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* From the Department of Medicine, National Jewish Medical and Research Center, Denver, CO.
Correspondence to: Richard J. Martin, MD, FCCP, National Jewish Medical and Research Center, 1400 Jackson St, Room J116, Denver, CO 80206; e-mail: martinr{at}njc.org
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Participants and measurements: We evaluated blood vessels
and edema by immunohistochemistry in endobronchial biopsy samples from
10 normal control subjects and 15 patients with mild-to-moderate asthma
before and after a 6-week treatment with clarithromycin (n = 8) or
placebo (n = 7). Type IV collagen and 
2-macroglobulin
were used to identify blood vessels and edema in the tissue,
respectively. Mycoplasma pneumoniae was evaluated by
polymerase chain reaction.
Setting: National Jewish Medical and Research Center.
Results: At baseline, the vascularity, the number of blood vessels, and the edematous area in the airway tissue were not significantly different between asthmatic patients and normal control subjects. However, asthmatic patients demonstrated increased blood vessel size compared with normal control subjects (p = 0.03). After clarithromycin treatment in asthmatic patients, the number of blood vessels was increased (p = 0.02), while edema decreased (p = 0.049). Asthmatic patients who tested positive for M pneumoniae showed a significant increase in vascularity than asthmatic patients who tested negative for M pneumoniae (p = 0.02).
Conclusion: Our data
suggest that angiogenesis and edema may not be significant features of
airway remodeling in patients with chronic, mild-to-moderate asthma.
Clarithromycin treatment in asthmatic patients could reduce the
edematous area as identified by 2-macroglobulin
staining, which may lead to airway tissue shrinkage and cause an
artificial increase in the number of blood vessels.
Key Words: asthma • blood vessel • edema • Mycoplasma
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Airway mucosal edema results from increased microvascular permeability
during the inflammatory process, which may further increase the airway
wall thickening and lead to more severe airway
obstruction.6
Although edema has been described in the
airway mucosa of asthma patients for many years,7
it is
very difficult to directly evaluate and quantify edema in the airways
of asthma patients. Several plasma proteins, such as albumin,
fibrinogen, and 2-macroglobulin, have been
used as indexes of microvascular leakage and plasma exudation in
asthma.8
Although increased plasma leakage as evaluated by
BAL was found in asthmatic patients after allergen
challenge,9
10
it is uncertain whether patients with
stable asthma have increased microvascular leakage.8
11
To
our knowledge, no study has been performed to evaluate edema or
microvascular leakage in endobronchial biopsy tissue from asthma
patients.
Previous studies12–14 have suggested that Mycoplasma infection may contribute to airway inflammation and remodeling. Airway inflammation and remodeling such as angiogenesis and goblet-cell metaplasia were induced in a murine model of respiratory infection with Mycoplasma pulmonis.12 Treatment with an antibiotic oxytetracycline in this animal model significantly reduced the airway inflammation, edema, and the number of blood vessels.12 We have reported that chronic asthma is associated with Mycoplasma pneumoniae in the airway.13 Additionally, improvement in lung function followed with macrolide antibiotic treatment.14
In the current study, we first asked whether patients with mild-to-moderate asthma demonstrate increased airway vascularity, number of blood vessels, vessel size, and edema as compared to normal control subjects. Then, we asked whether a macrolide antibiotic, such as clarithromycin, could reduce these parameters. Therefore, we evaluated blood vessels and edema by immunostaining endobronchial biopsy specimens from normal control subjects and asthmatic patients treated with clarithromycin or placebo.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bronchoscopy
Both asthmatic patients and normal control subjects underwent
bronchoscopy as previously described.16
Endobronchial
biopsy tissues were obtained for staining protocol and M
pneumoniae polymerase chain reaction analysis.13
The
location of the biopsies was randomized to the right or left lower
lobe. Two to four tissue specimens were obtained under direct
visualization from the fourth-generation or fifth-generation airways.
Asthmatic patients were then randomly divided into two subgroups with
different treatments. One subgroup of asthmatic patients (n = 8)
received oral clarithromycin, 500 mg bid, for 6 weeks. The remainder of
the asthmatic patients (n = 7) received placebo for 6 weeks.
