Abstract
The role of inflammation in the pathogenesis of severe asthma chronically treated with high doses of glucocorticoids is poorly understood. Despite this, treatment has been aimed at advancing anti- inflammatory and immunomodulator therapy. This study was designed to evaluate both the presence and type of airway inflammation in patients with severe asthma. A prospective bronchoscopic study evaluated 14 severe, high-dose oral glucocorticoid dependent asthmatics. Bronchoalveolar lavage fluid was analyzed for cytology and inflammatory mediators. Endobronchial and transbronchial biopsies were performed in selected patients for morphometric evaluation of macrophage/monocytes, neutrophils, eosinophils and lymphocytes. These results were compared with lavage and endo- and transbronchial biopsy studies in normal controls and patients with moderate asthma. The concentration of eosinophils in bronchoalveolar lavage fluid was highest in the moderate asthmatics not on glucocorticoids, with very little difference between normal controls and severe asthmatics (significant difference among the groups, p = 0.007). In contrast, the severe asthmatics demonstrated a twofold higher concentration of neutrophils in lavage than either the mild-moderate asthmatics, or the normal controls (p = 0.032 among the groups, p < 0.05 between the severe asthmatics and both controls). Similar results were obtained in the endobronchial and transbronchial biopsy specimens, which consistently showed significantly higher numbers of neutrophils in the severe asthmatics than in the control groups. The eicosanoid mediators, thromboxane and leukotriene B4, were also highest in the severe asthma group (differences among the groups, p = 0.019 and p = 0.023, respectively). These findings suggest that inflammation remains in severe symptomatic asthmatics despite treatment with high dose glucocorticoids which may be due to the severity of disease, glucocorticoid treatment, or other as yet undefined factors.
Asthma is increasing in both prevalence and severity throughout the world. Although the most severe asthmatics make up 10% or less of the asthmatic population, they expend up to 60–70% of the health care budget (1). In addition, their disease remains difficult to control, with little pathophysiologic data available to guide treatment options.
The importance of airway inflammation and/or fibrosis in symptomatic asthmatic patients with chronic airflow limitation on high dose glucocorticoids is currently unknown. Almost no clinical, physiologic or pathologic data exist on severe, steroid-dependent asthmatics; i.e., those with ongoing, partially reversible chronic airway obstruction despite glucocorticoid doses of greater than 20 mg/d of prednisone or the equivalent. Bronchoscopic evaluation is now considered a safe and useful procedure for the investigation of mild to moderate asthma. Even in these patients with mild-to-moderate asthma a strong inflammatory component to asthma has been noted, consisting of epithelial disruption, increases in mast cells, lymphocytes and eosinophils, and elevations in both eicosanoid and cytokine mediators (2). In these mild-to-moderate asthmatics, prolonged (6 wk to 6 mo) courses of inhaled steroids reduce airway inflammation (3-5). However, it appears that inflammation may persist at a low level despite moderate glucocorticoid use, as well as after prolonged inhaled dosages of up to 2,000 μg/d (5). In addition, isolated reports suggest that glucocorticoids can induce growth factors and/or collagen synthesis in certain conditions (6, 7), however the effect on pulmonary fibrosis, collagen formation and inflammation in severe asthmatics has not been evaluated.
The current study was designed to evaluate the pathologic and biochemical changes which distinguish airway inflammation in severe, glucocorticoid dependent asthmatics from selected control populations. For this purpose, 14 severe asthmatics with chronic, partially reversible, airway obstruction were evaluated for evidence of airway inflammation despite long-term high dose glucocorticoid treatment.
