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Alterations in Lung Mast Cell Populations in Patients with COPD
Cecilia K Andersson1, 2
, Michiko Mori2, Leif Bjermer
1, Claes-Göran Löfdahl
1, and Jonas
S Erjefält1, 2
1Department of Respiratory Medicine and Allergology, Lund University Hospital,
2Department of Experimental Medical Science, Lund University, Lund, Sweden
Corresponding author and requests for reprints should be addressed to:
Jonas Erjefält, Assoc. Prof., e-mail: [email protected]
Airway Inflammation Unit, Dept. of Experimental Medical Science,
BMC D12, Lund University, SE-22184, Lund, Sweden
Phone: +46 46 222 0960, Fax: +46 46 211 3417
Sources of support: The Heart & Lung Foundation, Sweden, The Swedish Medical Research
Council, The Swedish Asthma and Allergy Associations Research Foundation, and The Cra-
foord Foundation
Running head: Altered Mast Cell Populations in COPD
Word count: 5,385
Page 1 of 51 AJRCCM Articles in Press. Published on November 19, 2009 as doi:10.1164/rccm.200906-0932OC
Copyright (C) 2009 by the American Thoracic Society.
Andersson et al. - Altered Mast Cell Populations in COPD
2
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Mast cells are part of the resident immune system in the human lung. They have recently been
ascribed roles of potential importance to COPD. Yet, mast cells have only rarely been studied
in lungs from COPD patients.
What This Study Adds to the Field
This study shows that the mast cell populations in the lung are altered in COPD, as exempli-
fied by a change in the MCTC/MCT balance, altered tissue distribution, and modified morpho-
logical and molecular characteristics. Collectively, our data show alterations in lung mast
cells in COPD that correlate with lung function and may have significant pathophysiological
consequences.
Online Supplement
This article has an online data supplement, which is accessible from the issue’s table of con-
tent online at www.atsjournals.org
Page 2 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
3
ABSTRACT
Rationale: Mast cells have important roles in innate immunity and tissue remodeling but
have remained poorly studied in inflammatory airway diseases like COPD.
Objectives: To perform a detailed histological characterization of human lung mast cell popu-
lations at different severities of COPD, comparing with smoking and never-smoking controls.
Methods: Mast cells were analyzed in lung tissues from patients with mild to very severe
COPD, GOLD I–IV (n = 25, 10 of whom were treated with corticosteroids). Never-smokers
and smokers served as controls. The density, morphology and molecular characteristics of
mucosal and connective tissue mast cells (MCT and MCTC, respectively) were analyzed in
several lung regions.
Measurements and Main Results: In all compartments of COPD lungs, especially at severe
stages, the MCTC population increased in density while the MCT population decreased. The
net result was a reduction in total mast cell density. This phenomenon was paralleled by in-
creased numbers of luminal mast cells whereas the numbers of TUNEL+ apoptotic mast cells
remained unchanged. In COPD lungs, the MCT and MCTC populations showed alterations in
morphology and expression of CD88 (C5a-R), TGF-β, and renin. Statistically significant cor-
relations were found between several COPD-related mast cell alterations and lung function
parameters.
Conclusions: As COPD progresses to its severe stages, the mast cell population in the lung
undergoes changes in density, distribution, and molecular expression. In COPD lungs, these
novel histopathological features were found to be correlated to lung function and they may
thus have clinical consequences.
Word count: 242
Key words: mast cells, COPD, chymase, tryptase, apoptosis, luminal
Page 3 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
4
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is a common disease, which is projected to be
the third leading cause of death worldwide by 2020 (1). The most important risk factor for
developing COPD is tobacco smoke, and 15–20% of all smokers develop the disease (2).
Smokers who develop COPD have chronic inflammation with subsequent development of
bronchitis, bronchiolitis, and emphysema. Previous studies have shown that inflammation in
COPD is characterized by increased numbers of CD8-positive T-lymphocytes, neutrophils,
and macrophages (3-5). Recent studies have also implicated B-lymphocytes as being
potentially important (6). However, currently little is known about the overall cellular
composition and role of the individual cell types that infiltrate the lungs in COPD. Mast cells
have been less frequently explored in studies of COPD pathology, despite several indications
of their possible involvement.
In the human lung, at healthy baseline conditions mast cells are present in large numbers at all
levels of the airway including the alveolar parenchyma (7, 8). The discovery of several roles
of mast cells in, for example, innate immunity (9), blood flow regulation (10), antigen presen-
tation (11), and T-cell regulation (12) thus make them of possible relevance to the pathogene-
sis of COPD. To date, only a few COPD studies have measured mast cell density (4, 13, 14).
While these important studies confirm that there is a significant occurrence of mast cells in
COPD, no general picture has emerged as to how mast cell densities change in different re-
gions of the lungs and how mast cell density relates to disease severity. Furthermore, there is
no information on the phenotypes of the different mast cell populations in COPD.
In the present study, our aim was to explore patterns of mast cell density and distribution and
to perform phenotypic characterization of lung mast cells in COPD. To study the influence of
Page 4 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
5
disease severity, we collected human lung samples from three patient groups with different
severities of COPD and from corresponding smoking and never-smoking control groups.
Mast cells are highly heterogeneous cells that exist as two major subtypes, mucosal mast cells
(MCT) and connective tissue mast cells (MCTC), where MCT is most common in airways (15).
Among the key parameters selected was the proportion of the two subtypes, as the proportion
between the two has been shown to be altered in airway diseases such as asthma (16, 17).
Furthermore, since microlocalization of inflammatory cells may have functional
consequences, in the present study mast cells were analyzed at several anatomical
compartments of the lung (18, 19). We have recently shown that in the healthy human lung,
each of the MCT and MCTC subtypes can be further divided into additional site-specific
populations that are specific for each anatomical compartment of the lung (8). In the present
study, the activation status and morphological features of these novel subpopulations are
explored in detail using histological approaches. Some of the results of this study have been
previously reported in the form of abstracts (20, 21).
METHODS
Description of Patients
The present study involved 40 subjects divided into 3 COPD patient groups: patients with
mild COPD (GOLD I, n = 6), moderate to severe COPD (GOLD II–III, n = 9), and very se-
vere COPD (GOLD IV, n = 10). Two groups were used as controls: ex- or current smokers
without COPD (n = 7) and a control group with subjects who had never smoked (n = 8) (8).
The patient grouping was based on GOLD classifications (22). For patient characteristics, see
Table 1. Lung tissue from mild, moderate, and severe COPD was obtained in association with
lung lobectomy due to suspected lung cancer, a procedure that has been used repeatedly to
collect tissues from COPD patients (23-26). Only patients with solid tumors with visible bor-
Page 5 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
6
ders were included in the study, and tissue was obtained as far from the tumor as possible.
Smoking and never-smoking control tissue was obtained by the same procedure from other-
wise healthy non-atopic individuals. In patients with very severe COPD (GOLD IV), match-
ing lung tissue was collected in association with lung transplantation. For all patient groups,
care was taken to immerse the tissue in fixative immediately after surgical excision and mul-
tiple large tissue blocks were prepared for histological analysis. Due to the lack of tissue from
large airways from the resection material, bronchial biopsies from healthy controls were used
to perform a comparison of central airways in controls and in patients with very severe COPD
(for patient characteristics, see online supplement). All subjects gave their written informed
consent to participate in the study, which was approved by the local ethics committee in Lund,
Sweden.
Processing of Tissue for Immunohistochemistry and Electron Microscopy
Samples for immunohistochemistry were placed in 4% buffered formaldehyde, dehydrated,
and embedded in paraffin. From each block, a large number of sequential sections 3 µm in
thickness were generated. For electron microscopy, tissues were fixed in buffer supplemented
with 1% glutaraldehyde and 3% formaldehyde, post-fixed in 1% osmium tetroxide for 1 h,
and dehydrated in graded acetone solutions and embedded. Ultrathin sections (90 nm) were
cut and placed on 200-mesh copper grids. For details, see online supplement.
