Strong expression of Bcl-2 in pigmented villonodular synovitis of the knee with aggressive clinical behaviour.

OBJECTIVE: To determine the expression of bcl-2, p53, and caspase 3, and measure the Ki-67 proliferation index as well as DNA content and DNA fragmentation in a case of diffuse pigmented villonodular synovitis (PVNS) of the knee with aggressive clinical behaviour. METHODS: Expression of p53, Bcl-2 and Ki-67 was investigated using immunohistochemistry. In addition, multiparametric flow cytometry was performed for expression of p53, bcl-2, and caspase 3, as well as analysis of DNA content and distribution of cell cycle phases. DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL). RESULTS: A strong cytoplasmic positivity for Bcl-2 protein, a key factor in regulation of apoptosis, was found in the majority of proliferating synovial cells. No apoptotic cell fraction was found by analysis of DNA content. DNA fragmentation was observed in 6.8% of cells. No elevated expression of p53 and caspase 3 was detected. CONCLUSION: Our results indicate a possible role of dysregulation of apoptosis in this case of PVNS. This aspect in the pathogenesis of PVNS should be clarified in further studies.

Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

AIMS: Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. METHODS AND RESULTS: Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. CONCLUSIONS: In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

Expression pattern of cell cycle-related gene products in synovial stroma and synovial lining in active and quiescent stages of rheumatoid arthritis.

OBJECTIVE: To investigate the expression pattern of cell cycle related gene products in active and quiescent Rheumatoid arthritis (RA). METHODS: Synovial tissue from 20 patients with active proliferative RA and 28 patients with RA in remission was immunohistochemically examined for expression of p53, p63, p21, p27, p16, cyclin D1, CDK4, RB, E2F, Ki-67 on tissue microarrays and by DNA flow cytometry for cell cycle phases. RESULTS: Elevated expression of p53 and p27 was found in synovial lining and in stromal cells in proliferative active RA. In the remission stage this finding was confined to the synovial lining. Most of the cells were in the G0-phase. Ki-67 proliferation index was maximum 10% in synovial cells. CONCLUSION: The p53 pathway is activated in synovial cells in active RA as well as in quiescent stage of disease. Differences in the spatial expression pattern of proteins involved in the p53 pathway in RA in remission compared to actively proliferating RA reflect the phasic nature of the disease and support in our opinion the concept of adaptive role of p53 pathway in RA.

Apoptosis resistance in pigmented villonodular synovitis.

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.

Comparative analysis of cell populations involved in the proliferative and inflammatory processes in diffuse and localised pigmented villonodular synovitis.

The aim of the present study was a comparative quantitative evaluation of cell populations involved in the proliferative and inflammatory compartment in both localised and diffuse pigmented synovitis villonodularis (PVNS). 15 cases of each localised and diffuse PVNS were examined by flow cytometry, immunohistochemistry, double immuno-fluorescence and confocal microscopy with quantitative evaluation of CD3-, CD4-, CD8-, CD20-, CD57-, CD55-, CD68-, CD163- and h4Ph positive (+) cells. The proliferative compartment of localised and diffuse PVNS was mainly composed of double-positive CD68+/h4Ph+ (CD163+/CD55+) synoviocytes. The number of double-positive synoviocytes for macrophage and fibroblast markers was significantly higher in diffuse compared to localised PVNS. The accompanying inflammatory infiltrate showed a predominance of cytotoxic cells (CD8+, CD57+), whereby the number of CD3+ and CD20+ cells was significantly higher in localised PVNS. The number of CD57+ NK cells was significantly higher in diffuse PVNS. The proliferating macrophage- like synovial cells and the cytotoxic lymphocytes could contribute to the aggressive behaviour of localised and diffuse PVNS. Moreover, with regard to the quantitative differences in cell composition between diffuse and localised PVNS and their different clinical behaviour, further studies should continue to analyse localised and diffuse PVNS separately.