1 Center For Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill 27599; and 2 National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Little is known
about the functional capabilities of bronchial macrophages (BMs) and
their relationship to airway disease such as asthma. We hypothesize
that BMs from asthmatics may be modulated in their function compared
with similar cells from healthy individuals. BMs obtained by induced
sputum from mild asthmatics (n = 20) and healthy
individuals (n = 20) were analyzed using flow cytometry for
CD16, CD64, CD11b, CD14, and human leukocyte antigen-DR expression,
phagocytosis of IgG opsonized yeast, and oxidant production. Asthma
status was assessed by lung function [percent predicted forced vital
capacity and forced expiratory volume in 1 s
(FEV1)], percent sputum eosinophils, and nonspecific airway responsiveness [provocative concentration that produces a
20% fall in FEV1 (PC20,FEV1)]. Asthmatics
with >5% airway eosinophils (AEo+) had decreased BM CD64 expression
and phagocytosis compared with asthmatics with <5% eosinophils
(AEo). Among asthmatics, a significant correlation was found between
CD64 expression and BM phagocytosis (R = 0.7, P < 0.009). Phagocytosis was also correlated with
PC20,FEV1 (R = 0.6, P < 0.007), lung function (%predicted FEV1, R = 0.7, P < 0.002) and percent eosinophils (R
=
0.6, P < 0.01). In conclusion, BM from
asthmatics are functionally modulated, possibly by Th2 cytokines
involved in asthma pathology.
asthma; bronchial macrophages; induced sputum; flow cytometry analysis of surface receptors; CD64; CD11b; phagocytosis
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INTRODUCTION |
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THE IMMUNOPATHOLOGY OF
ALLERGIC asthma is characterized by peribronchial infiltration of
macrophages, eosinophils, and T cells in stable and symptomatic periods
of disease (2, 5, 37, 39). These cells may all be
functionally involved in asthma symptomatology. Among them, macrophages
are critical in removing inhaled particulates and microbes that have
the potential to exacerbate asthma (6, 11, 43). Previous
studies have suggested a link between decreased phagocytic function and
asthma (26, 27) as well as enhanced superoxide generation
(10, 20) and mediator release (31) by airway
cells in asthmatics. These studies, however, examined peripheral blood
phagocytes, not bronchial macrophages (BMs) proximal to the site of
disease pathology. T cells at the site of allergic-asthmatic disease
have been shown to preferentially produce the cytokines interleukin
(IL)-4 and IL-5 (31). IL-4 has been shown to modulate
surface receptors on macrophages in vitro (3), while IL-5
promotes the survival and activation of eosinophils both in vivo and in
vitro (1). Among the receptors modulated by IL-4 are
human leukocyte antigen (HLA)-DR and the phagocytic receptor FcRI,
i.e., CD64. HLA-DR expression is increased while CD64 expression
is decreased following exposure to IL-4 (3). Decreased
expression of CD64 and CD11b is associated with decreased
phagocytosis (3, 53). On the other hand, exposure to
interferon-
(IFN-
), also found to be increased in
asthmatics (38), upregulates CD64 and HLA-DR while
decreasing CD11b expression and phagocytosis (3).
Induced sputum has proven to be a safe and relatively noninvasive procedure that obtains cells and fluid phase components from the bronchial airways (33-35). As a result, induced sputum is well suited to examine bronchial airway cells from asthmatic subjects. Sputum eosinophilia, described cytologically as >2-5% eosinophils (18, 22), can serve as a reliable inflammatory marker of asthma severity in asthmatics with airway eosinophilia (32, 46, 49). However, many asthmatics do not have airway eosinophilia even following acute exacerbations (18, 23, 47). Asthmatics with eosinophilic sputum have been reported to have increased symptoms of coughing and wheezing, greater airway obstruction, and more severe airway hyperresponsiveness compared with asthmatics without sputum eosinophilia (18). Few studies have attempted to investigate the characteristics and functions of cells obtained by the induced sputum technique (19). In this study, we wanted to determine whether, in mild atopic asthmatics, lung function and sputum eosinophilia, which have been shown to be associated, also correlate with modulated macrophage phenotype and function. Flow cytometry was used to assess the expression of cell surface receptors known to be modulated by environmental factors as well as by phagocytosis in 20 mild asthmatic subjects and 20 controls.
