J. Anat. (2008) 213, pp452–463
doi: 10.1111/j.1469-7580.2008.00951.x
Comparative in vitro study of interactions between
particles and respiratory surface macrophages,
erythrocytes, and epithelial cells of the chicken and the rat
Blackwell Publishing Ltd
S. G. Kiama,1 J. S. Adekunle2 and J. N. Maina2
1
2
Department of Veterinary Anatomy and Physiology, University of Nairobi, P. O. Box 30197, Nairobi, Kenya
School of Anatomical Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa
Abstract
In mammals, surface macrophages (SMs) play a foremost role in protecting the respiratory system by engulfing and
destroying inhaled pathogens and harmful particulates. However, in birds, the direct defense role(s) that SMs
perform remains ambiguous. Paucity and even lack of SMs have been reported in the avian respiratory system. It has
been speculated that the pulmonary defenses in birds are inadequate and that birds are exceptionally susceptible
to pulmonary diseases. In an endeavour to resolve the existing controversy, the phagocytic capacities of the
respiratory SMs of the domestic fowl and the rat were compared under similar experimental conditions by exposure
to polystyrene particles. In cells of equivalent diameters (8.5 μm in the chicken and 9.0 μm in the rat) and hence
volumes, with the volume density of the engulfed polystyrene particles, i.e. the volume of the particles per unit
volume of the cell (SM) of 23% in the chicken and 5% in the rat cells, the avian cells engulfed substantially more
particles. Furthermore, the avian SMs phagocytized the particles more efficiently, i.e. at a faster rate. The chicken
erythrocytes and the epithelial cells of the airways showed noteworthy phagocytic activity. In contrast to the rat
cells that did not, 22% of the chicken erythrocytes phagocytized one to six particles. In birds, the phagocytic
efficiencies of the SMs, erythrocytes, and epithelial cells may consolidate pulmonary defense. The assorted cellular
defenses may explain how and why scarcity of SMs may not directly lead to a weak pulmonary defense. The
perceived susceptibility of birds to respiratory diseases may stem from the human interventions that have included
extreme genetic manipulation and intensive management for maximum productivity. The stress involved and the
structural–functional disequilibria that have occurred from a ‘directed evolutionary process’, rather than weak
immunological and cellular immunity, may explain the alleged vulnerability of the avian gas exchanger to diseases.
Key words bird; cellular defense; lung; phagocytosis; rat; surface macrophages.
Introduction
‘I assert, and I expect no disagreement, that more than
90% of lung diseases is either initiated by or at least
aggravated by inhalation of particles and gases.’ Brain
(1996)
Underscoring the expansive respiratory surface area and
the extreme thinness of the blood–gas barrier in the human
lung, Weibel (1983) and Gehr et al. (1990) aptly drew
analogies respectively to the square footage of a tennis
court and to a fractional measurement of the thickness of
Correspondence:
J. N. Maina, School of Anatomical Sciences, University of the
Witwatersrand, 7 York Road, Parktown 2193, Johannesburg,
South Africa. T: +27 (0)11 7172713; F: +27 (0)11 7172422;
E: [email protected]
Accepted for publication 10 June 2008
Article published online 14 July 2008
an air-mail paper. These confounding structural properties
bespeak how extensively and close the respiratory media,
air and blood, approximate to allow efficient flux of
respiratory gases by passive diffusion. Pulmonary bioengineering is inherently paradoxical: whereas vast surface
area and thin tissue barrier enhance gas exchange, on
the downside, the expansive and close proximity of the
respiratory media undesirably makes the gas exchangers
primary portals of entry of pathogenic microorganisms,
harmful particulates, and toxic gases. The role(s) that
particulates play in initiating or aggravating pulmonary
diseases is now well understood (e.g. Hayter & Besch,
1974; Dockery et al. 1992; Schwartz, 1993; Peters et al.
1997; Brunekreef & Holgate, 2002). In a 24-h day (e.g. Brain,
1996), the human lung filters about 20 000 L of air.
Depending on the severity of environmental pollution,
with each breath, air containing thousands of particles
and different kinds of allergens is inhaled. With annual
averages of 200–600 μg m3 and peak concentrations
that may exceed 1000 μg m3 (UN Environment Program and
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Interactions between particles and surface macrophages, S. G. Kiama et al. 453
WHO Report, 1994), most of the world’s large cities have
very high concentrations of particulates in ambient air.