Immunohistochemical Staining
Double Immunohistochemical Staining of MCTC and MCT
A double staining protocol was used for simultaneous visualization of MCTC and MCT cells
(see ref 8 and online supplement for details). Briefly, after rehydration and antigen retrieval,
chymase-containing mast cells were detected with an anti-chymase antibody and the non-
Page 6 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
7
permeable chromogen DAB. The remaining MCT subclass was visualized with an anti-
tryptase antibody and Permanent Red chromogen (Table 2). The immunostaining was per-
formed using an automated immunohistochemistry robot (Autostainer; DakoCytomation,
Glostrup, Denmark) with EnVision™ G|2 Doublestain System (K5361, Dako).
Identification of Mast Cell-Related Molecules using Immunofluorescence
Triple staining by using immunofluorescence was used to simultaneously visualize both
MCTC and MCT populations and the mast cell-related molecules CD88 (the C5a receptor, re-
cently identified as a broad activation marker on mast cells (27)), TGF-β (a major pro-fibrotic
growth factor), and renin (involved in vascular homeostasis and recently identified in mast
cells) in paraffin sections from all patient groups. See Table 2,(8), and online supplement for
antibody references and details of the protocol. Staining was absent in control sections using
isotype-matched control antibodies.
Detection of Apoptotic Mast Cells in Lung Tissues Using the TUNEL Technique
The extent of mast cell apoptosis was studied by combining anti-tryptase immunofluorescence
immunohistochemistry (see above), a pan DNA marker (Hoechst 3332; Sigma, Stockholm),
and apoptotic cell detection using the TUNEL technique (28) (ApopTag Fluorescein In Situ
Apoptosis detection kit, S7110; Chemicon/Millipore, Billerica, MA). For details of the proto-
col, see online supplement.
Morphological and Morphometric Measurements
For quantification of mast cell densities, mast cell-related molecules, and morphometric pa-
rameters, large (> 4 cm2) paraffin-embedded tissue blocks from three separate regions of the
lung were analyzed from each patient. In each block, mast cells were analyzed in the follow-
Page 7 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
8
ing anatomical structures. Small airways: bronchioles, average 5 per section, defined by ab-
sence of cartilage and diameter < 2 mm. In small airways, mast cells were quantified in the
wall (epithelium, lamina propria, smooth muscle, and adventitia) and the lumen. Pulmonary
vessels: mid-size or large pulmonary arteries in the broncho/bronchiovascular axis or with an
intra-acinar localization (7 per block on average). Mast cells in pulmonary vessels were quan-
tified in all distinct layers (i.e. intima, media, and adventitia). Alveolar parenchyma: 4 ran-
domly selected 0.5 mm2 alveolar regions were analyzed in each block. Bronchial mast cells:
densities in large airway tissue were analyzed in separate biopsies and lung resections from
never-smoking controls and from patients with very severe COPD (see online supplement and
Table E3). Bronchial biopsies were taken from the first and second bronchial division from
the lower and upper right lobe (generation 3–4) and central airways (lung resections) were
defined as airways with a diameter of 3–6 mm (generation 3–5) surrounded by cartilage.
Mast Cell Density
In sections double-stained for MCTC and MCT, the density of each population was quantified
manually in blind sections and related to the area of each type of tissue analyzed (8), which
was determined using a computerized image analysis algorithm that excluded any luminal
spaces, the same approach was applied also in the alveolar parenchyma (Image-Pro Plus; Me-
diaCybernetics, Silver Springs, MD, and NIS-Elements; Nikon, Kanagawa, Japan). The pro-
portion of the MCTC subtype was calculated according to (MCTC / [MCTC + MCT]) × 100.
Expression of Mast Cell-Related Molecules
In triple stained sections, MCTC and MCT were counted manually as described above. All
tryptase- and chymase-positive cells in 4 randomly selected small airway walls, 4 randomly
selected pulmonary vessel walls, and 4 randomly selected 0.5-mm2 alveolar regions per sec-
tion were counted. By subsequently dividing the number of tryptase and/or chymase cells that
Page 8 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
9
were co-positive for CD88, renin, and TGF-β, respectively, by the total numbers of MCT and
MCTC, the proportion (%) of each subtype and the total number of mast cells expressing each
mast cell-related molecule were obtained.
Ultrastructural Characteristics of Mast Cells
Lung samples were processed for electron microscopy using a standard protocol (29) and a
Philips CM-10 TEM microscope (Philips, Eindhoven, the Netherlands). Tissue from never-
smoking controls and patients with very severe COPD (GOLD IV) were used for ultrastruc-
tural analysis of lung mast cells. Apart from exploring the general ultrastructure, each indi-
vidual mast cell was assessed for signs of degranulation (see online supplement for definitions
of categories) according to previously described criteria (30), and the percentage (per patient)
of degranulated mast cells was calculated.
Quantification of Mast Cells, Epithelial Cells, and Leukocytes in the Airway Lumen
Using double-stained sections, the numbers of intra-luminal MCT and MCTC were determined
in all patient groups (2-3 tissue blocks per patient). Briefly, MCT and MCTC were counted in
each individual airway and related to the luminal area. The airway lumen area was measured
by manual cursor tracing and digital image analysis (Image-Pro Plus, Media Cybernetics, Sil-
ver Spring, MD) and intra-luminal mast cells were expressed as cells per mm2 lumen area. In
never-smoking controls and patients with very severe COPD intra-luminal epithelial cells was
quantified in the same way after immunohistochemical staining with the pan-epithelial cell
marker cytokeratin (see Table 2) and detection using the EnVision™ G|2 Doublestain System
(K5361; Dako). In these patient groups, the leukocyte profile of the small airway lumen was
evaluated in consecutive sections stained for neutrophils, monocytes/macrophages, eosino-
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Andersson et al. - Altered Mast Cell Populations in COPD
10
phils, CD4+
positive T-lymphocytes, CD8+ positive T-lymphocytes and B-lymphocytes (for
protocol details and markers, see table E2 in the online supplement).
Analysis of Apoptotic (TUNEL-Positive) Mast Cells in Paraffin Sections
Paraffin sections from never-smoking controls and patients with very severe COPD were ana-
lyzed using fluorescence microscopy with triple-band UV filters, and image analysis allowed
all individual tryptase-positive mast cells in a lung section to be categorized as either
TUNEL+ or TUNEL
-.
Statistical Analysis
Data were analyzed statistically using Kruskal-Wallis test with Bonferroni’s multiple com-
parisons test for comparison between three groups or more (mast cell densities, proportions,
and molecular expression) and Mann-Whitney rank sum test for comparison between two
groups (mast cell densities in central airways, TUNEL, and size analysis), using GraphPad
Prism v. 5 (GraphPad Software, Inc., La Jolla, CA). Differences between groups were consid-
ered significant at p ≤ 0.05. The Spearman rank correlation test (two-tailed) was used to study
the correlation between lung function values and mast cell parameters, or correlations be-
tween intra-luminal mast cells and luminal epithelial cells or leukocytes, Results were consid-
ered significant at p ≤ 0.05. To compensate for multiple testing, the false discovery rate pro-
cedure was applied to the correlation analysis, which guaranties that less than 5% of all posi-
tive results are false positive (31).
Page 10 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
11
RESULTS
Alteration of Density and Proportion of Mast Cells in COPD
Mast cells were identified in small airways (range from 0.44–1.98 mm in diameter), pulmo-
nary vessels (range from 0.42–4.21 mm in diameter), and alveolar parenchyma in sections
from all patient groups by immunohistochemical double staining. In both control and COPD
subjects, mast cells were present at all anatomical compartments of the lung. In small airways,
a significant reduction in total mast cell density was found in patients with very severe COPD
(GOLD IV) relative to the control groups (never-smokers and smokers) (Figure 1A, Table
E3). The reduction was also significant in patients with moderate to severe COPD (GOLD II–
III) relative to never-smoking controls (Figure 1A, Table E3). The total density of mast cells
in pulmonary vessel walls was significantly reduced in patients with very severe COPD when
compared to never-smokers, smokers and patients with GOLD I–III COPD (Figure 1D, Table
E3). No difference in total numbers was found in the alveolar septa of the parenchyma (Figure
1G, Table E3).
