New immunological approaches and cytokine targets in asthma and allergy REVIEW
New immunological approaches and cytokine targets in asthma and allergy REVIEW
Copyright #ERS Journals Ltd 2000 European Respiratory Journal ISSN 0903-1936 Eur Respir J 2000; 16: 1158±1174 Printed in UK ± all rights reserved 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, K.F. Chung. #ERS Journals Ltd 2000. ABSTRACT: The aims of current asthma treatment are to suppress airway inflammation and control symptoms, and corticosteroids maintain a commanding position in this role. Steroids effectively suppress inflammation in the majority of patients but have little impact on the natural history of this disease. In severe asthmatics, corticosteroids may have relatively less beneficial effects. Recent advances in understanding the inflammatory and immunological mechanisms of asthma have indicated many potential therapeutic avenues that may prevent or reverse abnormalities that underlie asthma. As the roles of effector cells, and of signalling and adhesion molecules are better understood, the opportunities to inhibit or prevent the inflammatory cascade have increased. In addition, there have been advances in the synthesis of proteins, monoclonal antibodies and new small molecule chemical entities, which may provide valuable flexibility in the therapeutic approach to asthma. The novel immunological approaches include the prevention of T-cell activation, attempts to influence the balance of T-helper cell (Th) populations to inhibit or prevent Th2-derived cytokine expression, 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 as highly specific targeting, and also prospects for prevention and reversal of 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 The chronic airway inflammation of asthma is characterized by infiltration of the airway wall and lumen by diverse effector cells, including T-lymphocytes, eosinophils, monocytes/macrophages, mast cells, and occasionally, neutrophils [1±3]. The mobilization, activation and trafficking of these effector cells to the airway are controlled by a complex cytokine milieu derived from activated CD4+ T-helper (Th) cells and also from other resident airway cells including airway smooth muscle and epithelial cells. T-helper cells of type 2 variety (Th2) secrete a Th2 profile of cytokines, after cognate stimulation of the naive T-cell by antigen presenting cells, such as the dendritic cell and the alveolar macrophage (fig. 1). The Th2 cytokines include interleukins (IL)-4, -5, -9, -10 and -13. These cytokines promote various elements of allergic inflammation (table 1), including propagation of the Th2 phenotype, isotype-switching from immunoglobulin (Ig)-G1 to IgE synthesis, eosinophil mobilization, maturation and activation and mast cell activation . Additionally, the airway wall undergoes chronic structural changes labelled as remodelling, which include thickening of the airway smooth muscle due to hypertrophy National Heart and Lung Institute, Imperial College School of Medicine and Royal Brompton and Harefield Hospital, London, UK. Correspondence: K.F. Chung Dept of Thoracic Medicine National Heart & Lung Institute Dovehouse Street London SW3 6LY UK Fax: 44 2073518126 Keywords: Adhesion molecules asthma cytokines gene therapy immunology T-cells Received: September 20 2000 Accepted after revision September 22 2000 and hyperplasia, myofibroblast activation with increase in subepithelial basement membrane collagen deposition, angiogenesis and increase in submucosal blood vessels and an increase in goblet cell numbers in the airway epithelium [5±8]. These changes are also variably influenced by Th2 cytokines, and certain growth factors [9, 10]. The exact link between the chronic inflammatory and remodelling processes and the clinical presentation of asthma is unclear in terms of the pathophysiological mechanisms and of the contribution of the components of these processes to bronchial hyperresponsiveness, airway narrowing and the acute exacerbations that characterize the disease. The overwhelming hypothesis is that these are intricately related such that inhibition or prevention of the inflammatory process should improve the control of asthma, an idea supported by the potent actions of topical corticosteroids in controlling the eosinophilic and lymphocytic inflammation of asthma, while being most effective in restoring lung function and bronchial hyperresponsiveness [9, 10]. However, identification of the key immunological and inflammatory processes that lead to functional abnormalities and symptoms remain to be elucidated. 1159 NEW APPROACHES TO ASTHMA THERAPY Table 1. ± Summary of Th2 cytokine effects as predicted by cytokine stimulation and ablation studies (animal and human data included) Th response IL-4 IL-5 IL-9 IL-10 IL-13 Th1 Th2 - + - BHR IgE Blood + + + + Mucus hyperplasia Eosinophils + - Airway Mast cell activation Surface markers Survival + + - + + + VCAM + MHC + + - MHC II + VCAM + IL: interleukin; BHR: bronchial hyperresponsiveness; IgE: immunoglobulin-E; VCAM: vascular cell adhesion molecule; MHC: major histocompatability complex. Limitations of current therapy Despite the introduction of new agents such as the leukotriene inhibitors, corticosteroids remain the antiinflammatory drug of choice for the majority in the treatment of chronic asthma [11, 12]. However, treatment failure and drug-related side-effects pose serious limitations to their use . Severe and difficult-to-treat asthmatics are characterized by ongoing symptoms and frequent exacerbations despite the use of existing antiinflammatory and bronchodilator therapies. These patients are frequently prescribed corticosteroids to which their disease is partially responsive and they endure the adverse consequences of high dose inhaled and systemic corticosteroid therapy [13, 14]. Amongst severe asthmatics, up to 25% demonstrate relative unresponsiveness to the therapeutic but not to the adverse effects of corticosteroids [15, 16]. The identification of novel therapeutic targets and subsequent development of specific and safe therapeutic agents therefore, represents a major challenge in asthma therapy. In addition to attempting to reverse established changes in the airways resulting from chronic inflammation and airway remodelling, the prevention of allergy and asthma remains an important consideration given the rising prevalence of these conditions in many industrialized countries . Immunological imbalance: T-helper cell balance The vast majority of asthmatics have an atopic background, in whom the inflammatory process of asthma is assumed to be driven following sensitization and reexposure, or challenge by common aeroallergens. However, the inflammatory abnormalities in nonatopic asthma also bear many similarities to those observed in the allergic inflammatory process, including the presence of eosinophils, activated CD4+ T-cells, and the expression of Th2derived cytokines . These findings suggest that most asthmatics may benefit from an approach that targets mechanisms of allergic sensitization and inflammation. Allergens are taken up by specialized cells within the mucosa such as dendritic cells (antigen-presenting cells), and subsequently processed, following which peptide fragments are presented to naive T-cells. The activation of naive T-cells requires direct signalling by two distinct pathways (fig. 1): firstly via the CD4+ T-cell receptor through the antigen-presenting cell (APC)-bound antigen to major histocompatability (MHC)-II complex and, secondly, via the costimulatory pathway linked by the B7 family and the T-cell bound CD28 . T-cells stimulated via the T-cell receptor (TCR) in the absence of costimulatory signalling are incapable of IL-2 secretion and subsequent activation and therefore, enter an anergic state . CD28 itself has two major ligands, B7.1, which inhibits Th2 cell activation and development, and B7.2, which induces T-cell activation, and Th2 development. An important third ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA4), is expressed on activated T-cells, binds CD28 with enhanced avidity and acts as a negative regulator of T-cell function by inhibiting Th2 differentiation [21, 22]. In those who later develop an allergic response, naive T-cells differentiate into the Th2 subtype. T-cell profiles in the newborn demonstrate a Th2 bias suggesting that prenatal influences are involved in T-cell priming . Th1 cells form a natural counterbalance to Th2 cells driving protective cell-mediated immunity (CMI), and are induced on exposure to foreign agents including protozoa and bacteria. Th1 responses are characterized by the induction of CMI responses and the synthesis of IgG2a, while Th2 responses are of the humoral-type, inciting the production of IgE and IgG4. Signal 2 B7.2 CD28 APC MHCII-Ag CD4 TCR/CD3 Signal 1 Th0 MHCII-Ag TCR/CD3 CD4 APC B7.2 CTLA4 Th0 T-cell activation IL-12 Clonal anergy CTLA4-Ig Fig. 1. ± T-helper cells differentiate from the naive state following interaction with the antigen-presenting cell. The Th2 phenotype is characterized by secretion of the cytokines interleukins -4, -5, -9 and -13, while the Th1 cells secrete interferon-c, and interleukin (IL)-2. IL-4 directly stimulates Th2 differentiation while IFN-c promotes the Th1 phenotype. APC: antigen presenting cell; MHC: major histocompatibility complex; TCR: T-cell receptor; CTLA4: cytotoxic T-lymphocyte associated protein-4; Ag: antigen. 1160 R.G. STIRLING, K.F. CHUNG Th1 responses inhibit Th2 responses through the production of cytokines such as IL-12 and interferon gamma (IFN-c) (fig. 2). Abnormal polarization of these responses may result in tissue pathology. The excessive expression of Th1 cytokines is noted in autoimmune conditions including experimental diabetes mellitus [24, 25] and multiple sclerosis [26, 27], while excessive Th2 expression is observed in the atopic diathesis. There is evidence for a preferential skewing to expansion of the CD4+ Th2 lymphocyte subset in allergic processes [3, 28] and this is a likely crucial forerunner to the development of allergic disease. During the course of maturation of the normal infant however, increased Th1 expression occurs, and the Th2 imbalance is overcome . Delay or failure of this Th1 response may result in Th2 persistence and atopy or atopic disease, and accordingly in infants destined to become atopic, an impaired production of IFN-c by circulating lymphocytes is observed . The "hygiene hypothesis" postulates a diminished induction of Th1 responses as a potential explanation for the rising prevalence of atopy and asthma [23, 31, 32]. Crosssectional surveys have identified inverse relationships between prior microbial exposure and development of atopy [23, 33, 34]. Further, respiratory allergy appears less frequently in those heavily exposed to orofaecal and foodborne microbes . Thus, improved hygiene, early infection and antibiotic use, and a westernized or semisterile diet may facilitate atopy by influencing exposure to commensals and pathogens that stimulate immune cell populations such as gut-associated lymphoid tissue [35, 36]. Thus, early environmental exposure may be a determinant of the development of atopy in the adult . The identification of ways to prevent, control or even reverse the process of Th2 immunodeviation has become a focus for the development of new strategies to control asthma and allergies. IL-12 IL-18 Mast cell NK IFN-γ IFN-γ IL-2 IL-10 Th1 Th0 Th2 T-cell immunomodulators Cyclosporin A and the functionally-related, tacrolimus (FK506), are powerful immunosuppressant agents used widely to prevent immune rejection in organ transplantation. These compounds inhibit T-cell growth by creating a block in the G0 phase of development through inhibition of T-cell growth factors