Interactions between Corticosteroids and -Adrenergic Agonists in Asthma Disease Induction, Progression, and Exacerbation

GARY P. ANDERSON

Lung Disease Research Laboratory, Department of Pharmacology, University of Melbourne, Parkville, Australia

	INTRODUCTION

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The therapeutic benefits and safety of inhaled ₂-adrenoceptor agonists and glucocorticosteroids place these drugs as the cornerstones of contemporary antiasthma therapy. Neither drug class can cure asthma, alone or in combination (1). However, clinical studies strongly support the following concepts:

1. Introducing steroid therapy earlier in disease progression produces better long-term outcomes for lung function (1) with acceptable safety.

2. Therapy with both long-acting -adrenoceptor agonists (LABAs) and inhaled glucocorticosteroids improves disease control and reduces disease exacerbations (2) with acceptable safety (5).

In contrast to concerns over short-acting -adrenoceptor agonist (SABA) safety, the objectively measured benefits of combined LABA/steroid therapy raise obvious questions:

1. Do -adrenoceptors and steroids interact in a fundamentally beneficial manner?

2. Do these interactions differ over the course of the natural history of the disease?

3. What, if anything, distinguishes the mode of action of LABAs from SABAs?

4. What molecular mechanism(s) are responsible for observed differences?

The pharmacology of -adrenoceptor agonists and of glucocorticosteroids has been exhaustively reviewedthis article concentrates therefore on mechanisms that may affect interactions of -agonists and steroids during primary disease induction, disease progression, in severe asthma, and during disease exacerbation.

	INDIVIDUAL PROPERTIES OF -ADRENOCEPTOR AGONISTS, CORTICOSTEROIDS, AND THEIR RECEPTORS

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Four subtypes of -adrenoceptors are known, of which three have been cloned. Both ₁- and ₂-adrenoceptors are found in the human lung but the ₂-adrenoceptors are of greatest importance and are widely expressed in the lung (9). Human airway smooth muscle is not relaxed by selective ₁-agonists, since ₁-adrenergic receptors are confined to glands and alveoli. ₃-Adrenoceptors are not expressed in human lung. It is unknown if the ₄ subtype, whose existence has been inferred from pharmacological studies of cardiac tissues (10), is present in the lung. ₂-Adrenoceptor-mediated effects therefore predominate in smooth muscle and in other cell types relevant to asthma.

₂-Adrenoceptors are transmembrane proteins whose architecture clusters seven helices of amino acids together to form a central ligand-binding core (11, 12). The receptor conformation is spontaneously flexible. When agonists bind to the active sites of the ₂-adrenoceptor core they change the conformational shape of the receptor, allowing binding and activation of cytoplasmic G proteins, which are signaling intermediates. Classically, this G protein is stimulatory G protein (G_s), which is activated when it binds to agonist-occupied receptors. G_s dissociates into  and subunits. G_s in turns binds to and activates effector molecules, typically the enzyme adenylyl cyclase. Adenylyl cyclase increases cyclic adenosine monophosphate (cAMP) concentrations, activating protein kinase A (PKA), which in turn promotes decreased contractility of airway smooth muscle by reducing calcium levels and inhibiting the phosphorylation of myosin light chain kinase. G_s may also activate bronchodilation by activating large conductance potassium channels (maxi-K channels). However, salmeterol, which is an effective bronchodilator in humans, lacks this property (13) and the role of maxi-K channels remains disputed.

It is possible that under some circumstances ₂-adrenoceptors may couple to G_i, an inhibitory G protein that activates biochemically pathways, especially inositol triphosphate (IP₃) kinase and PKC, which are functionally antagonistic to G_s-mediated responses. In vitro studies also predict that subunits may also cause deleterious effects by activating mitogen-associated protein (MAP) kinases implicated, for example, in airway wall thickening. These alternative forms of signal have not been identified in human asthma.

-Adrenoceptor agonists are classified by their selectivity, potency, and pharmacological efficacy (Table 1). Potency is less relevant to understanding the in vivo pharmacology than selectivity or efficacy because differences in potency are easily compensated for by adjusting inhaled doses whereas selectivity and efficacy are intrinsic properties of each drug. All of the synthetic -adrenoceptor agonist bronchodilators currently available are good to highly selective agonists at the ₂-adrenoceptor (with the exception of isoproterenol, which is approximately equiactive on ₁- and ₂-adrenoceptors).

