We have shown in nocturnal asthma that alveolar tissue eosinophils are increased at night as compared with the proximal airway, and that they correlate with the overnight decrement in lung function. As the CD4 + cell is thought to be the principal orchestrating cell in eosinophil recruitment, we evaluated its presence in the proximal and distal airways in nocturnal asthma. Eleven patients with nocturnal asthma (NA) and 10 patients with non-nocturnal asthma (NNA) underwent two bronchoscopies with proximal airway endobronchial and distal alveolar tissue transbronchial biopsy in a random order at 4:00 p.m. and at 4:00 a.m. separated by 1 wk. Immunohistochemical staining and morphometric analysis were used to determine the number of CD3 + , CD4 + , and CD8 + cells and EG2 + eosinophils per mm2 in the epithelium, lamina propria, and alveolar tissue. At 4:00 a.m., the NA group had a significantly greater number of CD4 + cells in the alveolar tissue than the NNA group (9.8 cells/ mm2 [5.6–30.8, interquartile (IQ)] versus 1.5 cells/mm2 [0–6.3, IQ], p = 0.04). Within the NA group, there were significantly greater numbers of CD3 + , CD4 + , CD8 + , and EG2 + cells in the proximal airway lamina propria than in the distal airway at both 4:00 p.m. and 4:00 a.m. There were no differences within the epithelium between the groups at either time point. Only alveolar tissue, not airway tissue, CD4 + cells correlated inversely with the percentage predicted FEV1 at 4:00 a.m. (r = − 0.68, p = 0.0018) and positively with the number of alveolar tissue EG2 + cells (r = 0.66, p = 0.01). These findings suggest that the CD4 + lymphocyte is increased in the alveolar tissue at night in nocturnal asthma as compared with non-nocturnal asthma.
T-lymphocytes are thought to play a pivotal role in the pathogenesis of asthma (1). The ability of eosinophils to migrate from the blood through the vascular endothelium is markedly increased by exposure to inflammatory cytokines produced by T-lymphocytes such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-5, and IL-13 (2-4). In particular, the CD4+ lymphocyte is an important source of these cytokines and possibly chemokines such as macrophage inflammatory protein-alpha (MIP-α), eotaxin, and regulated upon activation in normal T cells expressed and secreted (RANTES), all of which have possible roles in enhancing eosinophils' chemotaxis, survival, maturation, and activation (5, 6). Daytime studies have demonstrated the presence of activated T-lymphocytes in the airways of asthmatics during the day, but only proximal airway tissue has been evaluated (1, 7). We have previously reported that the number of alveolar tissue eosinophils and macrophages are increased at night in patients with nocturnal worsening of asthma, hereby referred to as nocturnal asthma (NA) (8). The increase in eosinophils also correlated with the nocturnal decrement in lung function, suggesting that alveolar tissue inflammation is an important aspect of nocturnal asthma pathogenesis. Although total lymphocyte counts were not increased, identification of particular subclasses of lymphocytes by immunohistochemistry was not performed. In view of the relationship between eosinophil influx into the airways and cytokine release by the T-lymphocyte, we hypothesized that particular subclasses of lymphocytes might be responsible for the recruitment of eosinophils into the alveolar tissue at night in subjects with nocturnal asthma as compared with asthmatics without nocturnal worsening of lung function.
The 21 subjects previously reported (8) and two additional subjects were evaluated in the present study. All had asthma as defined by the American Thoracic Society (9). Among these, 12 patients experienced NA, defined as a documented fall in the overnight peak expiratory flow rate (PEFR) of ⩾ 15% in at least four of seven nights of testing at home prior to study, and 11 subjects had non-nocturnal asthma (NNA), defined as a fall in the overnight PEFR of < 10% each night over seven nights of testing at home prior to study. Subjects who experienced decrements in their overnight PEFR between 10 and 15% were not included in the study. Exclusion criteria included the use of oral or inhaled corticosteroids within 4 wk of study, an upper respiratory infection within 4 wk of study, cigarette smoking of ⩾ 5 pack-years and/or any cigarette use within 3 yr of study, pregnancy and/or significant nonasthma medical problems as determined by the principal investigator. All subjects gave their informed consent to this study, which was approved by the National Jewish Medical and Research Center Institutional Review Board (Denver, CO).
