Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

The Dynamics of Articular Leukocyte Trafficking and the Immune Response to Self Heat-Shock Protein 65 Influence Arthritis Susceptibility

INTRODUCTION: Adjuvant arthritis (AA) shares several features with human rheumatoid arthritis, and it can be induced in the Lewis (LEW) rat but not the Wistar Kyoto (WKY) rat (both RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis (Mtb). We set out to unravel the mechanisms underlying the differential susceptibility to AA of these MHC-compatible rat strains. MATERIALS AND METHODS: We compared the levels of T-cell proliferative and cytokine response to the immunoregulatory self (rat) hsp65 (Rhsp65) after an arthritogenic (Mtb) challenge and the kinetics of migration of adoptively transferred, (111)Indium-labeled, Mtb-primed leukocytes into the hind paw joints of recipient rats. RESULTS AND DISCUSSION: The WKY rats raised a significantly higher level of T-cell proliferative response coupled with a temporally opposite cytokine profile against the disease-regulating Rhsp65 compared to that of LEW rats. Moreover, the arthritogenic leukocytes accumulated into the joints of WKY rats at significantly lower numbers than that in LEW rats. CONCLUSIONS: These results offer novel insights into the immune events influencing the pathogenesis of autoimmune arthritis.     

Tumour necrosis factor (TNF) production by T cell receptor-primed T lymphocytes is a target for low dose methotrexate in rheumatoid arthritis.

Methotrexate (MTX) is an effective immunosuppressive agent in various chronic inflammatory diseases such as rheumatoid arthritis (RA). However, its mechanisms of action are only partially understood. In this study, we assessed the effects of MTX on the differentiation of peripheral blood (PB) CD4+CD45RA 'naive' and CD4+CD45RO 'memory' T cells from healthy controls and patients with RA. Accordingly, purified T cells were primed and restimulated in vitro via the T cell receptor (TCR) in the presence of IL-2 to generate effector T cells secreting large amounts of Th1 and Th2 cytokines. We observed that low doses of MTX strongly suppress TNF and to a lesser extent interferon-gamma (IFN-gamma) production by T cells from both healthy donors and RA patients when present during T cell priming via the TCR. Similar data were obtained for TCR-primed synovial fluid mononuclear cells in RA. In contrast, production of IL-4 by TCR-primed CD45RA T cells was significantly increased upon MTX treatment. Interestingly, MTX did not enhance IL-4 production when present during restimulation of effector CD45RO T cells, although it still suppressed TNF production. The results indicate that MTX effects depend on the stage of T cell activation and identify TNF production by TCR-primed T lymphocytes as a target for low-dose MTX treatment in RA. These findings could explain the delayed clinical effects of MTX and may contribute to its potent anti-inflammatory and immunoregulatory properties.     

类风湿关节炎患者外周血Th细胞亚群的变化及依那西普的调节作用

目的:观察活动期类风湿关节炎(RA)患者外周血中辅助性T细胞亚群(Th1、Th2和Thl7)数量和比例的变化,探讨依那西普治疗对Th亚群的作用.方法:选择20例活动期中重度RA患者,随机分为依那西普加MTX联合治疗组、MTX治疗组,于治疗前后分离PBMC,采用流式细胞术(FCM)检测Th1、Th2、Th17细胞的数量及Th1/Th2比值,同步用ELISA测定培养上清中IFN-γ、IL-4、IL-17水平,与同期选择的健康对照组比较并与患者的病情活动指标进行相关性分析.结果:治疗前活动期RA患者PBMC中IFN-γ Th1细胞的量、IL-17 Th17细胞的量及平均荧光强度、Th1/Th2比值均高于健康对照组[(27.0±2.9)%vs(23.2±1.7)%,P<0.05;(36.78 ±2.6)%vs(2.35±0.9)%及(26.61±2.6)vs(11.4±1.2),P<0.01;(12.8±1.6)vs(8.60±1.9),P<0.01];依那西普加MTX联合治疗12周后,RA患者的病情改善,IFN-γ Th1细胞、Th1/Th2比值、IL-17 Th17细胞的量及平均荧光强度、培养上清中IL-17水平均低于治疗前,而与健康对照组相比无统计学意义(P>0.05),且联合治疗组降低的幅度均大于MTX治疗组;治疗前后Th17细胞的量与C反应蛋白(CRP)、疾病活动指数(DAS28)的消长呈正相关(P<0.05,r=0.38和P<0.05,r=0.42).结论:IL17 Th17细胞、IFN-γ Th1细胞及Th1/Th2比值的增高可能参与活动期RA的发病机制,依那西普治疗可改善RA病情并下调Th1和Th17细胞亚群.     

