![shows flow cytograms of leukocyte populations recovered from peripheral blood (Fig. 1A), BAL (Fig. 1B), BW (Fig. 1C), and induced sputum (Fig. 1D) from the same subject. Using FSC and SSC and positive expression for CD45, and keeping identical measurement settings on the flow cytometer for each fluid sample analyzed, populations of Mac, Mono, PMN, Lym, and Eos were distinguished and compared both within and between samples. Figure 2 shows mean light scatter results from peripheral blood, BAL, and sputum in neutrophils (Fig. 2A), macrophages (Fig. 2B), and monocytes (Fig. 2C). SPMN were smaller in size than PBPMN [FSC (MFI): 280 20 vs 385 10, P 0.0001]; SMac were significantly denser than AMac [SSC (MFI): 780 45 vs 600 37, P 0.0001], but were smaller in size on average. SMono were significantly larger than PBMono [FSC (MFI): 480 15 vs 275 9, p 0.01], but had very similar granularity [SSC (MFI): 200 8 vs 193 7] (data not shown).](https://www.researchgate.net/profile/Andrew-Ghio/publication/12325569/figure/fig1/AS:349617188294666@1460366641851/shows-flow-cytograms-of-leukocyte-populations-recovered-from-peripheral-blood-Fig-1A_Q320.jpg)
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Sputum Phagocytes from Healthy Individuals Are Functional and
Activated: A Flow Cytometric Comparison with Cells
in Bronchoalveolar Lavage and Peripheral Blood1
Neil Alexis,2Joleen Soukup,* Andrew Ghio,* and Susanne Becker*
Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, North Carolina 27599-7310; and
*Clinical Research Branch, Human Studies Division, U.S. EPA, Chapel Hill, North Carolina 27599
Cells in the bronchial airways of healthy individuals
are continuously exposed to inhaled particulates in
the size range 2–5
m, which preferentially deposit in
the bronchial rather than the alveolar lung. Induced
sputum obtains cells primarily from the surfaces of
bronchial airways. Using flow cytometry, we investi-
gated whether sputum phagocytes demonstrate phe-
notypes indicative of increased functional activation
and inflammation compared to phagocytes from the
alveolar airways and peripheral blood (PB) in healthy
subjects (Nⴝ17). Sputum macrophages demonstrated
increased levels of CD11b, increased oxidative burst,
and greater phagocytosis than autologous alveolar
macrophages. Expression of CD11b, CD64, and
HLA-DR in sputum monocytes was upregulated com-
pared to that in PB monocytes. Sputum neutrophils
showed increased expression of CD11b, CD64, CD14,
and HLA-DR and were more phagocytic than PB neu-
trophils. In conclusion sputum/bronchial phagocytes
from healthy individuals express an inflammatory
phenotype and are functionally more active than
phagocytes from the alveolar airways and peripheral
blood. © 2000 Academic Press
Key Words: sputum; phagocytes; functional flow
cytometry.
INTRODUCTION
The lung is constantly exposed to inhaled environ-
mental microorganisms and air pollutants including
combustion particles and resuspended dusts. The ma-
jority of these agents are in the size range of 2–10
m
in diameter (1) and preferentially deposit in the bron-
chial airways rather than the alveolar airways (2). As
a result, bronchial phagocytes may display a pheno-
type indicative of increased functional activation com-
pared to alveolar and peripheral blood phagocytes. In-
duced sputum cells are obtained primarily from the
surfaces of bronchial airways and together with flow
cytometric analyses can be characterized for surface
phenotype and function. Enhanced expression of sur-
face receptors associated with host defense and inflam-
mation (Fc
␥
RI, CD64; Fc
␥
RIII, CD16; complement re-
ceptor, CD11b; the LPS-receptor CD14; and HLA-DR),
increased phagocytosis, and greater oxidative burst ca-
pacity are good markers of functional activation and
are modified by inflammatory signals.
IgG-mediated phagocytosis is dependent on the
quantitative expression of CD64 while complement-
mediated phagocytosis is primarily dependent on the
expression of CD11b (3). CD64 and CD11b are thought
to act in a compensatory manner on mononuclear and
polymorphonuclear phagocytes (4), with some evidence
suggesting that macrophage (Mac) phagocytosis is pri-
marily driven by CD64 and that polymorphonuclear
neutrophil (PMN) phagocytosis is driven by CD11b (5).
