口服鸡源性Ⅱ型胶原蛋白诱导实验性CIA小鼠免疫耐受
作者：李卫东 冉国侠 沈建英 滕惠玲 林志彬
单位：李卫东 林志彬 沈建英 滕惠玲(北京大学基础医学院药理学系，北京 100083)；冉国侠(新疆维吾尔自治区人民医院)
关键词：关节炎；佐剂性；免疫耐受；胶原；鸡源性Ⅱ型胶原；白细胞介素-1
北京医科大学学报000306 [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250 μg.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。
, 百拇医药
[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)
, http://www.100md.com
RAN Gou-Xia
(People's Hospital, Xinjiang Weiwuer Autonomous Region)
ABSTRACT Objective： 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 50 μg.kg-1 of CCⅡ, but not at 250 μg.kg-1. The level of anti-collagen antibodies was also reduced at a dose of 5 μg.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.
, http://www.100md.com
KEY WORDS
KEY WORDS Arthritis, 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.
, 百拇医药
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.
, 百拇医药
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.
, http://www.100md.com 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.
, 百拇医药
1 MATERIALS AND METHODS
1.1 Animal
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.
1.2 Chicken type Ⅱ collagen
Native chicken type Ⅱ collagen was purified from lathyritic chicken preparations by the method of Cremer， et al[8].
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1.3 Induction 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 100 μg of type Ⅱ collagen in CFA injected intradermally at four to six sites on the back. Control mice were primed with acetic acid emulsified in CFA. Mice were challenged after 21 d with 100 μg of type Ⅱ collagen in 100 μl acetic acid, injected intraperitoneally. Control mice received 100 μl of 0.1 mol.L-1 acetic acid alone, intraperitoneally.
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1.4 Evaluation of arthritis[6]
The incidence of arthritis was defined as the number of mice that had clinical evidence of arthritis within 42 days after induction of disease. The severity of arthritis was graded according to standard methodology. Each of the four paws was graded as follows: 0 = normal, 1 = redness only, 2 = redness plus mild swelling, 3 = severe swelling, and 4 = joint deformity.The arthritis score for each animal was the sum of the score for each of the four paws. The maximum arthritis score was the highest score of an individual animal during the entire course of the disease.All evaluations were performed in a blinded fashion without knowledge of the treatment group.All mice were examined daily for the initial visual appearance of arthritis.
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1.5 Antibody quantitation
Mice were bled for sera on day 40.
1.6 Immunoassay of antibody to CⅡ[9]
Sera from mice were obtained by tail bleeding. Anti-collagen antibody was measured by an enzyme-linked immunosorbent assay (ELISA). Briefly, micro-ELISA plates were coated with CⅡ dissolved in phosphate-buffered saline(PBS) 0.2 mol.L-1(50 mg.L-1), 100 μl/well. Sera were diluted 1∶400, 100 μl/well. Peroxides-conjugated rabbit anti-rat IgG was added at a 1∶1 600 dilution. Levels of antibody were expressed as rectified absorbance(A490 nm).The passive sera obtained from CIA mice 40 d after immunization were pooled and diluted 1:400, and carried in each ELISA plate.
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1.7 T lymphocyte subsets assayed by flow cytometry method
Spleen cells were washed with PBS and diluted in RPMI 1640 medium supplemented with 5% fetal calf serum. Cells (2.5×105) were incubated with a 1∶100 dilution of mouse anti-rat L3T4 or anti-rat Lyt-2 monoclonal antibodies for 30 min at 4℃. After incubation, the cells were washed twice with PBS. The cells were incubated with FITC-conjugated goat anti-rabbit IgG for 30 min at 4℃ and analysed by flow cytometry(FACScan, Becton Dickinson). The percentages of cells positive for green (FITC) fluorescence were counted.
