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Rheumatology Advance Access originally published online on July 26, 2008
Rheumatology 2008 47(10):1469-1475; doi:10.1093/rheumatology/ken261
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The value of synovial cytokine expression in predicting the clinical response to TNF antagonist therapy (infliximab)

M. H. Buch1, R. J. Reece1, M. A. Quinn1, A. English1, G. Cunnane1, K. Henshaw1, S. J. Bingham1, V. Bejarano1, J. Isaacs1 and P. Emery1

1Academic Unit of Musculoskeletal Disease, University of Leeds, Leeds, UK.

Correspondence to: P. Emery, Leeds Teaching Hospitals NHS Trust, Academic Unit of Musculoskeletal Disease, Second Floor, Chapel Allerton Hospital, Chapeltown Road, Leeds LS7 4SA, UK. E-mail: p.emery{at}leeds.ac.uk


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
 References
 
Objectives. Clinical response to TNF-{alpha} blockade in the treatment of RA is heterogeneous. The study aims were to determine whether pre-treatment synovial cytokine expression predicted infliximab response and whether synovial changes after therapy correlated with response.

Methods. Fifty-one patients had arthroscopic biopsies of the knee joint prior to infliximab (3 mg/kg) treatment. Synovial tissue cell numbers (CD68 and CD3 positive) and cytokine expression (TNF-{alpha}, lymphotoxin-{alpha}, IL-1{alpha}, -β and receptor antagonist, and IL-6) pre-treatment was assessed using semi-quantitative immunohistochemistry. Changes in these parameters were assessed 16 weeks after infliximab in 32 patients who underwent repeat arthroscopic biopsy.

Results. Of the total patients, 47% (n = 24) achieved an ACR20 response; 53% (n = 27) did not. Baseline synovial TNF-{alpha}, IL-1{alpha} and -β expression did not differ between the two groups. No differences in baseline TNF-{alpha} levels were observed with ACR levels of response (ACR20 and ACR50/70 groups). Post-treatment biopsies (17 ACR responders, 15 ACR non-responders) revealed significant reductions in sub-lining layer TNF-{alpha} expression in both response and non-response groups with significant reduction in vascularity and membrane proliferation scores. The worst ACR non-responders (<20% CRP suppression) demonstrated no reduction in any of the parameters.

Conclusion. Pre-treatment synovial TNF-{alpha} or IL-1 expression does not predict TNF blockade response. Both ACR response and non-response was associated with reduction in synovial TNF-{alpha}-level expression. Suppression in TNF-{alpha} levels was not observed in the worst non-responders. The improvements (including in vascularity), independent of ACR clinical response, are compatible with the reduced structural damage documented in all groups of patients independent of response.

KEY WORDS: Rheumatoid arthritis, Infliximab, Synovial biopsy, Tumour necrosis factor, Cytokine


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
 References
 
Pro-inflammatory cytokines, especially TNF-{alpha} and IL-1, are pivotal in the RA inflammatory cascade [1]. Initial in vitro [1, 2] and subsequent in vivo studies demonstrated a hierarchical role of TNF-{alpha} in RA; development of TNF antagonist therapies with monoclonal antibodies or soluble receptor antagonists have been shown to dramatically suppress signs and symptoms of inflammation and prevent progression of structural damage in virtually all patients [3–5].

Despite the general effectiveness of anti-TNF-{alpha} therapies, a lack of response is evident in up to 40% of the patients treated with these agents [5]. Few studies have investigated the underlying basis of anti-TNF-{alpha} resistance. Variable synovial cytokine expression [6, 7] and variable synovial tissue TNF-{alpha} levels with treatment have been observed [8, 9]; however, studies have generally comprised few patients and no clear explanation has emerged.

The principal aims of this study were to examine the heterogeneity of synovial cytokine (particularly TNF-{alpha}) expression, correlate this with the level of response to infliximab and observe whether non-response represented a subgroup of RA that was mediated independently of TNF-{alpha}. Our findings indicate that baseline cytokine expression is not predictive, and changes do not correlate with ACR response. Importantly, TNF-{alpha} expression was significantly reduced in both ACR response and non-response patients, consistent with structural data. However, complete failure to respond to TNF-blockade in the small minority may be associated with unchanged tissue TNF-{alpha} expression. These findings have implications both for the therapeutics of RA and for understanding disease mechanisms.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
 References
 
Patients
The study was approved by Leeds Research Ethics Committee. Informed consent was obtained from each patient prior to the study. All patients fulfilled the revised 1987 American College of Rheumatology (ACR) criteria for RA [10]. These patients had all failed at least two disease-modifying drugs including MTX and met the British Society of Rheumatology guidelines for biological drug prescribing [11]. No patients had previously received a biologic agent. Infliximab infusions were administered at the recommended licensed dose of 3 mg/kg at weeks 0, 2, 6, 14 and continued 8-weekly infusions thereafter. All patients were on concomitant MTX (dose range between 7.5 and 25 mg weekly).