The data obtained suggest an intriguing correlation between eosinophilic inflammation, lung function, and decreased BM function, associated with decreased CD64 expression, in subjects with mild asthma.
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METHODS |
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Subjects.
Subject characteristics are shown in Table
1. Nonsmoking volunteers between 18 and
40 yr of age were recruited for the study. A medical screening exam
that included a medical history, psychological questionnaire, physical
exam, blood tests, and allergy scratch tests was performed on all
subjects on a separate day before the study. All asthmatics and 10 healthy subjects received a methacholine challenge test to assess
nonspecific airway responsiveness. The maximum concentration of
methacholine used was 40 mg/ml. Seven of ten healthy subjects were able
to achieve a nonspecific airway responsiveness [provocative
concentration that produces a 20% fall (PC20) in forced
expiratory volume in 1 s (FEV1;
PC20,FEV1)] by the maximum concentration. Pulmonary
function tests were performed between 8:00 and 9:00 AM on all subjects,
and with the exception of one subject, none of the asthmatics had taken
any 2-agonist within 24 h of pulmonary function
testing.
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Induced sputum.
The induction procedure of Pin et al. (32) was followed
with some modifications. Three 7-min inhalation periods of 3, 4, and
5% hypertonic saline were administered following baseline spirometry.
All asthmatics were premedicated with inhaled 2-agonist before saline inhalation, and FEV1 was checked following
each saline dose. At the end of each 7-min inhalation period, subjects performed a three-step cleansing procedure before a cough attempt as
follows: 1) the mouth was rinsed and gargled with water,
2) the back of the throat was cleared (without coughing),
and 3) the nose was blown. The subject was then instructed
to perform a "chesty-type" cough without clearing the back of the
throat. The sample was expectorated into a sterile specimen cup that
was placed on ice throughout the procedure.
Analysis of fluid-phase cytokines and eosinophil cationic
protein.
Supernatants from the soluble fraction of the spun-down sputum sample
were collected and analyzed for IL-4, IL-5, IL-8, IFN-, and
eosinophil cationic protein (ECP). Cytokines were measured by specific
and sensitive human immunoassays (ELISA, Quantikine; R&D Systems,
Minneapolis MN). The lower limits of detection for IL-4, IL-5, IL-8,
and IFN-
were as follows: 35, 3, 10, and 3 pg/ml,
respectively. ECP levels in sputum supernatant were measured by
means of specific radioimmunoassay (Pharmacia RIA, Uppsala, Sweden).
This method has been proven highly specific and sensitive (lower
detection limit <2 µg/l), with high intra- and interassay reproducibility.
Flow cytometry.
Flow cytometry was performed with a FACSORT (Becton Dickinson) using an
argon-ion laser (wavelength = 488 nm). Gain and amplitude settings
were set so as to analyze both blood and sputum samples from the same
subject to establish reference gates for sputum leukocyte
identification. Settings were kept the same throughout the study for
each subject. The FACSORT was calibrated with Calibrite (Becton
Dickinson) beads (no color, green, and red) before each use. Ten
thousand events were counted for all sample runs. Gating of white cells
in sputum was based on a combination of light-scatter characteristics
and positive/negative expression for relevant surface markers,
i.e., lymphocytes CD3+/CD14, monocytes
CD14+/CD3
, neutrophils
CD16+/HLA-DR
, macrophages
HLA-DR+/CD14+, and the use of reference gates
based on isolated (Percoll-separated) leukocytes from whole blood
preparation. Side light scatter reflects cell density/granularity and
forward light scatter reflects cell size (41). Using these
techniques, discrete populations of lymphocytes, monocytes,
neutrophils, macrophages, and eosinophils were observed. Fluorescein
(FITC)- and phycoerythrin-conjugated nonspecific antibodies of the same
isotype as the receptor antibodies were used as controls to establish
background fluorescence and nonspecific antibody binding. The mean
fluorescence intensity (MFI) of the cells stained with control antibody
was subtracted from the mean fluorescence of the cells stained
with receptor antibodies to provide a measure of receptor-specific fluorescence.