Attesting to the fact that the respiratory system is protected from infection by a formidable arsenal of mechanical
and cellular defenses supplemented when necessary by
inflammatory and immune responses, in a healthy person,
below the larynx, the respiratory system is practically sterile
(e.g. Skerret, 1994). The fortifications that maintain the high
degree of ‘cleanliness’ include: 1) aerodynamic filtration
that causes deposition of particles along the branched
airway system (e.g. Hayter & Besch, 1974; Brown et al. 1997;
Geiser et al. 2003), 2) a physical barrier that comprises a
subaqueous phase, a surfactant, and a blood–gas (tissue)
barrier (Welsch, 1983; Stearns et al. 1987; Schürch et al.
1990; van Iwaarden et al. 1990; Gehr et al. 1996, 2000;
Widdicombe, 2002; Maina & West, 2005), 3) efficient
mucociliary escalator system (e.g. Kilburn, 1968; Green,
1973; Lippmann & Schlesinger, 1984); 4) phagocytic and
freely motile surface macrophages (SMs) (e.g. Lehnert,
1992; Warheit & Hartsky, 1993; Nicod, 2005), 5) epithelial
lining well endowed with tight junctions (e.g. Breeze &
Wheeldon, 1977; Harkema et al. 1991; Godfrey, 1997; Nicod,
2005), 6) phagocytic respiratory epithelium, especially in
birds (Maina & Cowley, 1998; Nganpiep & Maina, 2002),
and 7) strategically placed mucosal and bronchial associated
lymphoid tissue that is involved in dissolution and antibody labeling of foreign particulates (Anderson et al. 1966;
Fagerland & Arp, 1990, 1993; Reese et al. 2006). Occasionally,
depending on the particular etiologic agent and specific
defects in the host defenses, with severe consequences,
the defenses are overwhelmed.
A meaningful understanding of how, where, and when
pulmonary defenses are breached and devastated by
various pathogenic agents and particulates now exists in
mammals (e.g. Green et al. 1977; Bowden, 1987; Dockery
et al. 1992; Oberdörster et al. 1994). Regarding birds, however, relatively less is known about the cellular defenses of
the respiratory system (e.g. Fulton et al. 1990; Brown et al.
1997; Reese et al. 2006). This disappointing state of affairs
exists albeit that: 1) in the poultry industry, respiratory
diseases cause immense economic losses (e.g. Currie, 1999;
Wideman et al. 2007), 2) a zoonotic disease like bird-flu
[caused by avian influenza A (H5N1) viruses] has reached
endemic levels in birds, especially in some South-Asian
countries (e.g. Beigel et al. 2005); and 3) investigators like
Hill & Hoffman (1984), Furness & Greenwood (1993), and
Brown et al. (1997) have relentlessly urged that because
of the unique anatomy and physiology of the avian
respiratory system, birds may offer better animal models
and more sensitive bioindicators of environmental pollution compared to the commonly used small laboratory
mammals.
As phagocytosis is the most important defense mechanism
in all phyla of the animal kingdom (e.g. van Oss, 1986; Nicod,
2005), it is particularly important that the contribution of
the SMs in the clearance of particles and neutralization of
pathogens in the lung should be well understood. Paucity
(e.g. Toth et al. 1988; Fulton et al. 1990; Maina & Cowley,
1998; Nganpiep & Maina, 2002) and even lack of SMs (e.g.
Stearns et al. 1987; Klika et al. 1996; Lorz & López, 1997)
have been reported in the healthy avian lung. Investigators
like Klika et al. (1996) and Spira (1996) have alleged that
birds are highly susceptible to pulmonary infections. Maina
& Cowley (1998) and Nganpiep & Maina (2002), however,
argued that scarcity of SMs should not ipso facto mean
that the pulmonary defenses are compromised. Multiple
lines of defense that include: 1) airway epithelial cells and
SMs that are well endowed with lytic enzymes, 2) presence
of subepithelial and intravascular pulmonary macrophages,
3) efficient translocation of subepithelial phagocytes onto
the respiratory surface, and 4) strategic location of SMs in the
atria and infundibulae, where they guard the respiratory
surface, were reported. Data on the phagocytizing attributes
of the avian SMs are, however, lacking. This study has
taken the inquiry further: the SMs of the chicken lung, the
airway epithelial cells, and the erythrocytes (harvested from
the respiratory system) were challenged with polystyrene
particles and comparison made with corresponding cells
from the rat lung to find out whether the avian cells differ
from those of mammals.
