Abstract
We reasoned that a prospective assessment of glucocorticoid withdrawal in subjects with asthma would provide insight into the basis for flares of the disease. We therefore enrolled 25 subjects with moderate persistent asthma and treated them for 30 days with inhaled fluticasone propionate (1,760 μg/day) followed by a withdrawal period that lasted until peak expiratory airflow decreased by 25% and FEV1 by 15% or 6 weeks elapsed. After glucocorticoid withdrawal, 13 of 25 subjects reached the target, whereas 12 subjects did not. The number of eosinophils in bronchial biopsies was increased by glucocorticoid withdrawal in both groups, but increases in airway T cells were found in only those with exacerbation. T-cell accumulation was a reflection of similar increases in both CD4+ and CD8+ T cells and was accompanied by increased expression of chemokine CCL5 (regulated upon activation, normal T cell expressed and secreted) in the airway epithelium without activation of the transcription factor nuclear factor-κB. The pattern of glucocorticoid-sensitive inflammation during an asthma exacerbation is more reminiscent of an antiviral response than an eosinophil-predominant response to allergen and implies an independent role for airway T cells in mediating asthma flares and in determining glucocorticoid efficacy in the treatment of this disease.
Despite widespread use of glucocorticoids to treat inflammatory diseases, the precise mechanism of action for this treatment remains uncertain. In the case of asthma treatment, glucocorticoids are delivered directly to the airway, and thus, immediate cellular targets include immune cells (e.g., T cells, macrophages, mast cells, and eosinophils) and resident parenchymal cells (e.g., airway epithelial and smooth muscle cells). In these cell types, the glucocorticoid action appears to proceed via the glucocorticoid receptor and its capacity to alter gene transcription (1). However, the precise cellular and molecular targets still need to be determined.
We reasoned that the action of glucocorticoids in asthma would be better understood if the precise inflammatory events that are sensitive to treatment were more fully defined in vivo. We further reasoned that flares of asthma are often triggered by treatment changes without clinically evident exposure to allergen or viral infection. Previous attempts to define the action of glucocorticoids have often used placebo compared with treatment groups (2–8). In recent studies (including those from our group), subjects have been compared with themselves before and after glucocorticoid treatment (9–13). However, several of these studies did not assess airway inflammation directly at the level of airway tissue, and even when this was done, the level of airway inflammation was not characterized completely and correlated precisely to airway function.
For this study, we developed a protocol for glucocorticoid withdrawal in asthma that allows for natural variations of disease and includes measurements of airway function and endobronchial biopsy and lavage during treatment and then after withdrawal of inhaled glucocorticoids using subjects as their own control subjects. To monitor airway function, we included measurements of subject symptoms as well as airway obstruction and reactivity. To monitor inflammation, we assessed immune cell accumulation and activation as well as alterations in airway epithelial pathways that have been implicated in asthmatic inflammation but not yet defined as sensitive to withdrawal of glucocorticoid treatment (14). We also paid special attention to signaling pathways that depend on nuclear factor-κB (NF-κB) as well as downstream gene targets such as the chemokine CCL5 (regulated upon activation, normal T cell expressed and secreted [RANTES]), as these may mediate airway inflammation in this setting (11, 15, 16). We especially aimed to determine how glucocorticoid-sensitive inflammation compares with other stimuli (especially allergy) and whether any specific aspect of the response might predict which subjects would be sensitive to discontinuing treatment. Despite previous suggestions that eosinophil recruitment and activation may be critical for the asthma pathogenesis and may be the target for glucocorticoid action (10, 17), we found that alterations in the other immune cell populations, that is, CD4+ and CD8+ T cells, correlated more closely with worsening asthma symptoms and associated alterations in airway obstruction and reactivity.
The extended version of the methods is available in the online supplement. Twenty-seven subjects (19 females and 8 males) with moderate persistent asthma who required treatment with inhaled glucocorticoids (18) were recruited using informed consent for a protocol approved by the University Human Studies Committee. The diagnosis of asthma was based on demonstration of reversible airway obstruction (i.e., 12% increase in FEV1) and was confirmed by airway hyperreactivity to methacholine (i.e., provocative concentration of methacholine producing a 20% drop in FEV1 [PC20] of 8 mg/ml or less) (19). Prick skin tests were performed with a panel of 28 regional allergens as well as negative and positive control reagents (14). Baseline characteristics for the 25 subjects completing the study are provided in Table 1
Characteristic | Value (n = 25) |
|---|---|
| Age ± SD (range), yr | 41 ± 14 (23–72) |
| Sex, male/female | 8/17 |
| Atopy | 17 |
| FEV1 ± SD, L/min | 2.60 ± 0.83 |
| FEV1% predicted ± SD | 83 ± 16 |
| FEV1% predicted, range | 41–111 |
| FEV1 PC20 ± SD (range), mg/ml | 1.4 ± 2.0 (0.2–7.5) |
At study entry, subjects discontinued previous medications and began treatment with inhaled fluticasone propionate at a dose of 1,760 μg/day for 30 days and albuterol as needed to control symptoms. After 30 days of treatment, each subject underwent spirometry, airway reactivity testing, and then bronchoscopy with endobronchial biopsy and brushing as described previously (14). After recovery from this procedure, subjects discontinued fluticasone and continued inhaled albuterol as needed to control symptoms. The withdrawal period lasted until the afternoon PEF decreased by 25% and FEV1 by 15% or 6 weeks elapsed. At the end of the withdrawal period, each subject underwent repeat spirometry, reactivity testing, and endobronchial biopsy and brushing. Primary study outcome consisted of the presence or absence of exacerbation, but other outcome measures included asthma symptom score, daily rescue medication use, airway function (PEF, FEV1% predicted, methacholine reactivity) as well as airway inflammation (levels of tissue immune cells, CCL5 expression, and NF-κB activation).