Fourteen severe, difficult to control asthmatics referred to the National Jewish Medical and Research Center in Denver, Colorado, were evaluated. Complete history and physical, routine laboratory testing and pulmonary function studies were performed on all patients. Subjects were classified with “severe asthma” if they were on the equivalent of 20 mg/d or greater of oral prednisone for greater than 1 yr, with evidence of ongoing reversible airway obstruction (FEV1 < 70% predicted and at least a 15% improvement in FEV1 post-bronchodilator or a positive methacholine challenge study). One patient had been on glucocorticoids but was weaned off when she was felt to be clinically and physiologically unresponsive to them. Pediatric patients (< 18 yr old) had documented evidence for FEV1 < 70% predicted during the previous year, but three of the five were bronchoscoped on high doses of prednisone resulting in FEV1 > 90% predicted. These patients all had < 15% response to bronchodilators, but a positive methacholine challenge with a provocation dose 20 of < 1 mg/ml. Patients were excluded if they had ever smoked, had evidence for a recent upper respiratory infection (URI), or other serious medical illness. Compliance was assessed by history and random early morning plasma cortisol concentration and subjects excluded if the morning plasma cortisol was > 1.0 μg/l. Informed consent for bronchoscopic evaluation was obtained from all subjects and was in accordance with the ethical standards of the institutional review board.
Selected controls for bronchoalveolar lavage (BAL) studies were obtained from a population of moderate asthmatics (n = 12) and normal subjects (n = 6). Biopsy controls were obtained from a second group of moderate asthmatics (n = 11) and normals (n = 6) enrolled in other ongoing asthma studies. No normal controls were available for transbronchial biopsy comparisons. All the asthma control patients were required to have evidence of ongoing reversible airway obstruction (prebronchodilator FEV1 < 85% predicted), but were maintained on β-agonists alone. None had taken glucocorticoids (oral or inhaled) within the last month. The normal subjects were required to have normal pulmonary function and no clinical history of allergic or respiratory disease. Similar to the severe asthmatics, none of the control subjects had a smoking history > 5 pack-years, a recent URI or any other serious medical problem.
Bronchoscopies were performed following previously published American Thoracic Society guidelines (8). All severe asthmatics took their daily dose of glucocorticoid prior to the bronchoscopy. In addition, all severe asthmatic subjects received a nebulized albuterol treatment (0.5 ml of the 0.083% solution) just prior to the bronchoscopy. The controls (including the normals) received an inhalation of either isoproterenol (252 μg) or terbutaline (400 μg) just prior to the bronchoscopy. Further premedication consisted of atropine 0.6 mg intramuscularly and codeine 60 mg intramuscularly. Severe asthmatics and controls were anesthetized with 2% lidocaine aerosolized to the naso- and oropharynx. An intravenous line was placed, supplemental oxygen delivered and patients monitored with pulse oximetry and blood pressure measurements. The bronchoscope (Olympus BF/P30, Lake Success, NY) was then passed through the nasopharynx and additional lidocaine (2%) applied to the vocal cords. After passage through the vocal cords, lidocaine (1%) was utilized as needed throughout the tracheobronchial tree. Midazolam (2–8 mg) and/or fentenyl (25–125 μg) were given to maintain light sedation.
Two pediatric subjects were bronchoscoped under general anesthesia with isofluorane. A rigid bronchoscope was used in one patient and a rigid bronchoscope through which a flexible bronchoscope was inserted was used in the other patient.
BAL was obtained from a subsegment of the right middle lobe in all subjects using four (60 ml) aliquots followed by manual aspiration. Four to six endobronchial biopsies were obtained from the subcarinae of the right lower lobes in 11 patients. Three were studied before biopsy became part of the protocol. Transbronchial biopsies (4-6) were obtained from peripheral tissue in the right lower lobe of six patients under fluoroscopic guidance. No pediatric patients underwent transbronchial biopsy. Controls and severe asthmatics were studied in a similar manner.
BAL samples were collected, placed on ice and then spun at 4° C (600 × g) for 10 min to separate fluid from cells. Cell counts were performed using a hemocytometer and trypan blue exclusion testing. Differentials were obtained on cytospin preparations, using a Diff-Quik™ (Scientific Products, McGraw Park, IL) stain, counting 300 cells. Fluid was processed for eicosanoids using a Sep-pak purification system, stored at −70° C until analysis using enzyme immunoassays (9).
Endobronchial and transbronchial biopsy specimens were collected in formalin. Inflammatory cells were counted using a point counting system and expressed as cells/mm3 (10). Tissue was stained with an HAE stain which allowed identification of macrophages, lymphocytes, eosinophils and neutrophils.
