Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia

Microglia, macrophage-like cells in the CNS, are multifunctional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration and are one of important cells in the CNS cytokine network to produce and respond to a variety of cytokines. The functions of microglia are regulated by inhibitory cytokines. We have reported the expression of interleukin (IL)-10, one of the inhibitory cytokines, and its receptor in mouse microglia; therefore, IL-10 may affect microglial functions. In this study, we investigated the effects of IL-10 on purified microglia in culture. IL-10 inhibited lipopolysaccharide-induced IL-1beta and tumor necrosis factor-alpha production, lysosomal enzyme activity, and superoxide anion production in a dose-dependent manner, but did not affect granulocyte/ macrophage colony-stimulating factor-dependent proliferation of microglia. IL-10 also decreased the expression of both IL-6 receptor and lipopolysaccharide-induced IL-2 receptor but not IL-4 receptor on microglia as measured by flow cytometric analysis with an indirect immunofluorescence technique. IL-10 also decreased mRNA expression of IL-2 and IL-6 cytokine receptors. These results suggest that IL-10 is a unique and potent inhibitory factor in the CNS cytokine network involved in decreasing the expression of cytokine receptors as well as cytokine production by microglia.     

Murine microglial cells produce and respond to interleukin-18

Interleukin (IL)-18 (interferon-gamma-inducing factor or IL-1gamma) belongs structurally to the IL-1 cytokine family and shares biological properties with IL-12. Expression, intracellular signaling, and functional relevance of IL-18 within the CNS are mostly unknown. We show that IL-18 protein is synthesized within mouse brain, preferentially during early postnatal stages, and that microglial cells but not astrocytes are a potential source. IL-18 is produced by cultured microglia on exposure to lipopolysaccharide (LPS). Microglia also express major components of the IL-1/IL-18 receptor system. On IL-18 stimulation, microglial IL-1 receptor-associated kinase (IRAK) can be coprecipitated with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) but not with IL-1 receptor type I, indicating that IRAK recruits TRAF6 during IL-18 signaling. IL-18 inhibits the LPS-induced release of IL-12 and attenuates that of TNF-alpha, whereas the production of IL-6 and macrophage inflammatory protein-1alpha is only marginally affected. IL-18 may play a role during CNS development and can be produced by activated microglia, thus probably contributing to immune and inflammatory processes in the brain.     

IL-2 and IL-15 manifest opposing effects on activation of nuclear factor of activated T cells

IL-2 and IL-15 are cytokines involved in T cell activation and death. Their non-shared receptors, IL-2Ralpha and IL-15Ralpha, are important in the homeostasis of lymphocytes as evidenced by gene deletion studies. How these cytokine/receptor systems affect T cell antigen receptor signaling pathways is poorly understood. Here, we show that the IL-2 and IL-15 cytokine/receptor alpha systems regulate activation of nuclear factor of activated T cells (NF-AT) in opposing ways. IL-15Ralpha increased while IL-2Ralpha decreased basal NF-AT activation status in a Jurkat transient transfection model. The effect of each of the alpha chain receptors on NF-AT activation was further opposed by addition of the respective cytokine. These effects were inhibited by anti-cytokine and anti-cytokine receptor reagents as well as by inhibitors of TCR signaling. These results suggest a novel pathway of cytokine action to regulate T cell signaling, activation, death, and homeostasis.     

Tubulin Polymerization Modulates Interleukin-2 Receptor Signal Transduction in Human T Cells

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R β -chain and enhanced the association of the IL-2R γ -chain with cytoskeletal tubulin. The IL-2R β -chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R α-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R α-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([³H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R α-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.     