Differentiation of MCT and MCTC populations revealed that in all anatomical compartments
of the lung, there was a gradual and significant reduction in MCT density (Figure 1B, E, H and
Table E3). In contrast, in the walls of small airways and in alveolar parenchyma, the density
of MCTC in patients with very severe COPD was significantly higher than in controls (Figure
1C, I, Figure 3A–F, and Table E3). In pulmonary vessels, the density of both MCT and MCTC
populations dropped significantly in patients with very severe COPD (Figures 1E-F, 3G-H
and Table E3). Calculation of the MCTC percentage of the total mast cell population revealed
that in lungs from patients with very severe COPD there was a several-fold increase in the
proportion of MCTC cells in small airways, small airway epithelium, pulmonary vessels, and
Page 11 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
12
alveolar parenchyma (Figure 2 A–D, Table E3). In lung tissue from patients with very severe
COPD, some larger airways of the peripheral lung containing submucosal glands were ana-
lyzed. Notably in these structures, MCTC comprised 95% of the total mast cell density (41
(28–56) mast cells per mm2; median and range).
Bronchial mast cell densities in large airway tissue were analyzed in separate biopsies and
lung resections from never-smoking controls and patients with very severe COPD. As for the
small airways, a reduction in the total numbers of mast cells and an increased proportion of
MCTC was also apparent in the central airways in patients with very severe COPD relative to
the controls (see online supplement and Table E4).
Redistribution of Mast Cells in the Small Airway Sub-compartments in Patients with
COPD
The distribution of mast cells was analyzed in more detail in sub-anatomical compartments
within the small airways and pulmonary vessels. In small airways from COPD patients, a shift
in relative mast cell densities from the outer to the inner wall layers (epithelium and lamina
propria) was observed (Figure 4A–B). For both MCT and MCTC, the percentage of intraepithe-
lial mast cells increased significantly in patients with very severe COPD while the proportion
of both subtypes decreased in the smooth muscle and adventitia layers (Figure 4 A–B). A re-
duction in airway smooth muscle-associated mast cells was also apparent in the smoking non-
COPD group (Figure 4A–B). No significant differences were found around pulmonary vessels
(data not shown).
Luminal Presence and Programmed Death of Lung Mast Cells
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13
In light of the reduction in total mast cell density in COPD, possible causes of mast cell
elimination were investigated. Mast cell elimination through apoptosis was explored through
TUNEL staining in combination with immunohistochemical staining for tryptase in paraffin
sections from controls and patients with very severe COPD. Egression into the airway lumen
and removal by the mucociliary escalator is a physiological mode of elimination for several
leukocyte types that infiltrate the lung (32). The occurrence of mast cells in the small airway
lumen was investigated in all patient groups.
Apoptosis
Scattered TUNEL-positive cell nuclei were present in all groups. In patients with very severe
COPD, a large proportion of the TUNEL-positive cells were identified as MPO-positive neu-
trophils (which were used as positive control cells; data not shown, Figure 6H). In each tissue
block an average of 5 small airways, 7 pulmonary vessels, and alveolar parenchyma were
screened for mast cells double positive for tryptase and nuclear TUNEL-staining. Through
this approach screening of about 6,000 mast cells per section revealed that TUNEL+ mast
cells were exceedingly rare. In patients with very severe COPD, the frequency of lung mast
cells positive for TUNEL staining was 0.035 ± 0.003% (mean ± SEM). The corresponding
value for never-smoking controls was 0.047 ± 0.005%, not significantly different to that for
COPD (p = 0.2).
Luminal Mast Cells
Exceedingly few mast cells were present among the luminal cells and secretions that occa-
sionally occurred in the never-smoking and smoking control groups (Table E4, Figure 4C-E).
Luminal mast cells were also rare in COPD patients in the GOLD I–III range despite the fact
that there was more luminal material present. In patients with very severe COPD (GOLD IV)
Page 13 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
14
whose small airways frequently contained cell-rich luminal plugs, luminal mast cells were
frequently observed in significantly increased numbers compared to the controls and the
COPD groups with milder disease (Table E3, Figure 4C–E). Interestingly, the luminal mast
cells in COPD patients were mostly MCT cells (77 ± 9% of the total, mean ± SEM). To de-
termine whether luminal mast cells could emerge from epithelial sloughing, double staining
was performed with a pan-cytokeratin antibody and mast cell tryptase. Exfoliated epithelial
cells increased in very severe COPD (229 (15-525) cells/mm2, median and range) compared
to controls (0 (0-37) cells/mm2, p=0.008). No statistical correlation was found between lu-
minal epithelial cells and luminal mast cells (see online supplement). Intra-luminal neutro-
phils, monocytes/macrophages, CD4+ and CD8
+ T-lymphocytes were all increased in COPD
whereas no change was found for eosinophils and B-lymphocytes (see online supplement and
Table E5). No significant correlations were found between intra-luminal mast cells and any of
the examined leukocytes (Table E5).
Altered MCT and MCTC Mast Cell Phenotypes in COPD
Ultrastructural Features
Ultrastructural examination of lung mast cells by transmission electron microscopy was per-
formed on lung samples from patients with very severe COPD lungs and from never-smoking
controls. MCTC and MCT subtypes were identified by their distinct granular morphology; MCT
from their scroll-rich granules and MCTC from their scroll-poor and crystalline/lattice granules
(33). In controls, the MCTC and MCT were easily identified according to these criteria (Figure
6A). Mast cells in healthy subjects were primarily of a non-degranulating phenotype, i.e. they
displayed filled granules lacking morphological signs of anaphylactic or piecemeal degranula-
tion. In the lungs of patients with very severe COPD, the distinction between MCTC and MCT
was less clear since mast cells that displayed the traditional morphology of MCTC frequently
Page 14 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
15
also contained scroll-filled granules. TEM analysis was performed on samples from 8 patients
with very severe COPD and 5 healthy controls (2–4 tissue blocks per patient, 5–15 mast cells
per patient). Based on granule appearance mast cells were classified into one of the following
categories: mild piecemeal degranulation, advanced piecemeal degranulation, or classical
anaphylactic degranulation (for definitions, see online supplement). Mast cells showing signs
of advanced piecemeal or anaphylactic degranulation were not found, neither in control tissue
nor in COPD lungs. Altogether, in COPD lungs, 20 ± 3% of the mast cells displayed signs of
mild to moderate piecemeal degranulation, which was a significant increase compared to the
controls (4 ± 4 %, p = 0.04).
Altered Expression of Mast Cell-Related Molecules
The expression of mast cell-related molecules was evaluated in the small airways, pulmonary
vessels, and alveolar parenchyma from all patient groups using immunofluorescence triple
staining. The levels of expression of CD88, TGF-β, and renin were changed in patients with
COPD compared to the controls (Figure 5). The percentage of mast cells that expressed the
C5a receptor (CD88) increased significantly in all COPD groups compared to the never-
smoking and smoking controls. The increase was present in both the MCTC and MCT popula-
tion and occurred in all anatomical compartments examined, i.e. the small airways, pulmonary
vessels, and alveolar parenchyma (Figure 5A–C). Also, there was a less clear but significant
increase in the proportion of mast cells that expressed TGF-β. In patients with COPD, the
most pronounced increase was observed for the MCTC subclass (Figure 5D–F). In contrast to
CD88 and TGF-β, the expression of mast cell renin was high at baseline conditions, particu-
larly in the MCTC subtype, which displayed almost 100% expression. Also in contrast to the
expression of CD88 and TGF-β, there was a significant decrease in renin expression (Figure 5
G-I) in the COPD lungs, particularly those affected by more severe disease (GOLD II-IV).
