Histological Patterns of Synovitis and Serum Chemokines in Patients with Rheumatoid Arthritis PIOTR ADRIAN KLIMIUK, STANISLAW SIERAKOWSKI, ROBERT LATOSIEWICZ, JAN SKOWRONSKI, JACEK PRZEMYSLAW CYLWIK, BOHDAN CYLWIK, and JUSTYNA CHWIECKO ABSTRACT. Objective. Studies indicate the genetic, biological, and clinical heterogeneity of rheumatoid arthritis (RA). Recently the histological diversity of RA has been postulated. We investigated whether serum concentrations of interleukin 8 (IL-8), RANTES (regulated upon activation normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) are correlated with histological appearance of the rheumatoid synovitis. Methods. Using ELISA we assessed IL-8, RANTES, and MCP-1 concentrations in serum of 47 patients with RA and 30 patients with osteoarthritis (OA). Results. Morphological analysis of synovial specimens distinguished 2 types of rheumatoid synovi- tis. Twenty-eight RA samples presented diffuse infiltrates of mononuclear cells with no specific microanatomical organization and were categorized as diffuse synovitis. In the remaining 19 speci- mens, classified as follicular synovitis, formation of lymphocytic follicles with germinal center-like structures was observed. Serum levels of studied chemokines were increased in patients with RA compared to the OA control group (p < 0.001 for all comparisons). Concentrations of IL-8, RANTES, and MCP-1 were highest in serum of RA patients with follicular synovitis in comparison with patients with diffuse synovitis (p < 0.01, p < 0.01, and p < 0.05, respectively) and could dis- tinguish RA patients with these 2 histological disease patterns. Serum levels of chemokines corre- lated with markers of disease activity such as erythrocyte sedimentation rate, C-reactive protein con- centrations, and Disease Activity Score. Conclusion. Distinct histological variants of rheumatoid synovitis associated with different serum levels of IL-8, RANTES, and MCP-1 reflect clinical activity of the disease and confirm the concept of RA heterogeneity. (J Rheumatol 2005;32:1666–72) Key Indexing Terms: INTERLEUKIN 8 RANTES MONOCYTE CHEMOATTRACTANT PROTEIN-1 RHEUMATOID ARTHRITIS SYNOVITIS HISTOLOGY Rheumatoid synovium is characterized by infiltration of The majority of rheumatoid synovia reveal only diffuse lymphocytes, macrophages, synoviocytes, and plasma cells. infiltrate of mononuclear cells, without additional micro- Enhanced angiogenesis and proliferation of the synovium anatomical organization, and may be classified as diffuse lining layer may also be observed. All these cells are sup- synovitis. In about one-third of RA synovia, formation of posed to contribute to the pathogenesis of rheumatoid arthri- lymphoid follicles was observed4-6. These T-B cell aggre- tis (RA) by several mechanisms, including chemokine pro- gates, which sometimes form germinal-like centers, are duction1,2. Chemokines have an important role in infiltra- thought to play a role in the pathogenesis of RA7,8. Such RA tion of rheumatoid synovium with mononuclear cells, lead- synovia may be categorized as follicular synovitis4. ing to initiation and progression of the disease3. Necrobiotic granulomas with a fibrinoid necrotic center lined by a collar of histiocytes were found only in individ- From the Department of Rheumatology and Internal Diseases, ual patients with RA. The presence of follicular structures Department of Rehabilitation Medicine, Department of Orthopaedics and and granulomatous necrobiosis was not observed in the Traumatology, and Department of Clinical Pathology, Medical University 4 of Bialystok, Bialystok, Poland. same patient . P.A. Klimiuk, MD, PhD, Assistant; S. Sierakowski, MD, PhD, DSc (Med), Further studies showed that patients with the formation Professor and Chair; J. Chwiecko, MA, PhD, Principal Technician, of lymphoid follicles tend to have a higher degree of Department of Rheumatology and Internal Diseases; R. Latosiewicz, MD, immunological activation and increased risk of joint PhD, Chair, Department of Rehabilitation Medicine; J. Skowronski, MD, 9,10 PhD, Professor and Chair, Department of Orthopaedics and destruction . Distinct histological variants of rheumatoid Traumatology; J.P. Cylwik, MD, Assistant; B. Cylwik, MD, PhD, synovitis were associated with the specific pattern of Assistant, Department of Clinical Pathology. cytokine production in synovium4, serum cytokine9, and Address reprint requests to Dr. P.A Klimiuk, Department of Rheumatology matrix metalloproteinase10 concentrations. Thus, in addition and Internal Diseases, Medical University of Bialystok, M.C. Sklodowskiej 24a, 15-276 Bialystok, Poland. E-mail: [email protected] to genetic, biological, and clinical heterogeneity, the histo- 4,5,12-14 Accepted for publication April 22, 2005. logical heterogeneity of RA has also been suggested . Personal non-commercial use only. The Journal of Rheumatology Copyright © 2005. All rights reserved. 