Effect of FTY720, a novel immunosuppressant, on adjuvant- and collagen-induced arthritis in rats

The anti-arthritic effect of FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride, a novel immunosuppressant which induces peripheral lymphocyte homing to peripheral lymph nodes, was compared with those of anti-rheumatic compounds, mizoribine and prednisolone in rat adjuvant-induced arthritis (AA) and collagen-induced arthritis (CIA). FTY720 at doses of 0.03-0.3 mg/kg, mizoribine at 3-30 mg/kg, and prednisolone at 1-10 mg/kg were orally administered to rats for 21 days from the day of inoculation with heat-killed Mycobacterium tuberculosis or type II collagen. Efficacy of FTY720 at 0.3 mg/kg was almost equal or higher as compared with those of mizoribine and prednisolone in both AA and CIA models. FTY720, but not mizoribine and prednisolone, decreased selectively lymphocyte counts in the peripheral blood in both models below the levels of the normal rats. Although FTY720 gave no other abnormal signs resulting in side effects, mizoribine was lethal to rats at 30 mg/kg and prednisolone inhibited body weight gain at 10 mg/kg, indicating that FTY720 has a wider margin of safety compared with these reference compounds. FTY720 also inhibited the production of anti-collagen antibody in CIA model, while neither mizoribine nor prednisolone did it. These results suggest that FTY720 is a promising compound for the treatment of arthritis with a unique profile.     

Effects of the novel immunosuppressant FTY720 in a murine rheumatoid arthritis model

We investigated the effects and mechanisms by which FTY720 (FTY) inhibits arthritis development in the SKG mouse rheumatoid arthritis (RA) model. FTY (1 mg/kg/day) administration suppressed the progression of laminarin-induced arthritis in SKG mice. FTY treatment decreased IL-6 and TNF-α expression in synovial fibroblast cells and the number of inflammatory cells overall. Bone destruction was also suppressed by treatment with FTY. The numbers of CD4⁺ and CD8⁺ T cells were significantly increased in the thymus and decreased in the spleen in FTY-treated SKG mice. FTY enhanced IL-4 production by CD4⁺ T cells stimulated by allogeneic spleen cells and inhibited prostaglandin E₂ (PGE₂) production by a TNF-α-stimulated synovial fibroblast cell line. These results indicate that FTY can inhibit arthritis in SKG mice via sequestration of autoimmune CD4⁺ T cells in the thymus, enhancement of Th2 immune responses, and inhibition of PGE₂ production by synovial cells.     

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats, III. Increase in frequency of CD62L-positive T cells in Peyer's patches by FTY720-induced lymphocyte homing.

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.     

A novel immunosuppressant, FTY720, increases the efficiency of a superantigen-induced peripheral T-cell deletion whilst inhibiting negative selection in the thymus.

A novel immunosuppressant, FTY720, was generated by chemical modification of ISP-I, an immunosuppressive compound purified from culture filtrates of Isaria sinclairii. FTY720 directly induces apoptotic cell death in lymphocytes, which is believed to be the mechanism by which this drug exerts its immunosuppressive effect. We examined the effect of FTY720 treatment on antigen-induced apoptotic cell death in peripheral T cells and thymocytes. A superantigen, staphylococcus enterotoxin B (SEB), induces T-cell antigen receptor (TCR) Vbeta-specific apoptotic cell death in mature T cells in vivo. In this well-documented experimental system, FTY720 administration significantly enhanced the efficiency of superantigen-induced T-cell deletion. We also determined that apoptotic cell death with DNA fragmentation induced in T-hybridoma cells after stimulation in vitro with anti-TCR antibodies was enhanced in the presence of non-cytolytic doses of FTY720. In sharp contrast, negative selection of T cells in the thymus, another example of antigen-induced apoptosis, was found to be inhibited by FTY720 treatment. A rescue effect was observed on clonal deletion in the H-Y-specific TCRalpha beta transgenic male thymus. In a chicken egg albumin (OVA)-specific TCRalphabeta transgenic system, OVA-induced apoptotic cell death of CD4+CD8+ thymocytes was also inhibited by FTY720 injection. Thus, FTY720 increased the susceptibility of mature T cells to TCR-mediated apoptosis but decreased that of immature thymocytes. The results in this report suggest that the potent immunosuppressive effect of FTY720 is, in part, a result of the augmentation of effects on antigen-induced apoptosis in mature T cells, and that two distinct apoptotic cell death pathways are operating in mature and immature T cells.     

