: K. Kanbe, Department of Orthopaedic Surgery, Tokyo Women's Medical University, Medical Center East, 2-1-10 Nishiogu, Arakawa, Tokyo 116-8567, Japan. Email: email@example.com
Aims: In order to investigate the histological change in effect attenuation cases of etanercept compared with methotrexate (MTX), we performed immunohistochemical examination by seven different molecules.
Methods: We histologically assessed synovial tissue from five MTX-treated rheumatoid arthritis (RA) patients as control and six etanercept and MTX-treated RA patients after synovectomy by arthroscopy. The synovium of both groups were assessed by hematoxylin and eosin (HE) and we also analysed the expression of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), B-cell precursors and mature B-cell transmembrane protein, CD20, macrophage marker, CD68, bromodeoxyuridine (BrdU) and vascular endothelial growth factor (VEGF) by immunohistochemistry.
Results: HE staining showed vascular and cell proliferations of the synovium of the RA patients who received etanercept compared with the control MTX group. TNF-α and IL-6 were expressed in both groups.MMP-3 and CD68 expressed less significantly in the etanercept group compared with the control (P < 0.05). CD20 strongly expressed in the etanercept group significantly (P < 0.05). BrdU expressed in the synovium in the etanercept group significantly (P < 0.05). VEGF was not found overall in both group.
Conclusion: Based on the histological change of synovium, treatment by etanercept may be involved in vascular and cell proliferations with inhibition of the expression of CD68 and MMP-3 in synovium of RA patients. These findings indicate immunohistochemical change of synovium with etanercept is one of the mechanism of efficacy of etanercept.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovitis and destruction of bone and cartilage in multiple joints.1 Although aetiological causes are still obscure, constitutive overproduction of tumour necrosis factor-α (TNF-α) or interleukin-6 (IL-6), a pleiotropic cytokine that regulates the immune response, inflammation, haematopoiesis, and bone metabolism, is thought to play a pathological role in RA.2,3 Overproduction of TNF-α or IL-6 augments the autoimmune reaction and causes systemic inflammatory manifestation. IL-6, synergistically with interleukin-1β (IL-1β) or TNF-α, induces the production of vascular endothelial growth factor (VEGF) – a potent inducer of angiogenesis necessary to oxygenate the hyperplastic synovial tissues in the affected joints.4 Recently, TNF-α therapy, such as etanercept (a fusion protein consisting of the extracellular ligand-binding domain of the 75-kDa receptor for TNF-α and the constant portion of human IgG1) is used in conjunction with methotrexate (MTX) to control inflammation and inhibit bone and joint destruction in RA patients.5 Even with TNF-α therapy, there are effect attenuation cases with joint swelling and tenderness with increasing C-reactive protein (CRP) concentrations. Lindberg etal.6 reported on the effect of infliximab on mRNA expression profiles in the synovial tissue of RA patients. They found that TNF-α expression decreased in non-responders of infliximab. However, the mechanisms of effect attenuation related to etanercept are unknown. To assess the mechanism of effect attenuation by etanercept therapy, we compared histological differences, including seven molecules of TNF-α, IL-6, MMP-3, CD20, CD68, bromodeoxyuridine (BrdU) and VEGF of synovial tissue obtained during arthroscopic synovectomy after the treatment of RA patients with etanercept at the time of effect attenuation.
Currently, several biologic therapies are being used to treat RA, such as adalimumab (a fully human monoclonal TNF-α antibody),7 tocilizumab (anti-IL-6 receptor),2,8 rituximab (anti-CD20),9 and abatacept (cytotoxic T-lymphocyte antigen 4 immunoglobulin).10 However, it is difficult to use applicable biologics to elucidate good response with continued efficacy for RA. On the basis of the hypothesis that the effect attenuation cases of TNF-α therapy induce different expression patterns of cytokines, we conducted a histological assessment of synovial tissue in RA patients treated with etanercept. This study presents the latest results of histological differences in synovial tissue of effect attenuation cases in response to etanercept.
