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Clinical Reviews in Allergy and Immunology © Copyright 2005 by Humana Press Inc. 1080-0549/05/257–269/$30.00
Pathological Role of IL-6 in the Experimental Allergic Bronchial Asthma in Mice Aysefa Doganci, Kerstin Sauer, Roman Karwot, and Susetta Finotto*
Abstract
Laboratory of Cellular and Molecular Immunology of the Lung, I. Medical Clinic, University of Mainz, Germany.
Although allergic asthma was described to be associated with the presence of mucosal Thelper (Th)2 cells, it is not entirely clear which factors are responsible for priming of T cells to differentiate into Th2 effector cells in this disease. Interleukin (IL)-6 has been recognized as important because it is secreted by cells of the innate immunity and induces the expansion of the Th2 effector cells, which are major players of the adaptive immune responses. Additionally, IL-6 released by dendritic cells (DCs) inhibits the suppressive function of CD4+CD25+ Tregulatory cells, thus inhibiting the peripheral tolerance. The signal transduction of IL-6 has recently taught us how this cytokine influences different aspects of the immune response, especially under pathological conditions. IL-6 can bind to the soluble IL-6R, increased after allergen challenge in asthmatic patients, and, through a mechanism called trans-signaling, induces proliferation of cells expressing the cognate receptor gp130. This mechanism appears to be used for proliferation by developed Th2 cells in the airways. In contrast, through the membrane-bound IL-6R, IL-6 controls CD4+CD25+ survival, as well as the initial stages of the Th2 cells development in the lung. These findings impact the establishment of new therapies for allergic diseases; indeed, blockade of the soluble IL-6R through the fusion protein gp130Fc reduces Th2 cells in the lung, and by blocking the membrane-bound IL-6R, anti-IL-6R antibody treatment induces the number of T-regulatory cells in the lung, thereby reducing the local number of CD4+ T-effector cells in experimental asthma.
Index Entries T-regulatory cells; Foxp3; T-bet; GATA-3; lung; allergic asthma; IL-6; IL-6R; gp130; antigenpresenting cells.
*
Author to whom all correspondence and reprint requests should be addressed. E-mail:
[email protected].
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Introduction Allergic asthma is a chronic inflammatory disease characterized by airway inflammation and airway hyperreactivity (AHR) that affects about 10% of the population in the United States (1) and other industrialized countries. This disease is also increasing in children. For example, in Sweden, the number of children with allergic rhinitis or asthma doubled over the last 12 yr. The term “allergy” was introduced in 1906 by von Pirquet, who applied this term to the “uncommitted” biological response that may lead either to immunity (a beneficial effect) or allergic disease (a harmful effect). Additionally, allergic diseases can be divided into two categories: atopic and nonatopic. The term “atopy” (from the Greek atopos, meaning out of place) is often used to describe immunoglobulin (Ig)E-mediated diseases. People with atopy have a hereditary predisposition to release IgE antibodies against common environmental allergens and suffer from one or more atopic diseases (i.e., allergic rhinitis, asthma, and atopic eczema). Some allergic diseases, such as contact dermatitis and hypersensitivity pneumonitis, develop through IgE-independent mechanisms and, in this sense, can be considered nonatopic allergic conditions (2). T-cells isolated from the blood of atopic persons respond to allergens in vitro by secreting type 2 T-helper (Th)2 cytokines (i.e., interleukin [IL]-4, -5, and -13) (3,4), rather than cytokines produced by Th1 cells (interferon [IFN]-γ and IL-2). It has been proposed that newborn infants have an immune system dominated by Th2 cells, and during subsequent development, the nonatopic infant’s immune system shifts in favor of a Th1-mediated response to inhaled allergens (a process termed “immune deviation”) (5). However, in the potentially atopic infant, there is a further increase in Th2 cells that were primed in utero. Much of the literature in the past century has focused on the interaction between the adaptive and innate immunity. Regarding Clinical Reviews in Allergy & Immunology