Bronchoscopy and biopsy were then repeated in both asthma groups. The
protocol was approved by the institutional review board, and all
subjects gave informed consent.
Tissue Processing and Staining
Endobronchial biopsy specimens were processed as previously
reported.17
Briefly, specimens were fixed in acetone at
- 20°C overnight and then embedded in glycol methacrylate resin.
Tissue blocks were stored at - 20°C. Serial 2-µm sections were
cut from the tissue blocks with a Reichert Ultracut E ultramicrotome
(Leica; Deerfield, IL) and placed on poly-L-lysine-coated slides for
immunostaining.
A mouse monoclonal antibody against human collagen type IV (1:50
dilution; Dako; Carpinteria, CA) was used to identify the blood
vessels.4
Since 2-macroglobulin
was shown to be a better edema marker in BAL fluid from patients with
allergic rhinitis,18
19
we decided to use a sheep
antihuman 2-macroglobulin polyclonal antibody
(1:1,000 dilution; Cortex Biochem; San Leandro, CA) to indirectly
identify the tissue edematous area. To reduce the variation of
immunostaining between different batches, the blood vessel or
2-macroglobulin staining was performed in the
same batch. Sections were treated with 0.3% hydrogen peroxide in 0.05
mol/L Tris buffered saline solution (TBS, pH 7.6) for 30 min to inhibit
endogenous peroxidase, followed by incubation with 1% normal horse or
rabbit serum for 30 min to block potential nonspecific binding sites.
The slides were then incubated with primary antibodies mentioned above
for 2 h at room temperature, followed by incubation with
biotinylated horse antimouse IgG or rabbit antisheep IgG for 1 h
at room temperature. Thereafter, avidin-biotin-peroxidase complex
(Vector Lab; Burlingame, CA) was added to the slides for 45 min at room
temperature. After rinsing the slides in TBS, 0.03%
aminoethylcarbazole in 0.03% hydrogen peroxide was used as a substrate
to develop a peroxide-dependent red color reaction. Slides were
counterstained with Mayer’s hematoxylin and covered with Crystalmount.
Negative control slides were similarly treated but with the primary
antibodies replaced by mouse or sheep serum or TBS.
Analyses of Blood Vessels and Edematous Area
Blood vessels and edematous area were examined in the submucosa
at x 400 magnification. A National Institutes of Health Scion image
analysis program was used to evaluate the area of blood vessels,
edematous area, and submucosal area.17
The area internal to the vessel endothelial basement
membrane was considered as the blood vessel area. The number of blood
vessels was counted in the submucosa excluding smooth muscle and
glands. The results of blood vessel evaluation in each
subject4
were expressed as follows: (1) vascularity
(percentage) = ([total vascular area/submucosal area] x 100);
(2) number of blood vessels per millimeters squared
submucosa = (total number of blood vessels/submucosal area); and (3)
mean vessel size (micrometers squared) = (total vascular
area/total number of blood vessels). To evaluate the
edematous area, the 2-macroglobulin staining
area was measured in the submucosa excluding blood vessels, smooth
muscle, and glands. The measurement was expressed as edematous area
(percentage) = ([2-macroglobulin staining
area/submucosal area] x 100).
All the slides were read and evaluated by the same observer who was completely blinded to subject status and treatment with a < 10% coefficient of variation for blood vessel and edematous area measurement on repeated measurement. We also performed a pilot study to test the interobserver variability for some randomly selected slides. The agreement between the two observers was < 10% coefficient of variation for the measurements.