Page 15 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
16
There was no evidence of a chymase-only positive mast cell population (MCC) in any of the
patient groups.
Correlations
For the COPD patient groups, several statistically significant correlations were found between
mast cell and lung-function parameters (FEV1/VC and FEV1 % of predicted). In all anatomi-
cal compartments, there was a positive correlation between reduced densities of total or MCT
mast cells and reduced lung function (Table 3). For MCTC, there was a correlation between
increased density within the small airways and the lung parenchyma on the one hand and re-
duced lung function on the other. Also, there was a correlation between the increased propor-
tion of MCTC in all compartments and worsening of lung function values. No correlations
were found between mast cell parameters and pack years. Regarding the expression of CD88,
TGF-β, and renin, the correlation to lung function was less clear although several statistically
assured correlations were found (Table 3). After compensation for multiple testing using the
false discovery rate (FDR) procedure, several correlations were still significant and these are
presented in Table 3 in bold.
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Andersson et al. - Altered Mast Cell Populations in COPD
17
DISCUSSION
The present study shows that the lung mast cell populations are altered at severe stages of
COPD. The alterations include changes in density, distribution, and cell phenotype. As mast
cells appear to be key players in both innate and adaptive immunity, the functional conse-
quences of these alterations are now emerging as an important field of research.
The modified double staining used in this study for detection of the two mast cell subpopula-
tions (MCT and MCTC) does not detect the tryptase-negative MCC population described by
Weidner and Austen in 1993 (34). Our triple staining using flourochrome-labelled antibodies
and specific broad-spectrum filters would detect chymase-only positive mast cells. Notably,
among the numerous individual mast cells examined in this study, not a single one was found
to belong to the chymase only (tryptase-, chymase
+) category. With the vast number of cells
analyzed we believe that the present modified double staining approach is adequate for detect-
ing the MCT and MCTC cells in this study.
There are only a few studies where mast cell densities in smokers, or smokers who have de-
veloped bronchitis and COPD, have been examined. Healthy smokers have been reported to
have increased numbers of bronchial mast cells compared to never-smoking controls (35).
Also, in COPD mast cells are present in high numbers, sometimes elevated compared to con-
trols (25, 36, 37). A recent study by Gosman et al. has suggested that the total mast cell den-
sity may also be reduced compared to control subjects (14). In the latter study, no distinction
was made between different severities of COPD. Our results agree with those of Gosman et
al. (14) and suggest that reduced mast cell density in COPD lungs is particularly associated
with severe stages of the disease. Our analysis of distinct mast cell populations revealed that
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Andersson et al. - Altered Mast Cell Populations in COPD
18
while the MCT population decreased in COPD, there was an increased density of MCTC in
both the small airways and the alveolar parenchyma. The resulting shift in the balance be-
tween MCTC and MCT represents a key finding in the present study. Interestingly, a similar
increased proportion of MCTC has been observed in severe asthma (17). It can be surmised
that a common feature of severely inflamed lungs is expansion of the MCTC population, while
the normally prevailing MCT population becomes reduced by as yet unknown mechanisms.
The process leading to altered MCTC/MCT balance in COPD is likely to be complex and mul-
tifactorial. One possible mechanism could be that the cellular inflammation and molecular
milieu in COPD lungs promote differentiation of mast cell progenitors into a MCTC pheno-
type. A more speculative possibility is that resident MCT start to produce chymase and thus
transform into an MCTC phenotype. Such transformation has previously been observed in vi-
tro (38, 39).
Another tentative mechanism behind the MCTC/MCT imbalance is selective elimination of
lung MCT. Mast cell apoptosis, a proposed key mechanism for clearance of tissue mast cells,
has been studied extensively in vitro but little is known regarding lung mast cell apoptosis
under in vivo conditions. In the present study, we double-stained for TUNEL+ (apoptotic) cell
nuclei and tryptase. Our results revealed that, in contrast to e.g. neutrophils, lung mast cells in
COPD lungs exhibit the same low frequency of apoptosis as control subjects. This finding
supports the general view of mast cells as being long-lived cells with a slow turnover. Al-
though we could not find any evidence of increased mast cell apoptosis in COPD, it cannot be
excluded that this mechanism has a role in the long-term regulation of cell numbers.
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Andersson et al. - Altered Mast Cell Populations in COPD
19
In light of the small number of apoptotic mast cells, our observation of increased numbers of
luminal mast cells in COPD indicates that loss of tissue mast cell to the airway lumen may
have contributed to the present decline in total mast cell numbers. Mast cells are regarded as
tissue-dwelling cells but they have been observed in luminal samples from asthmatics (41,
42). While patients with mild asthma have increased mast cell numbers, severe asthmatics
may have reduced numbers of tissue mast cells (43) and elevated numbers of luminal mast
cells (44). Although the present study cannot establish any mechanism of how mast cells enter
the lumen some potential processes should be considered. Active migration into the airway
lumen has been proposed as a physiological mode of elimination for several airway leuko-
cytes (32, 40). At present it cannot be excluded that in inflamed lungs also mast cells have the
capacity to actively egress into luminal compartments. Alternatively, intra-epithelial mast
cells may be deliberated into the lumen as a result of epithelial sloughing. The relative contri-
bution of these two alternatives in our study is hard to determine, especially since we failed to
find any correlation between luminal mast cells and exfoliated epithelial cells, or between
mast cells and other intra-luminal leukocyte populations. It is clear that more research is
needed to elucidate the phenomenon of luminal mast cells in inflamed human lungs.
It cannot be excluded that medical treatment contributed to the MCTC/MCT imbalance in our
COPD patients. Steroids, for example, have been demonstrated to reduce mast cell density in
central airways (45), and have been shown to mainly affect the MCT population in various
compartments of the respiratory system (17, 46, 47). However, several factors indicate that
mechanisms other than medical treatment contribute to mast cell abnormalities in COPD.
Firstly, reduced levels of total mast cells in the lung were observed even in patients with mod-
erate to severe COPD, of which only one subject had steroid treatment. Also, the most ad-
vanced reduction was observed in pulmonary vessels, a compartment that during steroid inha-
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Andersson et al. - Altered Mast Cell Populations in COPD
20
lation receives only a fraction of the drug concentration delivered to the airway mucosa. Fur-
thermore, the absolute numbers of MCTC cells increased, an event that is unlikely to be caused
by medications. Taken together, the reduction in mucosal mast cells that we describe in very
severe COPD may be partly caused by steroids, but should be noted as a significant feature of
late-stage—and thus extensively medicated—COPD. It should also be noted that currently it
is not clear whether anti-mast cell effects are solely beneficial. The potential effects of current
and emerging drugs on lung mast cells should thus be explored with regard to both the de-
structive and pro-inflammatory capacity of mast cells, as well as their potential beneficial role
in innate immunity, lung defense mechanisms, and vascular homeostasis.
The few previous studies on mast cells in non-allergic inflammatory respiratory diseases
make it difficult to speculate about the functional consequences of the present types of mast
cell alterations. Gosman et al. (14) hypothesized that mast cells may have a protective role in
COPD. In general, our data agree with this hypothesis but highlight the need to discriminate
between mast cell subtypes. For example, there was a positive correlation between increased
densities of MCTC in pulmonary vessels and alveolar parenchyma on the one hand and wors-
ening of both FEV1 % predicted and FEV1/VC on the other, indicating that MCTC may have a
negative role in COPD. Expansion of the MCTC population in COPD may have pathogenic
implications. A recent publication by Maryanoff et al. 2009 (48), supports this and shows
broad anti-inflammatory effects of a dual chymase and cathepsin G inhibitor in animal models
of allergic asthma and COPD. Chymase has been shown to increase secretion from serous
airway glands (49). This may be of relevance to our finding of a particularly high density of
MCTC in glands in COPD lungs. Chymase can also destroy TIMP-1, an inhibitor of the ma-
trix-degrading protease MMP-9 (50), and thereby contribute indirectly to emphysema forma-
tion and release of matrix-bound pro-fibrotic factors such as TGF-β (51).