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asthma or allergic diseases, it has become clear that cells of the innate immunity play an important role in orchestrating the downstream adaptive immunoresponses. One of the focuses has been defining the cellular sources of IL-4 responsible for driving the naive T-cells into the Th2 pathway. Therefore, invariant naturalkiller (iNK) cells have been identified as the most likely source of IL-4 and IL-13, which are responsible for the Th2 development in allergic diseases. Invariant T-cells (iNKTs) constitute a lymphocyte subpopulation that is abundant in the thymus, spleen, liver, and bone marrow, and they are also present in the lung. NKTs express surface markers characteristic of both NK cells (such as NK1.1 and CD161) and conventional T-cells (such as T-cell receptors [TCRs]). Several NKTs (referred to as Vα14 iNKT cells) recognize glycolipid antigens presented by the nonpolymorphic major histocompatibility complex (MHC) class I-like protein CD1d and express an invariant Vα14-Jα281 (also called Jα18 or Jα15) TCR in mice or an invariant Vα24-Jα15 TCR in humans (6). It was recently demonstrated in experimental asthma that IL-4 produced by iNKTs might be responsible for the priming of naïve CD4+ T-cells into CD4+ Th2 cells (7). The same cells secrete IL-13, which is responsible for allergen-induced airway hyperreactivity (AHR). Using NKT-deficient mice, the authors demonstrated that allergeninduced AHR, which is a cardinal feature of asthma, does not develop in the absence of Vα14 iNKTs. However, the failure of NKTdeficient mice to develop AHR is not caused by an inability of these mice to produce Th2 responses, because NKT-deficient mice that are immunized subcutaneously at nonmucosal sites produce normal Th2-biased responses. Moreover, macrophages are cells of the innate immunity present in high percentages in the lung and play a fundamental role in challenging downstream immune responses upon antigen exposure. These cells engulf microbes and secrete IL-12, which induces Th1 and NK Volume 28, 2005
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cells to produce IFN-γ, thereby shifting the immune system into an “allergy-protective” Th1-mediated response. Researchers should investigate the other type of antigen-presenting cells (APCs) and determine whether different subtypes of macrophages, which are situated at the gate of antigen exposure, drive different types of immune response. For example, it has been demonstrated that alveolar macrophages from atopic asthmatics—but not from nonatopic, nonasthmatics—enhance IL-5 production by CD4+ T-cells. Additionally, this study positively correlated the Th2-inducing capability of macrophages derived from atopic patients with their IL-1β and IL-6 secretion (8). Other factors may also influence whether Th1 or Th2 cells dominate the response, including the amount and type of allergen, the duration of exposure to the allergen, and the type of APCs and T-cells and their interaction. Although allergic asthma was described to be associated with the presence of mucosal Th2-cells, it is not entirely clear which factors are responsible for priming of T-cells to differentiate into Th2-effector cells in this disease. Moreover, Th2-driven immune responses are not sufficient to induce asthma per se. Additional elements—either structural cells or other cells of the immune system that are intrinsic to the respiratory tract—may be required for inducing the Th2 response in the lungs. A unique role is played by mast cells (MCs) and is defined not only by their extensive mediator profile but also by their ability to interact with the vasculature and to release chemotactic cytokines or chemokines for other inflammatory cells such as eosinophils. Acute MC activation is a feature of many types of tissue injury, as well as responses to numerous nonpathogenand pathogen-associated inflammatory stimuli. Histamine, which accounts for about 10% of MC granule content, can inhibit IL-12 secretion from monocytes that had been stimulated with bacterial products such as lipopolysaccharide or Staphylococcus aureus Cowon strain 1 (9,10). In contrast to DCs that matured in the absence Clinical Reviews in Allergy & Immunology
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of histamine, histamine-matured DCs polarize naive CD4+ T-cells toward a Th2 phenotype (11). Crosslinking of surface IgE by cognate antigens or direct binding of microbial products or complement components to cellular receptors induces MCs to release large amounts of preformed inflammatory mediators such as histamine, proteases, prostaglandins, and leukotrienes. Additionally, activated MCs produce numerous cytokines, including tumor necrosis factor-α, granulocyte macrophage colony-stimulating factor, and IL-3, -4, -5, -6, -10, and -13, which have the potential to influence T- and B-cell responses. Interestingly, IL-6 is secreted by MCs and, together with stem cell factor secreted by fibroblasts and endothelial cells, induces MC differentiation from progenitor cells. The MC–T-cell interaction is particularly relevant to asthma. In fact, T-cells secrete survival factor for MCs, and intra-epithelial MCs are depleted in the absence of T-lymphocytes (12).
