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Rheumatology 2001; 40: 1243-1255
© 2001 British Society for Rheumatology

Original Papers

Association of clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in rheumatoid arthritis

A. R. Pettit, H. Weedon1, M. Ahern1, S. Zehntner, I. H. Frazer, J. Slavotinek1, V. Au1, M. D. Smith1 and R. Thomas

Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102 and
1 Rheumatology Research Unit, Repatriation General Hospital, Adelaide, South Australia 5041, Australia


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the assessment of rheumatoid arthritis (RA) disease activity, synovial tissue (ST) biopsied arthroscopically was assessed from 18 patients before and after commencement of disease-modifying anti-rheumatic drug (DMARD) therapy.

Methods. Lymphocytes, macrophages, differentiated dendritic cells (DC), vascularity, tumour necrosis factor (TNF) {alpha} and interleukin-1ß levels were scored. Clinical status was scored using the American College of Rheumatology (ACR) core set and serial radiographs were scored using the Larsen and Sharp methods. Histopathological evidence of activity included infiltration by lymphocytes, DC, macrophages, tissue vascularity, and expression of lining and sublining TNF{alpha}. These indices co-varied across the set of ST biopsies and were combined as a synovial activity score for each biopsy.

Results. The change in synovial activity with treatment correlated with the ACR clinical response and with decreased radiological progression by the Larsen score. The ACR response to DMARD therapy, the change in synovial activity score and the slowing of radiological progression were each greatest in patients with high initial synovial vascularity.

Conclusions. The data demonstrate an association between clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in RA. High ST vascularity may predict favourable clinical and radiological responses to treatment.

KEY WORDS: Rheumatoid arthritis, Synovial vascularity, DMARD therapy.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Elucidation of the key factors involved in the perpetuation of chronic inflammation in diseases such as rheumatoid arthritis (RA) is essential to the design of effective therapeutic interventions. Previous reports indicate that immunopathological features of RA synovial tissue (ST) reflect clinical disease activity [1–4]. Therefore, the pathological assessment of arthroscopic synovial biopsy in RA patients may improve the assessment of therapeutic efficacy or potentially predict therapeutic or disease outcome [5]. To-date, immunohistochemical analysis of ST has not identified reliable prognostic indicators of outcome in RA. The advent of a number of novel therapies in RA highlights the need for robust and reliable measures of disease activity in RA ST to measure efficacy, examine mechanisms of action and predict outcomes to particular agents in clinical trials [6].

The pathology of the rheumatoid synovial inflammatory process is well characterized and includes synoviocyte proliferation, pannus formation (destructive ST at the bone and cartilage interfaces), angiogenesis, cytokine production and mononuclear cell infiltration [7–9]. In addition, the immune reaction is usually characterized by organized focal aggregates of lymphocytes, macrophages and dendritic cells (DC) that can progress to germinal centre-like structures in established disease [10–13]. It has been proposed that DC play a role in the formation and maintenance of such organized lymphoid tissue [14, 15]. The pro-inflammatory cytokines tumour necrosis factor (TNF) {alpha} and interleukin (IL)-1ß have also been proposed to be key contributors to the inflammatory cytokine cascade in RA, and specific monotherapies designed to inhibit their activity have proved beneficial [16, 17]. Expression of TNF{alpha} and IL-1ß and the synovial immune response may be additive or even synergistic in the perpetuation of clinical disease due to their roles in endothelial activation, differentiation and activation of cells involved in the immune response, and in bone and cartilage destruction [18, 19].

