вход по аккаунту


Occurence of lipid bodies in canine type II pneumocytes during hypothermic lung ischemia.

код для вставкиСкачать
Occurence of Lipid Bodies in Canine
Type II Pneumocytes During
Hypothermic Lung Ischemia
Department of Anatomy, Division of Electron Microscopy, Georg August University,
Göttingen, Germany
Clinical Research Group Chronic Airway Diseases, Department of Internal Medicine
(Respiratory Medicine), Philipps University, Marburg, Germany
Type II pneumocytes defend the pulmonary alveolus by synthesis and secretion of
surfactant and by contributing to alveolar epithelial regeneration. Lipid bodies are regarded
as intracellular domains for the synthesis of eicosanoid mediators that can be induced by
inflammatory stimuli. The aim of the present study was to establish whether hypothermic
ischemic lung storage without further preservation measures leads to an induction of lipid
body formation in canine type II pneumocytes. The lungs of 18 dogs were fixed for transmission electron microscopy (TEM) immediately after cardiac arrest (six double lungs) and after
ischemic storage in Tutofusin solution at 4°C for 20 min, 4 hr, 8 hr, and 12 hr (six single lungs,
respectively). Type II pneumocytes were analyzed qualitatively by conventional TEM
(CTEM) and quantitatively by stereology. The relative phosphorus content of surfactant
containing lamellar bodies, lipid bodies, and intermediate forms was investigated by energyfiltering TEM (EFTEM). By CTEM, lipid bodies as well as forms intermediate between lipid
bodies and lamellar bodies were already noted in the control group but were more pronounced
in the ischemia groups. Beginning at 20 min of ischemic storage, a significant increase in the
volume density of lipid bodies was noted in the ischemic groups as compared to the control
group. By EFTEM, the highest intracellular phosphorus signals were recorded over lamellar
bodies and lamellar areas of intermediate forms in all experimental groups, while lipid bodies
and homogeneous areas of intermediate forms did not show a clear phosphorus signal. These
results indicate that the formation of lipid bodies in canine type II pneumocytes is induced
early during ischemic lung storage. Anat Rec Part A 277A:287–297, 2004.
2004 Wiley-Liss, Inc.
Key words: lamellar body; surfactant; lung preservation; transmission electron microscopy; stereology
The structural and functional integrity of type II pneumocytes is of crucial importance for the maintenance of an
adequate lung function. Type II pneumocytes have two
main functions: they serve as the cellular source of pulmonary surfactant and they contribute to the regeneration of the alveolar epithelium under physiological and
pathological conditions. These properties form the basis of
the concept of the type II pneumocyte as the “defender of
the alveolus” (Mason and Williams, 1977; Fehrenbach,
Pulmonary surfactant is synthesized, stored, secreted,
and to a large extent recycled by type II pneumocytes
(Mason and Shannon, 1997; Fehrenbach, 2001). Surfactant prevents alveolar atelectasis by means of a surface
area-dependent reduction of the alveolar surface tension.
Moreover, certain surfactant components are considered
to have important immunomodulatory functions (Hawgood, 1997; Wright, 1997; Notter, 2000). Biochemically,
surfactant is a complex mixture, consisting of approxi-
Grant sponsor: the Deutsche Forschungsgemeinshaft; Grant
number: SFB 330, B 12.
*Correspondence to: Dr. Matthias Ochs, Department of Anatomy, Division of Electron Microscopy, Georg August University,
Kreuzbergring 36, D-37075, Göttingen, Germany. Fax: 49-551397004. E-mail:
Received 9 May 2003; Accepted 14 December 2003
DOI 10.1002/ar.a.20013
mately 90% lipids, mainly saturated phosphatidylcholine,
and 10% proteins, including the surfactant apoproteins
SP-A, SP-B, SP-C, and SP-D. Within type II pneumocytes,
the intracellular surfactant pool consists of characteristic
storage organelles termed lamellar bodies (Schmitz and
Müller, 1991; Weaver et al., 2002). The synthesis and
posttranslational processing of surfactant components involve endoplasmic reticulum, Golgi complex, and multivesicular bodies (Chevalier and Collet, 1972; Voorhout et al.,
1993). Surfactant material present in lamellar bodies is
secreted into the alveolar lumen via exocytosis (Ryan et
al., 1975; Kliewer et al., 1985; Dietl et al., 2001).