Antigen-Presenting Cells Upon antigen exposure, the activation of APCs results in the release of factors that are responsible for the activation and differentiation of CD4+ T-cells. DCs are capable of either inducing productive immunity or maintaining the state of tolerance to self-antigens and -allergens. The diverse functions of DCs have been attributed to distinct lineages of DCs, which arise from common immature precursor cells that differentiate in response to specific maturation-inducing factors. These subsets of DCs induce different lineages of T-cells, such as Th1, Th2, and T-regulatory (Treg)- cells (including Th1[reg] and Th2[reg] cells). The latter can regulate allergic diseases and asthma, thus providing anti-inflammatory responses and protective immunity (13,14). Moreover, DCs have the capacity to secrete a large number of cytokines and express different co-stimulatory molecules, influencing the subsequent immune response. For example, subsets of DCs Volume 28, 2005
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(DC1) that produce IL-12 and express high levels of co-stimulatory molecules, such as B7.1 and B7.2, induce Th1 responses associated with the production of IFN-γ and IL-2 (15). In contrast, other DC subsets (DC2) and possibly those expressing low levels of co-stimulatory molecules—particularly those in the pulmonary parenchyma during repeated exposure to low levels of antigen—induce Th2 responses associated with the production of IL-4, -5 and -13 (16–19). Additionally, DCs have the capacity to induce T-cell unresponsiveness to antigens—a phenomenon also known as “tolerance” (20–23). The mechanism by which respiratory antigens induce T-cell tolerance includes T-cell clonal deletion, anergy, and active suppression mediated by regulatory cells secreting IL-10 or transforming growth factor (TGF)-β (24-26) The development of respiratory tolerance is initiated by uptake of antigen into the lungs by immature DCs (27).Within 24 h after respiratory exposure to antigens, such as ovalbumin, the pulmonary DCs migrate to the draining bronchial lymph nodes, where they mature, transiently produce IL-10, and express high levels of B7.1 and B7.2 co-stimulatory molecules (28). In the bronchial lymph nodes, DCs induce an initial phase of allergen-specific Tcell activation, proliferation, and expansion, followed by depletion of these T-cells from the lymphoid organs, although a stable population of these cells survive but remain refractory (functionally disabled) to antigenic rechallenge (29). However, establishing a tolerant state in allergen-specific T-cells prevents the development of airway inflammation. The inhibition of inflammation by T-cell tolerance is partially mediated by allergen-specific CD4 T-cells exerting potent regulatory/suppressor cell activity (adaptive Treg-cells). These antigen-specific CD4+ Treg-cells produce IL-10 and can be generated by activating CD4 T-cells, with IL-10 producing DCs from bronchial lymph nodes of tolerized mice (30). These DCs express high levels of co-stimulatory molecules, including inducible co-stimulatory molecule ligand (ICOSL), Clinical Reviews in Allergy & Immunology
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and possess a low capacity for phagocytosis, all of which are characteristics of mature DCs. It has been suggested that production of IL-10 by these DCs plays a major role in the generation of Treg-cells.
Treg-Cells A suppressor cell is functionally defined as a T-cell that inhibits an immune response by influencing the proliferation of the main effector cell population responsible for the disease. In neonatal and adult animals, it has been observed that suppressor cells can inhibit autoreactive T-cells. If the suppressor lineage was deleted or compromised in the adult animal, then auto-immune disease developed. Sakaguchi et al. (31) demonstrated that a minor population of CD4+ T-cells (10%) that co-expressed the CD25 + antigen (the IL-2Rα-chain) function as regulatory cells in the adult mice. When the CD4+CD25+, CD45RB/RClow T-cells were depleted from a population of normal adult CD4+ T-cells, the remaining CD4+CD25– T-cells were transferred to an immunocompromised recipient that would develop a spectrum of autoimmune diseases, and cotransfer of CD4+CD25+ cells prevented the