American Journal of Respiratory and Critical Care Medicine

Our understanding of asthma pathogenesis has changed dramatically with time. Records, which date back to 1500 bc, indicate that asthma was considered to be a disease caused by “spirits.” This viewpoint remained until 1678, when Thomas Willis described asthma to be due to “obstruction of bronchi by thick humors, swelling of their walls and obstruction from without.” That asthma was due to spasm of bronchial smooth muscles was first suggested by Sir John Floyer in 1698. With the advent of fiberoptic bronchoscopy in the 1970s and the use of modern molecular biology tools, it became clear that asthma was a chronic inflammatory disorder of the airways mediated by a multitude of cell types and inflammatory mediators. Mast cells and eosinophils were initially believed to play a central role in driving the airway inflammation associated with asthma; however, the emphasis has now shifted to T lymphocytes. In particular, helper T type 2 (Th2) cells (a subset of T cells) are believed to play a central role in initiating and orchestrating the asthmatic airway inflammatory response (1).

The “Th2 hypothesis for asthma” was first suggested by Mosmann in 1989 (2), who had earlier discovered the presence of two distinct subtypes of helper T cells in mice, namely, Th1 and Th2 (3). These two subclasses of helper T cells differ in their production of cytokines and are reciprocally inhibitory. The Th2 lymphocytes produce interleukin 4 (IL-4), IL-5, IL-9, and IL-13, which activate mechanisms important in defense against parasites and in allergic inflammation. Such mechanisms include IgE production, mast cell differentiation, and eosinophil growth, migration, and activation. Th1 lymphocytes produce interferon γ (IFN-γ) and IL-2, which activate mechanisms important in defense against viruses and bacteria (2). A mechanism accounting for the predominance of Th2 cells in asthmatic airway was suggested by the discovery that the context in which antigen-presenting cells process and present allergens critically influences the pattern of helper T cell differentiation. The Th2 hypothesis for asthma describes that asthma is caused by a relative increase in Th2 cellular response in combination with a decrease in Th1 (helper T type 1) response. The consequent alteration in cytokine milieu (most likely in the lung), with excess Th2 products (e.g., IL-4, IL-5, and IL-13) in concert with decreased Th1 products (e.g., IFN-γ and IL-12), is predicted to drive the asthma phenotype (4). Evidence of such a shift in the Th1/Th2 balance derives from studies of asthma in cellular and murine models, where Th cell polarization and allergen dependence of Th2 responses are most easily defined and from human studies that profile cytokine production.

Although this viewpoint has remained popular over the last decade, more recent studies suggest that the Th2 hypothesis for asthma is too simplistic (5). We now provide evidence to argue against the Th2 hypothesis for asthma.

How Are Th1 and Th2 Defined in Humans?

In the mouse, a CD4+ cell that makes IL-4 and not IFN-γ is termed Th2 and a cell that makes IFN-γ and not IL-4 is termed Th1. This nomenclature has been extended to other cell types such as CD8+ T cells (Tc1, Tc2) and natural killer cells (NK1, NK2). Although definitions of such T cell subtypes are clear in mice, this distinction is less clear in humans. T cells that produce only Th1 or Th2 cytokines do not exist in humans. Depending on the stimulus, human T cells can make both IL-4 and IFN-γ (6). Because of this, most researchers use the ratio of IFN-γ to IL-4 to define the phenotype of the T cell response. However, such definitions do not take into account the quantity of cytokine produced or the strength of the signal that was used to stimulate cytokine production (6). In vivo, cytokines have an effect locally, and therefore the quantity of cytokines made is important and not the ratio. Moreover, use of such ratios to define the Th1/Th2 phenotype of the cell can be misleading. For example: If a group of T cells produces 20 units of IFN-γ and 2 units of IL-4 (ratio, 10:1), this would be labeled as Th1 response; if these cells when stimulated with antigen produced 40 units of IFN-γ and 20 units of IL-4 (ratio, 2:1), this would still be labeled as Th1 response, even when these cells produced a 2-fold increase in Th1 cytokine and a 10-fold increase in Th2 cytokines.

Historically the ratio of IFN-γ to IL-4 has been used to define the Th phenotype. IL-5 is also considered to be a Th2-specific product; however, levels of IL-4 and IL-5 after antigenic stimulation do not necessarily correlate with each other (7, 8). Therefore using IL-5 instead of IL-4 as a Th2 product would change the Th1/ Th2 ratio significantly, and hence likely alter the Th phenotype.

Are Levels of IFN- γ (Th1) Decreased in Asthmatic Airways?