, 百拇医药
1.8 IL-1 producing and assay
PM from the rats administrated by CCⅡ were obtained ip injected with 10% thioglycolate broth (Difco, 1 ml/rat, 4 d later). The peritoneal cavities were lavaged with RPMI1640 media. The cells were washed 3 times in RPMI1640 media. The cells placed in 24-well plates at 2×106 cells/well, and allowed to adhere at 37℃ in a 5% CO2 for 1 h. Nonadherent cells were washed out. IL-1 was induced with LPS (5 mg.L-1) from PM, then the plates were incubated at 37℃ in a 5% CO2 atmosphere for 24 h. After incubation, the supernatant was collected by centrifugation(600×g, 10 min) and stored at -25℃ until assay. IL-1 activity was evaluated by ELISA method.
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1.9 Statistical analysis
One-way analysis of variance and Student's t-test were used.
2 RESULTS
2.1 Effect of feeding chicken type Ⅱ collagen on CIA mice
The effects of orally administering chicken type Ⅱ collagen on CIA mice were studied.The effect was observed as measured by incidence of arthritis, incidence of arthritic limbs, and maximum arthritis score, in which 5 μg.kg-1.d-1, 50 μg.kg-1.d-1, or 250 μg.kg-1.d-1 of CCⅡ were administered on day 5 before immunization to day 14 of immunization. As shown in table 1, by 6 weeks post-immunization, the control groups, given vehicle, had an arthritic incidence of 100%(10/10); animals in groups fed 5 or 50 μg of CⅡ had low incidence. Suppression was not seen at 250 μg.kg-1.d-1.
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2.2 Effect of CCⅡ on production of serum antibodies of anti-CⅡ level in CIA mice
All of the animals were bled at 6 weeks post-immunization and tested for anti-body response to native CⅡ by ELISA method. The results were presented separately for animals with and without arthritis. The anti-CⅡ antibody levels in CCⅡ-fed mice (5 μg and 50 μg.kg-1.d-1)were significantly lower than those in arthritic control mice administered with vehicle(see table 2).
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Table 1 Suppression of arthritis with soluble chicken collagen at 6 weeks post-immunization ( n = 10,
±s) Antigen injected
Arthritic mice
Arthritic limbs
Maximum arthritis score
Control(vehicle)
10/10(100%)
34/40(85%)
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7.1±1.2
CCⅡ (5 μg.kg-1.d-1)
1/10(10%)
5/40(13%)
4.9±0.9**
CCⅡ (50 μg.kg-1.d-1)
3/10(30%)
16/40(40%)
5.4±0.7*
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CCⅡ(250 μg.kg-1.d-1)
9/10(90%)
33/40(83%)
6.6±1.3
Male Kunming mice, 5 weeks of age, were fed with either buffer(control group), or various doses of CCⅡ 20 times on days from -5 to 14, and intradermally injected on day 0 at the base of the tail with CFA containing 10 g.L-1 of MT(mycobacterium tuberculosis) for the induction of CIA. The arthritis was evaluated twice each week from day 20 to day 40. Data were expressed as the numbers of mice with arthritis and the numbers of arthritic limbs at 6 weeks post-immunization.Animals were examined for clinical signs of CIA and were scored individually,and the “arthritis score” reflected the average arthritis score(sum of the four paws). *P<0.05， **P< 0.01 compared with control using student's t test.Table 2 Effect of CCⅡ feeding on isotype distribution of serum
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anti-collagen on 6 weeks after immunization (n=10,
±s) Pretreatment
Dose
(μg.kg-1, i.g.)
