Corticosteroid Therapy in Asthma: Predictors of Responsiveness

Clin Chest Med 27 (2006) 119 – 132

Corticosteroid Therapy in Asthma: Predictors of Responsiveness Christopher M. Mjaanes, MDa,b, Glenn J. Whelan, PharmDa, Stanley J. Szef ler, MDa,b,T a b

National Jewish Medical and Research Center, Denver, CO, USA University of Colorado Health Sciences Center, Denver, CO, USA

Asthma is a chronic disease of the airways characterized by reversible airflow obstruction, airway hyperresponsiveness, and airway inflammation. As understanding of the pathogenesis of this chronic disease has grown, so to have the armamentarium of medication used to combat asthma’s ill effects. Glucocorticoids (GC) are potent anti-inflammatory agents, which are the mainstay of treatment for both chronic and acute asthma management [1,2]. These agents bind to their cytoplasmic receptor (GC receptor [GR]) and initiate a cascade of events, which are believed to result in the decreased migration of inflammatory cells to the lungs, diminished inflammatory cell survival, decreased airway mucous production, inhibition of proinflammatory cytokine production, and a variety of other anti-inflammatory effects by increased gene transcription (Fig. 1). Although most asthmatics have mild to moderate disease, up to 10% have severe disease that is often refractory to various therapies [3]. Although the mechanisms for severe asthma are not entirely understood, an area of growing interest is GC insensitivity or resistance. Because of the complex interactions between genetic and environmental factors, which give rise to asthma in an individual, and the unique, multiphasic pathways involved in the processing of GCs, several mechanisms for GC resistance or insensitivity

have been proposed. Before the possible causes of steroid resistance (SR) or steroid insensitivity (SI) can be examined, a definition of this entity is required.

Definition SR was first described in 1968 by Schwartz and coworkers [4] in six patients who failed to demonstrate the anticipated respiratory improvement and decrease in peripheral eosinophils after receiving intravenous cortisol. It is now believed that approximately 30% of asthmatics possess some degree of SI [5]. A formal definition of SR asthma was proposed in 1981 by Carmichael and coworkers [6], who defined this entity as an improvement in the am prebronchodilator forced expiratory volume in one second (FEV1) by less than 15% after a 7-day course of 20 mg/d of prednisone or its equivalent. More recently, a workshop [7] on SR asthma proposed that the condition be defined as a failure to improve baseline FEV1 by greater than 15% predicted following 7 to 14 days of 20 mg of oral prednisone given twice daily.

Steroid insensitive versus steroid resistant T Corresponding author. National Jewish Medical and Research Center, 1400 Jackson Street, Office J304, Denver, CO 80209. E-mail address: [email protected] (S.J. Szef ler).

The terms ‘‘SR asthma’’ and ‘‘SI asthma’’ are often incorrectly used interchangeably. The more likely scenario is that these two conditions exist as part

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Fig. 1. A schematic representation of the anti-inflammatory effects glucocorticoids have on the lungs. GC, glucocorticoids; GCR, glucocorticoid receptor; IL, interleukin.

of a spectrum of steroid responsiveness, with SI describing a variable response to GCs and SR describing a complete lack of response to GCs. It is important to understand the difference and the connotations implied by these terms, which explains why experts have recently urged the use of the term ‘‘SI asthma,’’ because this suggests a less absolute state [7]. SI asthma is a phenotype in which a suboptimal but variable response to GCs is seen. It is much more common than SR and asthmatics with SI demonstrate a relative insensitivity to GCs. In the purest sense, SI refers to a poor response to GCs when given in normal, age-appropriate, anti-inflammatory doses. Patients with this condition may respond to GCs when administered in higher doses, at increased frequency, and for prolonged periods of time [5]. Several clinical features have been identified as being associated with SI asthma. These patients are typically classified as having severe asthma with persistent respiratory symptoms; nocturnal symptoms; and evidence of chronic airflow obstruction (FEV1 < 70% of predicted) [8]. Chan and coworkers [9] conducted a retrospective analysis of 164 consecutive adolescents admitted to the National Jewish Medical and Research Center in Denver, Colorado, to identify features of SI asthma. This group identified several addi-

