Analysis of Regulatory T cells using a novel in vivo Human Cell Model for Rheumatoid Synovitis

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 14580802
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: St. Marianna University School of Medicine
• Principal Investigator: YAMADA Hidehiro St.Marianna University School of Medicine, Department of Internal Medicine, Associate Professor, 医学部, 助教授 (00174730)
• Project Period (FY): 2002 – 2004
• Keywords: rheumatoid arthritis / animal model / suppressor T cell / pannus

Research Abstract

Objective : To study a role of T-cells infiltrating in the rheumatoid synovial tissues on the development of pannus tissue and the subsequent joint destruction in our recently established in vitro and in vivo models for rheumatoid synovitis.
Methods : Synovial tissues were obtained from 9 patients with RA who underwent joint replacement. Single suspensions of synovial tissue-infiltrating cells(STICs) were obtained after 3 days culture of synovial tissues. T cells were depleted by negative selection using immunobeads-coupled anti-CD3/CD4/CD8 mAbs. In an in vitro model, STICs were cultured in medium for 3-4 weeks to evaluate the development of pannus-like inflammatory tissues in vitro under an inverted microscope. Cytokines produced in the cultured supernatents were measured by ELISA kit. In an in vivo model, single cell suspensions of STICs were injected into both the right knee joint and the right dorsal subcutaneoum of the foot of SCID.bg mice at the age of 7 weeks. Mice were sacrifice d after 7 weeks. Histologic characteristics of their joints were examined in a blinded manner.
Results : In an in vitro model, STICs contain lymphocytes, macrophages and fibroblastoid cells. When cultured in medium alone, they continuously proliferate and endogenously produce proinflammatory cytokines such as TNF-a, M-CSF, IL-6,IL-8, and MMP-9. Fibroblastoid cells grow rapidly and show transforming phenotype. These cells finally form a macroscopic organized inflammatory tissue after 3 weeks of cultures, Histologically they have many features of pannus seen in rheumatoid arthritis. Both the in vitro development of pannus-like tissue and the production of proinflammatory cytokines were enhanced by depleting T-cells from STICs. This enhancement was reproduced by depletion of CD25-positive cells, but not by the depletion of CD4,CD8 or CD45RO-positive cells. When monocytes/macrophages were depleted from STICs, the remaining cell population failed to develop pannus-like tissue. In an in vivo model, injecting STICs into SCID bg. mice resulted in the development of pannus tissue and its invasion of joint cartilage and bone, which was rather enhanced when T-cell-depleted STICs were injected. This enhancement was reproduced by injecting CD25-depleted STICs.
Conclusion : The present study indicates that the majority of T cells infiltrating synovial tissues play an autoregulatory role on the development of pannus tissue and the subsequent joint destruction in RA.