Statistical Analyses
Within-group comparisons were performed using the paired
t test or Wilcoxon rank sum test, depending on the
distribution of the data. Between group comparisons were performed
using the unpaired t test or a nonparametric Kruskal-Wallis
analysis of variance, depending on data distribution. All tests were
two sided, and a p value of 
0.05 was regarded as significant.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Edematous Area in Airway Tissue of Asthma Patients and Normal
Control Subjects
Edematous area was determined by
2-macroglobulin immunostaining in the
submucosa. At the baseline, the edematous area in asthmatic patients
was not significantly different from the normal control subjects (3.1%
[0.6 to 5.5%] vs 6.0% [2.1 to 16.7%]; p = 0.22). The edematous
area also did not differ (p = 0.22) between asthmatic patients
receiving (2.3% [0.4 to 3.7%]) or not receiving (5.0% [0.5 to
14.8%]) inhaled corticosteroids.
Effects of Clarithromycin Treatment on Airway Blood Vessels in
Asthma Patients
At the baseline, the vascularity and the vessel size were similar
between asthmatic patients treated with clarithromycin or placebo.
However, asthmatic patients treated with placebo had higher numbers of
blood vessels than those treated with clarithromycin (p = 0.02).
After clarithromycin treatment, as shown in Table 2 , the vascularity did not significantly change. However, the number of blood vessels in the submucosa was significantly increased (p = 0.02). However, the blood vessel size tended to be decreased (p = 0.09). Placebo treatment in asthmatic patients did not significantly alter the vascularity, number of blood vessels, and vessel size (Table 2) .
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= + 48.9/mm2 [- 10.8 to
109.5/mm2] vs
= - 37.5/mm2 [- 112.6 to
- 2.1/mm2]; p = 0.037; Fig 2
). The changes in vascularity (p = 0.12) and blood vessel size
(p = 0.16) were not significantly different between the two asthma
groups.
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= 2.9% [1.1 to
4.2%] vs = 0.2% [- 1.1 to 0.3%]; p = 0.02).
Effects of Clarithromycin Treatment on Airway Edema in Asthma
Patients
At baseline, the edematous area was similar between asthmatic
patients treated with clarithromycin or placebo. After clarithromycin
treatment, as shown in Table 2
, asthmatic patients tended to have
decreased edematous area (p = 0.12). Asthmatic patients treated with
placebo did not show a significant change in edematous area
(p = 0.95). When the changes (treatment - baseline) of edematous
area were compared, clarithromycin-treated asthmatic patients
demonstrated a significant decrease in edematous area as compared to
placebo-treated asthmatic patients ( = - 0.4% [- 4.2 to
0.0] vs = 0.5% [- 0.2 to 2.6%]; p = 0.049)
In contrast to the vascularity, the changes in edematous area were not
significantly different between M pneumoniae-positive and
M pneumoniae-negative clarithromycin-treated asthmatics.
Examples of airway tissue staining for blood vessels and
2-macroglobulin are given in Figure 3
.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study does not indicate that angiogenesis is one of many airway remodeling features in patients with mild-to-moderate asthma. Our data support an early study in which similar bronchial tissue vascularity and total number of blood vessels were found among patients with fatal asthma, nonfatal asthma, and control subjects.5 However, our results were contrary to those reported by Li and Wilson,4 who found increased vascularity and number of blood vessels (identified by collagen type IV staining) in patients with mild asthma. The methodology in these two studies was similar, except that we evaluated blood vessels in the whole submucosa while they only evaluated blood vessels 150 µm below the basement membrane where there are more blood vessels. This may explain that the number of blood vessels in both normal control subjects and patients with mild asthma in their study was higher than that in our study. However, we cannot explain why we failed to see a significant difference between asthmatic patients and normal control subjects as reported by Li and Wilson.4 Interestingly, a recent study by Vrugt et al20 also evaluated blood vessels (identified by another blood vessel marker, EN4) in the whole submucosa of endobronchial biopsy tissues from normal control subjects, patients with mild asthma, and patients with severe asthma. Similar to our study, they did not find any significant differences in the number of blood vessels between patients with mild asthma and normal control subjects. However, they demonstrated a significant increase in the number of blood vessels in patients with severe asthma compared with patients with mild asthma and normal control subjects, suggesting that angiogenesis might be a characteristic of airway remodeling in patients with severe asthma, but not in patients with mild asthma. Although angiogenesis was not found in the large airways of our patients with mild-to-moderate asthma, these patients showed an increase in blood vessel size. The significance and mechanisms of vasodilation in our asthmatic patients remain to be elucidated.