Data were analyzed using the JMP software program (SAS Institute, Cary, NC). As the data were not normally distributed, Wilcoxen's signed rank testing (Kruskal-Wallis modification for multiple comparisons) was used to determine differences among the three groups. When a statistical difference was detected among the groups, the Dunn's test for multiple comparisons was used to determine significance between two specific groups. Significance was accepted at p < 0.05.
Fourteen severe asthmatics were studied and their individual characteristics given in Table 1. Ages ranged from 6 to 48 yr old. All patients had asthma diagnosed for greater than 5 yr, and had been treated with oral steroids for 5 yr or more. They were compared to 23 moderate asthmatic controls on inhaled β-agonists alone and 12 normal controls (Table 1). Although there was a large difference in the medication history of the moderate asthmatics (β-agonists alone) versus the severe asthmatics (long term oral and inhaled glucocorticoids), there was no difference in baseline FEV1 for the BAL studies (Table 1). The moderate asthma controls for the biopsy studies were, in general, slightly less obstructed at 4:00 p.m. (p = 0.03) than the severe group, but there was no significant difference in FEV1 between the two moderate groups. Age did not affect results. Although the control groups were premedicated for the bronchoscopies with inhaled isoproteronol or terbutaline, while the severe asthmatics received nebulized albuterol, the overlapping mechanism of action of these drugs would appear to make the possibility that the β-agonist treatment influenced the results unlikely. All subjects were bronchoscoped within one hour of pretreatment. The mean glucocorticoid dose in the severe asthma group was 39 ± 6 mg/d. All severe asthmatics were using inhaled glucocorticoids with doses ranging from 800 μg/day to 4,000 μg/d, but compliance was difficult to verify. In addition to β-agonists and glucocorticoids, all severe asthmatics were currently taking, or had taken, another anti-asthma drug, such as theophylline or cromolyn. Four of the 14 had previously been on methotrexate, cyclosporin or intravenous gamma globulin. Eight of 14 had a history of near fatal asthma events. Both moderate and severe asthma subjects demonstrated a 15% or greater improvement in FEV1 after bronchodilators or a positive methachline challenge within the 6 mo prior to study (see Table 1).
| Severe Asthma Subjects | Age | Sex | FEV1(% predicted ) | Bronchodilator Response (%) | Current Steroid Dose (mg/day) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 6 | F | 57 | 39 | 80 | |||||
| 2 | 37 | F | 50 | 42 | 20 | |||||
| 3 | 17 | F | 97 | 9 | 20 | |||||
| 4 | 37 | M | 39 | 40 | 40 | |||||
| 5 | 18 | F | 90 | 5 | 20 | |||||
| 6 | 12 | F | 36 | 25 | 48 | |||||
| 7 | 11 | M | 95 | 6 | 40 | |||||
| 8 | 19 | M | 55 | 31 | 50 | |||||
| 9 | 48 | F | 37 | 52 | 36 | |||||
| 10 | 29 | F | 54 | 22 | 40 | |||||
| 11 | 27 | F | 62 | 28 | 0* | |||||
| 12 | 38 | F | 50 | 72 | 40 | |||||
| 13 | 47 | F | 53 | 18 | 40 | |||||
| 14 | 43 | F | 27 | 145 | 25 | |||||
| Mean ± SEM | 28 ± 4 | 11F/4M | 58 ± 6 | 38 ± 10 | 39 ± 6 | |||||
| Controls (mean ± SEM) | ||||||||||
| BAL | ||||||||||
| Normal (n = 6) | 29 ± 2 | 3F/3M | 104 ± 4 | 3 ± 1 | 0 | |||||
| Moderate asthma | ||||||||||
| (n = 12) | 32 ± 3 | 6F/6M | 65 ± 3 | 22 ± 4 | 0 | |||||
| range 44–77 | ||||||||||
| Biopsy | ||||||||||
| Normal (n = 6) | 33 ± 4 | 3F/3M | 103 ± 4 | 2 ± 2 | 0 | |||||
| Moderate asthma | ||||||||||
| (n = 11) | 34 ± 2 | 4F/7M | 75 ± 4 | 19 ± 3 | 0 | |||||
| range 53–92 | ||||||||||
BAL was obtained on all subjects and was well tolerated. No subject required more than one nebulized albuterol treatment post-procedure or prolonged observation. There were no desaturations to < 90% which did not respond to modest increases in liter flow of oxygen. The BAL return was significantly greater in the normal controls (166 ml [146–187 ml]) than either asthmatic group (p < 0.05). The return was less in the severe group (86 ml [66–131 ml]) than in the moderate group (120 ml, range 81–156 ml), but the difference did not reach statistical significance.
