Surg Endosc (1992) 6:277-282

surgical EncioscoPy 9 Spfinger-VerlagNew York Inc. 1992

Correlative histologic and arthroscopic evaluation in rheumatoid knee joints A. Zsch~ibitz 1, M. Neurath 1, J. Grevenstein 2, H. Koepp l, and E.

Stofft 1

1 Department of Anatomy, and 2 Department of Orthopaedic Surgery, Johannes Gutenberg-University, Saarstr. 19-21, W-6500 Mainz, Federal Republic of Germany

Summary. The correlation between arthroscopic observations and histologic changes in rheumatoid arthritis is still controversial. Synovial samples of 21 knee joints in rheumatoid arthritis patients were comparatively investigated by endoscopy and histology. Biopsies were scored by an endoscopist and subsequently dissected. Different histochemical and immunocytochemical staining techniques were used to define inflammatory activity. Arthroscopic and histological values were compared by rating scales and variance analysis. Our study indicates that synovial biopsy is of diagnostic value in rheumatoid arthritis. However, its usefulness depends on the histochemical methods used. The results revealed highly significant correlations of endoscopic features with the number of neutrophilic granulocytes, intravascular leukocytes, and peroxidase-positive macrophages. However, no relationship was found between the detection of lymphocytes or resident macrophages and inflammatory scores. The close correlation between endoscopic and histological findings suggests that arthroscopic evaluation allows a valuable classification of the inflammatory activity in rheumatoid synovitis.

toid arthritis (RA) has not yet been established. Some rheumatologists suggest that synovial biopsies are of limited validity in the management of patients with RA [18]. An alternative view is that knowledge of histologic changes gives significant clinical information [19]. This lack of agreement is probably due in part to the fact that the inflammatory involvement differs in distinct parts of the joint [16]. Because most investigations have been performed using randomly obtained biopsy specimens, the uncertainty regarding the clinical value of additional histopathological examination could also reflect fortuitous sampling differences. In this study the degree of inflammatory activity of synovial tissue in rheumatoid knee joints was classified during arthroscopy by using rating scales. The results were compared to a panel of microscopic patterns in corresponding synovial biopsies that were stained using different histochemical and immunocytochemical techniques.

Key words: Arthroscopy - Inflammation - Morphology - Synovial tissue - Rheumatoid arthritis - Synovectomy

Patients

Patients and methods

Twenty-one patients (Table 1) undergoing arthroscopic examination of an arthritic knee joint were entered into the study. All had definite or classic RA [ 1] and were receiving no corticosteroids or slow-acting antirheumatic drugs. All patients satisfied criteria for disease activity

[12]. Arthroscopic evaluation of the knee joint is an excellent means with which to macroscopically analyze the inflammatory variations of the synovial tissue. In contrast, the usefulness of histological analysis in rheuma-

Presented in part at the Congress of the Socidt6 Mddicale Internationale d'Endoscopie et de Radiologie (S.M.I.E.R.), Mannheim, November 27-30, 1991 Offprint requests to: E. Stofft

Arthroscopy and synovial biopsy Endoscopic evaluations were performed by the same investigator after local anesthesia. The joint was continuously irrigated with saline under low pressure. A Storz 1.2-mm arthroscope was used. Areas of different inflammatory involvement were selected and scored for increased vascularity, hyperemia, and villous formation (Table 2). The patients were ranked according to the severity of inflammatory activity. After macroscopic evaluation synovial biopsies were obtained from the same areas. White fibrotic pathes were not dissected.

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Histologic preparation Biopsy samples were either snap-frozen in liquid nitrogen and then stored at -70~ or fixed in formalin and embedded in paraffin. Seven-micron sections were stained with PAS, hematoxylin-eosin, or Goldner trichrome staining. Peroxidase, acid phosphatase, and nonspecific esterase enzyme reactions were done [2]. Monoclonal antibodies (mABs) and neoglycoproteins were applied to determine the distribution of lymphocytes (mABs: L 26, UCHL 1), neutrophilic granulocytes (mAB: M1), and macrophages (mABs: OKM 1, MAK 1, CD 14, M814, 25 F9, neoglycoproteins: galactose-BSA, sialic acidBSA). The streptavidine-biotin technique or the alkaline phosphatase antibody (APAAP) method was used to reveal antibody binding.

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Histologic sections were coded and morphologic features scored separately by an independent observer. With the exception of granulocytes a nine-level rating scale was used (Table 3). Granulocytes were classified into three groups. Where the results varied throughout the section, the score corresponding to the predominant picture was taken.

Fig. 1. Arthroscopic examination of the synovial inflammation. The number of patients is ranked according to the mean inflammatory scores

Lymphocytes Lining cells The score was increased according to the predominant cell number of the synovial lining layer.

Because of a heterogenous cell arrangement the scores had to be attributed subjectively. The distribution pattern was graded as diffuse, mixed, or focal.

Blood vessels

Neutrophilic granulocytes and macrophages

The number of vessels was quantified using objective 100 (oil immersion) on an Olympus VCX 100 microscope. The presence of intravascular leukocytes was estimated.