Materials and methods
Pulmonary lavage of the avian respiratory system
All experimental procedures were approved by the Animal Ethics
Committee of the University of the Witwatersrand (Clearance
number: 07/55/01). Surface macrophages (SMs) were obtained from
the respiratory system (lung–air sac system) of mature specimens
of the domestic fowl, Gallus gallus variant domesticus as described
by Maina & Cowley (1998) and Nganpiep & Maina (2002). Briefly,
chickens were killed by intravenous injection of an overdose of
pentobarbitone sodium (Euthanase®) into the brachial vein. The
trachea was then exposed and cannulated with a sterile cannula
attached to a funnel. Sterile pre-warmed (40 °C) phosphatebuffered saline (PBS) was poured down the respiratory system from
a height of 30 cm. During instillation, the coelomic cavity was gently
massaged to expel air to ensure penetration of PBS into all air spaces,
including the air sacs. The instilled fluid was left in the respiratory
system for 5 min and thereafter aspirated with a 50-mL syringe. The
recovered lavage fluid was centrifuged and the pelleted respiratory
macrophages re-suspended in sterile cell-culture medium.
Bronchoalveolar lavage of the rat
Six rats with a body mass of approximately 493 g were used for
this study. The animals were anesthetized with an intraperitoneal
injection of 0.2 mL ketamine hydrochloride (50 mg mL−1) followed
by sodium pentobarbital (50 mg mL−1). The dosage was adjusted
to reach deep anesthesia. The trachea was exposed, tracheotomy
performed, and a cannula introduced and tightly fixed with a
thread. A pneumothorax was created before the bronchoalveolar
lavage procedure was started. The procedure consisted of six
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
454 Interactions between particles and surface macrophages, S. G. Kiama et al.
washes (5 mL per wash) of the lungs with Ca+2 and Mg+2 free PBS.
The total recovered lavage fluid was centrifuged and the pellet
re-suspended in sterile medium (RPMI 1640).
Phagocytosis of polystyrene particles
The avian and the rat SMs were washed twice in PBS and resuspended in fresh RPMI 1640 medium at about 6.105 and
1.106 mL–1 for the chicken and the rat cells, respectively. The cells
were co-cultured with polystyrene microspheres of 1.5 μm diameter
in sterile Eppendorf tubes at a concentration of about 100 microspheres per cell for 5 and 24 h. The tubes were shaken regularly to
avoid sedimentation of the cells and the particles.
Processing of cells for transmission electron
microscopy (TEM)
Cells in suspension were washed three times in PBS and resuspended in 2.5% phosphate-buffered glutaraldehyde solution.
The glutaraldehyde-fixed cells were centrifuged and post-fixed in 1%
osmium tetroxide in 0.1 M sodium-cacodylate buffer. This was
followed by dehydration in graded series of ethanol (70%, 80%, 90%,
100% twice) and gradual replacement of ethanol with propylene
oxide before infiltrating and embedding the cells in epoxy resin.
Semithin and ultrathin sections were cut using a Reichert®
ultramicrotome. The ultrathin sections were picked on 200-wire
mesh copper grids, stained with uranyl acetate, counterstained
with lead citrate, and observed with a Philips 201C or a JEM-100S
TEM under an accelerating voltage of 60 kV. The semithin sections
were collected on glass slides, stained with 0.5% toluidine blue,
and viewed under a light microscope.
Estimation of the diameters of the SMs and the volume
density of the phagocytized particles
The diameters of the SMs of chicken and the rat lung were determined under an ocular graticule with a linear scale at a magnification
of ×100. In each field, to avoid bias, the cells were singled out at
random. Only the diameters of the cells at the four corners of the
fields and one at the middle were measured.