Lipid bodies are structurally distinct nonmembranebound cytoplasmic organelles present in a variety of cell
types (Galli et al., 1985; Murphy, 2001). They can be
viewed as one of the two major forms of macromolecular
lipid assemblies associated with biological systems, the
other form being membrane lipid bilayers (Murphy, 2001).
Lipid bodies are thought to represent specialized intracellular domains for the synthesis of eicosanoid mediators
(Weller and Dvorak, 1994; Weller et al., 1999). Lipid body
formation can be induced in several types of leukocytes by
inflammatory stimuli (Robinson et al., 1982; Triggiani et
al., 1995; Bozza et al., 1996, 1997; Chilton et al., 1996;
Weller et al., 1999; Pacheco et al., 2002). Thus, accumulation of cytoplasmic lipid bodies in these cells is regarded
as a highly inducible process that may play a key role in
the inflammatory response (Murphy, 2001).
In the process of lung transplantation, lungs are subjected to ischemic stress during the time interval between
explantation and implantation. Lung preservation is a
special situation in this respect since the lung has sufficient oxygen available for the generation of reactive oxygen species (ROS) already during ischemic storage. This is
in apparent contrast to other organs where ROS are usually not formed until the beginning of reperfusion (Fisher
et al., 1994; Heffner, 1998; de Perrot et al., 2003). ROS are
thought to play a significant role in the pathogenesis of
ischemia/reperfusion (I/R) injury (Lewis et al., 1997; Maurer, 1997; Heffner, 1998; Kelly, 2000; de Perrot et al.,
2003). Between 15% and 35% of all lung transplant recipients develop clinically relevant graft dysfunction in the
early postoperative period, mainly due to I/R injury (Trulock, 1997; King et al., 2000; de Perrot et al., 2003). Clinically, I/R injury is characterized by pulmonary edema
formation associated with an increased pulmonary artery
pressure and hypoxemia (Maurer, 1997; de Perrot et al.,
2003). The spectrum of I/R injury may range from mild
acute lung injury (ALI) to severe acute respiratory distress syndrome (ARDS) (Trulock, 1997; Kelly, 2000; de
Perrot et al., 2003). A complex inflammatory response is
part of the pathophysiological events that take place in I/R
injury (Heffner, 1998; Kelly, 2000; de Perrot et al., 2003).
Furthermore, I/R injury in experimental and clinical lung
transplantation is associated with alterations in the biochemical composition, biophysical function, and morphology of intra-alveolar surfactant (Lewis et al., 1997; Ochs,
2001; de Perrot et al., 2003).
Recent data have demonstrated that exposure to hypoxia induces the formation of lipid bodies in cultured aortic and pulmonary arterial endothelial cells (Scarfo et al.,
2001). Moreover, in a case of human single lung transplantation in which the recipient developed severe I/R
injury eventually leading to retransplantation, an accumulation of lipid bodies was observed in type II pneumo-
cytes of the contralateral nontransplanted donor lung at
the end of ischemic storage (Fehrenbach et al., 1998).
Therefore, the present study was undertaken to analyze
systematically whether cold ischemic storage without further preservation measures leads to the formation of lipid
bodies in type II pneumocytes of canine lungs. Ultrastructural analysis was performed by means of conventional
transmission electron microscopy (CTEM), energy-filtering transmission electron microscopy (EFTEM), and stereology. By EFTEM, the relative phosphorus content of
lipid bodies was determined in comparison to lamellar
bodies and forms transitional between lipid bodies and
lamellar bodies. Stereologically, the volume densities and
volume-to-surface ratios of lipid bodies and transitional
forms were estimated during the course of hypothermic
ischemic storage.