Biopsy tissues were frozen in Tissue-Tek Optimal Cutting Temperature (Sakura Finetek USA, Torrance, CA) or fixed in 10% neutral-buffered formalin and embedded in paraffin. Formalin-fixed tissue was prepared as previously described (14) and then incubated with anti-human CD3 monoclonal antibody (mAb) clone PS1 (20), anti-human CD68 clone KP1 (21), anti-human tryptase mAb (22), or anti-human EG2 mAb (23), each at 1 μg/ml. Primary antibody binding was detected with biotinylated horse anti-mouse IgG antibody, streptavidin-conjugated alkaline phosphatase, and red chromogenic substrate. T-cell subsets were assessed using double immunostaining with anti-human CD3 mAb clone SP7 (Biogenex Laboratories, San Ramon, CA) and anti-human CD4 mAb clone C/1F6 (Biogenex Laboratories) or anti-human CD8 mAb clone BC/1A5 (Biogenex Laboratories), each at 1 μg/ml. For each biopsy section, the number of immunopositive cells per mm2 of tissue was quantified by two independent reviewers in a blinded manner. Tissue sections were also subjected to immunofluorescence microscopy for major basic protein or NF-κB as described previously (24). Immunofluorescence was scored for cellular major basic protein (0 = no cells, 4 = more than 100 cells/mm2) and extracellular major basic protein (0 = no extracellular deposition, 4 = massive confluent areas of deposition).
Tissue sections were subjected to in situ hybridization for CCL5 (RANTES) mRNA as described previously (9). In brief, a human CCL5 cDNA fragment (nucleotides 1–410) was cloned into pBluescript to generate CCL5 riboprobe. Radiolabeled 35S-UTP sense and antisense cRNA transcripts were transcribed in vitro by T3 and T7 RNA polymerases, respectively, using the Gemini Riboprobe system. To quantify in situ hybridization levels, at least three separate sections were assigned a score using a 0–4 scale by two independent reviewers in a blinded manner.
Endobronchial brushings were used to prepare nuclear extracts for gel mobility shift assays as described previously (25). Binding of NF-κB to DNA was monitored by using a 32P-labeled oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) that contains the NF-κB site. As a positive control for transcription factor activation, we also assessed Stat1 binding to a probe (5′-GGGAAGGCGCGAGGTTTCCGGGAAAGCAGCACCGCCC-3′) with the γ-activation site (25, 26).
Within-group comparisons were made using paired t tests. When the data were not normally distributed, they were transformed before analysis. Correlation analysis was performed using the Pearson test. A p value of less than 0.05 was considered significant. All results are presented as mean ± SD.
From the group of 25 subjects with asthma, 13 met criteria for exacerbation of their disease after glucocorticoid withdrawal, whereas 12 did not. The group as a whole still exhibited a significant increase in asthma symptoms (1.3 ± 1.7, p < 0.01) after glucocorticoid withdrawal that correlated with increased self-reported and dosimeter-monitored albuterol use (r = 0.50, p = 0.03), decreased FEV1 (r = 0.55, p = 0.02), and decreased PC20 (r = 0.55, p = 0.02). The group also showed a significant decrease in PEF (p < 0.01), although this did not correlate with the decline in FEV1 (r = 0.37, p = 0.14). However, the subgroup of subjects with exacerbation exhibited the most marked changes in airway function and concomitant albuterol use (Table 2)
Subjects (−) Exacerbation (n = 12) | Subjects (+) Exacerbation (n = 13) | |||||
|---|---|---|---|---|---|---|
| Physiologic Parameter | (+) GC | (−) GC | (+) GC | (−) GC | ||
| A.M. PEF ± SD, L/min | 407 ± 107 | 389 ± 107* | 429 ± 103 | 374 ± 125* | ||
| P.M. PEF ± SD, L/min | 451 ± 125 | 425 ± 105 | 424 ± 88 | 387 ± 125 | ||
| FEV1 ± SD, L/min | 2.89 ± 0.83 | 2.80 ± 0.82 | 2.91 ± 0.75 | 2.16 ± 0.99* | ||
| FEV1% predicted ± SD | 87 ± 12 | 84 ± 13 | 94 ± 13 | 74 ± 24* | ||
| FEV1% predicted, range | 63–107 | 61–107 | 71–111 | 32–114 | ||
| FEV1 PC20 ± SD
(range), mg/ml | 3.70 ± 5.81
(0.13–16.00) | 2.23 ± 4.46
(0.16–16.00) | 3.43 ± 4.91
(0.05–16.00) | 1.74 ± 3.48*
(0.03–12.00) | ||
Baseline age, atopy, or asthma severity (as measured by FEV1, PC20, asthma symptom score, and albuterol use) was not predictive of an asthma exacerbation when analyzed as a dichotomous outcome. However, when time to exacerbation (days) was analyzed, there was a significant inverse relationship with baseline asthma symptom score in only those that experienced an exacerbation (r = −0.63, p = 0.04). Furthermore, when we analyzed time to the exacerbation, using a Cox proportional hazards model, the baseline asthma symptom score was predictive of an exacerbation (p = 0.03).