All synthetic -agonists are also partial agonists, which means they activate signal transduction, e.g., via cAMP, less efficiently and to a lower maximal extent than the natural catecholamines adrenaline or isoprenaline. The degree of efficacy varies markedly among -agonists and is less discernable in cell types with large numbers of ₂-adrenoceptors or where signal transduction coupling is highly efficient (14). For this reason, in humans, the spectrum of low to high efficacies is not detectable when direct bronchodilation is measured. However, less efficacious agents do not protect as well against induced bronchoconstriction and are less able to inhibit responses in some inflammatory cells with low surface numbers of receptors, such as the eosinophil (17).

The biophysical properties of -adrenoceptor agonists vary markedly between SABAs and LABAs. SABAs are short acting because they are easily soluble in water and diffuse away from airway smooth muscle rapidly after inhalation. LABAs (formoterol and salmeterol) are lipid soluble and partition readily into the outer phospholipid layer of cell membranes. Both LABAs therefore alter the fluidity of the cell membrane and this may in turn modulate the activity of membrane-bound signaling molecules. In the case of salmeterol, which has the highest membrane partition coefficient, a number of inhibitory effects that are not mediated by -adrenoceptors have been formally documented. The existence of a distinct exosite mediating the long duration of action of salmeterol is highly controversial but not excluded (12, 18, 19). It is possible that biophysical properties may be important in the differential interaction of SABAs and LABAs with glucocorticosteroid (GCS).

All of the clinically useful -agonists are enantiomeric mixtures, with the greatest pharmacological activity residing in the (R)-isomer. Concern has been raised about possible adverse effects of (S)-isomers but to date there is no convincing evidence that (S)-isomers are harmful, or adversely affect the activity of (R)-isomers or interact in any way with glucocorticosteroids (20). However, (S)-salbutamol [(S)-albuterol] has been shown to increase airway smooth muscle calcium via a muscarinic receptor-dependent mechanism (21). As muscarinic receptors are coupled to IP₃ and diacylglycerol (DAG)/PKC- dependent transduction via G_i it is possible that (S)-salbutamol would oppose the activity of (R)-salbutamol. However, in vitro (S)-salbutamol does not diminish the efficacy or potency of (R)-salbutamol and relaxes rather than contracts airway smooth muscle, indicating that any muscarinic activity of (S)- salbutamol must be trivial if it occurs at all in vivo. The (S)- isomers of LABAs are approximately 1,000-fold less potent than the (R)-isomers but do not inhibit or impair the activity of the (R)-isomers.

Glucocorticosteroid receptors (GCSRs) are best thought of as ligand-activated transcription factors that operate, either directly or indirectly, to alter gene transcription. Like ₂-adrenoceptors, GCSRs are almost ubiquitously expressed in cells and tissues relevant to asthma (22). The GCSR belongs to a superfamily containing retinoid, thyroxine, and vitamin D receptors. Unlike adrenoceptors only one GCSR gene is known; however, alternative splicing may give rise to a truncated form (GCSR) that lacks the ability to modulate gene transcription but may affect the activity of GCSR. GCSR is an intracellular receptor, so that CGS must diffuse through the cell membrane in order to bind. GCSs are therefore lipophilic molecules.

CGSRs have three functional domains:

1. A C-terminal ligand-binding domain

2. A small DNA-binding domain incorporating a central zinc finger domain that binds to glucocorticosteroid response elements (GREs) present in the regulatory regions of some genes

3. An N-terminal trans-activation domain

Ligand binding to GCSRs at the C-terminal domain causes a conformational change that frees the GCSR from associated heat shock proteins (HSP90, HSP70, HSP56) that serve as molecular chaperones. The ligand-occupied receptor translocates to the cell nucleus, where it may either form a DNA-binding homodimer (23) or bind to other nuclear transcription factors. Broadly, GCSRs can alter gene transcription by two processes. First, occupied GCSRs can direct binding to DNA and GREs in a process termed "transcriptional trans-activation." GREs are classically positive regulators of transcription; however, examples of negative GREs are known. Three important examples of positively regulated genes are the ₂-adrenoceptor; lipocortin, an endogenous antiinflammatory molecule; and the molecule I-B, the intrinsic inhibitor of the proinflammatory transcription factor NF-B. Negative regulation of gene transcription also occurs via the ability of GCSRs to physically bind to other transcription factors, thereby inhibiting gene function indirectly in a process termed "trans-repression." The most extensively researched interactions of this type are inhibitory interactions with the proinflammatory transcription factors AP-1 (a c-Jun/c-Jun homodimer or c-Fos/c-Jun heterodimer) and NF-B (Table 2). Subtle variations of trans-activation and trans-repression conveniently explain the diverse effects of GCS on genes but the full range of suspected molecular interactions has not been unequivocally proven to occur in vivo.