Levels of expression of these receptors are directly
correlated with phagocytic capacity (4, 6). CD64 and
CD11b are constitutively expressed on alveolar Mac
(AMac) and peripheral blood (PB) monocytes
(PBMono), but modulated by T-lymphocyte-derived cy-
tokines such as interferon (IFN) and interleukin 4
(IL-4) (7–9), bacterial endotoxin (10–17), environmen-
tal particulates (18), and antigen (19–21). PMN, on the
other hand, contain large intracellular pools of CD11b
that become rapidly externalized and expressed during
migration to the airways in response to an inflamma-
tory signal (22). Similarly, cell-surface receptor CD16
(the receptor for soluble immune complexes (23, 24)),
CD14 (part of the endotoxin receptor complex (25)),
and HLA-DR are constitutively expressed on mononu-
clear phagocytes. As PBMono migrate into the airways
and mature into Mac, CD16 and HLA-DR are upregu-
1This report has been reviewed by the National Health and En-
vironmental Effects Research Laboratory, United States Environ-
mental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and
policies of the Agency nor does mention of trade names and commer-
cial products constitute endorsement or recommendation for use.
2To whom correspondence should be addressed at Center for En-
vironmental Medicine and Lung Biology, University of North Caro-
lina, 104 Mason Farm Road, Chapel Hill, NC 27599-7310. Fax: (919)
966-9863. E-mail: alexis.neil@epamail.epa.gov.
Clinical Immunology
Vol. 97, No. 1, October, pp. 21–32, 2000
doi:10.1006/clim.2000.4911, available online at http://www.idealibrary.com on
1521-6616/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
21
lated while CD14 is downregulated (26, 27). On PMN,
CD16, CD14, and HLA-DR are upregulated in an in-
flammatory environment (28–30). Therefore, analysis
and comparison of levels of CD11b, CD64, CD16, and
HLA-DR expression on phagocytes in the airways can
reveal recent immigration as well as in situ inflamma-
tory activation.
In addition, certain morphologic and functional char-
acteristics distinguish newly recruited blood cells from
those with a longer residence time in the airway mu-
cosa. Identification, therefore, of recently recruited
cells in the airways would suggest local inflammatory
activation. For example, PMN newly migrated into the
airways have increased volume and higher cell density,
while older PMN are smaller and hypodense (31, 32).
On the other hand, PBMono increase in size and be-
come more granular as they enter the airways and
continue to mature. Resident tissue Mac that have
been stimulated become hypodense and vacuolated
and are functionally less responsive to stimuli (33–35).
It is of interest then to examine whether sputum cells
appear morphologically heterogeneous with respect to
cell size and granularity, compared to cells obtained
from the alveolar airways and peripheral blood.
Although phagocytes obtained from bronchoalveolar
lavage (BAL) have been well characterized, they reflect
a different lung compartment and cellular milieu than
sputum-derived cells, which examine exclusively
“bronchial” airway inflammation. Consequently, one
cannot assume sputum cells to be phenotypically or
functionally similar to BAL-derived cells. To date, no
study has clearly defined the functional capabilities of
sputum phagocytes or their possible inflammatory ac-
tivity in healthy individuals. In this study flow cytom-
etry was used to characterize sputum phagocytes and
determine whether they display phenotypes indicative
of increased activation and inflammation compared to
BAL and peripheral blood phagocytes. Cell-surface
phenotype analysis, phagocytosis of IgG opsonized
yeast, and intracellular oxidative burst data all sug-
gest that there is a persistence of functionally activated
phagocytes in the bronchial airways.
METHODS
Subjects
Subject characteristics are shown in Table 1. Seven-
teen nonsmoking volunteers between 18 and 40 years
of age were recruited for the study. A medical screen-
ing exam that included a medical history, psychological
questionnaire, physical exam, blood tests, and allergy
scratch tests was performed on all subjects on a sepa-
rate day prior to the study, as was an induced sputum
to ensure that all subjects could produce adequate spu-
tum samples. Pulmonmary function tests, induced spu-
tum, and BAL were all performed between 8:00 and
9:00 AM on all subjects. BAL was performed first on all
subjects and sputum induction (with blood draw) fol-
lowed 24–48 h later. A total of 17 (N⫽17) healthy
subjects underwent successful bronchoscopy and in-
duced sputum. No health complications were observed
on any subjects following bronchoscopies or induced
sputums. Subjects had no history of asthma or allergic
disease and were free of any symptoms of acute respi-
ratory illness for at least 4 weeks. They had FEV1/FVC
equal to or greater than 75%, as well as FVC and FEV1
greater than 90% of predicted normal. This study was
approved by the Committee on the Protection of the
Rights of Human Subjects at the University of North
Carolina, Chapel Hill.
