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Research in Veterinary Science 93 (2012) 393397

Contents lists available at ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

PAF increases phagocytic capacity and superoxide anion production in equine

alveolar macrophages and blood neutrophils
Luis Alexandre Muehlmann a,, Pedro Vicente Michelotto Jr. b, Everson Arajo Nunes c,
Fernanda Cristine Ceccon Grando d, Fabiana Tieme da Silva b, Anita Nishiyama d
a

Department of Genetics and Morphology, Instituto de Biologia, Campus Universitrio Darcy Ribeiro, Universidade de Braslia, Braslia, DF 70910-900, Brazil
Department of Agricultural and Environmental Sciences, Pontifcia Universidade Catlica do Paran, So Jos dos Pinhais, PR 80242-980, Brazil
Physiological Sciences Department, Biological Sciences Center, Federal University of Santa Catarina, Trindade Campus, Florianpolis, SC 88040-900, Brazil
d
Department of Physiology, Setor de Cincias Biolgicas, Campus Centro Politcnico, Universidade Federal do Paran, Av Cel Francisco H dos Santos, s/n, Jardim das Amricas,
PR 81530-900, Brazil
b
c

a r t i c l e

i n f o

Article history:
Received 29 April 2011
Accepted 13 July 2011

Keywords:
Platelet-activating factor
Neutrophil
Alveolar macrophage
Equine lower airways
Phagocytosis

a b s t r a c t
Phagocytosis exerted by alveolar macrophages and neutrophils is crucial in the clearance of exogenous
particles deposited in the airways. Therefore, substances that activate these phagocytes in the airways
can exert important effects on the particle clearance rate. PAF, particularly, was proved to be a potent
activator of several immune cells and was shown to be present in the equine lower airways in specic
conditions, such as after exercise. The present study aimed to investigate if PAF is able to increase the
phagocytic capacity and the production of superoxide anion in equine alveolar macrophage and blood
neutrophils. The results show that PAF increased these parameters in both phagocytes even in concentrations as low as 0.1 and 1.0 nM. On that ground, the present work suggests that PAF is involved in the process of particle clearance in equine lower airways.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The pulmonary conditioning signicantly affects the health and
the performance of horses. The stabling and the training tracks to
which the horses are exposed frequently lead the animal to inhale
a great amount of particulate matter. As the deposition of particles
on the alveolar epithelia impairs pulmonary function (Tetley,
2002), the lungs are endowed with a particle clearance system.
This system is highly dependent on phagocytosis and, therefore,
alveolar macrophages (AMs) and neutrophils are importantly involved in particle clearance in the lower airways.
Given the importance of these two phagocytes in particle clearance, the control of their activity may be a major mechanism of
adapting the clearance rate to the particle load, which may vary
signicantly depending on a myriad of factors e.g., season, environment, etc. In this context, several proinammatory mediators
have been shown to be released after particle deposition on the airways and they were proved to inuence the activity of both AMs
and neutrophils, altering the clearance rate (Lehnert, 1992;
Oberdrster, 1988).
The platelet-activating factor (PAF) is a proinammatory mediator that has raised the interest of several investigators because of
Corresponding author. Tel.: +55 61 8217 4852.
E-mail address: luismuehlmann@yahoo.de (L.A. Muehlmann).
0034-5288/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2011.07.008

its potent effects on a variety of inammatory cells (Zimmerman

et al., 1995; Marathe et al., 2001). Recently, it was shown that this
inammatory mediator was able to induce transient increases in
the intracellular Ca2+ concentration in equine blood neutrophil
(Muehlmann et al., 2010) and that PAF was present in the bronchoalveolar lavage uid (BALF) cells of exercised Thoroughbred colts
(Michelotto et al., 2010). Therefore, the question of whether PAF
could be involved in particle clearance in the equine lower airways
was raised. The present study shows that PAF activates, in both
AMs and blood neutrophils, key events related to particle
clearance.

