Lung lining liquid modifies PM2.5 in favor of
particle aggregation: a protective mechanism
Michaela
Kendall1,
Teresa D.
Tetley2,
Edward
Wigzell1,
Bernie
Hutton3,
Mark
Nieuwenhuijsen1, and
Paul
Luckham1
1 Imperial College of Science, Technology, and Medicine,
London SW7 2BP; 2 National Heart and Lung Institute,
Imperial College of Science, Technology, and Medicine, London
SW3 6LY; and 3 Christopher Ingold Laboratories,
Department of Chemistry, University College London WC1H 0AJ,
United Kingdom
 |
ABSTRACT |
The health effects of
particle inhalation including urban air pollution and tobacco smoke
comprise a significant public health concern worldwide, although the
mechanisms by which inhaled particles cause premature deaths remain
undetermined. In this study, we assessed the physicochemical
interactions of fine airborne particles (PM2.5) and lung
lining liquid using scanning electron microscopy, atomic force
microscopy, and X-ray photon spectroscopy. We provide experimental
evidence to show that lung lining liquid modifies the chemistry and
attractive forces at the surface of PM2.5, which leads to
enhanced particle aggregation. We propose that this is an important
protective mechanism that aids particle clearance in the lung.
fine particles; bronchoalveolar fluid; surface chemistry; interactions
 |
INTRODUCTION |
THERE IS STRONG
EPIDEMIOLOGICAL and toxicological evidence that demonstrates the
adverse health effects of atmospheric particulate matter having an
aerodynamic diameter of <2.5 µm, which is termed PM2.5
(5). Although the biological mechanism is not yet clear, particle size is believed to be a crucial factor in affecting health
(13, 18). Particle size determines the site of deposition, the surface area-to-volume mass ratios, and importantly, the clearance rates within the respiratory tract. Thus smaller particles are more
likely to reach the gas-exchange region of the lung and present a
greater interactive surface per unit mass of inhaled material. In
addition, although particles >5 µm in diameter undergo macrophage phagocytosis and mucociliary clearance, ultrafine particles (<0.1 µm
in diameter) are not readily phagocytosed and may access the pulmonary
interstitium via the epithelium (2, 9). Significantly, epidemiological studies of the effects of aerosols of different origins
imply that bulk particle composition is a relatively poor predictor of
health outcome compared with mass concentration (4) even though particle-surface chemistry is likely to be extremely significant in determining ultimate biological reactivity.
The first line of defense against inhaled particles is lung lining
liquid, which bathes the underlying epithelial cells and contains
important neutralizing agents including antioxidants, lysozyme
defensins, lipids, mucins, and proteins. This liquid helps to maintain
homeostasis of the airways through antimicrobial and immunologic
defense mechanisms. An essential property of pulmonary surfactant,
which is the predominant component of lung lining liquid in the
respiratory units, is the capacity to reduce surface tension in an
area-dependent way thereby preventing alveolar collapse. In addition,
surfactant has been shown to displace respirable particles (<6 µm in
diameter) into the hypophase of lung lining liquid, which makes the
particles available for clearance by the mucociliary escalator
(6, 15). Lung lining liquid proteins such as surfactant
proteins A and D appear to be important modulators of the
clearance of microorganisms by macrophage phagocytosis (3, 7, 12,
14, 22). Similar processes, possibly involving opsonization, may
also trigger uptake by macrophages and clearance of inhaled
PM2.5.
The factors that render individuals susceptible to increased ambient
PM2.5 are also unclear, although those with existing respiratory and cardiovascular disease are most at risk during pollution episodes (17). Certainly the large surface areas
presented to the peripheral lung by PM2.5 have enormous
potential to deliver toxic material to or deplete defensive material
from the lung lining fluid. If one role of the fluid is to prevent such
toxic processes, abnormalities in lung lining liquid composition, e.g., in smokers and asthmatics (7, 8), may contribute to the observed susceptibility to PM2.5 episodes. However, very
little is understood about the interaction between lung lining liquid and inhaled ambient particles.