Clinical response and non-response sub-types
Clinical data at baseline and follow-up (Week 16) was collected to evaluate ACR response on therapy. The ACR non-response group was divided into two groups based on our previous description of clinical non-response and CRP patterns [12]. The response groups were thus defined as:

  1. ACR response group: this includes all patients achieving ACR20, -50 and -70 responses.
  2. ACR non-response group: all patients failing to achieve an ACR20 response. This group is further subdivided into:
    1. CRP suppressed group: those patients not achieving ACR20 response but demonstrating ≥20% reduction in CRP from baseline to Week 16.
    2. CRP non-suppressed group: those patients not achieving ACR20 response and demonstrating no OR <20% reduction in CRP from baseline to Week 16.

Arthroscopy and synovial biopsy
Patients underwent medical arthroscopy of a swollen knee joint by one of the two arthroscopists (R.J.R. and M.A.Q.), blinded to the clinical response prior to commencing infliximab therapy and 2 weeks after the fourth infusion (Week 16). Visual analogue scores (VAS) for both vascularity and membrane proliferation were noted on both the occasions [13, 14]. Multiple synovial biopsies were obtained with 3.5 mm diameter grasping forceps from representative inflamed sites including the cartilage–pannus junction within the knee joint under direct vision using a Hopkins 2.7 mm 30° arthroscope. Repeat biopsies were obtained from sites adjacent to previous biopsy sites and avoided sampling of scarred areas. All biopsy samples were immediately embedded in optimum cutting tissue (OCT) snap-frozen in liquid nitrogen and stored at –80°C until sectioned for staining.

Immunohistochemistry
Serial cryostat sections (3 m thick) were mounted on superfrost slides (Surgipath Europe Ltd, Peterborough, UK) and dried overnight at 37°C. Sections were fixed for 20 min in acetone, washed in phospate-buffered saline (PBS) and endogenous peroxidase activity and blocked for 10 min before incubating with the following monoclonal antibodies for 1 h: anti-TNF-{alpha} [monoclonal mouse IgG1 clone 28 401, R&D Systems (Abingdon, UK)], anti lymphotoxin-alpha (LT-{alpha}) (monoclonal mouse IgG1 clone 5802.21, R&D Systems), anti-IL-1{alpha} (monoclonal mouse IgG1 clone 4414.141, R&D Systems), anti-IL-1β (monoclonal mouse IgG1 clone 8516.311, R&D Systems), anti-IL-1-receptor antagonist (ra) (monoclonal mouse IgG1 clone 10309.211, R&D Systems), anti-IL-6 (monoclonal mouse IgG1 clone 6708.111), anti-CD68 (monoclonal mouse IgG1 clone EBM11, Dakocytomation System, Ely, UK) and anti-CD3 (monoclonal mouse IgG1 clone UCHT1, Dakocytomation System).

A standard staining procedure using ChemMate (Dakocytomation System) was used. Secondary biotinylated antibody was added and incubated for 30 min followed by addition of horseradish peroxidase-conjugated avidin–biotin complex for another 30 min. 3,3'-Diaminobenzidine (DAB) was used to develop colour and was terminated by serial washings for 10–15 min. The slides were counterstained in haematoxylin (Sigma Poole), dehydrated in industrial methylated spirit and xylene and then mounted in acrytol (Surgipath Europe Ltd). Negative controls were used for each biopsy at each staining session by omitting the primary antibody.

Microscopic analysis
Sections were randomly analysed and the histological features scored in blinded fashion (M.H.B.). This included blinding to the sequence of biopsies as well as patient identification. Inter-individual variation was checked with a second, blinded scorer (A.E.). For all markers, all areas of each biopsy section were examined and scored on medium-power field with the use of a 1 mm graticule. Sub-lining layer (SL) CD3 and both SL and synovial lining layer (LL) expression of CD68, TNF-{alpha}, LT-{alpha}, IL-1{alpha}/-β/-ra and IL-6 was scored in each field semi-quantitatively on a 5-point scale (0–4; 0 = minimal infiltration, 4 = maximal infiltration) with a median value obtained [15]. In addition, LL hyperplasia (number of cell layer depth) was determined using a 4-point scale (0–3).