Their number was determined using objective 100 (oil immersion). Because of the low number of granulocytes (see Results), peroxidase, nonspecific esterase, or galactose-positive cells were counted as macrophageso

Table 1. Characteristics of the patients studied

Mean age Gender (masc./fern.) Mean disease duration Rheumatoid factors Rheumatoid nodules ESR

Statistical methods

46 years (18-69) 8 masc./13 fem. 6.5 years (2-17) 18 pos./3 neg. 4 pos./17 neg. 57 mm/hr (14-95)

The correlation coefficient was calculated using the linear regression analysis. The significance of the correlations was examined by Student's t-test and the SAS system.

Results

Table 2. Method of arthroscopical scoring the inflammatory activity

Criterion

Score

Vascularity Hyperemia/edema Villus formation

0 0 0

"Sum"

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U n d e r a r t h r o s c o p i c e x a m i n a t i o n o n l y in t h r e e p a t i e n t s w a s n o r m a l - a p p e a r i n g s y n o v i u m s e e n in s m a l l a r e a s . I n f l a m m a t o r y a c t i v i t y w a s h o m o g e n e o u s in n i n e p a t i e n t s ( s c o r e : m e d i a n --- 1), w h e r e a s in 12 p a t i e n t s e n d o scopic appearance was remarkably variable within the j o i n t ( F i g . 1). T h e t r a n s f o r m a t i o n s w e r e e i t h e r b r i s k or successive. The highest inflammatory scores were i n v a r i a b l y s e e n c l o s e t o m e n i s c i o r c a r t i l a g e ( F i g . 2).

Table 3. Method of histologically scoring the synovial samples

Parameter Number of synovial cell layers Number of vessels per HPF a Percentage of lymphocytes per HPF Number of granulocytes b per HPF Number of macrophages per HPF a HPF: High-power field b Granulocytes: maximum score of 3

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>40

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Arthroscopically different groups within the same joint showed similar scores for lymphocytes. The majority of cells were UCHLl-positive T-lymphocytes. Focal aggregates of lymphocytes were found in biopsy specimens of nine patients and four revealed perivascular infiltrates. In sections from five patients lymphocytes were evenly scattered. Both distribution patterns were detected in seven patients. However, no correlations were found between the proof of lymphocytes and the endoscopic findings (Fig. 5). More than two Ml-positive granulocytes per highpower field were only detected in specimens resected from areas with macroscopically significant inflammatory activity (Figs. 6, 7). Analysis showed that the relationship between arthroscopic findings and the proof of macrophages was extremely dependent on the specific markers applied. Correlations between the number of mAb 25F9 binding cells and endoscopically determined inflammatory activity did not reach statistical significance. Using mABs OKM 1 (Fig. 8), M814

Fig. 2. Arthroscopic appearance of the synovium shows congestion of blood vessels and slight edematous swelling of villi. Patient 4, inflammation score 3 (*) and 5 (*)

Fig. 4. Intravascular monocytes, perivascular macrophages, and lining-cell hyperplasia. Patient 18, inflammation score 7

Fig. 3. Numerous vessels in the subsynovium without perivascular inflammatory cells and lining-cell hyperplasia. Patient 1, inflammation score 2

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The majority of patients showed scores for liningcell hyperplasia spanning more than 3 points. No significant correlations were found between synovial lining-cells hyperplasia and arthroscopic grading. The distribution patterns of blood vessels also varied significantly in most patients. Their numbers only weakly correlated with macroscopic inflammation scores. There were clusters of vessels in nearly uninflamed synovial tissue (Fig. 3), whereas specimens resected from areas graded arthroscopically "high activity" revealed often few vessels. However, intravascular leukocytes detected in more than one vessel were indicative of active synovitis (Fig. 4).

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Fig. 5. Scores for lymphocytes compared to the results of the macroscopic evaluation of inflammatory activity

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Fig. 6. One granulocyte in a synovial biopsy endoscopically evaluated as "low inflammatory activity" (score 2). Patient 5

Fig. 8. OKM-1 positive macrophages in the synovial tissue. Lining cells are negative. Patent 7, inflammation score 6

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Fig. 9. Multiple M-814-positive macrophages in the subsynovium. Same area as in Fig. 6

(Fig. 9) or CD 14 a low but significant correlation was found. Highly significant statistical correlations between the inflammatory score and the detection ofmacrophages were observed, if endogenous peroxidase activity or galactose affinity (Figs. 10, 11) was applied as markers. No relationship was found between the arthroscopic evaluation and the activity of the enzyme acid phosphatase or nonspecific esterase. Also, the binding patterns of sialic acid was not correlated with endoscopic observations. Discussion

The aim of the present study was to elucidate the value of arthroscopically obtained biopsies in the classification of patients with RA. However, several difficulties are inherent in our effort to compare macroscopic signs of inflammation with histologic sections. In our opinion, the most significant limitation in the assessment of the inflammatory activity during arthroscopy is subjec-