In this study, we hypothesized that in healthy individuals the lung
lining liquid plays an important protective role by modifying the
physicochemical properties at the surface of particulate matter and
thus changing the surface chemistry and neutralizing its reactivity in
situ. We also hypothesized that these modifications act to enhance
pulmonary clearance by increasing the attractive forces between the
particles, thereby inducing aggregation and enhanced phagocytosis by
macrophages. To investigate this, we used scanning electron microscopy
(SEM), atomic force microscopy (AFM), and X-ray photon spectroscopy
(XPS) to show that lung lining liquid modifies the surface chemistry
and attractive forces at the surface of PM2.5, which leads
to enhanced particle aggregation. We used SEM to determine particle
morphology before and after immersion in human lavage fluid, AFM to
examine particle-liquid interactions, and XPS to examine changes in
particle-surface chemistry before and after immersion in lavage fluid.
 |
MATERIALS AND METHODS |
Lung lining liquid was collected using bronchoalveolar lavage.
Human bronchoalveolar lavage fluid (BALF) was collected during diagnostic fiber-optic bronchoscopy with a routine method as described previously (21). Briefly, the tip of the bronchoscope was
wedged into a segment of the right middle lobe. Warmed sterile 0.15 M NaCl (50 ml) was introduced and gently aspirated. This was repeated three times (total lavage, 200 ml), and the washings were pooled and
centrifuged at 300 g for 10 min to remove the cellular
component, which was used for diagnostic purposes. The supernatant was
stored at
40°C until used. Because a large volume of lavage fluid
was required to perform the whole experiment, the supernatant from two
subjects (who were subsequently diagnosed as normal) were pooled before
the study.
PM2.5 was collected from three sources, which represented
outdoor urban air, outdoor "clean" air, and indoor tobacco smoke pollution. Atmospheric or outdoor PM2.5 was collected near
London traffic ("urban PM2.5") and at a clean
atmosphere-monitoring station in Mace Head, Galway ("clean air
PM2.5"). "Indoor smoke PM2.5" was
collected in an indoor smoking area. Personal PM2.5
samplers (BGI-400 pump fitted with GK-2.05 cyclone; BGI, Waltham, MA)
were used to collect PM2.5 onto Nucleopore and Teflon
filters for subsequent SEM and XPS analysis, respectively
(11). In addition, a purpose-designed, direct
particle-liquid (DPL) system was configured to allow collection of
PM2.5 directly into liquid, in this case 0.15 M NaCl or
BALF. This system is based on an existing bioaerosol sampler design and
a more complete description can be found elsewhere (10). Table 1 summarizes the types of samples
taken as part of this study, the collection methods used, and the
associated analytic methods employed.
The filter samples collected with personal PM2.5 samplers
were stored in airtight containers in a cool dark room for up to 4 days. Using a scalpel, the filters were then divided into three parts.
One unchanged (dry) portion was analyzed using XPS, another portion was
immersed in sterile 0.15 M NaCl, and the third was immersed in BALF,
for 4 h. The saline- and lavage-treated samples were then bathed
in nanopure water and placed in a desiccating argon atmosphere for
24 h. Samples collected by DPL were aspirated from the collection
reservoir, and the supernatant was filtered through a
0.4-µm-pore-size Nucleopore filter under negative pressure. Samples
were centrifuged immediately after collection at 3,000 rpm for 30 min
to remove the largest particles. The filters were placed in a
desiccating argon atmosphere for 24 h to dry before SEM analysis.
AFM was performed on diesel exhaust particles of ~10 µm in
diameter, because particles collected as PM2.5 were too
small to manipulate accurately. Exhaust particles were collected onto a glass slide securely fixed inside a clean container. The container was
opened and held at the exhaust outlet of a diesel engine to allow
particles to strike the glass slide. The container was then sealed and
taken to the laboratory where the slide was examined under a microscope
to identify single compact 10-µm particles. Force-distance
measurements were carried out with a Topoetrix Explorer 2000 (ThermoMicroscopes) using an Ultralever contact tipless cantilever
(Park Scientific Instruments). A cantilever with the tip primed with a
small amount of epoxy resin adhesive was maneuvered using a
micromanipulator over a previously identified particle and lowered onto
it; the cantilever was then moved away from the slide and viewed under
a microscope to confirm that the particle was satisfactorily attached
to the cantilever. In addition, the particle was examined to ensure
that it was not too "flat" and that the contact area was not
covered in resin. The preparation was allowed to dry for 48 h
before study. A carbon graphite disc (20 mm in diameter) was used as
the contact surface.