While the relationship between clinical activity and synovial immunopathology has been examined in response to treatment with various anti-rheumatic agents, radiological damage has not been simultaneously assessed in this context [3, 4, 20–22]. The current study addressed prospectively two issues. The first was to examine, for patients presenting with active RA, whether there was a relationship between synovial immunopathology, clinical activity, and radiological progression, following optimal conventional disease-modifying anti-rheumatic drug (DMARD) therapy. Patients were evaluated clinically using the American College of Rheumatology (ACR) core set and their response to treatment determined by ACR response criteria. Serial radiographs were scored using the Larsen and Sharp methods. Serial synovial biopsies were evaluated for vascularity, infiltration by macrophages, differentiated DC and lymphocytes, and expression of TNF{alpha} and IL-1ß. The second aim was to evaluate possible immunohistological predictors of clinical and radiological responsiveness to DMARD therapy, by analysis of pre-treatment synovial biopsies for lymphocytes, DC, macrophages, tissue vascularity, and expression of lining and sublining TNF{alpha} and IL-1ß. The data demonstrate an interrelationship between clinical and radiological outcome and synovial immunohistological activity. Furthermore, they suggest that high tissue vascularity predicts favourable pathological, clinical and radiological responses to DMARDs.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
ST was obtained from a clinically active knee joint of each of 18 patients who fulfilled the ACR criteria for RA and who had active disease in at least one knee joint at the time of entry into the study [23]. However, all patients had at least five swollen and 10 tender joints at study entry (Table 1Go). Patients were recruited between 1991 and 1996 and were included only if the treating rheumatologist considered that commencement of a DMARD was necessary. Patients were ineligible if arthroscopy was contra-indicated for any reason. Ethical approval for the collection and study of arthroscopic biopsies was obtained from the Repatriation General and Princess Alexandra Hospitals. The characteristics of the patients entered into the study are shown in Table 1Go. Of the 18 patients, there were eight males, seven had previously taken one or more DMARD and 12 were seropositive. The ACR clinical core set of measurements [24, 25], as well as radiographs and a synovial biopsy were obtained before and at least 1 month after commencement of treatment considered optimal for each patient's clinical condition [24, 25]. While the objective was to achieve the optimal patient clinical response, the nature of the treatment was at the discretion of the treating rheumatologist. Treatments included i.m. gold, methotrexate, cyclosporin, sulphasalazine, hydroxychloroquine and D-penicillamine. The clinical response at the time of the second biopsy was evaluated by ACR criteria [24]. The interval between the serial assessments of clinical and synovial response varied between 1 and 28 months, with a median value of 8.5 months (Table 1Go). The interval between the serial radiographs was longer than that between the serial biopsies in eight cases and was the same in the other 10 cases. Two observers unaware of the clinical details of the patients assessed the serial radiographs. The Kaye-modified Sharp score, the Larsen score, and the change in score from the first to the second evaluation were recorded [26–28].


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  		TABLE 1. Patient variables, disease activity and response

 

ST processing and immunohistochemistry
Tissue was fixed in formalin and embedded in paraffin, and 4 µm serial sections were cut and mounted on glass slides. Double immunohistochemistry was carried out using an immunoperoxidase–immunoalkaline phosphatase technique exactly as described previously [29]. All incubations were carried out at room temperature. The sections were examined and photographed using a transmitted light microscope and camera (Leitz Diaplan, Leica, Solms, Germany).

Antibodies
The antibodies used included C-19 directed against RelB (Santa Cruz Biotechnology, CA, USA), TAL.1B5 directed against HLA-DR, L26 directed against CD20, polyclonal antibody directed against CD3, and Kp-1 directed against CD68, all from Dako (Carpinteria, CA, USA), 12E9 directed against precursor and active forms of IL-1ß (Oncogene Science, Manhassets, NY, USA) and M112 directed against TNF{alpha} (Genzyme, Cambridge, MA, USA). Biotinylated Ulex europaeus agglutinin I (Ulex), a lectin that specifically binds endothelial cells, was purchased from Vector Laboratories (Burlingame, CA, USA). Control antibodies included isotype-matched mouse and rabbit control immunoglobulins (Dako).

Analysis of DC, lymphocytes and vascularity in RA ST
Biopsy sections were randomized and coded prior to analysis by an observer unaware of the clinical details of the patients (AP). Serially cut sections were double stained for RelB and either HLA-DR, CD20 or CD68, or single stained for RelB. The percentage of DC in each tissue was calculated, as previously described, by counting the entire section and using the formula below [29]:


Interobserver variability was demonstrated to be minimal, as an average difference in score of 1.4% was demonstrated between two independent observers unaware of the patients’ clinical details (AP and SZ), and 80% of scores fell within 2% of each other. To quantitate differentiated DC infiltration, the number of nuclear RelB (nRelB)+ cells was enumerated in each tissue section at high power. For each biopsy, this number was corrected for the area of the section using a graticule, to obtain the number of nRelB+ cells/mm2. Tissue vascularity was quantitated by counting individual Ulex+ vascular structures in the entire section, corrected for the area of the section. Lymphocytic infiltration was evaluated using a semi-quantitative 10-point score assessed by two independent observers (AP and SZ) as shown in Table 2Go. Each high power field in the entire section was scored, and the scores averaged. The variability between scorers was minimal, with an average difference in final score of 0.2, and 98% of all scores fell within half a score of each other. The accuracy of similar quantitative and semi-quantitative methods of analysis of RA ST has been previously demonstrated [30, 31].