Statistical analysis
Independent t-testing assessed for statistically significant differences in baseline parameters [age, CRP, disease activity score (DAS28) score] between the two clinical groups while Mann–Whitney test (non-parametric testing) was employed for percentage RF positive. Baseline cytokine expression in the two ACR response groups were compared using Mann–Whitney testing with Holm correction in view of the multiple parameters (>5) assessed. This was also used for comparison of the two non-response sub-groups. One-way analysis of variance (ANOVA) (with Tukey multiple-comparison procedure) or Kruskal–Wallis test was applied to determine any differences between the two non-response sub-groups and the ACR response group with respect to baseline parameters and baseline cytokine expression.

Wilcoxon signed-rank test with Holm correction was applied to assess for significant reductions in the various parameters assessed following infliximab therapy. With the single figure numbers in each of the non-response sub-groups (see Results section), Holm correction was not applied; any changes at the P < 0.05 level representing trends for further evaluation. Finally, paired t-test was applied to observe for significant reductions in vascularity and membrane proliferation on arthroscopy.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
 References
 
Baseline characteristics
A total of 51 patients with RA were studied prior to infliximab. The clinical and demographic data of the patients are presented in Table 1. The baseline characteristics of the two ACR response groups were not statistically different (Table 1). The percentage female:male ratio and median prednisolone and MTX doses were also comparable. A greater percentage of non-responders were seropositive for IgM RF compared with responders but this did not reach statistical significance.


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  		TABLE 1. Baseline characteristics of all clinical non-response and response

 
Figure 1 summarizes patient numbers involved in this study both pre- and post-infliximab.


Figure 1
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  		FIG. 1. Diagram summarizing patient numbers for baseline and post-infliximab immunohistological analysis with response grouping. A total of n = 51 biopsies were available for baseline immunohistological assessment. After infliximab treatment, n = 32 of these had a further biopsy for analysis of change in cell and cytokine parameters.

 
Clinical response
At 16 weeks, 47% (n = 24) of the 51 patients treated with infliximab achieved an ACR20 response. Of the 24 responders, 13 achieved an ACR20 only, seven attained an ACR50 and four achieved an ACR70 response. Twenty-seven (53%) patients failed to achieve ACR20 (non-responders).

Clinical non-response subtypes
Eighteen patients demonstrated ACR non-response with CRP suppression at Week 16; nine patients with ACR non-response and no or minimal CRP suppression at Week 16. No differences in baseline characteristics were observed between these two non-response groups and the ACR response groups (data not shown).

Arthroscopy and synovial biopsy
Fifty-one patients had baseline arthroscopy, providing the pool of biopsies available for staining (with a subgroup of n = 30 based on tissue availability stained for IL-6). Thirty-two underwent a second arthroscopy. An average of 75–90% of the tissue sections stained for the various cell populations and cytokines described above were considered satisfactory and included for analysis. The numbers used for each analysis is included in the individual graphs later in the Results section. Reasons for rejecting slides included poor-quality biopsy (including in the post-treatment biopsies of good responders). Intra-observer and inter-observer variability of biopsy scoring was satisfactory (correlations of 0.92 and 0.87, respectively).


   Pre-infliximab histological analysis
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
Changes in histological and...
 Discussion
 Acknowledgements
 References
 
Baseline LL hyperplasia, CD3 and CD68 populations
ACR response and non-response
Median scores of SL CD3 and SL- and LL CD68 for both responders and non-responders are shown in Fig. 1a. There was no statistically significant difference in any of these three parameters between the two ACR response groups. LL hyperplasia was also comparable (Fig. 2a).


Figure 2
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  		FIG. 2. (a) Baseline median semi-quantitative scores for LL hyperplasia, SL CD3 and LL and SL CD68 infiltration in infliximab ACR responders (n = 24) and non-responders (n = 25). No statistical differences in the above parameters at baseline were observed between ACR response and non-response groups using Mann–Whitney analysis (and Holms correction). (b) Baseline median semi-quantitative score for LL and SL TNF-{alpha} and LT-{alpha} expression in infliximab ACR response and non-response groups. Biopsies in n = 24 ACR responders and n = 26 ACR non-responders were analysed. No statistical differences in the baseline expression TNF-{alpha} and LT-{alpha} were observed between ACR response and non-response groups using Mann–Whitney analysis (and Holms correction).