2. Material and methods

2.1. Chemicals
BN52021 was purchased from Biomol (Plymouth Meeting, PA).
PAF (b-acetyl-c-O-hexadecyl-l-a-phosphatidylcholine, P-4904)
was obtained from Sigma (St. Louis, USA). Phorbol myristate acetate (PMA) was purchased from InvivoGen Corp., (San Diego,
USA) and Acepromazine (Acepran 1%) from Univet Laboratory
(So Paulo, Brazil). Xylazine (Sedazine) and Butorphanol (Torbugesic) were obtained from Fort Dodge Laboratory (So Paulo,
Brazil). All others chemicals were purchased from Sigma.

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2.2. Study design

All procedures were approved by the Committee on Animal
Experimentation of the Pontifcia Universidade Catlica do Paran
(PUCPR), Curitiba, Brazil (protocol number 129). Blood neutrophils
and AMs were obtained from two clinically healthy 2-years old
Thoroughbred horses. These cells were suspended in phosphatebuffered saline (PBS), separately plated on 96-wells microplates
(1  105 cells/well) and left to adhere for 1 h at 37 C. Then, cells
were washed, kept in 100 lL PBS and received one of the following
treatments: PBS alone, PMA 400 nM, PAF 0.1 nM, PAF 1.0 nM, PAF
10.0 nM and PAF 100.0 nM. For each group of cells receiving PAF,
it was carried out a control group with the same concentration of
PAF plus 150 lM BN52021 (PAF antagonist). BN52021 150 lM
alone or PMA 400 nM plus BN52021 150 lM were also tested.

30 min. The absorbance was measured on a spectrophotometer

at 550 nm.

2.7. Superoxide anion assay

This measurement was carried out as previously described (Dyrynda et al., 1998). AMs and blood neutrophils were incubated in PBS
with nitro-blue tetrazolium (NBT) 0.2% (m/v) and their respective
treatment at 37 C for 1 h. Then, the plate was centrifuged and
the supernatant was discarded. The wells were washed twice with
PBS and the cells were incubated with methanol 50% (v/v, aqueous)
for 10 min. The supernatant was removed. Then, 140 lL dimethylsulfoxide and 120 lL KOH 2 M were added. After 30 min, the absorbance was measured on a spectrophotometer at 595 nm.

2.3. BALF collection and AM isolation

The horses were sedated with acepromazine (0.03 mg/kg, IM)
and, after 30 min, received xylazine (0.30.5 mg/kg, IV) and butorphanol (0.05 mg/kg, IV). For BALF collection, a silicon catheter
(V-PBAL 300, Cook Vet Products) was introduced through the right
nare. Once it was lodged in a bronchus, the cuff was insufated
with 10 mL of air. Four 120 mL aliquots of sterile PBS with sodium
heparine (5 IU/mL) at 37 C were infused into the lung and immediately retrieved. The BALF was kept in ice. In less than 1 h after the
collection, the BALF was centrifuged at 340g for 6 min at 4 C. The
cell pellet was resuspended in 2 mL PBS and the cellular density
was then determined in a Neubauer chamber. The cellular viability
was about 90%, as determined by trypan blue exclusion. The AMs
suspended in PBS were left to adhere on microplates and the
non-adherent cells were removed in washing steps.
2.4. Blood collection and neutrophil isolation
Blood was collected by jugular vein puncture and deposited in a
blood bag (CPDA-1, JP Pharmaceutical Industry SA). Neutrophils
were then isolated from the CPDA-1-anticoagulated blood by centrifugation on Ficoll-Paque Plus (Amersham Biosciences, USA) as
described elsewhere (Byum, 1976). The cellular viability was
about 90%, as measured by trypan blue exclusion. The percentage
of neutrophils was over 90% in the polymorphonuclear leukocytes
suspension, as determined by May-Grnwald/Giemsa staining.
2.5. Cell adhesion assay
After the rst incubation for 1 h at 37 C (cell isolation step), the
plated cells were xed with methanol for 10 min and then incubated with 100 lL Giemsa 0.2% (w/w, aqueous) per well for
10 min. Wells were washed twice with distilled water and the
Giemsa dye retained by the cells was solubilized with 200 lL
methanol. The absorbance of the resulting solution was then measured on a spectrophotometer at 550 nm (Rosen and Gordon,
1987). The absorbance value was used to calculate the Af (adherence factor) with the equation Af = (adhesion assay absorbance) 1. Af was used to correct the results obtained with
plated cells (Muehlmann et al., 2010).