XPS was used to determine the surface chemistry of PM2.5 to
a depth of ~5 nm. XPS measurements were performed on a VG ESCALAB 220i XL instrument using monochromatic Al-K
radiation (1,486.6 eV)
of 600-µm spot size. A magnetic objective lens was used for enhanced
sensitivity throughout this work, and the analysis chamber was
maintained at a pressure >10
9 Torr. Survey spectra of
the particulates were collected over a 1,100-eV range at a resolution
of 0.8 eV/step and 100 ms/step and a pass energy of 100 eV.
High-resolution spectra were collected for species of interest at a
resolution of 0.1 eV/step and 100 ms/step and a pass energy of 20 eV.
Charge compensation was achieved by placing a tantalum-conducting mask
over the sample to ensure good electrical contact before flooding the
sample with low-energy (4 eV) electrons. All peaks were referenced to
the C1s binding energy for hydrocarbons at 285.0 eV. Quantification was
performed using a Shirley background (19) and the
sensitivity factors described previously (23). Binding
energies were taken at peak maxima for all species. Particles were
collected on Teflon filters and analyzed before and after immersion in
normal saline and unprocessed lavage fluid. Displacement of some of the
atmospheric particles from the Teflon filters was detected after
immersion in saline and lavage fluid. Blank Teflon filters were
therefore used to establish the background Teflon spectra and to insure
that there was no appreciable surface contamination during liquid treatments.
 |
RESULTS |
Representative SEM images of the atmospheric PM2.5
samples collected onto Nucleopore filters are shown in Fig.
1. Figure 1, A-C, shows typical examples of urban
particles that are characteristic of those found in the U.K. urban
atmosphere as previously reported (1, 20). Figure 1,
D and E, shows typical indoor smoke particles. Both sample types are dominated by small particles comprised of loosely
agglomerated 35-nm spheres that form chains or transparent particles.
Figure 1F shows typical particles in the clean air sample;
compared with the urban particle samples, these particles tend to
appear as larger single crustal particles that are denser in structure
and occur at much lower concentrations.

View larger version (140K):
[in this window]
[in a new window]
|
Fig. 1.
Typical particles observed by scanning electron
microscopy (SEM) of particulate matter with <2.5-µm diameter
(PM2.5) samples collected directly onto Nucleopore filters
from the London atmosphere (A-C) and an indoor smoking area
(D-E). These two particle types are similar in appearance
and comprise agglomerated ~35-nm spherules generated during
combustion. Particles may even appear fibrous because of
single-particle chain agglomeration (C). Smallest particle
type found in the clean air sample is shown (F); compared
with combustion particles, the particles sampled at this site tended to
be larger in size, crystalline in structure, and tended to occur at
much lower concentrations. Dark holes on all images are the filter
pores.
|
|
When the urban particle samples were collected using the DPL sampler in
the presence of BALF, the most striking feature was the appearance of
increased numbers of dense conglomerates >5 µm (mostly in the
10-µm range) that consisted predominantly of agglomerated 35-nm
particles (Fig. 2, A and
B). This particle size (~10 µm) was largely absent in
ambient air samples and in the saline DPL control sample, where most of
the particles were 35-nm spheres and small-chain agglomerates (<2.5
µm) with the occasional less-dense larger particles that may have
formed during sample drying. In 10 randomly selected fields of view,
more than twice as many large- and medium-sized agglomerates appeared
in the BALF-collected sample. Figure 3
shows two low-magnification backscatter SEM images of the BALF- and the
saline-collected particles and shows the relative abundance of large,
densely packed agglomerates, which appear as dark patches. Clearly, the
BALF-collected particles agglomerated, whereas particles collected into
the saline sample remained dispersed.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 2.