Immunofluorescence staining.
Aliquots of 100 µl (100,000 cells/tube) or 200 µl (200,000 cells/tube) of sputum cell suspension (1 × 106
cells/ml) were stained with 10 or 20 µl, respectively, of saturating concentrations of monoclonal antibodies (Immunotech, Coulter, France)
for 60 min in the dark at 4°C. After they were stained, the cells
were washed with 2 ml of cold Hanks' balanced salt solution (HBSS) and centrifuged for 5 min at 1,000 rpm at 4°C, and
supernatants were decanted and blotted. The cells were then resuspended
in cold HBSS (250 µl) and fixed with paraformaldehyde (250 µl, 0.5%) for a final volume of 500 µl. The cells were then
stored at 4°C in the dark until analyzed on the flow cytometer within
24 h of staining. The following monoclonal antibodies were used:
CD11b to recognize the complement receptor (CR3), CD64 to recognize the
FcRI receptor, and CD45 to recognize all white cells.
Measurement of surface marker expression was done using a Becton
Dickinson FACSORT flow cytometer. Analysis of surface marker expression was done using the Cell Quest software (Becton Dickinson), which provided a calculation of MFI for the gated populations.
Phagocytosis. Saccharomyces cerevisiae zymosan A BioParticles (Molecular Probes, Eugene, OR) conjugated to FITC were opsonized with opsonizing reagent (IgG) for 45 min at 37°C and then washed with RPMI 1640 medium two times before the particle concentration was adjusted to 2 × 106/ml. Sputum cells (2 × 106/ml) from the same subject were exposed to the yeast cell walls at a ratio of 1:10 for 1 h at 37°C in the presence of human serum (20 µl) before tubes were placed on ice. Next, 200 µl of 2% paraformaldehyde were added to each tube, and the tubes were stored at 4°C in the dark until analyzed by flow cytometry (FACSORT) within 24 h of particle exposure. Particle uptake was identified on histogram analysis as a uniform rightward shift into the M2 region (MFI) from the control (non-particle-exposed) M1 region. Phagocytosis was determined by measuring the MFI of the cells in the particle-exposed region (M2) less the MFI of the cells in the control region (M1). A uniform shift in all sputum phagocyte populations was routinely observed when cells were exposed to particles such that at least 93% of the cells shifted (with particle exposure) into M2 compared with M1 (no particle exposure) on all occasions.
Statistical analyses. Statistically significant differences between subject groups were assessed by one-way ANOVA. Parametric statistics were applied on log-transformed PC20 data for tests of differences between means and for correlations. Simple linear regression analyses were performed to analyze associations between variables, and Pearson's correlation coefficients (R) were determined from these analyses. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Characterization of subjects, their lung function, and sputum
differentials.
Subject characteristics including baseline lung function and
nonspecific airway responsiveness are shown in Table 1. All the
asthmatics examined had mild, well-controlled disease, i.e., inhaled
bronchodilator was used as needed, no inhaled corticosteroids were taken within the previous 6 mo, and a normal range spirometry was
present. Sputum eosinophil counts are shown in Table
2. Asthmatic subjects with 5% or greater
sputum eosinophils were classified as eosinophilic (AEo+;
n = 9), and asthmatic subjects with <5% sputum
eosinophils were classified as noneosinophilic (AEo;
n = 11). The arbitrary 5% cutoff for designating
eosinophilia was based on prior reports describing sputum eosinophilia
in asthmatics (33, 18, 22, 46, 47). Asthmatics
demonstrated lower baseline lung function (%predicted FVC, %predicted
FEV1) and higher nonspecific airway responsiveness
(PC20,FEV1) than healthy controls, and AEo+ subjects
demonstrated lower baseline lung function, higher nonspecific airway responsiveness, and greater peripheral blood eosinophils than AEo
subjects. The AEo
subjects demonstrated significantly greater nonspecific airway responsiveness
than healthy controls (PC20,FEV1 = 2.3 vs. 11.9 mg/ml, P < 0.05), verifying their mild asthma status.