Initial analysis indicated that the number of eosinophils (using EG2 staining) and the level of eosinophil degranulation (using major basic protein staining) were increased in bronchial biopsies after glucocorticoid withdrawal (Figure 1)
Figure 1. Increased eosinophil levels in bronchial biopsies in subjects with asthma with and without exacerbation (Ex) after glucocorticoid withdrawal. (A) Endobronchial biopsies were obtained from subjects with asthma during treatment with inhaled glucocorticoid (+ GC) and after glucocorticoid withdrawal (− GC) and were subjected to immunostaining with anti–eosinophil granule 2 (EG2) or anti–major basic protein (MPB) antibodies. Representative photomicrographs are demonstrated for each condition. Control staining with nonimmune IgG gave no detectable signal above background (data not shown). Bar = 20 μm. (B) Corresponding quantitative analysis of results in A. Values for EG2 represent mean of cell number per mm2 of subepithelial tissue for subgroups with and without exacerbation (+ or − Ex) and entire group (Both). Values for major basic protein represent the mean of scoring (0–4 scale) for cellular and extracellular staining intensity. *p < 0.05.
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Figure 2. Increased T-cell levels in bronchial biopsies in subjects with asthma with exacerbation (Ex) after glucocorticoid withdrawal. (A) Endobronchial biopsies were obtained from subjects with asthma during treatment with inhaled GC and after treatment withdrawal and were immunostained with anti-CD3, anti-CD68, or antitryptase antibodies. Representative photomicrographs are shown for each condition. Control staining with nonimmune IgG gave no detectable signal above background (data not shown). Bar = 20 μm. (B) Corresponding quantitative analysis of changes in (A) as described in Figure 1. *p < 0.05.
[More] [Minimize]Further characterization of T-cell infiltration revealed a significant increase in both CD4+ and CD8+ T-cell subsets after glucocorticoid withdrawal (p = 0.01 and p = 0.04, respectively) (Figure 3)
Figure 3. Increased CD4+ and CD8+ T-cell subsets in bronchial biopsies in subjects with asthma with exacerbation (Ex) after glucocorticoid (GC) withdrawal. Endobronchial biopsies were obtained during treatment with inhaled GC and after treatment withdrawal from subjects with asthma with and without exacerbation and were double immunostained with anti-CD3, anti-CD4, and anti-CD8 antibodies. Closed arrows indicate CD3+ immunostaining alone; asterisks indicate CD4+ or CD8+ immunostaining alone, and an open arrow indicates double immunostaining for both CD3+ and CD4+ or CD3+ and CD8+. Representative photomicrographs are shown for each condition. Control staining with nonimmune IgG gave no detectable signal above background (data not shown). Bar = 20 μm. (B) Corresponding quantitative analysis of changes in A as described in Figure 1. Values for CD4+ or CD8+ T cells per mm2 represent double immunostaining CD4+ or CD8+ cells. *p < 0.05.
[More] [Minimize]We next examined which factors might be responsible for T-cell recruitment to the airway tissue after glucocorticoid withdrawal. Previous work indicated that CCL5 is selectively produced by airway epithelial cells at high levels in response to cytokine stimulation or paramyxoviral infection and is necessary for efficient transepithelial migration of T cells in vitro and in vivo (9, 27). In this setting, we found a significant increase in epithelial levels of CCL5 mRNA after glucocorticoid withdrawal (Figure 4)
Figure 4. Increased CCL5 (regulated upon activation, normal T cell expressed and secreted) expression in bronchial biopsies in subjects with asthma with exacerbation (Ex) after glucocorticoid (GC) withdrawal. Endobronchial biopsies were obtained during treatment with inhaled GC and after treatment withdrawal from subjects with asthma with and without exacerbation and were subjected to in situ hybridization for CCL5. Tissue sections were hybridized with 35S-labeled CCL5 antisense cRNA, autoradiographed, and imaged by darkfield microscopy. Hybridization with 35S-labeled CCL5 sense RNA gave no detectable signal above background (data not shown). Bar = 20 μm.