development of autoimmunity (31). This and other studies pointed to the role of CD4+CD25+ T-cells as a major subset of cells that play a central role in regulating immune responses. CD4+CD25+ cells represent 5 to 8% of the total population of CD4+ T-cells in the normal mouse lymph node. Compared with CD4+CD25–, CD4+CD25+ populations have a slightly higher proportion of CD4+CD62Llow cells, and they are absent from the CD4+CD45RBhigh population. Recently, other markers have been defined to characterize Treg-cells. The major transcription factor Foxp3 has been recognized as associated with the CD4+ Treg-phenotype (32,33). All the CD4+CD25+ have the TCR-α and -β and intracellularly express the cytotoxic Tlymphocyte-associated antigen 4 (34–36). Additionally, it was recently demonstrated that TGF-β can transform peripheral CD4 +CD25 – naïve T-cells into CD4+CD25+ through inducVolume 28, 2005
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tion of Foxp3 (37,38). CD4+CD25+ Treg-cells do not directly mediate the death of the responders but induce a cell cycle arrest at the G1-S phase, which is often followed by cell death. It has been shown that human CD4+CD25+ T-cells induce suppressor activity in the CD4CD25– responders cells, which then become Treg-cells capable of suppressing by producing IL-10 and TGF-β. Finally, it has been demonstrated in a murine model of asthma that TGF-β-induced suppressor cells prevented house dust mite-induced allergic pathogenesis in lung (37,39,40). However, the role of TGF-β in asthma is not completely understood. Indeed, TGF-β has both enhanced regulatory capability and is a strong airway remodeling inducer that contributes to the worsening of this disease. Other experiments have shown that DCs in the bronchial lymph nodes can induce the development of Treg-cells via the ICOS– ICOSL pathway. Additionally, transfer of Treg-cells in recipient mice can inhibit the development of the AHR and lung eosinophilia in an IL-10- and ICOS-dependent manner (30).
Role of IL-6 Secretion by DCs in Inducing Treg-Cells IL-6 is produced by APCs, such as B-cells, macrophages, and DCs. It can also be secreted by nonprofessional APCs, such as glial, epithelial, endothelial, and some tumor cells. IL-6 is crucial for the acute phase response in hepatocytes, is involved in the differentiation of B-cells and myeloid cells, and induces growth of osteoblasts, hematopoietic, and neural cells (41). One important advance regarding tolerization of the Treg-cells in the spleen by DCs was provided by the observation that DCs produce a cytokine or soluble factors capable of blocking the suppression of T-effector cells. In this respect, it has been demonstrated that IL-6 has a dual role in immune responses. On one hand, IL-6 derived from APCs is able to induce initial IL-4 production in naïve CD4+ spleen T-cells via activation of the transcription Clinical Reviews in Allergy & Immunology
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factor NFATc2, thereby polarizing these cells into Th2 cells (41,42). Additionally, IL-6 is a survival factor for resting T-cells but not for activated CD4+ T-cells (43,44), and it protects CD8+ T-cells from cell death (45). IL-6 induces the expression of suppressor of cytokine signaling (SOCS)1, which inhibits IFN-γ signaling (46), a function that results in inhibition of the pathway leading to a preferential development of the Th2 pathway. Finally, IL-6 and other unidentified factors produced by mDCs are able to break CD4+CD25+ Treg-cell anergy and trigger significant proliferation and detectable IL-2 production. These results suggest that DCs may be important regulators of CD4+CD25+ Treg-cells—predominantly through cell–cell interaction, inhibiting or facilitating their function as required for productive immunity. Tolllike receptors (TLRs) control activation of adaptive immune responses by APCs. Production of IL-6 by DCs in response to TLR ligation during infection is critical for T-cell activation, because it allows pathogen-specific T-cells to overcome the suppressive effect of CD4+CD25+ Treg-cells. However, removal of Treg-cells allows T-cell activation even in the absence of IL-6 (in IL-6-deficient mice), suggesting that induction of co-stimulation on DCs is sufficient for T-cell activation in the absence of Tregcells.