Studies that have measured levels of IFN-γ in asthmatic blood and airways have shown contradictory findings. Corrigan and Kay (9) demonstrated increased circulating blood levels of IFN-γ in patients with acute severe asthma during exacerbations, and these levels were reduced after treatment. Supernatants of resting and stimulated bronchoalveolar lavage (BAL) leukocytes from subjects with asthma have demonstrated elevated concentrations of IFN-γ (10), whereas T cell cytokine profile evaluated at the single cell level in bronchoalveolar lavage and blood in allergic asthma, using fluorescence-activated cell sorting (FACS) analysis, has clearly demonstrated significant increases in IFN-γ production, with no significant differences in IL-4 production (11). More recently, Magnan and coworkers (12) demonstrated increased IFN-γ-producing CD8+ T cells in asthmatic airways, and the levels of IFN-γ correlated with asthma severity, bronchial hyperresponsiveness, and blood eosinophilia. These observations therefore question the concept that levels of Th1 cytokines are decreased in patients with asthma.

Studies That Have Addressed Therapeutic Interventions with Th1-enhancing Activity in Animal Models of Asthma

Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma. Using an adoptive transfer system, Hansen and colleagues (13) assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)- specific T cell receptor transgenic mice and transferred into lymphocyte OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation as expected, but surprisingly, Th1 cells did not attenuate Th2 cell-induced airway hyperreactivity and inflammation in either SCID mice or OVA-immunized immunocompetent BALB/c mice. Moreover, introduction of Th1 cells worsened the underlying airway inflammation. The authors concluded that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease. On the contrary, they may cause further acute lung pathology. In a similar murine model of OVA-induced airway inflammation, Randolph and coworkers (14) demonstrated that Th1 cells predominate early in the response and Th2 cells predominate later, and that antigen-specific Th1 cells are not protective in this model of asthma, but rather potentiate the asthmatic inflammatory response.

More recently, in an ocular model of allergic inflammation in mice, it has been demonstrated that IL-12, a Th1 cytokine, was necessary for late-phase allergic inflammation, and that giving recombinant murine IL-12 further increased allergic inflammation (15).

Human Studies That Have Addressed Therapeutic Interventions with Th1-enhancing Activity or Anti-Th2 Cytokine Administration in Asthma

Two studies have addressed therapeutic interventions based on the Th2 hypothesis for asthma: (1) blocking monoclonal antibodies against IL-5, the Th2 cytokine most important in promoting the growth, attraction, and activation of eosinophils (16) and (2) administering human recombinant IL-12 to shift the helper T lymphocyte population in the airways toward Th1 cells (17). Both studies demonstrated significant decreases in blood and sputum eosinophils, suggesting that a biological response was noted, but this fall in eosinophils was not accompanied by changes in the early- or late-phase response to allergen challenge or in the allergen-induced increases in bronchial reactivity to histamine. Similar results were obtained earlier by Kips and coworkers (18), using a different anti-IL-5 monoclonal antibody, which also showed no clinical improvement despite a marked suppression of eosinophil numbers. Although it can be argued that these studies did not attempt to measure eosinophil numbers in bronchial tissue and that the sample sizes were small, they nevertheless suggest that deviating T cell responses away from Th2 or toward Th1 do not affect asthma outcome measures in humans.

Do Drugs Currently Proven to Be Effective in the Management of Asthma Affect the Th1/Th2 Balance?

Glucocorticoids, β2-adrenergic agonists, and phosphodiesterase enzyme inhibitors are the most effective drugs used in asthma treatment. These drugs have been shown to markedly improve asthma symptoms, quality of life, and lung function, as well as reduce the underlying airway inflammation associated with asthma. If the Th2 hypothesis for asthma is true, then these drugs should, intuitively, affect the Th1/Th2 imbalance and deviate the immune response toward the Th1 phenotype or away from the Th2 phenotype. At the least, these drugs should not affect the Th1/Th2 balance. However, studies do not support this viewpoint.

Glucocorticoids are the most potent anti-inflammatory drugs available in the treatment of asthma. Although they generally suppress innate immunity, their action on the cellular and humoral immune responses is more complex. The cellular immune response (mediated mainly by Th1 cells) is strongly suppressed by glucocorticoids, whereas the humoral immune response (mediated mainly by Th2 cells) is either poorly inhibited or even enhanced. A large number of studies have clearly indicated that glucocorticoids participate in guiding the differentiation of helper T cells toward the Th2 phenotype (19). The immunosuppressive effect of glucocorticoids after organ transplantation is mainly due to preferential blockade of Th1 cytokine expression and promotion of a Th2 cytokine- secreting profile (20). IL-12 is a cytokine that skews the immune response further towards the Th1 phenotype and away from the Th2 phenotype. Studies have demonstrated that glucocorticoids (1) inhibit IL-12 secretion from monocyte-macrophages and dendritic cells (21-24), (2) decrease IL-12 receptor β1- and β2-chain expression, thereby inhibiting IL-12 signaling (25), and (3) inhibit IL-12-induced STAT-4 (transcription factor that drives Th1 differentiation) phosphorylation without affecting STAT-6 (transcription factor that drives Th2 differentiation) phosphorylation (26), and thereby deviate the immune response predominantly toward the Th2 phenotype.