Experimental Groups
The lungs of 18 mongrel dogs of both sexes were used for
the study in combined neuroleptic analgesia (Kehrer et
al., 1989). Fixation, sampling, and processing were performed as described previously (Fehrenbach and Ochs,
1998; Ochs et al., 2001). Fixation was carried out either in
situ immediately after cardiac arrest (six double lungs) or
ex situ after storage of the lung in precooled Tutofusin
solution (Baxter, Unterschleißheim, Germany) at 4°C for
20 min, 4 hr, 8 hr, and 12 hr (six single lungs, respectively). Tutofusin solution is composed of electrolytes (sodium 140 mmol/l, potassium 5 mmol/l, calcium 2.5 mmol/l,
magnesium 1.5 mmol/l, chloride 153 mmol/l).
Lungs were fixed by airway instillation of 1.5% glutaraldehyde and 1.5% formaldehyde (prepared from freshly
depolymerized paraformaldehyde) in 0.1 M cacodylate
buffer (pH 7.35; buffer osmolality 300 mOsm/kg) at a
hydrostatic pressure of 25 cm. Lungs were stored in the
fixative with the airways clamped for at least another 2 hr
before further processing.
By the application of a systematic uniform random sampling procedure, tissue blocks representative of the whole
organ were obtained. The lungs were cut into horizontal
slices of 2 cm thickness starting at an apical position
chosen at random between 0 and 2 cm. Using a transparent grid with 34 holes with a distance of 4 cm between
each hole, which was then superimposed over the slices,
tissue samples of about 1 mm3 were taken whenever a
hole hit the cut surface of a slice.
Further processing was carried out with an automatic
tissue processor (Histomat, Bio-med, Theres, Germany).
Samples were osmicated in 1% OsO4 in 0.1 M cacodylate
buffer for 2 hr, stained en bloc in 1.5% aqueous uranyl
acetate overnight, dehydrated through an ascending series of acetone, and finally embedded in Araldite. Tissue
blocks were allowed to acquire random orientation in the
embedding capsules.
For CTEM analysis, ultrathin sections of 70 nm thickness were examined using an EM 10 transmission elec-
tron microscope (Zeiss, Oberkochen, Germany). Sections
were counterstained with uranyl acetate and lead citrate.
EFTEM analysis was performed with a CEM 902 transmission electron microscope (Zeiss, Oberkochen, Germany) equipped with an integrated electron energy spectrometer and image processing software for elemental
analysis (IBAS, Zeiss-Kontron). Ultrathin sections of 35
nm thickness were used without further counterstaining.
The relative phosphorus content of lipid bodies, lamellar
bodies, and transitional forms was investigated as described previously (Ochs et al., 1994; Ochs et al., 2001).
Briefly, the three-window method for phosphorus distribution calculation was performed. The element-specific
window beyond the PL2,3 edge was set at an energy loss of
155 eV and the two background windows below the PL2,3
edge were set at energy losses of 122 and 127 eV. The slit
width was 5 eV. The cutoff gray level for the background
was determined interactively by using ribosomes as an
internal reference. Phosphorus distribution images were
finally photographed from the computer screen. In addition, electron energy loss spectra were recorded over a
range of 50 –250 eV with an objective aperture of 90 ␮m
and a spectrometer aperture of 100 ␮m. Spectra recordings were performed with an integrated scintillator-photomultiplier system connected to an x/y plotter (BBC, SE
790) at a primary magnification of 50,500⫻. This ensured
that the whole recorded area of 0.05 ␮m2 was occupied by
the region of interest.