IL-6 Signal Transduction IL-6 binds to the surface IL-6 receptor-α (mIL-6Rα) , leading to the dimerization of gp130/ IL-6Rβ into tetra- or hexametric (two IL-6s, two mIL-6Rs, and two gp130s) structures, thereby forming the active IL-6R complex (Fig. 1; ref. 47). However, neither IL-6 nor IL-6R alone can bind or activate gp130. Although gp130 is expressed ubiquitously, the expression of mIL-6R is restricted to the membrane of hepatocytes and hematopoietic cells. However, cells lacking mIL-6R can also respond to IL-6 via the soluble IL-6 receptor (sIL-6R), because the IL-6–sIL-6R complex (hyperIL-6) can activate Volume 28, 2005
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Fig. 1. Interleukin (IL)-6 signal transduction. (A) IL-6 trans-signaling. Cells lacking mIL-6R can respond to IL-6 via soluble (s)IL-6R. The IL-6–sIL-6R complex can activate target cells expressing gp130. This process is called trans-signaling, which can be blocked by gp130Fc. (B) Membrane-bound IL-6R is expressed on hepatocytes and hematopoetic cells. IL-6 can bind to mIL-6Rα (light gray) forming the active IL-6R complex, which leads to the dimerization of gp130 (dark gray). The dimerization of gp130 by IL-6 causes the activation of two intracellular signaling pathways: a. Activation of Janus kinase (JAK)1 leads to phosphorylation and activation of signal transducers and activators of transcription (STAT)3, whereas activation of JAK2 and JAK1 leads to STAT1 phosphorylation. b. Activation of intracellular Ras-Raf signal cascade.
target cells expressing gp130 in a process termed IL-6 trans-signaling (47–50). Dimerization of gp130 by IL-6 causes the activation of two signaling pathways: the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway and activation of JAK1 and 2, and Tyk2 by IL-6 result in the phosphorylation and activation of STAT3 and, to much lesser extent, STAT1, leading to the induction of type-2 IL-6 responsive gene expression (51–54). IL-6 also intracellularly activates the Ras-Raf signaling cascade, which regulates phosphorylation of mitogen-activated protein kinase and, Clinical Reviews in Allergy & Immunology
ultimately, activation of the transcription factors NF-IL-6 (a C/EBP family member) and AP-1 (c-Jun and c-Fos). Trials on IL-6-deficient mice recently demonstrated that sIL-6R regulates leukocyte recruitment in a subcutaneous air pouch model of inflammation (55). Subsequently, researchers demonstrated that although endothelial cells lack the cognate IL-6R, they could be activated to phosphorylate STAT3, produce chemokines (IL-8, MCP-1, and, to a lesser extent, MCP-3), and upregulate adhesion molecules (intracellular adhesion molecule-1, vascular cell adhesion molecule-1) expression in Volume 28, 2005
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response to the [sIL-6R–IL-6] complex (55,56). This activation likely occurs via direct interaction with the gp130 signal-transducing element. Therefore, the [sIL-6R–IL-6] complex plays a crucial role in regulation of leukocyte recruitment and may serve a positive role in the prothrombotic/pro-inflammatory activation of endothelial cells. Consistent with this finding, it was recently reported that the degree of leukocyte infiltration into arthritic joints correlates with elevated sIL-6R levels in synovial fluid (57).
SOCS3 Negatively Regulates IL-6 Signaling in Allergic Diseases Some members of the SOCS family are potential key physiological negative regulators of IL-6 signaling. SOCS3 deficiency shows prolonged activation of STAT1 and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with IFN-γ, indicating an inhibitory role of SOCS3 in IL-6 activation of STAT1 and STAT3. Conversely, IL-6-induced STAT activation is normal in SOCS1-deficient cells, whereas STAT1 activation induced by IFN-γ is extended, indicating that SOCS-1 controls IFN-γ-induced STAT1 activation (58). The SOCS and cytokine-inducible SH2 protein are pivotal physiological regulators of the immune system. Principally, SOCS1 and SOCS3 regulate T-cells, as well as APCs, including macrophages and DCs. Cytokines, including ILs, IFNs, and hemopoietins, activate the JAKs (JAK1, JAK2, JAK3, and Tyk2), which associate with their cognate receptors. Once activated, JAKs phosphorylate the cytoplasmic domain of the receptor, creating a docking site for SH2signaling proteins. Among the substrates of tyrosine JAKs, members of the STAT family of proteins are most important for cytokine actions. For example, IFN-γ activates JAK1 and JAK2, which mainly induce STAT1 phosphorylation. Binding of the pro-inflammatory cytokine IL-6 to the IL-6R α-chain and gp130 mainly activates STAT3 through JAK1. The antiClinical Reviews in Allergy & Immunology
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inflammatory cytokine IL-10 also activates STAT3 (59). Some members of the SOCS family are involved in the pathogenesis of inflammatory diseases (60). SOCS3 is a protein related to SOCS1 that is transiently induced by both inflammatory and anti-inflammatory cytokines, such as IFN-γ, IL-3, -6, and -10. SOCS3 has been demonstrated to blocks the JAK–STAT pathway in various inflammatory diseases. Indeed, SOCS members have an important role in controlling Th1 and Th2 development and activation. For example, Th2 cells exclusively express SOCS3 messenger RNA (mRNA) and protein in mouse T-cells, which may be responsible for the enhancement of airway responsiveness. SOCS3 expression favors the secretion of Th2 cytokines, and the presence of IL-4 in the Th cell commitment process is crucial for the expression of SOCS3 in the Th2 subset. Increased Th2 responses caused by increased expression of SOCS3 indicate that SOCS molecules act as autocrine-negative feedback regulators. The literature shows that in mouse models, enhanced expression of SOCS3 increases the risk for the Th2-mediated type I allergic disease. Studies in humans demonstrated that there is a correlation between SOCS3 expression and the severity of atopic dermatitis and asthma. It is possible that high SOCS3 expression in patients can be attributed to the accumulation of Th2 cells in the periphery, resulting in exacerbation of allergic pathogenesis.
