Copyright #ERS Journals Ltd 2000 Eur Respir J 2000; 16: 1158±1174 European Respiratory Journal Printed in UK ± all rights reserved ISSN 0903-1936

REVIEW

New immunological approaches and cytokine targets in asthma and allergy

R.G. Stirling, K.F. Chung

New immunological approaches and cytokine targets in asthma and allergy. R.G. Stirling, National Heart and Lung Institute, K.F. Chung. #ERS Journals Ltd 2000. Imperial College School of Medicine and ABSTRACT: The aims of current asthma treatment are to suppress airway inflam- Royal Brompton and Harefield Hospital, mation and control symptoms, and corticosteroids maintain a commanding position in London, UK. this role. Correspondence: K.F. Chung Steroids effectively suppress inflammation in the majority of patients but have little Dept of Thoracic Medicine impact on the natural history of this disease. In severe asthmatics, corticosteroids may National Heart & Lung Institute have relatively less beneficial effects. Recent advances in understanding the inflam- Dovehouse Street matory and immunological mechanisms of asthma have indicated many potential London therapeutic avenues that may prevent or reverse abnormalities that underlie asthma. SW3 6LY As the roles of effector cells, and of signalling and adhesion molecules are better UK understood, the opportunities to inhibit or prevent the inflammatory cascade have Fax: 44 2073518126 increased. Keywords: Adhesion molecules In addition, there have been advances in the synthesis of proteins, monoclonal asthma antibodies and new small molecule chemical entities, which may provide valuable cytokines flexibility in the therapeutic approach to asthma. The novel immunological appro- gene therapy aches include the prevention of T-cell activation, attempts to influence the balance of immunology T-helper cell (Th) populations to inhibit or prevent Th2-derived cytokine expression, T-cells and the inhibition or blockade of the downstream actions of these cytokines such as effects on immunoglobulin-E and eosinophils. These approaches provide broad as well Received: September 20 2000 as highly specific targeting, and also prospects for prevention and reversal of Accepted after revision September 22 2000 immunological and inflammatory abnormalities associated with asthma. Hopefully, the development of effective antiasthma agents with effects beyond those provided by current therapies coupled with lesser side-effects will further address the unmet needs of asthma. Eur Respir J 2000; 16: 1158±1174.

Airway inflammation and remodelling and hyperplasia, myofibroblast activation with increase in subepithelial basement membrane collagen deposi- The chronic airway inflammation of asthma is char- tion, angiogenesis and increase in submucosal blood acterized by infiltration of the airway wall and lumen by vessels and an increase in goblet cell numbers in the diverse effector cells, including T-lymphocytes, eosino- airway epithelium [5±8]. These changes are also variably phils, monocytes/macrophages, mast cells, and occasion- influenced by Th2 cytokines, and certain growth factors ally, neutrophils [1±3]. The mobilization, activation and [9, 10]. trafficking of these effector cells to the airway are The exact link between the chronic inflammatory and controlled by a complex cytokine milieu derived from remodelling processes and the clinical presentation of activated CD4+ T-helper (Th) cells and also from other asthma is unclear in terms of the pathophysiological resident airway cells including airway smooth muscle mechanisms and of the contribution of the components of and epithelial cells. T-helper cells of type 2 variety (Th2) these processes to bronchial hyperresponsiveness, airway secrete a Th2 profile of cytokines, after cognate stimu- narrowing and the acute exacerbations that characterize the lation of the naive T-cell by antigen presenting cells, such disease. The overwhelming hypothesis is that these are as the dendritic cell and the alveolar macrophage (fig. 1). intricately related such that inhibition or prevention of the The Th2 cytokines include interleukins (IL)-4, -5, -9, -10 inflammatory process should improve the control of and -13. These cytokines promote various elements of asthma, an idea supported by the potent actions of topical allergic inflammation (table 1), including propagation of corticosteroids in controlling the eosinophilic and lym- the Th2 phenotype, isotype-switching from immunoglo- phocytic inflammation of asthma, while being most bulin (Ig)-G1 to IgE synthesis, eosinophil mobilization, effective in restoring lung function and bronchial hyper- maturation and activation and mast cell activation [4]. responsiveness [9, 10]. However, identification of the Additionally, the airway wall undergoes chronic structu- key immunological and inflammatory processes that lead ral changes labelled as remodelling, which include thic- to functional abnormalities and symptoms remain to be kening of the airway smooth muscle due to hypertrophy elucidated. NEW APPROACHES TO ASTHMA THERAPY 1159

Table 1. ± Summary of Th2 cytokine effects as predicted by cytokine stimulation and ablation studies (animal and human data included)

Th response BHR IgE Eosinophils Mucus hyperplasia Mast cell activation Surface markers Th1 Th2 Blood Airway Survival

IL-4 - + + + + VCAM + MHC IL-5 + + + IL-9 + + + + + IL-10 - - - - MHC II IL-13 - + + + VCAM IL: interleukin; BHR: bronchial hyperresponsiveness; IgE: immunoglobulin-E; VCAM: vascular cell adhesion molecule; MHC: major histocompatability complex.

