyde and stored at –70 C until analysed. Immunohistochemistry was used to detect synovial LT-α and TNF-α using a mouse monoclonal anti-LT-α antibody (clone 359-81-11, ab21506, Abcam, USA) and a mouse monoclonal anti-human TNF-α (2C8, Nordic Biosite AB, Sweden). Isotype controls using mouse immunoglobulin (Ig)G1 (X 0931 from DAKO Cytomation, Denmark) were included.
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P Neregård et al
Stained synovial biopsy sections were evaluated semiquantitatively in random order by two independent observers (SR and PN) using a four-point scale (0 ¼ no positive cells, 1 ¼ occasional positive cells, 2 ¼ scattered positive cells and/or focal positive cells, and 3 ¼ extensive positive cells). The observers were unaware of patient identity. The synovial expression of each marker was evaluated by computer-assisted image analysis by a single observer (PN) unaware of the identity of each section. The results were expressed as the percentage of positive stained area relative to the total tissue area. Statistical analysis Differences between paired samples were analysed by the Wilcoxon test and differences between groups of patients by the Mann–Whitney test. A p-value < 0.05 was considered statistically significant. Results Clinical findings Following 8 weeks of treatment, 10 out of 12 patients treated with etanercept were EULAR responders, either moderate (5/10) or good (5/10) responders. Ten out of 12 etanercepttreated patients reached ACR20 response criteria, with only six out of 12 patients reaching at least ACR50 response. In the infliximab-treated group, nine out of 11 patients were EULAR responders, either moderate (6/9) or good (3/9) responders. Eight out of 11 infliximab-treated patients reached ACR20 criteria, with only five out of 11 patients reaching at least ACR50 response.
Arthroscopy findings Macroscopic evaluation of the joints during arthroscopy and scoring of local inflammation on video pictures revealed a significant decrease of local inflammation mainly in responders. Scoring according to our previously published protocol revealed a decrease in the hypertrophy score from a median of 2.75 (range 1–3) to a median of 1.75 (range 1–3), a decrease in the vascularity score from a median of 1.5 (range 1–2.5) to a median of 1 (range 0–3), a decrease in the synovitis score from a median of 2 (range 1–3) to 1.25 (range 0–3). (10).
Characterization of the synovial expression of LT-α and TNF-α LT-α was present in a majority of the biopsies obtained from the etanercept-treated patients (11 out of 12 biopsies obtained at baseline and eight out of 12 biopsies obtained after 8 weeks of treatment) with large variations between different patients (median 1.5%, range 0.02–57.9%,). LT-α was mainly expressed in macrophages of both lining and sublining layer and vessels (Figure 1). TNF-α expression
Etanercept decreases synovial LT-α and TNF-α
A
87
C
B
Figure 1. Brown diaminobenzidine immunoperoxidase staining showing expression of LT-α (A) in the lining layer, and (B) in endothelial and (C) mononuclear cells of the sublining layer. Original magnification 250. Inserts show close-up pictures.
LT-α before treatment LT-α after treatment TNF-α before treatment TNF-α after treatment
SQ score
IA score
Correlation coefficient
1 (1–2) 1 (1–2) 2 (1–3) 1 (1–2)
1.5 (0.3–9.9) 0.3 (0.2–7.3) 11.7 (2.7–19) 5.6 (1.5–10.8)
0.8 0.9 0.9 0.8
was generally higher than that of LT-α (median 11.7%, range 0.2–86.8%). The same pattern of expression was observed in the infliximab-treated group, with LT-α being present in a majority of the biopsies (10 out of 11 biopsies obtained at baseline and six out of 11 obtained after 8 weeks of treatment) and with large inter-individual variations.
Etanercept down-regulates synovial LT-α and TNF-α expression in good clinical responders Semi-quantitative and quantitative computer-assisted image analysis revealed a general decrease in the levels of both LT-α and TNF-α at a group level, without
reaching statistical significance (Table 1 and Figure 2). However, when patients were divided according to their clinical response into good responders (at least ACR50 response) and non-responders, we observed a significant decrease in the levels of both LT-α and TNF-α in the responders (Figure 3) but no changes in the nonresponders (Figure 4). Baseline TNF-α expression tended to be higher in the responders (median 18.9%, range 0.6–86.8) than the non-responders (median 9.9, range 0.2–16), without reaching statistical significance. No difference was observed for LT-α (median of 1.5%, range 0.3–14.7 in responders; median of 2.5%, range 0.2–11.1 in non-responders). Levels of expression of neither LT-α nor TNF-α at baseline could predict clinical response after
Synovial LT-α expression
Synovial TNF-α expression
A
B
% of positive stained area
60 % of positive stained area
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Table 1. Semi-quantitative (SQ) and computer-assisted image analysis (IA) results.
25 20 15 10 5 0 Before etanercept
After etanercept
90 85 80 75 25 20 15 10 5 0 Before etanercept
After etanercept
Figure 2. Graphs showing results from computer-assisted image analysis of synovial expression of (A) LT-α and (B) TNF-α in etanercept-treated RA patients.
