Leukemia inhibitory factor induces endothelial differentiation in cardiac stem cells

The importance of interleukin 6 (IL-6)-related cytokines in cardiac homeostasis has been studied extensively; however, little is known about their biological significance in cardiac stem cells. Here we describe that leukemia inhibitory factor (LIF), a member of IL-6-related cytokines, activated STAT3 and ERK1/2 in cardiac Sca-1+ stem cells. LIF stimulation resulted in the induction of endothelial cell-specific genes, including VE-cadherin, Flk-1, and CD31, whereas neither smooth muscle nor cardiac muscle marker genes such as GATA4, GATA6, Nkx-2.5, and calponin were up-regulated. Immunocytochemical examination showed that about 25% of total cells were positively stained with anti-CD31 antibody 14 days after LIF stimulation. Immunofluorescent microscopic analyses identified the Sca-1+ cells that were also positively stained with anti-von Willebrand factor antibody, indicating the differentiating process of Sca-1+ cells into the endothelial cells. IL-6, which did not activate STAT3 and ERK1/2, failed to induce the differentiation of cardiac stem cells into the endothelial cells. In cardiac stem cells, the transduction with dominant negative STAT3 abrogated the LIF-induced endothelial differentiation. And the inhibition of ERK1/2 with the MEK1/2 inhibitor U0126 also prevented the differentiation of Sca-1+ cells into endothelial cells. Thus, both STAT3 and ERK1/2 are required for LIF-mediated endothelial differentiation in cardiac stem cells. Collectively, it is proposed that LIF regulates the commitment of cardiac stem cells into the endothelial cell lineage, contributing to neovascularization in the process of tissue remodeling and/or regeneration.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Differential effects of IL-27 on human B cell subsets

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.     

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.     

Two-sided roles of IL-27: induction of Th1 differentiation on naive CD4+ T cells versus suppression of proinflammatory cytokine production including IL-23-induced IL-17 on activated CD4+ T cells partially through STAT3-dependent mechanism

Recent lines of evidence have demonstrated that IL-27, a newly identified IL-12-related cytokine, has two apparently conflicting roles in immune responses: one as an initiator of Th1 responses and the other as an attenuator of inflammatory cytokine production. Although the IL-27-mediated Th1 initiation mechanism has been elucidated, little is known about the molecular basis for the suppression of cytokine production. In the present study, we demonstrated that IL-27 suppressed the production of various proinflammatory cytokines by fully activated CD4+ T cells while it had no effect on the cytokine production by CD4+ T cells at early phases of activation. IL-27 also suppressed IL-17 production by activated CD4+ T cells, thereby counteracting IL-23, another IL-12-related cytokine with proinflammatory effects. In fully activated CD4+ T cells, STAT3 was preferentially activated by IL-27 stimulation, whereas both STAT1 and 3 were activated by IL-27 in early activated CD4+ T cells. Lack of STAT3 in fully activated cells impaired the suppressive effects of IL-27. These data indicated that the preferential activation of STAT3 in fully activated CD4+ T cells plays an important role in the cytokine suppression by IL-27/WSX-1.     

Induction of IgG2a class switching in B cells by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-gamma-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-gamma-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.