Limitations of current therapy to major histocompatability (MHC)-II complex and, secondly, via the costimulatory pathway linked by the Despite the introduction of new agents such as the B7 family and the T-cell bound CD28 [19]. T-cells leukotriene inhibitors, corticosteroids remain the anti- stimulated via the T-cell receptor (TCR) in the absence of inflammatory drug of choice for the majority in the costimulatory signalling are incapable of IL-2 secretion treatment of chronic asthma [11, 12]. However, treatment and subsequent activation and therefore, enter an anergic failure and drug-related side-effects pose serious limita- state [20]. CD28 itself has two major ligands, B7.1, which tions to their use [13]. Severe and difficult-to-treat asth- inhibits Th2 cell activation and development, and B7.2, matics are characterized by ongoing symptoms and which induces T-cell activation, and Th2 development. frequent exacerbations despite the use of existing anti- An important third ligand, cytotoxic T-lymphocyte inflammatory and bronchodilator therapies. These pa- associated protein-4 (CTLA4), is expressed on activated tients are frequently prescribed corticosteroids to which T-cells, binds CD28 with enhanced avidity and acts as a their disease is partially responsive and they endure the negative regulator of T-cell function by inhibiting Th2 adverse consequences of high dose inhaled and systemic differentiation [21, 22]. In those who later develop an corticosteroid therapy [13, 14]. Amongst severe asth- allergic response, naive T-cells differentiate into the Th2 matics, up to 25% demonstrate relative unresponsiveness subtype. to the therapeutic but not to the adverse effects of T-cell profiles in the newborn demonstrate a Th2 bias corticosteroids [15, 16]. The identification of novel the- suggesting that prenatal influences are involved in T-cell rapeutic targets and subsequent development of specific priming [23]. Th1 cells form a natural counterbalance to and safe therapeutic agents therefore, represents a major Th2 cells driving protective cell-mediated immunity challenge in asthma therapy. In addition to attempting to (CMI), and are induced on exposure to foreign agents reverse established changes in the airways resulting from including protozoa and bacteria. Th1 responses are chronic inflammation and airway remodelling, the pre- characterized by the induction of CMI responses and vention of allergy and asthma remains an important con- the synthesis of IgG2a, while Th2 responses are of the sideration given the rising prevalence of these conditions humoral-type, inciting the production of IgE and IgG4. in many industrialized countries [17]. Signal 2 B7.2 CD28 T-cell Immunological imbalance: T-helper cell balance APC Th0 activation MHCII-Ag CD4 IL-12 The vast majority of asthmatics have an atopic TCR/CD3 background, in whom the inflammatory process of asthma Signal 1 is assumed to be driven following sensitization and re- exposure, or challenge by common aeroallergens. How- ever, the inflammatory abnormalities in nonatopic asthma MHCII-Ag TCR/CD3 also bear many similarities to those observed in the allergic CD4 Clonal anergy inflammatory process, including the presence of eosino- APC Th0 phils, activated CD4+ T-cells, and the expression of Th2- B7.2 CTLA4 derived cytokines [18]. These findings suggest that most asthmatics may benefit from an approach that targets mechanisms of allergic sensitization and inflammation. CTLA4-Ig Allergens are taken up by specialized cells within the Fig. 1. ± T-helper cells differentiate from the naive state following mucosa such as dendritic cells (antigen-presenting cells), interaction with the antigen-presenting cell. The Th2 phenotype is and subsequently processed, following which peptide characterized by secretion of the cytokines interleukins -4, -5, -9 and fragments are presented to naive T-cells. The activation of -13, while the Th1 cells secrete interferon-c, and interleukin (IL)-2. IL-4 directly stimulates Th2 differentiation while IFN-c promotes the Th1 naive T-cells requires direct signalling by two distinct phenotype. APC: antigen presenting cell; MHC: major histocompati- pathways (fig. 1): firstly via the CD4+ T-cell receptor bility complex; TCR: T-cell receptor; CTLA4: cytotoxic T-lymphocyte through the antigen-presenting cell (APC)-bound antigen associated protein-4; Ag: antigen. 1160 R.G. STIRLING, K.F. CHUNG

Th1 responses inhibit Th2 responses through the pro- Prevention of T-cell activation duction of cytokines such as IL-12 and interferon gamma (IFN-c) (fig. 2). Abnormal polarization of these responses Three main thrusts of research into new therapies have may result in tissue pathology. The excessive expression developed: 1) preventing T-cell activation; 2) prevention or of Th1 cytokines is noted in autoimmune conditions reversal of Th2 polarization, and; 3) ablation of the effects including experimental diabetes mellitus [24, 25] and of Th2 cytokines and downstream mediators (table 2). multiple sclerosis [26, 27], while excessive Th2 expres- Many of the potential ways of achieving these objectives sion is observed in the atopic diathesis. There is evidence have been investigated in animal (mainly murine) models, for a preferential skewing to expansion of the CD4+ Th2 and therefore, their applicability to human asthma and lymphocyte subset in allergic processes [3, 28] and this is allergic diseases remains unclear given the inherent a likely crucial forerunner to the development of allergic variability within varied species. disease. During the course of maturation of the normal infant however, increased Th1 expression occurs, and the Th2 imbalance is overcome [29]. Delay or failure of this T-cell immunomodulators Th1 response may result in Th2 persistence and atopy or atopic disease, and accordingly in infants destined to Cyclosporin A and the functionally-related, tacrolimus become atopic, an impaired production of IFN-c by (FK506), are powerful immunosuppressant agents used circulating lymphocytes is observed [30]. widely to prevent immune rejection in organ transplanta- The "hygiene hypothesis" postulates a diminished in- tion. These compounds inhibit T-cell growth by creating a duction of Th1 responses as a potential explanation for the block in the G0 phase of development through inhibition of rising prevalence of atopy and asthma [23, 31, 32]. Cross- T-cell growth factors including IL-2 [38]. Both agents sectional surveys have identified inverse relationships strongly inhibit mitogen-stimulated IL-5 release by between prior microbial exposure and development of inhibition of transcription [39, 40]. Cyclosporin A and atopy [23, 33, 34]. Further, respiratory allergy appears less frequently in those heavily exposed to orofaecal and Table 2. ± Novel strategies for the inhibition and preven- foodborne microbes [32]. Thus, improved hygiene, early tion of allergic asthma infection and antibiotic use, and a westernized or semi- Target Agent sterile diet may facilitate atopy by influencing exposure to commensals and pathogens that stimulate immune cell Prevention of T-cell activation Anti-CD4 populations such as gut-associated lymphoid tissue [35, CTLA4 36]. Thus, early environmental exposure may be a Prevention of reversal of determinant of the development of atopy in the adult [37]. Th2 expression The identification of ways to prevent, control or even Inhibition of Th2 cytokines/ Soluble phenotype reverse the process of Th2 immunodeviation has become rhu IL-4 mutant proteins a focus for the development of new strategies to control STAT-6 inhibition asthma and allergies. Anti-IL-5 monoclonal antibody GATA inhibition Soluble IL-13Ra Promotion of Th1 cytokines/ IFN-c phenotype IL-12 Mast NK IL-10 IL-18 IL-12 cell Immunotherapy SIT IL-18 Peptide immunotherapy M. vaccae vaccination IFN-γ IL-4 CpG APC APC Inhibition of downstream mediators Antiinflammatory cytokines IL-10 Th0 Th0 IL-1Ra Inhibition of eosinophil CCR3 antagonist migration and activation CCR3 antisense VLA4 inhibitor ICAM-1 inhibitor Th1 Th2 met-RANTES IFN-γ IL-4 met-Ckb7 IgE inhibition Monoclonal anti-IgE (E25) IL-2 IL-5 IL-13 CTLA4: cytotoxic T-lymphocyte associated protein-4; IL: interleukin; STAT-6: signal transduction and activation of Fig. 2. ± Activation of T-helper cells by the antigen presenting cell transcription-6; IFN: interferon; SIT: specific immunotherapy; (APC) requires dual signalling via the major histocompatibility complex and the T-cell receptor, and via B7.2:CD28 interaction and results in Th2 M. vaccae: Mycobacterium vaccae; CCR3: eotaxin receptor; deviation, activation and interleukin (IL)-2 secretion. T-cell receptor VLA4: very late antigen-4; ICAM-1: intercellular adhesion stimulation in the absence of costimulation via CD28 induces clonal molecule-1; Ig: immunoglobulin; E25: nonanaphylactogenic anergy. NK: natural killer cell; IFN-c: interferon-gamma. IgE monoclonal antibody. NEW APPROACHES TO ASTHMA THERAPY 1161

