Alterations in Lung Mast Cell Populations in Patients with Chronic Obstructive Pulmonary Disease

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: jonas.erjefalt@med.lu.se

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

of 51 AJRCCM Articles in Press. Published on November 19, 2009 as doi:10.1164/rccm.200906-0932OC

Andersson et al. - Altered Mast Cell Populations in COPD

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

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

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

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-

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-

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-

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

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-

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).

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

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

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

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)

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

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).

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.

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

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.

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-

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).

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

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.

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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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-

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.

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.

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

(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

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

Anti-TGF-β Mouse 1:40 TGFB17 Novocastra, Newcastle upon Tyne, UK

Anti-cytokeratin Mouse 1:100 MNF116 Dako, Glostrup, Den-mark

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.

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.

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

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

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

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

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

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

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.

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)

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)

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.

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).

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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in the airway mucosa at epithelial removal-restitution. Am J Respir Crit Care Med

1996;153:1666-1674.

E2. Dvorak AM. Ultrastructural studies of human basophils and mast cells. J

Histochem Cytochem 2005;53:1043-1070.

E3. Dvorak AM, Hammel I, Schulman ES, Peters SP, MacGlashan DW, Jr.,

Schleimer RP, Newball HH, Pyne K, Dvorak HF, Lichtenstein LM, et al. Differences in the

behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation.

J Cell Biol 1984;99:1678-1687.

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