Subjects underwent two bronchoscopies with endobronchial (EBBX) and transbronchial (TBBX) biopsies at 4:00 p.m. and 4:00 a.m. in a random order separated by 1 wk as previously described (8). The location of the biopsies was randomized to the right or left lower lobe for the first bronchoscopy, followed by biopsies in the opposite lobe during the second bronchoscopy. Prior to the bronchoscopies conducted at 4:00 a.m., subjects spent the night in the laboratory and underwent spirometry prior to bedtime. Prior to each bronchoscopy, spirometry was performed before and after 0.18 mg of albuterol from a metered-dose inhaler (MDI). Subjects then received 60 mg of codeine and 0.6 mg of atropine intramuscularly. Xylocaine (4%) was used to anesthetize the upper airway, and xylocaine (1%) was applied to the laryngeal area, trachea, and orifice of the right lower or left lower lobe bronchi via the bronchoscope. Four TBBX were performed under fluoroscopic guidance followed by four EBBX performed under direct visualization from the fourth and fifth generation airways. Supplemental oxygen was administered throughout the procedure along with monitoring of heart rate and oxygen saturation. An expiratory chest radiograph was performed after each procedure to rule out the presence of a pneumothorax. Subjects were monitored in the laboratory for at least 4 h after the procedure. One subject experienced a 10% pneumothorax, which rapidly resolved with conservative treatment.
Tissue samples were fixed in acetone containing protease inhibitors (iodoacetamide, 20 mM; phenyl sulphonyl fluoride, 2 mM) at −20° C overnight and processed into glycol methacrylate resin as previously described (10). Initially, a 2-μm section was cut and stained by a rapid toluidine blue method (11) to assess suitability of biopsies for immunohistochemistry. To qualify as an adequate sample in this study, the lamina propria must have been at least 0.5 mm2, excluding areas of muscle, glands, crush, cartilage, etc. All the cells of interest within a suitable area of a specimen were counted. In addition, all intact epithelia within a given specimen were also counted.
From suitable biopsies, sections 2 μm thick were cut and stained immunohistochemically using primary mouse antihuman monoclonal antibodies and the Strept-avidin biotin peroxidase detection system. The primary antibodies employed in this study were directed to CD3 (UCHT1; Dako, High Wycombe, UK), CD4 (Leu 3a+3b; Becton Dickinson, Oxford, UK), CD8 (DK25; Dako) lymphocytes and to eosinophils (EG2; Pharmacia & Upjohn Diagnostics, Milton Keynes, UK).
In the EBBX sections the number of positively stained nucleated cells were counted separately in the lamina propria and the epithelium. In the TBBX sections the number of positive cells were counted in the alveolar tissue. The area of EBBX lamina propria, the length of the EBBX epithelium, and the area of the TBBX alveolar tissue were measured using a computerized image analysis system and the colorvision software package 1.6.4SR (Improvision, Coventry, UK). The results were expressed as the number of positive cells per mm2 of EBBX and TBBX alveolar tissue. A second observer blinded to subjects' asthma status and time of day counted a random sample of biopsies to determine reproducibility via an intraclass correlation coefficient.
The physiologic outcome variables included FEV1, recorded at 4:00 p.m. and 4:00 a.m., and the percentage overnight fall in FEV1. The tissue morphometric variables included the number of CD3+, CD4+, CD8+, and EG2+ cells/mm2 of epithelial tissue or lamina propria. Tissue variables and FEV1 were compared between time, location, and group using a mixed-effects model with fixed effects for time, group, location, and the interactions between time, group, and location, plus a random subject effect (12, 13). If the interaction was significant, pairwise comparisons of least-squares means were made within groups and within times along with an adjustment for multiple comparisons using Tukey's multiple comparison test (14). If the data were not normally distributed, a log transformation was performed. If the log transformation did not normalize the data, then Wilcoxon's sign rank test was used to compare variables. Spearman's correlation coefficient was used to assess the relationship between CD4+ cells and the overnight lung function, EG2+ cells and overnight lung function, and the relationship between the CD4+ cells and the EG2+ cells. Values are expressed either as medians with the 25–75 interquartile ranges (IQ) or means ± the standard error of the mean (SEM), again depending on the distribution of the data. To determine reproducibility of cells counts, an intraclass correlation was calculated. A level of significance was defined as p ⩽ 0.05. The sample size reflected a power of 80% to detect a difference of 11.0 cells/mm2 with a type I error rate of 5% (15).
