A T suppressor cell cytokine regulates murine arthritis and T lymphocyte stimulation

Experimental immunotherapy of murine collagen-induced arthritis can be achieved by administration of specific T suppressor cell hybridomas. The present study examines the immunoregulation noted in this experimental immunotherapy by describing the immunomodulatory effects of a cytokine produced by a suppressor T cell line, T101N. The inhibition of the activation of splenic lymphocytes in response to T cell mitogens and the erythema and edema associated with arthritis were assayed. Mice given T101N ascites showed reduced inflammation (p<0.05). Lymphocytes derived from naive mice and cultured in the presence of T101N culture supernatant showed reduced response to concanavalin A. Therefore, this form of experimental immunotherapy of arthritis may be associated with cytokines secreted from T suppressor cells which modulate T cell activation.     

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen

Injection of native type II collagen (CII) to susceptible strains of mice (H-2^q) induces a rheumatoid arthritis-like disease. To study the role of CD8⁺ T cells in the collagen-induced arthritis (CIA),we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H.Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CH-pulsed macrophages and recognize different antigenic epitopes in association with K^q molecules. When these T cell clones were irradiated and inoculated into (C3H.Q × AKR)F₁ mice 21 days prior to priming with native CII/ complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.     

Cytotoxic T lymphocytes specific for murine type II collagen do not trigger arthritis in B10 mice

To investigate the role of cytotoxic T lymphocytes (CTL) in arthritis, we set out to induce CTL specific for murine type II collagen (mCII) in a mouse model. The primary protein sequence of the murine pro-α1(II) was screened for fragments bearing H-2 D^b or K^b binding motifs. Six fragments were identified and the corresponding peptides synthesized. One of these peptides, peptide P201 (amino acid 199–208 in the C-propeptide of the murine pro-α1(II)), was found to be a strong binder to H-2 D^b. When used to treat RMA-S cells at 26°C, peptide P201 induced a four-fold increase of surface expression of H-2 D^b. Administration of the P201-treated RMA-S cells into B10 mice (H-2^b) induced strong CTL responses against the immunizing collagen peptide. Despite the high frequencies of mCII-specific CTL precursors in the periphery, however, the immunized mice showed no sign of arthritis up to 16 weeks after immunization. Implications of these data for autoimmunity and arthritis are discussed.     

T细胞疫苗对CIA的免疫调节作用

目的探讨T细胞疫苗(TCV)对小鼠胶原诱导性关节炎(CIA)的免疫调节作用。方法通过TCV干预CIA模型,观察小鼠发病情况,运用免疫组化技术观察局部关节病变程度;FACs分析T细胞亚群变化;ELISA检测细胞培养上清中TGF-β、IFN-γ、IL-17水平;定量PCR方法检测Treg、Th1、Th17细胞相关转录因子表达水平。结果经过TCV干预,小鼠自身反应性T细胞的增殖能力被显著抑制,CII抗体水平也显著下降;相较CIA组,TCV组Treg细胞百分比上升,Th1、Th17细胞百分比明显降低;各细胞分泌细胞因子的能力也发生相应的变化,其中TGF-β上升,IFN-γ、IL-17降低;Treg细胞转录因子Foxp3基因水平提高,Th1、Th17细胞转录因子T-bet、RORα及RORγt的mRNA水平降低。结论 TCV干预可通过抑制CIA小鼠自身反应性T细胞增殖,上调小鼠体内Treg细胞并抑制Th1、Th17细胞的表达而发挥免疫调节作用,有效降低小鼠发病严重度和关节炎症。     