 
ACR non-response. CRP response and non-response
There were no statistically significant differences between these two groups (data not shown).

Baseline cytokine expression
ACR response and non-response
Baseline synovial TNF-{alpha} levels were not significantly different between ACR responders and ACR non-responders [LL median score 1.0 (range 0–3.3) and 1.15 (range 0–3.0), respectively and SL median scores 0.8 (range 0–2.0) and 1.0 (range 0–3.0), respectively, Fig. 2(b)]. No differences in TNF-{alpha} levels were observed with different levels of ACR response (ACR20 and ACR50/70 groups) (data not shown). No significant difference in expression of the other cytokines including IL-1{alpha} or -β was observed (data not shown).

ACR non-response. CRP response and non-response
Likewise, no significant differences in baseline cytokine expression between these two subgroups were observed (data not shown). No differences, including TNF-{alpha} expression was noted on comparing ACR responders with the worst ACR non-responders (data not shown). A previous case analysis [16] suggested LT-{alpha} expression may be an underlying basis for the opposing responses between infliximab and etanercept. Therefore, synovial LT-{alpha} expression was compared between 11 patients who achieved either an ACR50 or ACR70 response on infliximab and 5 patients who demonstrated clinical and biochemical non-response (no CRP suppression) to bring out a trend difference between infliximab responders and non-responders. There was no significant baseline LT-{alpha} difference between these two groups [median score in response group 0.48 (range 0–2.0) vs 0.75 (range 0.33–1.3) in the extreme non-response group].


   Changes in histological and macroscopic analysis post-infliximab
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
Discussion
 Acknowledgements
 References
 
Thirty-two patients underwent a second arthroscopy. This group comprised 17 ACR responders and 15 ACR non-responders (n = 11 with CRP suppression; n = 4 with no CRP suppression).

Change in LL hyperplasia and CD68, and SL CD3 and CD68 populations post-infliximab
ACR response and non-response
Holm correction determined P < 0.008 to be significant at the 5% level in the response group and P < 0.009 in the non-response group. Trends reduction in SL CD3 was observed in both ACR response (P < 0.017) and non-response groups (P < 0.013) (Fig. 3a) as well as reduction in SL CD68 infiltration (P < 0.03 in responders, P < 0.04 in non-response group) (Fig. 3a). The ACR response group also showed suggestion of reduction in LL hyperplasia (P < 0.03) (Fig. 3a). No reduction in LL CD68 infiltration was observed in either of the groups.


Figure 3
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  		FIG. 3. (a) Change in median semi-quantitative scores for LL depth, SL CD3 and LL and SL CD68 infiltration after four infliximab infusions (3 mg/kg) in infliximab ACR response (n = 17) and non-response groups (n = 15). Wilcoxon rank test with Holm correction was applied. In both the ACR response and non-response groups, clear trends for reduction in SL CD3 (P < 0.017 and 0.013, respectively) and SL CD68 (P < 0.03 and 0.04, respectively) infiltration was observed. In addition, the ACR response group suggested improvement reduction in LL hyperplasia (P = 0.03). (b) Change in median semi-quantitative scores for LL and SL LT-{alpha} and TNF-{alpha} after four infliximab infusions (3 mg/kg) in infliximab ACR response (n = 15) and non-response (n = 11) groups. Wilcoxon rank test with Holm correction was applied. (*P < 0.008 for ACR response group; P < 0.009 for ACR non-response group). In both the ACR response and non-response groups, significant reduction in SL TNF-{alpha} (P < 0.002 and 0.009, respectively) was observed. Trend in reduction in SL LT-{alpha} (P < 0.038 and 0.01) expression was noted. (c) Change in median semi-quantitative scores for LL and SL IL-1 cytokines and IL-6 after four infliximab infusions (3 mg/kg) in infliximab ACR response and non-response groups. IL-1 cytokines were assessed in n = 16 ACR responders and n = 14 ACR non-responders; change in IL-6 expression was evaluated in n = 11 ACR responders and n = 9 ACR non-responders. Wilcoxon rank test with Holm correction was applied. (*P < 0.008 for ACR response group; P < 0.009 for ACR non-response group). Significant reduction in SL IL-6 was observed in the ACR response group (P < 0.008) with similar trend in the non-response groups (P < 0.012). Suggestion of improvement in SL IL-1{alpha} (P < 0.05) in the ACR response group and SL IL-1ra (P < 0.01) in the ACR non-response group was noted.