Ligand-occupied GCSR has been shown to physically bind to AP-1, which is constitutively present in the nucleus, at least in vitro. However, this interaction is weak (24). Studies of the regulation of AP-1-dependent protease genes, the transcription of which is regulated by one AP-1 site, suggest a more complex interaction. The occupied GCSR, and possibly stabilizing proteins, may bind to AP-1 already on DNA, blocking access of the transcriptional machinery to the repressed gene. Alternatively, occupied GCSR may competitively bind an essential cofactor needed for AP-1-regulated gene transcription (25). This is unlikely to represent a unique mechanism as a number of genes are known to be regulated by composite binding of AP-1 dimers and GCSR at separate sites (26).

The proinflammatory transcription factor NF-B is held inactive in the cytoplasm by physical interaction with its endogenous inhibitor, I-B (27). Inflammatory stimuli cause I-B to be proteolytically cleaved after phosphorylation, allowing dimerized NF-B to translocate to the nucleus. Occupied CGSR is thought to inhibit NF-B by two mechanisms: physical binding to the p65 NF-B subunit (28) and induction of I-B gene transcription (29). However, it is thought unlikely that trans-repression of AP-1 and or NF-B can explain the ability of CGS to inhibit or attenuate the transcription of a large number of early response genes, whose induction is often caused by diverse stimuli acting through separate second-messenger/ transcription factor systems. The early response genes are essential for acute inflammatory reactions and include CC and CXC chemokines (including eotaxin), iNOS, and COX-2 (30).

An emerging layer of complexity in steroid regulation of gene transcription is the likely effect of steroid receptor complexes as regulators of condensed chromatin structure and regulators of initiation complex access to genes (31, 32). It is therefore likely that GCSRs are able to exert extremely varied and complex effects on gene transcription. This subtlety may also explain why some inhibitory effects of steroids are cell type specific. Furthermore, GCS decreases the stability of mRNA for a number of gene products, notably interleukin 1 (IL-1) and IL-6 (33, 34), by an unknown mechanism(s), presumably involving ribonuclease activity. The GCSR also has multiple phosphorylation sites on serine and threonine residues and is a substrate for numerous kinases including proline-dependent kinase, p34 cdc2 kinase, casein kinase II, and calmodulin. Phosphorylation seems not to affect trans-activation but does govern trafficking to and from the nucleus.

This diversity of action greatly increases the difficulty of identifying the molecular basis of GCSR interactions with ₂-adrenoceptors but the number of such possible interactions seems increasingly large.

	DISEASE INDUCTION

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As delaying introduction of inhaled corticosteroid has been shown to produce less objective improvement in lung function than earlier introduction (1) it is appropriate to consider how -adrenoceptor agonists and inhaled GCS might interact in the underlying processes that drive early disease induction. There is overwhelming evidence that allergic asthma is driven by CD4⁺ T cells that have been biased in their cytokine production toward helper T cell type 2 (Th2) pattern. Th2 immunity underlies atopy, a major risk factor for asthma and genes known to regulate Th2 immune bias are among the most consistently identified candidate genes in asthma genetic linkage studies. Th2-biased immune responses result in overproduction of IL-4, a factor essential for IgE antibody production; IL-5, a factor crucial for eosinophil differentiation and survival; and other cytokines including IL-9, IL-3, and IL-13, each linked to disease progression (35).

Induction of Th2 immunity is critically dependent on IL-4 and is opposed by IL-12 and interferon  (IFN-) (35). Animal models suggest the following general mechanisms. Aeroallergen is captured by dendritic cells that migrate from the mucosa to regional lymph nodes, by which time they have expressed processed antigen peptides on their surface MCH II molecules in the context of a panel of costimulation molecules, notably B7-2. Dendritic cells normally produce interleukin 12, a major inducer of Th1 immune response during antigen presentation to T cells, but in asthma this is less likely to occur. Epithelial-derived IL-6 is a possible determinant of suppressed IL-12 production. In addition, molecular defects in the IL-4 promotor may contribute to its overproduction. IL-4 actions are critically dependent on the transcription factors STAT-6 and c-maf as well as a panel of coinducing transcriptional regulators, but primary defects in these signaling molecules have not been identified to date.

