Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.     

Regulation of the IL-10 production by human T cells

Interleukin (IL)-10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL-10 production is therefore highly important for understanding the immunoregulation. The IL-10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha all had efficacious and independent costimulatory activities on the IL-10 production, while PGE2 was inhibitory. Dependence on autocrine IL-2 signalling was shown by the effects of anti-IL-2 and anti-IL-2R monoclonal antibodies (MoAb), but the IL-10 production proceeded partly IL-2-independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL-12 or rIFN-alpha, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL-10 (a cytokine-inhibitory interleukin) and IL-2 (a cytokine-inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL-10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.     

Regulation of tumor growth and metastasis by interleukin-10: the melanoma experience.

Because interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, including melanoma, we hypothesized that its production by tumor cells may contribute to the escape from immune surveillance. To test this hypothesis, we transfected human A375P melanoma cells that do not express IL-10 with the murine IL-10 gene and subsequently analyzed for changes in tumor growth and metastasis in nude mice. Surprisingly, IL-10 gene transfer resulted in a loss of metastasis and significant inhibition of tumor growth. In addition, the growth of other murine or human melanoma cells was also inhibited when they were admixed with IL-10-transfected cells before injection into nude mice. We provide evidence that IL-10 exerts its antitumor and antimetastatic activity by inhibiting angiogenesis in vivo. The in vivo decrease in neovascularization found in IL-10-secreting tumors is most likely due to the ability of IL-10 to downregulate the synthesis of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and matrix metalloproteinase-9 (MMP-9) in tumor-associated macrophages. Other studies have shown that IL-10 inhibits tumor metastasis through a natural killer (NK) cell-dependent mechanism. The inhibitory effects of IL-10 on tumor growth and metastasis were also demonstrated in other tumor models, including breast cancers. Furthermore, administration of rIL-10 into mice resulted in inhibition of tumor metastasis. Because IL-10 has little toxicity when given systemically to human volunteers, its efficacy as an antimetastatic agent should be further explored, both as an independent and in combination with other inhibitors of neovascularization.     

IL-10 is necessary for FasL-induced protection from experimental autoimmune thyroiditis but not for FasL-induced immune deviation

Ectopic expression of FasL on thyrocytes confers immune privilege status to the thyroid by inducing apoptosis of Fas-expressing autoimmune effector T cells and anti-thyroglobulin (Tg) immune deviation away from the T1 type. Fas-mediated apoptosis of lymphoid cells leads to rapid production of anti-inflammatory cytokines such as IL-10. On the other hand, cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis (EAT), and systemic and local administration of IL-10 has a curative effect on EAT. To test the effect of endogenous IL-10 production in EAT, and to find out whether IL-10 production could be involved in FasL-induced protection, EAT was induced in IL-10(-/-) and in IL-10(-/-)xFasL-transgenic CBA/J mice.The results demonstrated that wild-type and IL-10 knockout (KO) animals developed similar EAT. In contrast, lack of endogenous IL-10 abolished the protective effect of FasL. Polymorphonuclear cells were observed significantly more frequently in the inflammatory cell infiltrates from IL-10(-/-)xFasL animals compared to IL-10(-/-) animals, but they were never detected in wild-type or IL-10(+/+)/FasL-transgenic mice. A shift away from T1 response was observed in FasL-transgenic mice irrespective of their IL-10 status, demonstrating that in our model, endogenous IL-10 plays no part in the T1-towards-T2 anti-Tg immune balance induced by FasL. In summary, endogenous IL-10 is not essential in EAT, or for the immune deviation induced by thyroid FasL expression, whereas it is necessary for the immune privilege status of the thyroid conferred by FasL expression on thyrocytes.     

Prolonged survival of mice with Pseudomonas aeruginosa-induced sepsis by rIL-12 modulation of IL-10 and interferon-gamma

Interleukin-12 (IL-12) is thought to play an important role as a modulator of levels of IL-10 and interferon-gamma (IFN-gamma). To address the therapeutic effects of rIL-12 in an endogenous sepsis model in mice, which closely mimics the pathophysiology of septicaemia in man, the effects of rIL-12 on the levels of cytokines such as IL-10 and IFN-gamma, and on the survival of septic mice infected with Pseudomonas aeruginosa PAO1 were examined. First, in the endogenous sepsis model, the serum levels of IFN-gamma and IL-10 remained normal until days 8 and 10, respectively, when significant rises were seen. On day 11, levels of IFN-gamma returned to normal, but levels of IL-10 remained high. Interestingly, the IL-10 serum level reached a maximum 2 days later than the IFN-gamma serum level. In the light of these results, septic mice were given 0.01 microg of rIL-12 by intraperitoneal injection and the serum levels of endogenous cytokines and the survival times were examined. Mice treated with rIL-12 on days 5, 6 and 7 after infection survived significantly longer than control septic mice treated with saline only. Treatment with rIL-12 also led to a significant increase of the serum IFN-gamma level and a decrease of the serum IL-10 level on day 11. These results suggest that rIL-12 exerts therapeutic activity against endogenous sepsis caused by P. aeruginosa by stimulating proinflammatory responses and attenuating anti-inflammatory responses.     

