［摘要］目的：评价口服鸡源性Ⅱ型胶原蛋白(chicken type Ⅱ collagen, CCⅡ)对胶原性关节炎(collagen-induced arthritis, CIA)小鼠的抗炎及免疫作用的影响。方法：以CCⅡ蛋白与福氏完全佐剂免疫昆明种小鼠，并于第21天强化免疫1次。CCⅡ于致敏前第5天即开始灌胃给药，共20 d。每周两次评价多发性关节炎评分。以ELISA法测定CIA小鼠血清抗CⅡ抗体。以流式细胞测定法测定小鼠脾T淋巴细胞亚型。以ELISA法测定佐剂性关节炎大鼠腹腔巨噬细胞IL-1分泌水平。结果：口服CCⅡ 5 μg^．kg^－1和50μg^．kg^－1可明显降低多发性关节炎评分值，而250μg^．kg^－1剂量组则无作用；CCⅡ 5μg^．kg^－1剂量组可抑制CIA小鼠升高了的血清抗CⅡ抗体水平；口服CCⅡ可使CIA小鼠升高的L3T4⁺/Lyt-2⁺值有所降低。口服CCⅡ可使佐剂性关节炎大鼠升高了的IL-1水平有所降低。结论：口服CCⅡ可抑制CIA小鼠多发性关节炎的发生，此作用可能有体液免疫和细胞免疫两种机制参与。以上作用的发生为剂量依赖性的，即小剂量作用最强。

［中图分类号］ R979.5［文献标识码］A

［文章编号］ 1000-1530（2000）03-0214-05

Induction of immunologic tolerance to collagen induced arthritis

mice by oral administration of chicken type Ⅱ collagen

LI Wei-Dong，SHEN Jian-Ying，TENG Hui-Ling，LIN Zhi-Bin

（Department of Pharmacology, School of Basical Medical Sciences,

Peking University, Beijing 100083,China）

RAN Gou-Xia

（People's Hospital, Xinjiang Weiwuer Autonomous Region）

ABSTRACTObjective： To assess the immunological effects by orally administering chicken type Ⅱ collagen(CCⅡ) on collagen-induced arthritis（CIA）mice. To assess the effect on producing IL-1 of peritoneal macrophage in adjuvant arthritis rats by orally administering CCⅡ. Methods： Arthritis were induced in Kunming mice by immunization with chicken type Ⅱ collagen with Freund's complete adjuvant, followed by an interperitoneal injection of CCⅡ 3 weeks later.Chicken type Ⅱ collagen was orally administered from 5 days prior to the induction of arthritis to 14 days after inducing arthritis model. The animals were examined visually twice weekly for polyarthritic signs of swollen and erythemic limbs. Quantitation of antibody level of CIA mice was measured by ELISA method. Subpopulations of T lymphocytes in mice were evaluated by flow cytometry method. IL-1 assay was evaluated by ELISA method. Results: Joint swelling was significantly reduced at a dose of 5 μg^．kg^－1 and 50μg^．kg^－1 of CCⅡ, but not at 250 μg^．kg^－1.The level of anti-collagen antibodies was also reduced at a dose of 5μg^．kg^－1 and 50μg^．kg^－1 (OD value from CIA model control 0.242±0.073 to CCⅡ 5 μg^．kg^－1 0.123±0.029 and CCⅡ 50 μg^．kg^－1 0.110±0.075 respectively). Subpopulations of T-lymphocytes were changed by orally administering of CCⅡ, and the ratio of L3T4⁺/Lyt-2⁺ was lowered(the ratio from 1.71 of CIA model control to 1.21, 1.51 of administered CⅡ 5 μg^．kg^－1,50μg^．kg^－1 respectively.) after administering CCⅡ. IL-1 level can be reduced (the value from adjuvant arthritis model control 62.8±0.9 to 43.4±1.3, 49.7±0 ng^．L^－1 administered CⅡ 5 μg^．kg^－1,50μg^．kg^－1 respectively). Conclusion： Arthritis sign in CIA animal model can be suppressed by oral CCⅡ. The effects may be involved by influencing the mechanisms both humoral and cellular immunity. The effects occurred at lower doses of CCⅡ. These results demonstrated the biologic relevance of by-stander suppression associated with oral tolerance, and the potential use of this approach to treat human inflammatory joint diseases.

KEY WORDS

KEY WORDSArthritis, adjuvant; Immune tolerance; Collagen; Chicken type Ⅱ collagen; IL-1▲

Oral tolerance, a method of inducing antigen-specific tolerance, also called as a state of immunological unresponsiveness, has been shown to suppress autoimmunity in a number of animal models, including arthritis models^［1～3］.

Oral tolerance is an ideal therapy that decreases inflammation in the joint by a disease-specific mechanism and lacks toxicity. Administering antigen by oral leads to systemic hyporesponsiveness, and as we know, low doses antigen favor the generation of active suppression^［4］. Anergy may also be a mechanism for oral tolerance.

Rheumatoid arthritis(RA）， is a common chronic illness in which the synovial membrane of multiple joints becomes inflamed, causing damage to cartilage and bone.RA is associated with a variety of immune abnormalities of both T and B cells,which include decreased capacity for delayed hypersensitivity-type response and impairment of several T cell functions.In addition, RA is associated with excessive immunoglobulin production and formation of autoantibodies. Evidence suggests that, in some circumstances,excessive production of immunoglobulin and formation of autoantibodies may result from a defect in suppressor T cell function.Support for this concept is provided by animal models of autoimmune disease,primarily the New Zealand mice, in which the loss of suppressor T cell function precedes the development of autoantibodies^［5］.Dysfunction of suppressor T cells can be also found in the peripheral blood lymphocytes from patients with RA.

Immunization of animals by type Ⅱ collagen (CⅡ), a major component of cartilage, induced an arthritis, termed collagen-induced arthritis (CIA)^［6］,which clinically and histologically resembles rheumatoid arthritis (RA) in certain respects^［7］.The development of arthritis is associated with high levels of cell-mediated immunity as well as humoral immunity to type Ⅱ collagen.It has been regarded as an important model of these human diseases.

Native chicken type Ⅱ collagen was purified from lathyritic chicken articular cartilage preparations by the method of Cremer, et al^［8］.

Although oral tolerance has been studied for a number of antigens,the effect of oral administration of chicken type Ⅱ collagen on CIA mice has not been reported. The aim of this study was to investigate the immunopharmacologic effects of intragastric administration of soluble chichen type Ⅱ collagen on CIA mice.

1MATERIALS AND METHODS

1.1Animal

Kunming male mice, 4－6 weeks old, Wistar male and female rats, (200±40) g, were provided by the Department of Experimental Animals, Health Science Center, Peking University.

1.2Chicken type Ⅱ collagen

Native chicken type Ⅱ collagen was purified from lathyritic chicken preparations by the method of Cremer， et al^［8］.

1.3Induction of arthritis

Native type Ⅱ collagen was solubilized in 0.1 mol·L^－1 acetic acid at 1 g·L^－1, and its stable emulsion was emulsified in an equal volume of complete Freund's adjuvant （CFA）.Mice were primed with 100μg of type Ⅱ collagen in CFA injected intradermally at four to<