FK506 also have effects on other cells and markedly Inhibition of T-cell costimulation: cytotoxic reduce basophil histamine release [41]. In human studies, T-lymphocyte associated protein-4-immunoglobulin cyclosporin A by inhalation provides significant inhibi- tion of the allergen-induced late allergic reaction [42]. In Although corticosteroids inhibit antigen uptake and corticosteroid-dependent asthma, low-dose cyclosporin A processing, they do not appear to impact on antigen improved lung function [43] and allowed for a 62% presentation [65]. Therefore, the potential for disruption reduction in oral steroid dose requirement [44], but at of antigen presentation by specific inhibition of costi- the expense of adverse effects which would not prove mulatory molecule interaction has been recognized and tolerable in mild disease. studied in murine models. A recombinant fusion protein The folate antagonist, methotrexate, has well recognized consisting of the extracellular domain of CTLA4 linked anti-inflammatory effects in rheumatoid arthritis and is to the constant region of IgG1, known as CTLA-4-Ig, used as a steroid-sparing agent in asthma. Meta-analysis of binds B7 molecules with an affinity similar to that of controlled trials confirms steroid-sparing effects while membrane CTLA4. It therefore, acts as a powerful maintaining lung function or symptoms [45±47]. In clini- inhibitor of B7: CD28 mediated costimulation. T-cell cal practice, these immunomodulators are only modestly activation following B7:CD28 signalling may be blocked effective in a proportion of patients. A number of com- using the soluble protein ligand CTLA4-Ig, and this pounds, which show immunomodulatory activity, are can substantially reduce BHR, bronchoalveolar lavage currently under development. However, it is likely that (BAL) eosinophilia and specific IgE responses when compounds that have nonspecific immunosuppressive T- given either prior to sensitization or to allergen chal- cell activity may not be as effective as the more specific lenge [66]. CTLA4-Ig reduced IL-4 and IgE levels while Th2 cell inhibitors [48]. Suplatast tosilate, a compound IFN-c and IgG2a were unchanged, suggesting down- developed in Japan, selectively prevents the release of IL- regulation of Th2 response without upregulation of the 4 and IL-5 from Th2 cells and can reduce bronchial Th1 response [67]. Similar outcomes are observed fol- eosinophilia in animal models of bronchial hyperrespon- lowing monoclonal anti-B7.2 (anti-CD86) treatment in siveness (BHR) [49, 50]. It has been shown to improve allergen-challenged mice [68]. Thus, CTLA4-Ig is poten- pulmonary function and symptom control, and allows for tially a powerful immunomodulator with the potential a decrease in the dose of inhaled corticosteroids [51], and of suppressing Th2 based responses to allergens in has been launched for the treatment of asthma in Japan. humans.

T-cell depletion: anti-CD4 monoclonal antibody Modulation of T-helper 1/T-helper 2 differentiation

CD4+ T-cells appear central to the orchestration of Specific attempts to alter Th1/Th2 balance by enhancing allergen-mediated airway disease. Depletion of T-cells in Th1 and abrogation of Th2 responses have been the major murine sensitized and allergen-challenge models using thrust of immune approaches to the prevention and treat- recombinant monoclonal antibodies results in a complete ment of allergies and asthma. Induction of Th1 responses ablation of airway hyperresponsiveness and airway may have a direct suppressive effect on Th2 mediated eosinophilia [52]. Conversely, antibody-mediated CD8+ inflammatory processes (table 3). The most direct appro- T-cell depletion augments BHR and eosinophilic inflam- aches include administration of cytokines that will induce mation in the allergen-challenge model [53]. A prelimi- activation of Th1 pathways (e.g. IFN-c, IL-12 and IL-18) nary clinical trial in asthma has evaluated the use of a or of blocking antibodies that inhibit the effect of Th2- single dose of anti-CD4 monoclonal antibody in severe related cytokines (e.g. anti-IL-4, anti-IL-5, anti-IL-9, and corticosteroid dependent asthma [54]. This treatment led anti-IL-13). to a reduction in circulating CD4+ T-cell numbers and caused an improvement in morning and evening peak expiratory flows but did not significantly impact on asthma symptoms. Early studies have also suggested Table 3. ± Summary of T-helper 1 cytokine effects as predicted by cytokine stimulation and ablation studies some benefit of this approach in the CD4+ dependent (animal and human data included) processes of multiple sclerosis [55, 56] and collagen- arthritis [57]. Th response BHR IgE Eosinophils Mediator It is likely that other T-cell subsets may also be release important in asthma. Recent investigations have high- Th1 Th2 Blood Airway lighted the existence of CD8+ T-cells (i.e. TC2), which IFN-c + - - - - +TNF-a secrete Th2 type cytokines and their influence on the +IL-1b mediation of allergic airways disease is as yet to be IL-12 + - - - - - +TNF-a established [58±60]. Further uncertainty regarding the -IL-10 approach of CD4 depletion relates to the induction of IL-18 + - - + - +IFN-c CD4 lymphopenia and immunosuppression and the +Chemokines resultant risk of opportunistic infection and neoplasia -IL-10 [61±63]. Nondepleting CD4 antibodies have been IFN: interferon; IL: interleukin; BHR: bronchial hyperrespon- generated but are as yet untried in pulmonary disease siveness; IgE: immunoglobulin-E; TNF-a: tumour necrosis [64]. factor-alpha. 1162 R.G. STIRLING, K.F. CHUNG