Arthritis
D(490 nm)
Control
-
-
0
, http://www.100md.com
(Acetic acid-fed)
-
+
0.242±0.073
Collagen fed
5
+
0.123±0.029**
50
+
0.110±0.075*
250
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+
0.130±0.033
Male Kunming mice,5 weeks of age, were tolerized by oral administration of vehicle, CCⅡ 5 μg.kg-1, CCⅡ 50 μg.kg-1, CCⅡ 250 μg.kg-1, 5 d before immunization with 100 μg of native CⅡ. Mice were bled at 6 weeks post-immunization and tested for antibody to CⅡ using ELISA method. Each serum was diluted 1∶400, and values were expressed as mean units of activity. Statistical analysis was compared with control. *P<0.05， **P< 0.01 vs acetic acid fed arthritis control; D， optical density, represent anti-collagen antibodies level.2.3 Effect of feeding CCⅡ to the CIA mice on T cell subset from spleen cells
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Spleen cells from CIA mice were incubated with a 1∶100 dilution of mouse anti-rat CD4,or CD8 mAbs,and then FITC-conjugated goat anti-mouse IgG mAb was added.The result showed that the percent of CD4+ did not increase obviously by administration of CCⅡ 5 μg.kg-1, while the percent of CD8+ increased significiently with the same dose. The ratio of CD4+/CD8+ of splenocytes can be reduced by orally administering CCⅡ to CIA mice. The dosage of 5 μg.kg-1 was the most effective in this experiment(see table 3).
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2.4 Effect of feeding CCⅡ on LPS-induced IL-1 activity of peritoneal macrophages in adjuvant arthritis rats Oral feeding CCⅡ decreased the IL-1 production from peritoneal macrophage in vivo. CCⅡ 5 μg.kg-1.d-1 had a better effect(see table 4).
Table 3 Effect of feeding CCⅡ to the CIA mice on T cell subset from spleen cells at 3 weeks ( n=3,±s) Groups
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Dose
(μg.kg-1, i.g.)
CD4+(%)
CD8+(%)
CD4+/CD8+
Control
-
17.7±7.5
14.8±1.7
1.19
CIA model Control
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-
21.7±2.9
12.7±3.3
1.71
CCⅡ
5
21.8±5.7
17.9±5.7*
1.21
50
17.7±1.1
11.7±2.1
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1.51
250
20.9±2.6
10.1±1.7
2.07
*P<0.05 compared with control using student's t test. Spleen cells from CIA mice were incubated with a 1∶100 dilution of mouse anti-rat CD4, or CD8 mAbs for 30 min at 4℃, washed twice with PBS, and then added to a 1∶100 dilution of FITC-conjugated goat anti-mouse IgG mAb. The cells were incubated for 30 min at 4℃. The labeled cell samples were analyzed on a FACScan.
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3 DISCUSSION
We have investigated Ag-driven peripheral immune tolerance as a means to suppress autoimmune processes using the oral route of Ag exposure to the immune system because of its inherent clinical applicability, and oral administration of Ag has long been recognized to produce immunologic hyporesponsiveness or tolerance.
Table 4 Effects of feeding CCⅡ on LPS-induced IL-1 activity
of peritoneal macrophages in adjuvant arthritis rats( n=6,±s) Groups
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Dose(μg.kg-1, i.g.)
ρ(IL-1)/ng.L-1
Control
-
53.2±1.3
AA control
-
62.8±0.9**
Prednisone
25×103
, http://www.100md.com 54.6±3.2
CCⅡ
5
43.4±1.3☆☆
50
49.7±0☆☆
250
53.0±4.1
ρ, mass concentration; AA, adjuvant arthritis; CCⅡ ig from -d5 to d14 after injecting Freund's completed adjuvant， IL-1 activity assay were detected by d28. **P<0.01 compared with control；☆P<0.05, ☆☆P<0.01 compared with AA control. Our data demonstrated that oral administration of chicken collagen type Ⅱ effectively suppressed CIA when CCⅡ was administered before induction of disease. Our studies found that minute quantities of orally administering CCⅡ(5 to 50 μg.kg-1)were capable of suppressing CIA and that larger quantities(e.g. 250 μg.kg-1)were ineffective.
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It has been reported that CIA is an autoimmune disease initiated by the binding of antibody to autologous type Ⅱ collagen in the joint[10]. In the present study, the production of antibody could be inhibited effectively by oral administration of CCⅡ.