It is clear that T cells can make sufficient autocrine IL-4 to drive Th2 commitment if IL-12 and IFN- signals are weakened. Natural killer (NK) cells, including infrequently observed NK T cells, amplify this process, and at least in some strains of mice can provide high-level IL-4 secretion. Basophils and mast cells are potential sources of additional IL-4. To date no primary defects in IFN- or IL-12 pathways have been identified.

It is of considerable concern that -adrenoceptor agonists and GCS independently suppress IL-12 production in dendritic cells and other antigen-presenting cells (38, 39). Neutralization of IL-12 in vivo in mice intensifies Th2 immune responses and also converts mice that are genetically predisposed to the protective Th1 immune response into Th2 responders. In the case of -adrenoceptors suppression of IL-12 is via activation of G_s. This effect also prevents induction of IL-12 in responses to endotoxin from gram-negative bacteria and is completely independent of IL-10 since it occurs in IL-10 knockout mice. As such, histamine, which is able to activate G_s via histamine H2 receptors, and PGE₂, acting via EP₂ receptors, both of which are present on antigen-presenting cells, share this property. The ability of corticosteroids to effectively suppress IL-12 production has been widely overlooked. These observations are important because they raise the possibility that early intervention with current asthma therapy of -agonist and steroids, while it will effectively control overt symptoms and inflammation, may "hard wire" immune deviation toward Th2 responses. In this context, it is important that as Th2 immunity evolves, signal transduction from IL-12 and IFN- receptors be progressively extinguished by downregulation of essential signaling components such as the IL-12 receptor (IL-12R)  chain so that Th2-biased cells become irreversibly fixed into production of their distinctive panel of cytokines.

Biopsy studies have shown that GCS reduces the number of IL-4-secreting cells, and this has been interpreted as evidence that IL-4 is transcriptionally repressed by steroids. Furthermore, release of preformed IL-4 from both basophil and mast cells is efficiently suppressed by ₂-adrenoceptor agonists, particularly those with higher efficacy (40). However, the evidence that GCSs are efficacious repressors of IL-4 production is weak. IL-4 actually decreases the ability of steroids to function by decreasing receptor affinity through an obscure mechanism; steroids cannot suppress IL-4-induced upregulation of vascular cell adhesion molecule 1 (VCAM-1); IL-4 suppresses the ability of steroids to induce T cell apoptosis and greatly increases T cell autocrine IL-4 production and enhances B cell polyclonal IgE production (although there is some evidence that antigen-specific IgE might be reduced by GCS at the level of isotype-switched B cells) (43).

It is therefore clear that establishing the effects of early inhaled GCS therapy, alone and in combination with -agonists, on the persistence and intensity of underlying Th2 immunity is important. Steroids suppress IL-6 levels and also effectively suppress epithelial PGE₂ production, both of which are likely to protect against Th2 immunity. Steroids also reduce dendritic cell number in the airways (44) by induction of apoptosis (45). However, there are large gaps in our current understanding, as it is unclear whether steroids/-agonists can alter the balance of B7 family costimulation molecules or alter IFN-/ IL-12-inducing signals through IL-18; protect from downregulation of IL-12/IFN- signaling, or influence the effect of NK cells on disease induction. The net balance is likely to set the intensity of the atopic component of asthma. At present, it seems reasonable to propose that the reason steroids ameliorate but cannot cure or truly prevent asthma, alone or in combination with -agonists, is because they lack the ability to fundamentally suppress IL-4 induction and actions.

	DISEASE PROGRESSION

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The distinction between asthma disease induction and progression is arbitrary but convenient. In established asthma there is clear evidence of ongoing Th2-pattern cytokine production in the airway mucosa, predominantly from mast cells but also from metachromatic cells. During recurrent T cell stimulation armed CD4+ Th2-biased effector cells begin to express unique surface markers, such as T1/ST2 and IL-1 receptor family homologs. Cytokine secretion from Th2-biased T cell populations is broadly suppressed by steroids and there is evidence that -agonists, particularly those with higher efficacy or when combined with phosphodiesterase inhibitors to prevent cAMP hydrolysis, also inhibit cytokine production, e.g., IL-5 (46). However, it is important to reemphasize that even the most efficacious -agonists have almost no discernable antiinflammatory activity in human asthmatic airways, despite the extensive animal and in vitro evidence that -agonists have broad-ranging suppressive effects in models of acute inflammation (47). Similarly, SABAs and LABAs are essentially unable to block the rapid recrudescence of inflammation that follows withdrawal of inhaled steroid. The likely explanation for the lack of effect of -agonist on airway inflammation is that ₂-adrenoceptor populations on effector cells rapidly downregulate. Steroids attenuate this downregulation in receptor number but do not adequately restore inhibitory function. Inflammation induced by segmental allergen challenge tends to reduce -adrenoreceptor numbers in the airway epithelium and cAMP accumulation, and this effect should be prevented by steroids, but the direct downregulation induced by -agonist overwhelms any such positive interaction (48). Second, some inflammatory mechanisms, such as epithelial granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-8 production, are completely refractory to -agonists (49).