Enhanced and prolonged efficacy of superantigen-induced cytotoxic T lymphocyte activity by interleukin-2 in vivo

The bacterial superantigen, staphylococcal enterotoxin A (SEA) activates T cells with high frequency and directs them to lyse MHC-class-II-expressing cells in superantigen-dependent cell-mediated cytotoxicity (SDCC). Treatment of mice with SEA induced strong CD8⁺ T-cell(CTL)-mediated SDCC, as well as abundant cytokine production from CD4⁺ and CD8⁺ T cells. However, both cytotoxicity and cytokine release were transient. In contrast, combined treatment with SEA and recombinant interleukin-2 (rIL-2) increased peak levels and maintained CTL activity. These effects were concomitant with an increased number of SEA-reactive V11⁺ T cells. Both the CD4⁺ and CD8⁺ populations contained higher frequencies of cells expressing IL-2 receptor (IL-2R) , which suggests that continuous IL-2R signaling preserves its high expression and subsequently prevents loss of growth factor signal necessary for expansion of T cells. Although IL-2R expression was increased among both CD4⁺ and CD8⁺ cells, only the cytotoxic function of CTL, but not cytokine production from either CD4 or CD8, was augmented. These findings demonstrate that treatment with rIL-2 potentiates superantigen-induced cytotoxicity and maintains high CTL activity. rIL-2 might therefore be useful in improving superantigenbased tumor therapy.     

A cell-free Salmonella typhimurium extract modulates interleukin-2 (IL-2) receptor expression but not IL-2-stimulated responses of murine splenic lymphocytes

Abstract In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2). In this study, we found that a cell-free S. typhimurium extract modulated IL-2 receptor (IL-2R) expression on phytohemagglutinin (PHA)-stimulated murine spleen cells and this was a mechanism of T-cell non-responsiveness to IL-2, but did not affect IL-2 binding to IL-2R and the consequent responses. Western blotting using anti-phosphotyrosine antibodies showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in PHA-activated murine splenic T-cells, which express a high-affinity IL-2R (α- and β-chains), was not affected by treatment with the S. typhimurium cell-free extract. Furthermore, PHA-activated spleen T-cells responded to recombinant IL-2 and this was not inhibited by the extract. Surprisingly, IL-2R expression was augmented by treatment with the extract, although this was independent of IL-2 production. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract was associated with augmentation of IL-2R expression, rather than down-regulation of the IL-2 response. This may be a mechanism responsible for the Salmonella extract-evoked suppression of mitogen-induced T-cell proliferation.     

Interleukin-2 inhibits proliferation of HPV-associated tumor cells and halts tumor growth in vivo

Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.     

Astrocytes as antigen-presenting cells: expression of IL-12/IL-23

Interleukin-12 (IL-12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1-type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL-23, composed of the same p40 subunit as IL-12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen-presenting cell (APC) function can present antigen to myelin basic protein (MBP)-reactive T cells, and whether this presentation is blocked with antibodies against IL-12/IL-23p40. Interferon (IFN)-gamma-treated APC induced proliferation of MBP-reactive T cells. Anti-IL-12/IL-23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL-12/IL-23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL-12p70. Because IL-12 and IL-23 share p40, we wanted to determine whether astrocytes also express IL-12p35 and IL-23p19, as microglia were already shown to express them. Astrocytes expressed IL-12p35 mRNA constitutively, and IL-23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL-12/IL-23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti-IL-12/IL-23p40 antibodies.     