including IL-2 . Both agents strongly inhibit mitogen-stimulated IL-5 release by inhibition of transcription [39, 40]. Cyclosporin A and Table 2. ± Novel strategies for the inhibition and prevention of allergic asthma Target Agent Prevention of T-cell activation Anti-CD4 CTLA4 Prevention of reversal of Th2 expression Inhibition of Th2 cytokines/ phenotype Immunotherapy APC Th0 Three main thrusts of research into new therapies have developed: 1) preventing T-cell activation; 2) prevention or reversal of Th2 polarization, and; 3) ablation of the effects of Th2 cytokines and downstream mediators (table 2). Many of the potential ways of achieving these objectives have been investigated in animal (mainly murine) models, and therefore, their applicability to human asthma and allergic diseases remains unclear given the inherent variability within varied species. Promotion of Th1 cytokines/ phenotype IL-4 APC Prevention of T-cell activation CpG Inhibition of downstream mediators Antiinflammatory cytokines Inhibition of eosinophil migration and activation IL-4 IL-5 IL-13 Fig. 2. ± Activation of T-helper cells by the antigen presenting cell (APC) requires dual signalling via the major histocompatibility complex and the T-cell receptor, and via B7.2:CD28 interaction and results in Th2 deviation, activation and interleukin (IL)-2 secretion. T-cell receptor stimulation in the absence of costimulation via CD28 induces clonal anergy. NK: natural killer cell; IFN-c: interferon-gamma. IgE inhibition Soluble rhu IL-4 mutant proteins STAT-6 inhibition Anti-IL-5 monoclonal antibody GATA inhibition Soluble IL-13Ra IFN-c IL-12 IL-18 SIT Peptide immunotherapy M. vaccae vaccination IL-10 IL-1Ra CCR3 antagonist CCR3 antisense VLA4 inhibitor ICAM-1 inhibitor met-RANTES met-Ckb7 Monoclonal anti-IgE (E25) CTLA4: cytotoxic T-lymphocyte associated protein-4; IL: interleukin; STAT-6: signal transduction and activation of transcription-6; IFN: interferon; SIT: specific immunotherapy; M. vaccae: Mycobacterium vaccae; CCR3: eotaxin receptor; VLA4: very late antigen-4; ICAM-1: intercellular adhesion molecule-1; Ig: immunoglobulin; E25: nonanaphylactogenic IgE monoclonal antibody. 1161 NEW APPROACHES TO ASTHMA THERAPY Inhibition of T-cell costimulation: cytotoxic T-lymphocyte associated protein-4-immunoglobulin FK506 also have effects on other cells and markedly reduce basophil histamine release . In human studies, cyclosporin A by inhalation provides significant inhibition of the allergen-induced late allergic reaction . In corticosteroid-dependent asthma, low-dose cyclosporin A improved lung function  and allowed for a 62% reduction in oral steroid dose requirement , but at the expense of adverse effects which would not prove tolerable in mild disease. The folate antagonist, methotrexate, has well recognized anti-inflammatory effects in rheumatoid arthritis and is used as a steroid-sparing agent in asthma. Meta-analysis of controlled trials confirms steroid-sparing effects while maintaining lung function or symptoms [45±47]. In clinical practice, these immunomodulators are only modestly effective in a proportion of patients. A number of compounds, which show immunomodulatory activity, are currently under development. However, it is likely that compounds that have nonspecific immunosuppressive Tcell activity may not be as effective as the more specific Th2 cell inhibitors . Suplatast tosilate, a compound developed in Japan, selectively prevents the release of IL4 and IL-5 from Th2 cells and can reduce bronchial eosinophilia in animal models of bronchial hyperresponsiveness (BHR) [49, 50]. It has been shown to improve pulmonary function and symptom control, and allows for a decrease in the dose of inhaled corticosteroids , and has been launched for the treatment of asthma in Japan. Although corticosteroids inhibit antigen uptake and processing, they do not appear to impact on antigen presentation . Therefore, the potential for disruption of antigen presentation by specific inhibition of costimulatory molecule interaction has been recognized and studied in murine models. A recombinant fusion protein consisting of the extracellular domain of CTLA4 linked to the constant region of IgG1, known as CTLA-4-Ig, binds B7 molecules with an affinity similar to that of membrane CTLA4. It therefore, acts as a powerful inhibitor of B7: CD28 mediated costimulation. T-cell activation following B7:CD28 signalling may be blocked using the soluble protein ligand CTLA4-Ig, and this can substantially reduce BHR, bronchoalveolar lavage (BAL) eosinophilia and specific IgE responses when given either prior to sensitization or to allergen challenge . CTLA4-Ig reduced IL-4 and IgE levels while IFN-c and IgG2a were unchanged, suggesting downregulation of Th2 response without upregulation of the Th1 response . Similar outcomes are observed following monoclonal anti-B7.2 (anti-CD86) treatment in allergen-challenged mice . Thus, CTLA4-Ig is potentially a powerful immunomodulator with the potential of suppressing Th2 based responses to allergens in 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 allergen-mediated airway disease. Depletion of T-cells in murine sensitized and allergen-challenge models using recombinant monoclonal antibodies results in a complete ablation of airway hyperresponsiveness and airway eosinophilia . Conversely, antibody-mediated CD8+ T-cell depletion augments BHR and eosinophilic inflammation in the allergen-challenge model . A preliminary clinical trial in asthma has evaluated the use of a single dose of anti-CD4 monoclonal antibody in severe corticosteroid dependent asthma . This treatment led 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 some benefit of this approach in the CD4+ dependent processes of multiple sclerosis [55, 56] and collagenarthritis . It is likely that other T-cell subsets may also be important in asthma. Recent investigations have highlighted the existence of CD8+ T-cells (i.e. TC2), which secrete Th2 type cytokines and their influence on the mediation of allergic airways disease is as yet to be established [58±60]. Further uncertainty regarding the approach of CD4 depletion relates to the induction of CD4 lymphopenia and immunosuppression and the resultant risk of opportunistic infection and neoplasia [61±63]. Nondepleting CD4 antibodies have been generated but are as yet untried in pulmonary disease . Specific attempts to alter Th1/Th2 balance by enhancing Th1 and abrogation of Th2 responses have been the major thrust of immune approaches to the prevention and treatment of allergies and asthma. Induction of Th1 responses may have a direct suppressive effect on Th2 mediated inflammatory processes (table 3). The most direct approaches include administration of cytokines that will induce activation of Th1 pathways (e.g. IFN-c, IL-12 and IL-18) or of blocking antibodies that inhibit the effect of Th2related cytokines (e.g. anti-IL-4, anti-IL-5, anti-IL-9, and anti-IL-13). Table 3. ± Summary of T-helper 1 cytokine effects as predicted by cytokine stimulation and ablation studies (animal and human data included) Th response BHR IgE Th1 Th2 IFN-c + - IL-12 + - IL-18 + Eosinophils Blood Airway - - - - - - - - -+ - Mediator release +TNF-a +IL-1b +TNF-a -IL-10 +IFN-c +Chemokines -IL-10 IFN: interferon; IL: interleukin; BHR: bronchial hyperresponsiveness; IgE: immunoglobulin-E; TNF-a: tumour necrosis factor-alpha. 1162 R.G. STIRLING, K.F. CHUNG Inhibition of allergy and asthma by T-helper 1 related cytokines Interferon gamma IFN-c, released from CD4+(Th1) and CD8+(TC2) cells, is a critical factor controlling the balance of Th1/Th2 development, and exerts an inhibitory effect on Th2 cells . IFN-c is also a powerful and relatively specific inhibitor of IL-4-induced IgE and IgG4 synthesis by Bcells. A reduced production of IFN-c by the T-cells of asthmatic patients is seen and correlates with disease severity . Intra-tracheal IFN-c in allergen-sensitized and -challenged mice causes a dose-dependent reduction in BAL IL-5 levels and airway T-cells and eosinophils [70, 71]. Intraperitoneal IFN-c also reduced lung Th2 cytokine levels, attenuated allergen-induced BHR with concomitant reduction in BAL eosinophilia, while an IFN-c-blocking antibody led to an increase in airway CD4+ T-cells and BHR . The inhibition of eosinophil recruitment appears to be dependent on the inhibition of CD4+, but not on CD8+ T-cell accumulation within the airways [70, 73]. Inhalation of IFN-c by nonasthmatic humans increases epithelial lining and BAL fluid IFN-c levels but does not affect serum IFN-c levels and therefore, may avoid toxicity associated with systemic administration [74, 75]. However, in one study of subcutaneous IFN-c therapy in steroid-dependent asthma no effect on lung function or treatment requirement was observed despite a significant reduction in circulating eosinophil numbers . Interleukin-12 IL-12 is produced by antigen-presenting cells and enhances the growth of activated T- and natural killer (NK)-cells, stimulating them to produce IFN-c [77, 78]. IL-12 also promotes the differentiation of IFN-c-producing T-cells and inhibits the differentiation of T-cells into IL-4 secreting cells . Thus, IL-12 can regulate Th1 cell differentiation while suppressing the expansion of Th2 cell clones by early priming of undifferentiated Th0 cells for IFN-c secretion. IL-12 may play an important role in inhibiting inappropriate IgE synthesis and allergic inflammation as a result of allergen exposure. In murine asthma models, administration of IL-12 leads to a reduction in allergen specific IgE levels, ablation of airway hyperresponsiveness and inhibition of eosinophil recruitment [79±81]. However, further studies have suggested that this protective role may only be conferred when acting synergistically with IL-18 . In therapeutic trials, IL-12 levels increased during corticosteroid therapy  and during specific immunotherapy . In a phase-I trial of IL-12 in asthma, a significant reduction of peripheral eosinophils and a trend towards a reduction in airway eosinophils was observed without effect on allergen-induced early or late phase responses . IL-12 has proved a useful adjunct to cancer chemotherapy by induction of a protective Th1 response , but significant toxicity including arrhythmias, liver function abnormalities and flu-like illness will limit its potential utility in asthma . Interleukin-18 IL-18 (IFN-c-inducing factor) is a potent inducer of IFN-c production by T-, NK- and B-cells and plays an important part in the induction of Th1 responses [88, 89]. IL-18 receptors are expressed selectively on murine Th1 but not Th2 cells. Recombinant human IL-18 potently induces IFN-c production by mitogen-stimulated peripheral blood mononuclear cells and enhances NK-cell cytotoxicity , while increasing NK-cell granulocytemacrophage colony-stimulating factor (GM-CSF) release and CD8+ T-cell IL-10 production . IL-18 and IL-12 have synergistic effects on Th1 development, which may be due to reciprocal upregulation of their receptors. IL-18 may be important in the control of allergen-induced BHR by vaccination. Vaccination using heat-killed Listeria monocytogenes caused a marked inhibition of allergeninduced BHR and airway inflammation, associated with conversion to the Th1 phenotype in mice [92, 93]. This effect was IL-12-dependent and associated with a marked upregulation of IL-18 messenger ribonucleic acid (mRNA) expression. These studies also demonstrated that administration of an adjuvant after allergen exposure, was able to reverse established BHR. The direct administration of IL-18 in the murine asthma model provides more confusing results; although IL-18 appears to play a protective role when administered with allergen challenge, administration with sensitization paradoxically