The normal fate of antigen-activated T cells is to expand under the influence of IL-2 and then, in order to terminate immune responses, to die by activation-induced cell death (AICD) and/or apoptosis. It is important to note that cells that have entered the G₂/M phase of cell cycle, such as proliferating effector lymphocytes, are particular insensitive to GCS action because the N-terminal trans-activation domain is hyperphosphorylated at this point. Furthermore, inflammatory mediators, particularly interferons, upregulated by viral infection suspend T cell apoptosis (50). In cell lines and a number of leukocytes, including the eosinophil, -agonists lessen sensitivity to apoptosis but it is not known how -agonist and steroids affect human T cells in situ. Steroids, alone or in combination with -agonists, do not prevent T or B cell immunological memory, which accounts for the rapidity with which inflammation recurs when inhaled steroid therapy is withdrawn. A central and unanswered question is whether the demonstrated possibility of reducing steroid dose in protocols combining inhaled steroids with LABAs, without deterioration in lung function or worsening of objectively measured inflammatory parameters such as sputum eosinophils, represents a positive effect of steroid--agonist interaction on inflammation or is simply a benefit of well-controlled asthma (51).

Conditional depletion experiments have provided clear evidence that dendritic cells have an important role in reactivating antigen-experienced effector lymphocytes (52), but it is not clear if this reactivation is driven by mucosal or lymph node-localized dendritic cells. As steroids kill dendritic cells this may be one important mechanism for reduction of the disease exacerbation rate, but it is not known if dendritic cell death is enhanced or prevented by -agonists.

The perpetuation of inflammation requires coordinated expression of CC and CXC chemokine panels that direct leukocyte traffic in the mucosa and to regional lymph nodes. Induction of eotaxin is upregulated by epithelial IL-4 and its close congener, IL-13, and IL-13 may have a particularly important role in maintaining mucosal inflammation. IL-4 and IL-13 appear to be central mediators in the induction of goblet cell transdifferentiation (53), the process that underlies mucous hypersecretion in asthma. The relative insensitivity of IL-4-mediated effects to steroids may explain why steroids are only partially able to reduce goblet cell number in disease models (54).

An emerging body of early intervention studies and longer term cohort studies suggests that early treatment with steroids, alone or in combination with ₂-adrenoceptor agonists, may be highly beneficial in that this approach may prevent or reduce the extent of airway wall remodeling by limiting inflammation at an early stage. Airway wall thickening, which is largely due to smooth muscle proliferation and hypertrophy, is thought to be an important target for antiinflammatory drug action. As -agonists may interact favorably with corticosteroids, combination therapy may be particularly advantageous in preventing or lessening airway wall remodeling (55). The situation in long-established disease is, however, controversial. It has been discovered that airway smooth muscle undergoes phenotypic changes in asthma that alter its nature from a contractile tissue to a potentially proinflammatory structure, termed the "secretory phenotype." The secretory phenotype is accompanied by downregulation of contractile elements, but upregulation of the capacity to secrete a range of deleterious proinflammatory mediators. These mediators are known to include IL-1, tumor necrosis factor  (TNF-), IL-8, and probably also stem cell factor (SCF/c-KitL) active on mast cells and G-CSF active on neutrophils. The transition to a proinflammatory state explains the observation that human airway smooth muscle bundles become infiltrated with inflammatory mast cells in chronic asthma. In addition, there is direct evidence that secretory phenotype airway smooth muscle may deposit extracellular matrix components such as fibronectin and collagens. The effect of -agonist-steroid interactions on these processes is poorly understood.

Steroids do not uniformly produce beneficial effects on airway smooth muscle expansion because steroids suppress basal and COX-2-generated PGE₂ production; PGE₂, acting via G_s-coupled EP2 receptors, is a highly efficacious cAMP inducer (56). As -agonists suppress airway smooth muscle growth by signaling via the same G_s-coupled transduction pathway, and because airway smooth muscle ₂-adrenoceptor populations are privileged and refractory to complete downregulation (57), the combination of steroids and -agonists may have extremely beneficial effects in long-term disease. This may be especially true of LABAs, which biophysically partition into the airway smooth muscle cell bundles.