Significant inverse relationships were observed (data not shown)
between the percentage of sputum eosinophils and the percentage of
predicted FEV1 (R =
0.5,
P < 0.001), FVC (R =
0.5,
P < 0.001) and PC20,FEV1
(R =
0.5, P < 0.002) for all
asthmatics.
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Flow cytometric analysis of induced sputum samples.
The flow cytometer was used to quantify the expression of CD11b, CD14,
CD16, CD64, and HLA-DR on BM. The identification of the BM population
was easily achieved by the light-scatter properties (forward scatter
and side scatter) of the population with no overlap with
neutrophils or monocytes. Figure
1 shows the results of surface marker expression of CD11b, CD64, CD16, CD14, and HLA-DR on BMs in
healthy and asthmatic AEo+ and AEo subjects. If all the mild asthmatics were analyzed as one group, there was no difference between
healthy and asthmatic subjects for any of the markers. When subdivided
into AEo+ and AEo
asthmatics, however, Fc
RI (CD64,
P < 0.04) and CR3 (CD11b, P < 0.05)
expression (MFI) were significantly decreased in AEo+ subjects compared
with the AEo
subjects and controls (CD64, P < 0.03;
CD11b, P < 0.05). Expression of CD16, CD14, and HLA-DR
on BMs was not altered in subjects with eosinophilic inflammation.
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Sputum macrophage phagocytosis of IgG-opsonized
yeast particles.
No difference was observed in the percentage of BM-phagocytosing
particles between asthmatics and controls or between asthmatic subgroups. More than 94% of BMs took up particles in all subjects. When the number of particles taken up by the BM was measured as MFI of
the BM population, AEo+ subjects showed significantly decreased phagocytosis compared with the AEo subjects (P < 0.001) and controls (P < 0.001) (Fig.
2). BM phagocytosis was significantly
related to the expression of Fc
RI (CD64; R = 0.7, P < 0.01; Fig. 3).
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Association between sputum macrophage phagocytosis
and markers of asthma severity.
Sputum macrophage phagocytosis appeared to be predictive of
disease severity. Significant correlations were observed between sputum
macrophage phagocytosis and markers of disease severity, such as
percentage of eosinophils (R = 0.6, P < 0.01; Fig. 4A), percentage
of predicted FEV1 (R = 0.7, P < 0.002; Fig. 4B), percentage of
predicted FVC (R = 0.8, P < 0.0001;
Fig. 4C), and PC20,FEV1 (R = 0.6, P < 0.007), when all asthmatics were included.
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Fluid-phase components.
The subjects with eosinophilia (AEo+) demonstrated significantly higher
levels of ECP compared with the AEo subjects (P < 0.05) and healthy controls (P < 0.001), and ECP levels
were significantly related to the percentage of eosinophils in all
asthmatic subjects (R = 0.7, P < 0.001; data not shown) (Table
3). Cytokines IL-5 and IL-8 were
detectable in airway fluid but did not differ between AEo+ and AEo
subjects. IL-4 was not detectable in the same samples.
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DISCUSSION |
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This study has analyzed the phenotype and function of BMs obtained
from induced sputum from mild asthmatic and healthy subjects. Based on their composition of airway leukocytes, the asthmatic subjects could be separated into those with airway eosinophilia as
defined by the presence of >5% sputum eosinophils (mean = 32%) and those with noneosinophilic sputum (mean = 2%). The assessment of asthma severity by nonspecific bronchial responsiveness, baseline pulmonary function, and ECP levels supported the notion that increased levels of eosinophilic inflammation is associated with increased airway
responsiveness and therefore disease severity (22).
Accordingly, the AEo+ subject group was hyperreactive compared with the
AEo subject group. Our results showed that BMs from AEo+ subjects have decreased phagocytic function and decreased expression of Fc
RI
(CD64) and CR3 (CD11b) compared with AEo
subjects; however, as one
group, the mild asthmatics did not differ from normal subjects. This is
the first study to demonstrate that macrophages obtained from a site
proximal to asthma pathology are phenotypically and functionally
altered. Earlier observations on peripheral blood phagocytes from
subjects with asthma and chronic bronchitis have suggested that
phagocytic dysfunction may also be present in the circulation of
asthmatic individuals (26-28).