The volume density of the phagocytized particles in the SMs
[VV(p,c)] is the ratio of the total volume of phagocytized particles to
the total cell volume. The ratio was estimated by point-counting on
plane sections as described in Kiama et al. (2001). Briefly, one ultrathin
section was randomly sampled from each processed block of cell
pellets and 35 fields systematically sub-sampled from each section.
Then, corresponding micrographs of the sampled fields were recorded
on a 35-mm electron microscope film of which the individual negatives
were projected onto a screen at a final magnification of ×14 000.
A quadratic lattice grid was superimposed at a random position
onto each projected image. The total number of points falling
onto profiles of the phagocytized particles [P(p)] and on entire cell
[P(c)] were counted. An estimator of the volume density of the
phagocytized particles [VV(p,c)] was then calculated as follows:
VV(p,c) = P(p)P(c)−1
Time-lapse video microscopy
Cells (1 × 105 cells.cm−3) were allowed to attach on plastic dishes
in sterile RPMI-1640 with glutamine medium (BioWhittaker®)
supplemented with 10% fetal bovine serum (GibcoTM) and
100 U mL−1 penicillin/streptomycin (BioWhittaker®). The dishes
were placed on a Zeiss Axiovert-200 microscope and viewed at a
magnification of ×20 with phase contrast filters. The microscope
was equipped with a chamber pre-warmed to 40 °C (for the chicken
SMs) and 38 °C (for those of the rat) and ventilated with a slow
current of CO2 to ensure extended viability of the cells. Comparable
numbers of polystyrene particles were added to the dishes and
bright-field images were collected every 15 min for 1.5 h by a chargecoupled device camera connected to a computer. The cells which
were assessed for the rate of uptake of particles were randomly
chosen from the entire population, thus discounting potential
bias due to possible heterogeneity in macrophage size and the
differences in the uptake of the particles. Images were analysed
manually for the phagocytic events. Phagocytosis was confirmed
by reforming of the membrane boundary after internalization of the
particles. The cells blackened, presumably from reaction between the
particles and the lytic enzymes. The cells also appeared to merge
with each other as they progressively engulfed the particles.
Results
Morphological observations
Ultrastructurally, the chicken and the rat respiratory SMs
were similar. Typically, they had filopodial extensions,
variably electron dense vesicular cytoplasmic organelles
(presumed to be lysosomes), mitochondria, and sparsely
distributed rough endoplasmic reticulum (Fig. 1j,l). The
harvested chicken SMs (Fig. 1a–f,j–l), erythrocytes (Fig. 1g–I,
Fig. 2a–c), and airway epithelial cells (exfoliated/desquamated
from the trachea, secondary bronchi, and/or ostia) (Fig. 3b,
Fig. 4a,b) readily phagocytized the polystyrene particles.
The lavage fluid harvested from the rat lung contained
SMs (Fig. 2d,e,g,h, Fig. 3a) and erythrocytes (Fig. 2d–f): no
epithelial cells were observed. Although the rat SMs
avidly phagocytized the polystyrene particles (Fig. 2d,e,g,h,
Fig. 3a), in complete contrast to those of the chicken that
did so (Fig. 2a–c), the rat erythrocytes did not take up the
particles (Fig. 2d–f). Few polymorphonuclear leukocytes
were observed in the fluid lavaged from the chicken lung
(Fig. 3c). Such cells did not appear to phagocytize the
polystyrene particles. No polymorphonuclear leukocytes
were observed in the fluid collected from the rat lung. For
the chicken SMs (Fig. 5a,b) and those of the rat (Fig. 5c,d),
the number of polystyrene particles phagocytized by the
SMs increased with exposure time. In the chicken (Fig. 6a)
and the rat (Fig. 6b), some of the SMs were seen to have
burst and released the engulfed polystyrene particles,
presumably after having over-ingested them or the cells
having died. After exposing the cells to the polystyrene
particles for a period of 1.5 h, using time-lapse photography
(Fig. 7A,B), it was noted that the chicken SMs (Fig. 7a–f)
engulfed the polystyrene particles more efficiently, i.e. at
a faster rate, compared to the rat ones (Fig. 7g–l). This
was assessed qualitatively from progressive darkening of
the SMs as the cells took up the polystyrene particles, the
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Interactions between particles and surface macrophages, S. G. Kiama et al. 455
Fig. 1 a–f) Surface macrophages (M) of the chicken lung. The polystyrene particles are seen as small granules outside of the cells (circled: a–c,e,f) and
those that have been or are about to be phagocytized are shown by arrows. g–i) Erythrocytes (E) of the chicken lung. The polystyrene particles about
to be phagocytized are shown by arrows and those located in the culture medium are encircled (g). j–l) Surface macrophages of the chicken lung
showing their characteristic structural features and engulfed polystyrene particles. In j the open arrows show filopodia, thin arrows show lysosomes,
mitochondria are shown by M, and the encircled areas show rough endoplasmic reticulum. Nu, nucleus. In k and l, the arrows show phagocytized
polystylelne particles while the encircled area (l) shows lysosomes. Nu, (k), nucleus. The surface macrophages of the rat lung ultrastructurally resemble
those of the chicken.