Stereological Analysis
Established stereological methods were used to analyze
type II pneumocytes (Weibel, 1979). Whenever a systematic uniform random test field included a type II pneumocyte, a micrograph involving the whole cell profile was
recorded at a primary magnification of 5,000⫻. The final
magnification after photographic reproduction was
10,000⫻. By means of point (for volume estimators) and
intersection (for surface estimators) counting, the following stereological parameters were estimated using an M
168 multipurpose test system with a point distance of 1.4
cm (Weibel, 1979): the volume densities (VV) of lipid bodies (li) and intermediate forms (lilb) with reference to the
cell volume (, cell). The reference space (, cell) was known
to be constant in all groups since an earlier study using
the same material (Ochs et al., 2001) showed no changes
in the volume of type II pneumocytes during cold ischemic
storage of up to 12 hr by determining the volume densities
of type II pneumocytes with reference to the fixed number
of points on the test system, thereby representing a constant reference volume. In addition, the volume-to-surface
ratios (V/S) of lipid bodies and intermediate forms were
estimated. This stereological parameter is independent of
changes in the reference space and it is directly related to
the mean caliper diameter of particles provided their
shape remains constant (Weibel, 1979). On average, 120
type II pneumocytes were analyzed per experimental
group. Per individual lung, a mean of 350 test points
falling on type II pneumocytes were counted. This number
should ensure that the variability among measurements
contributes only to a minor extent to the total observed
experimental variability, which is then mainly dominated
by the biological variability among the individuals under
Data are presented as means (SEM). Parametric analysis of variance (ANOVA) was performed if the hypothesis
of normality and equal variance was not rejected at P ⬍
0.05. Otherwise, data were analyzed by nonparametric
analysis of variance using the Kruskal-Wallis rank sum
test. A P value of ⬍ 0.05 was considered to be significant.
All tests were performed using the SigmaStat 2.0 software
(Jandel Scientific, Erkrath, Germany).
An example for the typical ultrastructural appearance
of the alveolar septum in the control group is shown in
Figure 1. The most characteristic feature of type II pneumocytes is the presence of numerous lamellar bodies
within the cytoplasm. Already in the control group, but
considerably more pronounced in the ischemia groups,
lipid bodies with a matrix of homogeneous appearance and
low to medium electron density were noted as well as
intermediate forms between lipid bodies and lamellar bodies with homogeneous and lamellar areas (Fig. 2).
Whereas lipid bodies and homogeneous areas within intermediate forms were surrounded only by an osmiophilic
ring, lamellar bodies and intermediate forms were bound
by a trilaminar limiting membrane. The lamellae in intermediate forms were mostly arranged in densely packed
parallel or cap-like stacks with a lamellar periodicity of
4.3–5.4 nm, similar to lamellar bodies (Fig. 3). Occasionally, lipid bodies and intermediate forms were found in the
process of secretion into the alveolar space (Fig. 4). This
event was observed four times in the approximately 600
type II pneumocytes that were analyzed in total.
EFTEM analysis revealed the highest intracellular
phosphorus signals over lamellar bodies and lamellar areas of intermediate forms, thereby indicating a high relative phospholipid content. Distribution images using ribosomes as internal phosphorus reference showed highest
intensities over lamellar bodies and lamellar areas of intermediate forms in all experimental groups. In contrast,
no clear phosphorus signal was detectable over lipid bodies and homogeneous areas of intermediate forms. Correspondingly, these findings were confirmed by electron energy loss spectra (Fig. 5). No changes in the relative
phosphorus content were observed during the course of
ischemia (data not shown).
Stereological results are summarized in Table 1. Data
for lamellar bodies taken from an earlier study using the
same material (Ochs et al., 2001) are given for comparison. A significant increase in the volume density of lipid
bodies was noted in all ischemic groups compared to the
control group (P ⬍ 0.05). Because an earlier study has
shown that the volume of type II pneumocytes did not
change between groups (Ochs et al., 2001), an increased
volume density of lipid bodies can unambiguously be interpreted as an increase in total volume per cell. The
volume-to-surface ratio of lipid bodies, however, did not
change significantly. This indicates no significant changes
in the size of lipid bodies, based on the reasonable assump-
Fig. 1. Alveolar septum, control group. The alveolar space (A) and the capillary lumen (C) are separated
by the blood-air barrier consisting of the alveolar epithelium, an interstitial layer of varying thickness and the
capillary endothelium. The alveolar epithelium is composed of type I pneumocytes (PI) with their thin cell
extensions and type II pneumocytes (PII), which are characterized by their lamellar bodies. Scale bar ⫽ 2 ␮m.
tion that their shape did not change considerably. Therefore, it is safe to conclude that the increase in total volume
per cell can be attributed to an increase in the number of
lipid bodies per cell beginning with 20 min of ischemia.