tional distinguishing features among patients diagnosed with SI asthma including systemic GC therapy required at a younger age (5 versus 8 years, P = .025); a higher maintenance dose of oral GC therapy (20 versus 10 mg/d, P = .025); and more likely to be AfricanAmerican (38% versus 11.5%, P = .007). Asthmatics who are SR include those individuals with the most severe phenotype who exhibit a marginal response to even the highest oral doses of GCs. SR implies an inability to respond to GCs in any dose or at any time interval. SR asthma can be further subdivided into two types [10]. Type I SR asthma is acquired or cytokine-induced and may involve primary SR secondary to an immune response. This type may be associated with genetic polymorphisms leading to overproduction of various cytokines and key molecules, which can induce GC resistance. In addition, type I SR may be caused by allergen- or infection-induced cytokine activation or chronic exposure to b-agonists or GCs. Because the resistance in these patients lies at the level of their immune-inflammatory cells (lymphocytes), they present clinically with severe side effects including adrenal suppression and cushingoid facies. This indicates that although the immune cells remain relatively unresponsive to steroids, other tissue cell types remain susceptible to the unwanted effects of GCs.

corticosteroid therapy in asthma Table 1 Comparisons between Type I and Type II steroid resistance Constituents

Type I

Type II

Mechanism

Cytokine induced/ acquired Normal to high Reduced Lymphocytes Yes

Genetic Low Normal All cell types No

Suppressed Yes, reversible > 95%

Not present No, irreversible < 5%

GR number GR binding affinity Effector cells Corticoisteroid side effects? am Cortisol Reversibility Percent of SR patients

Data from Leung DYM, Spahn JD, Szefler SJ. Steroidresistant asthma: new insights and implications for management. In: Szefler SJ, Leung DYM, editors. Severe asthma: a multidisciplinary approach. 2nd edition. New York: Marcel Dekker; 2001. p. 602.

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medication, such as cyclosporine, or even surgical intervention for disease control. These patients with clinical resistance to steroids also were found to have in vitro T-cell resistance to steroids. In addition, SI has been reported in approximately 30% of patients with rheumatoid arthritis and systemic lupus erythematosus [12,13]. GCs are used extensively in the treatment of asthma. With approximately one third of all asthmatics demonstrating some degree of SI, it becomes exceedingly important for care providers to understand the features of SI, including how to manage and evaluate patients with known or suspected SI and develop a general understanding of the mechanisms underlying SI and several comorbid or co-existing conditions that may contribute to the development or persistence of SI.

Diagnosis of steroid resistance or insensitivity Type I SR likely accounts for more than 95% of SR asthma [5]. Type II SR affects all cell types, which is analogous to familial primary cortisol resistance. This type of resistance is likely secondary to a mutation in the GR gene, or in genes that modulate GR function. Because this type of resistance affects all tissues, global steroid effects are not seen (therapeutic or adverse). Lastly, type II GC resistance is an irreversible defect, accounting for less than 5% of all cases of GC-resistant asthma [5]. Unfortunately, the small number of these patients has not provided the opportunity to identify unique features in pathophysiology or insights for management. A summary of type I and type II SR asthma can be seen in Table 1.

Clinical importance Although the exact prevalence of SI is not known, it has been demonstrated in 24% to 42% of patients suffering from chronic inflammatory conditions including asthma [9,11 – 14]. Because GCs are extensively used to treat asthma, this disease provides an ideal model to investigate and observe the effects of SR providing a porthole for ever-growing investigations into the genetic, molecular, and environmental mechanisms contributing to SI. Other nonasthmatic conditions that demonstrate SI include ulcerative colitis, rheumatoid arthritis, and systemic lupus erythematosus [11 – 13]. Hearing and coworkers [11] reported that up to 30% of patients with ulcerative colitis do not respond to steroid therapy and require other anti-inflammatory

SR or SI asthma has several characteristic features including severe asthma with persistent respiratory symptoms; frequent nighttime symptoms; and chronic airflow obstruction (FEV1 < 70% of predicted) [8]. In addition, these patients tend to have required systemic GC therapy at a younger age, require higher daily maintenance doses of oral GCs, and are often African American. These features are summarized in Box 1. SI asthma should be suspected in any asthmatic who possesses a combination of these features; however, to meet the diagnostic criteria for SI patients must fail to demonstrate a greater than 15% improvement in their morning FEV1 value following a 14-day course of 20 mg of oral prednisone, given twice daily [7]. For diagnostic confirmation, such testing as a GC lymphocyte stimulation assay and steroid pharmacokinetics may also be considered to characterize fur-

Box 1. Summary of distinguishing features among steroid-insensitive/ steroid-resistant asthmatics Severe asthma Persistent respiratory symptoms Frequent nocturnal symptoms Chronic airflow obstruction (FEV1 <70% predicted) Systemic GC therapy at a young age Higher maintenance dose of oral GCs Tend to be African American