Inhibition of allergy and asthma by Interleukin-18 T-helper 1 related cytokines IL-18 (IFN-c-inducing factor) is a potent inducer of Interferon gamma IFN-c production by T-, NK- and B-cells and plays an important part in the induction of Th1 responses [88, 89]. IFN-c, released from CD4+(Th1) and CD8+(TC2)cells, IL-18 receptors are expressed selectively on murine Th1 is a critical factor controlling the balance of Th1/Th2 but not Th2 cells. Recombinant human IL-18 potently development, and exerts an inhibitory effect on Th2 cells induces IFN-c production by mitogen-stimulated periph- [4]. IFN-c is also a powerful and relatively specific eral blood mononuclear cells and enhances NK-cell inhibitor of IL-4-induced IgE and IgG4 synthesis by B- cytotoxicity [90], while increasing NK-cell granulocyte- cells. A reduced production of IFN-c by the T-cells of macrophage colony-stimulating factor (GM-CSF) release asthmatic patients is seen and correlates with disease and CD8+ T-cell IL-10 production [91]. IL-18 and IL-12 severity [69]. Intra-tracheal IFN-c in allergen-sensitized have synergistic effects on Th1 development, which may and -challenged mice causes a dose-dependent reduction be due to reciprocal upregulation of their receptors. IL-18 in BAL IL-5 levels and airway T-cells and eosinophils may be important in the control of allergen-induced BHR [70, 71]. Intraperitoneal IFN-c also reduced lung Th2 by vaccination. Vaccination using heat-killed Listeria cytokine levels, attenuated allergen-induced BHR with monocytogenes caused a marked inhibition of allergen- concomitant reduction in BAL eosinophilia, while an induced BHR and airway inflammation, associated with IFN-c-blocking antibody led to an increase in airway conversion to the Th1 phenotype in mice [92, 93]. This CD4+ T-cells and BHR [72]. The inhibition of eosinophil effect was IL-12-dependent and associated with a recruitment appears to be dependent on the inhibition of marked upregulation of IL-18 messenger ribonucleic CD4+, but not on CD8+ T-cell accumulation within the acid (mRNA) expression. These studies also demon- airways [70, 73]. Inhalation of IFN-c by nonasthmatic strated that administration of an adjuvant after allergen humans increases epithelial lining and BAL fluid IFN-c exposure, was able to reverse established BHR. The direct administration of IL-18 in the murine asthma model levels but does not affect serum IFN-c levels and provides more confusing results; although IL-18 appears therefore, may avoid toxicity associated with systemic to play a protective role when administered with allergen administration [74, 75]. However, in one study of sub- challenge, administration with sensitization paradoxically cutaneous IFN-c therapy in steroid-dependent asthma no increased IgE and IL-5 levels and promoted BAL effect on lung function or treatment requirement was eosinophilia [94, 95]. Thus, IL-18 appears to be involved observed despite a significant reduction in circulating in vaccination-mediated inhibition of Th2 responses and eosinophil numbers [76]. may have properties directly driving BHR in mice.