lytic enzymes acted on the particles, and the cells died.
In some areas, the cells appeared to form a continuum of
phagosomes.
Morphometric observations
The mean diameter of the chicken SMs (8.5 ± 0.90 μm SD)
was not significantly different (P < 0.001) from that of the
rat cells (9.0 ± 0.57 μm SD) (Table 1). Quantitative estimation
of the loading of the SMs with polystyrene particles
showed that the chicken cells, of which the phagocytized
particles formed a volume density of 22.5% of the volume
of the cell, took up significantly more particles. They
engulfed greater volume/mass of polystyrene particles
compared to those of the rat of which the volume density
of the particles formed only 5.3% (Table 1). The avian
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
456 Interactions between particles and surface macrophages, S. G. Kiama et al.
Fig. 2 a–c) Chicken erythrocytes (E) showing
phagocytized polystyrene particles (arrows). In
b (bottom right), PP shows a polystyrene
particle that has been engulfed by an epithelial
cell. d–f) Surface macrophages (arrows) and
erythrocytes (E in f and encircled areas in d,e of
the rat lung. While the macrophages engulfed
the particles, the erythrocytes did not. g,h)
Surface macrophages (M) of the rat lung that
have avidly engulfed polystyrene particles
(arrows). Nu, nucleus.
Table 1 Diameters of the surface macrophages and the volume densities of polystyrene particles phagocytized by chicken and rat cells after 24 h of
exposure
No. of
samples
Diameters of the chicken
surface macrophages (μm)
Volume density of
particles in chicken
surface macrophages (%)
Diameters of the
rat surface
macrophages (μm)
Volume density of particles
in rat surface macrophages (%)
1
2
3
4
Mean
9.3
9.1
8.4
7.3
8.5 ± 0.90 SD
24.0
26.3
19.2
20.5
22.5 ± 0.032 SD
8.5
9.8
9.1
8.7
9.0 ± 0.57 SD
4.5
5.3
6.6
4.7
5.3 ± 0.0094 SD
erythrocytes readily phagocytized the 1.5-μm diameter
polystyrene particles: 22% of the cells engulfed one to six
particles after 5 h co-incubation (Table 2). The number of
erythrocytes containing particles after 24 h could not,
however, be determined, as most cells had died on extended
incubation.
Discussion
Phagocytosis is a foremost function of the body’s innate
immunity. Macrophages and other antigen-presenting
cells interact and internalize particulate targets by actindependent mechanisms (e.g. Hartwig et al. 1977; Brain,
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Interactions between particles and surface macrophages, S. G. Kiama et al. 457
Fig. 3 a) Erythrocytes (E) and surface macrophages (M) of the of the rat
lung containing polystyrene particles (PP). Nu, nucleus. While the
macrophages engulfed particles, the erythrocytes did not. b) Erythrocytes
(E) and an epithelial cell (EC) from the chicken lung showing polystyrene
particles (PP) that have been engulfed by both erythrocytes and the
epithelial cell (arrows). Ci, cilia. c) A polymorphonuclear leukocyte (PL)
and erythrocytes (E) from the chicken lung showing that while the
leukocyte did not engulf particles, the erythrocytes did (arrows).
1985; Yamaya et al. 1995). From reported paucity (e.g.