This study demonstrated that spleen lymphocytes isolated from CIA mice activated especially CD4+cells, while ratio of CD4+/CD8+ decreased from 1.71 of CIA model control to 1.21, 1.51 of CⅡ 5 μg.kg-1, 50 μg.kg-1 administered respectively. As we all know, CD8+ cells increased might have prevented the sensitization of T cells directly involved in the initiation and maintenance of arthritis.As well as, in our previous study, the production of IL-1 in adjuvant arthritis rats was also reduced.
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The results indicated that immunologic effects both humoral and cellular with CIA mice were inhibited by orally administering chicken type Ⅱ collagen at lower dose.
(The authors thank Prof. TAO Jia-Ping for assistance in flow cytometric analysis.)■
REFERENCES
[1]Thompson HSG,Harper N,Bevan DJ, et al. Suppression of collagen induced arthritis by oral administration of type Ⅱ collagen:changes in immune and arthritic responses mediated by active peripheral suppression[J]. Autoimmunity， 1993， 16:189-199
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[2]Zhang JZ,Lee CSY,Lider O, et al. Suppression of adjuvant arthritis in Lewis rats by oral administration of type Ⅱ collagen[J]. J Immunol， 1993， 145:2489-2493
[3]Thompson SJ,Thompson HSG,Harper N, et al. Prevention of pristane-induced arthritis by the oral administration of type II collagen[J]. Immunology， 1993， 79:152-157
[4]Friedman A,Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage[J]. Proc Natl Acad Sci USA， 1994， 91:6688-6692
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[5]Klassen R， Krakauer RS， Steinberg AD. Selective loss of suppressor cell function in New Zealand mice induced by NTA[J]. J Immunol， 1977，19:830
[6]Trentham DE,Townes AS,Kang AS. Autoimmunity to type II collagen:an experimental model of arthritis[J]. J Exp Med， 1977，146:857-868
[7]Weiner HL,Friedman A,Miller A, et al. Oral tolerance:immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens[J]. Annu Rev Immunol， 1994，12:809-837
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[8]Cremer MA, Stuart JM, Townes AS, et al. Collagen-induced polyarthritis in rats:a study of native type collagen induces arthritis in mice[J]. J Immunol， 1980，124:2912
[9]Yu H,Xie SW,Yang GZ, et al.Technology of clinical immunity[M]. Shanghai:Shanghai Science Technology Press,1982.211-218
[10]Stuart JM,Tomoda K,Yoo TJ, et al. The role of collagen autoimmunity in animals and human diseases[J].Arthritis Rheum， 1983,26:1237-1244
Received:2000-03-09
Edited by JING Xia), 百拇医药
单位：李卫东 林志彬 沈建英 滕惠玲(北京大学基础医学院药理学系，北京 100083)；冉国侠(新疆维吾尔自治区人民医院)
关键词：关节炎；佐剂性；免疫耐受；胶原；鸡源性Ⅱ型胶原；白细胞介素-1
北京医科大学学报000306 [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250 μg.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。
, 百拇医药
[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)
, http://www.100md.com
RAN Gou-Xia
(People's Hospital, Xinjiang Weiwuer Autonomous Region)
ABSTRACT Objective： 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 50 μg.kg-1 of CCⅡ, but not at 250 μg.kg-1. The level of anti-collagen antibodies was also reduced at a dose of 5 μg.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.
, http://www.100md.com
KEY WORDS
KEY WORDS Arthritis, 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.
, 百拇医药
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.
, 百拇医药
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.
, http://www.100md.com 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.
, 百拇医药
1 MATERIALS AND METHODS
1.1 Animal
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.
1.2 Chicken type Ⅱ collagen
Native chicken type Ⅱ collagen was purified from lathyritic chicken preparations by the method of Cremer， et al[8].
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1.3 Induction 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 100 μg of type Ⅱ collagen in CFA injected intradermally at four to six sites on the back. Control mice were primed with acetic acid emulsified in CFA. Mice were challenged after 21 d with 100 μg of type Ⅱ collagen in 100 μl acetic acid, injected intraperitoneally. Control mice received 100 μl of 0.1 mol.L-1 acetic acid alone, intraperitoneally.