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Andersson et al. - Altered Mast Cell Populations in COPD
21
The MCTC population in our study had a particularly high level of renin, a key hormone in
vascular homeostasis that has been recently identified also in mast cells (52). This finding,
together with the capacity of chymase to act as an angiotensin converting enzyme and angio-
tensin II activator, suggests that MCTC may have a role in vascular regulation in the lung with
possible implications for COPD. The blood flow-modulating capacities of histamine and sero-
tonin (53, 54) further underscore the potential of mast cells in vascular regulation of the lungs.
As mast cells have been shown to regulate blood flow in several other organs (52, 55), loss of
lung vascular mast cells may thus contribute to the vascular changes and perfusion imbalance
observed in COPD (56). In further support of the clinical significance of reduced amounts of
vascular mast cells, the loss of vascular mast cells was strongly correlated to reduced lung
function in COPD patients.
The role of mast cells in COPD is dependent not only on their numbers in the diseased tissue
but also their activation status. Measurement of mast cell activation in tissues is complicated,
due to the lack of established parameters of activation. Electron microscopic assessment of
granule alterations reliably detects ongoing degranulation (57), but the time-consuming analy-
sis and small sample size limit its use. Despite the fact that we explored several blocks per
patient, in this study we failed to sample enough mast cells for detailed quantification. We
believe, however, that some important conclusions can be drawn from our ultrastructural
analysis. Of the more than 200 mast cells analyzed from COPD lungs, not a single one
showed signs of advanced piecemeal or anaphylactic degranulation. This observation suggests
that advanced degranulation is a rare phenomenon in very severe COPD, at least outside acute
exacerbations when the present surgical material was collected. In samples from patients but
not from controls, we did, however, observe scattered cells (20 ± 3%) involved in mild
Page 21 of 51
Andersson et al. - Altered Mast Cell Populations in COPD
22
piecemeal degranulation. Thus, we cannot exclude the possibility that in COPD also, mast
cells may cause pathogenic effects through degranulation. It should be noted that non-
degranulating mast cells might also contribute to inflammation through degranulation-
independent chemokine release (58), eicosanoid release, or just differentiation into a more
pro-inflammatory phenotype.
Our analysis of selected mast cell-related molecules revealed significantly increased mast cell
expression of the C5a receptor (CD88) and TGF-β in COPD. This observation shows for the
first time that mast cell expression profiles may be altered in COPD. It may, however, be too
early to speculate about any functional significance of these alterations. Clearly, a more sys-
tematic evaluation of expression patterns of a variety of key mast cell-associated molecules is
needed to define lung mast cell phenotypes at different stages of COPD.
In summary, the present study demonstrates that mast cell populations in COPD lungs are
altered on several counts. Apart from a marked decrease in numbers of mucosal mast cells, an
increase in connective tissue mast cells led to a shift in the relative proportion of MCTC and
MCT and each subtype showed a changed pattern of expression of mast cell-related mole-
cules. These alterations, which were found to be correlated to altered lung function and took
place in all major lung compartments, suggest that mast cells are part of the cellular inflam-
mation in COPD. The nature of this involvement and the influence of common medications
on mast cells now emerge as an important line of research.
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23
ACKNOWLEDGEMENTS
We thank Karin Janser and Britt-Marie Nilsson for skillful technical assistance with tissue
processing, immunohistochemical staining, and TEM procedures.
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24
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LEGENDS
Figure 1. Total mast densities (A, D, G) and densities of each subtype, MCT (B, E, H) and
MCTC (C, F, I), in lung tissue compartments of never-smoking and smoking control subjects
and patients with mild to very severe COPD presented as mast cells per mm2 lung tissue. Data
are presented for small airways (A–C), pulmonary vessels (D–F), and alveolar parenchyma
(G-I), and are expressed as scatter plots where the line denotes median. Statistical analyses
were performed using Kruskal-Wallis test with Bonferroni’s multiple comparisons test. Over-
all significance is shown in each picture. Asterisks show statistical difference when compared
to very severe COPD (GOLD IV) where * denotes p < 0.05, ** p < 0.01, and *** p < 0.001.
Significant differences between controls and moderate to severe COPD (GOLD II–III) are
shown as #
p < 0.05. SA: small airways; PV: pulmonary vessels; and AP: alveolar paren-
chyma.
Figure 2. The proportion of MCTC and MCT, expressed as the percentage of MCTC mast cells,
in anatomical lung compartments in never-smoking and smoking controls and patients with
mild to very severe COPD (GOLD I–IV). Data are presented for small airways (A), small
airway epithelium (B), pulmonary vessels (C), and alveolar parenchyma (D). Data are ex-
pressed as scatter plots, where the line denotes median. Statistical analyses were performed
using Kruskal-Wallis test with Bonferroni’s multiple comparison test. Overall significance is
shown in each panel. Asterisks show levels of statistical difference when compared to very
severe COPD (GOLD IV) where * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 3. Representative micrographs of immunohistochemical double staining of tryptase-
positive mast cells (MCT: permanent red) and chymase-positive mast cells (MCTC: DAB-
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32
brown) in different anatomical compartments of the lung. Panels A and B show representative
small airways at low magnification from a healthy control subject (A) and from a patient with
very severe COPD (B). Inset in B represents a close-up image (600×) of neighboring MCTC
and MCT cells. Panels C and D show small airway mast cells at higher magnification in a con-
trol subject (C) and a patient with very severe COPD (D). Note the higher proportion of
brown chymase-positive MCTC in COPD. The alveolar parenchyma is shown from a control
(E) and from a patient with very severe COPD (F); the increased proportion of MCTC in se-
vere COPD is highlighted in the insets. Panels G–H show mast cells in pulmonary vessels
from a control subject (G) and from a patient with very severe COPD (H). Scale bars: A, C–H
= 100 µm, B = 200 µm. SA: small airways; v: pulmonary vessel; ep: small airway epithelium;
sm: airway smooth muscle layer; lu: small airway lumen; and alv: alveolar parenchyma.
Figure 4. Distribution patterns of mast cell subtypes within distinct small airway compart-
ments. Bars show the mean percentage of total airway wall mast cells for MCT (A) and MCTC
(B) in each sub-anatomical compartment (epithelium, lamina propria, smooth muscle, and
adventitia). Significant differences between controls and very severe COPD (GOLD IV) are
shown in each figure. a denotes significant differences between very severe COPD and smok-
ing controls. Panels C–E show the density of airway lumen mast cells for total mast cells (C),
MCT (D), and MCTC (E). Data are presented as mast cells per mm2 lumen area and displayed
as scatter plots where the lines denote median. Statistical analyses were performed using
Kruskal-Wallis test with Bonferroni’s multiple comparison test. Overall significance is shown
above C–E. Asterisks show statistical significance compared to never-smoking controls where
* denotes p < 0.05.
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33
Figure 5. Molecular expression patterns of CD88 (A–C), TGF-β (D–F), and renin (G–I) in
small airways, pulmonary vessels, and alveolar parenchyma. In each graph, results are shown
as the percentage of total mast cells, and of MCTC and MCT subtypes, that are positive for the
respective mediators. Data are expressed as median with interquartile range. Statistical analy-
ses were performed using Kruskal-Wallis test with Bonferroni’s multiple comparison test
where * p < 0.05 and ** p < 0.01 when compared to controls. #
p < 0.05 and ##
p < 0.01 show
significant differences when compared to smoking controls. Overall significance (Kruskal-
Wallis) is presented for MCtot in each panel, except for E–F where overall significance for
MCTC is presented.