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  		TABLE 2. Lymphocytic infiltrate score

 

Analysis of CD68, TNF{alpha} and IL-1ß expression in RA ST
Immunostained sections were examined by an observer (HW) unaware of the patients' clinical details, using a computer-assisted colour video image analysis system (Video Pro 32, Leading Edge P/L, Adelaide, Australia), as previously described [32, 33]. At least five high power fields from each biopsy were analysed and the mean calculated. CD68 infiltration into either sublining or lining layers of ST was calculated from the area of CD68 staining. The total amounts of TNF{alpha} and IL-1ß present in the sublining and lining layers of ST were scored as the integrated optical density, which is calculated as the product of mean optical density and area of staining.

Statistical analysis
Associations between patient and ST pathological variables were determined using Pearson correlation coefficients (r). In some cases, scores were ranked before comparison. Differences were considered to be significant at P<0.05. The choice of comparison was made after the data were collected and analysed or scored, to test an underlying hypothesis that one or more immunohistochemical marker would be predictive of clinical or radiological outcome.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
The extent of mononuclear cell infiltration and ST vascularity correlate with TNF{alpha} expression
Serial ST biopsies taken before and after a period of DMARD treatment from 18 patients with RA were stained for lymphocytes, differentiated DC, macrophages, vascularity, and TNF{alpha} and IL-1ß expression. Differentiated DC were enumerated by the expression of nRelB. Double immunohistochemical staining for RelB and either HLA-DR, CD20 or CD68 confirmed the usefulness of nRelB as a marker of differentiated DC in RA ST, as previously described [29]. The mean percentage of nRelB+ cells that could be identified by double staining as differentiated DC in RA ST, regardless of treatment status, was 88±11% (n=12). ST biopsies taken from patients recruited with active RA demonstrated enrichment of differentiated DC in perivascular regions, within mononuclear cell aggregates containing CD3+ T cells, CD20+ B cells and CD68+ macrophages, as previously described (Fig. 1AGo–DGo and data not shown) [29]. ST biopsies from these patients also demonstrated expression of TNF{alpha}, predominantly in the synovial lining layer, but also in perivascular areas of the sublining (Fig. 1EGo, FGo), whereas IL-1ß was widely expressed in both ST sublining and lining layers (Fig. 1GGo, HGo).



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  		FIG. 1. Staining for synovial cellular infiltration and cytokines before and after treatment. ST biopsied before and after treatment was stained by double immunohistochemistry for RelB (brown) and Ulex (red) (A and B), CD3 (brown) and CD20 (red) (C and D), and for TNF{alpha} (E and F) and IL-1ß (G and H) by single immunohistochemistry (brown). This example is representative of 10 responding patients. Magnification x325.

 
Expression of TNF{alpha} and IL-1ß, tissue vascularity, and tissue infiltration of lymphocytes, macrophages and DC are all markers of inflammation expressed to varying extents in the RA ST biopsies. To determine the extent to which these markers were interrelated, Pearson correlation coefficients were determined between the scores obtained for each marker (Table 3Go). The extent of DC infiltration, lymphocytic infiltration, lining layer macrophage accumulation and vascularity co-varied significantly. Lymphocytic infiltration also co-varied with vascularity, macrophage infiltration and accumulation, and with sublining TNF{alpha} expression. Sublining TNF{alpha} further co-varied with lining layer TNF{alpha} and with vascularity. In contrast, lining and sublining IL-1ß both correlated negatively with vascularity. The data indicate that TNF{alpha} expression, mononuclear infiltrate and vascularity are all measures of a common pattern of inflammation, which is also characterized by lining layer macrophage accumulation. In contrast, IL-1ß expression did not correlate with TNF{alpha}, DC or monocyte infiltration and was negatively correlated with vascularity and lymphocyte infiltration. As shown in Fig. 1Go, this predominantly results from the lack of reduction of IL-1ß expression in ST after treatment, by comparison with mononuclear cells and TNF{alpha}.