Ficken et al. 1986; Toth et al. 1988; Fulton et al. 1990) and
even lack of SMs (e.g. Stearns et al. 1987; Klika et al. 1996;
Lorz & López, 1997), the enzymatic deficiencies of the SMs
Fig. 4 a,b) Exfoliated airway epithelial cells from the chicken lung that
have engulfed polystyrene particles (PP). E, erythrocytes; Ci, cilia;
Nu, nucleus.
in oxidative metabolism (e.g. Bellavite et al. 1977; Penniall
& Spitznagel, 1975), and differences in the enzymatic
systems of the avian lung tissue itself (Buckpitt & Boyd,
1983; Buckpitt et al. 1982), it has been claimed by, for
example, Klika et al. (1996) and Spira (1996) that the avian
respiratory system is exceptionally susceptible to diseases
and afflictions. However, work by other investigators (e.g.
Mensah & Brain, 1982; Ficken et al. 1986; Toth et al. 1987),
Table 2 Proportion of chicken erythrocytes that phagocytized the polystyrene particles
No. of samples
No. of erythrocytes containing particles No. of erythrocytes counted Percentage of erythrocytes containing particles
1
2
3
4
Mean
8
7
6
10
35
35
35
35
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
22.8
20.00
17.1
28.6
22.1 ± 4.9SD
458 Interactions between particles and surface macrophages, S. G. Kiama et al.
Fig. 5 Surface macrophages (arrows) of chicken lungs (a,b) and rat lungs (c,d) that have been exposed to polystyrene particles for 5 and 24 h.
The uptake of the particles increased with time of exposure. The inserts are Giemsa-stained cells photographed at the same times. E, erythrocytes;
Nu, nucleus; circles, erythrocytes (chicken) that have taken up polystyrene particles.
our earlier studies (Maina & Cowley, 1998; Nganpiep &
Maina, 2002), and this study indicate that the avian
respiratory system is adequately well defended. Except
for predisposing unnatural (human-caused) states and
conditions under which its defenses are overwhelmed, the
avian respiratory system may not be any more susceptible
to infections than, for example, the mammalian one. Some
of the studies that support our contention are: bacteria
introduced intratracheally are cleared from the avian
respiratory system within 24–48 h (e.g. Nagaraja et al.
1984) and injection of a suspension of incomplete Freund’s
adjuvant into the abdominal air sac (Ficken et al. 1986),
intratracheal inoculation of heat-killed Escherichia coli (Toth
et al. 1987) and exposure to live apathogenic Pasteurella
multocida vaccine (Toth et al. 1988) cause large production
of SMs. Furthermore, radioactive technetium particles
exposed to conscious chickens are cleared from the lungs
to the intestines within 1 h of exposure (Mensah & Brain,
1982).
Incontrovertibly, the remarkable difference between
the structure of the avian and the mammalian respiratory
systems (e.g. Weibel, 1984; Maina, 2005) has important
implications for the air-flow dynamics and the gas exchange
physiology (e.g. Scheid, 1979). This may translate into
fundamental differences in inhaled particle deposition
and clearance. Birds have larger tidal volumes and their
lungs have a flow-through system, i.e. the parabronchi
are ventilated continuously and unidirectionally in a
caudocranial direction (e.g. Scheid, 1979; Fedde, 1980). In
animals of equivalent body mass, the blood–gas barrier is
56–67% thinner and the respiratory surface area is 15%
more extensive in a bird compared to a mammal (e.g. Maina
et al. 1989). In species like the ostrich, Struthio camelus, the
air sacs extend out to lie subcutaneously (e.g. Bezuidenhout
et al. 2000) where they are susceptible to trauma and
infection. Owing to the complexity of its three-tiered
bronchial (airway) system (e.g. Maina, 2005), aerodynamic
filtration may be more efficient in the avian compared to
the mammalian respiratory system, where dichotomous
bifurcation essentially occurs (e.g. Weibel, 1984; Maina &
van Gils, 2001). Moreover, the avian lung is defended by
strategically placed lymphoid tissue scattered along the
airways (e.g. Reese et al. 2006) and SMs located in the atria
and the infundibulae (Maina & Cowley, 1998), where they
stop pathogens and particulates reaching the respiratory
surface.