Impaired phagocytosis by BMs was found to be associated with cellular
and spirometric markers of asthma severity. Relatively strong positive
correlations were observed between sputum macrophage phagocytosis and
percent predicted FEV1 (R = 0.7), FVC
(R = 0.8) and PC20,FEV1
(R = 0.8), and an inverse correlation was observed between sputum macrophage phagocytosis and sputum eosinophils (R = 0.6). Although it is unlikely that eosinophils
directly inhibit macrophage phagocytosis or that phagocytosis directly impacts lung volumes, these correlations do not likely represent an
epiphenomenon, but rather the pathogenesis of asthma severity may be
related to the ability of the airways to mount a basic host defense response.
We hypothesized that changes in BM phenotype and function were the
result of the cellular environment in asthmatic airways. T cells in
allergic asthma preferentially produce Th2-type cytokines including
IL-4 and IL-5, and cytokine-producing cells can be identified in the
mucosa of asthmatic individuals and in bronchoalveolar lavage fluid
following allergen provocation (9, 31, 42, 44). In vitro
experiments have shown that small concentrations of IL-4 (10 pg/ml) can
significantly downregulate the expression of CD64 and inhibit
FcR-mediated phagocytosis by human mononuclear phagocytes
(42). Unfortunately, the attempts to measure IL-4 in the
sputum supernatant were unsuccessful because most measurements were
below the lower limit of detection of the immunoassay (35 pg/ml). Other
investigators have also reported difficulty in recovering IL-4 in
sputum supernatant (23). Even spike experiments with exogenous purified IL-4 have yielded poor results, suggesting that the
sputum contains components interfering with IL-4 immunoreactvity. On
the other hand, both IL-4 and IL-5 mRNAs have been detected in the
sputum cells of asthmatic subjects, and the number of cells expressing
these cytokines was significantly higher in asthmatics than in controls
(31). In the present study, immunoreactive IL-5, IL-8, and
IFN-
could be detected in the sputum samples of most of the
subjects, but no difference was found in the levels of cytokines
between normal subjects and asthmatics or, with the exception of
IFN-
, between the AEo+ and AEo
groups. While induced sputum is a
good technique that gives reproducible and valid results on
inflammatory cells and most soluble markers of inflammation (IL-8, ECP)
(13, 17, 31a, 34, 50), it may not recover cytokines (IL-4,
IL-5) that are known to be synthesized and secreted by cells
predominantly found in the airway wall, i.e., lymphocytes (14,
25, 54).
The data showed that, along with decreased FcRI expression, BMs from
AEo+ subjects also demonstrated significantly decreased CR3 (CD11b)
expression. Since these two receptors interact in the phagocytic
process (4, 48, 52), the decrease in uptake of opsonized
yeast may be the result of decreased expression of both these
receptors. What then may be the significance of decreased BM phagocytic
function in asthmatic individuals? They are not known to have increased
fungal or bacterial airway infections. However, many microbes, both
opportunistic pathogenic and nonpathogenic organisms, are frequently
found in asthmatic airways where their presence may lead to increased
asthma severity (7). The presence of protease-producing
fungi has been shown to lead to asthma symptoms, and histamine release
induced by bacteria exacerbates the disease (21, 29).
Aspergillus fumigatus is an opportunistic pathogen to which
asthmatic subjects are particularly susceptible (21). Furthermore, decreased microbial clearance due to impaired
bronchial macrophage phagocytosis may require the involvement of
neutrophil inflammation to resist subsequent infection. This process
may, in turn, exacerbate asthma. Interestingly, some of studies have implicated bacterial infections as causative in the development of
asthma severity (30).
In conclusion, this study has demonstrated that, in a population of mild asthmatics, eosinophil inflammation, airway function, and macrophage function and phenotype are interrelated. How these markers of asthma severity link to and affect each other is still an open question.