Bronchoalveolar Lavage and Cell Preparation
A fiberoptic bronchoscope was wedged into a segmen-
tal bronchus of the lingula. One 20-ml aliquot and five
50-ml aliquots of sterile saline were sequentially in-
stilled and immediately aspirated. The procedure was
repeated on the right middle lobe. Samples were put on
ice immediately after aspiration and centrifuged at
300gfor 10 min at 4°C to pellet. The first 20-ml aliquot
was labeled the bronchial wash (BW) and reflects fluid
rich in airway cells (58). Cells from the final five ali-
quots were pooled (BAL) and washed once with RPMI/
0.25% gentamicin. Cells were counted and viability
TABLE 1
Subject Characteristics and Spirometry
Subject Age
(years) Sex Atopy % PRED
FEV1
% PRED
FVC
1 24 M None 110 127
2 24 M None 140 136
3 26 M None 102 112
4 30 M None 82 88
5 30 F None 120 122
6 24 M None 111 116
7 33 F None 97 119
8 28 M None 110 127
9 24 M None 99 106
10 30 M None 80 93
11 23 M None 112 106
12 26 M None 103 110
13 30 M None 81 85
14 21 F None 110 110
15 28 M None 121 112
16 24 M None 104 107
17 24 M None 107 108
Mean (SEM) 26.6 (0.8) 105 (3.7) 111 (3.3)
Range 21–33 81–140 85–136
Note. % PRED FEV1denotes the percentage predicted forced ex-
piratory volume in 1 s, and % PRED FVC denotes the percentage
predicted forced vital capacity.
22 ALEXIS ET AL.
was determined by Trypan blue exclusion; mean via-
bility was 91% for the alveolar fraction (Table 2). Cell
differentials were also performed on cytocentrifuged
slides stained with a modified Wright stain (Leukostat
Solution, Fisher Scientific). At least 500 cells per slide
were counted.
Induced Sputum
The induction procedure of Pin (36) was followed
with some modifications. Three 7-min inhalation peri-
ods of 3, 4, and 5% hypertonic saline were administered
following baseline spirometry. At the end of each 7-min
inhalation period subjects performed a 3-step cleans-
ing procedure prior to a cough attempt to reduce squa-
mous cell contamination: (i) Rinse the mouth and gar-
gle with water. (ii) Clear the back of the throat (but no
coughing). (iii) Blow his or her nose. The subject was
then instructed to perform a “chesty type” cough with-
out clearing the back of the throat. The sample is
expectorated into a sterile specimen cup that is placed
on ice throughout the procedure. A separate specimen
cup was used for the separate collection of saliva dur-
ing the induction procedure to reduce the effects of
dilution on the collected sample.
Sample processing begins immediately according
to method of Pizzichini (37). In brief, mucus plugs
are manually selected, weighed, and incubated (15
min at room temperature) in 0.1% dithiothreitol
(DTT) (Calbiochem Corp., San Diego, CA), an effec-
tive way of separating leukocytes from the remaining
saliva expectorate containing ⬎90% squamous epi-
thelial cells. DTT has been demonstrated to cause no
deleterious effects on sputum cells or interfere with
surface marker measurements using flow cytometry
(59, 60). Following DTT incubation, the sample is
washed with Dulbecco’s phosphate-buffered saline
(DPBS) and gravity filtered through a 48-
m pore
mesh filter (BBSH Thompson, Scarborough, Ontario,
Canada). Total cell counts were performed with the
use of a Neubauer hemocytometer. Visually identifi-
able squamous epithelial cells were not counted or
included in the total cell count. Cell viability was
determined using Trypan blue exclusion staining.
Differential leukocyte analysis of nonsquamous cells
(Diff Quik stained) was performed on a minimum of
400 cells provided that squamous cells were less
than 40% of the total cells. Differential cell counts
(lymphocytes, neutrophils, eosinophils, monocytes,
macrophages) were expressed as a percentage of to-
tal nonsquamous nucleated cells.
Flow Cytometry
Flow cytometry was performed with a FACSORT
(Becton Dickinson) using an argon-ion laser (wave-
length of 488 nm). Gain and amplitude settings were
set so as to analyze blood, sputum, and BAL samples
from the same subject in order to establish reference
gates for leukocyte identification. Settings were kept
the same throughout the study for each subject. The
FACSORT was calibrated with Calibrite (Becton
Dickinson) beads (noncolor, green, and red) before
each use. A total of 10,000 events were counted for
all sample runs. Gating of healthy Mac, monocytes
(Mono), PMN, eosinophils (Eos), and lymphocytes
(Lym) in sputum was based on light scatter proper-
ties, positive/negative expression for relevant anti-
bodies such as CD45 (pan leukocyte marker), CD3
(Lym), and CD14 (Mono), as well as using reference
gates based on whole-blood leukocyte preparations.
Based on these criteria, the following leukocyte pop-
ulations were easily distinguished: Lym, Mono,
PMN, Mac, and Eos. Fluorescein (FITC) and phyco-
erythrin (PE) conjugated nonspecific antibodies of
the same isotope as the receptor antibodies were
used as controls to establish background fluores-
cence and nonspecific antibody binding. The (arith-
metic) mean fluorescence intensity (MFI) of the cells
stained with control antibody was subtracted from
the MFI of the cells stained with receptor antibodies
to provide a measure of receptor-specific MFI. Rela-
tive cell size and density/granularity were quantified
by analyzing light scatter properties, namely, for-
ward scatter (FSC) for cell size and side scatter
(SSC) for cell density/granularity (38), and recording
the mean fluorescence intensities for each.