Whereas the insoluble particles deposited on the airways
of the avian respiratory tract are mainly cleared by the
mucociliary escalator action (Fedde, 1998), those that enter
the parabronchi (where a ciliated epithelium is lacking)
are removed by epithelial cells that line the atria and
infundibulae (e.g. Mensah & Brain, 1982; Stearns et al.
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Interactions between particles and surface macrophages, S. G. Kiama et al. 459
Fig. 6 Giemsa-stained surface macrophages from the rat (a) and
chicken (b) lungs showing cells that appear to have taken up too many
polystyrene particles and consequently burst. Arrows show internalized
and released polystyrene particles; Nu, nucleus; dashed line, site where
the cell membrane has broken; circled area (b), a particle in the culture
medium. In both cells, the nucleus (Nu) has been pushed to one side,
presumably from the intracellular pressure of accumulated particles. The
insert (b) shows a chicken surface macrophage at the initial stages of
engulfing polystyrene particles (arrows).
1986, 1987; Maina & Cowley, 1998; Nganpiep & Maina,
2002). From structural and functional considerations, it is
impractical for the SMs to exist if they do not operate
effectively in the air capillaries. The diameters of the air
capillaries range from 3 to 20 μm (Duncker, 1974; Maina
& Nathaniel, 2001). Recent three-dimensional studies
(Woodward & Maina, 2005, 2008) further showed that the
passageways that connect the globular parts of the air
capillaries range in diameter from a mere 0.5 μm to 2 μm.
The SMs would obstruct the air capillaries, hindering gas
exchange. Surface macrophages have not been observed
on the respiratory surface of normal/healthy lungs of birds
(Maina, unpublished observations). Unlike in mammals,
where phagocytes migrate over a relatively short distance
of 200–300 μm to reach the terminal bronchioles from
where the cells and the engulfed particles are mechanically
transported by the mucociliary escalator system to the
pharynx, as the parabronchial lumen is lined by a nonsecretory, non-stratified, non-ciliated epithelium (e.g.
McLelland, 1989; López, 1995; Pastor & Calvo, 1995), an SM
would have to travel over a distance of about 10 000 μm
to reach the nearest parts of the lung (the secondary
bronchi) that have a ciliated epithelium. To survive such a
distance, the cell would have to be exceptionally motile
and resilient.
In the mammalian lung, macrophage overloading causes
particle uptake by the epithelial cells (Oberdörster et al.
1994). This suggests that macrophages can only phagocytize
up to a certain maximum volume of particles (Morrow,
1988; Oberdörster et al. 1994). Morrow (1988) noted that
alveolar macrophages function began to be impaired when
an average of 6% of its volume was filled by phagocytized
particles. Chen & Morrow (1989) noted that a macrophage
maintained its mobility up to a certain particle burden
beyond which the mobility was drastically compromised.
In this study, for cells of comparable diameters and by
extrapolation cell volumes, the chicken SMs phagocytized
substantially more particles, i.e. four times more (22.5% of
the cell volume in the chicken compared to 5.3% of that
of rat cells). Moreover, the chicken SMs engulfed the
particles at a faster rate. Notable phagocytic differences
have been reported in the pulmonary macrophages of
various mammalian species (e.g. Nguyen et al. 1982; Warheit
et al. 1988; Warheit & Hartsky, 1993; Molina & Brain, 2007).
In the cat, pulmonary SMs manifested a slower rate of in
vivo phagocytosis, higher cell motility, and faster particle
clearance, whereas among rodents, the rat had the most
efficient SMs. Yamaya et al. (1995) observed that phagocytic
activity of the alveolar macrophages of the dog lung
corresponded with motility. It remains to be shown whether
the apparently more phagocytic avian SMs are outstandingly
agile. Although they were not observed in the fluid
harvested from the rat lung, ciliated epithelial cells were
reported in lavages of rat (Maudley, 1977) and dog lungs
(Rebar et al. 1980). The ciliated epithelial cells harvested
from the chicken had dislodged from the trachea, the
primary bronchus, the secondary bronchi, and/or from the
ostia (e.g. Hodges, 1974). Well endowed with lytic enzymes
(Nganpiep & Maina, 2002), the epithelial cells were seen to
avidly phagocytize the polystyrene particles. In life, these
cells may play the significant role of removing and preventing harmful particulates and pathogenic microorganisms
from reaching the lung and the air sacs.