[More] [Minimize]Recognizing that NF-κB may mediate glucocorticoid efficacy in general and may influence expression of CCL5 in particular (11, 15, 16), we next determined the status of NF-κB in this setting. We previously showed that, in contrast to Stat1, activation of NF-κB is not evident in subjects with stable asthma (14), but the status of both factors during glucocorticoid withdrawal still needed to be determined. Indeed, we confirmed that epithelial Stat1 but not NF-κB was activated in this group of subjects with asthma, and we also established that neither factor was altered by glucocorticoid withdrawal. Thus, Stat1 but not NF-κB translocated to the nucleus (based on confocal immunofluorescent microscopy) in samples of bronchial biopsies, and these findings were unaffected by treatment with glucocorticoids (Figure 5)
Figure 5. Activation status of nuclear factor-κB (NF-κB) and Stat1 in airway epithelial cells from subjects with asthma with and without glucocorticoid (GC) withdrawal. (A) Endobronchial biopsies from subjects with asthma during treatment with inhaled GC and after treatment withdrawal were subjected to immunostaining with anti-p65 antibody (Ab) and imaging with laser confocal microscopy (top row) or to immunostaining with anti-p65 Ab (green fluorescence) plus propidium iodide (PI; orange fluorescence) and standard immunofluorescent microscopy (bottom row). As a positive control for p65 nuclear translocation, biopsy tissues were incubated with tumor necrosis factor-α and subjected to the same procedures for immunostaining. Bar = 20 μm. (B) Endobronchial brushings of airway epithelial cells were used to prepare nuclear proteins that were subjected to gel mobility shift assay using radiolabeled probes for NF-κB (left panel) and Stat1 (right panel). As a positive control for NF-κB binding, human tracheobronchial epithelial (hTBE) cells were incubated with tumor necrosis factor-α and subjected to the same assay as well as supershift analysis with anti-p65 Ab. Subjects with asthma exhibited little NF-κB but significant Stat1 binding, and each was uninfluenced by treatment with GC.
[More] [Minimize]The traditional scheme for asthma pathogenesis (often designated the “Th2 hypothesis”) is based on a relative increase in Th2 cellular responses in combination with a decrease in Th1 responses. The consequent alteration in cytokine milieu, with excess Th2 products (e.g., interleukin [IL]-4, IL-5, and IL-13) and decreased Th1 products (e.g., IFN-γ and IL-12), is predicted to drive the asthma phenotype. Evidence for such a shift in the Th1/Th2 balance derives from studies of asthma in cellular and murine models, where T helper cell polarization and allergen dependence of Th2 responses are most clearly defined and from human studies that profile cytokine production and immune cell infiltrate. Thus, in the murine system, IL-4, IL-5, and IL-13 promote Th2-cell differentiation and B-cell–dependent IgE production, tissue eosinophilia, goblet cell hyperplasia, and airway hyperreactivity (28–31). Furthermore, these responses are downregulated by Th1 cytokines such as IFN-γ and IL-12 (32, 33). In humans, asthma is tied to this paradigm by association with atopy and in turn on increased production of IgE and Th2 cell cytokines as well as genetic linkage to polymorphisms in the IgE receptor, IL-4, IL-4 receptor, and IL-13 genes (34–37). Similarly, eosinophils and mast cells are characteristic of asthmatic airway inflammation (38, 39) and may act as critical effector cells at least under some circumstances (40, 41).
However, several lines of evidence in model systems and in humans raise questions for the Th2 hypothesis as a sole explanation for asthma. For example, Th1 as well as antigen-specific Th2 cells may be necessary for initiating the allergic response in mouse models of asthma (42, 43). Furthermore, endpoints of the allergic response, such as airway hyperreactivity and mucus production, may develop without IgE production and eosinophil influx (29, 44, 45). In fact, in human subjects, the development of allergy and asthma is often dissociated as well (46). Furthermore, in model systems and in humans, the Th2 hypothesis does not incorporate the intrinsic abnormality in cellular programming of the airway epithelium toward an antiviral Th1 response (47).
This study bears directly on this previous work and brings new insight into the relationship between the allergic and antiviral responses and the manifestations of asthma. In particular, we show that subjects with asthma exhibit eosinophilia in bronchial biopsies that is sensitive to treatment with inhaled glucocorticoid. However, this change in eosinophil infiltration does not necessarily correlate with other critical aspects of the asthma phenotype, that is, airway obstruction and hyperreactivity. Similarly, we find no relationship of mast cell infiltration under these conditions despite reports that infiltration of airway smooth muscle by mast cells is selectively associated with asthma (48). Instead, we find that the airway infiltration by T cells is sensitive to glucocorticoid withdrawal and that this aspect of inflammation occurs more often in the setting of asthma exacerbation. Moreover, we find that both CD8+ as well as CD4+ T-cell infiltration is associated with asthma exacerbation, and this event is linked to increased epithelial expression of CCL5. As developed later here, each of these findings raises the possibility that critical components of the antiviral response are also glucocorticoid-sensitive components of the asthma phenotype.