Densely agglomerated 35-nm particle conglomerates (>5
µm) found in particle samples collected by sampling PM2.5
directly into lung lining liquid. Samples were subsequently filtered
onto 0.4 µm Nucleopore filters for SEM analysis.
|
|

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 3.
Low-magnification images of particles sampled directly into lung
lining liquid (A) and saline solution (B).
Particles were subsequently filtered onto 0.4-µm Nucleopore filters
for SEM analysis to illustrate the relative abundance of large
particles in air sampled directly into lung lining.
|
|
AFM-measured force-distance values for the interaction forces between
the particle and the surface in different media are presented in Fig.
4 under atmospheric conditions
(A), in nanopure water (B), and in processed BALF
(C). When a particle was brought toward the graphite surface
in air, there was a short-range attraction due to van der Waals forces.
The adhesive force observed on separation in air likely reflects van
der Waals forces and/or capillary bridging. In water, there was a
marked reduction in these forces, possibly due to a slight repulsion
between the surfaces and reduced van der Waals interaction as would be
expected in liquid. When BALF was examined without processing (i.e.,
centrifugation), the opaque, lipid-rich fluid interfered with the force
measurements by attenuating the path of the laser in the AFM.
Consequently, the lavage sample was centrifuged to remove most of the
lipid component, which left a protein-rich supernatant. Treatment of
particles with processed BALF resulted in longer range attraction and
adhesion forces (Fig. 4C) than was observed in air or water.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
"Force in" graph shows the particle attached to the
atomic force microscopy (AFM) cantilevered tip being brought toward a
surface at 0 nm separation. As the tip pushes against the solid
surface, an increasingly positive (repulsive) force is recorded whereby
the tip remains stationary and the cantilever arm continues to be
forced toward the surface. When the applied force reaches +2 nN, the
force direction is reversed and the tip is pulled away from the surface
(represented by the "force out" graph). A negative (attractive)
force is recorded when the tip sticks to the surface during either
adhesion or spontaneous attraction to the surface, causing curvature of
the cantilever away from the surface. The "force out" graph shows
particle attraction to the surface (A); at approximately 3
nN, the particle is separated from the surface abruptly and springs
back to a distance of ~170 nm. In nanopure water (B), the
attractive forces are eliminated. However, in centrifuged
bronchoalveolar lavage (C), the AFM tip experiences a
spontaneous attraction toward the surface at 100 nm, and the particle
jumps to within 40 nm of the surface. As the tip is pulled away, an
attractive force resists particle separation from the surface. This
long-range attraction and adhesion is absent in the nanopure water
control.
|
|
XPS analysis confirmed that very low particle loads were present in the
clean air sample. Particles from this site consisted of
graphite-hydrocarbon, Cl
, and oxide species only. Higher
particle loads were observed for the outdoor urban and indoor smoke
samples. Graphite-hydrocarbon species dominated at the particle
surface, although lower levels of SiO2, oxide, and amide
species were also present. In addition, trace species on urban
outdoor-particle surfaces included C---O/N and C==O/COO
,
Cl
, NO3
,
NH4+, and SO4
. After
immersion in saline, Cl
, NO3
,
NH4+, and SO4
were no
longer observed at the surface and were deemed bioavailable. After
immersion in BALF, a strong amide signal was observed on the particles,
which indicates that significant quantities of protein had adsorbed
onto the surface of these particles. No amide signal was recorded on
the blank filter control or the saline control. Figure
5 shows the detected amide signal from
urban PM2.5: dry PM2.5 (A),
saline-soaked PM2.5 (B), and lung lining
liquid-soaked PM2.5 (C).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Amide signal detected from the surface of dry
(A), saline-soaked (B), and bronchoalveolar
lining fluid-soaked (C) urban PM2.5, using X-ray
photon spectroscopy.