One area that is particularly poorly understood is the potential interaction of -agonists and steroids at the level of growth factors, their receptors, and their kinase transduction systems. A large number of growth factors are implicated in epithelial repair and mucous secretion (epidermal growth factor [EGF] family molecules/transforming growth factor  [TGF-], basic fibroblast growth factors [bFGFs], hepatocyte growth factor [HGF]/ SCF, c-KitL/SCF), airway smooth muscle growth (EGF, platelet-derived growth factors [PDGFs], insulin-like growth factors [IGFs]), neovascularization (vascular endothelial growth factors [VEGFs], angiopoietins, PDGFs), nerve phenotype change (nerve growth factor [NGF], leukocyte inhibitory factor [LIF], oncostatin M [OSM]), and connective tissue deposition (connective tissue growth factor [CTGF], TGF-, bFGF, HB-EGFs, PDGFs). G protein-coupled receptor signaling has been shown to converge on growth factor MAP kinase signaling pathways via G protein subunits, but this biochemical cross-talk has not been demonstrated to occur for the -adrenoceptor. Steroids transcriptionally suppress JE, an early response gene induced by PDGF (58), and attenuate some PDGF-induced effects, such as proliferation of airway smooth muscle in vitro, and may also lessen the expression of immunoreactive EGF.

	SEVERE DISEASE AND DISEASE EXACERBATION

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Infection by respiratory tract virus is an extremely common cause of disease exacerbation. Evidence suggests that persistent subclinical infection with pathogens such as Chlamydia and Mycoplasma pneumonii are likely to worsen underlying disease and may also increase the risk of viral exacerbation. Neither -agonists nor corticosteroids have any known antiviral effect. Steroids may reduce expression of intercellular adhesion molecule 1 (ICAM-1), a coreceptor for rhinovirus infection of respiratory epithelium, although ICAM-1 is only weakly regulated by steroids.

IL-1 is upregulated by almost all airway infections and may contribute to disease worsening because it is able to rapidly reactivate antigen-experienced "memory" (CD45RO⁺, CD4⁺) Th2 effector cells independently of their cognate antigen. Sudden T cell reactivation contributes to exacerbations of inflammation. IL-1 is effectively suppressed by steroids, whose use in such exacerbations is underscored by the observations that IL-1 is unusually regulated in respiratory epithelium because of defective IL-1 Type II receptor expression (59). It is of interest that salmeterol has been shown to reduce epithelial damage induced by a number of respiratory tract pathogens (60, 61). However, LABAs alone do not reduce the frequency of disease exacerbations, have no ability to suppress controlled exacerbations in volunteers, and may mask patient perception of deteriorating lung function (62, 63).

Genetic defects in ₂-adrenoceptors and GCSRs are risk factors for severe asthma (64). Long-standing severe asthma may also impede -agonist function by a number of mechanisms that are at least partially improved by steroids. Human airway smooth muscle has a large receptor reserve of ₂-adrenoceptors, which largely obscures the difference in efficacy between formoterol and salmeterol unless tissues are strongly precontracted (14). The concept that severe airway inflammation fundamentally impairs the ability of ₂-adrenoceptor agonists to relax airway smooth muscle was confirmed in studies by Bai (65, 66), who demonstrated that the -adrenoceptor uncouples from its transduction pathway in fatal asthma by an unknown mechanism. These changes are reflected in the observations by Lefkowitz and coworkers that the ₂-adrenoceptor affinity state shift caused by GTP measures the degree of receptor uncoupling [ternary complex model and extended ternary complex model (67, 68)]. Pharmacological theory predicts that as the degree of uncoupling caused by inflammation increases, weaker partial agonists will be progressively less able to relax airway smooth muscle and may completely lose their ability to relax tissue. It therefore is of great interest to determine whether concomitant steroid therapy reduces the degree of uncoupling that might occur in severe disease exacerbations.

The molecular basis of uncoupling is incompletely understood but it is mimicked, at least in part, by mediators, notably IL-1 and TNF- (69), likely to be overproduced during sudden exacerbations, particularly if the worsening is precipitated by infection. Decreases in ₂-adrenoceptor mRNA, deficient adenylyl cyclase activity, and atypical coupling of the ₂-adrenoceptor to G_i rather than G_s and receptor uncoupling from G_s are all predicted to occur from animal models (66). In severe asthma inhibitory cross-talk between G_i/G_q-coupled inflammatory and contractile mediators that signal via IP₃/PKC/ DAG, such as cysteinyl leukotrienes, acetylcholine, substance P, or histamine, can biochemically impair G_s-mediated signal transduction (70, 71). This cross-talk impairs the ability to sustain cAMP accumulation. Hence functional antagonism of this type causes a measurable fall in efficacy of all -agonists and can convert near-full agonist to weak partial agonists (72).