In particular, the CD8+ T-cell population is the critical effector for the antiviral response. Activation of CD8+ T cells is characteristic of viral infection, and the loss of this cell population severely compromises host defense, especially against the type of respiratory viruses that have been linked to bronchiolitis and asthma pathogenesis (49). Furthermore, effector memory CD8+ T cells may persist in the airways and retain the potential to mediate recall responses to infection (50, 51). Moreover, nonspecific activation may also trigger CD8+ T-cell recruitment to the airway, similar to that found for CD4+ T cells (52, 53). Indeed, two recent studies also report an association of CD8+ T-cell accumulation in the airways with asthma severity (54, 55). In both reports, the CD8+ T-cell population expresses IL-4 and IFN-γ and does so at a higher ratio than control subjects. Experimental work indicates that virus-specific CD8+ T cells can also switch to IL-5 production (28). Thus, the mechanism whereby CD8+ T cells might alter airway function will require further study but may not follow strict rules for the Th1/Th2 dichotomy that has been proposed for the CD4+ T-cell system in an experimental (mostly murine) or human context.
To refine this pathway for asthma pathogenesis further, it will also be necessary to characterize the mechanism for T-cell recruitment to the airway. These results begin to address this issue by identifying an increase in epithelial CCL5 expression that correlates with exacerbation. This finding is consistent with a model for T-cell traffic that was developed in vitro and in vivo whereby polarized secretion of CCL5 is chiefly responsible for establishing a chemical gradient for transmigration of T cells across the airway epithelium (47). Because other T-cell chemotaxins and cell adhesion molecules are expressed by the epithelium and are increased in asthma, it seems likely that CCL5 works in concert with other factors in this setting. For example, epithelial intercellular adhesion molecule-1 is increased in this setting and its counterreceptor (i.e., CD11a) is upregulated on memory CD8+ T cells when they enter the airway tissue (14, 50). In addition, other mechanisms may be playing a role in the observed increase in T cells such as decreased apoptosis of the T cells, redistribution of the T cells from regional lymph nodes, or altered glucocorticoid receptor expression (56–58).
Nonetheless, the increase in CCL5 expression offers additional insights into mechanisms of asthma. In particular, CCL5 is the most potently induced gene expression found in experimental paramyxoviral infection of mice and respiratory syncytial virus infection of human airway epithelial cells (27, 59). In other systems, CCL5 expression has been attributed to NF-κB–dependent transcription, but in the case of respiratory syncytial virus infection, expression also depends on virus-dependent mRNA stabilization (27). Similar to the case of viral infection, we find no evidence of epithelial NF-κB activation and no change in activation with glucocorticoid withdrawal in asthma, consistent with two previous studies with less complete analysis (11, 13). This finding implies that mRNA stabilization may underlie the increase in CCL5 expression in asthma as well and is therefore consistent with our earlier reports that antiviral pathways are activated in this disease (12, 14). Other studies in animal models indicate that NF-κB activation is critical for eosinophilic airway inflammation (16). However, these results implicated NF-κB activation in immune cells that release IL-5 and eotaxin, rather than abnormalities in the epithelial compartment that we propose drive the current abnormalities in airway behavior.
Although surprising in view of the Th2 hypothesis and allergic paradigm, our findings that eosinophil infiltration does not correlate with physiologic outcomes are not without precedent. For example, in monkeys with Ascaris-induced asthma, a mAb against IL-5, almost completely eliminated eosinophilia and airway hyperreactivity (60). However, a subsequent study in patients with mild asthma showed that a high-affinity humanized IgG1 mAb against IL-5 abolished eosinophils in blood and reduced the number of eosinophils in sputum but had no apparent effect on the allergen-induced late-phase asthmatic reaction or nonspecific airway hyperreactivity (61). Other studies of animal models and humans show that exposure to nonallergic stimuli, such as air pollutants or viruses, may induce the asthmatic phenotype without eosinophilia (47). Nonetheless, some studies demonstrate that increases in sputum eosinophils may predict an asthma exacerbation during glucocorticoid withdrawal (10, 62, 63). However, one of these studies found only moderate predictive value for sputum eosinophil levels unless combined with airway reactivity measurements, and none of these previous studies assessed inflammation directly in airway tissue. Thus, studies, which solely rely on measurement of eosinophils in sputum, may not fully reflect eosinophil behavior in the tissue. Eosinophil degranulation (as reflected by major basic protein release) and apoptosis in tissue might contribute to inflammation that would not necessarily be detected in cellular analysis of sputum. In contrast to these previous studies (10, 63), this study suggests that the baseline asthma severity (as reflected by the asthma symptom score) is predictive of subsequent exacerbation of asthma. In contrast to Leuppi and colleagues (63), we did not find that baseline airway reactivity was predictive of a subsequent exacerbation.