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1.4 Evaluation of arthritis[6]
The incidence of arthritis was defined as the number of mice that had clinical evidence of arthritis within 42 days after induction of disease. The severity of arthritis was graded according to standard methodology. Each of the four paws was graded as follows: 0 = normal, 1 = redness only, 2 = redness plus mild swelling, 3 = severe swelling, and 4 = joint deformity.The arthritis score for each animal was the sum of the score for each of the four paws. The maximum arthritis score was the highest score of an individual animal during the entire course of the disease.All evaluations were performed in a blinded fashion without knowledge of the treatment group.All mice were examined daily for the initial visual appearance of arthritis.
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1.5 Antibody quantitation
Mice were bled for sera on day 40.
1.6 Immunoassay of antibody to CⅡ[9]
Sera from mice were obtained by tail bleeding. Anti-collagen antibody was measured by an enzyme-linked immunosorbent assay (ELISA). Briefly, micro-ELISA plates were coated with CⅡ dissolved in phosphate-buffered saline(PBS) 0.2 mol.L-1(50 mg.L-1), 100 μl/well. Sera were diluted 1∶400, 100 μl/well. Peroxides-conjugated rabbit anti-rat IgG was added at a 1∶1 600 dilution. Levels of antibody were expressed as rectified absorbance(A490 nm).The passive sera obtained from CIA mice 40 d after immunization were pooled and diluted 1:400, and carried in each ELISA plate.
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1.7 T lymphocyte subsets assayed by flow cytometry method
Spleen cells were washed with PBS and diluted in RPMI 1640 medium supplemented with 5% fetal calf serum. Cells (2.5×105) were incubated with a 1∶100 dilution of mouse anti-rat L3T4 or anti-rat Lyt-2 monoclonal antibodies for 30 min at 4℃. After incubation, the cells were washed twice with PBS. The cells were incubated with FITC-conjugated goat anti-rabbit IgG for 30 min at 4℃ and analysed by flow cytometry(FACScan, Becton Dickinson). The percentages of cells positive for green (FITC) fluorescence were counted.
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1.8 IL-1 producing and assay
PM from the rats administrated by CCⅡ were obtained ip injected with 10% thioglycolate broth (Difco, 1 ml/rat, 4 d later). The peritoneal cavities were lavaged with RPMI1640 media. The cells were washed 3 times in RPMI1640 media. The cells placed in 24-well plates at 2×106 cells/well, and allowed to adhere at 37℃ in a 5% CO2 for 1 h. Nonadherent cells were washed out. IL-1 was induced with LPS (5 mg.L-1) from PM, then the plates were incubated at 37℃ in a 5% CO2 atmosphere for 24 h. After incubation, the supernatant was collected by centrifugation(600×g, 10 min) and stored at -25℃ until assay. IL-1 activity was evaluated by ELISA method.
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1.9 Statistical analysis
One-way analysis of variance and Student's t-test were used.
2 RESULTS
2.1 Effect of feeding chicken type Ⅱ collagen on CIA mice
The effects of orally administering chicken type Ⅱ collagen on CIA mice were studied.The effect was observed as measured by incidence of arthritis, incidence of arthritic limbs, and maximum arthritis score, in which 5 μg.kg-1.d-1, 50 μg.kg-1.d-1, or 250 μg.kg-1.d-1 of CCⅡ were administered on day 5 before immunization to day 14 of immunization. As shown in table 1, by 6 weeks post-immunization, the control groups, given vehicle, had an arthritic incidence of 100%(10/10); animals in groups fed 5 or 50 μg of CⅡ had low incidence. Suppression was not seen at 250 μg.kg-1.d-1.
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2.2 Effect of CCⅡ on production of serum antibodies of anti-CⅡ level in CIA mice
All of the animals were bled at 6 weeks post-immunization and tested for anti-body response to native CⅡ by ELISA method. The results were presented separately for animals with and without arthritis. The anti-CⅡ antibody levels in CCⅡ-fed mice (5 μg and 50 μg.kg-1.d-1)were significantly lower than those in arthritic control mice administered with vehicle(see table 2).