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ACKNOWLEDGEMENTS |
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This study has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, and mention of trade names and commercial products does not constitute endorsement or recommendation for use.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Alexis, Center For Environmental Medicine and Lung Biology, Univ. of North Carolina, Chapel Hill, 104 Mason Farm Rd., Chapel Hill, NC 27599-7310 (E-mail: Neil_Alexis{at}med.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 May 2000; accepted in final form 29 August 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arm, JP,
and
Lee TH.
The pathobiology of bronchial asthma.
Adv Immunol
51:
323-382,
1992[ISI][Medline].
2.
Beasley, R,
Roche WR,
Roberts JA,
and
Holgate ST.
Cellular events in the bronchi in mild asthma and after bronchial provocation.
Am Rev Respir Dis
139:
806-817,
1989[ISI][Medline].
3.
Becker, S,
and
Daniel EG.
Antagonistic and additive effects of IL-4 and interferon- on human monocytes and macrophages: effects on Fc receptors, HLA-D antigens, and superoxide production.
Cell Immunol
129:
351-362,
1990[ISI][Medline].
4.
Berger, M,
Norvell TM,
Tosi MF,
Emancipator SN,
Konstan MW,
and
Schreiber JR.
Tissue-specific Fc gamma and complement receptor expression by alveolar macrophages determines relative importance of IgG and complement in promoting phagocytosis of Pseudomonas aeruginosa.
Pediatr Res
35:
68-77,
1994[Abstract].
5.
Bousquet, J,
Chanez P,
Lacoste JY,
Barneon G,
Ghavanian N,
Enander I,
Benge P,
Ahlstedt S,
Simon-Lafontaine J,
Godard P,
and
Michel FB.
Eosinophilic inflammation in asthma.
N Engl J Med
323:
1033-1039,
1990[Abstract].
6.
Brain, JD.
Macrophages in the respiratory tract.
In: Handbook of Physiology. The Respiratory System. Circulation and Nonrespiratory Functions. Bethesda, MD: Am. Physiol. Soc, 1985, sect. 3, vol. I, chapt. 14, p. 447-471.
7.
Cazzola, M,
Matera MG,
and
Rossi F.
Bronchial hyperresponsiveness and bacterial respiratory infections.
Clin Ther
13:
157-171,
1991[ISI][Medline].
9.
Clutterbuck, EJ,
Hirst EM,
and
Sanderson CJ.
Human interleukin 5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparisons and interactions with IL-1, IL-3, IL-6 and GM-CSF.
Blood
73:
1504-1512,
1989[Abstract].
10.
Cluzel, M,
Damon M,
Chanez P,
Bousquet J,
Crastes de Paulet A,
Michel FB,
and
Godard P.
Enhanced alveolar cell luminol-dependent chemiluminescence in asthma.
J Allergy Clin Immunol
80:
195-201,
1987[ISI][Medline].
11.
Dockery, D,
Pope C, III,
Xu X,
Spengler J,
Ware J,
Fay M,
Ferris B,
and
Speizer F.
An association between air pollution and mortality in six US cities.
N Engl J Med
329:
1753-1759,
1993
12.
Doerschug, KC,
Peterson MW,
Dayton CS,
and
Kline JN.
Asthma guidelines.
Am J Respir Crit Care Med
159:
1735-1741,
1999
13.
Efthimiadis, A,
Pizzichini MMM,
Pizzichini E,
Dolovich J,
and
Hargreave FE.
Induced sputum cell and fluid-phase indicies of inflammation: comparison of treatment with dithiothreitol vs. phosphate buffered saline.
Eur Respir J
10:
1336-1340,
1997
14.
Fahy, JV,
Kwang WK,
Liu J,
and
Boushey HA.
Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation.
J Allergy Clin Immunol
95:
843-852,
1995[ISI][Medline].
17.
Girgis-Garbado, A,
Kanai N,
Denburg JA,
Hargreave FE,
Jordana M,
and
Dolovich J.
Immunocytochemical detection of granulocyte-macrophage colony-stimulating factor and eosinophil cationic protein in sputum cells.
J Allergy Clin Immunol
93:
945-947,
1994[ISI][Medline].
18.
Grootendorst, DC,
van den Bos J-W,
Romeijn JJ,
Veselic-Charvat M,
Duiverman EJ,
Vrijlandt EJ,
Sterk PJ,
and
Roldaan AC.