Effect of Cornus officinalis glycoside on adjuvant-induced arthritis in rats

The aim of this study was to explore the effect of Cornus officinalis glucosides (COG) on adjuvant-induced arthritis in rats and its mechanism. Seventy-two rats were divided into six groups of normal, model, Dexasone (0.125 mg/kg), high-dose COG (240 mg/kg), mid-dose COG (120 mg/kg), and low-dose COG (60 mg/kg). Rat arthritis was induced by injection of Freund's complete adjuvant in the hind paws. All treatment started from the day the arthritis was induced. The edema degree of the adjuvant injection location was determined on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 23 and the opposite side was observed on days 11, 13, 15, 17, 20, 23 after the injection of adjuvant. All rats were sacrificed on day 24 after the injection of adjuvant for microscopic examination of the ankle, and for the study of the immunological molecular mechanism. The results showed that the COG significantly suppressed both the primary and secondary edema, improved pathological injuries of adjuvant arthritis (AA) rat ankles, significantly suppressed the proliferation of T lymphocytes and DTH reaction. It significantly suppressed IL-1, IL-6 and TNF-αproduction from peritoneal macrophages and PGE2 in plasma. In conclusion, the Cornus officinalis glucosides (COG) is able to prevent and cure the rat adjuvant-induced arthritis, and can suppress the production of pro-inflammatory cytokine IL-1, IL-6, TNF-αand PGE2.     

A new immunosuppressant, FTY720, induces bcl-2-associated apoptotic cell death in human lymphocytes.

FTY720 is a unique immunosuppressive drug produced by modification of a metabolite from Isaria sinclairii. In vitro treatment of human mononuclear cells with FTY720 resulted in a dose-dependent reduction of cell viability. These treated cells demonstrated characteristic DNA ladder formation on agarose gel electrophoresis. Jurkat cells transfected with human bcl-2 gene were resistant to FTY720; their neo type was susceptible to the drug. A rapid acceleration of cell death in human mononuclear cells was seen as early as 2 hr after incubation with FTY720. The intracellular Bax protein increased remarkably 1 hr after the culture; it markedly decreased in the surviving cells at 2 and 3 hr. Coincidental to the Bax decrease. Bcl-2 progressively decreased beginning 2 hr after the culture. Thus, the ratio of Bcl-2 to Bax was decreased by the enhanced expression of Bax immediately after FTY720-treatment, resulting in rapid cell death acceleration. The surviving cells (FTY720-resistant cells) at 2 and 3 hr after culture showed a similar ratio of Bcl-2 to Bax as was observed in the control cells. These results suggest that FTY720 displays bcl-2-associated apoptotic cell death in human mononuclear cells.     

Effect of antiserotonin antibodies on pain sensitivity in rats with adjuvant-induced arthritis

Active immunization with a serotonin—protein conjugate inducing the formation of antiserotonin antibodies exerts an analgesic effect in rats with adjuvant-induced arthritis and inhibits the development of arthritis in early stages after injection of Freund's complete adjuvant. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 9, pp. 292–295, September, 1997     

Inhibitory effects of ZSTK474, a phosphatidylinositol 3-kinase inhibitor, on adjuvant-induced arthritis in rats

Objective

We examined the effects of ZSTK474, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on adjuvant-induced arthritis (AIA).

Methods

AIA was induced in Lewis rats by subcutaneous administration of Freund’s complete adjuvant at the base of the tail on day 0. ZSTK474 was orally administered once daily from day 10. The severity of AIA was assessed by measuring the hind paw volume. The number of lymphocytes in inguinal lymph nodes (ILN) was determined by flow cytometry. The in vitro effects of ZSTK474 on the cell proliferation, and the cytokines and prostaglandin E₂ (PGE₂) production were evaluated by BrdU method, ELISA and cytometric beads array.

Results

ZSTK474 ameliorated the progression of AIA. The temporary increases in the number of T cells in ILN, which occurred along with the appearance of arthritis, were inhibited in the ZSTK474-treated groups. In vitro studies revealed that ZSTK474 inhibited the production of IFNγ and IL-17 in concanavalin A-activated T cells. In vitro studies further revealed that ZSTK474 inhibited the proliferation and PGE₂ production by fibroblast-like synovial cells (FLS).

Conclusion

ZSTK474 demonstrated prophylactic efficacy in a rat model of rheumatoid arthritis (RA) through inhibition of T cell and FLS functions. It was suggested that the inhibitors of PI3K have therapeutic potential for RA.     