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.     

Bacterial extract (OM-89) specific and non specific immunomodulation in rheumatoid arthritis patients

The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-gamma, TNF-alpha), Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production while TNF-alpha and IFN-gamma production tended to decrease during the study.Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release suggestive of an induced Tr1 response to OM-89.     

Synovial fluid T cells from patients with rheumatoid arthritis are refractory to the T helper type 2 differentiation-inducing effects of interleukin-4

The balance between T helper type 1 (Th1) and Th2 cytokines is thought to be important in the initiation and outcome of autoimmune diseases. The goal of the present study was to compare the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by synovial fluid (SF) and peripheral blood (PB) CD4+ and CD8+ cells from patients with rheumatoid arthritis (RA) using three-colour immunofluorescence staining and flow cytometry, and to investigate the capacity of IL-4, IL-10 and IL-12 to modify the cytokine production profile of SF T cells. The frequency of IFN-gamma-producing CD4+ and CD8+ cells was significantly increased in SF when compared with PB. In contrast to IFN-gamma, the expression of IL-4 in SF and PB T cells was comparable. The majority of IL-4-producing cells in SF belonged to Th0/T cytotoxic (Tc) type 0 phenotype, whereas there were significantly more Th2/Tc2 cells in PB than in SF. Interestingly, IL-4 was unable to induce differentiation of non-adherent SF mononuclear cells (SFMC) into Th2 cells, whereas PB mononuclear cells (PBMC) under similar culture conditions differentiated into cells producing high levels of IL-4, IL-10 and IL-13. In contrast, there were no major differences in the effects of IL-10 and IL-12 on the cytokine production profile of SFMC when compared with PBMC. Taken together, the present results suggest that SF T cells from patients with RA are terminally differentiated into Th1/Tc1-like phenotype, and Th2/Tc2 differentiation-inducing agents, such as IL-4, may not be able to reverse the inflammatory process occurring in the joints.     

Th17 cytokines and arthritis

Th17 cells are implicated in human autoimmune diseases, such as rheumatoid arthritis (RA), although it has not been established whether this persistent destructive arthritis is driven by Th1 and/or Th17 cells. Interleukin-17A (IL-17A) contributes to the pathogenesis of arthritis as has been shown in several experimental arthritis models. Importantly, recent data from first clinical trials with anti-IL-17A antibody treatment in psoriatic arthritis patients and RA patients looks promising. This review summarizes the findings about the role of Th17 cells in arthritis and discusses the impact of the different Th17 cytokines in the pathogenesis of this disease. However, further studies are needed to unravel the interplay between IL-17A and other Th17 cytokines such as IL-17F, IL-22, and IL-21 in the pathoimmunological process of this crippling disease, in particular, whether regulating Th17 cell activity or specific combinations of Th17 cytokines will have additional value compared to neutralizing IL-17A activity alone. Moreover, tumor necrosis factor-positive Th17 cells are discussed as potential dangerous cells in driving persistent arthritis in human early RA.     

Intracytoplasmic Th1 and Th2 cytokines in rheumatoid arthritis blood and synovial tissue

In rheumatoid arthritis (RA), T cells have been proposed either as a main actor or as an epiphenomenon in such a primarily synoviocyte-driven disease. A major issue remains the remarkable paradox between the T cell infiltrate and the relative failure to detect definite markers of their activity. To determine the Th1/Th2 cytokine profile in RA synovium, we used a single cell flow cytometric assay for interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4 and IL-10 in paired peripheral blood (PB) and synovial tissue (ST) lymphocytes from RA and osteoarthritis (OA) patients and PB lymphocytes from healthy controls. Cytokines were undetectable in unstimulated PB and ST lymphocytes. More stimulated PB and ST CD4(+)lymphocytes produced IFN-gamma than IL-4, for all individuals tested. RA PB CD4(+)lymphocytes showed the same Th1 cytokine pattern as normal controls. No increase of such a Th1 profile was observed for ST lymphocytes. A specific recruitment of T CD4(+)lymphocytes in the rheumatoid inflamed synovium could not be concluded on the basis of these results.     