 
ACR non-response. CRP response and non-response
The ACR non-response group with significant CRP suppression demonstrated a trend reduction in SL CD3 (–0.4, range of –1.2 to +0.3; P < 0.025) and SL CD68 (–1.6, range of –2.3 to +1.8; P < 0.04). The worst non-responders (with no CRP reduction) failed to demonstrate any such changes.

Change in cytokine expression post-infliximab
ACR response and non-response
Both the ACR response and non-response groups demonstrated significant reductions in SL TNF-{alpha} (P < 0.002 and P < 0.009, respectively) (Fig. 3b). Significant reduction in SL IL-6 was observed in the ACR response group (P < 0.008) with a similar trend in the non-response group (P < 0.01) (Fig. 3c). Trend reduction in SL LT-{alpha} expression was observed in both the response groups (Fig. 3b). ACR responders demonstrated a tendency for reduction in SL IL-1{alpha} (P < 0.049); ACR non-responders demonstrated similar trend in SL IL-1ra only (P < 0.01) (Fig. 3c).

ACR non-response. CRP response and non-response
The ACR non-response group with CRP suppression (n = 11) showed trend reduction in SL TNF-{alpha} expression with a change of –0.4 (range of –1.4 to +0.2; P < 0.04) (Fig. 4a). Similar declines in expression of SL LT-{alpha} (–0.8, range of –2.7 to +0.3; P < 0.012) and SL IL-6 (–1.0, range of –2.2 to +0.3; P < 0.012) was observed. Reductions in SL IL-1β of –0.4 (range of –1.1 to +0.6; P < 0.04) and SL IL-1ra of –0.7 (range of –2.0 to +0.5; P < 0.023) were noted.


Figure 4
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  		FIG. 4. (a) Change in median semi-quantitative score for synovial SL TNF-{alpha} after four infliximab infusions (3 mg/kg) in infliximab ACR response group (n = 16) and non-response (NR) with high CRP (n = 10) and low CRP groups (n = 4). Wilcoxon rank test was applied. (*P < 0.04; **P < 0.002—significant by Holm correction). In comparison with the ACR response group with significant SL TNF-{alpha} reduction after four infliximab infusions, the ACR non-response group with ≥20% CRP suppression suggested a similar trend. The ACR non-response group with <20% CRP suppression did not show any suggestion of decrease. (b) TNF-{alpha} expression in paired synovial tissue samples from a patient with RA who responded to infliximab. Panels A and B are the negative controls for the patient at baseline and post-infliximab, respectively. Panels C and D demonstrate TNF-{alpha} staining at baseline and post-infliximab, respectively. Considerable lining and SL TNF-{alpha} expression is observed in the baseline biopsy with clearly noticeable reduction in the post-infliximab biopsy. (c) Arthroscopic images before and after four infliximab infusions from a patient failing to achieve an ACR response (but with ≥20% CRP suppression). The post-infliximab arthroscopic image clearly demonstrates improvement with reduced villous proliferation and vascularity. Despite failing to achieve an ACR response, the majority of patients (with ≥20% CRP suppression) demonstrated similar findings to the ACR response group, with improvement in arthroscopic VAS as represented by the images and significant reduction in SL TNF-{alpha} expression Fig. 4b).

 
Evaluation of the worst ACR non-response group, failing to show CRP suppression (n = 4) revealed no trend reduction in any of the parameters assessed. Comparison of the magnitude of change in cytokine expression was not significantly different between the response and non-response groups (data not shown).

(See Fig. 4b for representative sections pre- and post-infliximab demonstrating reduction in TNF-{alpha} expression and Fig. 4a for boxplot of baseline sub-lining scores and change in scores for the response and non-response groups.)

Change in VAS at arthroscopy and response (n = 32)
ACR response and non-response
Both ACR response (n = 17) and non-response groups (n = 15) demonstrated significant reduction in mean VAS vascularity after infliximab therapy (ACR response: reduction from 49 to 35 mm, P < 0.02; ACR non-response: reduction from 45 to 29 mm, P < 0.003). Although both response groups demonstrated reduction VAS membrane proliferation, interestingly, this only reached statistical significance for the ACR non-response group (ACR response: reduction from 47 to 35 mm, P < 0.18; ACR non-response: from 35 to 22 mm, P < 0.006).