Cell counts/differentials. The data for cells/ml and differentials in the three groups are presented in Table 2. The neutrophil and eosinophil data are also graphically presented in Figure 1. Although severe asthmatics had higher cell concentrations, there was considerable overlap among the groups (p = 0.25). However, a significant difference existed among the eosinophil concentrations in the three groups (p = 0.007), with eosinophils greater in the mild asthmatics than in both the normal controls and 2 times greater than the numbers in severe asthmatics. There was also a significant difference for neutrophils among the three groups (p = 0.032). In contrast to eosinophils, neutrophil concentrations from patients with severe asthma were twofold higher than normals or moderate asthmatics. Pairwise comparisons were not significant among the groups for eosinophils only.
| Group | Total Cells (×104 ) | Macrophages (×104 ) | Lymphocytes (×103 ) | Eosinophils*(×103 ) | Neutrophils†(×103 ) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Normal (n = 6) | 10.2 (7.9–11.8) | 9.2 (7.1–10.9) | 6.5 (4.8–10.0) | 0 (0–0.3) | 0.5 (0–1.5) | |||||
| Mild–moderate | ||||||||||
| asthma (n = 12) | 10.0 (7.6–10.8) | 7.8 (7.0–9.2) | 3.0 (1.3–6.9) | 2.3 (1.0–3.8) | 1.0 (0–2.1) | |||||
| Severe asthma | ||||||||||
| (n = 14) | 11.5 (8.2–21.3) | 7.9 (6.6–17.2) | 5.8 (3.9–16.7) | 1.2 (0–2.1) | 2.6 (1.2–8.7) |
Fig. 1. Eosinophil and neutrophil numbers among BAL cells in controls, moderate and severe asthmatics. Neutrophils were significantly increased in severe asthmatics compared with both moderate asthmatics and normal controls. Eosinophils were significantly different among the groups (p = 0.007), but specific intergroup comparisons were not significant.
[More] [Minimize]Despite the high (> 35 mg/day) steroid dose, the eicosanoid mediators (leukotriene [LT] B4 and thromboxane [TX]) remained elevated, with TX levels significantly higher (p < 0.05 for pairwise comparisons) in patients with severe asthma (34 pg/ml BAL fluid [range 9–70 pg/ml]) than either normals (7 pg/ml [3–9 pg/ml]) or mild-moderate asthmatics (11 pg/ml [7– 23 pg/ml]), p = 0.019 (Figure 2b). LTB4 levels were significantly different among the three groups, with severe asthmatics having the highest levels (10 pg/ml, range 5–39 pg/ml) (p = 0.023), but intergroup comparisons were not significant (Figure 2a). Although several severe asthmatics had the highest LTE4 levels measured during the study, the levels were not significantly different among the groups (p = 0.01) (Figure 2c). Similar to LTE4, histamine levels were sporadically highly elevated, but the variability in levels precluded a significant difference from normals. Histamine levels had not been obtained in the BAL fluid from the asthmatic controls (Figure 2d). Interestingly, elevations in one mediator did not predict elevations in a second mediator.
Fig. 2. (a) Leukotriene B4 levels were significantly different among the groups (p = 0.032), with the highest levels measured in severe asthmatics. (b) Thromboxane levels were significantly different among the three groups with significantly higher levels measured in severe asthmatics than either control group, (c) Leukotriene E4 levels were not different among the groups (p = 0.1). (d ) Histamine levels were not different between normals and severe asthmatics, although very high histamine levels were measured in several severe asthmatics.