Immunofluorescence Staining
Aliquots of 100
l (100,000 cells/tube) of sputum cell
suspension, BAL cell suspension (1 ⫻106cells/ml), and
EDDA-anticoagulated whole blood were stained with
10
l of saturating concentrations of monoclonal anti-
bodies (Immunotech, Coulter Corp., Paris, France) for
60 min in the dark at 4°C (30 min in the dark at room
temperature for whole blood). Following staining, the
sputum and BAL cells were washed with 2 ml of cold
Hanks’ balanced salt solution (HBSS) and centrifuged
for 5 min at 1000 rpm and 4°C, and supernatants were
decanted and blotted. Whole blood samples were
treated with BD FACS lysing solution (2 ml) for 20 min
in the dark at room temperature. Sputum and BAL
cells were then resuspended in cold HBSS (250
l) and
fixed with paraformaldehyde (250
l, 0.5%) for a final
volume of 500
l. The cells were then stored at 4°C in
the dark until analyzed on the flow cytometer within
24 h of staining. Following cell lysis, whole blood sam-
ples were centrifuged (1000 rpm, 5 min) and superna-
tants were decanted and blotted. The samples were
resuspended with 2 ml DPBS and centrifuged (1000
rpm, 5 min) and supernatants were decanted. The
23SPUTUM PHAGOCYTES ARE FUNCTIONAL: A FLOW CYTOMETRIC STUDY
whole blood samples were fixed with 1 ml of 0.5%
paraformaldehyde and stored at 4°C in the dark until
analyzed on the flow cytometer with the sputum and
BAL samples. FITC or PE conjugated monoclonal an-
tibodies used for sputum and blood were CD11b, CD14,
CD64, CD16, HLA-DR, and CD3. Measurement of sur-
face marker expression was done using a BD
FACSORT flow cytometer. Analysis of surface marker
expression was done using the Cell Quest software
(BD), which provided a calculation of MFI for the gated
populations.
Phagocytosis
Saccharomyces cerevisiae zymosan A BioParticles
(Molecular Probes, Inc., Eugene, OR) conjugated to
FITC were opsonized with opsonizing reagent (IgG) for
45 min at 37°C and then washed with RPMI 1640 two
times before the particle concentration was adjusted to
2⫻106/ml. Purified (Percoll separated) blood mononu-
clear cells (2 ⫻106/ml) and PMN (2 ⫻106/ml), sputum
cells (2 ⫻106/ml), and BAL cells (2 ⫻106/ml) from the
same subject were exposed to the yeast cell walls at a
ratio of 1:10 for1hat37°C in the presence of human
serum (20
l) before tubes were placed on ice. Next,
200
l of 2% paraformaldehyde was 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 and
displayed on histogram plots as a rightward shift in
side scatter (SSC, xaxis) in the phagocyte populations,
i.e., PMN, Macs, and Mono. Phagocytosis was deter-
mined by assessing the proportion of cells in the zymo-
san-exposed population showing increased mean fluo-
rescence compared to cells that had not been exposed
(i.e., control population) to zymosan particles.
Intracellular Oxidative Burst
Aliquots (1 ml) of purified (Percole separated) blood
mononuclear cells, PMN (2 ⫻106/ml), sputum cells
(2 ⫻106/ml), and BAL cells (2 ⫻106/ml) were incu-
bated with 123-dihydrorhodamine stain (50
M) (Mo-
lecular Probes, Inc.) for 20 min at 37°C to allow the
stain to enter the cell. After several washings to re-
move unincorporated stain, phorbol myristate acetate
(PMA) (100
g/ml) was added to the cells for 30 min at
37°C. Following PMA stimulation, cells were immedi-
ately analyzed on the FACSORT where a fluorescent
signal indicated intracellular formation of reactive ox-
ygen intermediates. A control tube did not include
PMA stimulation and was used as background MFI
and was subtracted from the MFI of the PMA stimu-
lated cells.
Statistical Analysis
Statistically significant differences between multiple
study end-points were assessed using a repeated-mea-
sures one-way analysis of variance (ANOVA) followed
by Turkey’s multiple comparison post hoc analysis.
Analysis between two study end-points was assessed
using Student’s Ttest (two-tailed). Welche’s correction
was applied when equal variances between two study
end-points could not be assumed or were found to be
significantly different. Correlation coefficients (R)
were determined by simple linear regression analysis.
APvalue ⬍0.05 was considered statistically signifi-
cant.