Although the process by which they enter the air spaces
(from the pulmonary vasculature) is presently uncertain,
the presence of erythrocytes in the lavage fluid of the
chicken was not unexpected: the cells were reported in
earlier studies (e.g. Toth & Siegel, 1986; Maina & Cowley,
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
460 Interactions between particles and surface macrophages, S. G. Kiama et al.
Fig. 7 Time-lapse photographs (taken at intervals of 15 min) showing surface macrophages of the chicken (a–f) and the rat (g–l) lungs exposed to
polystyrene particles (the cells were examined under an inverted microscope at a magnification of ×20). The inserts are pictures (of the chicken
macrophages) taken by an inverted microscope using different filters at the same time intervals to show how the cells progressively darkened and
formed phagocytic continua (arrows). The chicken surface macrophages took up the polystyrene particles at a faster rate compared to the rat ones.
Scale bars (inserts), 10 μm.
1998; Nganpiep & Maina, 2002). Unexpectedly, however,
the cells were observed to avidly phagocytize the polystyrene
particles: about 22% of them internalized between one
and six particles. A similar property was reported in the
nucleated erythrocytes of the rainbow trout, Salmo gairdneri
by Passantino et al. (2002). Mammalian erythrocytes are
indeed used as model cells that display non-phagocytic
property: they lack phagocytic receptors on their surface
and do not have an actin–myosin system (e.g. Geiser et al.
2005; Rothen-Rutishauser et al. 2006) that occurs on
specialized phagocytes such as SMs (e.g. Strossel & Hartwig,
1976; Hartwig et al. 1977; Underhill & Ozinsky, 2002).
The cells (mammalian erythrocytes) have, however, been
observed to internalize ultrafine particles of less than
0.2 μ m in diameter (e.g. Geiser et al. 2005; RothenRutishauser et al. 2006). Little is, however, known about
the interaction between such extremely small particles
and the cells: the particles are thought to be transported
into cells by mechanisms that are different from those of
phagocytosis and endocytosis. In this study, compared to
the rat cells that did not, the avian erythrocytes engulfed
polystyrene particles that were about eight times larger in
diameter than those that the mammalian erythrocytes
are known to engulf. Pertinent questions arise from this
observation: 1) Is the uptake of the larger particles by the
chicken cells an intrinsic feature of nucleated erythrocytes?
2) What mechanism(s) is involved in the uptake of such
relatively large particles? And, perhaps most importantly:
3) What role(s), if any, might the inherently phagocytic
erythrocytes play, particularly in the defense of the
pulmonary system and the body in general? Further studies
are needed to answer these interesting questions. For
now, it is supposed that the avian erythrocytes may clear
particles from the lung and deliver them to organs like
the liver and the spleen where they may be destroyed or
sequestered. In the cat lung, particles cleared from the
pulmonary intravascular macrophages (but not from the
SMs) were delivered to the liver and elsewhere in the body
(Molina & Brain, 2007).
In battery chicken production, birds are kept in closed,
crowded spaces which may contain high concentrations of
particulates and toxic gases (e.g. Anderson et al. 1964;
Conceição et al. 1989; Madelin & Wathes, 1989). Viable
particle concentrations as high as 2.4.105 m−3 have been
reported in poultry units (e.g. Anderson et al. 1964).
Additionally, in many cases, commercial birds are kept in
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Interactions between particles and surface macrophages, S. G. Kiama et al. 461
crowded conditions and may well be placed on a strict
feeding regimen that entails force-feeding. Stress under
such unnatural conditions may compromise the immunological robustness and cellular body defenses. The sweeping
anecdotal assertion that pulmonary defense in birds is
wanting and the inference that birds are exceptionally
susceptible to respiratory diseases may be a deduction
based on an artificial (human-induced) effect that could
have been provoked by intense breeding for attributes of
commercial value. The organs and organ systems of the
body and particularly the immunological and cellular
defense efficiencies may not have had time to adjust, i.e.
to adapt to the environmental and behavioural change(s).
The innate defenses of the avian respiratory system need
not necessarily be poor or inferior in all species of birds.
Acknowledgements
We are most grateful to the National Research Foundation (NRF)
which funded this study. Excellent technical support was provided
by Ms S. Rogers and H. Ali.
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