IL-6 Family Members and Asthma As a pleiotropic cytokine, IL-6 exerts (61) important biological effects on inflammation, immunity, and stress (62,63). Accumulating evidence reveals that IL-6 levels increase in blood (64), bronchoalveolar lavage fluid (BALF) (65), and lung tissues (66) of asthmatic patients. The receptor complex mediating the biological activities of IL-6 consists of two distinct membrane-bound glycoproteins, an 80-kDa cognate receptor subunit (IL-6R, CD126), and Volume 28, 2005
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a 130-kDa signal-transducing element (gp130, CD130). Expression of the transmembranespanning gp130 is ubiquitous and was found to be expressed in different organs, including heart, kidney, spleen, liver, lung, placenta, and brain (67). In contrast, cellular distribution of the cognate IL-6R is limited, and its expression is predominantly confined to hepatocytes and leukocyte subpopulations (monocytes, neutrophils, T-cells, and B-cells). Although gp130 was initially identified as the signal-transducing component of IL-6R, it is now clear that cognate receptors for IL-11, oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1, leukemia inhibitory factor (LIF), and novel neurotrophin-1/B-cell-stimulating factor-3 all transmit activation signals via gp130 (68–71). Therefore, each of these cytokines possess overlapping activities, and because of compensatory mechanism, the phenotypic characteristics of mice lacking either IL-6, IL-11, LIF, or CNTF (72–74) are less severe than is suggested by the apparent pleiotropic properties of these mediators. Conversely, targeted disruption of the gp130 gene is embryonically lethal (75). Despite numerous reports implicating OSM as a key modulator of chronic inflammatory diseases, there are limited studies demonstrating the role of this cytokine in lung diseases— particularly asthma. There are studies suggesting that OSM may play an important role in the development of airway wall remodeling; consequently, OSM may be a suitable target for further investigation in the pathogenesis of asthma and its treatment. Moreover, OSM gives rise to vascular endothelial growth factor (VEGF) expression in human airway smooth muscle cells by a transcriptional mechanism involving STAT3. IL-1β also synergizes with OSM to increase VEGF release, likely resulting from effects of IL-1β on VEGF mRNA stability, as well as from effects of OSM on IL-1R1 expression (76). The cytokine LIF is known to be produced by both inflamed peripheral autonomic nerves and several cell types involved in the regulaClinical Reviews in Allergy & Immunology
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tion of the immune response. It has been demonstrated that several structural cell types in human airways produce LIF in response to inflammatory stimuli and that LIF augments contractile responses to tachykinins in airway explants. Moreover, peripheral blood eosinophils express LIF and mRNA for LIF and LIF receptor. Serum LIF levels are higher in atopic patients with mild asthma than in nonatopic normal donors, indicating a pro-inflammatory role that is eosinophil-dependent of LIF in airway disorders. Indeed, LIF is synthesized and released by human eosinophils and modulates activation state and chemotaxis (77). Although in vivo animal models may provide useful information, most of the currently developed animal models of allergic airway inflammation are restricted to acute inflammatory changes following relatively short periods of allergen exposure, which do not simulate the clinical course of airway remodeling in chronic asthma. Additionally, it is difficult to separate the effects of IL-6 from the other cytokines in these situations. Moreover, because IL-6 is multifunctional cytokine, it may exert distinct effects, depending on the phase of airway inflammation. The in vivo chronic effects of IL-6 and IL-11 in the airways could be observed in transgenic mice that overexpressed these cytokines under the control of the Clara cell 10-kD protein promoter (78,79). The phenotypes of these animals had numerous similarities, including the accumulation of lymphoid nodules in peribronchiolar regions and airway wall thickening (78,79). Moreover, the IL-11 mice had emphysema-like airspace enlargement resulting from the inhibition of septation during acinar development (80) and airway remodeling with subepithelial fibrosis (79) as well as increased baseline AHR. Conversely, the animals that overexpressed IL-6 manifested normal baseline airway resistance and were hyporesponsive. Histologically, IL-6 overexpression causes alveolar airspace enlargement and subepithelial fibrosis, and similar degrees of airspace Volume 28, 2005
