類風溼性關節炎病人Chemokines及Chemokine Receptors在不同TH細胞亞型上的表現

行政院國家科學委員會專題研究計畫報告

計畫編號：NSC89-2314-B006-098

執行期限：88 年 8 月 1 日至 89 年 7 月 31 日

主持人：王崇任 成功大學醫學院學士後醫學系

中文摘要

類風濕性關節炎（RA）是一種滑膜 發炎性疫病，屬於自體免疫性成因，會造成 關節的破壞與變形。由於關節滑膜腔內某些 抗原被具有 MHC Class II 抗原提示細胞提 供給 CD4 T 細胞，進而活化這些 T 細胞。 一些釋放出的趨化因子（chemokines）可吸 引更多的 T 細胞、B 細胞、單核球等至發炎 部位擴大反應。 近來的報告指出 Th1 及 Th2 細胞由 於具有不同的 chemokine receptors 會與不同 的 chemokine 產生反應。Th1 細胞表現出 CCR5 receptor，Th2 細胞表現出 CCR3 及 receptor，兩者則皆有 CXCR4 及 receptor。 因此 T 細胞上的 chemkine receptors 的表現 種類可以分析侵襲關節滑膜的 Th 細胞亞 群。 本研究計劃將檢查 150 位 RA 病人周 邊血液及其中一些病人的關節液及關節組 織中 T 細胞上 chemokine receptors CCR3、 CXCR4、CCR5 的分佈情形，並以 110 位健 康正常人做為對照組。同時檢查具有 CCR5 表面標誌的 CD4 T 淋巴球以 PMA/inomycin 刺激後其 cytokine 產生的變化情形。此外白 種人被報告帶有 CCR5 欠損變異型基因，本 研究抽取病人及正常人周邊血的 DNA，利 用 PCR 來做 CCR5 基因型的鑑定。結果顯 示 RA 病人周邊血液 CD4 T 細胞上 CCR5 的表現較正常人低，而其關節液及關節組織 內的 T 細胞則有顯著的量的 CCR5 表現。 CCR5 陽性的 CD4 細胞經刺激後產生 IFNγ 而非 IL4。總共 260 位中國人並未找到 CCR5 delt 32 的變異型基因。經由這些研究發現 RA 病人關節發炎時 Th1 細胞亞群利用其細 胞上的 chemokine receptors CCR5，造成細 胞移動而侵襲滑膜。希望此項研究能針對類 風濕性關節炎的病態生理及未來之免疫療 法有所助益。 Introduction

Rheumatoid arthritis (RA) is an inflammatory disease involving synovium and causing destruction of joint. Some antigen peptides within synovial cavity are presented by MHC class II antigen presenting cells to CD4 T cells. Activated CD4T cells can be further recruited to the inflammatory sites by some humoral factors released from synovial mononuclear cells and fibroblasts. The migration of T cells depends on the combined action of adhesion molecules and chemotactic factors (chemokines). Chemokines released from inflammatory tissue can recruit T cells with some specialized chemokine receptor

cells with different chemokine receptors can response to different chemokines. Accumulated data show that Th1-type response is related to the pathogenesis of RA. In addition, the animal model of RA demonstrated an inflammatory role of Th1 cells and anti-inflammatory role of Th2 cells. However, the control mechanism for Th1 cells recruiting to the inflammatory sites remains unknown. CCR5 is preferentially expressed on Th1 cells, and CCR3 is preferentially expressed on Th2 cells. However, CXCR4 receptors is expressed both on Th1 and Th2 cells. Data concerning the presence of chemokine receptors on T cells and their response to T cells are limited in RA patients. In this research, we will examine the expression of chemokine receptors on T cells from peripheral blood, synovial fluid, and synovial tissue of RA patients. The CCR5+ CD4 T cells will be stimulated with PMA/ ionomycin and the change in the expression of cytokine, IL4 and IFNγ will be measured. In addition, genomic DNA prepared from RA patients will be analyzed for CCR5 genotype by polymerase chain reaction (PCR). Through this study we can understand how different subsets of Th cells recruit into synovium by using different surface expression of chemokine receptors in RA patients.

Mater ials and Methods

One hundred and fifty RA patients, with diagnosis according to the revised criteria of the American College of Rheumatology, were enrolled into the study project. Their clinical

severity and associated laboratory finding were recorded. Synovial fluids were available from therapeutic aspiration of inflammatory joint and synovial tissue was taken during operation. Peripheral blood were drawn simultaneously. Another 110 healthy individuals were served as a control group.