β2-Adrenergic agonists are potent bronchodilators used in the treatment of asthma, and in addition, possess anti-inflammatory properties. Human peripheral blood mononuclear cells when stimulated in vitro with β2-adrenergic agonists have demonstrated decreased levels of IFN-γ (Th1 cytokine) and increased levels of IL-4, IL-5, and IL-10 (Th2 cytokines), an effect that is thought to be mediated by decreasing IL-12 production (27); thereby suggesting that β2-agonists promote Th2 cytokine production. These findings are in accordance with previous reports by Fedyk and coworkers (28) and Borger and coworkers (29). Similarly, prostaglandin E2 (PGE2), a potent bronchodilator and anti-inflammatory agent, which also acts by increasing intracellular levels of cAMP (similar to β2-agonist), has also been shown to promote Th2 cytokine production in maturing human naive helper T cells by impairing the ability of dendritic cells to produce IL-12 (30).

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. Although 10 different isoforms of PDE have been identified, immune cells predominantly express the isoforms PDE3, PDE4, and to a lesser extent PDE7. Although an initial report demonstrated that PDE inhibitors suppress Th2 responses in humans (31), others have reported that selective PDE4 inhibitors as well as nonselective PDE inhibitors decrease the secretion or expression of Th1 cytokines by human mononuclear cells and favor the cytokine milieu toward the Th2 phenotype (31, 32). In animal studies, PDE inhibitors have been shown to inhibit antigen-mediated T cell proliferation and skew the T cell cytokine profile toward a Th2 phenotype by downregulating the expression or production of Th1 cytokines (33, 34) and having no effect or even augmenting the production of Th2 cytokines (35). More recently, Bielekova and coworkers (36) have demonstrated that the selective PDE4 inhibitor rolipram and the PDE3 inhibitor cilostamide produce predominantly inhibitory effects on Th1-mediated immune responses in humans, without having any effect on, or even inducing, a Th2-type immune response.

If the current paradigm for the Th2 hypothesis of asthma was true, then all the drugs used in the treatment of asthma (glucocorticoids, β2-agonists, and phosphodiesterase enzyme inhibitors), which skew the cytokine profile toward the Th2 phenotype, should invariably worsen the underlying disease state in asthma. However, the marked clinical efficacy of these drugs in asthma management suggests otherwise.

The Th2 hypothesis for asthma was based on the assumption that IgE and eosinophils play a major and crucial role in asthma pathogenesis, and that factors that regulated IgE synthesis and eosinophil numbers and activity (namely, Th2 cytokines) played a major role in driving asthma pathogenesis. Murine models of asthma supported this concept by showing that regulators of IgE and eosinophils, namely, IL-4 and IL-5, respectively, were major contributors to asthma pathogenesis. Subsequent animal studies using monoclonal antibodies against Th2 cytokines or factors that deviated T cell immune responses toward Th1 and away from Th2 showed promising results. However, these observations have not been consistent and some studies have reported contrasting observations. Extrapolating studies from animal models of asthma to human asthma has often been disappointing. Therapeutic strategies aimed at deviating T cell responses toward Th1 or away from Th2 have not been shown to be effective in treating asthma in humans. It has been argued that IgE does not appear crucial for driving the asthma phenotype and that raised levels of IgE, while contributing to the inflammatory response in asthma, are likely to represent an associated parallel rather than sequential event (37). It is premature to consider that eosinophils do not play an important role in asthma pathogenesis; however, evidence from more recent studies suggests a less major role for eosinophils. Bronchial biopsy specimens and induced sputum samples from patients with asthma do not always reveal excess numbers of eosinophils (38), and in some mouse models of asthma, airway responses to antigen challenge require neither IL-5 nor IgE (39).

Although we have learned a great deal about cellular and molecular events in the pathogenesis of asthma, the Th2 hypothesis as an answer seems an oversimplification. Future research will need to focus on other possible underlying mechanisms for asthma pathogenesis. It has been suggested that the exaggerated inflammation and remodeling that occur in the airways of patients with asthma are due to the consequence of an abnormal injury and repair response arising from the susceptibility of the bronchial epithelium to components of the inhaled environment (40). Atopy (i.e., increased propensity to produce allergen-specific IgE) is likely a parallel event in addition to the altered tissue response rather than a sequential event, and thereby provides an explanation for the failure of a significant proportion of atopic individuals to develop asthma. By functionally interacting with the altered tissue response, the atopic predisposition serves to magnify or prolong events in this sequence. The existence of these parallel pathways leading to inflammation and remodeling can then account for the variable nature of this chronic and relapsing disease.

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Correspondence and requests for reprints should be addressed to Sundeep S. Salvi, M.D., Ph.D., University Medicine, Level D Centre Block, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK. E-mail: sundeepsalvi @hotmail.com

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