[More] [Minimize]Endobronchial biopsies. Endobronchial biopsies were obtained from 11 of the 14 asthmatics, with 7 having sufficient tissue for analysis. The endobronchial biopsies were very well tolerated by all the subjects. There were no cases of excessive bleeding, induced bronchospasm or need for prolonged observation. Although considerable variability existed in the level and type of tissue inflammation in the severe asthmatics, an inflammatory cell influx was present in all the patients, despite the high oral steroid dose. Although the difference among groups did not reach significance (p = 0.078), large numbers of eosinophils were only noted in two of seven patients, with the remainder undetectable (Figure 3a). In contrast, neutrophils were consistently present in significantly higher numbers than in the biopsy specimens from the moderate asthmatics or normals (overall p = 0.01, p < 0.05 between severe asthma patients and the two control groups) (Figure 3a). Interestingly, macrophages were significantly increased (p < 0.05) in the moderate asthmatics compared to the severe asthmatics and normals (overall p = 0.02) (Table 3). Lymphocytes were not present in differing amounts in the severe group than in the two control groups. Mast cells were not specifically evaluated in this study.
Fig. 3. (a) Neutrophils were significantly greater in endobronchial biopsy samples from severe asthmatics than either control group. Differences among the groups for eosinophils did not achieve significance (p = 0.078). (b) Neutrophils were significantly increased in transbronchial biopsy specimens from severe asthmatics as compared to moderate asthmatic controls (p = 0.03).
[More] [Minimize]| Group | Macrophages* | Lymphocytes | Eosinophils | Neutrophils† | ||||
|---|---|---|---|---|---|---|---|---|
| Normal (n = 6) | 57.7 (41.9–71.0) | 0 (0–1.5) | 0 (0–0) | 0 (0–0.7) | ||||
| Moderate asthma (n = 11) | 79.4 (65.7–107.7) | 0 (0–1.7) | 1.7 (1.0–30.0) | 0 (0–1.7) | ||||
| Severe asthma (n = 7) | 54.9 (41.9–75.5) | 1.5 (0–1.6) | 0 (0–3.0) | 3.0 (1.6–12.9) |
Transbronchial biopsies. Transbronchial biopsies were obtained from six patients, while five had sufficient distal tissue for analysis. Similar to the endobronchial biopsies, no complications were observed with these studies. Specifically, no pneumothoraces, decreased oxygen saturation or excessive bleeding were observed. Although sampling a different section of the lung, a similar pattern existed as for the endobronchial biopsies (Table 4, Figure 3b). Occasional patients had predominant eosinophils in their tissues, but neutrophils were consistently and significantly elevated compared with the moderate asthmatics (p = 0.03). Similar to the endobronchial biopsies, the macrophages were significantly less in the severe asthmatics than in the moderate asthmatics (p = 0.01).
Our current data demonstrate for the first time inflammation in the airways of asthmatics who have poor airway function despite very high doses of oral and inhaled glucocorticoids. The most consistent cellular finding in the severe asthmatics was the high numbers and percentages of neutrophils on BAL, endobronchial and transbronchial biopsy specimens. These differences were seen in conjunction with significantly lower numbers of eosinophils compared to moderate asthmatics on BAL and on biopsy. Preliminary data also suggest that mediators, including lipid derived and mast cell derived, may remain elevated over normal controls, despite the high doses of glucocorticoids.
The patients evaluated in the current study were all severe asthmatics, referred to the National Jewish Medical and Research Center for evaluation and treatment of their disease. These patients were bronchoscoped in an effort to specifically guide clinical decisions on anti-inflammatory therapy, as all had reached a point where the effects from chronic steroid use were highly debilitating. If inflammation were absent, but fibrosis predominated, aggressive anti-inflammatory therapy could potentially cause more risk than benefit. However, the data reported here suggest that a novel form of inflammation is persistent in the majority of severe asthmatics on high doses of steroids. In this regard, the inflammation present in these severe asthmatics is different from the inflammation seen in moderate asthmatics. Despite the fact that asthma has generally been regarded as a “syndrome of reversible airflow limitation” rather than a singular “disease,” clinicians tend to treat all asthmatics in a similar manner. This is partially due to the paucity of pathophysiologic data which could discriminate certain subgroups from each other. In contrast, restrictive lung diseases are pathologically subtyped into a wide variety of specific diagnoses. However, in asthma, the currently popular belief suggests that most asthma is a T-lymphocyte/eosinophil initiated and driven process. This hypothesis is based upon studies from very selected patient populations, usually mild– moderate allergic asthmatics, often treated with beta agonists alone. The present study would suggest that differences may exist in the inflammatory pattern among individual patients.