Hodgkin's disease and anaplastic large cell lymphoma revisited

In cultures, and in tissues as well, Hodgkin's and Reed-Sternberg (H-RS) cells and anaplastic large cell lymphoma (ALCL) cells are known to express a variety of cytokines, including IL-1, -5, -6, -8, -9, TNF-, GM-CSF, M-CSF, TGF-, CD70, CD80, and CD86. Various numbers of H-RS/ALCL cells may express cytokine receptors (R), such as CD30, CD40, IL-2R (CD25/CD122), IL-6R (CD126), IL-7R (CD127), TNF-R (CD120), TGF--R (CD105/endoglin), M-CSF-R (CD115), and SCF-R (CD117/c-kit receptor). All of these cytokines and cytokine receptors are implicated in the growth regulation of H-RS/ALCL cells, the histopathologic alterations in tissues, and the clinical manifestations in patients with Hodgkin's disease (HD) or ALCL. Many of these cytokines or cytokine receptors also play an important role in the pathogenesis of other types of lymphomas. In this review, we describe the cytokine or cytokine-receptor expression that is diacritic for H-RS/ALCL cells. The identification of such unique cytokine-cytokine receptor interactions is likely to explain the biologic property that distinguishes HD/ALCL from other types of lymphomas. These interactions include those of CD30L-CD30, CD40L-CD40, CD70-CD27, CD80/CD86-CD28, SCF-CD117, IL-9-IL9R, and IL-7-IL-7R. The H-RS/ALCL cells express IL-9 and two cytokine receptors, CD30 and CD117, which are observed infrequently in NHLs. Although IL-7 expression is not restricted to H-RS/ALCL cells, the expression of IL-7 in conjunction with IL-9 and/or CD117 may be regarded as unique for HD/ALCL because of an unusual combination and a synergistic activity among these cytokines. The expression of CD70 and CD80/CD86 (as cytokines) may exert a unique effect in HD because of intimate contact between H-RS cells and CD27/CD28-positive T cells. The expression of these costimulators (CD70 and CD80/CD86) and other adhesion/constimulator molecules such as CD54 and CD58, along with the secretion of soluble cytokines such as Il-1, IL-6, IL-7, or TNFs by H-RS/ALCL cells, could result in the profound T-cell proliferation often seen in lymph nodes involved by HD and some ALCL. On the other hand, the expression of CD30L and CD40L by surrounding T cells may affect the proliferation of H-RS/ALCL cells. The cytokine-cytokine receptor interaction between H-RS cells and T cells via direct cell-cell contact is bidirectional, a situation not commonly seen in NHLs.     

Short-term culture with IL-2 is beneficial for potent memory chimeric antigen receptor T cell production

Interleukin-2 (IL-2) has been extensively used to boost the body's immune cells, especially T cells. IL-2 is a cytokine that for many years was used to activate and amplify T cells. Due to its potent T cell growth-inducing functions in vitro, for many years, IL-2 was used for the culture and expansion of various T cell products, including tumor-infiltrating lymphocytes (TIL), T cell receptors T cells (TCR T), or genetically engineered cells with chimeric antigen receptors T cells (CAR T). Despite its positive effect on T cell production, the side-effect is not well studied. Here, we reported that long-term culture with IL-2 promotes terminal differentiation and impairs rather than boosts the function of chimeric antigen receptor T cells. However, short-term culture with IL-2 predominantly generates memory CAR T cell favorable for cancer treatment.     

Interleukin-34 Restores Blood–Brain Barrier Integrity by Upregulating Tight Junction Proteins in Endothelial Cells

Interleukin-34 (IL-34) is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R), and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS) and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood–brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood–brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer''s disease.     

Interleukin-19 Acts as a Negative Autocrine Regulator of Activated Microglia

Activated microglia can exert either neurotoxic or neuroprotective effects, and they play pivotal roles in the pathogenesis and progression of various neurological diseases. In this study, we used cDNA microarrays to show that interleukin-19 (IL-19), an IL-10 family cytokine, is markedly upregulated in activated microglia. Furthermore, we found that microglia are the only cells in the nervous system that express the IL-19 receptor, a heterodimer of the IL-20Rα and IL-20Rβ subunits. IL-19 deficiency increased the production of such pro-inflammatory cytokines as IL-6 and tumor necrosis factor-α in activated microglia, and IL-19 treatment suppressed this effect. Moreover, in a mouse model of Alzheimer’s disease, we observed upregulation of IL-19 in affected areas in association with disease progression. Our findings demonstrate that IL-19 is an anti-inflammatory cytokine, produced by activated microglia, that acts negatively on microglia in an autocrine manner. Thus, microglia may self-limit their inflammatory response by producing the negative regulator IL-19.     

白细胞介素2新的功能位点及其中枢镇痛作用

白细胞介素２（ＩＬ－２）,不仅是重要的免疫调节因子,而且具有重要的中枢调节作用。本工作表明：（１）ＩＬ－２具有中枢镇痛作用;（２）ＩＬ－２除具有免疫调节作用功能位点外,还存在着另一新的与之相互独立的镇痛功能位点;（３）ＩＬ－２的中枢镇痛作用,主要是由ＩＬ－２第４５位Ｔｙｒ残基以及空间结构上邻近的４４、１１７位Ｐｈｅ等残基共同构成的镇痛功能位点与阿片受体直接结合所介导。本工作提示,细胞因子的多功能性,可能是其相互独立的功能位点作用于不同的受体或受体亚型所致。