	KNOWN MOLECULAR MECHANISMS OF GLUCOCORTICOSTEROID-2-ADRENOCEPTOR ACTIVATION

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There are several possible interactions between -agonists and corticosteroids (Figures 1 and 2).

View larger version (42K): [in this window] [in a new window]   Figure 1.   Overview of sites of known and possible interactions between 2-adrenoceptor agonists and glucocorticosteroids in asthma disease induction, progression, and exacerbation. The diagram shows a conceptual compartmental model in which -adrenoceptor agonists and steroids affect (or are suspect to affect) asthma disease induction, progression, and exacerbation. (1) Primary allergic sensitization to aeroallergen, (2) expansion of CD4+ effector lymphocytes biased to secrete net Th2 cytokines (e.g., IL-4 and IL-5), (3) reexpansion of effector CD4+ cells from memory pools after reexposure to allergen and/or as a consequence of concomitant infection during an asthma exacerbation, (4) long-term allergen-specific T cell memory, (5) production of cytokines and regulation of net activation state of effector cells, such as the eosinophil (eos), in the inflamed airway mucosa, (6) production of antiapoptotic, T cell-reactivating factors, such as IL-1, that may also uncouple -adrenoceptors from Gs in airway smooth muscle, during viral exacerbations, (7) regulation of receptor number, signaling efficiency, and degree of functional antagonist between contractile and relaxant agonists at the level of airway smooth muscle; regulation of smooth muscle bulk. Detailed descriptions of individual molecular mechanisms are presented in the text.

View larger version (40K): [in this window] [in a new window]   Figure 2.   Known and putative molecular interactions between glucocorticosteroids and -adrenoceptors on immune deviation in allergic asthma. The diagram shows a figurative CD4+ naive T cell population (Thp/o) undergoing immune deviation toward Th2- biased cytokine production (left-hand side) or Th1-biased immune responses. Elevated intracellular cyclic AMP (cAMP) in response to 2-adrenoceptor activation (B2), histamine H2 receptor activation, or prostaglandin E2 (EP2) receptor activation predisposes toward Th2-biased immunity, as does suppression of IL-12 by steroids and -agonists. Cofactors contributing to induction of Th2 immunity (B7-2, IL-4, and ICAM-2) or Th1 immunity (IL-12, IFN-, and IL-18 acting via their respective receptor systems) are shown in italics. Note that both 2-agonists and steroids have been shown in in vitro studies to strongly suppress IL-12 and bias toward allergic-type Th2-biased immunity, but steroids also suppress PGE2 levels and, indirectly via actions on mast cells, also reduce histamine levels. The net effects on immune deviation in humans are unknown. The diagram illustrates that the in vivo situation may be more complex because of the multiple levels of regulation exerted by these agents (e.g., suppression of PGE2 and histamine release) that may counterbalance effects on IL-12. Arrows represent stimulation and arrows terminating in a bar represent inhibition.

₂-Adrenoceptor Transcriptional Regulation by Steroids: Tolerance and Subsensitivity

₂-Adrenoceptors are transcriptionally upregulated by glucocorticosteroids and steroids can reverse, at least partially, the degree of loss of surface receptors that occurs during homologous desensitization to sustained agonist stimulation, even in the case of the weakest partial agonists (73). In vitro studies have demonstrated that steroids protect from the loss of receptors and function that follows long-term agonist incubation with human bronchial smooth muscle (74). However, although steroids restore the ₂-adrenoceptor number of the cell surface this does not uniformly improve the attenuation (or nearly complete loss in some cell types) of signaling ability in vivo. Airway smooth muscle is among the tissues least susceptible to homologous downregulationbronchodilator responses are retained unimpeded over years of treatment with LABAs. However, all -agonists produce detectable losses in the degree of protection afforded against induced bronchoconstriction, which cannot be overcome by higher doses of agonist (75). There is contradictory evidence on the degree of protection offered against this loss of protection by inhaled steroids. In the case of formoterol, oral but not inhaled steroids were shown to protect against loss of protection to methacholine-induced bronchospasm (76, 77). Conversely, inhaled beclomethasone attenuated tolerance to the loss of protective effect of salmeterol against allergen challenge (78).

Can -Agonists Directly Activate Glucocorticoid Receptors?