1666 The Journal of Rheumatology 2005; 32:9 Downloaded on September 28, 2021 from www.jrheum.org We investigated whether serum concentrations of chemo- RESULTS kines are associated with different histological appearances Histological findings. Synovial samples from RA patients of the RA synovitis. were characterized by mononuclear cell infiltrates, and con- sisted mainly of lymphocyte- and macrophage-like cells and MATERIALS AND METHODS plasma cells. Twenty-eight specimens revealed diffuse lym- Study groups. Forty-seven patients fulfilling the American College of phocyte infiltration, without any further microanatomical 15 Rheumatology 1987 revised criteria for RA and 30 patients with organization, and were categorized as diffuse rheumatoid osteoarthritis (OA) constituting the control group were recruited for study. Synovial samples were obtained during hip or knee joint orthopedic surgery synovitis. Lymphocyte follicular aggregates, sometimes from all RA and OA patients. Study procedures were approved by the insti- with germinal center-like structures, were observed in 19 tutional ethics committee. Characteristics of patient populations are shown samples. Such RA synovia were classified as follicular syn- in Table 1. ovitis. In rheumatoid specimens, synovial lining layer pro- Clinical and laboratory evaluation. Clinical and laboratory evaluations liferation, rare giant-like cells, and neoangiogenesis were were performed prior to surgery. The analysis included the number of ten- also observed. Formation of necrobiotic granulomas was not der joints (Ritchie index)16, number of swollen joints, erythrocyte sedi- mentation rate (ESR), Disease Activity Score (DAS)17, C-reactive protein detected. OA synovial samples revealed only mild mononu- (CRP) concentration, and rheumatoid factor level. Joint destruction was clear cell infiltrates. Representative examples of OA and 2 evaluated according to Steinbrocker criteria18. different forms of rheumatoid synovitis are presented in Histopathological analysis. Synovial specimens underwent routine staining Figure 1. with hematoxylin and eosin. Histological analyses included assessment of Demographic and clinical data. We did not observe differ- the mononuclear cell infiltrate density and their microanatomical organiza- tion, as described4,9,10. ences in sex profile, age, or disease duration between Serum samples. Blood specimens were obtained prior to surgery and were patients with both histological types of RA and with OA. clotted for 30 min and then centrifuged for 10 min at 1000 g. Serum ESR and CRP levels were enhanced in RA compared to the aliquots were frozen at –80°C immediately after collection. OA group (in all cases p < 0.001), especially in patients with Chemokine assays. Concentrations of chemokines [interleukin 8 (IL-8), follicular rheumatoid synovitis (Table 1). RA patients with RANTES (regulated upon activation normal T cell expressed and secreted), the follicular form of synovitis were also characterized by and monocyte chemoattractant protein-1 (MCP-1)] were assessed with higher numbers of swollen joints and DAS than those with ELISA kits (R&D Systems, Wiesbaden-Nordenstadt, Germany). Measurements were carried out strictly according to the manufacturer’s diffuse synovitis (p < 0.01 and p < 0.05, respectively). directions. Sensitivities of the assays were 10 pg/ml (IL-8), 8 pg/ml Roughly 64% and 79% of RA patients with diffuse and fol- (RANTES), and 5 pg/ml (MCP-1). licular synovitis, respectively, were seropositive (Table 1). Statistical analysis. Normally distributed data were analyzed by unpaired All patients had been using nonsteroidal antiinflammatory Student t test. Mann-Whitney U test was used to evaluate differences drugs (data not shown). Disease modifying antirheumatic between non-normally distributed data for ESR, RANTES, and MCP-1 val- drugs (DMARD) were more often taken by patients with the ues. The probability of differences in frequency distributions was deter- mined by chi-square test. Correlations between study variables were follicular histological type of RA, but the difference was defined using Spearman’s rank order test. P values less than 0.05 were con- significant only in the case of methotrexate (MTX; Table 1) sidered statistically significant. (p < 0.05). More advanced joint destruction (Steinbrocker Table 1. Patient characteristics. Data presented as means ± SD unless stated otherwise. OA Diffuse Follicular p Rheumatoid Rheumatoid Synovitis (A) Synovitis (B) OA vs A OA vs B A vs B Female/male 22/8 23/5 16/3 NS NS NS Age, yrs 57.4 ± 13.7 52.2 ± 12.1 57.8 ± 13.5