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ther the nature or etiology of the SI [15,16]. These specialized tests are covered in greater detail in the following sections. Management of steroid-insensitive asthma Management of SI and SR patients proves challenging because GCs are the cornerstone of asthma therapy. Based on current data (24% – 42% estimated prevalence of SI), 58% to 76% of patients with chronic inflammatory conditions including asthma should be responsive to GC therapy. Management of all cases should proceed in an organized, stepwise manner. The initial phase in management of the SI asthmatic involves confirmation of the underlying diagnosis. Performing a thorough history and physical examination and ordering appropriate laboratory tests and pulmonary function testing is necessary to confirm the diagnosis of asthma, and identify or rule out other comorbid or ‘‘masquerading’’ conditions, which can complicate the treatment of asthma. A list of comorbid conditions can be found in Box 2. Particular attention should be given to focusing on the identification of the previously mentioned risk factors or features of patients with SR asthma as listed in Box 1. The next step in assessing these patients is to ensure adequate medical treatment adherence. A multitude of studies have demonstrated that medication adherence rates among asthmatics average around 50% [17 – 22]. Milgrom and coworkers [23] measured adherence to inhaled corticosteroids by using electronically monitored metered dose inhalers in children 8 to 12 years of age with asthma. The authors demonstrated that after 13 weeks of treatment

Box 2. Comorbid conditions complicating and/or masquerading as difficult-to-treat or steroid-resistant asthma Gastroesophageal reflux disease (GERD) Vocal cord dysfunction (VCD) Hypersensitivity pneumonitis (HSP) Cystic fibrosis (CF) Allergic bronchopulmonary mycoses (eg, allergic bronchopulmonary aspergillosis [ABPA]) B1-Antitrypsin deficiency Tracheomalacia Bronchomalacia

the children who required a burst of oral GCs during the study had the lowest median adherence (13.7%), compared with the group who did not require oral GCs (median adherence 68.2%, P = .008). Similarly, Spahn and coworkers [24] determined that out of eight subjects with a history of requiring daily oral GCs on study enrollment, only 50% of these patients were found to have abnormal or suppressed serum cortisol concentrations (indicating 50% adherence to the use of oral steroids in the group). Once the treatment was conducted in a supervised medical setting, all serum cortisol concentrations became significantly suppressed, reflecting optimal adherence. The underlying etiologies of nonadherence can be quite complex ranging from simple forgetfulness, to lack of monetary resources, to severe psychologic problems including depression. With children in whom the diagnosis of SI asthma remains in the differential diagnosis, identifying nonadherence and implementing appropriate corrective measures are essential for a successful outcome. Simplifying medication regimens, providing medication diaries or check-sheets, directing patients and families toward financial assistance programs if available, monitoring patient’s technique of medication administration, and referring patients in need to psychosocial services for counseling can have a tremendous impact on improving adherence and improving disease control [22,25]. Following the assurance of adequate adherence to the medical regimen, asthmatic patients should be evaluated for possible microbial infection of the airways. This may be most important for patients on chronic oral steroids or on high doses of inhaled steroids who have local airways immune responses that may be compromised to the point of predisposing these airways to colonization by opportunistic organisms, including Mycoplasma and Chlamydia species, that can activate chronic antibacterial (nonallergic) inflammation [26,27]. These patients may respond to a prolonged course of antimicrobial therapy, such as clarithromycin [27]. Clarithromycin possesses antiinflammatory properties and has been observed in a small subset of asthmatics to increase GC sensitivity [28]. Furthermore, the combination of macrolide antibiotics (erythromycin, troleandomycin, and clarithromycin, but not azithromycin) is able inhibit the clearance of methylprednisolone by the mechanism of drug metabolizing enzyme (CYP3A4) inhibition. This inhibition of metabolism creates a steroidsparing effect in which the patient receives greater benefit by a generalized increased exposure to the corticosteroid. This effect is seen to a lesser degree with prednis(ol)one, because of its lower affinity for the CYP3A4 enzyme [29,30].