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Table 1 Suppression of arthritis with soluble chicken collagen at 6 weeks post-immunization ( n = 10,
±s) Antigen injectedArthritic mice
Arthritic limbs
Maximum arthritis score
Control(vehicle)
10/10(100%)
34/40(85%)
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7.1±1.2
CCⅡ (5 μg.kg-1.d-1)
1/10(10%)
5/40(13%)
4.9±0.9**
CCⅡ (50 μg.kg-1.d-1)
3/10(30%)
16/40(40%)
5.4±0.7*
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CCⅡ(250 μg.kg-1.d-1)
9/10(90%)
33/40(83%)
6.6±1.3
Male Kunming mice, 5 weeks of age, were fed with either buffer(control group), or various doses of CCⅡ 20 times on days from -5 to 14, and intradermally injected on day 0 at the base of the tail with CFA containing 10 g.L-1 of MT(mycobacterium tuberculosis) for the induction of CIA. The arthritis was evaluated twice each week from day 20 to day 40. Data were expressed as the numbers of mice with arthritis and the numbers of arthritic limbs at 6 weeks post-immunization.Animals were examined for clinical signs of CIA and were scored individually,and the “arthritis score” reflected the average arthritis score(sum of the four paws). *P<0.05， **P< 0.01 compared with control using student's t test.Table 2 Effect of CCⅡ feeding on isotype distribution of serum
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anti-collagen on 6 weeks after immunization (n=10,
±s) PretreatmentDose
(μg.kg-1, i.g.)
Arthritis
D(490 nm)
Control
-
-
0
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(Acetic acid-fed)
-
+
0.242±0.073
Collagen fed
5
+
0.123±0.029**
50
+
0.110±0.075*
250
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+
0.130±0.033
Male Kunming mice,5 weeks of age, were tolerized by oral administration of vehicle, CCⅡ 5 μg.kg-1, CCⅡ 50 μg.kg-1, CCⅡ 250 μg.kg-1, 5 d before immunization with 100 μg of native CⅡ. Mice were bled at 6 weeks post-immunization and tested for antibody to CⅡ using ELISA method. Each serum was diluted 1∶400, and values were expressed as mean units of activity. Statistical analysis was compared with control. *P<0.05， **P< 0.01 vs acetic acid fed arthritis control; D， optical density, represent anti-collagen antibodies level.2.3 Effect of feeding CCⅡ to the CIA mice on T cell subset from spleen cells
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Spleen cells from CIA mice were incubated with a 1∶100 dilution of mouse anti-rat CD4,or CD8 mAbs,and then FITC-conjugated goat anti-mouse IgG mAb was added.The result showed that the percent of CD4+ did not increase obviously by administration of CCⅡ 5 μg.kg-1, while the percent of CD8+ increased significiently with the same dose. The ratio of CD4+/CD8+ of splenocytes can be reduced by orally administering CCⅡ to CIA mice. The dosage of 5 μg.kg-1 was the most effective in this experiment(see table 3).
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2.4 Effect of feeding CCⅡ on LPS-induced IL-1 activity of peritoneal macrophages in adjuvant arthritis rats Oral feeding CCⅡ decreased the IL-1 production from peritoneal macrophage in vivo. CCⅡ 5 μg.kg-1.d-1 had a better effect(see table 4).
Table 3 Effect of feeding CCⅡ to the CIA mice on T cell subset from spleen cells at 3 weeks ( n=3,
±s) Groups, 百拇医药
Dose
(μg.kg-1, i.g.)