However, the total number of lipid bodies per cell can
directly be estimated only by the application of the physical dissector method (Sterio, 1984) at the EM level, which
allows unbiased and assumption-free direct estimation of
total particle number. For reasons of workload and efficiency (precision of a stereological estimate per time or per
unit cost), this method would not have been justified in the
present study where differences between groups were of
interest rather than total number. No significant changes
in the volume density or volume-to-surface ratio of intermediate forms were noted.
The general morphological appearance of lipid bodies
has been well described (Fawcett, 1981; Ghadially, 1997).
They are usually present within the cytoplasm as spheri-
Fig. 2. Type II pneumocyte, 20-min cold ischemic storage. Beside many lamellar bodies, an intermediate
form between a lipid body and a lamellar body (arrow) is visible. Scale bar ⫽ 1 ␮m.
cal droplets. Lipid bodies may vary in size and electron
density, partly depending on their chemical composition
and the mode of tissue preparation. Characteristically,
they are surrounded by an osmiophilic ring instead of a
trilaminar limiting membrane. This osmiophilic ring is
thought to represent a monolayer of amphipathic phospholipids, glycolipids, sterols, and specific proteins that
encircles a hydrophobic core of neutral lipids (Murphy,
2001). The presence of lipid bodies in cells may be much
more common than has been previously anticipated. They
should no longer be regarded as a rare ultrastructural
oddity but rather as ubiquitous cell components playing
important roles in various aspects of lipid trafficking
(Murphy, 2001). This is in agreement with the present
study where lipid bodies were already observed to a certain degree in the control group. However, the present
study has also shown that lipid bodies in canine type II
pneumocytes are increased significantly already in the
early phase of cold ischemic lung storage without further
preservation. This finding is in line with the recent con-
Fig. 3. Intermediate form between a lipid body and a lamellar body,
control group. a: While the homogeneous area within the intermediate
form is surrounded only by an osmiophilic ring (arrows), the intermediate
form itself is bound by a trilaminar limiting membrane (arrowheads).
Scale bar ⫽ 0.2 ␮m. b: Higher magnification. The lamellar area within the
intermediate form is arranged in parallel stacks with a periodicity of
4.3–5.4 nm. Scale bar ⫽ 50 nm.
Fig. 4. Intermediate form between a lipid body and a lamellar body, 20-min cold ischemic storage. The
intermediate form is shown in the process of secretion into the alveolar space. Scale bar ⫽ 0.2 ␮m.
cept of lipid bodies as early response structures involved
in the production of lipid mediators of inflammation (Pacheco et al., 2002).
The presence of lipid bodies in type II pneumocytes of
several species has occasionally been reported. So far,
lipid bodies have been described by means of TEM in type
II pneumocytes of the human (Dvorak et al., 1992; Fehrenbach et al., 1998), dog (Barkett et al., 1969; Modry and
Chiu, 1979), pig (Fehrenbach et al., 1991), goat (Atwal and
Sweeny, 1971; Kahwa et al., 1997), ferret (Miller et al.,
1982), guinea pig (Valdivia et al., 1966), and opossum lung
(Krause et al., 1976) under normal and pathological conditions. In the first report on lipid bodies in type II pneumocytes, Valdivia et al. (1966) described their appearance
in guinea pigs exposed to severe hypoxia and suggested
that this fatty change might represent a metabolic alteration interfering with surfactant synthesis. In canine
lungs, lipid bodies in type II pneumocytes were detected in
hemorrhagic shock (Barkett et al., 1969) and in pulmonary reperfusion injury following 5 hr of pulmonary artery
occlusion (Modry and Chiu, 1979). The concept that type II
pneumocyte lipid bodies are involved in the formation of
eicosanoid mediators was supported by the findings of
Dvorak et al. (1992), who localized prostaglandin endoperoxide synthase (cyclooxygenase) in lipid bodies in cultured
human lung type II pneumocytes. Appropriately, increased concentrations of eicosanoid mediators have been
found in several models of lung I/R injury (Heffner, 1998;
de Perrot et al., 2003).