increased IgE and IL-5 levels and promoted BAL eosinophilia [94, 95]. Thus, IL-18 appears to be involved in vaccination-mediated inhibition of Th2 responses and may have properties directly driving BHR in mice. Inhibition of T-helper 2 related cytokines Interleukin-4 IL-4 has a central role in Th cell development as a potent inducer of Th2 maturation from the naive T-lymphocyte [96, 97]. Additionally, IL-4 induces the isotype-switch necessary for IgE synthesis , upregulates IgE receptors [99, 100] and vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelium, thus facilitating endothelial passage and accumulation of eosinophils . IL-4 is therefore, an attractive target to inhibit, resulting in downregulation of Th2 activation. Anti-IL-4 monoclonal antibody treatment of mice prior to allergic sensitization markedly reduces IgE synthesis , but does not appear to inhibit airway eosinophilia or BHR . A recombinant soluble IL-4 receptor (sIL4R) has been designed as a mimic of the cell-surface receptor, which thus binds and sequestrates free IL-4, but because these soluble receptors lack transmembrane and cytoplasmic domains, they act as an IL-4 receptor blocker. In murine studies, sIL-4R reduces allergenspecific IgE responses, airway hyperresponsiveness, VCAM-1 expression and eosinophil accumulation . Preliminary human asthma studies with single doses of sIL-4R have shown acceptable tolerance, improvement in lung function and a reduction in rescue b2-agonist requirement . Additionally, a trend towards a reduction in serum eosinophilic cationic protein (ECP) and exhaled nitric oxide (NO) levels suggests suppression of inflammation by this agent. A recombinant 1163 NEW APPROACHES TO ASTHMA THERAPY mutant human protein (BAY 16-9996) binds the IL-4 receptor a- but not cc-chain, and therefore, antagonises receptor transduction. This protein substantially reversed allergen-induced BHR with a reduction in airway inflammation in a primate model . 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 region of IL-4 inducible genes and this transcription factor is expressed at abnormally high levels in the epithelium of severe asthmatics . STAT-6 knockout mice demonstrate a defect in IL-4 and IL-13 mediated signal transduction . The potential utility of STAT-6 targeted therapies is highlighted by the ability of STAT-6 directed antisense oligonucleotides to markedly downregulate germline Ce mRNA levels, reflecting inhibition of IL-4-dependent IgE isotype switching [109, 110]. IL-5 α β JAK2 JAK1 Ras Lyn Fyn STAT1 STAT5 Syk MEK MAPK Interleukin 5 Eosinophil mobilization and trafficking, their maturation and maintenance are largely promoted by the Th2 cytokine, IL-5, making it an attractive therapeutic target in eosinophilic conditions such as asthma and rhinitis. Ablation of the effects of IL-5 has been accomplished with antisense oligonucleotides in rodent models where a reduction in IL-5 protein levels is followed by a reduction in allergen-induced eosinophilia and BHR . Importantly, this suppression of lung eosinophilia was maintained for 17 days following a single intravenous treatment. Monoclonal antibodies used to block IL-5 in mouse, guinea-pig, rabbit and monkey studies provided a sustained reduction in antigen-induced airway eosinophilia but had little effect on antigen-induced BHR [112±114]. Two humanized forms of anti-IL-5 (SB-240563 and Sch 55700) are now available with the potential for clinical studies [115, 116]. In mild asthmatics, SB-240563 had a marked inhibitory effect on both airway and peripheral blood eosinophil levels and on allergen-induced sputum eosinophilia following single subcutaneous administration . However, as with the studies in mouse, rabbit and monkey, no significant effect on bronchial responsiveness or the late phase response to allergen-challenge was observed. Phase-I trials in severe persistent asthma have provided similar results . IL-5 blocking antibodies prevent the mobilization and subsequent trafficking of eosinophils to the lung in allergic asthma, but are unable to influence BHR or the late phase response. Currently, studies are in progress to determine whether anti-IL-5 blocking antibodies can improve the control of asthma, despite their apparent lack of protection against BHR. Recent studies on IL-5 signalling (fig. 3) suggest that various IL-5-dependent functions are mediated by distinct and separate secondary messenger systems . Thus, targetting such pathways may lead to inhibition of IL-5 effector functions. The anti-apoptotic effects of IL-5 on eosinophils are dependent on the activation of lyn-, Janus kinase-2 (Jak2) and Raf-1 kinase, of these, only Raf-1 is necessary for eosinophil activation and degranulation [119, 120]. Lyn-kinase dependent signalling may be specifically inhibited by a peptide inhibitor, Raf-1 Gene transcription Fig. 3. ± Binding of IL-5 activates the IL-5 receptor and triggers a cascade of secondary messengers. The secondary signalling pathway is a linear system in which cell surface associated receptor protein tyrosine kinases (e.g. Jak-1/2 and Lyn) activate the signal transducers and activators of transcription (STAT1 and STAT5) and Ras family of serine/ threonine protein kinases (e.g. Raf-1) respectively. Ras/Raf activation may function as a relay switch, positioned upstream from a further cytoplasmic cascade of kinases that include the mitogen-activated protein kinases (MAPK) and mitogen-activated protien kinases/ extracellular regulated protein kinase (MEK). Activated MAPKs in turn regulate the activities of nuclear transcription factors such as activator protein-1 (AP-1, of which c-jun and c-fos are important components). resulting in inhibition of lyn-dependent IL-5 signalling without affecting Jak2 dependent IL-5 signalling. This inhibitor blocks allergen-driven airway eosinophilia . The transcription factor GATA-3 is critical for IL-5 expression in Th2 cells, and increased GATA-3 gene expression in association with IL-5 mRNA positivity has been shown in airway cells isolated from patients with asthma. These findings support a causal association between augmented GATA-3 expression and dysregulated IL-5 expression and asthma [122, 123]. Specific inhibition of GATA-3 expression using dominant negative GATA-3 transgenic mice led to a reduction in Th2 cytokine expression and marked attenuation of airway eosinophilia, mucus production, and IgE synthesis . These outcomes could also potentially be achieved using antisense oligonucleotide technology. Interleukin-9 IL-9 has been identified in airway tissue in asthma and is known to induce BHR, elevated serum IgE, mucin gene transcription and epithelial CC chemokine release [125± 128], and thus is a broadly attractive therapeutic target in asthma . One mechanism by which IL-9 may induce airway eosinophilia is by enhancing IL-5 receptor expression, thereby increasing the differentiation and survival of eosinophils . Specific blockade of IL-9 1164 R.G. STIRLING, K.F. CHUNG activity has been accomplished in the mouse by intratracheal instillation of monoclonal anti-IL-9 antibody . Anti-IL-9 significantly inhibited BHR, airway eosinophilia, serum IgE, airway inflammatory cell infiltration and mucin production induced by allergen, demonstrating a surprisingly broad inflammatory effect for this cytokine. As yet there are no reported clinical trials adopting IL-9 as a therapeutic target. Interleukin-13 This cytokine shares 70% of its sequence homology with IL-4 and despite a degree of functional redundancy due to sharing of the IL-4Ra subunit, a specific role in the development of asthma has been demonstrated in murine models [132±134]. Notably IL-13 does not share the IL-4 influence on specific induction of the Th2 phenotype from naive Th0 cells. By using a soluble form of the IL13Ra chain of the IL-13 receptor, known to bind IL-13 exclusively, these groups demonstrated a significant reduction in airway hyperreactivity, airway eosinophilia and mucus hyperplasia. Curiously, when administered after the initial allergen sensitization however, this agent had no effect on serum IgE levels [133, 134]. Notably, an IL-13 blocking antibody reduced BHR independently of IL-5 . T-helper 1/T-helper 2 modulation by vaccination An indirect way of modulating Th1/Th2 balance has been to boost innate immunity by the use of vaccines, 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 preventing the development of asthma and allergic diseases. The beneficial effects of specific immunotherapy may result from increasing Th1 immune responses while the development of peptide immunotherapy may lead to a more effective and safer treatment. Mycobacterium species The potential benefit of Bacille Calmette-Guerin (BCG) vaccination in atopic diseases was first raised by a study in Japanese school children, in whom an association between BCG vaccination and a diminished incidence of atopy and allergic disease was observed, suggesting a role for early mycobacterial exposure in the subsequent development of atopic responsiveness . Experimental models have supported this concept using the nonpathogenic mycobacterial products of Mycobacterium bovis and Mycobacterium vaccae. Mice vaccinated with BCG prior to allergen sensitization had increased IFN-c and decreased IL-4 and IL-5 expression along with reduced levels of airway T-cells and eosinophilia and bronchial reactivity [136, 137]. Mycobacterium vaccae is ubiquitously present in the soil as a saprophyte and can evoke a strong production of IFN-c. A suppression of Th2 activation has been demonstrated using heat-killed M. vaccae in mice [138±140], and these studies have opened the way to clinical studies in human asthma . Deoxyribonucleic acid vaccines: cytosine-guanosine repeat motifs The cytosine-guanosine dinucleotide repeat, when present in a particular base context, is known as a CpG motif and has been recognized as an important procaryotic immunomodulatory effector whose role is probably that of a warning or priming agent against bacterial infection . This motif is expressed at a very low frequency in vertebrates, in a nonfunctional methylated form, and is without function. Rodent studies highlight a potential function of synthetic CpG motifs in vertebrates, where a potent effect on lymphocyte function has been found . CpG vaccination directly induces antigen-presenting cells and B-lymphocytes to release IL-12, IL-18 and tumour necrosis factor-alpha (TNF-a), effectively suppressing Th2 responses by inducing the Th1 phenotype. These CpG oligodinucleotides (ODN) are effective in preventing the development of eosinophilic airway inflammation, allergen-induced elevation of serum IgE and BHR in murine asthma models . These ODN not only effectively prevent Th2 type responses by pre-administration and co-administration with allergen-challenge but also reduce established inflammation [142±144]. Further, these motifs show a large degree of flexibility towards the dose and the route of administration, being active following transmucosal , inhaled and parenteral administration . As an added potential, a deoxyribonucleic acid (DNA) vaccine containing this motif may provide a more effective adjuvant than the widely used, alum. The clinical efficacy and safety of such CpG motifs needs evaluation  and such trials are currently being considered. Potential drawbacks of manipulating T-helper 1/T-helper 2 balance The mounting evidence for Th2 cell activation in allergic asthma suggests downregulation or ablation of the Th2 response to be an appropriate aim in treating asthma and allergic disorders. However, the consequences of such a strategy are as yet unclear. Two potential outcomes need consideration; first the consequences of Th2 ablation and second, those of allowing a Th1 dominated immune activation. Parasite exposure is known