|
|
 |
DISCUSSION |
In this study, we have shown that when PM2.5 is
collected directly into normal lung lining liquid, the particles
aggregate into larger (>5 µm) dense structures compared with samples
collected in air or into saline. The control showed that the
agglomeration effects were not due to drying per se but were
specifically associated with the protein-rich solution, which is in
line with the AFM study that showed enhanced attraction between
surfaces in BALF. The XPS studies of surface chemistry for urban and
smoking PM2.5 showing significant modification by BALF
together with the AFM findings of increased attractive and adhesive
forces in BALF suggest that aggregation is enhanced by components of
lung lining liquid. The transition of PM2.5 surface
chemistry from a principally organic carbon layer with trace soluble
species to an organic layer with no trace soluble species and a strong
amide signal indicates that the surface adsorption of protein from lung
lining liquid may be responsible for this change. The long-range
molecular attractions shown to occur between a particle and a graphite
surface in protein-rich BALF may be attributable to particle
surface-protein interactions. Opsonization of inhaled particles by
surfactant proteins [which are known to have opsonizing properties
(12, 22)], antioxidants, and serum-derived and other
locally produced proteins may change the surface-charge forces in favor
of aggregation. The processed BALF used in this study may represent the
hypophase of lung lining liquid (described in Refs. 7 and
16) into which the surface surfactant film has been shown to submerge
particles <6 µm (15, 16). It is suggested that when the
particles reach the hypophase, they are then more readily cleared by
normal clearance mechanisms.
This aggregation mechanism is highly significant because macrophages do
not readily phagocytize the smaller agglomerates of 35-nm spheres that
dominate urban air in developed countries (1); epithelial
cells have been demonstrated to internalize these ultrafine particles
(9). We hypothesize that in susceptible subjects, the
inability of PM2.5 to aggregate in lung lining liquid,
which is possibly due to low opsonization, reduces the chances of
particle clearance by macrophages and enhances the possibility of
epithelial cell uptake and transfer to the interstitium (2,
9). In addition, particle adsorption and depletion of lung
lining components, for example surfactant components such as proteins
and antioxidants, may compromise lung defense mechanisms. Such
processes may contribute to susceptibility to PM2.5 in
elderly patients with existing cardiovascular and respiratory diseases
and may result in the acute increased mortality rates observed during
PM2.5 pollution episodes.
 |
ACKNOWLEDGEMENTS |
We thank John Watt (Middlesex University) for producing Fig. 3,
A and B.
 |
FOOTNOTES |
We gratefully acknowledge the support of the U.K. Department of Health
for funding this project.
Address for reprint requests and other correspondence and
present address of M. Kendall: EPA Particulate Matter Center, Nelson Institute of Environmental Medicine, New York Univ., 57 Old Forge Rd.,
Tuxedo, NY 10987 (E-mail:
Michaela.Kendall{at}env.med.nyu.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 25 April 2001; accepted in final form 1 August 2001.
 |
REFERENCES |
1.
Berube, KA,
Jones TP,
Williamson BJ,
Winters C,
Morgan AJ,
and
Richards RJ.
Physicochemical characterisation of diesel exhaust particles: factors for assessing biological activity.
Atmos Environ
33:
1599-1614,
1999[ISI].
2.
Churg, A,
and
Brauer M.
Human lung parenchyma retains PM2.5.
Am J Respir Crit Care Med
1555:
2109-2111,
1997.
3.
Crouch, EC.
Modulation of host-bacterial interactions by collectins.
Am J Respir Cell Mol Biol
21:
558-561,
1999[Free Full Text].
4.
Department of Health, Committee on the Medical Effects of Air Pollution.
Non-Biological Particles and Health. London: HMSO, 1995.
5.
Dockery, DW,
and
Pope CA.
Epidemiology of particle effects.
In: Air Pollution and Health, edited by Holgate ST,
Koren HS,
Maynard RL,
and Samet JM.. London: Academic, 1999, p. 673-705.
6.
Gehr, P,
Geiser M,
Im Hof V,
Schürch S,
Waber U,
and
Baumann M.
Surfactant and inhaled particles in the conducting airways: structural, stereological, and biophysical aspects.
Microsc Res Tech
26:
423-426,
1993[ISI][Medline].
7.
Gehr, P,
Green FHY,
Geiser M,
Im Hof V,
Lee MM,
and
Schürch S.