2.8. Statistical analysis

All values are expressed as the mean standard error of the
mean (S.E.M.) for four replicates. Statistical differences were assessed by analysis of variance (ANOVA) one way, followed by Tukeys post test (a = 0.05). All the values were multiplied by Af
before statistical analysis and graphic representation.

3. Results
3.1. PAF increases the phagocytic capacity in equine alveolar
macrophages
All the tested PAF concentrations increased the phagocytic
capacity in equine AM and this effect was signicantly reduced
by the PAF antagonist BN52021, evidencing that this effect was
in fact due to PAF. The results presented in Fig. 1 show that even
the subnanomolar concentration of PAF (0.1 nM) was able to signicantly increase the phagocytic capacity of AM (97.1%). The maximum response was obtained at 100 nM PAF (355.3% 13.82), but
this was not signicantly different in comparison to the response
obtained at 10 nM PAF (325.6% 8.79). The effect exerted by
PMA, a potent macrophage activator, at 400 nM was signicantly
lower even when compared to that exerted by PAF 0.1 nM. Treatments with BN52021 150 lM alone or PMA 400 nM plus
BN52021 150 lM did not present signicant differences in comparison to their respective controls, i.e., PBS or PMA 400 nM alone
(data not shown).

2.6. Phagocytic capacity assay

It was carried out as previously described (Dyrynda et al., 1998).
Briey, 10 lL neutral red stained zymosan (1  108 particles/mL)
were added along with the treatment, to the plated cells. After
1 h incubation at 37 C, cells were xed with Bakers solution for
30 min and washed twice with water. The neutral red retained
by the cells was solubilized with 0.1 mL acidied ethanol for

Fig. 1. Phagocytic capacity of AMs under different PAF concentrations with or

without 150 lM PAF antagonist BN52021 (BN). Data are presented as mean S.E.M.
a
p < 0.0001 vs. Control and p < 0.05 vs. PMA; bp < 0.0001 vs. Control and PMA, and
p < 0.05 vs. PAF 1 nM + BN; cp < 0.0001 vs. Control, PMA, PAF 1 nM and PAF
10 nM + BN; dp < 0.0001 vs. PMA, PAF 0.1 nM, PAF 1 nM and PAF 100 nM + BN.

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395

3.2. PAF induces a strong production of superoxide anion in equine

alveolar macrophages
PAF induced a massive production of superoxide anion by AM as
shown in Fig. 2. The production of superoxide anion obtained in
alveolar macrophages by the treatment with PAF 0.1 and 1.0 nM
was statistically equal to that obtained by the treatment with
PMA 400 nM. It is worth noting that even PAF 0.1 nM promoted
a strong increase in superoxide production (almost 840-fold in
comparison to control), evidencing that the equine AM is very sensible to this inammatory mediator. At 100 nM PAF, the inhibition
mediated by BN52021 was only partial, which can be due to the
competitive antagonism exerted by BN52021 and points to the
need of higher concentrations of this antagonist for obtaining a
complete inhibition of the PAF effects. BN52021 150 lM alone or
PMA 400 nM plus BN52021 150 lM did not promote signicant
differences in comparison to their respective controls (data not
shown).

Fig. 3. Phagocytic capacity of blood neutrophils under different PAF concentrations

with or without PAF antagonist BN52021 (150 lM). As positive control, 400 nM
PMA was used. Data are presented as mean S.E.M. ap < 0.0001 vs. Control;
b
p < 0.0001 vs. Control, PMA 400 nM, and PAF 0.1 nM + BN; cp < 0.0001 vs. Control,
PAF 0.1 nM and PAF 1 nM + BN; dp < 0.0001 vs. Control, PMA, and PAF 10 nM + BN;
e
p < 0.0001 vs. Control, PAF 0.1 nM, PAF 1 nM, PAF 10 nM and PAF 100 nM + BN.