RESULTS
Cell Recovery from Induced Sputum and BAL
Differential and total cell counts and cell viability for
sputum cells, BAL, and PB are shown in Table 2. With
the exception of monocytes, differential leukocytes
were determined from microscopic examination of cy-
tospin preparations. Monocytes were determined from
flow cytometric analysis of gated monocytes. All spu-
tum samples had ⬍40% squamous epithelial cells and
⬎60% viability and were acceptable for study analysis.
Differential cell counts did not differ significantly be-
TABLE 2
Mean (⫾SEM) Total and Differential Leukocyte Counts in Peripheral Blood, BAL, and Sputum
Sample % PMN % Mac % Monoa% Eos % Lym Total cells
(⫻106)Cell viability
(%)
Peripheral blood (N⫽17) 63 (2.0) N/AP 10 (1.0) 1 (0.8) 25 (3.0) 5.3 ⫻103/
lN/AV
BAL (N⫽17) 0.8 (0.1) 87 (1.3) 7 (0.9) 0.3 (0.2) 6 (1.3) 24.5 (2.3) 91 (1)
Sputum (N⫽17) 51 (6.4)* 40 (4.2)* 7 (1.2) 0.7 (0.35) 2 (0.5)* 5.7 (0.7)** 74 (2.9)*
Note. N/AP, not applicable; N/AV, not available; PMN, polymorphonuclear neutrophil; Mac, macrophage; Mono, monocyte; Eos, eosinophil;
Lym, lymphocyte.
aMonocytes determined from flow cytometric analysis.
* Significantly different from BAL.
** Significantly different from BAL (P⫽0.0001).
24 ALEXIS ET AL.
tween the sputum performed on the screen day and the
post-BAL study sputum for all subjects (data not
shown). A significantly greater percentage of PMN
were recovered in sputum than BAL (51% ⫾6.4 vs
0.8% ⫾0.1) (P⫽0.001), while BAL had a significantly
greater percentage of Mac (87% ⫾1.3 vs 40% ⫾4.2)
(P⫽0.001) and Lym (6% ⫾0.5 vs 2.0% ⫾0.5) (P⫽
0.001) than sputum. BAL had significantly higher
mean cell viability (91%) than sputum (74%) (P⫽
0.001).
Flow Cytometric Analysis of Peripheral Blood, BAL,
and Induced Sputum Cells: Morphological
Observations
Figure 1 shows flow cytograms of leukocyte popula-
tions recovered from peripheral blood (Fig. 1A), BAL
(Fig. 1B), BW (Fig. 1C), and induced sputum (Fig. 1D)
from the same subject. Using FSC and SSC and posi-
tive expression for CD45, and keeping identical mea-
surement settings on the flow cytometer for each fluid
sample analyzed, populations of Mac, Mono, PMN,
Lym, and Eos were distinguished and compared both
within and between samples. Figure 2 shows mean
light scatter results from peripheral blood, BAL, and
sputum in neutrophils (Fig. 2A), macrophages (Fig.
2B), and monocytes (Fig. 2C). SPMN were smaller in
size than PBPMN [FSC (MFI): 280 ⫾20 vs 385 ⫾10,
P⫽0.0001]; SMac were significantly denser than
AMac [SSC (MFI): 780 ⫾45 vs 600 ⫾37, P⫽0.0001],
but were smaller in size on average. SMono were sig-
nificantly larger than PBMono [FSC (MFI): 480 ⫾15 vs
275 ⫾9, p⫽0.01], but had very similar granularity
[SSC (MFI): 200 ⫾8vs193⫾7] (data not shown).
Cell-Surface Phenotype Analysis
Surface phenotypes on gated cell populations were
determined for peripheral blood, BAL, and induced
sputum leukocytes. There was no significant differ-
ence for any surface marker between the sputum
performed on screen day and the post-BAL study
sputum (data not presented). Figure 3A shows that
SMono express higher levels of CD11b than AMono
(P⫽0.05) and PBMono (P⫽0.01). SMono also
expressed more CD64 and HLA-DR than AMono
(P⫽0.05) and PBMono (P⫽0.001). Figure 3B
shows increased levels of CD11b (P⫽0.01) and
decreased levels CD64 (P⫽0.001) on SMac versus
AMac. Figure 3C shows that SPMN express higher
levels of CD11b (P⫽0.0001), CD14 (P⫽0.0005),
CD64 (P⫽0001), and HLA-DR (P⫽0.0001) than
PBPMN. In a separate analysis (results not pre-
sented in Fig. 3), PMN-A demonstrated increased
surface expression for CD11b (MFI ⫽32 ⫾2vs25⫾
3, P⫽0.1), CD14(MFI⫽17 ⫾4vs7⫾1, P⫽0.03),
CD64 (MFI ⫽9⫾2vs6⫾1, P⫽0.2), and CD16
(MFI ⫽284 ⫾30 vs 154 ⫾17, P⫽0.02) relative to
PMN-B. Our data show that relative to blood leuko-
cyte surface expression, CD11b expression on spu-
tum and BAL cells is relatively constant across cell
types with sputum cells demonstrating approxi-
FIG. 1. (A) Flow cytogram of peripheral blood leukocytes from a
healthy subject (subject 1). Gated populations based on light scatter
properties, i.e., forward scatter (FFS) and side scatter (SSC) and
include lymphocytes (LYM), monocytes (MONO), polymorphonuclear
neutrophils (PMN), and eosinophils (EOS). (B) Flow cytogram of
bronchoalveolar lavage (BAL) cells from subject 1. Gated populations
include lymphocytes, monocytes, and macrophages. (C) Flow cyto-
gram of bronchial wash (BW) cells from subject 1. Gated populations
include lymphocytes, monocytes, polymorphonuclear neutrophils,
and macrophages. BW cells reflect the cell recovery from the first
20-ml aliquot of BAL fluid. (D) Flow cytogram of induced sputum
cells from subject 1. Gated populations based on light scatter prop-
erties and positive expression for CD45 (pan leukocyte marker).