Figure 6. Panels A–C show transmission electron micrographs exemplifying different levels
of degranulation in mast cells in very severe COPD: non-degranulated phenotype (A), minor
piecemeal degranulation (B), and substantial piecemeal degranulation (C). High-power im-
ages of intact and degranulating granules are shown as insets in A and C, respectively. Scale
bars in A–C = 1 µm. Panels D and E are immunofluorescence images with double staining for
TUNEL+ apoptotic cells and tryptase, and the neutrophil marker myeloperoxidase, respec-
tively (Alexa F 488-green). Cell nuclei are stained blue with the pan-DNA marker Hoechst
3332. Arrowhead in D exemplifies a TUNEL+, tryptase
- cell. Apoptotic neutrophils (see also
inset in E) were readily found in lung tissues, and lumen of COPD patients was used as a
positive control. Panel F represents a bright-field micrograph of tryptase-stained mast cells
and demonstrates a mast cell-rich luminal plug in a patient with GOLD IV COPD. Scale bars:
D–E = 25 µm, F = 100 µm. Ep: epithelium.
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TABLE 1. CHARACTERISTICS OF PATIENTS WITH COPD AND HEALTHY CONTROLS
Controls Smokers GOLD I GOLD II+III GOLD IV
Sex (M/F, n) 2/6, 8 3/4, 7 5/1, 6 7/2, 9 4/6, 10
Agea
(years) 63 (33–76) 56 (47–68) 65 (56–72) 67 (58–76) 58.5 (53–66)
Pack yearsa (years) 0 43 (20–80) 43 (25–66) 50 (34–65) 40.7 (25–60)
Ex-smokers/current smokers 0 3/4 2/4 8/1 10/0
Inhaled GCS (y/n/unknown) 0 0 0/6/0 1/8/0 9/0/1b
Oral GCS (y/n/unknown) 0 0 0/6/0 0/9/0 1/8/1b
B2 agonist (y/n/unknown) 0 0 2/4/0 1/8/0 9/0/1b
Anticholinergics (y/n/unknown) 0 0 1/5/0 0/9/0 8/1/1b
Mucolytic (y/n/unknown) 0 0 3/3/0 0/9/0 5/4/1b
Lung function
FEV1a 2.7 (1.7–5.1) 2.9 (1.9–3.5) 2.8 (1.6–3.2) 1.8 (1.2–2.6) 0.6 (0.4–1.0)
FEV1/(F)VCa 85.9 (66–121) 78.1 (71–88) 67.5 (65–70) 51.4 (41–68) 30.2 (17–39)
FEV1 % of pred.a 109.8 (82–141) 97.4 (82–120) 86.3 (78–95) 61.8 (43–74) 21.4 (13–27)
a Data are given as mean (range).
b n = 10; 1 subject with missing medical history. M = male, F = female, GCS =
glucocorticosteroid, FEV1 = forced expiratory volume in 1 second, VC = vital capacity.
TABLE 2. ANTIBODIES USED FOR IMMUNOHISTOCHEMISTRY
Antibody Species Dilution Clone Origin Secondary antibody
Anti-tryptase Mouse 1:12 000 G3 Chemicon, Temecula, CA
EnVision™ G|2 Doublestain System
Anti-chymase Mouse 1:100 CC1 Novocastra, Newcastle upon Tyne, UK
EnVision™ G|2 Doublestain System
Anti-renin Mouse 1:50 Swant Scientific, Bel-linzona, Switzerland
Biotinylated Horse anti-mouse IgG
Anti-CD88 Mouse 1:500 P12/1 Acris Antibodies, Hiddenhausen, Germany
Biotinylated Horse anti-mouse IgG
Anti-TGF-β Mouse 1:40 TGFB17 Novocastra, Newcastle upon Tyne, UK
Biotinylated Horse anti-mouse IgG
Anti-cytokeratin Mouse 1:100 MNF116 Dako, Glostrup, Den-mark
EnVision™ G|2 Doublestain System
a
Heat-induced antigen retrieval was performed in citrate buffer, pH 6, in a pressure cooker (see online sup-plement).
a Heat-induced antigen retrieval was performed in EDTA buffer, pH 9.
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35
TABLE 3. CORRELATIONS BETWEEN LUNG FUNCTION AND MAST CELL PARAMETERS
IN COPD PATIENTS
FEV1/VC FEV1 % of pred.
Parameter Anatom. MC rs p-value rs p-value
Density S. Airway MCtot 0.43 0.05 0.33 0.1
Density S. Airway MCT 0.48 0.03 0.46 0.03
Density S. Airway MCTC -0.13 0.3 -0.17 0.3
Density Pul. Vessel MCtot 0.70 0.0003 0.76 <0.0001
Density Pul. Vessel MCT 0.72 0.0002 0.75 <0.0001
Density Pul. Vessel MCTC 0.29 0.1 0.42 0.03
Density Parenchyma MCtot 0.59 0.003 0.63 0.001
Density Parenchyma MCT 0.69 0.0004 0.74 <0.0001
Density Parenchyma MCTC -0.75 <0.0001 -0.70 0.0002
Proportion S. Airway MCTC -0.38 0.05 -0.40 0.04
Proportion Parenchyma MCTC -0.80 <0.0001 -0.76 <0.0001
Proportion Pul. Vessel MCTC -0.59 0.003 -0.55 0.005
Proportion SA epithelium MCTC -0.44 0.04 -0.43 0.04
CD88 S. Airway MCtot 0.25 0.2 -0.05 0.4
CD88 S. Airway MCT -0.14 0.3 -0.40 0.07
CD88 S. Airway MCTC 0.59 0.01 0.36 0.1
Renin S. Airway MCtot 0.50 0.02 0.53 0.02
Renin S. Airway MCT 0.53 0.02 0.54 0.02
Renin S. Airway MCTC 0.43 0.07 0.55 0.03
TGF-β S. Airway MCtot 0.50 0.02 0.40 0.06
TGF-β S. Airway MCT 0.42 0.05 0.36 0.08
TGF-β S. Airway MCTC -0.38 0.08 -0.42 0.05
Correlation analysis was performed using Spearman rank correlation test on pooled COPD groups, GOLD
I–IV (number of patients: 25). rs = Spearman rank correlation coefficient, p ≤ 0.05 is considered significant.
SA = small airway. Non-significant p-values are shown in italics. The correlations that remained significant
after performing FDR procedure are shown in bold.
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Andersson et al. - Altered Mast Cell Populations in COPD
Alterations in Lung Mast Cell Populations in Patients with COPD
Cecilia K Andersson, Michiko Mori, Leif Bjermer, Claes-Göran Löfdahl, and Jonas S
Erjefält
ONLINE DATA SUPPLMENT
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2
ONLINE SUPPLEMENTARY DATA
METHODS
Patient description
Multiple large tissue blocks were obtained from 10 patients with severe COPD (GOLD IV)
undergoing lung transplantation at Lund University Hospital (Table 1). Six patients with mild
COPD (GOLD I) and 9 patients with moderate-to-severe COPD (GOLD II–III) undergoing
surgery for suspected lung cancer at Lund University Hospital were used. Eight non-smoking,
non-atopic patients and 7 smoking patients without COPD were used as controls. Only
patients with solid, well-defined tumors were included, and tissue was obtained as far from
the tumor as possible (Table 2). Controls had no symptoms of infections at least four weeks
prior to the beginning of the study, and none were treated with oral or inhaled steroids. For all
groups, multiple tissue slices (around 15 mm thick) were immediately immersed in fixative
after surgery. After fixation for 24 h, the tissues were trimmed into blocks with the aim of
obtaining blocks for paraffin embedding that contained most anatomical regions of the lungs.