corticosteroid therapy in asthma

The next step is to maximize anti-inflammatory and bronchodilator therapy. The recent release of combination inhaled corticosteroids and long-acting b-agonist in a single device both in the United States and Europe has shown profound beneficial effects for asthmatics of varying degrees of severity. A multitude of studies have shown that the addition of longacting b-agonist to low-dose inhaled corticosteroids resulted in increased pulmonary function, decreased daytime and nocturnal asthma symptoms, decreased rescue medicine usage, and decreased mild and severe exacerbations when compared with high-dose inhaled corticosteroids alone [31 – 33]. Furthermore, evaluating a fixed versus an adjustable dosing regimen using the budesonide-formoterol combination Turbohaler (AstraZeneca, Lund, Sweden) in patients experiencing intermittent asthma deteriorations has been undertaken. The results of these studies confirmed that patients on the adjustable regimen experienced fewer asthma exacerbations, less rescue medication use, less nocturnal asthma symptoms, fewer severe asthma exacerbations, and increased likelihood of attaining a well-controlled asthma week than those on a fixed dosing regimen [34 – 37]. The addition of a long-acting b-agonist to an inhaled corticosteroid regimen for uncontrolled asthma seems not only to have additive benefits but also to be synergistic. b2-agonists have been found to enhance nuclear translocation of the GR [38], whereas oral GCs have been shown to enhance b2-adrenergic receptor expression [39,40]. In patients who have not tried leukotriene-modifying agents or theophylline, yet continue to display poorly controlled symptoms, one might consider a trial of these agents in addition to the patient’s current regimen because they have been shown to possess steroid-sparing effects [41,42]. Following maximization of therapy, an evaluation of systemic GC pharmacokinetics and assessment of lymphocytic corticosteroid sensitivity (pharmacodynamics) may be considered in those patients with uncontrolled asthma. Performing pharmacokinetics helps determine if the asthmatic patient is absorbing and eliminating the GC in an aberrant way, giving insight into the patient’s overall exposure to the GC. Pharmacodynamic testing involves isolation of lymphocytes and stimulation of these cells with phytohemaggluttin. Lymphocytes are subsequently tested against several different GCs at a range of concentrations to determine the IC50. The IC50 helps determine the patient’s lymphocyte responsiveness to the GC being tested, giving insight to the patient’s overall response to the GC [16,43,44]. Measurement of plasma cortisol levels may also be used to assess compliance. These studies are most useful in patients

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who lack typical or expected steroid-adverse effects despite long-term high-dose corticosteroid therapy. In patients who demonstrate poor gastrointestinal absorption of oral prednisone, a liquid oral steroid preparation should be considered. In those patients found to have rapid corticosteroid elimination a split-dosing regimen may be incorporated with two thirds of the patient’s total daily dose being given in the morning and one third of the patient’s total daily dose being given in the afternoon. In addition, the practitioner may consider the use of methylprednisolone orally, which has a greater penetration and retention in the lung tissue [45] and a high volume of distribution [46,47]. For patients identified as having decreased in vitro sensitivity to a given GC, changing to an alternative GC may be considered; increasing the dosage of the original GC is another option. The following step in evaluating these patients involves assessing for the presence of persistent tissue inflammation despite treatment with high-dose GCs. This may be carried out through an evaluation for biomarkers known to be associated with inflammation including exhaled nitric oxide and plasma eosinophilic cationic protein [48]. This particular method of analysis may be most useful if conducted before and after a 7- to 14-day course of oral prednisone therapy. Persistently increased levels of markers of inflammation demonstrate a failure to respond to GCs and provide strong evidence for the diagnosis of SR asthma and the use of alternative therapies [49]. Although there is considerable debate on how exhaled nitric oxide values should be interpreted, it is generally accepted that exhaled nitric oxide levels greater than 30 ppb (at a flow rate of 50 mL/s) are considered indicative of persistent inflammation in the lungs. Comprehensive guidelines on the testing and interpretation may be seen in recently published American Thoracic Society guidelines [50]. Consideration of the use of alternative antiinflammatory and immunomodulatory agents should be the final step in the approach to the uncontrolled asthmatic. For patients with the rare type II form of SR asthma associated with generalized GC resistance, this is of particular importance but may also apply to patients with poorly controlled type I SI asthma. There have unfortunately been no well-controlled specific studies evaluating the use of these alternative therapies in SR asthma. Treatment with anti-IgE (omalizumab), intravenous immunoglobulin, and cyclosporine has been reported to have steroid-sparing side effects and is potentially useful in patients in whom steroid therapy fails [51 – 53]. Response to this set of treatments, however, is highly variable. The rationale for the use of cyclosporine comes from