ACR non-response: CRP response and non-response
The ACR non-response group with CRP suppression at week 12 (n = 11) also demonstrated improvement in mean VAS vascularity (from 46 to 24 mm, P < 0.0001) and mean VAS membrane proliferation (from 31 to 17 mm, P < 0.008) (see Fig. 4c for representative arthroscopic pictures). The non-response group without CRP suppression (n = 4) did not demonstrate such a trend, with a deterioration in vascularity score (mean VAS vascularity from 45 to 51 mm) and an essentially unchanged VAS membrane proliferation (from 52 to 48 mm).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
Acknowledgements
 References
 
The objective of this study was to investigate the basis of non-response to TNF-{alpha}-blockade by assessing synovial tissue cellular and cytokine expression with immunohistochemistry in the largest UK TNF antagonist predictive biopsy cohort to date. The results show that ACR non-response to infliximab could not be predicted by baseline synovial TNF-{alpha} levels or explained by the absence of TNF-{alpha} before treatment. Furthermore, in most cases of non-response there was a significant improvement in TNF-{alpha} expression and vascularity (discussed later). The small number of patients with a poor clinical response and continued inflammation (raised CRP) suggested failure to suppress TNF-{alpha} levels.

This study specifically recruited patients receiving intravenous infliximab; biopsies were taken at baseline and after 4 months of treatment, permitting classification of patients as responders or non-responders. Previous synovial biopsy studies in similar but smaller cohorts have been conducted to identify the immediate effects of anti-TNF-{alpha} therapy, determine their mechanisms of action in RA and to explore impact on further cytokine expression [8, 9]. The study by Ulfgren et al. [9] in eight patients, all responding to infliximab contrasted with our findings by suggesting that higher TNF-{alpha} production prior to treatment predicted a better response, with an improved response associated with reduced TNF-{alpha} synthesis after therapy. Another study assessing cytokine changes and response to therapy following a single infliximab infusion found no change in synovial TNF-{alpha} and IL-1β expression but reductions in peripheral blood IL-1β mRNA and endogenous IL-1 and TNF-{alpha} antagonists [8]. An article by Smeets et al. [17] aimed to examine early (48 hrs) effects of a standard infliximab infusion (3 mg/kg) but also compared cellularity changes in responders and non-responders. This comparison, however, was only after two infusions with response measured at Day 28 and showed a rapid decrease in synovial macrophage numbers following therapy, without evidence of increased apoptosis—the basis of these findings is unclear. A more recent study evaluated circulating TNF bioactivity, demonstrating level of bioactivity to be predictive of clinical response to infliximab [18]; it remains unclear how such bioactivity correlates to synovial tissue expression of TNF-{alpha} in the context of plasma and synovial TNF levels, differing micro-environments and interactions.

The intention of this study was to explore clinical response and non-response by testing the hypothesis that non-responders to TNF-{alpha} antagonists represent a separate disease entity in terms of synovial cytokine profile. It is acknowledged that higher numbers would have provided greater power to the overall results; as has been evidenced by previous published biopsy-based studies, recruiting large numbers often proves difficult in practice, with the majority of cited studies to date in this area comprising less than half the numbers in this study or even single figures. Employing a semi-quantitative scoring system has been associated with it being insufficiently sensitive to detect biologically significant differences between responders and non-responders [19]; changes detected in our non-response subsets, however, suggests otherwise. Whilst immunohistochemistry studies cannot fully appreciate the dynamic system within which TNF-{alpha} operates, information on overall ‘available’ protein can be gained. This study aimed to test our hypothesis and guide on best approach for further lines of investigation.

A slightly lower percentage of patients than would be expected achieved an ACR20 response (47%); all patients were biologic naïve, chosen to avoid sub-selection of a group of TNF blockade non-response patients. This response is likely to reflect the longer duration, aggressive disease cohort selected for TNF blockade in initial practice. Importantly, the patient cohort was based on the British Society for Rheumatology (BSR) criteria for biological therapy eligibility and is in line with other data [3]. The findings would still be relevant to a more recent cohort as this study has investigated the common phenomenon of non-response on a synovial level. The level of clinical response was correlated with macroscopic arthroscopic visual assessment, which scored both vascularity and membrane proliferation [13, 14]. This is the first such study to report these two parameters as independent VAS scores in such a comprehensive cohort and to assess non-response with no CRP suppression and TNF-{alpha} expression.