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  		TABLE 3. Mononuclear cell infiltration and tissue vascularity correlate with the level of TNF{alpha} in ST biopsies

 
To measure change in these parameters of inflammation with treatment, pre- and post-treatment biopsies were scored for the above-mentioned indices of histological activity, and the change in score (delta) calculated. Pearson correlation coefficients between the delta scores were calculated to assess the relationship between the changes (Table 4Go). Changes in vascularity, DC and lymphocyte infiltration with treatment were co-associated. The change in lymphocytes correlated with that in sublining TNF{alpha} and negatively with that in lining IL-1ß. The change in lining layer IL-1ß also negatively correlated with the change in vascularity, thus confirming that, among the parameters assessed, there is a common pattern to inflammation in RA, characterized by TNF{alpha} expression, cellular infiltration, and increased vascularity.


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  		TABLE 4. Correlation between the change in pathological variables with serial measurement

 

Relationship between the change in synovial activity on biopsy and response
The preceding data suggest that the co-associated pathological variables could be combined to generate an index of synovial activity for each ST biopsy analysed, comprising the sum of CD68 lining, CD68 sublining, lining layer TNF{alpha}, vascularity, DC and lymphocytic infiltrate scores. For each patient, three outcomes in response to treatment for RA were therefore of interest: the ACR clinical response; the change in radiological score; and the change in synovial activity score on biopsy. An interrelationship between these outcomes would suggest that a change in synovial activity may reflect a clinical or radiological response to treatment. Because the number of patients was relatively small, scores were ranked to avoid assumptions regarding normality of the distribution of the data. The change in synovial activity score correlated with the ACR clinical response (Table 5Go, Fig. 2AGo). When the measures that comprised the synovial activity score were examined individually, there was a significant association for each of these, except the DC score, with the ACR clinical response. The strongest associations were with the changes in lining layer TNF{alpha} (Fig. 2BGo) and lining layer CD68 (r=0.65, P<0.005), while the association with the change in DC represented a trend only (r=0.43, P=0.079). There was no association of the ACR clinical response with the change in IL-1ß expression in the lining or sublining layers (r=-0.25, P=0.3).


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  		TABLE 5. Relationship between the change in clinical, radiological and synovial activity scores

 


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  		FIG. 2. The interrelationships between the change in clinical, radiological and pathological measurements with treatment were examined by linear regression analysis of the ACR response, the change in the Larsen or the Sharp score or the change in synovial activity score. The synovial activity score comprised the sum of CD68 lining, CD68 sublining, lining layer TNF{alpha}, vascularity, DC and lymphocytic infiltrate scores.

 
The change in radiological activity with treatment was assessed by the Larsen and modified Sharp methods. Radiographs were taken at the time of the initial biopsy and as close as possible to the time of the second clinical evaluation. However, in eight cases, the post-treatment biopsy and clinical evaluation preceded that of the radiographic evaluation by>6 months. The change in scores by the two methods co-varied (Table 5, Fig. 2CGo). Slowed progression of the Larsen but not the modified Sharp score correlated with the ACR clinical response (Table 5, Fig. 2DGo, P<0.004), measured either at the time of biopsy or radiological re-evaluation (data not shown), indicating that patients responding clinically also demonstrated a slowing of radiological progression, at least as assessed by the Larsen method. Furthermore, slowed progression of the Larsen score was also associated with a reduction in synovial activity scores (Table 5, Fig. 2EGo). When the measures that comprised the synovial activity score were examined individually, the correlation with the decrease in the Larsen score was significant for each of the measures (data not shown). In addition, the change in lining layer IL-1ß was weakly associated with the change in the Larsen score (r=0.48, P<0.05). These data suggest that changes in synovial activity on biopsy following treatment, ACR clinical response, and radiological progression as assessed by Larsen scoring, are all measuring similar underlying pathological changes with treatment, at least in this group of 18 RA patients.