3.3. PAF increases the phagocytic capacity in equine blood neutrophils

PAF increased the phagocytic capacity of equine blood neutrophils at all tested concentrations (Fig. 3). The responses elicited
by PAF at concentrations ranging from 1 to 100 nM were comparable to that induced by PMA 400 nM. At 0.1 nM, PAF increased the
phagocytic capacity by 49% in comparison to the control, evidencing that even picomolar PAF concentrations are able to affect the
neutrophil activity. The highest response was obtained with PAF
100 nM (increase of 157% in comparison to control), however, this
result was only slightly higher than those obtained with PAF 1 and
10 nM (increase of 116% and 98%, respectively, in comparison to
control), taking into account the great difference among these concentrations. This result suggests that the optimal PAF concentration for inducing an increase in the phagocytic capacity
in neutrophils seems to be in the subnanomolar range, as observed
also for AM. BN52021 150 lM alone or PMA 400 nM plus BN52021
150 lM did not signicantly modify the phagocytic capacity in
comparison to their respective controls (data not shown).

was comparable to that obtained with the treatment with PMA

400 nM. In comparison to the results obtained with the AM, this
important event involved in the oxidative burst in neutrophils
seems to be less sensible to PAF, as PAF 0.1 nM was not able to induce an increase in superoxide production. The response obtained
with PAF 100 nM was about 3-fold higher than that obtained with
PAF 10 nM. The same 3-fold difference was observed between the
results of neutrophils treated with 0.1 and 1 nM. Taken together,
these results suggest that the optimal PAF concentration for inducing an increase in the production of superoxide anion seems to be
at the nanomolar range for horse blood neutrophil. BN52021
150 lM alone or PMA 400 nM plus BN52021 150 lM did not signicantly modify this parameter in comparison to their respective
controls (data not shown).

4. Discussion

PAF, at nanomolar concentrations, promoted a signicant increase in the superoxide production in equine blood neutrophils,
as shown in Fig. 4. The increase observed at PAF 1 and 10 nM

This work shows that PAF, at the range of pico- to nanomolar

concentrations, is a potent promoter of phagocytosis and oxidative
burst in equine neutrophils and AMs. These ndings are important
in the context of the maintenance of the lung functionality, since
the phagocytosis exerted by these two cells is the main mechanism
for the elimination of particles in the airways (Oberdrster, 1988).
Although the response to PAF in these two phagocytes can be af-

Fig. 2. Superoxide anion production by AMs under different PAF concentrations

with or without PAF antagonist BN52021 (150 lM). As positive control, 400 nM
PMA was used. Data are presented as mean S.E.M. ap < 0.0001 vs. Control;
b
p < 0.0001 vs. Control and p < 0.001 vs. PAF 0.1 nM + BN; cp < 0.0001 vs. PAF 0.1 nM
and PAF 1 nM + BN; dp < 0.0001 vs. PMA, PAF 1 nM and PAF 10 nM + BN; ep < 0.0001
vs. PMA and PAF 100 nM + BN.

Fig. 4. Superoxide anion production by blood neutrophils under different PAF

concentrations with or without 150 lM PAF antagonist BN52021 (BN). As positive
control, 400 nM PMA was used. Data are presented as mean S.E.M. ap < 0.001 vs.
Control; bp < 0.0001 vs. PAF 0.1 nM and PAF 1 nM + BN; cp < 0.0001 vs. PAF
10 nM + BN; dp < 0.0001 vs. PMA, PAF 10 nM and PAF 100 nM + BN.