As additional control tissue, we also collected bronchial biopsies from young, healthy, non-
atopic individuals (Table E1) during a study period from May 2007 to February 2008 at the
Department of Respiratory Medicine, Lund University Hospital. Bronchoscopy was
performed after local anesthesia, with a flexible bronchoscope (Olympus IT160, Tokyo,
Japan). Before bronchoscopy, the subjects received oral Midazolam (1 mg per 10 kg) and i.v
Glykopyrron (0.4 mg). Local anesthesia was given as Xylocain spray: local and through-spray
catheter. Just before the procedure, alphentanyl (0.1–0.2 mg per 10 kg body weight) was
given intravenously and extra i.v. Midazolam was given when needed. Central airway
biopsies were taken from the segmental or sub-segmental carina in the lower and upper right
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3
lobe. Oxygen was given as needed during and after the procedure. All subjects gave their
written informed consent and the study was approved by the local ethics committee.
TABLE E1. SUBJECT CHARACTERISTICS AND OVERVIEW OF TISSUE SAMPLES FROM
CENTRAL AIRWAYS OF CONTROLS
Variable Bronchial biopsies Lung resections
Patients (n) 5 3
Males/females 4/1 1/2
Age (years) 29.6 (25–41) 70 (63–73)
FEV1 % of predicted 107 (95–116) 103 (98–109)
Smoking (pack years) 0 0
Data are presented as median (range).
Tissue Processing for Immunohistochemistry and for Electron Microscopy
Paraffin-embedded tissue
After fixation (4% formalin in phosphate buffer, pH 7.2) tissue blocks were dehydrated
through a series of increasing ethanol solutions, cleared in xylene, and embedded in paraffin.
From each block a large number of sequential sections of 3 µm thickness were generated
using a microtome (HM 350 SV; Microme International, Walldorf, Germany).
Transmission Electron Microscopy
After fixation in buffer supplemented with 1% glutaraldehyde and 3% formaldehyde
overnight, the samples were rinsed in buffer, post-fixed in 1% osmium tetroxide for 1 h, and
dehydrated in graded acetone solutions and embedded in Polarbed 812. One-µm thick plastic
sections were examined by bright field microscopy and areas with a well-preserved
morphology were selected for electron microscopic analysis. Ultrathin sections (90 nm) were
cut and placed on 200-mesh, thin bar copper grids before staining with uranyl acetate and lead
citrate (1). The specimens were examined in a Philips CM-10 transmission electron
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4
microscope (Philips, the Netherlands). The degranulation status of each individual mast cell
was analyzed according to established ultrastructural characteristics (2) and the results were
scored according to the following categories: no degranulation, completely filled granules
with characteristic scroll or crystalline pattern; mild piecemeal degranulation, presence of
granules with loss of structural organization and electron density, and absence of granule
fusions to the plasma membrane (2); advanced piecemeal degranulation, the majority of
granules partially or completely empty and abundant secretory vesicles; and classical
anaphylactic degranulation, granule swelling, fusion, or degranulation channel formation and
occurrence of extracellular granules (3).
Immunohistochemical staining
Double IHC Staining of MCT and MCTC
To stain for mast cell subtypes in the same section, mucosal mast cells (MCT, positive for the
protease tryptase) and connective tissue mast cells (MCTC, positive for proteases chymase and
tryptase), a double-staining technique was developed. In running this protocol, paraffin
sections were pretreated with the high-temperature antigen unmasking technique (pressure
cooking, DIVA buffer, pH 6, for 20 min; Biocare Medical, Concord, CA). The
immunohistochemical staining was performed with an automated immunohistochemistry
robot (Autostainer; DakoCytomation, Glostrup, Denmark) with EnVision™ G|2 Doublestain
System (K5361; Dako). Sections were blocked with dual endogenous enzyme block for 10
minutes. They were then incubated with primary antibody (mouse monoclonal anti-mast cell
chymase; see Table 2) for 1 hour, then incubated with an HRP-conjugated polymer for 30 min
and developed using a DAB+ chromogen. By saturating all chymase-positive mast cells with
a dark brown DAB (3,3’-diaminobenzidine) precipitation staining, they become inert to
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Andersson et al. - Altered Mast Cell Populations in COPD
5
further mast cell tryptase staining (the precipitated DAB complex constitutes a steric
hindrance for any further antibody binding). Next, sections were blocked using double stain
block reagent (5 min) and they were incubated with mouse monoclonal anti-mast cell tryptase
(see Table 1) for 1 hour at room temperature. This was followed by Rabbit/Mouse link, 10
min, and 30 min of incubation with an AP-polymer. Sections were visualized with permanent
red chromogen. Background staining was visualized with hematoxylin and sections were
mounted in Kaiser’s mounting medium (Merck, Darmstadt, Germany). The resulting staining
was MCTC cells in dark brown and the MCT population appeared bright red (Figure 3B,
insert).
Immunohistochemical Identification of Mast Cell-related Molecules
Slides were pretreated using high-temperature antigen unmasking technique (pressure
cooking, DIVA buffer, pH 6, for 20 min; Biocare Medical, Concord, CA). Paraffin sections
were blocked for unspecific binding in 5% dry milk mixed with 20% normal horse serum
(Vector Laboratories, Burlingame, CA) at ambient temperature for 30 min. They were
blocked for endogenous streptavidin and biotin with avidin/biotin blocking kit (Vector
Laboratories, Burlingame, CA). The sections were incubated with primary antibodies to the
molecules of interest (the dilutions are presented in Table 2). All antibodies had been tested
and validated for immunohistochemical use on formalin-fixed tissues and paraffin sections:
renin, TGF-β, and CD88 (see also Table 2). After a rinsing step, sections were incubated for 1
hour at ambient temperature with the biotinylated secondary antibody (Table 2). Next, they
were incubated with Alexa Fluor 555-conjugated streptavidin (1:200, 10 µg/ml, S21381;
Molecular Probes, Eugene, OR) for 30 minutes at ambient temperature. To stain the same
sections for mast cell subtypes also (MCT and MCTC), the mouse monoclonal antibodies used
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6
in the double staining described above, anti-mast cell tryptase and anti-mast cell chymase,
were directly labeled with Alexa Fluor 488 and Alexa Fluor 350, respectively, using Zenon®
Mouse IgG labeling kit (Invitrogen, Molecular Probes, Eugene, OR). Sections were incubated
with the mixed solutions for 1 hour at ambient temperature and mounted in Vectashield
mounting medium (H-1000; Vector Laboratories, Burlingame, CA). All rinse steps were in
TBS buffer. The microscopic examination was performed on a Nikon 80i fluorescence
microscope equipped with specific multiple-wavelength UV filters.
Detection of Apoptotic Mast Cells in Lung Tissues with the TUNEL Technique
Paraffin-embedded sections (3 µm) were deparaffinized and pretreated with proteinase K
(20 µg/ml; Sigma, Stockholm, Sweden) for 15 min at room temperature. Apoptotic cells were
visualized using the TUNEL technique according to the manufacturer’s instructions (ApopTag
Fluorescein In Situ Apoptosis Detection Kit, S7110; Chemicon/Millipore, Billerica, MA) (4).
Apoptotic cells were visualized with a sheep anti-digoxigenin fluorescein (FITC) antibody.
No staining was evident in negative controls when the terminal deoxynucleotidyl transferase
(TdT) enzyme was omitted. Mast cells were detected using primary antibody to tryptase (1 h
at ambient temperature; see Table 2) and visualized with an Alexa-555 conjugated secondary
goat anti-mouse antibody (Invitrogen, Molecular probes, Eugene, OR). Slides were
counterstained with the DNA-binding stain Hoechst 33342 (Sigma) to show the total number
of cell nuclei. As positive control, dexametasone-treated thymus was used.
Morphological and Morphometric Measurements
Density Analysis
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The densities (cells per tissue area, mm2) of MCT and MCTC were calculated in the walls of
small airways and pulmonary vessels, as well as in the alveolar lung parenchyma. In each
tissue region, a large number of cells were quantified and related to the tissue area analyzed.
The alveolar tissue area was calculated by image analysis (Image-Pro Plus; MediaCybernetics
Inc., Silver Springs, MD, and NIS-Elements; Nikon, Kanagawa, Japan). Air spaces were
excluded from the quantifications to give a more relevant tissue density in this compartment.