Subjects' characteristics, FEV1, and percent overnight fall in FEV1 measured in the laboratory are shown in Table 1. The mean age and medications were similar in both groups. The male:female ratio was approximately 2:1. Reproducibility of counts was assessed by calculating an intraclass correlation (ICC). The ICC was 0.97 (0.92 to 0.99, 95% confidence interval [CI]).
|Nocturnal Asthma||Non-Nocturnal Asthma||p Value|
|Sex||8 M:4 F||7 M:4 F|
|Age, yr||33.3 ± 1.9||34.1 ± 2.5||0.82|
|Medicines||β2-agonists (12) Theophylline (2)||β2-agonists (11)|
|4:00 p.m. FEV1, L||3.1 ± 0.2||3.2 ± 0.2||0.49|
|4:00 p.m. FEV1, % pred||71.1 ± 4.8||85.6 ± 3.5||0.03|
|4:00 a.m. FEV1, L||2.6 ± 0.2||3.2 ± 0.2||0.07|
|4:00 a.m. FEV1, % pred||61.5 ± 4.3||84.8 ± 3.1||0.0003|
|% Overnight fall in FEV1, %†||−26.3 ± 1.2||−4.5 ± 1.2||0.0004|
Endobronchial (airway) biopsies. Of the airway biopsies, 19 of 23 from 4:00 p.m. were adequate for analysis, nine from the NA group and 10 from the NNA group (Table 2). At 4:00 a.m., 17 of 23 biopsies were adequate samples, nine from the NA group and eight from the NNA group. Comparing the number/mm2 of CD3+, CD4+, CD8+ and EG2+ cells of the NA and NNA groups, at 4:00 p.m. and at 4:00 a.m., there were no significant differences in either the epithelial or submucosal compartments (Table 2).
|4:00 p.m.(number of cells/mm2 )||4:00 a.m.(number of cells/mm2 )|
|CD3||87.6 (48.7–114.2)†||71.4 (52.8–91.2)‡|
|CD4||45.6 (35.9–68.7)§||56.1 (25.9–79.8)‖|
|CD8||38.7 (17.7–49.8)¶||38.5 (16.9–54.7)**|
|EG2||22.0 (13.4–38.2)†,†||28.6 (22.1–48.1)‡|
|CD3||29.8 (20.3–36.1)†||38.7 (17.7–49.8)‡|
|CD4||19.0 (8.6–28.7)§||9.8 (5.6–30.8)∥, ∥ ∥|
|CD8||9.7 (5.5–12.8)¶||5.9 (1.0–19.9)**|
|EG2||12.4 (2.5–19.2)†,†||14.6 (5.5–21.5)‡|
|CD3||40.0 (21.8–133.9)||58.2 (4.9–119.9)|
|CD4||19.6 (9.6–89.8)||45.0 (7.7–57.7)|
|CD8||20.4 (4.9–29.3)§§||39.9 (5.8–79.8)|
|EG2||24.1 (14.3–57.5)||15.8 (6.2–30.8)|
|CD3||22.5 (6.6–43.6)||20.4 (4.9–28.3)|
|CD4||9.9 (2.1–19.7)||1.5 (0–6.3)‖, ‖|
|CD8||6.2 (2.8–15.7)§§||5.7 (0.6–11.6)|
|EG2||14.3 (1.6–18.4)||5.4 (2.6–13.9)|
Transbronchial (alveolar tissue) biopsies. Of the alveolar tissue biopsies, 19 of 23 from 4:00 p.m. were acceptable for analysis, nine from the NA group and 10 from the NNA group. At 4:00 a.m. 14 of 23 biopsies were adequate, eight from the NA group and six from the NNA group. Examples of alveolar tissue biopsies stained for CD3+, CD4+, and CD8+ cells are shown in Figure 1. Analysis revealed an increase in the number of CD3+ cells at 4:00 a.m. in the NA group as compared with that in the NNA group, and this difference approached significance (p = 0.07) (Table 2). CD4+ cells were significantly greater in the NA alveolar tissue at 4:00 a.m. as compared with NNA (p = 0.04). No significant difference in the CD8+ cells was noted between the groups at either time point. There were also greater numbers of eosinophils in the alveolar tissue of the NA group at 4:00 a.m. than in the NNA group, which approached statistical significance (p = 0.07) (Table 2).