to induce Th2 type cytokine synthesis [147±149], and mice with Th2 cytokine deletions demonstrate impaired parasite clearance [150±152]. Although early Th1 expression may be a more important response to acute phase parasitaemia, subsequent parasite clearance appears to be more Th2 cytokine dependent [152, 153]. Autoimmune diseases including experimental allergic encephalomyelitis, myasthenia gravis and hypothyroidism may be derived from an exuberance of Th1 activity [154±156], raising the concern that increased Th1 cell activity may induce these autoimmune diseases. Several reports note associations between IFN-c, IFN-b and IL-12 exposure and the development or exacerbation of autoimmune processes [154, 157±159]. Th1 cells may contribute to asthmatic inflammation. In adoptive transfer studies of Th1 cells in mice, a prolonged enhancement of cell-mediated immune responses was observed , and passive transfer of Th1 cells with Th2 cells induces enhanced NEW APPROACHES TO ASTHMA THERAPY tissue eosinophilia compared to when Th2 cells alone were administered [161±163]. However, in other studies in mice and rats, transfer of allergen-specific Th1 cells  inhibited the effects of allergen-specific Th2 cells, namely BAL eosinophilia and BHR in rats, without itself inducing any inflammation . Allergen-specific Tc1 cells also diminished allergen-induced BHR and BAL eosinophilia . Nevertheless, IFN-c produced by Th1 cells may activate epithelial cells to induce the production of pro-inflammatory cytokines and chemokines [166± 168]. Two further observations appear important to the contribution of Th1 type processes to asthma. First, the prominent expression of the Th1 dependent transcription factor, STAT1, in stable asthma  and second, the association between respiratory viral infections and asthma [170, 171]. Viral infections induce Th1 expression with upregulation of TNF-a and VCAM1 expression supporting a role for Th1 mediated processes in asthma exacerbation [169, 172]. Therefore, caution is necessary in studies that aim towards altering the balance of the Th1/Th2 in the treatment of asthma and allergic diseases. Specific immunotherapy Specific immunotherapy (SIT) is a treatment aimed at the induction of specific unresponsiveness, or anergy, in peripheral T-cells to peptide epitopes. Peripheral T-cells following SIT are characterized by reduced cytokine production and attenuated proliferative responses to specific allergens . This process is initiated by an autocrine effect of IL-10 produced by antigen-specific Tcells [174±176]. IL-10 induces anergy by inhibition of CD28-costimulatory molecule signalling [177, 178] and also by anti-inflammatory effects on basophils, mast cells and eosinophils . There are reduced levels of IL-4 and IL-5 and increased IFN-c production, indicating a shift of the T-cells towards an increased Th1 response at the expense of Th2 responses [179, 180]. This treatment has been successfully used in asthma and allergic rhinitis and for venom allergy [181, 182], but is usually most useful in subjects allergic to single allergens such as grass pollens, and in subjects with mild forms of allergic diseases [183, 184]. Recently, cloning of epitopes of allergens has provided a higher level of epitope purity than previously available, and there has been a greater understanding of the specific mechanisms of tolerance induction. These factors may lead to a wider application of SIT in atopic disease, using better characterized peptides which are more effective but with a lesser risk of side-effects, in particular anaphylaxis. 1165 specific T-lymphocytes. Such constructs have been demonstrated to induce IgE-independent late-phase bronchoconstrictor responses in cat-sensitive mild asthmatics . Ex vivo studies subsequently demonstrated a dose-dependent reduction in T-cell IL-4 production following cat peptide, not observed following whole cat hair extract . In vitro cat-peptide studies also showed a reduction in IL-2, IL-4, IL-5 and IFN-c release from peripheral blood T-cells and a reduction in CD40 ligand (CD40L) expression, resulting in a diminished ability of these cells to give help to B-cells for the production of IgE [187, 188]. In a six-week trial of cat peptide immunotherapy in asthma, a reduction in IL-4 levels was again observed and was accompanied by an improvement in the provocative dose causing a 20% fall in (PD20) forced expiratory volume in one second (FEV1) , although there was no effect on IFN-c or IgE, suggesting the possible induction of a Th2 to Th0 change. The possibility of selective induction of clonal anergy is invited by this approach and phase I and II studies of cat peptide immunotherapy are currently being launched. Anti-immunoglobulin-E antibody Studies of the recombinant human anti-IgE antibody (rhu MAb-E25, Xolair) are now in phase IV in asthma and allergic rhinitis. E25 binds to free IgE at the high affinity receptor-binding site (FceR1), preventing the crosslinking of IgE bound to effector mast cells and basophils. This strategy ensures that the antibody is nonanaphylactogenic. The antibody may be delivered by subcutaneous injection on a 2±4 weekly schedule, dosing based on weight and serum IgE levels. Tolerance and adverse effect profiles are thus far acceptable with no reports of serum sickness, hypersensitivity reaction or the development of anti-E25 antibodies. E25 induces a dose-dependent reduction in serum free IgE associated with a reduction in basophil FceR1 expression and histamine release [189, 190]. A significant improvement in both early  and late phase  bronchoconstrictor responses to allergenchallenge and a reduction in methacholine-induced hyperresponsiveness, with a reduction in sputum eosinophil numbers have been reported following E25 administration. Results of several clinical trials show subjective improvement and significant effects on corticosteroid reduction in moderate to severe asthma , and allergic rhinitis . This treatment should become available in the near future, and its initial role 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 of T-cell activation independent of IgE-mediated inflammatory processes. Synthetic MHC-restricted oligopeptides mimicking recognized epitopes of the major cat allergen dI (Fel dI), modified by single amino acid substitution have been produced and are noted to have variable effects on T-cell proliferation and cytokine production by Fel dI Inhibition of eosinophil activation and chemoattraction IL-5 is involved in the maturation and mobilization of eosinophils and cooperates with chemoattractant cytokines (chemokines) in chemoattraction and tissue activation of eosinophils. 1166 R.G. STIRLING, K.F. CHUNG Chemokine receptor antagonists Chemokines are inducible pro-inflammatory proteins whose key function is leukocyte chemoattraction and activation. Three families are currently described, distinguished on a structural basis into C, CC and CXC families . Chemokine effects are mediated by chemokine receptors and differential cell receptor profiles allow cellspecific attraction [196±198]. Thus, the CC chemokine eotaxin has an eosinophil selective role in eosinophil recruitment due to selective expression of the eotaxin receptor CCR3 on the eosinophil [199, 200]. Chemokines are however, also involved in the recruitment of monocytes, dendritic cells, T-lymphocytes, NK cells, Blymphocytes and basophils. Antagonism of chemokine receptor signalling has been accomplished by modification of endogenous agents to create high-affinity receptor antagonists. These agents, which include met-regulated upon activation normal T-cell expressed and secreted (RANTES) and met-Ckb7 [201± 203], are potent CCR3 specific antagonists which inhibit CCR3 receptor signalling and consequent eosinophil migration. To date several small-molecular weight CCR3 antagonists have been developed and await clinical evaluation. One potential strategy combines the inhibition of both IL-5 and eotaxin, a double-pronged approach that would reduce both mobilization, chemoattraction and activation of eosinophils more effectively given the cooperation between IL-5 and eotaxin demonstrated in in vivo models [204, 205]. Such a combined approach may lead to clinical improvement. Blocking adhesion molecules and integrins Eosinophil migration to sites of allergic inflammation is specifically mediated by interactions between endothelium, epithelium and eosinophil adhesion molecules including the a4-integrin very late antigen-4 (VLA-4) . a4-Integrins are also implicated in human lymphocyte coactivation . An a4-integrin monoclonal antibody inhibited inflammation and BHR in a sensitized mouse model , and also eosinophilic inflammation and BHR induced by eotaxin in IL-5 transgenic mice . Peptido-mimetic small molecule VLA-4 inhibitors, administered prior to allergen challenge, also prevent increases in VLA-4+ leukocytes (eosinophils, lymphocytes and macrophages) in lung tissue while significantly inhibiting early and late phase allergic responses . Similar approaches inhibiting interactions between leukocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) have also proven effective in blocking eosinophil adhesion and transmigration . Inhibition of pro-inflammatory cytokines and pathways Several pro-inflammatory cytokines such as TNF-a  and IL-1b  have been shown to be overexpressed in asthma, and exposure to environmental endotoxin such as that present in house dust  may lead to further activation of a panel of such pro- inflammatory cytokines. Activation of the transcription factor, NFkB, which can regulate the production of a range of pro-inflammatory cytokines including IL-1, IL6, IL-8 and TNF-a but also adhesion molecules ICAM-1 and VCAM-1, has been shown in the airway epithelium and macrophages of patients with mild asthma . Similarly, increased expression of the transcription factor, AP-1, has also been reported . Tumour necrosis factor-a TNF-a increases airway responsiveness in BrownNorway rats  and in humans, together with an increase in sputum neutrophils . TNF-a also potently stimulates airway epithelial cells to produce cytokines including regulated upon activation, normal Tcell expressed and secreted (RANTES), IL-8 and GMCSF, and increases the expression of the adhesion molecule, ICAM-1. TNF-a has a further synergistic effect with IL-4 and IFN-c to increase VCAM-1 expression on endothelial cells [217, 218]. These factors indicate that TNF-a may be an important mediator in initiating chronic inflammation in the airways. Several approaches to inhibition of TNF-a synthesis and effects are now under investigation in asthma, including monoclonal antibodies to TNF-a and soluble TNF-a receptors. An anti-TNF-a antibody has retarded the progression of severe rheumatoid arthritis with significant clinical amelioration , but has not been tried in asthma. This antibody may have 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 potential anti-inflammatory compounds that may be used in asthma, and are currently under development. Recent attention has also been focused on intracellular signalling cascades such as inhibitors of p38 MAP kinase which inhibit the synthesis of pro-inflammatory cytokines in vivo [220, 221]. In this mouse model, inhibition of p38 kinase activity markedly reduced cytokine-associated inflammation and inhibited lipopolysaccharid (LPS)induced TNF-a production while allergen-induced airway eosinophilia was all but abolished. Interleukin-1 IL-1 co-induces CD4+ T-cell proliferation and IL-2 secretion following interaction of T-cells with antigen presenting cells, and is an important growth factor for antigen primed Th2 cells . IL-1 also potently induces the synthesis and release of multiple pro-inflammatory 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 been isolated from multiple tissues including alveolar macrophages , and inhibits most of the activities of IL-1 . IL1-Ra blocks Th2 but not Th1 clonal proliferation in vitro and in the guinea pig reduces allergen induced BHR and pulmonary eosinophilia . Manipulation of this endogenous control mechanism may therefore, impact on asthma and needs to be evaluated in the clinical setting. 