Although broadly speaking the results can be viewed as ‘negative’, they provide important learning points. It is worth highlighting first, however, that this study employed more rigorous statistical P-values for significance as determined by Holm correction (contrasting with previously published studies that accepted P > 0.05 as significant despite testing for multiple parameters). Using this method, the significant findings were as follows. First, the synovial membranes of non-responders express comparable levels of TNF-{alpha} to responders suggesting that the role of TNF-{alpha} in both these groups’ disease is similar. Second, non-responders did not express greater IL-1{alpha} or -β at baseline. This is important as IL-1β in particular has an important role in RA, distinct to that of TNF-{alpha}, and is consistent with the previous clinical finding that patients who fail to respond to anti-TNF-{alpha} agents also do not respond to IL-1ra [20]. Third, the results from the post-therapy biopsies indicate that both the ACR response and non-response groups show evidence of biological improvement with significant reduction in TNF-{alpha}. The ACR response group also confirmed significant reduction in IL-6, consistent with CRP reduction observed in this group. Both groups revealed trends for reduction in most of the other cellular and cytokine parameters assessed. In addition, both the ACR response group patients and the majority of the ACR non-response patients (with also CRP suppression) showed clear suggestion of improvement in macroscopic VAS for vascularity and membrane proliferation after therapy. Synovial tissue macrophages have been reported to be a sensitive biomarker for response to treatment in patients with RA [21]. Our study included a different patient cohort that may explain a lack of correlation seen.

Overall, where there was no CRP suppression post-infliximab, TNF-{alpha} expression seems to remain unchanged. Excessive TNF-{alpha} levels have been suggested; however, no baseline differences were observed. The issue of poor entry by the monoclonal into the synovium has also been proposed; however, systemic blockade of TNF-{alpha} at the hepatic level should at least lead to a CRP reduction that was not observed in this group. One other possibility includes failure of the monoclonal to actually bind and block TNF-{alpha}—the development of anti-globulins seems unlikely, as a (biochemical nor clinical) response even after the first infusion was not observed in this group. Finally, we have previously described the presence of synovial LT-{alpha} in a patient resistant to infliximab but responsive to etanercept [16] raising the possibility of a role for LT-{alpha}. In this present study, no significant difference in expression of synovial LT-{alpha} at baseline (pre-biologic) was observed compared with infliximab ACR50/70 responders. Larger synovial studies may be of merit to address this definitively.

In conclusion, our results challenge the concept that non-response to TNF-{alpha}-blockade represents a TNF-{alpha}-independent disease and confirm that baseline synovial TNF-{alpha} or IL-1 expression cannot be used as predictors of response to TNF blockade. This contrasts with previously (smaller number) published findings. The clear reduction in TNF-{alpha} (including vascularity) observed in the ACR non-response group as well as the ACR response group is compatible with the reduced structural damage previously seen in all response groups. Other areas of investigation would clearly be of interest including, for example, the impact of TNF blockade impact on lymphoid architecture; more extensive work as part of a separate study, however, would be needed to properly address this question. There remains the possibility of involvement of other cytokines (e.g. IL-15 and -6) in the TNF-{alpha} network, but it is clear that further, more sophisticated studies are required to fully clarify the mechanisms of differing response to TNF-{alpha}-blocking therapy.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
References
 
M.H.B. was supported by an Arthritis Research Campaign (ARC) Clinical Research Fellowship, UK. P.E. is an ARC Professor. We would like to thank Dr E. Hensor for her statistical input.

Disclosure statement: M.A.Q. has received honoraria and participated in advisory boards for Abbott and Schering-Plough and has received an unrestricted grant from Abbott. P.E. has provided expert advice and clinical trials for Schering-Plough, Wyeth and Abbott. All other authors have declared no conflicts of interest.


   Notes
 
Present addresses: G. Cunnane, St. James's Hospital, James's Street, Dublin 8, Ireland; J. Isaacs, Musculoskeletal Research Group, School of Clinical Medical Sciences, University of Newcastle upon Tyne, UK.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Pre-infliximab histological...
 Changes in histological and...
 Discussion
 Acknowledgements
 References
 

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Submitted 15 October 2007; revised version accepted 17 June 2008.
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