T cell responses induced by allergen-specific immunotherapy

Allergen‐specific immunotherapy is recognized as a highly effective practice in the treatment of patients with severe allergic rhinitis and/or asthma and is recommended by World Health Organization as an integrated part of allergy management strategy. Several studies have shown that allergen‐specific immunotherapy, based on the administration of increasing doses of allergen, achieves a hyposensitization and reduces both early and late responses occurring during the natural exposure to the allergen itself. This is the unique antigen‐specific immunomodulatory treatment in current use for human diseases. Successful immunotherapy is associated with reductions in symptoms and medication scores and improved quality of life. After interruption it usually confers long‐term remission of symptoms and prevents the onset of new sensitizations in children up to a number of years. Subcutaneous immunotherapy usually suppresses the allergen‐induced late response in target organs, likely due to the reduction of the infiltration of T cells, eosinophils, basophils, mast cells and neutrophils. In addition to the reduction of cells of allergic inflammation, immunotherapy also decreases inflammatory mediators at the site of allergen exposure. This review provides an update on the immunological T cell responses induced by conventional subcutaneous and sublingual immunotherapy, and gives a unifying view to reconciling the old dualism between immunoredirecting and immunoregulating mechanisms.     

Abnormalities of circulating T cell subsets in atopy: influence of specific immunotherapy

Blood samples of patients with severe respiratory allergic diseases contain increased numbers of T cells bearing surface HLA-DR antigens, indicating the presence of activated T cells. In the same group of patients, MLR3 and MLR4, two monoclonal antibodies (Mab) directed to subsets of activated peripheral T cells, recognize T cell percentages within the normal range. Thus, it seems possible that specialized subsets of activated T cells (HLA-DR+/MLR3-MLR-) are represented in the peripheral blood of atopic patients. Such cells are lacking in patients after specific immunotherapy. Similar results--an increased percentage of 5/9+ T cells in untreated patients and normal counts of 5/9+ T cells in treated ones--were obtained in the two groups of patients by using another Mab, 5/9, which serves as a reliable marker of helper T cells in resting peripheral T lymphocytes. These data further support the concept of a T cell imbalance in allergic patients and suggest a possible role of specific immunotherapy in correcting the modification of peripheral T cell abnormalities.     

T suppressor lymphocytes regulation of adjuvant arthritis in two inbred strains of rats.

Adjuvant arthritis can be induced by a single injection of Freund's complete adjuvant (FCA) in the highly susceptible Lewis (LEW) rat strain, but not the resistant Wistar A.G. (WAG) strain. This strain-dependent susceptibility to the disease is correlated with differences in T suppressor cells regulation. In WAG rats, indeed, the in vitro response in LEW alloantigens was highly inhibited 11 days after FCA injection, while LEW rats in vitro response to WAG alloantigens was slightly increased. Furthermore, spleen cells from WAG rats given FCA 4 days before exhibited T cell-mediated active suppression of WAG in vitro response to LEW alloantigens when they were co-cultured with WAG normal spleen cells. This suppression was abolished by removal of T cells on nylon wool column. A previous irradiation of these T cells also inhibited their suppressive effect, suggesting that FCA-induced suppression might be due to soluble suppressor factor(s). On the other hand T cells from FCA treated LEW rats did not produce any modification of LEW in vitro response to WAG alloantigens. This suggests that the severe arthritis induced in LEW rats could be correlated with a defect of their suppressor cells functions, while in WAG rats FCA activated suppressor T cells could control the disease.     

Efficient promotion of collagen antibody induced arthritis (CAIA) using four monoclonal antibodies specific for the major epitopes recognized in both collagen induced arthritis and rheumatoid arthritis

An antibody response to defined epitopes located on the triple helical portion of type II collagen (CII) is associated with the development of collagen-induced arthritis (CIA) and rheumatoid arthritis (RA). Monoclonal antibodies to epitopes associated with arthritis, but not antibodies specific for epitopes not associated with arthritis, induce arthritis in mice, the so-called collagen antibody induced arthritis (CAIA) model. We have selected monoclonal IgG antibodies specific for four well-defined major epitopes on triple helical CII, the C1, J1, D3 and U1 epitopes. These antibodies bind the epitopes specifically as determined using recombinant or synthetic triple helical epitopes. They are encoded from somatically mutated V genes. They all bind cartilage in vivo in normal mice. All of the antibodies induce mild arthritis after injection intravenously and if injected as a cocktail they induce severe clinical arthritis. Intravenous injection of a total of 4 mg antibodies (0.5 mg antibodies per clone) induced arthritis in several different mouse strains without any secondary immune stimulus and intraperitoneal injection of LPS 7 days later dramatically raised the severity. Thus, this method is recommended as a new protocol for the induction of CAIA.     