Pre-treatment variables predictive of outcome
The previous data demonstrate that, for patients with RA, changes in the three outcome variables following treatment with DMARDs (clinical, radiological, and synovial activity scores) were co-associated. The data were next analysed to determine whether any patient (Table 1Go), clinical (components of the ACR core set), radiological or histopathological variables measured at the time of entry into the study predicted a favourable outcome following treatment. Ranked scores were analysed. No single patient or clinical variable predicted a favourable clinical or histopathological outcome. However, high lining layer TNF{alpha} (r=0.49, P<0.05) and high vascularity (r=0.53, P<0.03) in initial synovial biopsies predicted the ACR response (Fig. 3AGo, BGo). High vascularity in initial synovial biopsies also predicted a favourable histopathological outcome (r=0.76, P<0.0003, Fig. 3DGo). The initial synovial activity score predicted a favourable histopathological outcome less strongly (r=0.57, P<0.02). Considering predictors of favourable radiological responses, a low initial Sharp score was predictive of a small change in the Larsen score (r=0.51, P<0.03) and a low initial Larsen score similarly predicted a small change in the Sharp score (r=0.8, P<0.0001). In addition, a short disease duration prior to study entry predicted a favourable radiological response (Larsen: r=0.47, P<0.05, Sharp: r=0.82, P<0.0001). As expected, both the initial Larsen (r=0.72, P<0.0009) and Sharp scores (r=0.47, P<0.05) increased with disease duration. High vascularity in initial synovial biopsies strongly predicted a favourable radiological response as measured by the change in the Larsen score (r=-0.88, P<0.0001, Fig. 3CGo). Finally, to determine predictors of treatment outcome from the patient, clinical, radiological and histopathological variables, the ACR score and the change in the Larsen, Sharp and synovial activity scores were combined as an overall outcome score. Initial synovial vascularity (r=0.8), disease duration at study entry (r=-0.5) and Sharp score at study entry (r=-0.49) each predicted the overall outcome, and the best correlation for outcome following treatment was with the initial synovial vascularity (Fig. 3).



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  		FIG. 3. The relationship between the treatment outcome measurements and initial tissue lining layer (LL) TNF{alpha} or vascularity were examined by linear regression analysis.

 


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  		FIG. 4. Model for the interrelationship between patient, clinical, radiological and histopathological variables at study entry and clinical, radiological and histopathological responses.

 


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
In the current small study of 18 patients with RA, arthroscopic biopsy of ST was employed to compare immunohistochemistry with clinical scoring and radiology for the assessment of disease activity in response to treatment and to evaluate possible histological predictors of responsiveness to DMARD therapy. Histopathological evidence of disease activity included ST infiltration by lymphocytes, nRelB+ differentiated DC, macrophages, tissue vascularity, and expression of lining and sublining TNF{alpha}. Despite the relatively small number of patients, these indices of disease activity correlated across the set of ST biopsies and co-varied in response to treatment in individual patients. The indices measured were therefore combined to generate a synovial activity score for each biopsy. The change in synovial activity with treatment correlated with the ACR clinical response. The decrease in radiological progression with treatment as assessed by a change in the Larsen score also correlated with the ACR response and with the change in synovial activity score. Thus, clinical, radiological or immunohistochemical measures can be used to assess RA disease severity and to predict the likely response to treatment, although each defines a different aspect of the underlying pathology.

The current study is limited in its capacity to determine prognostic factors for outcome for several reasons. In particular, variability in patient factors at study entry, including disease duration, previous DMARD treatment and severity of radiological damage could all influence outcome. In addition, the patients were treated with a variety of drugs and were assessed after varying periods of time. Therefore, the current data highlight the need for a study of patients with RA receiving their first DMARD within the first 12 months of disease, to test the hypothesis that tissue vascularity predicts treatment response over a defined period of time. It is also possible that histopathological prognostic factors may vary depending on the interval between serial measurements and the intervening treatment. A previous study suggested that lining layer thickness may predict clinical outcome of RA after 1–3 yr. In that study, patients were not given specific therapeutic intervention between serial measurements [5]. Finally, in the current study, crude associations between variables were made using Pearson correlation coefficients and no statistical correction has been made for the number of variables compared. However, correlations in which the P value is <0.001 would be significant even allowing for multiple comparisons.