Gated populations include lymphocytes, monocytes, polymorphonu-
clear neutrophils, subpopulation of activated PMN (PMN-A), sub-
population of less active PMN (PMN-B), and macrophages.
25SPUTUM PHAGOCYTES ARE FUNCTIONAL: A FLOW CYTOMETRIC STUDY
mately 3–5⫻that of blood and BAL cells expressing
approximately 2⫻that of blood (CD11b:Mono, 3:2;
CD11b:Mac, 4:2.5; CD11b:PMN, 5:2.5). This pattern
was also observed with the other surface markers
examined in the study. The variability in surface
expression on blood leukocytes in healthy subjects
was relatively low on all surface markers examined
in this study as demonstrated by relatively small SE
bars in Figs. 2A and 2C. With the exception of
SPMN, we observed a uniform shift in MFI of gated
sputum leukocytes with respect to all surface mark-
ers analyzed. This was represented as a uniform
shift in a single population of cells on histogram
analysis. For these cells, MFI represented the sur-
face marker expression on the majority of cells. For
SPMN, two populations of cells were observed on
histogram analysis, one a high surface-marker-ex-
pressing population (PMN-A) and one a low-surface-
marker-expressing population (PMN-B). For SPMN,
MFI reflected an averaging of a bright population
FIG. 2. (A) Mean (⫾SEM) forward scatter (FSC) results analyzed by flow cytometry expressed as mean fluorescence intensity (MFI) for
peripheral blood and sputum neutrophils in 17 healthy subjects. Sputum neutrophils have significantly lower FSC than peripheral blood
neutrophils (P⫽0.0001). (B) Mean (⫾SEM) side scatter (SSC) results analyzed by flow cytometry expressed as MFI for BAL and sputum
macrophages in 17 healthy subjects. Sputum macrophages have significantly higher SSC than BAL macrophages (P⫽0.0001). (C) Mean
(⫾SEM) FSC results analyzed by flow cytometry expressed as MFI for blood and sputum monocytes in 17 healthy subjects. Sputum
monocytes have significantly increased FSC compared to peripheral blood monocytes (P⫽0.01).
26 ALEXIS ET AL.
(PMN-A) with a dull subpopulation (SPMN-B). We
observed no overlap in gated sputum leukocyte pop-
ulations with respect to neutrophil and lymphocyte
expression of CD64 and HLA-DR. SPMN were CD3⫺/
CD64⫹and CD3⫺/HLA-DR⫹, whereas sputum lym-
phocytes were CD3⫹/CD64⫺and CD3⫹/HLA-DR⫹.
With respect to CD64 expression, some overlap may
have occurred with SPMN and monocytes. But this
was likely minimal, since compared to SPMN, mono-
cytes had very distinguishable SSC properties and
FIG. 3. (A) Mean (SEM) cell-surface marker expression analyzed by flow cytometry on blood (solid bar), BAL (hatched bar), and sputum
(clear bar) monocytes in 17 healthy subjects. Surface markers CD11b (CR3), CD14 (LPS), CD64 (Fc
␥
RI), and HLA-DR. (B) Mean (SEM)
cell-surface marker expression analyzed by flow cytometry on BAL (solid bar) and sputum (clear bar) macrophages in 17 healthy subjects.
Surface markers: CD11b (CR3), CD14 (LPS), CD64 (Fc
␥
RI), and HLA-DR. (C) Mean (SEM) cell-surface marker expression analyzed by flow
cytometry on blood (solid bar) and sputum (clear bar) neutrophils in 17 healthy subjects. Surface markers: CD11b (CR3), CD14 (LPS), CD64
(Fc
␥
RI), and HLA-DR.