Induction of murine T cell lymphoma expressing specific cytotoxic activity

The present work reports the establishment of an antigen-specific cytotoxic T cell lymphoma line after immortalization with a murine leukemia virus. Lymph node cells from mice bearing a transplanted syngeneic MCA sarcoma were infected in vitro with radiation leukemia virus and injected intrathymically into cogeneic recipient mice. Some lymphomas of donor origin were established as permanent continuous cell lines in vitro. One of them, NS8, expressed Thy-1.2, Lyt-1, Lyt-2 and peanut agglutinin surface markers. These cells were cytotoxic in vitro for the tumor cell line corresponding to the immunizing MCA sarcoma. No significant cytolytic activities against other syngeneic or allogeneic sarcomas or lymphomas, nor against the mastocytoma P815 were observed. After several months of in vitro propagation, the specificity of the cytotoxic activity had degraded and the level of Lyt-2 or peanut agglutinin receptors dropped. These characteristics were restored with a single in vivo passage. Thus, murine leukemia virus can be used to immortalize antigen-specific cytotoxic T cells.     

Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis

Heat shock proteins (hsp) are highly conserved, immune-dominant microbial proteins, whose expression is increased at sites of inflammation. In the experimental model of adjuvant arthritis (AA) immune responses to hsp determine the outcome of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response directed against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic arthritis. Next to hsp60, other hsp have similar protective effects in arthritis, while other conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in suppressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candidates for immune therapy in chronic arthritis.     

Suppressor T cells and soluble suppressor factors in allergy: effect of immunotherapy

Suppressor-cell activity of Concanavalin-A-stimulated lymphocytes was studied in allergic patients by inhibition of one-way mixed lymphocyte culture reactions before and after allergy immunotherapy. This activity was compared with twelve healthy controls. In preliminary experiments, six out of eight allergic patients had no detectable T suppressor activity. In the second prospective group, eight out of eleven patients had much reduced suppressor-cell activity before immunotherapy, and seven out of eleven patients had much reduced activity after immunotherapy. The data suggest that non-specific T suppressor-cell activity is reduced in allergic patients but immunotherapy does not restore such activity.     

The regulation of T cell responses by spontaneously active suppressor cells.

In studying T cell regulation, peripheral blood mononuclear cells from normal subjects were examined for 'spontaneous', rather than mitogen-induced, suppressor cell activity. Normal blood leucocytes from 30 subjects included a subpopulation of cells capable of suppressing the response of lymphocytes to the T cell mitogen phytohaemagglutinin by 21-35%. The indicator system for these studies consisted of fresh normal lymphocytes stimulated by three concentrations of PHA in the presence or absence of normal but mitomycin C treated peripheral blood lymphocytes. To measure accurately the spontaneous suppressor cell activity, additional cultures were needed to control for the suppressive effects of crowding and metabolic competition. Allogeneic, cryopreserved 'B' cell enriched populations, supplied satisfactory control cells for this purpose. While allogeneic culture systems could induce significant suppressor cell activity after 7 days of co-culture, they could not induce this activity in the 3 days required to assay spontaneous suppressor cell effects. In developing this assay we noted that (a) crowding became a factor in the cellular response to mitogens with concentrations higher than 2 X 10(4) cells/well, (b) spontaneous suppressor cell activity decreased rapidly once cells were placed in culture and (c) both spontaneous and concanavalin A (Con A) activated suppressor cells could significantly reduce the response to PHA even when added to cultures established with mitogens 72 hr earlier. The ability to measure spontaneous suppressor cell activity in vitro will allow more physiological studies of the membrane markers and functional characteristics of these cells than is possible in conventional studies utilizing Con A. In addition, this assay allows the detection of enhanced in vivo activity of suppressor cells not easily detected in assays relying on mitogen induction of suppression. Such increased activity is thought to be an important factor in the pathogenesis of a number of human diseases.     