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an in vitro study in which Corrigan and coworkers [54] provided limited information that T cells from SR asthmatics may actually respond to the immunosuppressive actions of this agent. In addition, a more recent study demonstrated that treatment of steroid-dependent asthmatics with intravenous immunoglobulin was associated with an increase in GR binding affinity [53]. The primary concern regarding SR asthmatics arises not simply from their day-to-day control but from the fact that they are at an increased risk of morbidity and mortality because of asthma and the adverse effects of therapy [5]. It is of paramount importance for the clinician to realize that these patients do respond to bronchodilator therapy; these medications should be instituted as early as possible in the course of an exacerbation. Additionally, type I SR asthmatics often require much higher doses of intravenous GCs to attain control of their asthma exacerbations than their steroid-sensitive counterparts. Lastly, type II SR asthmatics (a very rare form of asthma) on rare occasion may respond to highdose GCs, but may also require alternative antiinflammatory therapy in addition to aggressive bronchodilator therapy to attain control of their exacerbation. The key, as with all asthmatics, continues to be early intervention in the face of any exacerbation. A new and potentially useful medication in patients suffering from SI asthma is anti-IgE therapy. Al-

though the exact role of this therapy in the management of asthmatics remains to be determined, it is clear that a potential role lies in the treatment of patients with SI or SR asthma who have exhausted most other therapeutic options.

Molecular mechanisms Glucocorticoid action The process of how GCs exert their antiinflammatory effects is complicated and involves several signaling, enzymatic, and genomic pathways. The GR is mainly a cytosolic protein that, when not bound to ligand, remains in a large protein complex to prevent the GR from entering the nucleus. The protein complex includes the GR; several heat shock proteins (70 and 90); and other related proteins. Once a GC enters the cell, it binds to the GR, phosphorylating it, breaking-up the complex, and allowing it to enter the nucleus. The new GR-ligand complex forms a homodimer (which is required for most of its interaction with the nuclear material; however, some interaction is allowed without dimerization), and when interacting with GC responsive elements, then inhibits the transcription of inflammatory proteins including tumor necrosis factor-a (TNF-a), nuclear

Fig. 2. A schematic representation of the glucocorticoid effector pathway describing activation and mechanism of glucocorticoid action. GC, glucocorticoids; GRa, glucocorticoid receptor alpha (ligand binding isoform); GRE, glucocorticoid responsive element; Hsp, heat shock protein (70 and 90); mRNA, messenger RNA; P, phosphate group; Pol II, RNA polymerase II; TAFs, TATA binding protein (TBP) associated factors; TATA, transcription initiation site; TFIIs, transcription elongation factors.

corticosteroid therapy in asthma

factor kappa B (NF-kB), and interleukin (IL)-1 to -6, -11, -13, and -16 [5], or may induce the transcription of anti-inflammatory proteins including lipocortin-1 and IL-1 receptor decoy by the acetylation of lysine residues on histone complexes in the chromatin [55]. Schematic representations of the pathways through which GCs affect cells and through which NF-kB and GCs interact can be seen in Figs. 2 and 3, respectively. The GR may physically block the binding of nuclear factors that up-regulate inflammatory proteins, including NF-kB, and activating protein-1. More recently, it has been shown that GCs exert their anti-inflammatory actions upstream, by the mitogen activating protein kinase (MAPK) pathway [56]. Several molecular mechanisms have been investigated for GC resistance. This section focuses on the ones that are thought to be most influential, and most thoroughly investigated. Such mechanisms include the b-isoform of the GR, NF-kB pathway, and the MAPK pathway. Glucocorticoid receptor isoforms and genetics The schematic of the GR structure is shown in Fig. 4. The GR protein is comprised of the transactivation domain, DNA-binding domain, and the

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hormone-binding domain. The different isoforms of the GR, a and b, are derived from the hormonebinding domain. As the GR undergoes transcription, alternative splicing may occur in exon 9, giving formation to the a or b mRNA, and the respectively expressed protein. The different isoforms of the GR are identical up until amino acid 727; the a isoform has 50 additional amino acids that bind ligand, whereas the b isoform has 15 additional amino acids that do not bind ligand. Because the GR gene contains information for both a and b isoforms, different tissues or cell types may express each isoform differently. The b isoform associates with steroidresistant and fatal asthma [57 – 60], is expressed in different amounts in different cell types, and exists primarily in the nucleus [61,62]. The GR is susceptible to down-regulation after ligand binding. On ligand binding, the a isoform’s half-life decreases from 24 hours to 10 hours, whereas the half-life of the b isoform, which does not bind ligand, remains stable at 48 hours [63]. The b isoform may contribute to SR-SI by mechanisms other than simply not binding ligand. The b isoform also interferes at a cellular level with the actions of the GRa isoform [61,62,64 – 66]. The b isoform has been shown to have a dominant-

Fig. 3. A diagrammatic representation of the nuclear factor kappa B pathway, describing activation, and nuclear factor kappa B dynamic with the ligand-bound glucocorticoid receptor. GCR, glucocorticoid receptor; IkB, inhibitor of NFkB; IKK, IkB kinase; IL-1, interleukin-1; KARY, karyopherin chaperone protein; LPS, lipopolysaccharide; NFkB, nuclear factor kappa B; NIK, NFkB-inducing kinase; TNF, tumor necrosis factor.