Airway surfactant, a primary defense barrier: mechanical and immunological aspects.
J Aerosol Med
9:
163-181,
1996[ISI][Medline].
8.
Griese, M.
Pulmonary surfactant in health and human lung disease.
Eur Respir J
13:
1455-1476,
1999[Abstract/Free Full Text].
9.
Griese, M,
and
Reinhardt D.
Smaller sized particles are preferentially taken up by alveolar type II pneumocytes.
J Drug Target
5:
471-479,
1998[ISI][Medline].
10.
Griffiths, WD,
Stewart IW,
Futter SJ,
Mark D,
and
Upton SL.
The development of sampling methods for the assessment of indoor bioaerosols.
J Aerosol Sci
28:
437-457,
1997[ISI].
11.
Kendall M, Nieuwenhuijsen M, Cullinan P, Ashmore M, and Jantunen
M. Methodology and preliminary results of EXPOLIS Oxford:
determinants and distribution of personal indoor airborne
pollutants in urban populations. Proc Indoor Air Conf Edinburgh
UK 1999, vol. 2, p. 806-811.
12.
Mason, RJ,
Greene K,
and
Voelker DR.
Surfactant protein A and surfactant protein D in health and disease.
Am J Physiol Lung Cell Mol Physiol
275:
L1-L13,
1998[Abstract/Free Full Text].
13.
Oberdorster, G,
Ferin J,
and
Lehnert BE.
Correlation between particle size and in vivo particle persistence and lung injury.
Environ Health Perspect
102, Suppl5:
173-179,
1994[ISI][Medline].
14.
Restrepo, CI,
Dong Q,
Savov J,
Mariencheck MI,
and
Wright JR.
Surfactant protein D stimulates phagocytosis of Pseudomonas aeruginosa by alveolar macrophages.
Am J Respir Cell Mol Biol
21:
576-585,
1999[Abstract/Free Full Text].
15.
Schürch, S,
Gehr P,
Im Hof V,
Geiser G,
and
Green FHY
Surfactant displaces particles toward the epithelium in airways and alveoli.
Respir Physiol
80:
17-32,
1990[ISI][Medline].
16.
Schürch, S,
Geiser M,
Lee M,
and
Gehr P.
Particles at the airway interfaces of the lung.
Colloids Surfaces B Biointerfaces
15:
339-353,
1999[ISI].
17.
Schwartz, J.
What are people dying of on high pollution days?
Environ Res
64:
26-35,
1994[ISI][Medline].
18.
Seaton, A,
MacNee W,
Donaldson K,
and
Godden D.
Particulate air pollution and acute health effects.
Lancet
345:
176-178,
1995[ISI][Medline].
19.
Shirley, DA.
High-resolution X-ray photoemission spectrum of the valence bands of gold.
Phys Rev B
5:
4709-4715,
1972[ISI].
20.
Sitzmann, B,
Kendall M,
Watt J,
and
Williams ID.
Characterisation of airborne particles in London by computer-controlled scanning electron microscopy.
Sci Total Environ
241:
63-73,
1999[ISI].
21.
Smith, SF,
Guz A,
Burton GH,
Cooke NT,
and
Tetley TD.
Comparison of alastolytic activity in lung lavage from current, ex- or non-smokers.
Life Sci
43:
459-464,
1998.
22.
Van Iwaarden, JF,
van Strijp JA,
Ebskamp MJ,
Welmers AC,
Verhoef J,
and
van Golde LM.
Surfactant protein A is an opsonin in the phagocytosis of herpes simplex virus type I by rat alveolar macrophages.
Am J Physiol Lung Cell Mol Physiol
261:
L204-L209,
1991[Abstract/Free Full Text].
23.
Wagner, CD,
Davis LE,
Zeller MV,
Taylor JA,
Raymond RM,
and
Gale LH.
Empirical atomic sensitivity factors for quantitative analysis by electron spectroscopy for chemical analysis.
Surface Interface Analysis
3:
211-225,
1981[ISI].
Am J Physiol Lung Cell Mol Physiol 282(1):L109-L114
1040-0605/02 $5.00
Copyright © 2002 the American Physiological Society