Lactobacillus casei potentiates induction of oral tolerance in experimental arthritis

Probiotics have been shown to exert beneficial effects on modulation of diverse diseases. However, no information is available for the effect of probiotics in the induction of oral tolerance in autoimmune diseases. The main purpose of this study was to elucidate whether Lactobacillus casei (L. casei) affect the induction of oral tolerance in experimental rheumatoid arthritis (RA). Type II collagen (CII) alone or together with L. casei was orally administered into collagen-induced arthritis (CIA) rats, and its effects on the clinical and histopathological aspects of RA were investigated. Co-administration of L. casei with CII more effectively suppressed clinical symptoms, paw swelling, lymphocyte infiltration and destruction of cartilage tissues of experimental arthritis than the rats treated with CII alone. The enhanced therapeutic efficacy was associated with an increase in anti-inflammatory cytokines (IL-10 and TGF-beta) while decreasing pro-inflammatory cytokines (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma and TNF-alpha). Co-administration of L. casei with CII more effectively suppressed CII-reactive T cell proliferation and the levels of Th1-type IgG isotypes (IgG2a and IgG2b), while up-regulating Foxp3 expression levels and the population of Foxp3(+) CD4(+) T cells. Our study provides evidence that L. casei could potentiate antigen-specific oral tolerance and suppress Th1-type immune responses of arthritic inflammation.     

Predominance of Th1-type T cells in synovial fluid of patients with Yersinia-induced reactive arthritis.

The pathogenetic mechanisms underlying the development of reactive arthritis and the functional capacities of synovial T cells specific for Yersinia enterocolitica are still unclear. In this study we have determined the cytokine secretion patterns of 24 CD4+ synovial fluid (SF)-derived T cell clones from 2 patients with Yersinia-induced reactive arthritis, 16 clones specific for different Yersinia antigens and 8 clones as controls. The clones specific for Yersinia antigens predominantly belong to the T helper cell 1 (Th1) subset with production of interferon (IFN)-gamma and interleukin (IL)-2, but no IL-4, whereas SF T cells not reactive with Yersinia antigens produce IL-2, IL-4 and IFN-gamma and thus belonged to the Th0 subset. Moreover, short-term T cell lines established from SF and peripheral blood showed the same pattern. To further analyze the functional relevance of these data we investigated the influence of IFN-gamma and IL-4 on the intracellular killing of Yersinia in a human glioblastoma cell line. Our data show that the Th1 cytokine IFN-gamma promotes intracellular killing of Yersinia, whereas this effect is antagonized by the Th2 cytokine IL-4. Furthermore, the Th2 cytokine IL-10 inhibited the antigen-specific proliferative response and IFN-gamma and IL-2 production by the Th1 cells. These results provide insight into the antibacterial mechanisms at work in reactive arthritis after infection with Yersinia enterocolitica and, for the first time, reveal the cross-regulatory properties of cytokines derived from Th1 and Th2 cells in a human immune response to bacterial antigens.     

T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Th 1 Subset

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.     

Enhanced cytokine generation by peripheral blood mononuclear cells in allergic and asthma subjects.

BACKGROUND: Although preferential expression of the Th2 cytokines, IL-4 and IL-5, has been described in atopic asthma, the role of IFN-gamma and IL-10 are less clear. OBJECTIVE: To determine the cytokine pattern of T cell mitogen-induced peripheral blood mononuclear cells obtained from atopic asthmatic (AA) subjects. METHODS: Peripheral blood mononuclear cells obtained from AA (n = 24), allergic rhinitis (AR) (n = 9), and normals (NL) (n = 9) were stimulated with phytohemagglutinin (PHA) and the generation of IL-4, IL-5, IFN-gamma, IL-10, and GM-CSF was quantified using ELISA. RESULTS: Compared with NL subjects, peripheral blood mononuclear cells from the atopic groups had increased generation of both IL-5 (AA, P = .001 and AR, P = .024) and IFN-gamma (AA, P = .037 and AR, P = .048) and decreased generation of IL-10 (AA, P = .038 and AR, P = .036). The absolute levels of cytokines did not differ between the two atopic groups; however, the ratio of IL-5/IL-10 was significantly higher in AA (P < .05), but not in AR when compared with NL subjects. CONCLUSION: The concomitant increase in the generation of IL-5 and IFN-gamma, with a decrease in IL-10 in the atopic groups suggests that in, at least a subset of these patients, there is potential expression of both Th2- and Th1-type cytokines. Furthermore, the increased IL-5 to IL-10 ratio could represent a key feature that distinguishes atopic asthmatic from non-asthmatic atopic subjects.     