27SPUTUM PHAGOCYTES ARE FUNCTIONAL: A FLOW CYTOMETRIC STUDY
much greater expression of CD14 to confirm their
gated identity as monocytes.
Phagocytosis of Opsonized Yeast Particles
Phagocytosis by Mac and PMN was assessed by their
ability to phagocytize IgG opsonized yeast particles in
the presence of human serum. Figure 4A shows that
SPMN demonstrated significantly greater phagocyto-
sis than PBPMN (MFI ⫽608 ⫾138 vs 336 ⫾74, P⫽
0.05). SMac showed a trend toward increased phago-
cytosis compared to AMac but the difference was not
statistically significant (MFI ⫽1544 ⫾414 vs 1143 ⫾
169, P⫽0.4). PMN-A demonstrated increased phago-
cytosis compared to PMN-B (results not presented in
Fig. 4A) (MFI ⫽305 ⫾14 vs 145 ⫾7, P⫽0.001).
Relative to blood leukocytes, BAL and sputum cells
had greater phagocytic activity with sputum cells dem-
onstrating consistently higher phagocytosis than BAL
cells (MAC, 4.8:4.2; PMN, 2:1).
Intracellular Oxidative Burst
In response to PMA, healthy phagocytes will release
large amounts of intracellular reactive oxygen inter-
mediates (oxidative burst) as part of their host defense
function to destroy ingested microorganisms. Figure
4B demonstrates that SMac exhibit greater oxidative
burst activity than AMac (MFI ⫽36 ⫾10vs8⫾1, P⫽
0.04). The MFI values represented a 720% vs 160%
increase over control values for SMac and AMac, re-
spectively. PMN-A demonstrated an increased oxida-
tive burst compared to PMN-B (results not presented
in Fig. 4B) (MFI ⫽65 ⫾6, 1300% over control, vs 13 ⫾
2, 260% over control, P⫽0.02).
DISCUSSION
Unlike BAL, induced sputum obtains cells primarily
from the surfaces of the bronchial airways and as a
result provides an excellent opportunity to test hypoth-
eses involving cells specifically from this lung region.
The incorporation of flow cytometry to examine surface
phenotypes and cell function can extend the level of
sputum cell characterization beyond microscopic exam-
ination of cytospin preparations. In this study we hy-
pothesized that due to the exposed nature of the bron-
chial airways to environmental particulate matter and
infectious microorganisms, cells (phagocytes) from this
lung region will be more functionally active than
phagocytes obtained from less directly exposed regions
of the lung (BAL) and peripheral blood. The present
study provides evidence through a comparative exam-
ination with BAL and peripheral blood that sputum
phagocytes have increased cell function and inflamma-
tory surface phenotypes.
In agreement with previous studies (39–42), cells
obtained by induced sputum contained 40% macro-
phages and 51% neutrophils. Flow cytometric exami-
nation revealed that sputum macrophages appeared
more heterogeneous than alveolar macrophages with
respect to cell size (FSC) and cell granularity (SSC). On
the average, sputum macrophages were smaller than
alveolar macrophages but had increased granularity.
This could be explained by the presence of younger
macrophages in the bronchial airways, since macro-
phages with longer residence times in the lung are
large cells with hypodense, vacuolated cytoplasms (34)
and are functionally less responsive to stimuli (30). The
presence of newly recruited macrophage-like mono-
cytes in the bronchial airways suggest an active in-
flammatory state in this lung region.
Comparing phagocytosis and oxidant generation (in
response to PMA) in sputum and alveolar macro-
phages, we found sputum macrophages equally phago-
cytic but with the ability to generate fourfold more
oxygen radicals than alveolar macrophages. The
phagocytic capability of bronchial macrophages is
likely crucial in preventing inhaled PM-associated mi-
croorganisms to gain a foothold in the lower airways. If
bronchial phagocytes have poor phagocytic capacity,
this may place an increased phagocytic burden on the
alveolar macrophages, resulting in dysfunctional oxi-
dative responses by the cell. One can speculate, there-
fore, that healthy airway status (distal and central)
will be compromised if an imbalance occurs in the
functional ability of phagocytes in the bronchial and
alveolar airways. This suggestion is supported by ear-
lier reports of a link between impaired phagocytosis
and airway diseases like asthma (43, 44) and chronic
bronchitis (45). The increased reactive oxygen species
(ROS) generation by sputum macrophages may be ex-
plained by the presence of high-density macrophages
since earlier studies report that high-density macro-
phages have potentiated superoxide release following
stimulation (46). Increased ROS generation by airway
macrophages is an intracellular host defense response
against exogenous pathogenic stimuli and supports the
notion that the bronchial airways are indeed a site of
persistent immuno-inflammatory activation.