Interleukin-12 Inhibition of T-helper 2 related cytokines IL-12 is produced by antigen-presenting cells and Interleukin-4 enhances the growth of activated T- and natural killer (NK)-cells, stimulating them to produce IFN-c [77, 78]. IL-4 has a central role in Th cell development as a potent IL-12 also promotes the differentiation of IFN-c-produ- inducer of Th2 maturation from the naive T-lymphocyte cing T-cells and inhibits the differentiation of T-cells into [96, 97]. Additionally, IL-4 induces the isotype-switch IL-4 secreting cells [77]. Thus, IL-12 can regulate Th1 necessary for IgE synthesis [98], upregulates IgE rec- cell differentiation while suppressing the expansion of eptors [99, 100] and vascular cell adhesion molecule-1 Th2 cell clones by early priming of undifferentiated Th0 (VCAM-1) expression on vascular endothelium, thus cells for IFN-c secretion. IL-12 may play an important facilitating endothelial passage and accumulation of eosinophils [101]. IL-4 is therefore, an attractive target role in inhibiting inappropriate IgE synthesis and allergic to inhibit, resulting in downregulation of Th2 activation. inflammation as a result of allergen exposure. In murine Anti-IL-4 monoclonal antibody treatment of mice prior to asthma models, administration of IL-12 leads to a allergic sensitization markedly reduces IgE synthesis reduction in allergen specific IgE levels, ablation of [102], but does not appear to inhibit airway eosinophilia airway hyperresponsiveness and inhibition of eosinophil or BHR [103]. A recombinant soluble IL-4 receptor (sIL- recruitment [79±81]. However, further studies have 4R) has been designed as a mimic of the cell-surface suggested that this protective role may only be conferred receptor, which thus binds and sequestrates free IL-4, when acting synergistically with IL-18 [82]. In ther- but because these soluble receptors lack transmembrane apeutic trials, IL-12 levels increased during corticosteroid and cytoplasmic domains, they act as an IL-4 receptor therapy [83] and during specific immunotherapy [84]. In blocker. In murine studies, sIL-4R reduces allergen- a phase-I trial of IL-12 in asthma, a significant reduction specific IgE responses, airway hyperresponsiveness, of peripheral eosinophils and a trend towards a reduction VCAM-1 expression and eosinophil accumulation in airway eosinophils was observed without effect on [104]. Preliminary human asthma studies with single allergen-induced early or late phase responses [85]. IL-12 doses of sIL-4R have shown acceptable tolerance, has proved a useful adjunct to cancer chemotherapy by improvement in lung function and a reduction in rescue induction of a protective Th1 response [86], but signi- b2-agonist requirement [105]. Additionally, a trend tow- ficant toxicity including arrhythmias, liver function ards a reduction in serum eosinophilic cationic protein abnormalities and flu-like illness will limit its potential (ECP) and exhaled nitric oxide (NO) levels suggests sup- utility in asthma [87]. pression of inflammation by this agent. A recombinant NEW APPROACHES TO ASTHMA THERAPY 1163 mutant human protein (BAY 16-9996) binds the IL-4 IL-5 receptor a- but not cc-chain, and therefore, antagonises receptor transduction. This protein substantially reversed αβ allergen-induced BHR with a reduction in airway inflammation in a primate model [106]. Another approach to inhibition of IL-4 production is to target the control of transcription factors of the IL-4 gene. Signal transduction and activation of transcription-6 (STAT-6) responsive elements are found in the promoter Ras region of IL-4 inducible genes and this transcription JAK2 JAK1 factor is expressed at abnormally high levels in the epithelium of severe asthmatics [107]. STAT-6 knockout Lyn mice demonstrate a defect in IL-4 and IL-13 mediated Raf-1 signal transduction [108]. The potential utility of STAT-6 Fyn targeted therapies is highlighted by the ability of STAT-6 STAT5 directed antisense oligonucleotides to markedly down- STAT1 Syk MEK regulate germline Ce mRNA levels, reflecting inhibition of IL-4-dependent IgE isotype switching [109, 110]. MAPK Interleukin 5 Eosinophil mobilization and trafficking, their matura- tion and maintenance are largely promoted by the Th2 cytokine, IL-5, making it an attractive therapeutic target in Gene transcription eosinophilic conditions such as asthma and rhinitis. Fig. 3. ± Binding of IL-5 activates the IL-5 receptor and triggers a Ablation of the effects of IL-5 has been accomplished cascade of secondary messengers. The secondary signalling pathway is a with antisense oligonucleotides in rodent models where a linear system in which cell surface associated receptor protein tyrosine kinases (e.g. Jak-1/2 and Lyn) activate the signal transducers and reduction in IL-5 protein levels is followed by a reduction activators of transcription (STAT1 and STAT5) and Ras family of serine/ in allergen-induced eosinophilia and BHR [111]. Impor- threonine protein kinases (e.g. Raf-1) respectively. Ras/Raf activation tantly, this suppression of lung eosinophilia was main- may function as a relay switch, positioned upstream from a further tained for 17 days following a single intravenous cytoplasmic cascade of kinases that include the mitogen-activated protein kinases (MAPK) and mitogen-activated protien kinases/ treatment. extracellular regulated protein kinase (MEK). Activated MAPKs in Monoclonal antibodies used to block IL-5 in mouse, turn regulate the activities of nuclear transcription factors such as guinea-pig, rabbit and monkey studies provided a sus- activator protein-1 (AP-1, of which c-jun and c-fos are important tained reduction in antigen-induced airway eosinophilia components). but had little effect on antigen-induced BHR [112±114]. Two humanized forms of anti-IL-5 (SB-240563 and Sch resulting in inhibition of lyn-dependent IL-5 signalling 55700) are now available with the potential for clinical without affecting Jak2 dependent IL-5 signalling. This studies [115, 116]. In mild asthmatics, SB-240563 had a inhibitor blocks allergen-driven airway eosinophilia marked inhibitory effect on both airway and peripheral [121]. The transcription factor GATA-3 is critical for blood eosinophil levels and on allergen-induced sputum IL-5 expression in Th2 cells, and increased GATA-3 gene eosinophilia following single subcutaneous administra- expression in association with IL-5 mRNA positivity tion [117]. However, as with the studies in mouse, rabbit has been shown in airway cells isolated from patients and monkey, no significant effect on bronchial respon- with asthma. These findings support a causal association siveness or the late phase response to allergen-challenge between augmented GATA-3 expression and dysregu- was observed. Phase-I trials in severe persistent asthma lated IL-5 expression and asthma [122, 123]. Specific have provided similar results [118]. IL-5 blocking anti- inhibition of GATA-3 expression using dominant nega- bodies prevent the mobilization and subsequent traffick- tive GATA-3 transgenic mice led to a reduction in Th2 ing of eosinophils to the lung in allergic asthma, but are cytokine expression and marked attenuation of airway unable to influence BHR or the late phase response. eosinophilia, mucus production, and IgE synthesis [124]. Currently, studies are in progress to determine whether These outcomes could also potentially be achieved using anti-IL-5 blocking antibodies can improve the control of antisense oligonucleotide technology. asthma, despite their apparent lack of protection against BHR. Recent studies on IL-5 signalling (fig. 3) suggest that Interleukin-9 various IL-5-dependent functions are mediated by dis- tinct and separate secondary messenger systems [118]. IL-9 has been identified in airway tissue in asthma and is Thus, targetting such pathways may lead to inhibition known to induce BHR, elevated serum IgE, mucin gene of IL-5 effector functions. The anti-apoptotic effects of transcription and epithelial CC chemokine release [125± IL-5 on eosinophils are dependent on the activation of 128], and thus is a broadly attractive therapeutic target lyn-, Janus kinase-2 (Jak2) and Raf-1 kinase, of these, in asthma [129]. One mechanism by which IL-9 may only Raf-1 is necessary for eosinophil activation and induce airway eosinophilia is by enhancing IL-5 receptor degranulation [119, 120]. Lyn-kinase dependent signal- expression, thereby increasing the differentiation and ling may be specifically inhibited by a peptide inhibitor, survival of eosinophils [130]. Specific blockade of IL-9 1164 R.G. STIRLING, K.F. CHUNG activity has been accomplished in the mouse by intra- Deoxyribonucleic acid vaccines: tracheal instillation of monoclonal anti-IL-9 antibody cytosine-guanosine repeat motifs [131]. Anti-IL-9 significantly inhibited BHR, airway eosinophilia, serum IgE, airway inflammatory cell infil- The cytosine-guanosine dinucleotide repeat, when tration and mucin production induced by allergen, de- present in a particular base context, is known as a CpG monstrating a surprisingly broad inflammatory effect motif and has been recognized as an important procaryotic for this cytokine. As yet there are no reported clinical immunomodulatory effector whose role is probably that of trials adopting IL-9 as a therapeutic target. a warning or priming agent against bacterial infection [141]. This motif is expressed at a very low frequency in vertebrates, in a nonfunctional methylated form, and is Interleukin-13 without function. Rodent studies highlight a potential function of synthetic CpG motifs in vertebrates, where a This cytokine shares 70% of its sequence homology potent effect on lymphocyte function has been found with IL-4 and despite a degree of functional redundancy [142]. CpG vaccination directly induces antigen-present- due to sharing of the IL-4Ra subunit, a specific role in the ing cells and B-lymphocytes to release IL-12, IL-18 and development of asthma has been demonstrated in murine tumour necrosis factor-alpha (TNF-a), effectively sup- models [132±134]. Notably IL-13 does not share the IL-4 pressing Th2 responses by inducing the Th1 phenotype. influence on specific induction of the Th2 phenotype These CpG oligodinucleotides (ODN) are effective in from naive Th0 cells. By using a soluble form of the IL- preventing the development of eosinophilic airway in- 13Ra chain of the IL-13 receptor, known to bind IL-13 flammation, allergen-induced elevation of serum IgE and exclusively, these groups demonstrated a significant re- BHR in murine asthma models [142]. These ODN not duction in airway hyperreactivity, airway eosinophilia only effectively prevent Th2 type responses by pre-admi- and mucus hyperplasia. Curiously, when administered nistration and co-administration with allergen-challenge after the initial allergen sensitization however, this agent but also reduce established inflammation [142±144]. had no effect on serum IgE levels [133, 134]. Notably, an Further, these motifs show a large degree of flexibility IL-13 blocking antibody reduced BHR independently of towards the dose and the route of administration, being IL-5 [135]. active following transmucosal [145], inhaled and par- enteral administration [142]. As an added potential, a deoxyribonucleic acid (DNA) vaccine containing this T-helper 1/T-helper 2 modulation by vaccination motif may provide a more effective adjuvant than the widely used, alum. The clinical efficacy and safety of An indirect way of modulating Th1/Th2 balance has such CpG motifs needs evaluation [146] and such trials been to boost innate immunity by the use of vaccines, are currently being considered. particularly for redirection of the Th2 response in favour of the Th1 response. Several approaches are currently under investigation, and these may even raise the possibility of Potential drawbacks of manipulating preventing the development of asthma and allergic dise- T-helper 1/T-helper 2 balance ases. The beneficial effects of specific immunotherapy may result from increasing Th1 immune responses while the The mounting evidence for Th2 cell activation in development of peptide immunotherapy may lead to a allergic asthma suggests downregulation or ablation of the more effective and safer treatment. Th2responsetobeanappropriateaimintreatingasthma and allergic disorders. However, the consequences of such a strategy are as yet unclear. Two potential outcomes need Mycobacterium species consideration; first the consequences of Th2 ablation and second, those of allowing a Th1 dominated immune acti- The potential benefit of Bacille Calmette-Guerin (BCG) vation. Parasite exposure is known to induce Th2 type vaccination in atopic diseases was first raised by a study in cytokine synthesis [147±149], and mice with Th2 cyto- Japanese school children, in whom an association between kine deletions demonstrate impaired parasite clearance BCG vaccination and a diminished incidence of atopy and [150±152]. Although early Th1 expression may be a more allergic disease was observed, suggesting a role for early important response to acute phase parasitaemia, subse- mycobacterial exposure in the subsequent development of quent parasite clearance appears to be more Th2 cytokine atopic responsiveness [34]. Experimental models have dependent [152, 153]. Autoimmune diseases including supported this concept using the nonpathogenic myco- experimental allergic encephalomyelitis, myasthenia gra- bacterial products of Mycobacterium bovis and Myco- vis and hypothyroidism may be derived from an exu- bacterium vaccae. Mice vaccinated with BCG prior to berance of Th1 activity [154±156], raising the concern allergen sensitization had increased IFN-c and decreased that increased Th1 cell activity may induce these auto- IL-4 and IL-5 expression along with reduced levels of immune diseases. Several reports note associations be- airway T-cells and eosinophilia and bronchial reactivity tween IFN-c, IFN-b and IL-12 exposure and the [136, 137]. Mycobacterium vaccae is ubiquitously pre- development or exacerbation of autoimmune processes sent in the soil as a saprophyte and can evoke a strong [154, 157±159]. Th1 cells may contribute to asthmatic production of IFN-c. A suppression of Th2 activation has inflammation. In adoptive transfer studies of Th1 cells been demonstrated using heat-killed M. vaccae in mice in mice, a prolonged enhancement of cell-mediated [138±140], and these studies have opened the way to immune responses was observed [160], and passive clinical studies in human asthma [138]. transfer of Th1 cells with Th2 cells induces enhanced NEW APPROACHES TO ASTHMA THERAPY 1165 tissue eosinophilia compared to when Th2 cells alone specific T-lymphocytes. Such constructs have been dem- were administered [161±163]. However, in other studies onstrated to induce IgE-independent late-phase broncho- in mice and rats, transfer of allergen-specific Th1 cells constrictor responses in cat-sensitive mild asthmatics [164] inhibited the effects of allergen-specific Th2 cells, [185]. Ex vivo studies subsequently demonstrated a namely BAL eosinophilia and BHR in rats, without itself dose-dependent reduction in T-cell IL-4 production inducing any inflammation [165]. Allergen-specific Tc1 following cat peptide, not observed following whole cells also diminished allergen-induced BHR and BAL cat hair extract [186]. In vitro cat-peptide studies also eosinophilia [163]. Nevertheless, IFN-c produced by Th1 showed a reduction in IL-2, IL-4, IL-5 and IFN-c release cells may activate epithelial cells to induce the production from peripheral blood T-cells and a reduction in CD40 of pro-inflammatory cytokines and chemokines [166± ligand (CD40L) expression, resulting in a diminished 168]. ability of these cells to give help to B-cells for the Two further observations appear important to the production of IgE [187, 188]. In a six-week trial of cat contribution of Th1 type processes to asthma. First, the peptide immunotherapy in asthma, a reduction in IL-4 prominent expression of the Th1 dependent transcription levels was again observed and was accompanied by an factor, STAT1, in stable asthma [169] and second, the improvement in the provocative dose causing a 20% fall association between respiratory viral infections and in (PD20) forced expiratory volume in one second (FEV1) asthma [170, 171]. Viral infections induce Th1 expression [187], although there was no effect on IFN-c or IgE, with upregulation of TNF-a and VCAM1 expression suggesting the possible induction of a Th2 to Th0 change. supporting a role for Th1 mediated processes in asthma The possibility of selective induction of clonal anergy is exacerbation [169, 172]. Therefore, caution is necessary invited by this approach and phase I and II studies of cat in studies that aim towards altering the balance of the peptide immunotherapy are currently being launched. Th1/Th2 in the treatment of asthma and allergic diseases.