Antigen specific,suppressor cells induced by the immunomodulator FCE 20696 in aged NZB/WF1 mice

FCE 20696 is a new synthetic immunomodulating agent, active on some manifestations of the autoimmune disease of NZB/WF₁ mice, in which a disorder of T suppressor cells (SC) has been described to be of pathogenetic relevance. The ability of FCE 20969 to induce SC in NZB/WF₁ mice, which are then able to specifically inhibit the production of anti autologous red blood cells antibodies (aRBC Ab), was investigated. NZB/WF₁ mice were chronically treated with the compound starting from the ninth week of age, sacrificed at different times and their spleen cells transferred to 9 weeks old, syngeneic mice. In donor animals SC were induced by rat RBC, whereas in the recipients the suppressive activity of transferred cells was evaluated from the appearance of aRBC Ab induced by the same antigen. The results show that antigen specific SC were present in both FCE 20696 treated and control young animals. In older controls, SC couldn't be induced, whereas in treated animals SC were present and able to transfer suppression into the recipents. FCE 20696 was active at 1.5 mg/Kg.     

Increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: isoform specific regulation of inflammation

Pro-inflammatory cytokines and chemokines play critical roles in autoimmune diseases including rheumatoid arthritis (RA). Recently, it has been reported that β-arrestin 1 and 2 are involved in the regulation of inflammation. We hypothesized that β-arrestin 1 and 2 play critical roles in murine models of RA. Using a collagen-induced arthritis (CIA) and a human TNFα transgenic (TNFtg) mouse model, we demonstrated that β-arrestin 1 and 2 expression are significantly increased in joint tissue of CIA mice and TNFtg mice. In fibroblast-like synoviocytes (FLS) isolated from hind knee joint of CIA mice, we observed an increase of β-arrestin 1 and 2 protein and mRNA levels in the early stage of arthritis. In FLS, low molecular weight hyaluronan (HA)-induced TNFα and IL-6 production was increased by overexpression of β-arrestin 1 but decreased by overexpression of β-arrestin 2 demonstrating isoform specific regulation. TNFα and HA induced an increase of β-arrestin 1 and 2 expression in FLS, while high mobility group box (HMGB)-1 only stimulated β-arrestin 1 expression. TNFα- or HA-induced β-arrestin 2 expression was blocked by a p38 inhibitor. To examine the in vivo role of β-arrestin 2 in the pathogenesis of arthritis, WT and β-arrestin 2 KO mice were subjected to collagen antibody-induced arthritis (CAIA). β-Arrestin 2 KO mice exhibited more severe arthritis in CAIA. Thus β-arrestin 2 is anti-inflammatory in CAIA. These composite observations suggest that β-arrestin 1 and 2 differentially regulate FLS inflammation and increased β-arrestin 2 may reduce experimental arthritis severity.     

Combination therapies that inhibit cyclooxygenase-2 and leukotriene synthesis prevent disease in murine collagen induced arthritis

Objective and design:  To determine the effect of combinations of cyclooxygenase (COX) inhibitors and inhibitors of leukotriene (LT) syntheses on collagen induced arthritis (CIA) in mice. Methods:  The CIA model was evaluated for the presence of eicosanoids in the paw tissue. Several selective cyclooxygenase 2 (COX-2) inhibitors or non-selective non-steroidal anti inflammatory drugs (NSAIDs) were evaluated alone or in combination with leukotriene (LT) synthesis inhibitors in the CIA model. Results:  Arthritic paw tissue showed increased levels of prostaglandins and leukotrienes in comparison to normal paws. Analysis of mRNA levels indicated the inducible form of the COX-2 enzyme to be the source of prostaglandins. NSAIDs, COX-2 or leukotriene synthesis inhibitors administered alone in CIA decreased severity but had little effect on disease incidence. However, the combination of selective COX-2 inhibitors with leukotriene synthesis inhibitors produced significant decreases in both incidence and severity, suggesting an additive or synergistic effect. This effect was reversible with removal of drug. Little decrease in incidence was observed with the NSAID/5-LO inhibitor combinations. Conclusions:  These results suggest that the induction of the disease in CIA is mediated by products of the COX-2 enzyme and LTB4 production, and that blockade of both pathways is required to prevent CIA. Received 9 July 2008; returned for revision 20 August 2008; received from final revision 19 September 2008; accepted by J. A. Battista 15 October 2008