Alveolar and airway tissue: 4:00 p.m. versus 4:00 a.m . Within the NA group, eight of 12 pairs of airway tissue and seven of 12 pairs of alveolar tissue were found adequate for analysis. When the NA group was compared separately, there were no differences in the number of CD3+, CD4+, CD8+, and EG2+ cells in either the alveolar tissue from 4:00 p.m. to 4:00 a.m. or airway tissue from 4:00 p.m. to 4:00 a.m. (Table 2).
Endobronchial (airway) versus transbronchial (alveolar tissue) biopsies. Comparing EBBX (airway) with TBBX (alveolar tissue) at the two time points within the NA group, the numbers of CD3+, CD4+, CD8+, and EG2+ cells were greater in the proximal airway tissue than in the alveolar tissue at both 4:00 p.m. and 4:00 a.m. (4:00 p.m. CD3, p = 0.02; 4:00 a.m. CD3, p = 0.02; 4:00 p.m. CD4, p = 0.008; 4:00 a.m. CD4, p = 0.05; 4:00 p.m. CD8, p = 0.008; 4:00 a.m. CD8, p = 0.03). EG2+ cells were also greater in the proximal airway tissue than in the alveolar tissue at 4:00 p.m. and 4:00 a.m. (4:00 p.m. EG2, p = 0.02; 4:00 a.m. EG2, p = 0.02) (Table 2).
Alveolar and airway tissue: 4:00 p.m. versus 4:00 a.m . Within the NNA group, eight of 11 pairs of airway tissue and six of 11 pairs of alveolar tissue were found adequate for analysis. When the NNA group was compared separately, there were no differences in the number of CD3+, CD4+, CD8+, and EG2+ cells in either the alveolar tissue from 4:00 p.m. to 4:00 a.m. or airway tissue from 4:00 p.m. to 4:00 a.m. (Table 2).
Endobronchial (airway) versus transbronchial (alveolar tissue) biopsies. Within the NNA group, only CD8+ cells were increased in the airways as compared with the alveolar tissue, and only at 4:00 p.m. (p = 0.004) (Table 2).
There was a significant inverse correlation between the percentage predicted FEV1 measured at 4:00 a.m. and the number of CD4+ cells/mm2 in the alveolar tissue at 4:00 a.m. (r = −0.77, p = 0.0014) (Figure 2). This relationship was not seen in the airway tissue, where the percentage fall in FEV1 overnight did not correlate with the numbers of CD4+ cells present in the airway at 4:00 a.m. (r = −0.25, p = 0.34) (Figure 2). A significant and strongly positive correlation (r = 0.66, p = 0.01) was present between the number of eosinophils/mm2 in the alveolar tissue at 4:00 a.m. and the number of CD4+ cells/ mm2 in the alveolar tissue at 4:00 a.m. No significant correlation was present between the number of eosinophils/mm2 in the airway tissue at 4:00 a.m. and the number of CD4+ cells/ mm2 in the airway tissue at 4:00 a.m. (r = 0.25, p = 0.37) (Figure 3). Finally, the relationship between the percent overnight fall in FEV1 and the 4:00 a.m. alveolar tissue EG2+ cells was significant (r = −0.57, p = 0.03). Again, this relationship was not significant when the percent overnight fall in FEV1 and the airway EG2+ cells were correlated (r = −0.39, p = 0.12).
When the cell numbers in the airway and alveolar tissue were correlated, a significant relationship was present in the numbers of EG2+ cells at both 4:00 p.m. and 4:00 a.m. when the groups were combined (a.m.: r = 0.67, p = 0.03; p.m.: r = 0.57, p = 0.015). The CD3, CD4, and CD8 airway and alveolar count correlations were not significant. When the groups were analyzed separately, the relationship between CD3+, CD4+, CD8+, and EG2+ cells in the airway and alveolar tissue were not significant.