Previous serial and cross-sectional studies of synovial immunopathology have suggested that at least some immunopathological features of RA ST inflammation, including macrophages, lymphocytic infiltration, IL-6, and adhesion molecules, correlate with clinical activity [1–4, 21, 22]. Clinical studies have demonstrated the role of TNF{alpha} in cellular recruitment to the synovium and in synovial angiogenesis [21, 34, 35]. Previously, the vascularity of ST derived from active joints has been shown to be increased in comparison with ST derived from clinically unaffected joints in patients with RA or normal control ST [36], although the relationship of tissue vascularity to treatment outcome has not been examined. The current study shows a clear relationship between synovial vascularity and the overall inflammatory activity of the tissue and demonstrates that the response to treatment is associated with decreased tissue vascularity.

The association of the change in synovial activity score with clinical response highlights the pathogenetic role of the mediators of synovial inflammation in RA perpetuation. The interplay between nRelB+ DC and TNF{alpha} may be central to the development and maintenance of perivascular aggregates in synovial inflammation. Similarities between the phenotypes of RelB-/- and TNF-/- mice suggest that DC and TNF{alpha} both contribute to the formation of organized lymphoid structures [37, 38]. Further evidence has been obtained in a murine model of diabetes mellitus in which continued antigen presentation by DC may drive the formation of lymphoid aggregates at the autoimmune effector site [15]. DC and macrophages have been demonstrated to be the first cells to infiltrate the pancreas in NOD mice, suggesting their role in the generation of this local organized lymphoid tissue. The infiltrating DC expressed TNF{alpha} [39, 40]. Similar infiltration of ST by macrophages and the expression of macrophage-derived cytokines in asymptomatic joints of patients with RA have been noted [9]. In the current study, TNF{alpha} was expressed in the region of perivascular aggregates. In this regard, TNF{alpha} activates endothelial cells and stimulates angiogenesis and TNF{alpha} blockade reduces the level of mononuclear cell infiltration into RA ST, as well as angiogenesis [21, 35, 41]. The relationship of differentiated DC infiltration of ST to clinical and radiological activity has not been previously studied. Our recent studies demonstrated a close correlation between differentiated DC infiltration, lymphocytic infiltration and vascularity in ST from patients not only with RA, but also with osteoarthritis and spondyloarthropathy, suggesting common factors mediating the migration of DC and lymphocytes into damaged joints [42]. While the role of DC in the initiation of autoimmune diseases such as RA is highly likely based on a number of animal models, the current study also implicates DC in the perpetuation of RA inflammatory and erosive activity [43, 44].

The influence of RA clinical activity on radiological outcome has previously been demonstrated [45–47]. In the current study, clinical and radiological responses were found to be associated, in that radiological progression was slowed when clinical improvement occurred. This is in keeping with a number of clinical trials demonstrating slowing of radiological progression with various DMARDs [48–53]. Our study demonstrates an additional association of both clinical and radiological response with the change in synovial activity score, strengthening the evidence that clinical, radiological and histopathological responses to treatment are linked. It is important to mention that in at least some patients, the interval between radiographic assessments was longer than the interval between biopsies and the associated clinical assessments. Furthermore, the change in the Larsen score correlated better with the changes in ACR and synovial scores than the change in the Sharp score. The two radiological scores were not closely correlated, probably because the information obtained from these scores differs, and the sample size was small. Thus, the Larsen score measures changes in hands, wrists and feet, including erosions and joint space narrowing, while the modified Sharp score measures erosions only, in hands and wrists but not feet. Furthermore, Larsen grade 1 includes measures of soft tissue swelling and localized osteoporosis that are not included in the Sharp scoring system. Previous studies comparing the Steinbrocker radiographic stage, the modified Sharp score and the Larsen score have shown a close correlation between the measurements. However, these studies were cross-sectional and included over 100 patients [28]. Therefore, the correlation between the change in clinical, radiological and synovial activity noted in this longitudinal study suggests confirmation in a larger group of patients over a fixed response time.