Ribavirin shows immunomodulatory effects on activated microglia

Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.     

A new carboxamide compound exerts immuno-suppressive activity by inhibiting dendritic cell maturation

The immunosuppressive properties of a benzamide derivative, JM34, previously characterized as an anti-inflammatory compound are described. The immunosuppressive potential of JM34 was evidenced by inhibition of PBMC proliferation in vitro with an IC50 of 20 microM. In contrast with classical immunosuppressive drugs, JM34 affected neither cytokine production nor IL-2R expression from activated T cell clones, and displayed only moderate inhibition of IL-2-induced or anti-CD3/anti-CD28-induced proliferation. We investigated its effects on dendritic cells (DC) in vitro. Addition of JM34 during DC maturation inhibited the expression of some maturation markers: specifically, MHC molecule up-regulation was totally inhibited and CD83 expression was significantly reduced, while up-regulation of CD86, CD80 or CD40 was less affected. Moreover, JM34-treated DC showed impaired IL-12 but not IL-10 secretion, and a markedly reduced ability to present antigens to naive T lymphocytes in vitro. We provide evidence that these JM34-induced alterations of DC were associated with a marked inhibition of NF-kappaB nuclear translocation. Finally, JM34 inhibited delayed type hypersensitivity dose dependently in mice. In conclusion, our data suggest that JM34 inhibited T lymphocyte activation mainly by targeting DC, and thus may represent a new class of therapeutic agents in the fields of transplantation and autoimmune diseases.     

Interleukin-10 differentially regulates B7-1 (CD80) and B7-2 (CD86) expression on human peripheral blood dendritic cells

Most of the immunosuppressive effects of interleukin-10 (IL-10) are related to functional inhibition of antigen-presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL-10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL-10 inhibited in a dose-dependent manner the proliferative responses as well as the production of IL-2 and interferon-γ (IFN-γ) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL-10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL-10 were found to be deficient in the induction of alloreactive T cells even when anti-IL-10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL-10 did not modify the expression of ICAM-1 (CD54) and B7-1 (CD80), but decreased HLA-DR and B7-2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL-10 on primary alloreactive T cell responses involves down-regulation of class II MHC and B7-2 expression at the DC surface.     

The beneficial and detrimental effects of linoleic acid on autoimmune disorders

Type 1, or cellular, immune response is characterized by overproduction of IL-1, IL-2, IFN-gamma and TNF-alpha and is the underlying immune mechanism of some autoimmune disorders such as psoriasis, alopecia areata, rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes mellitus and experimental autoimmune uveitis. Type 2 immune response is seen in allergic and antibody-mediated autoimmune diseases and is characterized by IL-4, IL-6 and IL-10 overproduction. Linoleic acid is a precursor of prostaglandin E2 (PGE2) and its intake results in tissue production of PGE2, especially in the absence of other polyunsaturated fatty acids (PUFAS) which inhibit this conversion. PGE2 decreases the production of IL-1, IL-2, IFN-gamma and TNF-alpha and proliferation of TH1 cells and increases the production of IL-4, leading to suppression of the type 1 immune response. Taken together, linoleic acid, the major PUFA of maize oil, could have therapeutic efficacy against cellular autoimmune disorders. On the other hand, excessive intake of linoleic acid may aggravate type 2 autoimmune disorders.     

IL-10 has a distinct immunoregulatory effect on naive and active T cell subsets.

Interleukin-10 (IL-10) has been identified as a key immunomodulatory cytokine on T cells. However, both immunosuppressive and immunostimulatory effects of IL-10 on T cells also have been reported. The discrepancy between these in vitro effects of IL-10 may be due to the different T cells (naive vs. active or resting active T cells) used under various experimental conditions in these studies. Therefore, it is necessary to clearly define the IL-10 effect on T cell subsets in their different statuses. In this study, we used a molecularly defined T cell system, the ovalbumin (OVA)-specific CD4(+) and CD8(+) T cells from transgenic OT-I and OT-II mice expressing OVA-specific T cell receptor (TCR). We investigated the effect of IL-10 on these OVA-specific T cell subsets in their different statuses (i.e., naive and active T cells). Our data demonstrate that IL-10 has distinct immunoregulatory effects on naive and active T cell subsets. IL-10 inhibits active CD4(+) T cell proliferation, whereas it stimulates and suppresses active CD8(+) T cell proliferation and cytotoxicity, respectively. IL-10-treated dendritic cells (DCs) stimulate anergic cytotoxic T lymphocyte-associated molecule-4 (CTLA)-4-expressing CD4(+) T cell responses possibly through downregulation of major histocompetibility complex (MHC) class II and costimulatory molecule expression on DCs. The anergic CD4(+) T cells suppress T cell proliferation mainly through a CTLA-4-mediated pathway. The distinct role of IL-10 on T cell subsets may be useful in designing T cell-based immunotherapy of cancer and infectious diseases.     