Specific immunotherapy Anti-immunoglobulin-E antibody Specific immunotherapy (SIT) is a treatment aimed at the induction of specific unresponsiveness, or anergy, in Studies of the recombinant human anti-IgE antibody peripheral T-cells to peptide epitopes. Peripheral T-cells (rhu MAb-E25, Xolair) are now in phase IV in asthma and following SIT are characterized by reduced cytokine allergic rhinitis. E25 binds to free IgE at the high affinity production and attenuated proliferative responses to spe- receptor-binding site (FceR1), preventing the crosslinking cific allergens [173]. This process is initiated by an of IgE bound to effector mast cells and basophils. This autocrine effect of IL-10 produced by antigen-specific T- strategy ensures that the antibody is nonanaphylactogenic. cells [174±176]. IL-10 induces anergy by inhibition of The antibody may be delivered by subcutaneous injection CD28-costimulatory molecule signalling [177, 178] and on a 2±4 weekly schedule, dosing based on weight and also by anti-inflammatory effects on basophils, mast cells serum IgE levels. Tolerance and adverse effect profiles are and eosinophils [4]. There are reduced levels of IL-4 and thus far acceptable with no reports of serum sickness, IL-5 and increased IFN-c production, indicating a shift of hypersensitivity reaction or the development of anti-E25 the T-cells towards an increased Th1 response at the antibodies. E25 induces a dose-dependent reduction in expense of Th2 responses [179, 180]. This treatment has serum free IgE associated with a reduction in basophil been successfully used in asthma and allergic rhinitis and FceR1 expression and histamine release [189, 190]. A for venom allergy [181, 182], but is usually most useful in significant improvement in both early [191] and late subjects allergic to single allergens such as grass pollens, phase [192] bronchoconstrictor responses to allergen- and in subjects with mild forms of allergic diseases [183, challenge and a reduction in methacholine-induced 184]. Recently, cloning of epitopes of allergens has hyperresponsiveness, with a reduction in sputum eosi- provided a higher level of epitope purity than previously nophil numbers have been reported following E25 available, and there has been a greater understanding of administration. Results of several clinical trials show the specific mechanisms of tolerance induction. These subjective improvement and significant effects on factors may lead to a wider application of SIT in atopic corticosteroid reduction in moderate to severe asthma disease, using better characterized peptides which are [193], and allergic rhinitis [194]. This treatment should more effective but with a lesser risk of side-effects, in become available in the near future, and its initial role particular anaphylaxis. may be in severe atopic asthma, although it is not known whether E25 is also effective in nonatopic asthma. Peptide immunotherapy