Expression of sphingosine kinase 2 in synovial fibroblasts of rheumatoid arthritis contributing to apoptosis by a sphingosine analogue, FTY720

Gene expression profiles in synovial tissues from rheumatoid arthritis (RA) patients have yielded useful information on the pathogenetic process of the synovitis. In one group of them, sphingosine kinase 2 (SPHK2), a nuclear protein regulating cell proliferation, seemed to be highly expressed, undergoing a different pathogenetic process of synovitis. In the present study it was clarified that SPHK2 was expressed in the synovial fibroblasts of the synovial tissues obtained from the knee joints of the RA patients. In the cultured synovial fibroblasts from these patients, SPHK2 was more highly expressed than that in the human macrophage cell line, THP-1 and human dermal fibroblasts. SPHK2 was expressed in and around the nucleus and transferred to the cytoplasm and cell surface by the administration of epidermal growth factor, associated with the increased expression of sphingosine-1-phosphate. A sphingosine analogue, FTY720, which is activated by phosphorylation specifically by SPHK2, mediated apoptotic signaling of the cultured synovial fibroblasts. These findings suggest that SPHK2 may regulate the autonomous proliferation of synovial fibroblasts as one of the predisposing genes to RA and could be a target for a novel therapeutic strategy for RA.     

The effect of immunomodulating drugs on adjuvant-induced arthritis in Lewis rats

The purpose of this study was to investigate the effects of cyclosporine A (CSA) and methotrexate (MTX) as potential immunomodulators in a nonestablished adjuvant arthritis (AA) model. Non-injected hind paw volumes were reduced when AA rats were treated for 18 dayswith CSA (100% at 10 mg/kg) or MTX (100% at 0.1 mg/kg). Body weights of drug treated AA rats were increased above untreated AA rats and were similar to non-arthritic controls. AA rats show elevated T helper (W3/25⁺)/T suppressor (OX 8⁺) cell ratios (2.0 vs. 3.1,p<0.01). The immunomodulators tested all returned these elevated ratios to control non-arthritic levels. Similarly, these drugs returned the reduced mitogen responses and elevated blood granulocyte numbers toward normal non-arthritic control values.     

Changes in zinc,copper and selenium status during adjuvant-induced arthritis in rats

Trace elements such as zinc, copper and selenium are involved in the pathogenesis of inflammatory diseases. In order to obtain more information about the overall movements of these minerals during the evolution of an experimental chronic inflammatory process, trace element levels were determined in five body compartments of the rat at several time intervals after induction of adjuvant arthritis. Rapid and significant changes in plasma zinc and copper levels and in liver zinc levels were observed. These modifications occurred as early as those in biochemical parameters of inflammation such as serum fibrinogen and ceruloplasmin, and preceded the appearance of any clinical symptom of the disease. Inverse correlations were found between plasma zinc levels and these two biochemical indices. Other modifications in trace element levels were observed two weeks after disease induction, the most important being a considerable increase in liver copper levels. Although food intake of affected animals decreased with the progression of the disease, there was no evidence of depletion in zinc and copper levels over the study period. A redistribution of body zinc between different biological compartments (mainly plasma and liver) occurred simultaneously with an accumulation of copper in several organs. The decreasing selenium status of animals was not clearly related to the inflammatory process.     

联合应用FTY720和Cs A对小鼠淋巴细胞的影响

为探讨联合应用FTY72 0和环孢素A (CsA )对外周血及淋巴器官的影响 ,选用昆明小鼠分别给予FTY72 0、CsA或FTY72 0加CsA 2周 ,在服药期间和停药后观察外周血象淋巴细胞数、粒细胞数、血红蛋白、胸腺和脾脏重量以及骨髓象的变化。结果发现 ,FTY72 0组和联合用药组的淋巴细胞在服药后 1周和 2周均明显减少 ,停药后有不同程度的恢复。各组的粒细胞、血红蛋白、胸腺重量和骨髓象在服药期间或停药后与对照组相比均无明显差异。FTY72 0组和联合用药组的脾脏重量在服药 2周后或停药后 4周均明显减轻。该实验表明FTY72 0可选择性地清除成熟的外周淋巴细胞 ,CsA对FTY72 0的作用无明显影响 ,联合应用FTY72 0和CsA的免疫抑制效果可能是通过不同的机制起作用。     

Susceptibility to adjuvant-induced arthritis among germfree, specific-pathogen-free, and conventional rats.