MATERIALS AND METHODS
The six patients (1 man, 5 women) of effect attenuation cases of etanercept were treated with etanercept for an average of 8 months (range: 6–12) and had a mean age of 65.1 years (range: 57–76), with a mean duration of 11 years (range: 5–17). All etanercept-treated patients are initially responders but tolerated after treatment by etanercept. They received 25 mg of etanercept twice a week plus 5.4 mg/day of MTX (4–8 mg) and 5.5 mg/day of prednisolone (2.5–10 mg). According to Steinbrocker's criteria,11 two patients and four patients in the etanercept group were classified with class II and class III RA, respectively. Control samples were obtained from five RA patients who did not receive etanercept or other biologic therapy. These five control patients (1 man, 4 women) had an average age of 61.6 years (range: 59–78) and received 5.8 mg/week of MTX (4–8 mg) and 5.7 mg/day of prednisolone (2.5–10 mg) at the time of arthroscopic synovectomy. According to Steinbrocker criteria,11 one patients and four patients in the control group were classified with class II and class III RA, respectively. We selected the effect attenuation cases in two groups (C-reactive protein [CRP] > 3.0 mg/dL and disease activity score [DAS28] > 3.2) (Table 1). All patients who took part in this study were diagnosed according to the criteria of the American College of Rheumatology.12 All patients underwent general and physical examinations and routine blood and urinary analysis at baseline and at the time of arthroscopic surgery after the initial treatment. To evaluate disease activity, erythrocyte sedimentation rate (ESR), CRP, and DAS28 scores13 were measured.
Table 1. Patient backgrounds for comparison of histology of synovium in etanercept treatment
Serial paraffin sections of the synovium (2 µm) were stained with hematoxylin and eosin (HE). For immunohistochemical examination, the tissue sections were blocked for 10 min in phosphate-buffered saline containing 20% rabbit serum and then incubated overnight at 4°C with the following antibodies: anti-TNF-α mouse monoclonal antibody (1 : 1000; Biogenesis, Pool, UK) and anti-human IL-6 rabbit polyclonal antibody (Rockland Inc., Gilbertsville, PA, US), anti-human MMP-3 monoclonal antibody (1 : 100; Biogenesis, Poole, UK), anti-human CD20 monoclonal antibody (1 : 1000; DAKO, Glostrup, Denmark), anti-human CD68 monoclonal antibody (1 : 1000; DAKO), anti-BrdU mouse monoclonal antibody (1 : 500; Chemicon. Temecula, CA, US), and anti-human VEGF monoclonal antibody (1 : 800; Upstate Biotechnology, Lake Placid, NY, US). After treatment with the second antibody, we compared the expression pattern of TNF-α, IL-6, MMP-3, CD20, CD68, BrdU and VEGF with that of the control group. After treating with a second antibody at room temperature for 10 min, sections were then incubated for 10 min with appropriate Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, US), using 3,3′-diaminobenzidine-4HCL (DAB) (Sigma, St Louis, MO, US) for the colour reaction for 5 min, which resulted in brown staining of antigen-expressing cells. Microscopic evaluations were performed as described previously.14 The immunohistochemically stained samples were evaluated by estimating the number of positive staining cells in the 10 different areas of the synovium. Positivity was noted when complete staining of the cells was observed in all synovial samples at a magnification of ′200 by HE. Ten random readings per high-power field were recorded, and the results were expressed as a mean percentage (positive fields out of 10 random fields) with standard deviation (SD) in each of the 10 samples.
The Mann–Whitney U-test was used for continuous variables between two groups. The chi-square test was used for categorical variables between the two groups. P-value < 0.05 was considered significant.
Hematoxylin and eosin staining showed that the synovium of the RA patients who underwent etanercept therapy showed significantly greater vascular proliferation than that of the MTX control group (mean [SD] 15 [2.1]vs. 47 [7.2], respectively; P < 0.05) (Table 2) (Fig. 1a,b). TNF-α and IL-6 were expressed in both groups and there was no significant difference (Table 2) (Fig. 1c–f). The percentages of positive staining cells of MMP-3, CD20, CD68, and BrdU in synovium were different significantly in the etanercept group compared with the control (mean [SD] 32 [4.5], 73 [9.1], 15 [3.5], and 83 [7.8]vs. 67 [8.2], 2.5 [0.8], 81 [9.5], and 7.5 [1.7], respectively; P < 0.05) (Table 3). Therefore, the expression pattern of IL-6 and TNF-α in synovium of effect attenuation cases by etanercept were almost the same as controls. On the other hand, MMP-3 (Fig. 2a,b) and CD68 (Fig. 3a,b) were expressed less significantly in the etanercept group compared with the control (P < 0.05). CD20 strongly expressed in etanercept significantly (Fig. 2c,d) (P < 0.05). BrdU expressed all around cells in the synovium in the etanercept group significantly (Fig. 3c,d) (P < 0.05). VEGF was not found overall in either group (Fig. 3e,f). Therefore, histological changes in etanercept were found in those immmunohistochemical examinations.