Peptide immunotherapy can be considered as a refinement of SIT, with the specific intent of inhibition Inhibition of eosinophil activation and of T-cell activation independent of IgE-mediated inflam- chemoattraction matory processes. Synthetic MHC-restricted oligopeptides mimicking recognized epitopes of the major cat allergen IL-5 is involved in the maturation and mobilization of dI (Fel dI), modified by single amino acid substitution eosinophils and cooperates with chemoattractant cytokines have been produced and are noted to have variable effects (chemokines) in chemoattraction and tissue activation of on T-cell proliferation and cytokine production by Fel dI eosinophils. 1166 R.G. STIRLING, K.F. CHUNG

Chemokine receptor antagonists inflammatory cytokines. Activation of the transcription factor, NFkB, which can regulate the production of a Chemokines are inducible pro-inflammatory proteins range of pro-inflammatory cytokines including IL-1, IL- whose key function is leukocyte chemoattraction and 6, IL-8 and TNF-a but also adhesion molecules ICAM-1 activation. Three families are currently described, distin- and VCAM-1, has been shown in the airway epithelium guished on a structural basis into C, CC and CXC families and macrophages of patients with mild asthma [213]. [195]. Chemokine effects are mediated by chemokine Similarly, increased expression of the transcription factor, receptors and differential cell receptor profiles allow cell- AP-1, has also been reported [214]. specific attraction [196±198]. Thus, the CC chemokine eotaxin has an eosinophil selective role in eosinophil recruitment due to selective expression of the eotaxin Tumour necrosis factor-a receptor CCR3 on the eosinophil [199, 200]. Chemokines are however, also involved in the recruitment of mono- TNF-a increases airway responsiveness in Brown- cytes, dendritic cells, T-lymphocytes, NK cells, B- Norway rats [215] and in humans, together with an lymphocytes and basophils. increase in sputum neutrophils [216]. TNF-a also Antagonism of chemokine receptor signalling has been potently stimulates airway epithelial cells to produce accomplished by modification of endogenous agents to cytokines including regulated upon activation, normal T- create high-affinity receptor antagonists. These agents, cell expressed and secreted (RANTES), IL-8 and GM- which include met-regulated upon activation normal T-cell CSF, and increases the expression of the adhesion mole- expressed and secreted (RANTES) and met-Ckb7[201± cule, ICAM-1. TNF-a has a further synergistic effect with 203], are potent CCR3 specific antagonists which inhibit IL-4 and IFN-c to increase VCAM-1 expression on CCR3 receptor signalling and consequent eosinophil endothelial cells [217, 218]. These factors indicate that migration. To date several small-molecular weight CCR3 TNF-a may be an important mediator in initiating chronic antagonists have been developed and await clinical inflammation in the airways. Several approaches to evaluation. One potential strategy combines the inhibition inhibition of TNF-a synthesis and effects are now under of both IL-5 and eotaxin, a double-pronged approach that investigation in asthma, including monoclonal antibodies would reduce both mobilization, chemoattraction and to TNF-a and soluble TNF-a receptors. An anti-TNF-a activation of eosinophils more effectively given the antibody has retarded the progression of severe rheuma- cooperation between IL-5 and eotaxin demonstrated in in toid arthritis with significant clinical amelioration [219], vivo models [204, 205]. Such a combined approach may but has not been tried in asthma. This antibody may have lead to clinical improvement. therapeutic effects in severe asthma. Inhibitors of TNF-a converting enzyme (TACE), or of the cysteine protease caspase-1 (IL-1 converting enzyme, ICE) are also Blocking adhesion molecules and integrins potential anti-inflammatory compounds that may be used in asthma, and are currently under development. Recent Eosinophil migration to sites of allergic inflammation is attention has also been focused on intracellular signalling specifically mediated by interactions between endothe- cascades such as inhibitors of p38 MAP kinase which lium, epithelium and eosinophil adhesion molecules inclu- inhibit the synthesis of pro-inflammatory cytokines in ding the a4-integrin very late antigen-4 (VLA-4) [206]. vivo [220, 221]. In this mouse model, inhibition of p38 a4-Integrins are also implicated in human lymphocyte kinase activity markedly reduced cytokine-associated coactivation [204]. An a4-integrin monoclonal antibody inflammation and inhibited lipopolysaccharid (LPS)- inhibited inflammation and BHR in a sensitized mouse induced TNF-a production while allergen-induced air- model [207], and also eosinophilic inflammation and way eosinophilia was all but abolished. BHR induced by eotaxin in IL-5 transgenic mice [204]. Peptido-mimetic small molecule VLA-4 inhibitors, administered prior to allergen challenge, also prevent increases in VLA-4+ leukocytes (eosinophils, lympho- Interleukin-1 cytes and macrophages) in lung tissue while significantly inhibiting early and late phase allergic responses [208]. IL-1 co-induces CD4+ T-cell proliferation and IL-2 Similar approaches inhibiting interactions between leu- secretion following interaction of T-cells with antigen kocyte function associated antigen-1 (LFA-1) and inter- presenting cells, and is an important growth factor for cellular adhesion molecule-1 (ICAM-1) have also proven antigen primed Th2 cells [222]. IL-1 also potently induces effective in blocking eosinophil adhesion and transmi- the synthesis and release of multiple pro-inflammatory gration [209]. cytokines and chemokines. The IL-1 receptor antagonist (IL-1Ra) polypeptide shows significant homology with IL-1a and IL-1b and binds the IL-1 receptor. IL-1Ra has Inhibition of pro-inflammatory cytokines and been isolated from multiple tissues including alveolar pathways macrophages [223], and inhibits most of the activities of IL-1 [224]. IL1-Ra blocks Th2 but not Th1 clonal Several pro-inflammatory cytokines such as TNF-a proliferation in vitro and in the guinea pig reduces [210] and IL-1b [211] have been shown to be over- allergen induced BHR and pulmonary eosinophilia [225]. expressed in asthma, and exposure to environmental Manipulation of this endogenous control mechanism may endotoxin such as that present in house dust [212] may therefore, impact on asthma and needs to be evaluated in lead to further activation of a panel of such pro- the clinical setting. NEW APPROACHES TO ASTHMA THERAPY 1167