In summary, these results indicate that asthma exacerbations in the setting of glucocorticoid withdrawal are associated with increases in CD4+ and CD8+ T cells but not eosinophil recruitment to the airway or evidence of mast cell activation. The findings therefore serve to dissociate the asthma phenotype from the typical allergic response that is linked to eosinophil influx and/or mast cell activation. The implication is that critical components of asthma pathogenesis lie in other endogenous components of the immune and inflammatory response system. Precisely defining the mechanism of T cell infiltration in the airway is a goal of future experiments, but these results already indicate that the T-cell recruitment occurs in concert with induction of epithelial CCL5 expression. Earlier work indicates that CCL5 expression may be driven by viral replication and mRNA stabilization even without significant increases in NF-κB activation. In addition, there is clear evidence that other aspects of the epithelial immune response (i.e., Stat1 activation, Stat1-dependent gene expression, and IL-12p40 expression) are all activated in asthma but are insensitive to treatment with glucocorticoids (47). The basis for this change in epithelial behavior is not fully defined, but in all cases, the observed changes are similar to those found during the antiviral response, thereby further linking this type of response to asthma pathogenesis. These results therefore further support an altered paradigm in which epithelial immune response genes are specially programmed for innate immunity and abnormally expressed in asthma and are specially oriented to a T-cell effector response. These glucocorticoid-sensitive T cells appear to have an independent and direct role in mediating asthma flares and in determining glucocorticoid efficacy in the treatment of this disease. This system taken in conjunction with the one underlying Th2-dependent allergy has been designated as an epithelial–viral–allergic paradigm (47) and may serve to explain asthma pathogenesis more fully and response to antiinflammatory treatment.
The authors gratefully thank Dr. Kristin Lieferman (Mayo Clinic, Rochester, MN) for assistance in major basic protein immunostaining and David Misselhorn, Mike Henderson, and Karen McBride for assistance with the performance of fiberoptic bronchoscopy.
| 1. | Newton R. Molecular mechanisms of glucocorticoid action: what is important? Thorax 2000;55:603–613. |
| 2. | Trigg CJ, Manolitsas ND, Wang J, Calderon MA, McAulay A, Jordan SE, Herdman MJ, Jhalli N, Duddle JM, Hamilton SA, et al. Placebo-controlled immunopathologic study of four months of inhaled corticosteroids in asthma. Am J Respir Crit Care Med 1994;150:17–22. |
| 3. | Haahtela T, Jarvinen M, Kava T, Kiviranta K, Koskinen S, Lehtonen K, Nikander K, Persson T, Selroos O, Sovijarvi A, et al. Effects of reducing or discontinuing inhaled budesonide in patients with mild asthma. N Engl J Med 1994;331:700–705. |
| 4. | Laitinen A, Altraja A, Kampe M, Lindek M, Virtanen I, Laitinen L. Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid. Am J Respir Crit Care Med 1997;156:951–958. |
| 5. | Oliveri D, Chetta A, Del Donno M, Bertorelli G, Casalini A, Pesci A, Testi R, Foresi A. Effect of short-term treatment with low-dose inhaled fluticasone propionate on airway inflammation and remodeling in mild asthma: a placebo-controlled study. Am J Respir Crit Care Med 1997;155:1864–1871. |
| 6. | Druilhe A, Wallaert B, Tsicopoulos A, Lapa e Silva J, Tillie-Leblond I, Tonnel A, Pretolani M. Apoptosis, proliferation, and expression of Bcl-2, Fas, and Fas ligand in bronchial biopsies from asthmatics. Am J Respir Cell Mol Biol 1998;19:747–757. |
| 7. | Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. Am J Respir Crit Care Med 1999;159:22–30. |
| 8. | Lim S, Jatakanon A, John M, Gilbey T, O'Connor B, Chung K, Barnes P. Effect of inhaled budesonide on lung function and airway inflammation: assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 1999;159:22–30. |
| 9. | Taguchi M, Sampath D, Koga T, Castro M, Look D, Nakajima S, Holtzman M. Patterns for RANTES secretion and ICAM-1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo. J Exp Med 1998;187:1927–1940. |
| 10. | Jatakanon A, Lim S, Barnes P. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 2000;161:64–72. |
| 11. | Hart L, Lim S, Adcock I, Barnes P, Chung K. Effects of inhaled corticosteroid therapy on expression and DNA-binding activity of nuclear factor κB in asthma. Am J Respir Crit Care Med 2000;161:224–231. |
| 12. | Walter M, Kajiwara N, Karanja P, Castro M, Holtzman M. IL-12p40 production by barrier epithelial cells during airway inflammation. J Exp Med 2001;193:339–351. |