One of the most controversial, and potentially important, studies of the molecular basis of possible positive interactions between GCS and ₂-adrenoceptor agonists has been done by Eickelberg and co-workers (79), who observed that salmeterol promoted GRE binding of GCSRs in the absence of ligand, in human vascular endothelial smooth muscle cells and fibroblasts. Moreover, these authors demonstrated that the translocated receptor inhibited transcription of a p21^WAF1-CIP luciferase reporter gene construct. The effect of salmeterol was mimicked by the stable cAMP analogs dibutyryl cAMP and 8-bromo-cAMP and was blocked by propranolol, a nonselective -adrenoceptor antagonist, and partially suppressed by anti-PKA peptides.

This effect is not without precedent, as heat shock and stress can cause translocation of GCSRs. Moreover, ursodeoxycholic acid has been reported to produce similar ligand-independent activation of GCSR, although again, the effect is weak. The proposed involvement of PKA is not consistent with the observations of Orti and colleagues (80), who found that PKA-dependent kinases were not involved either directly of indirectly with phosphorylating the GCSR in vivo. However, on the basis of precedents at the 17-estradiol receptor, it is possible that PKA activation of calmodulin provides an intermediate pathway to GCSR ligand-independent activation (81, 82). Again, if calmodulin is the route to -agonist-induced ligand-independent GCSR activation it is difficult to see why leukotrienes, acetylcholine, and histamine do not produce the same effect, as G_i/G_q-coupled receptors have previously been shown to activate calmodulin (and Pyk2 and Src kinases) in a Ras-dependent manner.

Nonetheless, the -agonist-induced GCSR activation observed by Eickelberg and coworkers (79) may provide a starting point for dissecting the molecular basis of reduced disease exacerbations and better disease control observed in large, blinded clinical trials combining LABAs with inhaled GCS. Furthermore, these observations may also provide insights into the findings of the NIH SLIC trial demonstrating that steroid dose may be safely halved, but not discontinued, in patients whose asthma is well controlled by inhaled triamcinolone/salmeterol therapy without causing a deterioration in disease control, impairment of lung function, or recrudescence of inflammation.

Do ₂-Adrenoceptor Agonists Impair Corticosteroid Action?

High concentrations of -adrenoceptor agonists cause the transcription factor CREB (cyclic AMP response element-binding protein) to be phosphorylated by PKA. Activated CREB can sequester AP-1, a potentially beneficial interaction. However, CREB can also complex ligand-occupied GCSR and prevent trans-activation by impeding binding to GRE sites. -Agonist-mediated decreases in GCSR binding have been observed in vitro in a number of cell types (83). This has led to the suggestion that high doses of -agonists may progressively impair steroid action. This proposed adverse interaction would predict that LABAs, which reach high topical concentrations and also persist in tissues because of their biophysical properties, would be particularly likely to impair the action of steroids, but long-term clinical trials have revealed a trend to progressive improvement in lung function and no worsening of inflammation assessed by biopsy, induced sputum, or lavage. More recently the interaction of -agonist and steroids has been assessed in human monocytes ex vivo. Salbutamol was found not to impair the activity of dexamethasone on suppression of GM-CSF or TNF-, and salmeterol was found to enhance the activity of dexamethasone (84, 85); it seems clear that if any adverse effect occurs, it is extremely difficult to detect.

	IMPORTANT QUESTIONS

TOP INTRODUCTION INDIVIDUAL PROPERTIES OF -... DISEASE INDUCTION DISEASE PROGRESSION SEVERE DISEASE AND DISEASE... KNOWN MOLECULAR MECHANISMS OF... IMPORTANT QUESTIONS REFERENCES

• What is the net balance of influence on Th2 commitment processes when -agonists and steroids are combined in early disease?
• To what extent do steroids protect from impairment of -adrenoceptor signaling in severe disease? Is steroid suppression of IL-1 a key mechanism linking reduced inflammation with retained -adrenoceptor function?
• What molecular mechanism(s) explain the reduction in disease exacerbations when LABAs are used in conjunction with steroids?
• Is the ability of LABAs to partition into cell membranes an important determinant of how they may interact with steroids?
• What is the real significance of direct activation of steroid receptors by -agonists?
• Is -agonist pharmacological efficacy clinically relevant?

	Footnotes

Correspondence and requests for reprints should be addressed to G. P. Anderson, Ph.D., Lung Disease Research Laboratory, Department of Pharmacology, University of Melbourne, Parkville 3052, Australia. E-mail: g.anderson{at}pharmacology.unimelb.edu.au

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