CD4+(%)
CD8+(%)
CD4+/CD8+
Control
-
17.7±7.5
14.8±1.7
1.19
CIA model Control
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-
21.7±2.9
12.7±3.3
1.71
CCⅡ
5
21.8±5.7
17.9±5.7*
1.21
50
17.7±1.1
11.7±2.1
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1.51
250
20.9±2.6
10.1±1.7
2.07
*P<0.05 compared with control using student's t test. Spleen cells from CIA mice were incubated with a 1∶100 dilution of mouse anti-rat CD4, or CD8 mAbs for 30 min at 4℃, washed twice with PBS, and then added to a 1∶100 dilution of FITC-conjugated goat anti-mouse IgG mAb. The cells were incubated for 30 min at 4℃. The labeled cell samples were analyzed on a FACScan.
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3 DISCUSSION
We have investigated Ag-driven peripheral immune tolerance as a means to suppress autoimmune processes using the oral route of Ag exposure to the immune system because of its inherent clinical applicability, and oral administration of Ag has long been recognized to produce immunologic hyporesponsiveness or tolerance.
Table 4 Effects of feeding CCⅡ on LPS-induced IL-1 activity
of peritoneal macrophages in adjuvant arthritis rats( n=6,
±s) Groups, http://www.100md.com
Dose(μg.kg-1, i.g.)
ρ(IL-1)/ng.L-1
Control
-
53.2±1.3
AA control
-
62.8±0.9**
Prednisone
25×103
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CCⅡ
5
43.4±1.3☆☆
50
49.7±0☆☆
250
53.0±4.1
ρ, mass concentration; AA, adjuvant arthritis; CCⅡ ig from -d5 to d14 after injecting Freund's completed adjuvant， IL-1 activity assay were detected by d28. **P<0.01 compared with control；☆P<0.05, ☆☆P<0.01 compared with AA control. Our data demonstrated that oral administration of chicken collagen type Ⅱ effectively suppressed CIA when CCⅡ was administered before induction of disease. Our studies found that minute quantities of orally administering CCⅡ(5 to 50 μg.kg-1)were capable of suppressing CIA and that larger quantities(e.g. 250 μg.kg-1)were ineffective.
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It has been reported that CIA is an autoimmune disease initiated by the binding of antibody to autologous type Ⅱ collagen in the joint[10]. In the present study, the production of antibody could be inhibited effectively by oral administration of CCⅡ.
This study demonstrated that spleen lymphocytes isolated from CIA mice activated especially CD4+cells, while ratio of CD4+/CD8+ decreased from 1.71 of CIA model control to 1.21, 1.51 of CⅡ 5 μg.kg-1, 50 μg.kg-1 administered respectively. As we all know, CD8+ cells increased might have prevented the sensitization of T cells directly involved in the initiation and maintenance of arthritis.As well as, in our previous study, the production of IL-1 in adjuvant arthritis rats was also reduced.
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The results indicated that immunologic effects both humoral and cellular with CIA mice were inhibited by orally administering chicken type Ⅱ collagen at lower dose.
(The authors thank Prof. TAO Jia-Ping for assistance in flow cytometric analysis.)■
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[2]Zhang JZ,Lee CSY,Lider O, et al. Suppression of adjuvant arthritis in Lewis rats by oral administration of type Ⅱ collagen[J]. J Immunol， 1993， 145:2489-2493
[3]Thompson SJ,Thompson HSG,Harper N, et al. Prevention of pristane-induced arthritis by the oral administration of type II collagen[J]. Immunology， 1993， 79:152-157
[4]Friedman A,Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage[J]. Proc Natl Acad Sci USA， 1994， 91:6688-6692
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[8]Cremer MA, Stuart JM, Townes AS, et al. Collagen-induced polyarthritis in rats:a study of native type collagen induces arthritis in mice[J]. J Immunol， 1980，124:2912
[9]Yu H,Xie SW,Yang GZ, et al.Technology of clinical immunity[M]. Shanghai:Shanghai Science Technology Press,1982.211-218
[10]Stuart JM,Tomoda K,Yoo TJ, et al. The role of collagen autoimmunity in animals and human diseases[J].Arthritis Rheum， 1983,26:1237-1244
Received:2000-03-09
Edited by JING Xia), 百拇医药