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Fig. 2. Chronic overexpression of interleukin (IL)-6 in the airways leads to airway remodeling. IL-6 levels are increased in blood, bronchoalveolar lavage fluid, and lung tissue of asthmatic patients. (A) Mice overexpressing IL-6 in the airways (IL-6tg mice) exhibit subepithelial fibrosis, airway thickening, and airway lymphatic inflammation with mucus and eotaxin production (78). (B) The suggested pathway is the endogenous secretion of IL-6 by dendritic cells, which induces naïve CD4+ T-cells to produce IL-4, thereby polarizing these cells into T-helper-2 cells (84).
enlargement are seen in the IL-6 and IL-11 transgenic animals. Additionally, there was a significant increase in the caliber of the remodeled bronchioles in the hyporesponsive IL-6 animals. In summary, the lungs of both groups of transgenic mice showed similar peribronchiolar nodules, alveolar enlargement, and airway remodeling with subepithelial fibrosis (81). The severity of the alveolar airspace enlargement was similar in both groups of transgenic mice and, therefore, could not account for their different physiological profiles. Morphometry demonstrated that the wall thickening was more robust in the IL-11 transgenic mice Clinical Reviews in Allergy & Immunology
and airway caliber was increased in the IL-6 transgenic mice. Therefore, normal airflow and airway hyporesponsiveness occurred in conjunction with airway remodeling in enlarged airways; AHR was noted when remodeling occurred in normal-sized airways and was associated with wall thickening that was not proportionate to airway caliber. These studies demonstrate that airway remodeling and alveolar enlargement do not uniformly lead to AHR (81). These observations demonstrate the involvement of IL-6 in the airway remodeling process, with no involvement on the AHR (Fig. 2). However, the precise roles of endogenous IL-6 Volume 28, 2005
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in the airway remodeling process and AHR following chronic allergen challenge remain unclear.
Different Role of the MembraneBound IL-6R and Soluble IL-6R in Lung Immune Responses In our studies, we delineated a different role of the soluble and membrane-bound IL-6R in asthma (82). Indeed, we found increased levels of the sIL-6 in the airways of patients with allergic asthma compared with controls, especially after allergen challenge. In humans, baseline concentrations of sIL-6R in the BALF of asthmatic patients before allergen challenge were significantly increased compared to those of normal subjects. Furthermore, 24 h after antigen challenge, sIL-6R concentrations in the BALF of asthmatics were increased compared with baseline levels and were significantly higher than those of control patients. These findings demonstrate a possible role of the shedding of the membrane-bound IL-6R during inflammation and a switch of functions between the membrane-bound IL-6R on the membrane of naïve or Treg-cells and the function related to the sIL-6R—namely, growth and survival of the CD4+ Th2 cells during airway inflammation. Indeed, a positive correlation was observed between the number of CD4+ cells, IL-5, and IL-13 in the BALF of asthmatic subjects after allergen challenge and the BALF concentration of sIL-6R, suggesting a potential role of sIL-6R in controlling Th2 function in asthmatic subjects after allergen challenge (Fig. 3A). Additionally, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130Fc led to suppression of Th2 cells in the lung (Fig. 3B). Conversely, local treatment in the lung with anti-IL-6R antibody induced Th1–CD4+ cells as well as Foxp3-positive IL-6R-positive CD4 +CD25 + Treg-cells with immunosuppressive functions Clinical Reviews in Allergy & Immunology