1167 NEW APPROACHES TO ASTHMA THERAPY Interleukin-10 Although IL-10 is a Th2-derived cytokine, it has antiinflammatory properties that could be used to control asthmatic inflammation. IL-10 is derived largely from mononuclear cells, alveolar macrophages and both naive and committed T-cells. It reduces MHC and costimulatory molecule expression, reduces pro-inflammatory cytokine release and increases IL-1Ra expression [4, 226]. T-cell and macrophage IL-10 synthesis is significantly reduced in asthmatic subjects compared with nonasthmatics [227, 228] but IL-10 expression in alveolar macrophages is increased by corticosteroid therapy , suggesting in vivo relevance of this cytokine. A polymorphism in the promoter sequence of IL-10 has been associated with attenuated IL-10 expression and has been identified at increased frequency in severe asthma , while promoter polymorphisms have been demonstrated in asthma probands and are associated with elevation of IgE levels . This latter observation is of particular interest given a proposed suppressive effect of IL-10 on IL-4-induced IgE isotype switching by B-cells . IL10 administration to mice reduces airway eosinophilia and allergen-induced late responses . When given to normal volunteers, IL-10 reduced circulating CD3, CD4 and CD8 lymphocyte numbers and the proliferative response and reduced TNF-a and IL-1 production . Further studies in human asthma are awaited. Use of antisense oligonucleotides Oligonucleotide sequences forming a complementary copy of normal, or sense, messenger (mRNA) are able to bind to specific mRNA, and this dimer formation prevents mRNA translation and protein synthesis. The effectiveness of this technique was demonstrated in a rabbit model of asthma where aerosol inhalation of adenosine-A1 receptor antisense protected the animals from subsequent challenge with either adenosine or dust-mite allergen, with effects persisting up to 7 days [235, 236]. In a model in which the late allergic reaction could be transferred in the rat by ovalbumin sensitized T-cells , IL-4 antisense-treated T-cells caused a significant reduction in the late allergic reaction, airway eosinophilia, and IL-4 and IL-5 expression. However, when IL-5 antisense treated T-cells were administered, only BAL IL-5 expression was reduced suggesting a key role for IL-4 in CD4+ T-cell mediated late allergic responses. In murine models, an IL-5 antisense oligonucleotide reduced IL-5 protein levels, airway eosinophilia and allergen-induced late allergic responses . Similar approaches targetting CCR3 and the cytokine receptor a-subunits of IL-4, IL-13 and GM-CSF have also been reported with promising results [238± 240]. Antisense based therapies are currently in clinical use in the treatment of CMV retinitis and for advanced cancer [241±243] and are undergoing further safety and tolerability evaluation. where mucosal transfer of IL-12  and IFN-c  significantly reduced Th2 cytokine expression and inhibited BHR. Transfer of the glucocorticoid receptor to the epithelial cell line A549, lead to repression of transcription factor mediated gene transcription and may provide a therapeutic approach in steroid-resistant asthma . Gene transfer to the lungs in man is still in a developmental phase, and the efficiency of gene transfer using liposomal transfer technology is thus far suboptimal, as judged from the experience of the transfer of the cystic fibrosis transmembrane regulator DNA to patients with cystic fibrosis . Use of the more efficient transfer system, the adenovirus vector, may be detrimental in inducing lung damage and inflammation, and is no longer a viable option in man. The potential genes that may be delivered to the airway epithelium of patients with asthma for therapeutic effects are numerous. Candidates for gene transfer might include Th1 cytokines IL-12 or IFN-c, or of the anti-inflammatory cytokine, IL-10, or of IL-1Ra, or of the corticosteroid receptor may be contemplated, but gene therapy approaches for the treatment of asthma remain in the distant future. Conclusion Recent advances in the techniques for the synthesis and manufacture of monoclonal antibodies, synthetic peptides and peptidomimetic small molecules has increased the potential for the creation of specific inhibitors of immune processes in allergic inflammation. These agents have enabled specific intervention in the inflammatory cascade and allow a clearer understanding of the roles of specific agents in this cascade, while at the same time providing the possibility of therapeutic intervention in asthma. In the first instance, it is likely that these strategies will be aimed at those with severe difficult-to-treat asthma as it is here that the failings of current therapies are most evident. Whether new therapies may provide remission of disease with shortterm treatment cannot be predicted from the current understanding of asthma. While preliminary data for many of these agents appears most promising, these agents will have to endure rigorous evaluation of efficacy, long-term safety and cost-effectiveness. Several agents targeted to specific immunological or cytokine pathways may become available and may be more effective in certain types of asthma. Genetic pharmacological profiling may be needed to identify the best responders to particular types of specific drugs . The future of asthma therapy looks bright, but identifying targets is only a first step. The development of these new approaches is very exciting but the task remains daunting. References 1. Use of gene therapy 2. The technique of gene therapy entails the specific augmentation of gene expression by transfer of single genes using viral vectors or liposome transfer [244±247]. Gene therapy has been explored in asthma models in mice 3. Azzawi M, Bradley B, Jeffery PK, et al. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am Rev Respir Dis 1990; 142: 1407±1413. Bousquet J, Chanez P, Campbell AM, Vignola AM, Godard P. Cellular inflammation in asthma. Clin Exp Allergy 1995; 25: (Suppl. 2), 39±42. Robinson DS, Hamid Q, Ying S, et al. Predominant Th2like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992; 326: 298±304. 1168 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. R.G. STIRLING, K.F. CHUNG Chung KF, Barnes PJ. Cytokines in asthma. Thorax 1999; 54: 825±857. Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000; 161: 1720±1745. Kips JC, Pauwels RA. Airway wall remodelling: does it occur and what does it mean? Clin Exp Allergy 1999; 29: 1457±1466. Vignola AM, Chanez P, Siena L, Chiappara G, Bonsignore G, Bousquet J. Airways remodelling in asthma. Pulm Pharmacol Ther 1998; 11: 359±367. Wilson JW. What causes airway remodelling in asthma? Clin Exp Allergy 1998; 28: 534±536. Redington AE, Howarth PH. Airway wall remodelling in asthma. Thorax 1997; 52: 310±312. Holgate ST, Lackie PM, Davies DE, Roche WR, Walls AF. The bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma. Clin Exp Allergy 1999; 29: (Suppl. 2), 90±95. Barnes PJ. Efficacy of inhaled corticosteroids in asthma. J Allergy Clin Immunol 1998; 102: 531±538. Barnes PJ, Pedersen S, Busse WW. Efficacy and safety of inhaled corticosteroids. New developments. Am J Respir Crit Care Med 1998; 157: S1±S53. Chung KF, Godard P, Adelroth E, et al. Difficult/therapyresistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. ERS Task Force on Difficult/Therapy-Resistant Asthma. European Respiratory Society (see comments). Eur Respir J 1999; 13: 1198±1208. Chung KF. Management of difficult asthma. Br J Hosp Med 1994; 51: 80±81. Leung DY, Spahn JD, Szefler SJ. Immunologic basis and management of steroid-resistant asthma. Allergy Asthma Proc 1999; 20: 9±14. Leung DY, de Castro M, Szefler SJ, Chrousos GP. Mechanisms of glucocorticoid-resistant asthma. Ann NY Acad Sci 1998; 840: 735±746. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 1998; 351: 1225±1232. Humbert M, Menz G, Ying S, et al. The immunopathology of extrinsic (atopic) and intrinsic (non-atopic) asthma: more similarities than differences. Immunol Today 1999; 20: 528±533. Green JM. The B7/CD28/CTLA4 T-cell activation pathway. Implications for inflammatory lung disease. Am J Respir Cell Mol Biol 2000; 22: 261±264. Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature 1992; 356: 607±609. Walunas TL, Lenschow DJ, Bakker CY, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994; 1: 405±413. Oosterwegel MA, Mandelbrot DA, Boyd SD, et al. The role of CTLA-4 in regulating Th2 differentiation. J Immunol 1999; 163: 2634±2639. Prescott SL, Macaubas C, Smallacombe T, Holt BJ, Sly PD, Holt PG. Development of allergen-specific T-cell memory in atopic and normal children. Lancet 1999; 353: 196±200. Rabinovitch A, Suarez-Pinzon WL. Cytokines and their roles in pancreatic islet beta-cell destruction and insulin- 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. dependent diabetes mellitus. Biochem Pharmacol 1998; 55: 1139±1149. Hirai H, Kaino Y, Ito T, Kida K. Analysis of cytokine mRNA expression in pancreatic islets of nonobese diabetic mice. J Pediatr Endocrinol Metab 2000; 13: 91±98. Gallo P, Piccinno MG, Tavolato B, Siden A. A longitudinal study on IL-2, sIL-2R, IL-4 and IFN-gamma in multiple sclerosis CSF and serum. J Neurol Sci 1991; 101: 227±232. Olsson T. Cytokine-producing cells in experimental autoimmune encephalomyelitis and multiple sclerosis. Neurology 1995; 45: (Suppl. 6), S11±S15. Del Prete GF, De Carli M, D'Elios MM, et al. Allergen exposure induces the activation of allergen-specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders. Eur J Immunol 1993; 23: 1445±1449. Prescott SL, Macaubas C, Holt BJ, et al. Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the Th2 cytokine profile. J Immunol 1998; 160: 4730±4737. Doffinger R, Jouanguy E, Altare F, et al. Inheritable defects in interleukin-12- and interferon-gamma-mediated immunity and the TH1/TH2 paradigm in man. Allergy 1999; 54: 409±412. Hertzen LC. The hygiene hypothesis in the development of atopy and asthma: still a matter of controversy? QJM 1998; 91: 767±771. Matricardi PM, Rosmini F, Rapicetta M, Gasbarrini G, Stroffolini T. Atopy, hygiene, and anthroposophic lifestyle. San Marino Study Group. Lancet 1999; 354: 430. Gerrard JW, Geddes CA, Reggin PL, Gerrard CD, Horne S. Serum IgE levels in white and metis communities in Saskatchewan. Ann Allergy 1976; 37: 91±100. Shirakawa T, Enomoto T, Shimazu S, Hopkin JM. The inverse association between tuberculin responses and atopic disorder. Science 1997; 275: 77±79. Gustafsson D, Andersson K, Fagerlund I, Kjellman NI. Significance of indoor environment for the development of allergic symptoms in children followed up to 18 months of age. Allergy 1996; 51: 789±795. van Halteren AG, van der Cammen MJ, Cooper D, Savelkoul HF, Kraal G, Holt PG. Regulation of antigenspecific IgE, IgG1, and mast cell responses to ingested allergen by mucosal tolerance induction. J Immunol 1997; 159: 3009±3015. Holt PG. Primary allergic sensitization to environmental antigens: perinatal T cell priming as a determinant of responder phenotype in adulthood. J Exp Med 1996; 183: 1297±1301. Bonham CA, Thomson AW. Immunosuppressants. In: Kay AB, ed. Allergy and Allergic Disease. London, Blackwell Science, 36, 1997; pp. 1512±1542. Rolfe FG, Valentine JE, Sewell WA. Cyclosporin A and FK506 reduce interleukin-5 mRNA abundance by inhibiting gene transcription. Am J Respir Cell Mol Biol 1997; 17: 243±250. Mori A, Suko