| 13. | Wilson S, Wallin A, Della-Cioppa G, Sandstrom T, Holgate S. Effects of budesonide and formoterol on NF-κB, adhesion molecules, and cytokines in asthma. Am J Respir Crit Care Med 2001;164:1047–1052. |
| 14. | Sampath D, Castro M, Look D, Holtzman M. Constitutive activation of an epithelial signal transducer and activator of transcription (Stat) pathway in asthma. J Clin Invest 1999;103:1353–1361. |
| 15. | Ray P, Yang L, Zhang D-H, Ghost S, Ray A. Selective up-regulation of cytokine-induced RANTES gene expression in lung epithelial cells by overexpression of IκBR. J Biol Chem 1997;272:20191–20197. |
| 16. | Yang L, Cohn L, Zhang D-H, Homer R, Ray A, Ray P. Essential role of nuclear factor κB in the induction of eosinophilia in allergic airway inflammation. J Exp Med 1998;188:1739–1750. |
| 17. | Bousquet J, Chanez P, Lacoste JY, Barneon G, Ghavanian N, Enander I, Venge P, Ahlstedt S, Simony-Lafontaine J, Godard P, et al. Eosinophilic inflammation in asthma. N Engl J Med 1990;323:1033–1039. |
| 18. | National Asthma Education and Prevention Program. Expert panel report 2: guidelines for the diagnosis and management of asthma. Bethesda, MD: National Institutes of Health; National Heart, Lung, and Blood Institute; 1997. |
| 19. | Cherniak R. Pulmonary function testing. Philadelphia: W.B. Saunders; 1977. |
| 20. | Campana D, Thompson J, Amlot P, Brown S, Janossy G. The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage. J Immunol 1987;138:648–655. |
| 21. | Parwaresch M, Radzun H, Kreipe H, Hansmann M, Barth J. Monocyte/macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am J Pathol 1986;124:141–151. |
| 22. | Djukanovic R, Wilson J, Britten K, Wilson S, Walls A, Roche W, Howarth P, Holgate S. Quantitation of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry. Am Rev Respir Dis 1990;142:863–871. |
| 23. | Tai P-C, Ackermann S, Spry C, Dunnette S, Olsen E, Gleich G. Deposits of eosinophil granule proteins in cardiac tissue of patients with eosinophilic endomyocardial disease. Lancet 1987;1:643–647. |
| 24. | Peters M, Schroeter A, Kephart G, Gleich G. Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Dermatol 1983;81:39–43. |
| 25. | Look D, Roswit W, Frick A, Gris-Alevy Y, Dickhaus D, Walter M, Holtzman M. Direct suppression of Stat1 function during adenoviral infection. Immunity 1998;9:871–880. |
| 26. | Nelson P, Ortiz B, Pattison J, Krensky A. Identification of a novel regulatory region critical for expression of the RANTES chemokine in activated T lymphocytes. J Immunol 1996;157:1139–1148. |
| 27. | Koga T, Sardiña E, Tidwell R, Pelletier M, Look D, Holtzman M. Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization. Proc Natl Acad Sci USA 1999;96:5680–5685. |
| 28. | Coyle A, Erard F, Bertrand C, Walti S, Pirchert H, Le Gros G. Virus-specific CD8+ cells can switch to interleukin 5 production and induce airway eosinophilia. J Exp Med 1995;181:1229–1233. |
| 29. | Wills-Karp M. Interleukin-12 as a target for modulation of the inflammatory response in asthma. Allergy 1998;53:113–119. |
| 30. | Grunig G, Warnock M, Wakil A, Venkayya R, Brombacher F, Rennick D, Sheppard D, Mohrs M, Donaldson D, Locksley R, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998;282:2261–2263. |
| 31. | Webb D, McKenzie A, Koshknen A, Yang M, Mattes J, Foster J. Integrated signals between IL-13 and IL-4, and IL-5 regulate airways hyperreactivity. J Immunol 2000;165:108–113. |
| 32. | Lack G, Bradley K, Hamelmann E, Renz H, Loader J, Leung D, Larsen G, Gelfand E. Nebulized IFN-γ inhibits the development of secondary allergic responses in mice. J Immunol 1996;157:1432–1439. |
| 33. | Gavett S, O'Hearn D, Li X, Huang S, Finkelman F, Wills-Karp M. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice. J Exp Med 1995;182:1527–1536. |
| 34. | Burrows B, Martinez F, Halonen M, Barbee R, Cline M. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989;320:271–277. |
| 35. | Ying S, Durham S, Corrigan C, Hamid Q, Kay A. Phenotype of cells expressing mRNA for TH2-type (interleukin 4 and interleukin 5) and TH1-type (interleukin 2 and interferon γ) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell Mol Biol 1995;12:477–487. |
| 36. | Kay A. Allergy and allergic diseases: part 1. N Engl J Med 2001;344:30–37. |
| 37. | Ober C, Tsalenko A, Parry R, Cox N. A second-generation genomewide screen for asthma-susceptibility alleles in a founder population. Am J Hum Genet 2000;67:1154–1162. |
| 38. | Broide D, Paine M, Firestein G. Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics. J Clin Invest 1992;90:1414–1424. |
| 39. | Poston R, Chanez P, Lacoste J, Litchfield T, Lee T, Bousquet J. Immunohistochemical characterization of the cellular infiltration in asthmatic bronchi. Am Rev Respir Dis 1992;145:918–921. |