One potential limitation of our comparative analysis
with BAL cells is that the sputum cells may have been
activated from the bronchoscopy procedure that oc-
curred 24–48 h prior to induced sputum, thereby in-
validating their phenotype comparisons. Although this
is a possibility, it is unlikely this had a pronounced
effect on our outcome measures. Our data compared
surface marker expression on postbronchoscopy spu-
tum samples with sputum that was done on a screen
28 ALEXIS ET AL.
day at least 1 week prior to bronchoscopy. The results
showed no difference in any surface marker analyzed.
Our data showed that sputum macrophages ex-
pressed twice the levels of CD11b but less CD64 than
alveolar macrophages. Since CD11b is the primary sur-
face receptor mediating complement-associated phago-
cytosis, this may reflect the constant phagocytic bur-
den bronchial phagocytes face as they respond
continually to inhaled infectious microorganisms (en-
dotoxin) present in the upper airways. Lower levels of
CD64 reflect the less mature status of the bronchial
macrophage pool as it consists of newly recruited cells
from the peripheral blood that have migrated to the
airways in response to chemotactic signals.
Sputum cells contained a distinct population of
monocytes, which were increased in size compared to
peripheral blood monocytes and were likely to be re-
cent recruits from the peripheral blood. Two factors
can contribute to increased sputum monocyte size,
namely, cell maturation and phagocytic uptake of par-
ticles (18, 38). Both of these events are likely to occur in
the bronchial airways. Sputum monocytes demon-
FIG. 4. (A) Mean (SEM) phagocytosis results analyzed by flow cytometry expressed as mean fluorescence intensity (MFI) for BAL (solid
bar), sputum (clear bar), and blood (shaded bar) macrophages and neutrophils in 17 healthy subjects. FITC labeled Saccharomyces cerevisiae
zymosan A BioParticles were used and opsonized with IgG. (B) Mean (SEM) oxidative burst results analyzed by flow cytometry expressed
as the MFI in BAL (solid bar), sputum (clear bar), and blood (shaded bar) macrophages and neutrophils in 17 healthy subjects. PMA was used
to generate the oxidative burst.
29SPUTUM PHAGOCYTES ARE FUNCTIONAL: A FLOW CYTOMETRIC STUDY
strated increased surface expression of CD11b, CD64,
and HLA-DR compared to peripheral blood monocytes,
reflecting inflammatory activation as well as activation
due to maturation.
Examination of the light scatter characteristics of
sputum neutrophils revealed two discrete populations
of cells, one having higher SSC and FSC properties
(PMN-A) than the other (PMN-B). Previous in vitro
studies have suggested that following activation, neu-
trophils become hypodense and smaller, while newly
recruited neutrophils appear larger in size and more
granular, containing their full complement of secretory
granules prior to stimulation (17). The PMN-A popu-
lation therefore likely consisted of newly recruited
cells, while the PMN-B population consisted of cells
with longer residence times in the bronchial airways,
possessing necrotic characteristics, such as decreased
cell volume and hypodense cytoplasms (47).
As a group, sputum neutrophils expressed higher
levels of CD11b, CD14, CD64, and HLA-DR than pe-
ripheral blood neutrophils, were more phagocytic, and
produced a stronger oxidative response to PMA. Our
data revealed, however, that the activated phenotype
and function were associated mainly with the PMN-A
cells. Recent reports show that enhanced CD64 expres-
sion on blood PMNs can result from the presence of
gram-negative bacterial infection (endotoxin) and
INF-
␥
(56, 57). It is possible then that failure to main-
tain a regular pool of newly recruited neutrophils in
the bronchial airways may render this lung region less
capable of defending against invading environmental
pathogens. Upregulated surface expression of CD11b,
CD14, CD64, and HLA-DR on sputum neutrophils,
along with increased phagocytosis, reflects local im-
muno-inflammatory activation (48–51, 56, 57) and
supports the view that phagocytes recovered from the
surface of the bronchial airways are needed to be func-
tional and active in order to maintain healthy airway
status. Although we did not measure endotoxin in our
sputum samples, our data are consistent with previous
reports that suggest that endotoxin may have been
among the more likely agents to have generated the
inflammatory responses observed in our subjects. In
vitro studies on BAL and peripheral blood neutrophils
from humans have shown that endotoxin administra-
tion significantly increases CD14, CD11b, and CD64
surface expression as well as enhances certain func-
tional activities such as increased ROS generation
(52–57).
In conclusion, this study has shown that sputum
phagocytes from the bronchial airways of healthy indi-
viduals display a more active inflammatory phenotype
than phagocytes from BAL and peripheral blood. Fur-
ther, this study demonstrates the power of using the
flow cytometric approach to characterize surface phe-
notypes and functional capacity of sputum phagocytes.
These techniques may add essential information to the
examination of host defense responses in the bronchial
airways.
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Received March 15, 2000; accepted with revision June 30, 2000
32 ALEXIS ET AL.