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P Neregård et al A
B
C % of positive stained area
*
LT-a
Before etanercept
4 2 0
After etanercept
Before etanercept
E
F % of positive stained area
D
TNF-α
Before etanercept
After etanercept *
90 80 20 10 0
After etanercept
Before etanercept
After etanercept
Figure 3. Brown diaminobenzidine immunoperoxidase staining showing a significant decrease in synovial expression of LT-α (A) before treatment and (B) after treatment and of TNF-α (D) before treatment and (E) after treatment in a responder to etanercept therapy. Graphs show results from computerassisted image analysis of synovial (C) LT-α and (F) TNF-α expression in responders to etanercept therapy.
B
C % of positive stained area
A
LT-α
Before etanercept D
60 50 40 20 10 0
After etanercept
Before etanercept
After etanercept
Before etanercept
After etanercept
E F % of positive stained area
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16 14 12 10
TNF-α
Before etanercept
After etanercept
90 80 70 20 10 0
Figure 4. Brown diaminobenzidine immunoperoxidase staining showing no significant changes in synovial expression of LT-α (A) before treatment and (B) after treatment and of TNF-α (D) before treatment and (E) after treatment in a non-responder to etanercept therapy. Graphs show results from computer-assisted image analysis of synovial expression of (C) LT-α and (F) TNF-α in non-responders to etanercept therapy.
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Etanercept decreases synovial LT-α and TNF-α
A
B
89
Goodresponders % of positive stained area
C
LT-α
Before infliximab
40 20 0
After infliximab E
D
60
Before infliximab
After infliximab
Before infliximab
After infliximab
Non-responders % of positive stained area
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F
LT-α
Before Infliximab
After infliximab
80 60 40 20 0
Figure 5. Brown diaminobenzidine immunoperoxidase staining showing synovial expression of LT-α in a responder (A before treatment and B after treatment) and a non-responder (D before treatment and E after treatment) to infliximab treatment. Graphs show results from computer-assisted image analysis of synovial LT-α infliximab responders (C) and non-responders (F).
8 weeks. In contrast to the etanercept-treated patients, the decrease in synovial levels of LT-α (from a median of 13.2%, range 6.1–52.4 to a median of 5.7%, range 0.9–23.5) observed in the good responders to infliximab did not reach statistical significance (Figure 5).
Discussion This is the first study to investigate the effect of etanercept on synovial cytokine expression in relation to clinical response to therapy. We have demonstrated that etanercept decreases synovial expression of both TNF-α and LT-α and that this effect is restricted to clinical responders. Clinical response to etanercept could not be predicted by the differential synovial expression of these cytokines at baseline. Etanercept is the only TNF-α antagonist acting as a soluble receptor and able to block not only TNF-α but also LT-α, at least in vitro. High levels of expression of synovial LT-α have therefore been proposed as a possible mechanism to explain why certain patients might respond to etanercept while being resistant to treatment with TNF-α antagonists that block TNF-α exclusively, such as infliximab (4). However, this original speculation was dismissed in a subsequent study that demonstrated no significant differences in synovial baseline expression of
LT-α in responders and compared to non-responders to infliximab. We further confirm this by demonstrating similar baseline expression of synovial LT-α in both responders and non-responders to etanercept treatment. LT-α is as effective as TNF-α in promoting inflammatory changes in synovial-derived fibroblasts, and has been proposed as an important modulator of synovial inflammation in RA (11). In the current study, LT-α was found to be present in both lining and sublining layers in a majority of RA patients, with high interindividual variations, similar to what is described for other cytokines (12). Etanercept treatment resulted in significant down-regulation of both LT-α and TNF-α in good responders to therapy. A similar trend was also observed in the good responders to infliximab treatment, but without reaching statistical significance. Larger studies are needed to determine whether the observed reduction in LT-α levels is a drug-specific effect. Along with other studies, we previously demonstrated that treatment with both etanercept and infliximab decreases synovial cellularity in general, and the number of CD68positive macrophages in particular, through decreased cell recruitment and apoptosis induction (13–15). It is therefore possible that the decrease in the levels of synovial TNF-α and LT-α found in the current study might reflect this cell number reduction. However, normalization of the synovial TNF-α and LT-α levels to the total cell surface area (an indirect measurement of the cell numbers) in the computer-
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assisted image analysis also suggests a direct reduction in the synovial cytokine levels. Previously it was suggested that baseline levels of synovial TNF-α might predict a good response to TNF-α antagonists (16, 17), a finding that was not confirmed in another study (18). In our study we observed a tendency towards higher baseline levels of synovial TNF-α expression in responders compared to nonresponders to etanercept, but the difference was not statistically significant. In conclusion, we have demonstrated that etanercept treatment modulates in vivo synovial expression of both TNF-α and LT-α, an additional mechanism that might explain the clinical efficacy observed with this drug in clinical practice.
Acknowledgements This research has received funding from the Initial Training Networks 7th Framework Programme Osteoimmune (289150), the European 7th Framework Programme (FP7/2007-2013) Euro-TEAM (305549), the Innovative Medicines Initiative ‘Be The Cure’ (115142-2), and the Swedish Research Council.
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