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Fig. 4. Schematic of the glucocorticoid receptor (GR) isoforms, a and b. DBD, DNA binding domain; HBD, hormone binding domain; t1, t2, transactivation domains. (Adapted from Oakley RH, Madhabananda S, Cidlowski JA. The human glucocorticoid receptor beta isoform. J Biol Chem 1996;271:954; with permission.)

negative effect on the a isoform, with several mechanisms proposed, including competing for GC responsive elements, and formation of a b inactive heterodimers [67]. Very recently, the dominantnegative property of the b isoform over the a isoform has been shown not only to be dose dependent, but also steroid specific [67]. b Isoform expression in many tissue cells is increased by stimulation of proinflammatory cytokines. NF-kB is a transcription factor that induces the expression of TNF-a, which consequently activates NF-kB contributing to propagation of inflammation. Webster and coworkers [68] have shown that in HeLaS3 and CEMC7 cells (lymphoid line), NF-kB, following stimulus by TNF-a, is able to bind to the GR promoter region and induce GR transcription. Moreover, this specific transcription results in a twofold increase in GRb mRNA and a 1.5-fold increase in GRa mRNA expression [68]. Lastly, they found that SR in these cells was likely caused by cellular accumulation of the dominant isoform (GRb), which is not subject to normal negative down-regulation in the presence of GC and perpetuates its own survival during GC administration. Once the GR complex interacts with the GC responsive elements, it disassociates from the chromatin, followed by the GC breaking away from the GR. The GR either remains in the nucleus or is transported outside of the nucleus back to the cytosol, where it is recycled [5]. This process involves several shuttling proteins, which may be influenced to alter their function, resulting in changes in GR processing and hence GC responsiveness [69,70]. Currently, five single nucleotide polymorphisms have been identified as having functional consequence in the GR. These single nucleotide polymorphisms translate into the following protein changes: arginine to lysine at position 23 (Arg23Lys); Asn363Ser; Ile559Asn; Asp641Val; and Val729Ile. Investigation of these amino acid changes, in concert with the a and b isoforms of the GR, has resulted in

variable in vitro and in vivo responses to GCs, and variable in vitro responses to NF-kB and activating protein-1 transcription repression [71 – 76]. Another possible contributor to GC resistance includes decreased histone acetylation, which has been observed in asthmatics who are steroid sensitive, resulting in a decrease in the transactivation of anti-inflammatory proteins [77]. Nuclear factor-kappa B NF-kB includes a family of proteins that primarily exist in the heterodimer form and contain transactivation domains for gene activation. With inflammatory diseases, such as asthma, NF-kB is comprised of p50 and p65 subunit heterodimers. This form of NF-kB is involved in the transactivation of IL-1, IL-2, IL-6, granulocyte-macrophage colony – stimulating factor, TNF-a, inducible nitric oxide synthase, and cyclooxygenase-2, which are of concern in the pathogenesis of asthma [68,78,79]. The activation of NF-kB requires transactivation by IL-1 and TNF-a. NF-kB is complexed by the inhibitor protein, IkBb. TNF-a, IL-1, lipopolysaccharide, or NF-kB inducing kinase activate the IkB kinases a,b,g. The IkB kinases then phosphorylate IkB, and the complex breaks apart (with subsequent IkB degradation), allowing for nuclear entry, and inflammatory gene induction by NF-kB (see Fig. 3 for a schematic representation of the NF-kB pathway) [80]. GCs are observed to attenuate the actions of NF-kB by at least two mechanisms: GCs increase the expression of IkBb [81], and by physically blocking the p65 subunit of NF-kB from binding to GC responsive elements. This relationship is reciprocal, because NF-kB also blocks GCs from binding to GC-responsive elements, reducing their anti-inflammatory effect [68]. It has also been proposed that GCs and the p65 subunit of NF-kB may also compete for a common transcriptional cofactor [82]. In the case of NF-kB con-

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tributing to GC resistance, Bantel and coworkers [83] observed an increase in constitutive expression of NF-kB in epithelial cells of GC-resistant patients with Crohn’s disease.