Preparation of mononuclear cells

Peripheral heparinized blood will be subjected to Ficoll-hypaque gradient and mononuclear cells are purified through this procedure. Synovial fluids are to be incubated with 15 units / ml hyaluronidase for 30 mins at 370 C and then passed through a wire mesh. After spinning down and resuspending in RPMI-1640, the mononuclear cells are separated by Ficoll-hypaque gradient.

Flow cytometr y

The purified mononuclear cells were stained with Cychromec-conjugatted anti-CD3 and PE-conjugated anti-CD4 or anti-CD8 and FITC-conjugated anti-chemokine receptor CCR3, CXCR4 and CCR5 Abs for 30 mins on ice in the dark. After washing the stained cells are analysed with FACSort (Becton Dickison) and associated software programs for three colored stainings. Mononuclear cells were stimulated with PMA/inmycin and subjected to CD4 and CCR5 surface staining and followed by IFN γ and IL-4 cytoplasmic stainings.

PCR for CCR5 genotype

fragment of the CCR5 gene containing the potential 32 bp deletion will be amplified by a 35-cycle PCR using 5, -CCT-GGC-TGT-CGT-CCA-TGC-TG (sense) and 5, -CAA-GCA-GCG-GCA-GGA-CCA-GC (antisense) primers. The products of the PCR are electrophoresed on ethidium bromide-stained agarose gels.

Results

CCR5 expression on peripheral T cells from RA patients was lower on CD4 and double negative T cells than those from normal individuals (Fig. 1). Expression of CCR5 on CD4 T cells were much higher in synovial fluid than in peripheral blood. However, no significant difference was found in expression of CXCR4 and CCR3. IFNγ but not IL-4 production from stimulated CCR5+ CD4 T cells of RA patients was demonstrated in Fig. 2. Although CCR5 delta 32 gene deletion has been reported in Caucasian, there was no such polymorphism in Chinese (Fig. 3).

Dicussion

Patients with RA are one of most frequently encountered diseases in rheumatological clinics in Taiwan. RA is an autoimmune disease characterized by lymphocytes acculmulation within the synovial compartment and activated CD4 T cells predominate in the infiltrating mononuclear cells of inflamed joints. CD4 T cells have been classified into 2 major types

on the basis of their cytokine profiles and functional characteristics. Th1 cells secrete IFNγ and are associated with cellular immunity, whereas Th2 cells produce IL-4 and are involved in humoral immunity. Th1-type immune responses have been suggested to be important for the pathogenesis of organ-specific autoimmune diseases. Infiltration of Th1 phenotype into target organs is a characteristic of RA patients.

Chemokines are a superfamily of small structually-related cytokine molecules which induce directional migration of cells. The chemokine receptors comprise two major groups; the CC and CXC chemokine receptors. Recent studies show that chemokines receptors are expressed on T lymphocytes depending on their state of differentiation; CCR5 is preferentially expressed on Th1 cells, while CCR3 is linked to expression on Th2 cells. These data suggest that the differential expression of chemokines receptors may be useful for discriminating pathogenic Th cell types. In addition, chemokine may be part of effector and amplication mechanisms of polarized Th1 and Th2 ediated immune responses. From results of the present study, the decrease of CCR5+ CD4 T cells in peripheral blood from RA patients may be due to preferential accumulation of these cells in inflamed joint. This subset of T cells, by producing IFNγ can regulate Th1-mediated immune responses in synovial sites.

Caucasian popluations. Several reports have indicated that homozygosity for this deletion allele protect almost completely from human immunodeficiency virus transmission . No 32 base pair deletion of CCR5 gene was found in Chinese RA patients and healthy individuals. Therefore, the role of delta 32 CCR5 mutant allele in protection of RA remains unclear in Chinese population.

A subset of human DN T cells is CD1 restricted and shows preferential Vα24 T cell receptor usage analogous to Vα14 usage in the murine NK 1.1 subset. Upon activation, IL-4 and IFNγwere detected in supernatants of DN T cells from healthy individuals. No difference in number of DN T cells of peripheral blood was found between RA patients and healthy controls. Increased CCR5+ DN T cells infiltration in synovial cavity may play a role in pathogenesis of RA.

In conclusion, infiltration of IFN γ secreting CCR5+CD4 T cells into the joint cavity of RA patients suggests a role of Th1 cells in pathogenesis of inflamed synovium.

Acknowledgements

This work was supported by grants from the National Science Council, R.O.C. (NSC 89-2314-B006-098). I thank Ms. Wan-ching Cathy Lin for technical assistance.

Fig.1

Fig.2