The CD4+ T-lymphocyte is considered to be a key cell orchestrating “daytime asthma” by the production of cytokines, including those of the IL-4 gene cluster on chromosome 5 and TNF-α, that result in eosinophil recruitment into the lung (16, 17). In our present study of alveolar tissue inflammation at night, this concept of a link between the T cell and the eosinophil influx is supported. However, during the night, the location of the CD4+ cells is also important with regard to the physiologic response. We have demonstrated that although the number of CD4+ lymphocytes are greater in the proximal airway tissue, the correlation of this cell type to the overnight decrement in FEV1 and to the number of eosinophils identified by their content of eosinophil cationic protein only occurs in the alveolar tissue. As far as we are aware, no previous studies have evaluated the presence of T-lymphocytes and activated eosinophils in the alveolar tissue of asthmatics with chronic stable asthma.
Lymphocytes expressing the CD4 receptor are known to be important in asthma pathogenesis (1) through their production of particular cytokines such as IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor, which enhances eosinophil survival, maturation, and activation (18-20). The relationship between Th-2-like T-lymphocytes and eosinophils is thought to result in eosinophil accumulation in tissue independent of IgE, whereas chemokines, particularly RANTES, MIP-1α, and eotaxin, are considered important for local eosinophil chemoattraction (21-23). The T-lymphocyte also plays a pivotal role both in initiating and in sustaining immunologically driven inflammation of chronic asthma. To support these observations, CD4+ lymphocytes have been shown to be increased in the airway tissue of patients with stable asthma during the day, with further increases after both natural seasonal exposure and allergen challenge (7, 24-27).
The importance of our findings of alveolar tissue alterations is that asthma is not just characterized by inflammation of the large airways. This and our previous study (8) illustrate that the distal alveolar tissue participates in the asthmatic inflammatory process and plays a significant role in the worsening of lung function at night. Although the two studies cannot be directly compared because of differences in the morphometric analysis, the relationship between the percent overnight fall in FEV1 and the 4:00 a.m. alveolar tissue EG2+ cells (or eosinophils in the previous study) were nearly identical (r = −0.57 in this study, and −0.54 in the previous study; p values were both 0.03). Additionally, these findings support the physiologic construct of distal airway involvement in asthma, which involves both small airways and lung parenchyma, brought forth by Woolcock and colleagues (28), Wagner and colleagues (29), and Kaminsky and colleagues (30). Woolcock and colleagues have shown a frequency dependence of dynamic compliance in patients with asthma despite normal resting pressure– volume relationships consistent with nonuniform obstruction of the peripheral airways (28). In addition, Wagner and colleagues have measured peripheral airway resistance directly via bronchoscopy and have found a significant increase in subjects with mild asthma as compared with normal subjects, which also correlated with bronchial hyperresponsiveness (29). Kaminsky and colleagues also showed that peripheral resistance was increased in asthmatic subjects and further increased in response to cool, dry air (30).
The contribution of the lung parenchyma to asthma pathophysiology as compared with the small airways is more difficult to dissect. Work by Ludwig and collegues (31) in a canine model suggests that the histamine concentration response of the lung parenchyma, as assessed by the alveolar capsule technique, is remarkably similar to the behavior of the airways. Specifically, when the changes in lung resistance during histamine responsiveness were partitioned into an airway and tissue component using the alveolar capsule technique, a significant correlation was noted between changes in airway resistance and changes in tissue viscance, the resistive drop across the tissues. A follow-up study by the same group comparing specifically the peripheral airways and lung parenchyma suggests a heterogeneity in the histamine responsiveness (32). They concluded that either may dominate responsiveness in the peripheral lung. These findings are supported in our own ongoing study assessing peripheral airways resistance via bronchoscopy in nocturnal asthmatics at 4:00 p.m. and 4:00 a.m. (33). We found that some subjects exhibited an “airway” response, with low closing pressures. Subjects exhibiting the “parenchymal” response exhibited a high closing pressure consistent with loss of parallel, conducting units. Given these data, we feel that the presence of alveolar tissue inflammatory cells can contribute to the distal airway physiologic changes appreciated in asthma.