Interleukin-10 where mucosal transfer of IL-12 [248] and IFN-c [249] significantly reduced Th2 cytokine expression and inhi- Although IL-10 is a Th2-derived cytokine, it has anti- bited BHR. Transfer of the glucocorticoid receptor to the inflammatory properties that could be used to control epithelial cell line A549, lead to repression of transcrip- asthmatic inflammation. IL-10 is derived largely from tion factor mediated gene transcription and may provide a mononuclear cells, alveolar macrophages and both naive therapeutic approach in steroid-resistant asthma [250]. and committed T-cells. It reduces MHC and costimulatory Gene transfer to the lungs in man is still in a develop- molecule expression, reduces pro-inflammatory cytokine mental phase, and the efficiency of gene transfer using release and increases IL-1Ra expression [4, 226]. T-cell liposomal transfer technology is thus far suboptimal, as and macrophage IL-10 synthesis is significantly reduced judged from the experience of the transfer of the cystic in asthmatic subjects compared with nonasthmatics [227, fibrosis transmembrane regulator DNA to patients with 228] but IL-10 expression in alveolar macrophages is cystic fibrosis [251]. Use of the more efficient transfer increased by corticosteroid therapy [229], suggesting in system, the adenovirus vector, may be detrimental in vivo relevance of this cytokine. A polymorphism in the inducing lung damage and inflammation, and is no longer promoter sequence of IL-10 has been associated with a viable option in man. The potential genes that may be attenuated IL-10 expression and has been identified at delivered to the airway epithelium of patients with asthma increased frequency in severe asthma [230], while for therapeutic effects are numerous. Candidates for gene promoter polymorphisms have been demonstrated in transfer might include Th1 cytokines IL-12 or IFN-c,or asthma probands and are associated with elevation of IgE of the anti-inflammatory cytokine, IL-10, or of IL-1Ra, or levels [231]. This latter observation is of particular of the corticosteroid receptor may be contemplated, but interest given a proposed suppressive effect of IL-10 on gene therapy approaches for the treatment of asthma IL-4-induced IgE isotype switching by B-cells [232]. IL- remain in the distant future. 10 administration to mice reduces airway eosinophilia and allergen-induced late responses [233]. When given to normal volunteers, IL-10 reduced circulating CD3, CD4 Conclusion and CD8 lymphocyte numbers and the proliferative response and reduced TNF-a and IL-1 production [234]. Recent advances in the techniques for the synthesis and Further studies in human asthma are awaited. manufacture of monoclonal antibodies, synthetic peptides and peptidomimetic small molecules has increased the potential for the creation of specific inhibitors of immune Use of antisense oligonucleotides processes in allergic inflammation. These agents have enabled specific intervention in the inflammatory cascade Oligonucleotide sequences forming a complementary and allow a clearer understanding of the roles of specific copy of normal, or sense, messenger (mRNA) are able to agents in this cascade, while at the same time providing the bind to specific mRNA, and this dimer formation prevents possibility of therapeutic intervention in asthma. In the first mRNA translation and protein synthesis. The effectiveness instance, it is likely that these strategies will be aimed at of this technique was demonstrated in a rabbit model of those with severe difficult-to-treat asthma as it is here that asthma where aerosol inhalation of adenosine-A1 receptor the failings of current therapies are most evident. Whether antisense protected the animals from subsequent challenge new therapies may provide remission of disease with short- with either adenosine or dust-mite allergen, with effects term treatment cannot be predicted from the current persisting up to 7 days [235, 236]. In a model in which the understanding of asthma. While preliminary data for many late allergic reaction could be transferred in the rat by of these agents appears most promising, these agents will ovalbumin sensitized T-cells [237], IL-4 antisense-treated have to endure rigorous evaluation of efficacy, long-term T-cells caused a significant reduction in the late allergic safety and cost-effectiveness. Several agents targeted to reaction, airway eosinophilia, and IL-4 and IL-5 expres- specific immunological or cytokine pathways may become sion. However, when IL-5 antisense treated T-cells were available and may be more effective in certain types of administered, only BAL IL-5 expression was reduced asthma. Genetic pharmacological profiling may be needed suggesting a key role for IL-4 in CD4+ T-cell mediated to identify the best responders to particular types of late allergic responses. In murine models, an IL-5 anti- specific drugs [252]. The future of asthma therapy looks sense oligonucleotide reduced IL-5 protein levels, airway bright, but identifying targets is only a first step. The eosinophilia and allergen-induced late allergic responses development of these new approaches is very exciting but [111]. 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