Table 2. Comparison of histological finding of synovium in etanercept treatment
Hematoxylin and eosin
Results expressed as mean (SD) percentage of positive fields of staining cell numbers by immunohistochemistry; HV, hypervascularity; CP, cell proliferationTNF-α, tumor necrosis factor alpha; IL-6, interleukin – 6; *is significant difference (P < 0.05).
Effect attenuation cases in the etanercept group responded to the first treatment with MTX, which led to the discontinuation of treatment with biological agents. Treatment continuation after 12 months was reported to be similar in the etanercept (68.6%) group.15 In our institute, 21% of cases (out of 71) showed effect attenuation cases of etanercept. CRP has been shown to correlate well with IL-6 and MMP-3 in the sera of RA patients.16 Thus, high CRP concentrations may be induced by MMP-3 or IL-6. However, VEGF was not increased in the etanercept group compared with the control. Even in etanercept effect attenuation cases, VEGF expression was blocked, and the other mechanisms such as TNF-α or MMP-3 seemed to be responsible for hypervascularity of synovium.
It has been reported that an overall decrease in synovial cellularity after TNF is blocked is consistent with previous studies in response cases.17 We found that vascular proliferation as well as synovial infiltration in cases of effect attenuation with etanercept compared with the MTX control group. Therefore increasing vascularity induced inflammation, as reflected by the increase in CRP and DAS28. Interestingly CD68 and MMP-3 expression pattern were similar in terms of decreasing positive cells after using etanercept compared with thecontrol. This might indicate the production of MMP-3 from mononuclear cells with positive CD68 in synovial tissue may be blocked even in effect attenuation cases of etanercept. To confirm this, the double stains of MMP-3 and CD68 have to be performed in this study.
In the treatment of effect attenuation, there are choices to be made. Should treatment with the same biologics, such as steroids, be continued? Should the dose of MTX be increased? Should disease-modifying antirheumatic drugs be added to the treatment regimen? We previously reported that arthroscopic synovectomy is effective in ameliorating the effect attenuation associated with infliximab.18 The removal of synovium with abnormalities in TNF-α production in the etanercept group may decrease TNF-α concentrations in serum and joint fluid and result in a reduction in CRP concentrations.
The synovium of osteoarthritis patients produces significantly lower concentrations of several cytokines, including TNF-α and IL-6, than does the synovium of RA patients.14 RA synovium is considered to have an abnormal cytokine profile, which induces joint destruction. We previously reported serum matrix metalloproteinase activity relating to cartilage destruction in rheumatoid arthritis.19 In the present study, synovial tissues treated with etanercept decreased MMP-3 expression. These data mean etanercept was effective to prevent cartilage destruction even in effect attenuation cases. On the other hand, etanercept treatment resulted in no increase in TNF-α-positive cells in the synovium compared with control. Because etanercept is a fusion protein consisting of the extracellular ligand-binding domain to the receptor of TNF-α, it is possible that etanercept cannot block TNF-α expression in synovial cells. On the other hand, etanercept blocked CD68 expression to the same degree as did the control treatment. Therefore, the pattern of cytokine and cell marker expressions were different between the etanercept and MTX groups in effect attenuation cases. These results indicate that the effect attenuation cases of etanercept, which produced TNF-α, need to be blocked rather than another biologic, infliximab that decreases TNF-α production. It is possible to determine which biologics are the most advantageous based on TNF-α and IL-6 expression patterns, determined by immunohistochemistry. In this study we used seven different molecules to analyse the change of synovium after using anti-TNF therapy. If a different pattern is seen by immunohistochemistry, response or non-response can be predicted in etanercept for the future.