Examination of in vivo gelatinolytic activity in rheumatoid arthritis synovial tissue using newly developed in situ zymography and image analyzer.

Objective: The aim of this study was to examine in vivo gelatinolytic activity of rheumatoid arthritis (RA) synovium using a newly developed in situ zymography (ISZ) method and pathological image analyzer, and to evaluate the relationship between this activity and several features on RA.
Methods: A total of 8 samples of synovium were obtained from RA patients during surgery, and 8 samples from osteoarthritis (OA) patients were examined as controls. Furthermore, total 14 samples of syovium were obtained for comparison among radiographical classifications as Larsen grade (4 cases of grade III, 5 cases of grade IV and 5 cases of grade V). These specimens were frozen with OCT compound immediately after surgery. Frozen sections were applied to a newly developed gelatin-coated FIZ film (Fuji Film Co.Tokyo.Japan) designed for use ISZ, and incubated at 37 degrees C for 6 hours. Using an image analyzer (image processor for analytical pathology; IPAP), two variables were measured as indicators of in vivo gelatynolytic activity: optical density of gelatinolyzed area (ODG), and ratio of gelatinolyzed area (RGA). Also, we investigated the relationship between these indicators and the following variables: radiographic changes (Larsen grades), clinical data (C-reactive protein concentration), histological score of synovial tissue (modified Rooney's score), and expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 (assessed by immunohistochemistry).
Results: RA synovium had significantly higher RGA and lower ODG than OA, indicating higher gelatinolytic activity in RA. Synovium from cases with Larsen grade IV or V had significantly lower ODG than cases with grade III, but there was no significant difference in RGA between grades. There was no significant correlation between gelatinolytic activity (ODG or RGA) and either CRP or modified Rooney's Histological Score. The results of ISZ indicate that the gelatinolyzed areas were mainly localized in the lining area, with a small amount scattered throughout the stroma. The results of immunohistochemistry indicate that MMP-2, MMP-9, TIMP-1 and TIMP-2 were expressed in areas of gelatinolysis.
Conclusions: The present results indicate that in vivo gelatinolytic activity of synovium is stronger in RA than in OA. They also indicate that gelatinolytic activity of RA synovial cells is stronger in cases with Larsen grade IV or V than in cases with grade III, although the gelatinolyzed area is similar. Gelatinolytic activity, as indicated by optical density and the gelatinolyzed area, differed between regions, even within the same specimen, suggesting an imbalance between production of proteinases and their inhibitors. We believe that the present zymography method can contribute to the elucidation of biological enzymatic activity of RA synovium.