The significance of alveolar tissue inflammation correlating with FEV1, thought to be primarily a large airway measurement, merits discussion. Macklem and Mead (34) have reported that maximal expiratory flows such as the FEV1 are dependent upon three factors: the elastic recoil of the lung, the resistance in the small airways, and the cross-sectional area of the large airways. When the FEV1 decreases, as it did in our subjects with nocturnal asthma, it may be the result of changes in one or in all of these entities. Thus, one may hypothesize that alveolar tissue inflammation not only increases the small airways resistance, but decreases elastic recoil, the latter potentially caused by an uncoupling of the airways and parenchyma caused by the alveolar tissue inflammation. These changes may result in a fall in the FEV1 regardless of large airway changes. Thus, despite greater numbers of proximal airway T-lymphocytes and activated eosinophils, the distal alveolar tissue cells may play a more important role in altered lung function during sleep, as illustrated by the correlation between CD4+ lymphocytes and FEV1.
As discussed in our previous study (8), electron microscopic examination of a random sampling of our transbronchial biopsies was performed and revealed that approximately 10% of the cells seen are in blood vessels and the remainder in alveolar tissue. Cells within blood vessels of the airway biopsies were easily seen and excluded from analysis. Although some inflammatory cells are seen in the microvasculature and not the alveolar tissue, their distribution does not reflect the differential seen in blood. Martin and colleagues (35) showed that the percentages of neutrophils, eosinophils, and lymphocytes in peripheral blood in subjects with nocturnal asthma and those with non-nocturnal asthma were similar at 4:00 a.m.: neutrophils, 43 to 45%; eosinophils, 5 to 6%; lymphocytes, 44 to 46%. This distribution is quite different from what is seen in our alveolar tissue samples. The fact that the cell numbers did not correlate with peripheral blood cell differentials suggests that specific cells are being drawn to the lung parenchyma, especially at night in nocturnal asthma (8).
Of interest, CD4+ lymphocytes decreased, although not significantly, in the alveolar tissue overnight in both groups. However, they decreased less in the asthmatics with nocturnal asthma such that compared with those without nocturnal deterioration they were significantly higher in alveolar tissue. The dynamics of T-lymphocyte circulation within the lungs over a 24-h period are not known. However, peripheral blood CD4+ T-lymphocytes do exhibit a circadian variation in blood, with a peak around midnight and nadir around 8:00 a.m. (36, 37). This CD4 circadian rhythm is inverse to the circadian rhythm of the endogenous anti-inflammatory hormone cortisol, which peaks at 7:00 a.m. and reaches its nadir at approximately 11:00 p.m. (38). Whether a similar decrease in CD4+ lymphocytes in the lungs as a consequence of changes in cortisol levels occurs in normal subjects is unclear. Although we have shown that there are no differences in overnight levels of serum cortisol between nocturnal asthmatics, non-nocturnal asthmatics, and control subjects (39), we have shown that glucocorticoid receptor binding affinity is reduced in nocturnal asthmatics at night, as compared with non-nocturnal asthmatics and control subjects (40). As a functional correlate, we have also shown that bronchoalveolar lavage lymphocytes from nocturnal asthmatics exhibit decreased inhibition of proliferation by hydrocortisone at night as compared with daytime (40). These findings suggest that reduced steroid responsiveness may be one mechanism of enhanced inflammation in nocturnal asthmatics.
One consequence of reduced steroid responsiveness may be enhanced local cytokine production at night that results in increased expression of leukocyte endothelial adhesion molecules to facilitate selective recruitment of CD4+ cells and eosinophils into the lung periphery (41). It seems likely that the reduction in lung function observed at 4:00 a.m. is the consequence of activated eosinophils, especially since nocturnal asthmatics exhibit increased urinary excretion of leukotriene E4 (42).
In conclusion, T cells, particularly CD4+ cells, are present in the proximal and distal lung in patients with asthma. It is the distal CD4+ and EG2+ cells, not the proximal CD4+ and EG2+ cells, that correlated with overnight lung function in nocturnal asthma. The distal CD4+ cells also correlated with the presence of alveolar tissue EG2+ cells. Continued study of both the structure and function of the distal lung units is necessary, as they appear to play a significant role in the pathogenesis of asthma.
Supported by grants HL36577 and HL03343 from the National Heart, Lung and Blood Institute, and Grant no. y 860 4034 from the Medical Research Council of Great Britain.
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