It has been difficult to quantify tissue edema in a small endobronchial
biopsy specimen. In this study, we for the first time attempted to
evaluate airway tissue edema using
2-macroglobulin as a marker and measured the
edematous area by image analysis. In contrary to our expectation, we
did not find increased edema in biopsy tissue from asthmatic patients,
which suggests that edema may not exist in patients with
mild-to-moderate, stable asthma. Airway edema usually results from
acute inflammatory response to allergen exposure or
challenge.9
10
Our patients with asthma were all in stable
condition without any signs of acute exacerbation or recent respiratory
tract infections. Therefore, it may not be surprising that there was no
significant tissue edema in patients with mild-to-moderate, stable
asthma.
Since M pneumoniae has been shown to be associated with chronic asthma,13 a macrolide antibiotic (clarithromycin) was tested in asthmatic patients to see whether it could reduce blood vessels and edema. We found that, as compared to placebo-treated asthmatic patients, those treated with clarithromycin demonstrated an increased number of blood vessels and reduced edema. We also subgrouped the clarithromycin-treated asthmatic patients into M pneumoniae-positive and M pneumoniae-negative asthmatic patients. Although reduction in edema area was not significantly different between the two groups, M pneumoniae-positive asthmatic patients had a significant increase in the vascularity after clarithromycin treatment compared with M pneumoniae-negative asthmatic patients. This suggests that M pneumoniae-positive asthmatic patients might be more responsive to clarithromycin treatment. The significance and mechanisms of increased blood vessel number, vascularity, and reduced edema after clarithromycin treatment remain unclear. We speculate that increased numbers of blood vessels or vascularity in asthmatic patients may be an artifact of reduced edema by clarithromycin treatment. This artificially increased number of blood vessels or vascularity did not worsen the pulmonary functions in asthma patients, since 75% of clarithromycin-treated asthmatic patients had an improvement in FEV1 percent predicted and airway responsiveness to methacholine. Airway wall thickening has been suggested to contribute to asthma pathophysiology.21 The thickened airway wall consists of a mixture of many tissue components, including smooth muscle, mucous glands, cartilage, extracellular matrix, inflammatory cells, mesenchymal cells, blood vessels, and fluid. A change in any of these airway wall components could alter the airway wall thickness and thus affect the airflow in patients with asthma. Although no difference of edema was found at baseline between asthmatic patients and normal control subjects, a significant decrease in edema was seen in clarithromycin-treated asthmatic patients. This decrease in edema may overall reduce the airway wall thickness and therefore potentially increase the airflow in patients with asthma. Future studies combining the biopsy tissue analysis and whole airway wall thickness measurement by CT could be helpful to better understand the effects of clarithromycin treatment on airway tissue edema and other structure components. As for the mechanisms of tissue edema reduction by clarithromycin treatment, both antimicrobial (eg, Mycoplasma) and direct anti-inflammatory effects of clarithromycin could be involved.22 23 24 25 26 To further elucidate the effects of clarithromycin treatment on airway edema and blood vessels, future studies in an animal model with M pneumoniae infection are needed.
In summary, our data suggest that angiogenesis and edema may not be significant characteristics of airway remodeling in patients with chronic, mild-to-moderate, stable asthma. Clarithromycin treatment in asthma patients could reduce airway tissue edematous area, which might lead to an artificial increase in the number of blood vessels.
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Footnotes |
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Supported by the American Lung Association-ARC, and the National Heart, Lung, and Blood Institute, HL 36577.
Drs. Martin and Kraft have received grant funding from Abbott Laboratories.
Received for publication August 2, 2000. Accepted for publication January 9, 2001.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin and tryptase into human nasal and bronchial airways. J Allergy Clin Immunol 96,239-246[CrossRef][ISI][Medline]
2-Macroglobulin and eosinophil cationic protein in the allergic airway mucosa in seasonal allergic rhinitis. Eur Respir J 13,633-637[Abstract]
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