Germfree F344 rats developed severe arthritis with 100% incidence after a single intradermal inection of either squalane containing 0.5 mg of heat-killed Mycobacterium bovis BCG or a water-in-oil emulsion containing 0.2 mg of peptidoglycan derived from Staphylococcus epidermidis. Conventional F344 rats developed less-severe arthritis with 20% incidence for heat-killed BCG and 0% incidence for peptidoglycan. Specific-pathogen-free rats showed an intermediate susceptibility between germfree and conventional rats. Interestingly, both unimmunized specific-pathogen-free and conventional rats. but not unimmunized germfree rats, showed weak delayed-type hypersensitivity reactions to peptidoglycans derived from either S. epidermidis or Lactobacillus plantarum, suggesting that a bacterial flora may furnish a stimulus for induction of cell-mediated immunity to ubiquitous bacterial peptidoglycans. It is thus possible that although a bacterial flora is not necessary for development of adjuvant arthritis, it may have some suppressive effect on the development of the disease in specific-pathogen-free and conventional F344 rats, possibly through modulation of the immune response.     

High-density proteoglycan induces specific suppression of adjuvant-induced arthritis in rats.

We compared the levels of serum antibodies in male and female C57Bl/6 mice during the primary and after challenge infection with Giardia muris. Male mice began passing cysts in their faeces earlier than females, and were shedding cysts for over 60 days, while females stopped shedding cysts by day 20 after infection. In both males and females there were significant increases in parasite-specific IgM 10 and 20 days after infection. No differences in parasite-specific serum IgA were observed until 40 days after infection. Parasite-specific IgG (whole) levels were elevated on days 20 and 40 in females, while males showed no significant increases. In addition, females had a much stronger IgG2b and IgG3 response than males. After challenge with either cysts or soluble parasite protein only the females had significant increases in specific anti-parasite IgG2b. Our data show differential ability of males and females to control the infection with G. muris is paralleled by a difference in the anti-parasite serum IgG response of the mice.     

Cis-dichlorodiammineplatinum (II): Suppression of adjuvant-induced arthritis in rats

Cis-Dichlorodiamminepaltinum(II) (cis-PtII) suppressed adjuvant-induced arthritis in rats as assessed by paw volume, paw temperature, and microscopic appearance. Treated animals incurred lymphocytopenia and thymic atrophy. Paws from a number of rats with arthritis which were treated withcis-PtII showed focal bone marrow congestion and hemorrhages not found in rats with untreated arthritis.     

Effects of different dose of FTY720 on lymphocyte cell cycle arrest in cardiac transplantation model of rats

Purposes: To probe into immunosuppressive effect and cell cycle arrest effect of different dose of FTY720.

Method: we study immunosuppressive effect and cell cycle arrest effect of different dose of FTY720 and time and dose dependence of FTY720 with control and random method in vivo, respectively.

Results: (i) survival time of graft in rat cardiac transplantation model is prolonged in group treated with FTY720. It is longer in group treated with FTY720 10?mg/kg than that in group treated with FTY720 2?mg/kg. (ii) The mean sum of lymphocyte in periphery blood decrease obviously 2?h after administration, reaches its peak 4?h after administration, and then maintain at low level stably. The mean sum of lymphocyte shows a linear relationship with dose of FTY720 and decrease with increasing of dose of FTY720. (iii) The percent of cell in G1-G0 increased in group treated with FTY720, not only in thymus but also in lymph node; this is more prevalent in the group treated with FTY720 10?mg/kg than that in group treated with FTY720 2?mg/kg. Similarly, the percent of cell in S and G2-M decreased in group treated with FTY720, not only in thymus but also in lymph node, and this decrease is more associated with group treated with FTY720 10?mg/kg than the one treated with FTY720 2?mg/kg.

Conclusion: FTY720 is an effective immunosuppressant and immunosuppressive effect of FTY720 show a tendency of time and dose dependent. Effect of cell cycle arrest of FTY720 is related to dose of this new immunosuppressant.     

大鼠佐剂性关节炎模型表现特征及评价指标

目的:描述大鼠佐剂性关节炎(Adjuvant-induced arthritis,AA)临床表现主要指标的评价方法。方法:完全弗氏佐剂免疫SD大鼠诱导AA模型,进行全身及关节炎指数评分、计算关节肿胀数、测定足爪肿胀度、进行足爪X线摄片、踝关节病理检查及评分。结果:大鼠免疫后11天(d11)出现足爪红肿,全身及关节炎指数评分和关节肿胀数增加。炎症高峰期在免疫后d19～d28。大鼠出现活动障碍,可见耳朵、鼻和尾根部"关节炎"结节。X线可见关节软组织肿胀、骨密度降低、骨侵蚀病灶以及关节间隙狭窄甚至消失。踝关节病理见滑膜组织增生,炎细胞浸润,血管翳出现等。结论:大鼠AA是兼有局部关节炎症和全身表现的类风湿性关节炎动物模型。关节炎指数、关节肿胀数、足爪肿胀度、足爪X线摄片、踝关节病理等是评价AA模型临床表现的主要指标。