Unexpectedly, the only cellular pattern found to be consistently present in BAL and biopsy in the severe asthmatics was the preponderance of neutrophils. These results were consistently seen, no matter which compartment was analysed. At the present time, only limited studies from autopsies of asthmatics who died suddenly of asthma and sputum from asthmatics in status asthmaticus have suggested that the neutrophil may be important in these more severe forms of asthma (11, 12). Although the role of glucocorticoids in the neutrophil predominance of severe asthma is not clear, recent reports have suggested that this class of drugs may enhance neutrophil function through increased LT and superoxide production, as well as inhibition of apoptosis (13, 14). A further preliminary report suggests that treatment of severe asthmatics with oral glucocorticoids for one year dramatically increased the numbers of neutrophils in biopsy and lavage specimens, while effectively eliminating eosinophils (15). No improvement in FEV1 was seen in these asthmatics, and many worsened. It is conceivable that glucocorticoids, in general, could substantially reduce the eosinophil/lymphocyte driven process, while leaving behind, or even augmenting, a neutrophil mediated process. However, the current data do not allow us to determine whether the neutrophils are present because of (1) a different pathologic form of asthma; (2) a direct response to high dose glucocorticoids; or (3) an indirect response to an altered mileau, perhaps infectious, in the airways. These hypotheses await further testing. Finally, it is also conceivable that although the neutrophil is present in the airways, it is not contributing in any way to the pathologic process. Future evaluation of neutrophil markers of activation, such as neutrophil elastase and myeloperoxidase, should help to clarify its contribution to the pathophysiologic changes of severe asthma.
The second important result of the study was the ongoing release of eicosanoids into BAL fluid despite high dose glucocorticoid therapy. It is assumed that glucocorticoids effectively inhibit phospholipases through induction of lipocortins, and hence, decrease prostanoid and LT production (16). However, this induction may be limited to certain conditions and/or cells (17). In fact, numerous studies, both in vitro and in vivo, have demonstrated minimal suppression of LT production by glucocorticoids, and in some cases an enhancement of LT production. Recent studies suggest that in certain situations, glucocorticoids may enhance production of 5 lipoxygenase activating protein which modulates LT production, and may also upregulate cyclooxygenase I in cells exposed to stem cell factor concurrently with glucocorticoids (18, 19). Interestingly, the most consistent finding among the lipid mediators was the elevation of thromboxane. Thromboxane, produced predominantly by platelets and monocyte/macrophages, has been linked to the production of nonspecific airway hyperresponsiveness (20). This continued augmentation of TX levels could limit the improvement in airway reactivity seen after treatment with inhaled glucocorticoids (21). Whether these increases in eicosanoids are secondary to inadequately controlled inflammation, or are in response to the chronic high dose glucocorticoids, remains unknown.
Finally, data from the biopsy studies suggests that macrophages are increased in moderate asthmatics compared to normals and severe asthmatics. The role of the macrophage remains unclear in asthma, but several investigators have suggested that macrophages may have a suppressive effect on inflammation, which may be lost in asthma (22, 23). The normal levels of macrophages in the presence of a neutrophilic inflammation in severe asthmatics could support that hypothesis.
In conclusion, this study is the first to document the pathologic changes present in the airways of chronic, severe, asthmatics requiring high-dose oral glucocorticoids for control of airway function. It suggests that inflammation is present in the lungs of these individuals despite treatment, but that the inflammation in severe asthma may be distinct from the inflammation seen in moderate asthma. These findings are not without limitations, including overlap among the groups and the unclear relationship to type and severity of disease. However, we believe these preliminary data have considerable potential regarding therapeutic implications for the treatment of severe asthma.
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