In vivo activated T cells in rheumatoid synovitis. Analysis of Th1- and Th2-type cytokine production at clonal level in different stages of disease

T-cell cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). Their detection in the joint, however, is impaired by the complex network present in the synovium. Although many synovial T cells show signs of previous activation, only a few express interleukin (IL)-2 receptor, marker of recent activation. The aim of this study was to analyse the cytokine production by in vivo activated (IL-2R +) T cells from RA at different stages of the disease. For this purpose, T cells were isolated from peripheral blood and synovial fluid of four patients with active RA, two at the onset of the disease, one in the early phase during treatment, one in long-lasting chronic phase. One patient was studied at the onset of the disease and 52 months later. Cells were initially expanded with a low dose of IL-2, cloned and analysed for cytokine production. The results showed a strong predominance of T helper (Th) 1 clones in the blood and a slight prevalence of Th0 clones in the joint of all the four patients. Interferon-gamma and IL-2 production was higher in the long-lasting RA, whereas IL-4 synthesis was prevalent in early RA. Enrichment in IL-10-producing clones was present only in the joint of the untreated patients. The longitudinal study confirmed the differences in cytokine production between early and late phases of disease. These data confirm that RA is mainly a Th1-driven condition. However, in vivo activated synovial T cells produce also Th2-type anti-inflammatory cytokines, such as IL-4 and IL-10. The synthesis of both cytokines is a feature of the very early phase of RA, although the selective recruitment of IL-10-producing T cells is quickly lost.     

The expression and significance of intracellular T helper cytokines in systemic lupus erythematosus

To analyze the Th1 and Th2 paradigm of peripheral T helper cells in patients with systemic lupus erythematosus (SLE). The intracellular Th1 and Th2 cytokines were analyzed in fresh blood T cells from 20 SLE patients who had not yet received any treatment. Th1 and Th2 cells were quantitated based on their intracellular cytokine content as assessed by flow cytometry. Cytokine expressions were correlated with clinical features, laboratory findings, and disease activities. There was no difference in the expression of intracellular IFN-y, or IL-4 between SLE patients and healthy controls. However, the IL-2 and IL-10 levels were significantly higher and lower respectively in the lupus patients than in the control group. In addition, patients with arthritis had higher IFN-gamma expression than patients without arthritis. Moreover, patients with serositis or CNS involvement had higher IL-4 expression than in patients without these manifestations. There was no correlation between the SLEDAI scores and the cytokine expression levels. However, patients with serum anti-ds DNA antibodies had higher IL-10 levels than in those without these antibodies. The present study demonstrates that a Th1 pattern of intracellular cytokines predominates in patients with SLE prior to treatment. The pattern of particular intracellular T cell cytokines may suggest specific clinical manifestations and disease progression of SLE.     

Essential role of TGF-beta in the natural resistance to experimental allergic encephalomyelitis in rats

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non-MHC genes since congenic BN-1L rats, with LEW MHC on a BN-derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non-MHC-encoded resistance to develop clinical EAE in BN-1L rats is associated with a decreased production of IFN-gamma. This may be due to a difference between LEW and BN-1L rats in their ability to produce regulatory cytokines such as IL-4, IL-10 and TGF-beta. In comparison to LEW rats, immune lymph node cells from BN-1L rats express an increased amount of IL-4 mRNA but produce less IL-10. Furthermore, the sera from BN-1L rats contain higher amounts of active TGF-beta1. Therefore, we have investigated the involvement of IL-4 and TGF-beta in the resistance of BN-1L rats to develop EAE using neutralizing mAb. Neutralization of TGF-beta, but not IL-4, renders BN-1L rats susceptible to clinical EAE without affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF-beta production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RC(low) phenotype.