Induced sputum in adolescents with severe stable asthma. Safety and the relationship of cell counts and eosinophil cationic protein to clinical severity.
Eur Respir J
13:
647-653,
1999
19.
In', T,
Veen JCCM,
Grootendorst DC,
Bel EH,
Smits HH,
Van Der Keur M,
Sterk PJ,
and
Hiemstra PS.
CD11b and L-selectin expression on eosinophils and neutrophils in blood and induced sputum of patients with asthma compared with normal subjects.
Clin Exp Allergy
28:
606-615,
1998[ISI][Medline].
20.
Jarjour, NN,
and
Calhoun WJ.
Enhanced production of oxygen radicals in asthma.
J Lab Clin Med
123:
134-137,
1994.
21.
Kauffman, HF,
Tomee JF,
van der Werf TS,
deMonchy JG,
and
Kloeter GK.
Review of fungus-induced asthma.
Am J Respir Crit Care Med
151:
2109-2115,
1995[Abstract].
22.
Keisaku, F,
Kieshi K,
Matsuzawa Y,
and
Sekiguchi M.
Eosinophil cationic protein levels in induced sputum correlate with the severity of bronchial asthma.
Chest
112:
1241-1247,
1997
23.
Kotsimbos, TC,
Ghaffar O,
Minshall EM,
Humbert M,
Durham SR,
Pfister R,
Menz G,
Kay AB,
and
Hamid QA.
Expression of the IL-4 receptor -subunit is increased in bronchial biopsy specimens from atopic and nonatopic asthmatic subjects.
J Allergy Clin Immunol
102:
859-866,
1998[ISI][Medline].
25.
Maestrelli, P,
Saetta M,
Di Stefano A,
Calcagni PG,
Turato G,
Ruggieri MP,
Roggeri A,
Mapp CE,
and
Fabbri LM.
Comparison of leukocyte counts in sputum, bronchial biopsies, and bronchoalveolar lavage.
Am J Respir Crit Care Med
152:
1926-1931,
1995[Abstract].
26.
Matusiewicz, R,
Lebiedowski K,
Kowalczyk M,
Czajkowski M,
and
Stempniak M.
Ability of peripheral blood neutrophils from patients with infectious bronchial asthma to engulf latex particles and reduce nitroblue tetrazolium.
Arch Immunol Ther Exp (Warsz)
36:
55-59,
1988[ISI][Medline].
27.
Matusiewicz, R,
and
Rusiecka-Matusiewicz K.
The ability of granulocytes from patients with atopy to engulf neutral latex particles and Staphylococcus aureus.
Arch Immunol Ther Exp (Warsz)
35:
781-785,
1987[ISI][Medline].
28.
Nielson, HJ,
and
Bonde J.
Association of defective monocyte chemotaxis with recurrent acute exacerbations in chronic obstructive lung disease.
Eur J Respir Dis
68:
200-205,
1986[ISI][Medline].
29.
Norn, S,
Skov PS,
Jensen CM,
Espersen F,
Jarlov JO,
and
Koch C.
Bacteria and their products release histamine and potentiate mediator release: new aspects of airways disease.
Eur J Respir Dis
S147:
230-234,
1986.
30.
Oehling, A.
Bacterial immunotherapy in bronchial asthma.
J Investig Allergol Clin Immunol
7:
14-19,
1997[ISI][Medline].
31.
Olivenstein, R,
Taha R,
Minshall EM,
and
Hamid QA.
IL-4 and IL-5 mRNA expression in induced sputum of asthmatic subjects: comparison with bronchial wash.
J Allergy Clin Immunol
103:
238-245,
1999[ISI][Medline].
31a.
Ordonez, CL,
Shaughnessy TE,
Matthay MA,
and
Fahy JV.
Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma: clinical and biologic significance.
Am J Respir Crit Care Med
161:
1185-1190,
2000
32.
Pin, I,
Freitag AP,
O'Byrne PM,
Girgis-Gabardo A,
Watson RM,
Dolovich J,
and
Hargreave FE.
Changes in the cellular profile of induced sputum after allergen-induced asthmatic responses.