Type I interferons as immunoregulatory molecules; implications for therapy in experimental autoimmune uveoretinitis

Clinical trials have shown that the type I interferon (IFN)-alpha/beta have some beneficial effects on organ-specific autoimmune diseases, such as Behcet's diseases and multiple sclerosis, although the precise mechanisms remain largely unresolved. T helper cells 1 (Th1)-mediated autoimmune responses are involved in the initiation and/or progression of human uveitis, such as Behcet's disease. The animal model of experimental autoimmune uveoretinitis (EAU), characterized by a monophasic clinical course, has contributed to the understanding of the pathogenesis of human uveitis. Th1 producing IFN-gamma induce EAU development, while Th2 producing IL-4/IL-10 prevent the disease. However, depending on the cytokine milieu, the pro-inflammatory cytokine IFN-gamma may attenuate the autoimmune responses and anti-inflammatory cytokine IL-4 exacerbates it. Chemokines also play a crucial role in EAU development, which might be resolved by Th2-mediated immune responses. The administration of IFN-alpha/beta prevents EAU development, accompanied by a diminished production of IFN-gamma/IL-10. Interestingly, however, IFN-alpha/beta also have some beneficial effects on patients with Th2-like phenotype in addition to Th1-like phenotypes. Thus, the immuno-modulatory action of IFN-alpha/beta may be dependent on the context of cytokine combination and/or their concentrations.     

Regulation of immune responses by interleukin-27

Summary: Cytokine-mediated immunity plays a crucial role in the pathogenesis of various diseases including autoimmunity. Recently, interleukin-27 (IL-27) was identified, which, along with IL-12, IL-23, and IL-35, belongs to the IL-12 cytokine family. These family members play roles in the regulation of T helper (Th) cell differentiation. IL-27 is unique in that while it induces Th1 differentiation, the same cytokine suppresses immune responses. In the absence of IL-27-mediated immunosuppression, hyper-production of various pro-inflammatory cytokines concomitant with severe inflammation in affected organs was observed in IL-27 receptor α chain (WSX-1)-deficient mice infected with Trypanosoma cruzi. Experimental allergic or inflammatory responses were also enhanced in WSX-1-deficient mice. The immunosuppressive effects of IL-27 depend on inhibition of the development of Th17 cells (a newly identified inflammatory T-helper population) and induction of IL-10 production. Moreover, administration of IL-27 or augmentation of IL-27 signaling suppresses some diseases of autoimmune or allergic origin, demonstrating its potential in therapy of diseases mediated by inflammatory cytokines. In this review, we discuss recent studies on the role of IL-27 in immunity to parasitic and bacterial infections as well as in allergy and autoimmunity in view of its pro- and anti-inflammatory properties.     

Anti-inflammatory action of alpha-melanocyte stimulating hormone (alpha-MSH) in anti-CD3/CD28-mediated spleen and CD4(+)CD25(-) T cells and a partial participation of IL-10

alpha-Melanocyte stimulating hormone (alpha-MSH) has been shown to inhibit the production and the effects of pro-inflammatory cytokine by inflammatory cells in innate immunity. We have determined whether alpha-MSH inhibits anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation and its mechanism of action. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells markedly were suppressed by 50-100 nM and 5-100 nM alpha-MSH, respectively. alpha-MSH (100 nM) increased the production of the anti-inflammatory cytokine IL-10 and decreased the production of the pro-inflammatory cytokine IL-2 and IFN-gamma from CD4(+)CD25(-) T cells. Moreover, anti-IL-10 blocking Ab decreased the inhibitory effects of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation by alpha-MSH, indicating a partial participation of IL-10 in its mechanism of inhibitory action. These results suggest that alpha-MSH may be useful for treatment of autoimmune diseases and transplantation involving innate and adaptive immunity.     

IL-4-induced immune deviation as antigen-specific therapy for inflammatory autoimmune disease

Organ-specific autoimmune diseases arc mediated by interferon γ (IFN-γ)-producing T helper 1 (Th1) cells. Here, Martin Röcken and colleagues review the experimental basis for an antigen-specific therapeutic approach to inflammatory autoimmune diseases. This strategy involves selective deviation of harmful Th1 responses towards an anti-inflammatory, interleukin 4 (IL-4)-producing Th2 phenotype.