3.4. PAF increases the superoxide anion production in equine blood

neutrophils

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fected by other bioactive molecules present in the treatment medium, released by the plated cells, the results presented here clearly
show that PAF affects the activity of these phagocytes.
The large volume and alveolar surface area of the equine lungs
require a large number of cells specialized in the elimination of the
inhaled particulate matter that can eventually reach the lung epithelia. Therefore, it is not surprising that the equine lungs are endowed with about 48 billion AMs (Stone et al., 1992). This
phagocyte, due to its location and particular phenotype, is the main
orchestrator and effector of the elimination of particles reaching
the airways (Geiser, 2010) and its activity is controlled accordingly
to the particle load. When inhaled particles are deposited on airways epithelia, several proinammatory mediators are released
that modify the activity of AM and neutrophils (Sibille and Reynolds, 1990; Geiser, 2010). Therefore, it is reasonable to suggest
that PAF could exert important effects on phagocytes, thus affecting the rate of particle clearance in the airways.
The present work shows that the phagocytic capacity and the
production of superoxide anion are signicantly increased by PAF
in equine AMs, in a concentration-dependent fashion. It was already shown that PAF increases the in vitro phagocytic capacity
of murine peritoneal macrophages (Ichinose et al., 1994). It is
worth noting that this effect was exerted by PAF even at the concentration of 0.1 nM, a proof of the high intensity of the signal elicited by PAF in this phagocyte.
The AM is not the only phagocyte responsible for particle clearance. The neutrophil, particularly, is attracted to the lung epithelia
by alveolar macrophage-derived chemoattractants and, in a quite
interesting fashion, participates in the engulfment and inactivation
of the inhaled particles (Oberdrster, 1988; Sibille and Reynolds,
1990; Lehnert, 1992; Geiser, 2010). This is the reason why this
work also investigated the effects of PAF on the neutrophil activity.
The role played by the neutrophil in the particle clearance depends on the activity of the alveolar macrophage, from the beginning to the end of the whole process. When particles reach the lung
epithelia, chemotactic factors released by local cells, such as the
AMs (Warheit et al., 1986), recruit neutrophils (Hunninghake
et al., 1978). The recruited neutrophils engulf the deposited particles and are then phagocytosed by the AMs (Lehnert, 1992). Within
hours to days after the instillation almost all the inhaled particles
are associated with AMs (Lehnert, 1992). These particle-laden AMs
are able to reach the mucociliary escalator (Oberdrster, 1988;
Geiser, 2010) and are then transported up the conducting airways,
being swallowed or expectorated (Lehnert, 1992; Tetley, 2002).
The present study shows that the phagocytic capacity and the
production of the superoxide anion were both increased in neutrophils by PAF, even at 0.1 and 1.0 nM PAF, respectively. These results showed that, as for equine AM, PAF is a potent activator of
the equine blood neutrophil.
In fact, taking into account the mechanisms involved in phagocytosis, PAF was expected to increase the phagocytic capacity.
Phagocytosis is generally mediated by membrane receptors present on immune cells. These receptors bind ligands present on the
particle surface, promoting the cytoskeletal and membrane remodeling necessary to internalize the particle (Nunes and Demaurex,
2010). These ligands are present on the zymosan particles used
in this work, for example. Although the receptor binding is the
main initiator of phagocytosis, the phagocytic capacity can be
inuenced by several inammatory mediators through specic
second messengers. The Ca2+ cation, particularly, is involved in
critical steps of the phagocytosis (Vieira et al., 2002). PAF induces
transient increases in intracellular Ca2+ concentration (Muehlmann
et al., 2010). Therefore, the increased phagocytic capacity observed
in PAF-treated AMs and neutrophils may be directly related to the
transient increase of the intracellular Ca2+ concentration after PAF
treatment.

In addition to phagocytosis, other important immune processes,

such as the oxidative burst, are affected by the rise in intracellular
Ca2+ concentration (Segal, 1996). The oxidative burst is dependent
on the generation of several reactive species, derived from oxygen
and nitrogen, named ROS and RNS, respectively. The superoxide
anion is one of the main substrates for the generation of both
ROS and RNS and it is mainly produced by the transmembrane
enzymatic complex named NADPH oxidase (Rahman et al., 2006).
The Ca2+ cation is involved in the activation of the NADPH complex
as a second messenger (Dewitt et al., 2003). Therefore, as expected,
the production of superoxide anion was increased by PAF in equine
blood neutrophils and AMs. The increased capacity of generating
reactive species may increase the ability of the phagocyte to destroy engulfed particles.
Concluding, all these results suggest that PAF may be importantly involved in the equine airways homeostasis, since it signicantly affects the activity of equine blood neutrophil and AM, the
most important cells involved in particle clearance in the equine
lower airways. Moreover, given the essential role played by alveolar macrophages in microbial clearance in the lower airways (Sibille and Reynolds, 1990), PAF can also be an important mediator
involved in the immune response to microbial pulmonary infections. Therefore, studies on the effect of PAF in lung functionality
can lead to a better understanding of its role in the equine airways
and to better therapeutic approaches in pathological conditions
involving the airways.
5. Conict of interest statement
All authors disclose that they have no nancial or personal relationships with other people or organizations that could inappropriately inuence the work.
Acknowledgment
We thank Mr. Lucas Ferrari de Andrade for helping on the design of pilot studies.
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