Am Rev Respir Dis
145:
1265-1269,
1992[ISI][Medline].
33.
Pizzichini, E,
Pizzichini MMM,
Dolovich J,
and
Hargreave FE.
Measuring airway inflammation in asthma: eosinophils and ECP in induced sputum compared with peripheral blood.
J Allergy Clin Immunol
99:
539-544,
1997[ISI][Medline].
34.
Pizzichini, E,
Pizzichini MM,
and
Efthimiadis A.
Measurement of inflammatory indices in induced sputum: effects of selection.
Eur Respir J
9:
1174-1180,
1996
35.
Pizzichini, MMM,
Pizzichini E,
Clelland L,
Efthimiadis A,
Pavord I,
Dolovich J,
and
Hargreave FE.
Prednisone-dependent asthma: inflammatory indices in induced sputum.
Eur Respir J
13:
15-21,
1999
37.
Poston, RN,
Chanez P,
Lacoste JY,
Litchfield T,
Lee TH,
and
Bousquet J.
Immunohistochemical characterization of the cellular infiltration of asthmatic bronchi.
Am Rev Respir Dis
145:
918-921,
1992[ISI][Medline].
38.
Poulter, L,
and
Burke CM.
Macrophages and allergic lung disease.
Immunobiology
195:
574-587,
1996[ISI][Medline].
39.
Poulter, LW,
Power C,
and
Burke C.
The relationship between bronchial immunopathology and hyperresponsiveness in asthma.
Eur Respir J
3:
792-799,
1990[Abstract].
41.
Rossman, MD,
Chen E,
Chien P,
Rottem M,
Cprek A,
and
Schreiber AD.
Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages.
Am J Respir Cell Mol Biol
1:
211-220,
1989[ISI][Medline].
42.
Salzman, G,
and
Mullaney PP.
Light scattering approaches to cell characterizations.
In: Flow Cytometry and Sorting, edited by Melamed M,
Mullaney M,
and Mendelsohn P.. New York: Wiley, 1979, p. 105-124.
43.
Sanderson, CJ.
Interleukin 5, eosinophils, and disease.
Blood
79:
3101-3109,
1992[ISI][Medline].
44.
Schwartz, J.
Air pollution and daily mortality: a review and meta analysis.
Environ Res
64:
36-52,
1994[ISI][Medline].
46.
Te Velde, A,
de Waal M,
Huijbens J,
de Vries J,
and
Figdor C.
IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4 and IL-10.
J Immunol
149:
4048-4053,
1992
47.
Turner, MO,
Hussack P,
Sears MR,
Dolovich J,
and
Hargreave FE.
Exacerbations of asthma without sputum eosinophilia.
Thorax
50:
1057-1061,
1995[Abstract].
48.
Unkeless, JC.
Function and heterogeneity of human Fc receptors for immunoglobulin G.
J Clin Invest
83:
355-361,
1989[ISI][Medline].
49.
Vicksman, M,
Liu M,
Bickel C,
Schleimer RP,
and
Bochner BS.
Phenotypic analysis of alveolar macrophages and monocytes in allergic airway inflammation.
Am J Respir Crit Care Med
155:
858-863,
1997[Abstract].
50.
Virchow, JC, Jr,
Holscher U,
and
Virchow C, Sr.
Sputum ECP levels correlate with parameters of airflow obstruction.
Am Rev Respir Dis
146:
604-606,
1992[ISI][Medline].
52.
Wright, S.
Receptors for complement and the biology of phagocytosis.
In: Inflammation: Basic Principles and Clinical Correlates, edited by Gallin J,
Goldstein M,
and Snyderman R.. New York: Raven, 1992, p. 477.
53.
Wright, SD,
Detmers PA,
Jong MTC,
and
Meyer BC.
Interferon- depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin.
J Exp Med
163:
1245-1259,
1986[Abstract].
54.
Yamaguchi, Y,
Suda T,
Suda J,
Eguchi M,
Miura Y,
Harada N,
Tominaga A,
and
Takatsu K.
Purified interleukin 5 supports the terminal differentiation and proliferation of murine eosinophilic precursors.
J Exp Med
167:
43-56,
1988[Abstract].