Animal models suggest that synovial inflammation and bone erosion may be regulated by TNF{alpha} and IL-1ß, respectively [54, 55]. Surprisingly, in contrast to the animal models, improvement in both clinical and radiological responses was here associated with a fall in synovial TNF{alpha} but not in IL-1ß expression. The current data suggest that the conventional DMARDs employed here have little impact on the expression of IL-1ß in ST, while still impacting on clinical and radiological activity. Thus, while IL-1ß may play an important role in the pathogenesis of bone erosion [56], its expression may not always correlate with radiological damage. In this regard, it should be noted that the anti-IL-1ß antibody used in this study to stain ST recognizes both precursor and active forms of IL-1ß. Therefore, it is not clear whether the IL-1ß staining after treatment relates entirely to active IL-1ß. Further analysis of the activity of IL-1ß and IL-1 receptor antagonist is necessary to evaluate fully the relationship between disease outcomes and IL-1ß expression [12, 54, 55]. Finally, while radiological damage slowed in the current study, it did not stop. It is possible that IL-1ß expression in ST may serve as a very particular and specific indicator of drug action that is not affected by conventional DMARDs. Future studies combining IL-1ß inhibition with drugs and biologics that affect the ‘TNF{alpha} axis’ illustrated here will be of considerable interest.

The advent of many novel anti-rheumatic agents has highlighted the need for prognostic indices to assess the likelihood of treatment efficacy. Two possible factors that may influence disease outcome in response to treatment were apparent from the analysis of factors associated with clinical, radiological and synovial outcome. First, in keeping with previous reports, radiological score worsened with longer disease duration prior to assessment and treatment [46, 47]. Of interest, long disease duration and higher initial radiological score were associated with faster radiological progression despite treatment in the current study. While this may not have been predicted in a model in which radiological progression is constant over time [57, 58], several studies have suggested that progression appears to be retarded in early disease by therapeutic suppression of disease activity [59–61]. Furthermore, a recent meta-analysis of 14 trials of disease-modifying drug therapy showed that short disease duration is an important predictor of a favourable clinical response to treatment [62]. Radiological outcome was not assessed in that study [63]. Previous studies have shown that synovial macrophages but not T cells correlated with radiological joint damage [12, 64]. It should be noted that in those studies, serial radiographic scores were taken, but synovial pathology was examined only at follow-up, and serial measurements were not taken before and after treatment.

In the current study, the ACR response to treatment, the change in synovial activity score and the slowing of radiological progression were greatest in patients with high tissue vascularity scores. Figure 4Go shows a model of the data, using the overall treatment outcome score. The current data suggest at least two predictive factors for overall treatment outcome in this group of patients with RA. The best predictor was initial synovial vascularity. Disease duration and initial radiological score were also predictive. These two factors were co-associated and they significantly influenced the radiological outcome component of the overall score. There was no association between vascularity of the initial biopsy and either disease duration or initial radiological score. This suggests that vascularity and disease duration at study entry may be independent predictive factors for the response to treatment. In support of this, a comparison of ST biopsied from patients with RA for less than 1 yr or for greater than 5 yr duration, suggests that the association of clinical activity with histopathological inflammation does not wane over time [65–67].

There are several possible reasons why high tissue vascularity may be of prognostic significance. As shown here, vascularity is a marker of histological and clinical inflammation and should therefore be susceptible to suppression by DMARDs. A related possibility is that the ability of patients with highly vascular tissue to respond to treatment may result from better drug penetration into the synovium and reduced local hypoxia [68]. Other synovial pathological abnormalities, perhaps resulting from damage rather than inflammation, may be more resistant to current DMARDs [69]. In this regard, the consistent lack of response of a subgroup of patients to conventional disease-modifying drugs or to biologics, including TNF{alpha} blockade, suggests possible differences in the underlying synovial pathology of the patient subgroup with low tissue vascularity. A more detailed comparison of the pre-treatment biopsies of RA patients making a poor response and those making a favourable response to conventional DMARDs may reveal novel pathological subgroups [70], with therapeutic implications.


   Acknowledgments
 
The authors thank Dr G. Strutton for assistance with pathological sections, Dr J. Hay for statistical advice and Dr M. Handel and Dr N. Bellamy for helpful discussions. This research was supported by the National Health and Medical Research Council of Australia and the Arthritis Foundation of Australia. Dr Thomas is supported by the Arthritis Foundation of Queensland.


   Notes
 
Correspondence to: R. Thomas. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 20 November 2000; Accepted 9 May 2001


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