M, Nishizaki Y, et al. IL-5 production by CD4+ T cells of asthmatic patients is suppressed by glucocorticoids and the immunosuppressants FK506 and cyclosporin A. Int Immunol 1995; 7: 449±457. Sperr WR, Agis H, Semper H, et al. Inhibition of allergen-induced histamine release from human basophils by cyclosporine A and FK-506. Int Arch Allergy Immunol 1997; 114: 68±73. Sihra BS, Kon OM, Durham SR, Walker S, Barnes NC, NEW APPROACHES TO ASTHMA THERAPY 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. Kay AB. Effect of cyclosporin A on the allergen-induced late asthmatic reaction. Thorax 1997; 52: 447±452. Alexander AG, Barnes NC, Kay AB. Trial of cyclosporin in corticosteroid-dependent chronic severe asthma. Lancet 1992; 339: 324±328. Lock SH, Kay AB, Barnes NC. Double-blind, placebocontrolled study of cyclosporin A as a corticosteroidsparing agent in corticosteroid-dependent asthma. Am J Respir Crit Care Med 1996; 153: 509±514. Davies H, Olson L, Gibson P. Methotrexate as a steroid sparing agent for asthma in adults. Cochrane Database Syst Rev 2000; 2: CD000391. Hedman J, Seideman P, Albertioni F, Stenius-Aarniala B. Controlled trial of methotrexate in patients with severe chronic asthma. Eur J Clin Pharmacol 1996; 49: 347±349. Marin MG. Low-dose methotrexate spares steroid usage in steroid-dependent asthmatic patients: a meta-analysis (see comments). Chest 1997; 112: 29±33. Huang TJ, Newton R, Haddad EB, Chung KF. Differential regulation of cytokine expression after allergen exposure of sensitized rats by cyclosporin A and corticosteroids: relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol 1999; 104: 644±652. Asano K, Mizutani T, Shimane T, Hisano M, Hisamitsu T, Suzaki H. The inhibitory effect of anti-allergic agent suplatast tosilate (IPD- 1151T) on methacholine- and allergen-induced bronchoconstriction in sensitized mice. Mediators Inflamm 2000; 9: 77±84. Zhao GD, Yokoyama A, Kohno N, Sakai K, Hamada H, Hiwada K. Effect of suplatast tosilate (IPD-1151T) on a mouse model of asthma: inhibition of eosinophilic inflammation and bronchial hyperresponsiveness. Int Arch Allergy Immunol 2000; 121: 116±122. Tamaoki J, Kondo M, Sakai N, et al. Effect of suplatast tosilate, a TH2 cytokine inhibitor, on steroid-dependent asthma: a double blind randomised study. Lancet 2000; 356: 273±278. Gavett SH, Chen X, Finkelman F, Wills-Karp M. Depletion of murine CD4+ T lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia. Am J Respir Cell Mol Biol 1994; 10: 587±593. Huang TJ, MacAry PA, Kemeny DM, Chung KF. Effect of CD8+ T-cell depletion on bronchial hyperresponsiveness and inflammation in sensitized and allergen-exposed Brown-Norway rats. Immunology 1999; 96: 416±423. Kon OM, Sihra BS, Compton CH, Leonard TB, Kay AB, Barnes NC. Randomised, dose-ranging, placebo-controlled study of chimeric antibody to CD4 (keliximab) in chronic severe asthma. Lancet 1998; 352: 1109±1113. Lopez E, Racadot E, Bataillard M, Berger E, Rumbach L. Interferon gamma, IL2, IL4, IL10 and TNFalpha secretions in multiple sclerosis patients treated with an anti-CD4 monoclonal antibody. Autoimmunity 1999; 29: 87±92. van Oosten BW, Lai M, Hodgkinson S, et al. Treatment of multiple sclerosis with the monoclonal anti-CD4 antibody cM-T412: results of a randomized, double-blind, placebocontrolled, MR- monitored phase II trial. Neurology 1997; 49: 351±357. Breedveld FC. Monoclonal antibodies to CD4. Rheum Dis Clin North Am 1998; 24: 567±578. Dearman RJ, Moussavi A, Kemeny DM, Kimber I. Contribution of CD4+ and CD8+ T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory chemical allergens. Immunology 1996; 89: 502±510. Kemeny DM, Noble A, Holmes BJ, Diaz-Sanchez D, Lee 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 1169 TH. The role of CD8+ T cells in immunoglobulin E regulation. Allergy 1995; 50: (Suppl. 25), 9±14. Vukmanovic-Stejic M, Vyas B, Gorak-Stolinska P, Noble A, Kemeny DM. Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes. Blood 2000; 95: 231±240. Mackall CL. T-cell immunodeficiency following cytotoxic antineoplastic therapy: a review. Stem Cells 2000; 18: 10±18. Duse AG. Nosocomial infections in HIV-infected/AIDS patients. J Hosp Infect 1999; 43: (Suppl), S191±S201. Laurence J. T-cell subsets in health, infectious disease, and idiopathic CD4+ T lymphocytopenia. Ann Intern Med 1993; 119: 55±62. Schulze-Koops H, Davis LS, Haverty TP, Wacholtz MC, Lipsky PE. Reduction of Th1 cell activity in the peripheral circulation of patients with rheumatoid arthritis after treatment with a non-depleting humanized monoclonal antibody to CD4. J Rheumatol 1998; 25: 2065± 2076. Holt PG, Thomas JA. Steroids inhibit uptake and/or processing but not presentation of antigen by airway dendritic cells. Immunology 1997; 91: 145±150. Van Oosterhout AJ, Hofstra CL, Shields R, et al. Murine CTLA4-IgG treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma. Am J Respir Cell Mol Biol 1997; 17: 386±392. Krinzman SJ, De Sanctis GT, Cernadas M, et al. Inhibition of T cell costimulation abrogates airway hyperresponsiveness in a murine model. J Clin Invest 1996; 98: 2693±2699. Haczku A, Takeda K, Redai I, et al. Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice. Am J Respir Crit Care Med 1999; 159: 1638±1643. Koning H, Neijens HJ, Baert MR, Oranje AP, Savelkoul HF. T cell subsets and cytokines in allergic and nonallergic children. I. Analysis of IL-4, IFN-gamma and IL13 mRNA expression and protein production. Cytokine 1997; 9: 416±426. Nakajima H, Iwamoto I, Yoshida S. Aerosolized recombinant interferon-gamma prevents antigen-induced eosinophil recruitment in mouse trachea. Am Rev Respir Dis 1993; 148: 1102±1104. Lack G, Bradley KL, Hamelmann E, et al. Nebulized IFN-gamma inhibits the development of secondary allergic responses in mice. J Immunol 1996; 157: 1432±1439. Huang TJ, MacAry PA, Wilke T, Kemeny DM, Chung KF. Inhibitory effects of endogenous and exogenous interferon-gamma on bronchial hyperresponsiveness, allergic inflammation and T-helper 2 cytokines in Brown-Norway rats. Immunology 1999; 98: 280±288. Iwamoto I, Nakajima H, Endo H, Yoshida S. Interferon gamma regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells. J Exp Med 1993; 177: 573±576. Jaffe HA, Buhl R, Mastrangeli A, et al. Organ specific cytokine therapy. Local activation of mononuclear phagocytes by delivery of an aerosol of recombinant interferon-gamma to the human lung. J Clin Invest 1991; 88: 297±302. Martin RJ, Boguniewicz M, Henson JE, et al. The effects of inhaled interferon gamma in normal human airways. Am Rev Respir Dis 1993; 148: 1677±1682. Boguniewicz M, Schneider LC, Milgrom H, et al. Treatment of steroid-dependent asthma with recombinant interferon-gamma. Clin Exp Allergy 1993; 23: 785±790. 1170 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. R.G. STIRLING, K.F. CHUNG Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 1995; 154: 5071±5079. Trinchieri G, Gerosa F. Immunoregulation by interleukin12. J Leukoc Biol 1996; 59: 505±511. Kips JC, Brusselle GJ, Joos GF, et al. Interleukin-12 inhibits antigen-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med 1996; 153: 535±539. Lee Y, Fu C, Chiang B. Administration of interleukin-12 exerts a therapeutic instead of a long-term preventive effect on mite der p I allergen-induced animal model of airway inflammation. Immunology 1999; 97: 232±240. Lee YL, Fu CL, Ye YL, Chiang BL. Administration of interleukin-12 prevents mite Der p 1 allergen-IgE antibody production and airway eosinophil infiltration in an animal model of airway inflammation. Scand J Immunol 1999; 49: 229±236. Hofstra CL, Van AI, Hofman G, Kool M, Nijkamp FP, Van Oosterhout AJ. Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels. J Immunol 1998; 161: 5054±5060. Naseer T, Minshall EM, Leung DY, et al. Expression of IL-12 and IL-13 mRNA in asthma and their modulation in response to steroid therapy. Am J Respir Crit Care Med 1997; 155: 845±851. Hamid QA, Schotman E, Jacobson MR, Walker SM, Durham SR. Increases in IL-12 messenger RNA+ cells accompany inhibition of allergen-induced late skin responses after successful grass pollen immunotherapy. J Allergy Clin Immunol 1997; 99: 254±260. O'Connnor B, Hansel T, Holgate S, Barnes P. Effects of recombinant human IL-12 on allergen induced airway inflammation and the late response. Am J Respir Crit Care Med 2000; 161: A592. Song K, Chang Y, Prud'homme GJ. Regulation of Thelper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Ther 2000; 7: 481±492. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997; 3: 409±417. Yoshimoto T, Tsutsui H, Tominaga K, et al. IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA 1999; 96: 13962±13966. Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation. J Exp Med 1998; 187: 2103±2108. Fehniger TA, Shah MH, Turner MJ, et al. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J Immunol 1999; 162: 4511±4520. Micallef MJ, Tanimoto T, Kohno K, Ikeda M, Ikegami H, Kurimoto M. Augmentation of in vitro interleukin 10 production after in vivo administration of interleukin 18 is activated macrophage-dependent and is probably not involved in the antitumor effects of interleukin 18. Anticancer Res 1998; 18: 4267±4274. Hansen G, Yeung VP, Berry G, Umetsu DT, DeKruyff 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. RH. Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T cells and IL-18. J Immunol 2000; 164: 223±230. Yeung VP, Gieni RS, Umetsu DT, DeKruyff RH. Heatkilled Listeria monocytogenes as an adjuvant converts established murine Th2-dominated immune responses into Th1-dominated responses. J Immunol 1998; 161: 4146± 4152. Kumano K, Nakao A, Nakajima H, et al. Interleukin-18 enhances antigen-induced eosinophil recruitment into the mouse airways. Am J Respir Crit Care Med 1999; 160: 873±878. Wild JS, Sigounas A, Sur N, et al. IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma. J Immunol 2000; 164: 2701±2710. Maggi E, Parronchi P, Manetti R, et al. Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones. J Immunol 1992; 148: 2142±2147. Seder RA, Paul WE, Davis MM, Fazekas dS. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J Exp Med 1992; 176: 1091±1098. Lebman DA, Coffman RL. Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures. J Exp Med 1988; 168: 853±862. Pawankar R, Okuda M, Yssel H, Okumura K, Ra C. Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells. J Clin Invest 1997; 99: 1492±1499. Vercelli D, Jabara HH, Lee BW, Woodland N, Geha RS, Leung DY. Human recombinant interleukin 4 induces Fc epsilon R2/CD23 on normal human monocytes. J Exp Med 1988; 167: 1406±1416. Schleimer RP, Sterbinsky SA, Kaiser J, et al. IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1. J Immunol 1992; 148: 1086±1092. Zhou CY, Crocker IC, Koenig G, Romero FA, Townley RG. Anti-interleukin-4 inhibits immunoglobulin E production in a murine model of atopic asthma. J Asthma 1997; 34: 195±201. Tanaka H, Nagai H, Maeda Y. Effect of anti-IL-4 and antiIL-5 antibodies on allergic airway hyperresponsiveness in mice. Life Sci 1998; 62: L169±L174. Henderson WRJ, Chi EY, Maliszewski CR. Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma. J Immunol 2000; 164: 1086±1095. Borish LC, Nelson HS, Lanz MJ, et al. Interleukin-4 receptor in moderate atopic asthma. A phase I/II randomized, placebo-controlled trial. Am J Respir Crit Care Med 1999; 160: 1816±1823. Harris P, Lindell D, Fitch N, Gundel R. The IL-4 receptor antagonist (BAY 16-9996) reverses airway hyperresponsiveness in a primate model of asthma. Am J Respir Crit Care Med 2000; 159: A230. Mullings RE, Wilson SJ, Djukanovic R, et al. Increased Stat6 expression in bronchial epithelium of severe asthmatic subjects. Am J Respir Crit Care Med 2000; 63: A46. Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res 1999; 19: 75±120. NEW APPROACHES TO ASTHMA THERAPY 109. Hill S, Herlaar E, Le Cardinal A, van Heeke G, Nicklin P. Homologous human and murine antisense oligonucleotides targeting stat6. Functional effects on germline cepsilon transcript. Am J Respir Cell Mol Biol 1999; 21: 728±737. 110. Wang LH, Yang XY, Kirken RA, Resau JH, Farrar WL. Targeted disruption of stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4-driven T(H)2 cell response. Blood 2000; 95: 1249±1257. 111. Karras JG, McGraw K, McKay RA, et al. Inhibition of antigen-induced eosinophilia and late phase airway hyperresponsiveness by an IL-5 antisense oligonucleotide in mouse models of asthma. J Immunol 2000; 164: 5409± 5415. 112. Egan RW, Athwal D, Bodmer MW, et al. Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity. Arzneimittelforschung 1999; 49: 779±790. 113. Nagai H, Yamaguchi S, Inagaki N, Tsuruoka N, Hitoshi Y, Takatsu K. Effect of anti-IL-5 monoclonal antibody on allergic bronchial eosinophilia and airway hyperresponsiveness in mice. Life Sci 1993; 53: L243±L247. 114. Nagai H, Yamaguchi S, Tanaka H. The role of interleukin5 (IL-5) in allergic airway hyperresponsiveness in mice. Ann NY Acad Sci 1996; 796: 91±96. 115. Zhang J, Kuvelkar R, Murgolo NJ, et al. Mapping and characterization of the epitope(s) of Sch 55700, a humanized mAb, that inhibits human IL-5. Int Immunol 1999; 11: 1935±1944. 116. Zia-Amirhosseini P, Minthorn E, Benincosa LJ, et al. Pharmacokinetics and pharmacodynamics of SB-240563, a humanized monoclonal antibody directed to human interleukin-5, in monkeys. J Pharmacol Exp Ther 1999; 291: 1060±1067. 117. Leckie M, ten Brinke A, Lordan J, et al. IL-5 monoclonal antibody, SB-240563, single dose therapy: initial safety and activity in patients with asthma. Am J Respir Crit Care Med 1999; 159: A624. 118. Kips JC, O'Connor BJ, Langley SJ, et al. Results of a phase I trial with SCH55700, a humanised anti-IL-5 antibody, in severe persistent asthma. Am J Respir Crit Care Med 2000; 161: A505. 119. Adachi T, Choudhury BK, Stafford S, Sur S, Alam R. The differential role of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in eosinophil functions. J Immunol 2000; 165: 2198±2204. 120. Adachi T, Alam R. The mechanism of IL-5 signal transduction. Am J Physiol 1998; 275: C623±C633. 121. Adachi T, Pazdrak K, Stafford S, Alam R. The mapping of the Lyn kinase binding site of the common beta subunit of IL-3/granulocyte-macrophage colony-stimulating factor/IL-5 receptor. J Immunol 1999; 162: 1496±1501. 122. Nakamura Y, Christodoulopoulos P, Cameron L, et al. Upregulation of the transcription factor GATA-3 in upper airway mucosa after in vivo and in vitro allergen challenge. J Allergy Clin Immunol 2000; 105: 1146±1152. 123. Nakamura Y, Ghaffar O, Olivenstein R, et al. Gene expression of the GATA-3 transcription factor is increased in atopic asthma. J Allergy Clin Immunol 1999; 103: 215± 222. 124. Zhang DH, Yang L, Cohn L, et al. Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 1999; 11: 473±482. 125. Shimbara A, Christodoulopoulos P, Soussi-Gounni A, et al. IL-9 and its receptor in allergic and nonallergic lung 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 1171 disease: increased expression in asthma. J Allergy Clin Immunol 2000; 105: 108±115. Longphre M, Li D, Gallup M, et al. Allergen-induced IL9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 1999; 104: 1375±1382. Dong Q, Louahed J, Vink A, et al. IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice. Eur J Immunol 1999; 29: 2130±2139. McLane MP, Haczku A, van de Rijn M, et al. Interleukin9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice. Am J Respir Cell Mol Biol 1998; 19: 713±720. Levitt RC, McLane MP, MacDonald D, et al. IL-9 pathway in asthma: new therapeutic targets for allergic inflammatory disorders. J Allergy Clin Immunol 1999; 103: S485±S491. Gounni AS, Gregory B, Nutku E, et al. Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils. Blood 2000; 96: 2163±2171. McLane M, Tepper J, Weiss C, et al. Lung delivery of blocking IL9 antibody inhibits airway hyperresponsiveness, BAL eosinophilia, mucin production and serium IgE elevation to natural antigens in a murine model of asthma. Am J Respir Crit Care Med 2000; 161: A593. Grunig G, Warnock M, Wakil AE, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998; 282: 2261±2263. Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282: 2258±2261. Zhu Z, Homer RJ, Wang Z, et al. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999; 103: 779±788. Eum S, Maghni K, Hamid Q, Eidelman D, Martin J. Reduction of allergen induced airway hyperresponsiveness by anti-IL13 is independent of IL5. Am J Respir Crit Care Med 2000; 161: A591. Erb KJ, Holloway JW, Sobeck A, Moll H, Le Gros G. Infection of mice with Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) suppresses allergen-induced airway eosinophilia. J Exp Med 1998; 187: 561±569. Herz U, Gerhold K, Gruber C, et al. BCG infection suppresses allergic sensitization and development of increased airway reactivity in an animal model. J Allergy Clin Immunol 1998; 102: 867±874. Camporota L, Corkhill A, Long H, et al. Effects of intradermal injection of SRL-172 (killed Mycobacterium Vaccae suspension) on allergen induced airway responses and IL-5 generation by PBMC in asthma. Am J Respir Crit Care Med 2000; 161: A477. McMillan S, Escott K, Webber S, Foster M, Sargent C. Effect of heat-killed Mycobacterium Vaccae on murine antigen induceed airway inflammation. Am J Respir Crit Care Med 2000; 161: A477. Wang CC, Rook GA. Inhibition of an established allergic response to ovalbumin in BALB/c mice by killed Mycobacterium vaccae. Immunology 1998; 93: 307±313. Krieg AM. An innate immune defense mechanism based on the recognition of CpG motifs in microbial DNA. J Lab Clin Med 1996; 128: 128±133. Kline JN, Waldschmidt TJ, Businga TR, et al. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 1998; 160: 2555± 2559. 1172 R.G. STIRLING, K.F. CHUNG 143. Broide D, Raz E. DNA-Based immunization for asthma. Int Arch Allergy Immunol 1999; 118: 453±456. 144. Serebrisky D, Tepper A, Kattan M, Sampson H, Li X-M. Systemic or local CpG administration inhibits ongoing allergic airway inflammation. Am J Respir Crit Care Med 2000; 161: A591. 145. Shirota H, Sano K, Kikuchi T, Tamura G, Shirato K. Regulation of T-helper type 2 cell and airway eosinophilia by transmucosal coadministration of antigen and oligodeoxynucleotides containing CpG motifs. Am J Respir Cell Mol Biol 2000; 22: 176±182. 146. Askenase PW. Gee whiz: CpG DNA allergy therapy! J Allergy Clin Immunol 2000; 106: 37±40. 147. Pearce EJ, Caspar P, Grzych JM, Lewis FA, Sher A. Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J Exp Med 1991; 173: 159±166. 148. Loke P, MacDonald AS, Allen JE. Antigen-presenting cells recruited by brugia malayi induce Th2 differentiation of naive CD4(+) T cells. Eur J Immunol 2000; 30: 1127±1135. 149. Chatelain R, Varkila K, Coffman RL. IL-4 induces a Th2 response in Leishmania major-infected mice. J Immunol 1992; 148: 1182±1187. 150. Li C, Corraliza I, Langhorne J. A defect in interleukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice. Infect Immun 1999; 67: 4435±4442. 151. Mohrs M, Ledermann B, Kohler G, Dorfmuller A, Gessner A, Brombacher F. Differences between IL-4- and IL-4 receptor alpha-deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J Immunol 1999; 162: 7302±7308. 152. Bancroft AJ, McKenzie AN, Grencis RK. A critical role for IL-13 in resistance to intestinal nematode infection. J Immunol 1998; 160: 3453±3461. 153. Chen D, Copeman DB, Burnell J, Hutchinson GW. Helper T cell and antibody responses to infection of CBA mice with Babesia microti. Parasite Immunol 2000; 22: 81±88. 154. Sitaraman S, Metzger DW, Belloto RJ, Infante AJ, Wall KA. Interleukin-12 enhances clinical experimental autoimmune myasthenia gravis in susceptible but not resistant mice. J Neuroimmunol 2000; 107: 73±82. 155. Wang HB, Shi FD, Li H, van der Meide PH, Ljunggren HG, Link H. Role for Interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis. Clin Immunol 2000; 95: 156±162. 156. Drugarin D, Negru S, Koreck A, Zosin I, Cristea C. The pattern of a T(H)1 cytokine in autoimmune thyroiditis. Immunol Lett 2000; 71: 73±77. 157. McDonald N, Pender M. Autoimmune hypothyroidism associated with interferon beta-1b treatment in two patients with multiple sclerosis. Au NZ J Med 2000; 30: 278±279. 158. Tran EH, Prince EN, Owens T. IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines. J Immunol 2000; 164: 2759±2768. 159. Caturegli P, Hejazi M, Suzuki K, et al. Hypothyroidism in transgenic mice expressing IFN-gamma in the thyroid. Proc Natl Acad Sci USA 2000; 97: 1719±1724. 160. Ohta A, Sato N, Yahata T, et al. Manipulation of Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells. J Immunol Methods 1997; 209: 85±92. 161. Hansen G, Berry G, DeKruyff RH, Umetsu DT. Allergenspecific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J Clin Invest 1999; 103: 175±183. 162. Randolph DA, Stephens R, Carruthers CJ, Chaplin DD. Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation. J Clin Invest 1999; 104: 1021±1029. 163. Randolph DA, Carruthers CJ, Szabo SJ, Murphy KM, Chaplin DD. Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma. J Immunol 1999; 162: 2375±2383. 164. Huang TJ, Eynott P, Massain A, et al. Inhibitory effects of allergen-specific TH1 cells on effector functions of allergen-specific TH2 cells. J Immunol 2000; (in press). 165. Cohn L, Homer RJ, Niu N, Bottomly K. T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production. J Exp Med 1999; 190: 1309± 1318. 166. Schwiebert LM, Mooney JL, Van Horn S, Gupta A, Schleimer RP. Identification of novel inducible genes in airway epithelium. Am J Respir Cell Mol Biol 1997; 17: 106±113. 167. Stellato C, Beck LA, Gorgone GA, et al. Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids. J Immunol 1995; 155: 410±418. 168. Berkman N, Robichaud A, Krishnan VL, et al. Expression of RANTES in human airway epithelial cells: effect of corticosteroids and interleukin-4, -10 and -13. Immunology 1996; 87: 599±603. 169. Sampath D, Castro M, Look DC, Holtzman MJ. Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma. J Clin Invest 1999; 103: 1353±1361. 170. Schwarze J, Gelfand EW. The role of viruses in development or exacerbation of atopic asthma. Clin Chest Med 2000; 21: 279±287. 171. Grunberg K, Sterk PJ. Rhinovirus infections: induction and modulation of airways inflammation in asthma. Clin Exp Allergy 1999; 29: (Suppl. 2), 65±73. 172. Papi A, Johnston SL. Respiratory epithelial cell expression of vascular cell adhesion molecule-1 and its upregulation by rhinovirus infection via NF-kappaB and GATA transcription factors. J Biol Chem 1999; 274: 30041±30051. 173. Akdis CA, Blaser K. Immunologic mechanisms of specific immunotherapy. Allergy 1999; 54: (Suppl. 56), 31±32. 174. Akdis CA, Blesken T, Akdis M, Wuthrich B, Blaser K. Role of interleukin 10 in specific immunotherapy. J Clin Invest 1998; 102: 98±106. 175. Becker JC, Czerny C, Brocker EB. Maintenance of clonal anergy by endogenously produced IL-10. Int Immunol 1994; 6: 1605±1612. 176. Enk AH, Saloga J, Becker D, Madzadeh M, Knop J. Induction of hapten-specific tolerance by interleukin 10 in vivo. J Exp Med 1994; 179: 1397±1402. 177. Del Prete G, De Carli M, Almerigogna F, Giudizi MG, Biagiotti R, Romagnani S. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol 1993; 150: 353±360. 178. Punnonen J, de Waal M, van Vlasselaer P, Gauchat JF, de Vries JE. IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes. J Immunol 1993; 151: 1280±1289. 179. Jutel M, Pichler WJ, Skrbic D, Urwyler A, Dahinden C, Muller UR. Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures. J Immunol 1995; 154: 4187±4194. NEW APPROACHES TO ASTHMA THERAPY 180. Secrist H, Chelen CJ, Wen Y, Marshall JD, Umetsu DT. Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals. J Exp Med 1993; 178: 2123±2130. 181. Muller U, Akdis CA, Fricker M, et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 1998; 101: 747±754. 182. Bousquet J, Lockey RF, Malling HJ. WHO position paper. Allergen immunotherapy: therapeutic vaccines in allergic diseases. Allergy 1998; 44 (Suppl): 1±42. 183. Abramson M, Puy R, Weiner J. Immunotherapy in asthma: anupdatedsystematicreview.Allergy1999;54:1022±1041. 184. Durham SR, Walker SM, Varga EM, et al. Long-term clinical efficacy of grass-pollen immunotherapy. N Engl J Med 1999; 341: 468±475. 185. Haselden BM, Barry KA, Larche M. Immunoglobulin Eindependent major histocompatibility complex- restricted T cell peptide epitope-induced late asthmatic reactions. J Exp Med 1999; 189: 1885±1894. 186. Marcotte GV, Braun CM, Norman PS, et al. Effects of peptide therapy on ex vivo T-cell responses. J Allergy Clin Immunol 1998; 101: 506±513. 187. Pene J, Desroches A, Paradis L, et al. Immunotherapy with Fel d 1 peptides decreases IL-4 release by peripheral blood T cells of patients allergic to cats. J Allergy Clin Immunol 1998; 102: 571±578. 188. Fasler S, Aversa G, de Vries JE, Yssel H. Antagonistic peptides specifically inhibit proliferation, cytokine production, CD40L expression, and help for IgE synthesis by Der p 1- specific human T-cell clones. J Allergy Clin Immunol 1998; 101: 521±530. 189. MacGlashan DWJ, Bochner BS, Adelman DC, et al. Down-regulation of Fc(epsilon)RI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody. J Immunol 1997; 158: 1438±1445. 190. Fick R, Rohane P, Gupta N, et al. Anti-inflammatory effects of a recombinant monoclonal anti-IgE (E25) in asthma. Am J Respir Crit Care Med 2000; 161: A199. 191. Boulet LP, Chapman KR, Cote J, et al. Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response. Am J Respir Crit Care Med 1997; 155: 1835±1840. 192. Fahy JV, Fleming HE, Wong HH, et al. The effect of an anti-IgE monoclonal antibody on the early- and latephase responses to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med 1997; 155: 1828±1834. 193. Milgrom H, Fick RBJ, Su JQ, et al. Treatment of allergic asthma with monoclonal anti-IgE antibody. rhuMAb- E25 Study Group. N Engl J Med 1999; 341: 1966±1973. 194. Casale TB, Bernstein IL, Busse WW, et al. Use of an antiIgE humanized monoclonal antibody in ragweed-induced allergic rhinitis. J Allergy Clin Immunol 1997; 100: 110± 121. 195. Luster AD. Chemokines: chemotactic cytokines that mediate inflammation. N Engl J Med 1998; 338: 436±445. 196. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol 2000; 18: 217±242. 197. Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood 2000; 95: 3032±3043. 198. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000; 12: 121±127. 199. Heath H, Qin S, Rao P, et al. Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J Clin Invest 1997; 99: 178±184. 1173 200. Ponath PD, Qin S, Post TW, et al. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils (see comments). J Exp Med 1996; 183: 2437±2448. 201. Nibbs RJ, Salcedo TW, Campbell JD, et al. C-C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine-methionine swap. J Immunol 2000; 164: 1488±1497. 202. Elsner J, Petering H, Hochstetter R, et al. The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3. Eur J Immunol 1997; 27: 2892±2898. 203. Proudfoot AE, Buser R, Borlat F, et al. Amino-terminally modified RANTES analogues demonstrate differential effects on RANTES receptors. J Biol Chem 1999; 274: 32478±32485. 204. Hisada T, Hellewell PG, Teixeira MM, et al. Alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice. Am J Respir Cell Mol Biol 1999; 20: 992±1000. 205. Collins PD, Marleau S, Griffiths-Johnson DA, Jose PJ, Williams TJ. Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo. J Exp Med 1995; 182: 1169±1174. 206. Lobb RR, Abraham WM, Burkly LC, et al. Pathophysiologic role of alpha 4 integrins in the lung. Ann NY Acad Sci 1996; 796: 113±123. 207. Henderson WRJ, Chi EY, Albert RK, et al. Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J Clin Invest 1997; 100: 3083±3092. 208. Abraham WM, Ahmed A, Sielczak MW, Narita M, Arrhenius T, Elices MJ. Blockade of late-phase airway responses and airway hyperresponsiveness in allergic sheep with a small-molecule peptide inhibitor of VLA-4. Am J Respir Crit Care Med 1997; 156: 696±703. 209. Kelly TA, Jeanfavre DD, McNeil DW, et al. Cutting edge: a small molecule antagonist of LFA-1-mediated cell adhesion. J Immunol 1999; 163: 5173±5177. 210. Ying S, Robinson DS, Varney V, et al. TNF alpha mRNA expression in allergic inflammation. Clin Exp Allergy 1991; 21: 745±750. 211. Hallsworth MP, Soh CP, Lane SJ, Arm JP, Lee TH. Selective enhancement of GM-CSF, TNF-alpha, IL-1 beta and IL-8 production by monocytes and macrophages of asthmatic subjects. Eur Respir J 1994; 7: 1096±1102. 212. Michel O, Ginanni R, Duchateau J, Vertongen F, Le Bon B, Sergysels R. Domestic endotoxin exposure and clinical severity of asthma. Clin Exp Allergy 1991; 21: 441±448. 213. Hart LA, Krishnan VL, Adcock IM, Barnes PJ, Chung KF. Activation and localization of transcription factor, nuclear factor- kappaB, in asthma. Am J Respir Crit Care Med 1998; 158: 1585±1592. 214. Demoly P, Basset-Seguin N, Chanez P, et al. c-fos protooncogene expression in bronchial biopsies of asthmatics. Am J Respir Cell Mol Biol 1992; 7: 128±133. 215. Kips JC, Tavernier J, Pauwels RA. Tumor necrosis factor causes bronchial hyperresponsiveness in rats. Am Rev Respir Dis 1992; 145: 332±336. 216. Thomas PS, Yates DH, Barnes PJ. Tumor necrosis factoralpha increases airway responsiveness and sputum neutrophilia in normal human subjects. Am J Respir Crit Care Med 1995; 152: 76±80. 217. Briscoe DM, Cotran RS, Pober JS. Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. 1174 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. R.G. STIRLING, K.F. CHUNG Correlation with CD3+ T cell infiltration. J Immunol 1992; 149: 2954±2960. Thornhill MH, Wellicome SM, Mahiouz DL, Lanchbury JS, Kyan-Aung U, Haskard DO. Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and independent binding mechanisms. J Immunol 1991; 146: 592±598. Elliott MJ, Maini RN, Feldmann M, et al. Repeated therapy with monoclonal antibody to tumour necrosis factor alpha (cA2) in patients with rheumatoid arthritis. Lancet 1994; 344: 1125±1127. Badger AM, Bradbeer JN, Votta B, Lee JC, Adams JL, Griswold DE. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 1996; 279: 1453±1461. Underwood DC, Osborn RR, Kotzer CJ, et al. SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence. J Pharmacol Exp Ther 2000; 293: 281±288. Greenbaum LA, Horowitz JB, Woods A, Pasqualini T, Reich EP, Bottomly K. Autocrine growth of CD4+ T cells. Differential effects of IL-1 on helper and inflammatory T cells. J Immunol 1988; 140: 1555±1560. Galve-de RB, Nicod LP, Chicheportiche R, Lacraz S, Baumberger C, Dayer JM. Regulation of interleukin-1ra, interleukin-1 alpha, and interleukin-1 beta production by human alveolar macrophages with phorbol myristate acetate, lipopolysaccharide, and interleukin-4. Am J Respir Cell Mol Biol 1993; 8: 160±168. Hannum CH, Wilcox CJ, Arend WP, et al. Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature 1990; 343: 336±340. Watson ML, Smith D, Bourne AD, Thompson RC, Westwick J. Cytokines contribute to airway dysfunction in antigen-challenged guinea pigs: inhibition of airway hyperreactivity, pulmonary eosinophil accumulation, and tumor necrosis factor generation by pretreatment with an interleukin-1 receptor antagonist. Am J Respir Cell Mol Biol 1993; 8: 365±369. Spits H, de Waal M. Functional characterization of human IL-10. Int Arch Allergy Immunol 1992; 99: 8±15. Koning H, Neijens HJ, Baert MR, Oranje AP, Savelkoul HF. T cells subsets and cytokines in allergic and nonallergic children. II. Analysis and IL-5 and IL-10 mRNA expression and protein production. Cytokine 1997; 9: 427±436. Takanashi S, Hasegawa Y, Kanehira Y, et al. Interleukin10 level in sputum is reduced in bronchial asthma, COPD and in smokers. Eur Respir J 1999; 14: 309±314. John M, Lim S, Seybold J, et al. Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colonystimulating factor, and interferon-gamma release from alveolar macrophages in asthma. Am J Respir Crit Care Med 1998; 157: 256±262. Lim S, Crawley E, Woo P, Barnes PJ. Haplotype associated with low interleukin-10 production in patients with severe asthma. Lancet 1998; 352: 113. Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L. Interleukin-10 and transforming growth factor-beta promoter polymorphisms in allergies and asthma. Am J Respir Crit Care Med 1998; 158: 1958±1962. Jeannin P, Lecoanet S, Delneste Y, Gauchat JF, Bonnefoy JY. IgE versus IgG4 production can be differentially regulated by IL-10. J Immunol 1998; 160: 3555±3561. 233. Zuany-Amorim C, Haile S, Leduc D, et al. Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice. J Clin Invest 1995; 95: 2644± 2651. 234. Chernoff AE, Granowitz EV, Shapiro L, et al. A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses. J Immunol 1995; 154: 5492±5499. 235. Metzger WJ, Nyce JW. Oligonucleotide therapy of allergic asthma. J Allergy Clin Immunol 1999; 104: 260±266. 236. Nyce JW, Metzger WJ. DNA antisense therapy for asthma in an animal model. Nature 1997; 385: 721±725. 237. Molet S, Ramos-Barbon D, Martin JG, Hamid Q. Adoptively transferred late allergic response is inhibited by IL4, but not IL-5, antisense oligonucleotide. J Allergy Clin Immunol 1999; 104: 205±214. 238. Allakhverdi Z, Allam M, Renzi P. Inhibition of CCR3 expression by antisense phosphothioate oliginucleotides. Am J Respir Crit Care Med 2000; 161: A285. 239. Allam M, Renzi P. Antisense oligonucleotides targeting the common beta chain of GM-CSF receptor inhibits eosinophil response to GM-CSF, IL-3 and IL-5. Am J Respir Crit Care Med 2000; 161: A285. 240. Bouchaib L, Renzi P. Inhibition of IL-4a and IL-13a receptors by antisenes phosphothioate oligonucleotides suppresses IL-4-induced human IgE production and TH2 differentiation. Am J Respir Crit Care Med 2000; 161: A284. 241. de SM, Meenken CJ, van den Horn GJ. Fomivirsen - a phosphorothioate oligonucleotide for the treatment of CMV retinitis. Ocul Immunol Inflamm 1999; 7: 189±198. 242. Cunningham CC, Holmlund JT, Schiller JH, et al. A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer. Clin Cancer Res 2000; 6: 1626±1631. 243. Yuen AR, Halsey J, Fisher GA, et al. Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 1999; 5: 3357±3363. 244. Demoly P, Mathieu M, Curiel DT, Godard P, Bousquet J, Michel FB. Gene therapy strategies for asthma. Gene Ther 1997; 4: 507±516. 245. Albelda SM, Wiewrodt R, Zuckerman JB. Gene therapy for lung disease: hype or hope? Ann Intern Med 2000; 132: 649±660. 246. Ennist DL. Gene therapy for lung disease. Trends Pharmacol Sci 1999; 20: 260±266. 247. Prince HM. Gene transfer: a review of methods and applications. Pathology 1998; 30: 335±347. 248. Hogan SP, Foster PS, Tan X, Ramsay AJ. Mucosal IL-12 gene delivery inhibits allergic airways disease and restores local antiviral immunity. Eur J Immunol 1998; 28: 413±423. 249. Dow SW, Schwarze J, Heath TD, Potter TA, Gelfand EW. Systemic and local interferon gamma gene delivery to the lungs for treatment of allergen-induced airway hyperresponsiveness in mice. Hum Gene Ther 1999; 10: 1905±1914. 250. Mathieu M, Gougat C, Jaffuel D, et al. The glucocorticoid receptor gene as a candidate for gene therapy in asthma. Gene Ther 1999; 6: 245±252. 251. Alton EW, Stern M, Farley R, et al. Cationic lipidmediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebocontrolled trial. Lancet 1999; 353: 947±954. 252. Drazen JM, Yandava CN, Dube L, et al. Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment. Nat Genet 1999; 22: 168±170.