Fig. 10. Proof of galactose-binding macrophages. Patient 13, inflammation score 6

tive interpretation of the synovium. Although strict criteria were used, results can be influenced by variations in the physical conditions during endoscopical investigation - - such as water pressure, temperature,

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Fig. l l . S c o r e s for galactose-positive m a c r o p h a g e s c o m p a r e d to the results of the m a c r o s c o p i c e v a l u a t i o n o f i n f l a m m a t o r y activity

and the intensity of lightning [7]. Therefore, the possibility cannot be excluded that the same material will be interpreted in a contrasting fashion by different observers. In view of the limited knowledge concerning pathogenetic mechanisms in RA, the choice of parameters representing inflammatory activity has also to be critically discussed. In accordance with the studies of Ikeuchi [9], capillary hyperemia and edematous changes were chosen as indicators of acute inflammation. However, no significant correlations were found between the inflammatory activity and the number of small blood vessels. This finding could indicate that only distinct areas of the synovial vasculature are engaged in inflammatory reaction. We were not able to confirm the findings of Kurosaka et al. 111] that the vascular network was not detectable in most cases of RA. Because they were not ubiquitously present in the inflamed synovial membrane, other findings such as hemorrhages or exudation were omitted. On the other hand, it is certain that fibrin deposits are an important facet of the inflammatory reaction in RA [4]. The present endoscopic investigation demonstrates that numerous stages of inflammatory activity exist within a single joint in RA. The highest rating scales were often but not always confined to areas in the periphery of cartilage. Thus, the cartilage seems to be crucial for the propagation of synovitis. In contrast to hyperemia and edema, villus formation is part of chronic pathomechanisms propagating the immune response in RA. A generalized proliferation of synovial cells results in a 10-fold to 100-fold increase in cell numbers. Nonetheless, there is no direct relationship between inflammation and the development of aggressively invading "pannus." The main reasons for this may be a marked cellular heterogeneity in different areas of the rheumatoid subsynovium or differences in the inflammatory activity of lining cells [17]. This is in accordance with our finding that the number of lining cells were unsuitable as a marker of local inflammatory activity. Lindblad et al. and Rooney et al. also found no

correlations between endoscopic signs of inflammatory activity and the scores for synoviocyte hyperplasia [13, 16]. Numerous observations support the concept that there is no association between inflammation and the number of synoviocytes and cartilage damage in RA [17]. In contrast, the number of activated macrophages showed a close correlation with the endoscopic findings. Peroxidase activity, or the proof of endogenous lectins binding galactose, is indicative of macrophages that have recently arrived in tissues by emigration from the blood [20]. It was shown that in sections of active synovitis, synovial macrophages bear epitopes that are largely restricted to blood monocytes [6]. In contrast, mAB 25F9 hinds to inactive, resident histiocytes. The mABs M814, OKM 1, and CD 14 react with most of the monocytes/macrophages. Therefore, subpopulations with divergent cell dynamics and heterogenous stages of activation are detected. Our observation is consistent with the view that the degree of significance between endoscopic findings and the proof of macrophages is critically dependent on the panel of antimacrophage-ABs used. Neutrophilic granulocytes comprise over 90% of all cells found in the synovial fluid of patients with RA. It has been estimated that the turnover of these cells in a 30-ml rheumatoid joint effusion is greater than one billion [8]. However, only a few cells are detected in synovial articular tissue in RA. Therefore, a close relationship exists between the number of emigrating granulocytes and the release of inflammatory chemoattractants [10]. According to our results, the constant proof ofgranulocytes is the most reliable histologic marker of an acute inflammatory flare in RA. However, although these cells are thought to cause local destructions of surface structures, there is little evidence that they have a major involvement in destruction of cartilage [31. Conflicting opinions exist about the function of synovial lymphocytes. It has been suggested that lymphocytes are crucial in the pathogenesis of RA. In contrast, Muirden et al. found an inverse correlation between the degree of lymphocyte infiltration and joint damage [15]. In the present study, the number of these cells showed no correlation with endoscopic paramaters of inflammation. This is in accordance to the results of Lindblad and Hedfors, who frequently found "infiltrations of lymphocytes" in healthy synovium [14]. Also, absence of these cells has been reported in active inflammatory synovitis [5]. This provides additional evidence that lymphocytes and inflammatory activity are not directly correlated. However, these observations need to be interpreted with some care in view of current knowledge concerning lymphocyte subpopulations. In conclusion, the present investigation revealed a close correlation between arthroscopic findings and synovlal morphology. However, the relationship between the endoscopic findings and histopathological results was critically dependent on the parameters used. Our results indicate that endoscopic evaluation allows a valuable classification of the inflammatory ac-

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tivity in RA. Multiple biopsy is likely to give more information than resection of a single site. Acknowledgments. This research was supported by grants from the Deutsche Forschungsgemeinschaft (Zsch 7/2-1), The Bundesministerium far Forschung und Technologie (BMFT 01VM88205) and the Kemkes-Stiftung (Neu 18/91). Its a pleasure to recognize the expert technical assistence of Mrs. A. Bastelberger.

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