Mitogen activating protein kinase pathway The MAPK pathway is an intracellular signaling cascade, which is involved in several biologic processes. In asthma and other inflammatory diseases, activation of the MAPK pathway ultimately results in the expression and production of many proinflammatory mediators. This cascade is considered as another mechanism by which GCs exert their anti-inflammatory effects, although it is not specific to GC action. MAPK kinase is activated by TNF-a, IL-1, lipopolysaccharide, and UV radiation. Once activated, MAPK kinase then phosphorylates certain types of MAPK (p38, c-Jun N-terminal kinase [JNK]); these MAPK enzymes in turn activate MAPKactivated protein kinase enzymes, which stabilize mRNA that contain adenosine-uridine – rich elements. The stabilized adenosine-uridine – rich elements then allow for translation of proinflammatory proteins. GCs demonstrate their anti-inflammatory actions by activation of MAPK phosphatases [56]. The MAPK (p38 and JNK) are inactivated by removal of the phosphate groups by MAPK phosphatases [56]. MAPK p38 participates in the expression of NF-kB, and MAPK JNK participates in the expression of activating protein-1. The MAPK enzyme systems are up-regulated in inflammatory diseases, including asthma [84,85]. The MAPK pathway can induce SI. Szatma´ry and coworkers [86], observed a modest inhibition in GR-mediated signaling by p38 and JNK MAPK when HeLa cells were treated with TNF-a, which is suggested to contribute to the antagonistic relationship between TNF-a and GC. Additional supporting evidence for the MAP kinase pathway altering response to GCs was reported by Tsitoura and Rothman [87], who treated naive CD4+ T lymphocytes with anti-CD28, anti-CD23, and IL-2 in the presence of dexamethasone. CD28 and IL-2 signals were transduced by the MAPK family. The investigators observed a decrease in cell suppression in response to dexamethasone, which was caused by the JNK and extracellular signal-regulated kinase MAPK enzymes. These examples of molecular mechanisms of SI-SR underscore the complexity in which GCs exert their anti-inflammatory action and the many pathways for cellular resistance to GCs. Further re-

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search into the processes described previously will lead to more insight into identifying discrete mechanisms of GC resistance, and the possible discovery of more contributing enzymatic systems.

Contributing factors Allergen exposure Investigators studying the effects of ragweed pollen on GR binding affinity found a reduction in peripheral blood mononuclear cells’ (PBMCs) binding affinity for the GR after the ragweed season when compared with before the season [88]. Spahn and coworkers [24] demonstrated a significant decrease (P = .002) in serum eosinophilic cationic protein in 10 out of the 12 poorly controlled chronic asthmatics with ongoing allergic inflammation studied, who underwent a high-dose course of oral GCs and a marked improvement in GR binding affinity (P < .001) in 11 out of 12 of the subjects following the steroid course. Microbial superantigens There is significant evidence accumulating regarding the role that microbial superantigens play in the induction of SI in human PBMCs. Hauk and coworkers [89] stimulated PBMCs from seven healthy subjects with several prototypical superantigens. Essentially, superantigens are antigens that may bind to the type II major histocompatibility complex on antigen-presenting cells without being processed beforehand. This leads to prompt T-cell activation. After stimulating PBMCs with superantigens, the investigators then assessed the ability of dexamethasone to inhibit stimulation or proliferation of these PBMCs by immunocytochemistry. The investigators found that microbial superantigens, such as staphylococcal enterotoxin-B, toxic shock syndrome toxin-1, and staphylococcal enterotoxin-E, induced significantly less suppression of PBMC proliferation compared with phytohemaggluttin (P < .01) when exposed to dexamethasone (110 6 M), confirming the generation of SI by these microbial superantigens. Furthermore, stimulation of these normal PBMCs with staphylococcal enterotoxin-E produced a significant increase in GRb isoform expression versus phytohemaggluttin and unstimulated cells (P < .01), suggesting a mechanism (increased GRb isoform expression) for the new-found SI in the subjects’ PBMCs [89].