| 40. | Williams C, Galli S. Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice. J Exp Med 2000;192:455–462. |
| 41. | Bandeira-Melo C, Herbst A, Weller P. Eotaxins: contributing to the diversity of eosinophil recruitment and activation. Am J Respir Cell Mol Biol 2001;24:653–657. |
| 42. | Randolph D, Carruthers C, Szabo S, Murphy K, Chaplin D. Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma. J Immunol 1999;162:2375–2383. |
| 43. | Hansen G, Berry G, DeKruyff R, Umetsu D. Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J Clin Invest 1999;103:175–183. |
| 44. | Melhop P, van de Rijn M, Goldberg A, Brewer J, Kurup V, Martin T, Oettgen H. Allergen-induced bronchial hyperreactivity and eosinophilic inflammation occur in the absence of IgE in a mouse model of asthma. Proc Natl Acad Sci USA 1997;94:1344–1349. |
| 45. | Hogan S, Mould A, Kikutani H, Ramsay A, Foster P. Aeroallergen-induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins. J Clin Invest 1999;99:1329–1339. |
| 46. | Holtzman M, Sampath D, Castro M, Look D, Jayaraman S. The one-two of T helper cells: does interferon-γ knock out the Th2 hypothesis for asthma? Am J Respir Cell Mol Biol 1996;14:316–318. |
| 47. | Holtzman M, Holtzman MJ, Morton JD, Shornick LP, Tyner JW, O'Sullivan MP, Antao A, Lo M, Castro M, Walter MJ. Immunity, inflammation, and remodelling in the airway epithelial barrier: the epithelial-viral-allergic (epi-vir-all) paradigm. Physiol Rev 2002;82:19–46. |
| 48. | Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID. Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002;346:1699–1705. |
| 49. | Hou S, Doherty P, Zijlstra M, Jaenisch R, Katz J. Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells. J Immunol 1992;149:1319–1325. |
| 50. | Ely K, Roberts A, Woodland D. Effector memory CD8+ T cells in the lung airway retain the potential to mediate recall responses. J Immunol 2003;171:3338–3342. |
| 51. | Ostler T, Hussell T, Suth C, Openshaw P, Ehl S. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus. Eur J Immunol 2001;31:2574–2578. |
| 52. | Stephens R, Randolph D, Huang G, Holtzman M, Chaplin D. Antigen-nonspecific recruitment of Th2 cells to the lung as a mechanism for viral infection-induced allergic asthma. J Immunol 2002;169:5458–5467. |
| 53. | Ely K, Cauley L, Roberts A, Brennan J, Cookenham T, Woodland D. Nonspecific recruitment of memory CD8+ T cells to the lung airways during respiratory virus infection. J Immunol 2003;170:1423–1430. |
| 54. | O'Sullivan S, Cormican L, Faul JL, Ichinohe S, Johnston SL, Burke CM, Poulter LW. Activated, cytotoxic CD8+ T lymphocytes contribute to the pathology of asthma death. Am J Respir Crit Care Med 2001;164:560–564. |
| 55. | Magnan AO, Mely LG, Camilla CA, Badier MM, Montero-Julian FA, Guillot CM, Casano BB, Prato SJ, Fert V, Bongrand P, et al. Assessment of the Th1/Th2 paradigm in whole blood in atopy and asthma. Am J Respir Crit Care Med 2000;161:1790–1796. |
| 56. | Gagliardo R, Chanez P, Vignola A, Bousquet J, Vachier I, Godard P, Bonsignore G, Demoly P, Mathieu M. Glucocorticoid receptor α and β in glucocorticoid dependent asthma. Am J Respir Crit Care Med 2000;162:7–13. |
| 57. | Gagliardo R, Vignola A, Mathieu M. Is there a role for glucocorticoid receptor β in asthma? Respir Res 2001;2:1–4. |
| 58. | Irusen E, Matthews J, Takahashi A, Barnes PJ, Chung K, Adcock I. p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: Role in steroid-insensitive asthma. J Allergy Clin Immunol 2002;109:649–657. |
| 59. | Tyner J, Uchida Y, Kajiwara N, Kim E, O'Sullivan M, Walter M, Cook D, Danoff T, Holtzman M. Chemokine CCL5 signals required for survival during viral infection. J Allergy Clin Immunol (In press). |
| 60. | Mauser PJ, Pitman AM, Fernandez X, Foran SK, Adams GK III, Kreutner W, Egan RW, Chapman RW. Effects of an antibody to interleukin-5 in a monkey model of asthma. Am J Respir Crit Care Med 1995;152:467–472. |
| 61. | Leckie M, ten Brinke A, Khan J, Diamant Z, O'Connor B, Walls C, Mathur A, Cowley H, Fan Chung K, Djukanovic R, et al. Effects of interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–2148. |
| 62. | Gibson P, Wong B, Hepperle M, Kline P, Girgis-Gabardo A, Guyatt G, Dolovich J, Denburg J, Ramsdale E, Hargreave F. A research method to induce and examine a mild exacerbation of asthma by withdrawal of inhaled corticosteroid. Clin Exp Allergy 1992;22:525–532. |
| 63. | Leuppi J, Salome C, Jenkins C, Anderson S, Xuan W, Marks G, Koskela H, Brannan J, Freed R, Andersson M, et al. Predictive markers of asthma exacerbation during stepwise dose reduction of inhaled corticosteroids. Am J Respir Crit Care Med 2001;163:406–412. |