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in the lung in vivo (Fig. 3B). Therefore, our results demonstrate differential roles of sIL-6R and mIL-6R in experimental asthma: sIL-6R regulates Th2 cell functions in CD4 +CD25 – T-effector cells lacking the mIL-6R chain. However, mIL-6R controls the cell fate at the beginning of T-cell differentiation by directing CD4+-naïve cells toward the Th2 pathway and inhibiting the Treg-cell differentiation in the lung. Consistent with this concept, blockade of mIL-6R signaling induced expansion and immunosuppressive capacities of CD4+CD25+ T-regulatory cells in the lung in vivo and induced CD4+ IFN-γ-producing cells in the lung, ameliorating AHR in experimental asthma. These Treg-cells are similar to those previously described in the spleen (30,83). Unlike Tregcells isolated from the spleen, our Treg-cells isolated from the lung produced IFN-γ in addition to TGF-β and IL-10; however, we believe that additional experiments are required to define whether two different populations of Treg-cells are present in our CD4+CD25+ Tregpopulations in the lung. Collectively, these data indicate an important role for IL-6 trans-signaling via the sIL-6R in controlling Th2 T-cell function in asthma. In contrast to mIL-6R, sIL-6R induced CD4 Th2-effector cells in the lung, and its blockade through gp130Fc treatment led to an inhibition of Th2 expansion without affecting the CD4+ Th1 number. These results are consistent with a Th2-promoting potential of pulmonary DCs resulting from the inhibition of Th1 differentiation caused by IL-6 production (84). The signaling pathways that control Treg-cell development in the periphery remain poorly understood. Our data provide evidence for a key role of IL-6 signaling via mIL-6R in preventing Treg-cell responses in experimental asthma. Specifically, we found that local application of anti-IL-6R antibodies leads to expansion of lung Foxp3+ CD4+CD25+ T-cells (30,83, 85–89) producing IL-10. Therefore, signaling Volume 28, 2005
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Fig. 3. Interleukin (IL)-6 signaling controls the balance between T-effector cells and T-regulatory (reg)cells in the lung through different receptor molecules. (A) Dendritic cells (DCs) secreting IL-6 (84) lead to IL-6 trans-signaling via soluble (s)IL-6R, thereby supporting lung T-helper (Th)2 T-cell cytokine production, whereas IL-6 signaling via the mIL-6R suppresses the development of CD4+CD25+ Treg-cells in the lung. (B) Respiratory tolerance. Repeated antigen exposure (i.e., OVA) stimulates DCs to express high levels of B7.1 and B7.2 co-stimulatory molecule and inducible co-stimulatory molecule ligand. DCs then also produce IL10, leading to an acitvation of Treg-cells, which expand and produce IL-10 and transforming growth factorβ. These immunosuppressive cytokines downregulate the CD4+ T-effector cells. Similarly, blockade of sIL-6R with gp130Fc downregulates Th2 development. Conversely, anti-IL-6R antibodies treatment leads to expansion of the Treg- population in the lung and suppresses Th2 development.
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via the mIL-6R present on T-cells apparently suppresses the function of CD4+CD25+ Tregcells in the lung. Additionally, we demonstrated that the IL-6R is functionally active in CD4+CD25+ T-cells, because we showed phosphorylation and activation of STAT3 upon culture of these cells with IL-6 that is inhibited by anti-IL-6R antibody co-incubation. These data extend to previous studies, indicating that IL-6 production by spleen DCs enhances effector T-cell responses by overcoming CD4+CD25+ Treg-cell suppression (90). However, our data suggest that this process occurs in the peripheral immune system in the lung in vivo and involves signaling via the membrane-bound IL-6R. The functional relevance of this concept for asthma was underlined by adoptive transfer studies in which spleen CD4+CD25+ T-cells were able to suppress CD4+CD25– T-cell-induced allergic airway inflammation in Rag1 knockout mice. In summary, IL-6 signaling in the lung tightly controls the critical balance between T-effector and Treg-cell function in the lung via differential signaling events involving sIL-6R and mIL-6R, respectively. Although these data are obtained from an experimental model of asthma, they suggest a potential therapeutic use of antibodies to IL-6R as a novel molecular approach for the induction of local Tregresponses in patients with allergic asthma in humans. Additionally, the fusion protein gp130Fc could be used as an anti-inflammatory therapy to neutralize sIL6R that is increased during airway inflammation.
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