Detection of leucocytes in small airway lumen
Leukocyte densities of the small airway lumen were detected with immunohistochemical
staining using specific antibodies on consecutive tissue sections (see Table E2). The staining
was performed with an automated immunohistochemistry robot (Autostainer;
DakoCytomation, Glostrup, Denmark) with Dako REALTM
EnvisionTM
Detection System
(K5007; Dako).
TABLE E2. ANTIBODIES USED FOR DETECTION OF LUMINAL CELLS
Antibody Species Dilution Clone Origin Secondary antibody
Anti-MPO Rabbit 1:20 000 Dako, Glostrup, Denmark
Dako REALTM
Envision TM
Detection System
Anti-CD68 Mouse 1:1200 PG-M1 Dako, Glostrup, Denmark
Dako REALTM
Envision TM
Detection System
Anti-ECP Mouse 1:1000 EG2 Pharmacia, Uppsala, Sweden
Dako REALTM
Envision TM
Detection System
Anti-CD4 Mouse 1:100 1F6 Novocastra, Newcastle upon Tyne, UK
Dako REALTM
Envision TM
Detection System
Anti-CD8 Mouse 1:640 C8/144B Dako, Glostrup, Denmark
Dako REALTM
Envision TM
Detection System
Anti-CD20 Mouse 1:1200 L26 Dako, Glostrup, Denmark
Dako REALTM
Envision TM
Detection System
Heat-induced antigen retrieval was performed in citrate buffer, pH 6, in a pressure cooker.
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8
RESULTS
Alteration in the Density and Proportion of Mast cells in COPD
Mucosal and connective tissue mast cells were identified in small airways, small airway
epithelium, small airway luminal plugs, pulmonary vessels, and alveolar parenchyma in
sections from all patient groups by immunohistochemical double staining. In both control and
COPD subjects, mast cells were present in all anatomical compartments of the lung with a
gradual and significant decrease in total mast cell density at the severe stages of COPD and an
increase in the density and proportion of the MCTC population in very severe COPD (GOLD
IV) (Figure 1A, Table E2).
TABLE E3. DENSITY AND PROPORTIONS OF MAST CELL SUBTYPES IN ANATOMICAL
COMPARTMENTS OF THE LUNG, PRESENTED AS MEDIAN AND RANGE
Controls Smokers GOLD I GOLD II–III GOLD IV p-
valuea
Density (MC/mm2)
MCtot 350 (243–530) 358 (228–436) 250 (87–399) 228 (29–275) 216 (144–250) 0.0006
MCT 315 (204–500) 328 (182–391) 225 (71–329) 204 (28–258) 104 (84–171) 0.0002 Small
Airways MCTC 22 (1–73) 36 (15–46) 17 (14–70) 16 (2–171) 83 (27–150) 0.0031
MCtot 213 (122–730) 180 (135–235) 229 (38–567) 179 (102–385) 48 (16–140) 0.0007
MCT 146 (38–521) 92 (79–148) 157 (11–466) 108 (70–255) 14 (6–65) 0.0005 Pulmonary
Vessels MCTC 86 (28–209) 62 (56–119) 84 (27–101) 42 (11–135) 36 (9–74) 0.0137
MCtot 329 (167–611) 178 (120–381) 360 (199–427) 301 (231–602) 251 (136–323) ns (0.06)
MCT 310 (139–571) 170 (114–375) 335 (160–417) 280 (197–534) 112 (25–219) 0.0019 Alveolar
Parenchyma MCTC 19 (10–40) 6 (5–7) 20 (0–40) 34 (2–77) 84 (27–202) <0.0001
MCtot 31 (0–88) 53 (0–292) 64 (0–290) 18 (0–113) 41 (6–201) ns (0.5)
MCT 31 (0–76) 53 (0–243) 62 (0–256) 18 (0–113) 37 (2–193) ns (0.6)
Small
Airway
Epithelium MCTC 0 (0–22) 0 (0–49) 4 (0–34) 0 (0–12) 4 (0–74) ns (0.2)
MCtot 0 (0–2) 0 (0–8) 0 (0–17) 0 (0–41) 17 (0–55) 0.0198
MCT 0 (0–2) 0 (0–8) 0 (0–15) 0 (0–34) 15 (0–33) ns(0.07)
Small
Airway
Lumen MCTC 0 (0–0) 0 (0–0) 0 (0–1) 0 (0–7) 0.4 (0-27) 0.0455
Proportion (%) MCTC
S. Airways MCTC 6 (4–26) 10 (5–20) 15 (5–19) 9 (1–75) 42 (15–62) 0.0011
Pul. Vessels MCTC 46 (13–70) 40 (35–59) 31 (18–70) 24 (4–50) 62 (40–84) 0.0060
Alveolar P. MCTC 6 (3–20) 4 (2–5) 6 (0–20) 11 (1–21) 45 (12–85) 0.0001
SA epithel. MCTC 0 (0–17) 0 (0–17) 10 (0–18) 0 (0–17) 5 (0–72) ns
aSignificance calculated between controls, smokers and mild-to-very severe COPD (Kruskal-Wallis
test). Data are presented as median (range). SA = small airway. P = parenchyma. Proportion
presented as percentage MCTC of total numbers of mast cells.
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Mast cell density is reduced in the central airways compared to controls
When analyzing the two major mast cell subtypes, a decrease in the MCT population was
found in the bronchi of patients with very severe COPD relative to the controls. An increase
in the MCTC population in terms of both density and proportion was, however, observed in
very severe COPD (see Table E3). No significant differences were observed in control tissue
from lung resection compared to bronchial biopsies from healthy individuals.
TABLE E4. MAST CELL DENSITIES FOR CENTRAL AIRWAYS IN CONTROLS AND PATIENTS
WITH VERY SEVERE COPD (GOLD IV)
Controls
(Lung resections)
Controls
(Bronchial biopsies)
COPD (GOLD IV)
Transplantation p-value
MCtot 195 (77–345) 157 (77–601) 69 (29–307) 0.02
MCT 194 (69–345) 153 (69–566) 34 (12–108) 0.0005
MCTC 6 (0–22) 7 (0–35) 44 (16–199) 0.0008
% MCTC 5 (0–10) 7 (0–11) 60 (31–80) 0.0002
Data are presented as median (range).
Presence of Mast Cells, Epithelial Cells, and Leukocytes in the Airway Lumen
Exfoliated epithelial cells increased in very severe COPD (229 (15-525) cells/mm2, median
and range) compared to controls (0 (0-37) cells/mm2, p=0.008). No statistical correlation was
found between luminal epithelial cells and luminal mast cells (MCtot; rs=0.1, p=0.95, MCT;
rs=-0.2, p=0.8 and MCTC; rs=0.8, p=0.1). Intra-luminal neutrophils, monocytes/macrophages,
CD4+ and CD8
+ T-lymphocytes were all increased in COPD whereas no change was found
for eosinophils and B-lymphocytes (Table E5). No significant correlations were found
between intra-luminal mast cells and any of the examined leukocytes (Table E5).
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TABLE E5. DENSITIES OF LUMINAL CELLS IN CONTROLS AND PATIENTS WITH VERY
SEVERE COPD (GOLD IV) WITH CORRELATION TO TOTAL MAST CELL DENSITIES
Correlation
Controls
(Cells/mm2)
COPD (GOLD IV)
(Cells/mm2)
p-value rS p-value
MPO 0.0002 (0-0.002)* 7911 (803-68777)* <0.0001 -0.4 0.3
CD68 2 (0-60) 43 (8-226) 0.03 0.3 0.3
EG2 0 (0-16) 1 (0-21) 0.09 0.3 0.4
CD4 0 (0-2) 2 (0-248) 0.002 -0.2 0.6
CD8 0 (0-18) 6 (0-24) 0.04 0.2 0.6
CD20 0 (0-3) 0 (0-16) 0.1 -0.2 0.7
Data presented as median (range). *Density as pixels/mm2.
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