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Neutrophilia The idea of a high neutrophil presence in the pulmonary tissue of severe, difficult-to-treat asthmatics was given substantial support when Wenzel and coworkers [90] examined the bronchoalveolar lavage fluid from patients with severe asthma who were oral steroid-dependent, and compared these specimens with those from patients with moderate asthma and normal controls. The severe, oral steroid – dependent asthmatics had the highest concentration of neutrophils in the bronchoalveolar lavage fluid than both other groups and demonstrated higher levels of leukotriene B4 (induces neutrophil chemotaxis) and thromboxane (promotes airway hyperreactivity) than the moderate asthmatics or the normal controls. It has been established that neutrophils are constitutively SR. One mechanism for resistance is by increased GRb isoform expression in the neutrophil cytosol as demonstrated by Strickland and coworkers [91]. This concept is still somewhat controversial, however, because neutrophils may be left in the tissues after steroid-sensitive cells have left the area (eosinophils, T cells). Sinusitis It has been known that a relationship exists between rhinosinusitis accompanied by nasal polyps and severe asthma. Many details pertaining to the exact nature of this relationship remain to be determined. What has been demonstrated is that the GRb isoform expression inversely correlated with steroid responsiveness when reduction in tissue eosinophils (by staining) and reduction in immunostaining for endothelial vascular cell adhesion molecule-1 and CCL5 (RANTES) were used to demonstrate steroid responsiveness in the prebiopsy and postbiopsy nasal polyp specimens. Hamilos and coworkers [92] showed that despite chronic treatment with fluticasone, nasal polyp biopsies obtained 1 week before therapy was initiated and 4 weeks after treatment termination showed marked signs of SR including increased GRb isoform expression, reduced staining for vascular cell adhesion molecule-1, and RANTES when comparing the prebiopsies with postbiopsies. Nocturnal asthma A recent finding central to the understanding of nocturnal asthma is that PBMCs from patients with nocturnal asthma seem to demonstrate diminished GC responsiveness at 4 am versus 4 pm [93]. Kraft

and coworkers [94] demonstrated diminished suppression of lymphocytic IL-8 and TNF-a production in patients at 4 am versus 4 pm with nocturnal asthma who also showed significantly increased IL-13 expression by bronchoalveolar lavage macrophages. The authors may have identified a means by which the pulmonary macrophage through increased IL-13 production perpetuates nocturnal airway inflammation and diminished steroid responsiveness. Fatal asthma SI may be a contributing factor to fatal asthma because many of these patients die in the emergency department despite receiving high doses of intravenous GCs. Christodoulopoulos and coworkers [59] observed GRb isoform expression in lung tissue from seven patients who died of fatal asthma, six who died of emphysema, and eight who died from nonpulmonary diseases, with significantly higher numbers of GRb immune cells in the patients with fatal asthma than all other groups. GRb expression was increased in both the large and small airways of these patients. This study suggests a link between decreased steroid responsiveness and fatal asthma, which could possibly be secondary to increased inflammation.

Summary As new pathways into the field of difficult-to-treat asthma continue to be identified and new insights as to the underlying mechanisms of decreased steroid responsiveness gained, the challenge of predicting who responds to GCs and who requires alternative therapy becomes increasingly important. Insofar as predictors of responsiveness to inhaled GCs in children are concerned, recent studies [95] have identified several key features of asthma, which may correlate with an increased likelihood of responding to inhaled GCs. A study from the Childhood Asthma Research and Education Network evaluated the response to inhaled corticosteroid therapy in children 6 to 17 years of age with mild-to-moderate asthma. Lower prebronchodilator FEV1% of predicted, lower prebronchodilator FEV1 – forced vital capacity, lower PC20, higher serum eosinophilic cationic protein, and higher total serum IgE level were all significantly predictive (P < .01) of patients responding more favorably to inhaled fluticasone versus oral montelukast, a leukotriene receptor modifier. Previously, a similar group of investigators sought to identify whether predictors of responsiveness to GCs

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could also be identified for adults through the Asthma Clinical Research Network. In this study, Szefler and coworkers [96] examined asthmatics aged 18 to 55 years of age. Thirty patients were initially enrolled with 26 completing the full study. Subjects were treated with beclomethasone dipropionate metered dose inhaler or fluticasone propionate metered dose inhaler for 18 weeks followed by 3 weeks of high-dose fluticasone propionate dry powder. The investigators found that 5 out of 12 patients in the beclomethasone dipropionate group and three out of nine patients in the fluticasone propionate group had a ‘‘good response’’ to therapy, as determined by a > 15% improvement in FEV1. Higher median exhaled nitric oxide levels, greater median maximal bronchodilator reversibility, and lower median FEV1 – forced vital capacity pretreatment, were all found to be predictors of a ‘‘good pulmonary response’’ to GC treatment in this study. Further studies are needed to validate these potential predictors of steroid response in both children and adults. A recent report by Smith and coworkers [97] suggests that measurements of exhaled nitric oxide can be useful in guiding treatment with inhaled corticosteroids for asthma. Insights regarding response to GC therapy will continue to emerge with careful investigation of individual response to treatment. The challenge is then to apply these measures to clinical practice to predict and monitor response to treatment. The benefits of such an approach are to limit the escalation of therapy for patients who are unlikely to respond to GCs and limit the risk of adverse effects. Another advantage of pursuing studies in this area will be the discovery of new forms of treatment or new medications that are more helpful in achieving maximum response to a selected treatment regimen.

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