The present study has shown that lamellar bodies and
lamellar areas of intermediate forms differ markedly in
their phosphorus content from lipid bodies and homogeneous areas of intermediate forms. Lamellar bodies are
known to have a high phospholipid content (Gil and Reiss,
1973; Eckenhoff and Somlyo, 1988), but a detailed analysis of the lipid composition of lipid bodies is missing so far,
although they are believed to contain mainly neutral lipids (Fawcett, 1981; Murphy, 2001). Depending on the cell
type and the environmental conditions, their heterogeneous lipid composition might be variable. Moreover, inter- and intraspecies differences also have to be taken into
account. However, the present results indicate that, in
comparison to lamellar bodies, considerably fewer phospholipids are present in lipid bodies of canine type II
pneumocytes. This is in line with our earlier findings in
pig lungs (Fehrenbach et al., 1991).
In the present study, EFTEM analysis was performed
on chemically fixed and plastic-embedded samples because this allows the unambiguous differentiation of the
various inclusion bodies of type II pneumocytes based on
ultrastructural criteria. A previous quantitative elemental analysis of rat lung type II pneumocytes (Eckenhoff
and Somlyo, 1988) was based on electron probe microanalysis of freeze-dried cryosections from cryofixed samples.
However, this approach failed to demonstrate any lamellar ultrastructure while freeze-fracture studies of cryofixed type II pneumocytes (Williams, 1982) clearly confirmed the lamellar fine structure of lamellar bodies seen
after chemical fixation and plastic embedding. Different
types of inclusion bodies in type II pneumocytes would
therefore have been indistinguishable in freeze-dried cryosections, thus making this approach unsuitable for the
aim of the present study.
So far, forms that are intermediate between lipid bodies
and lamellar bodies have been described in type II pneumocytes of the ferret lung (Miller et al., 1982). In addition,
the present study for the first time provides morphological
evidence for secretion of intermediate forms by exocytosis.
The content of secreted lipid bodies or homogeneous parts
of intermediate forms is then part of the extracellular
lining layer covering the alveolar epithelium where it
might further interact with intra-alveolar surfactant components or alveolar macrophages.
In several cell types, the accumulation of lipid bodies
appears to be induced specifically in response to stress.
This has also been demonstrated for other cell types in the
lung. According to observations in isolated human lung
mast cells, lipid bodies are regarded as morphological
evidence of an activated cell (Peters et al., 1989). In genetargeted mice deficient in surfactant protein D, numerous
giant foamy alveolar macrophages containing lipid bodies
are present (Hawgood et al., 2001). Surfactant protein D
contributes to the regulation of oxidant metabolism and
inflammatory responses within the lung (Crouch, 2000).
Furthermore, in patients with ARDS, cytoplasmic lipid
bodies have been found in high numbers in neutrophils
isolated from bronchoalveolar lavage (Triggiani et al.,
1995). The pathophysiological features of the surfactant
alterations seen in ALI/ARDS are strikingly similar to
those observed in I/R injury and are partly mediated by
ROS (Lewis et al., 1997; Ochs, 2001; de Perrot et al., 2003).
It is well documented that ROS cause surfactant lipid
peroxidation in ALI/ARDS (Gilliard et al., 1994; Notter,
2000; Günther et al., 2001). ROS also inhibit acetylhydrolase (Ambrosio et al., 1994; Triggiani et al., 1997), the
enzyme that degrades platelet-activating factor (PAF).
Fig. 5. Lamellar bodies and lipid bodies, control group. a: Picture
obtained by CTEM. Scale bar ⫽ 0.5 ␮m. b: EFTEM. Phosphorus distribution image showing highest intensities over lamellar bodies with no
detectable signal over lipid bodies. c: EFTEM. Electron energy loss
spectra. The spectrum on the right was recorded over a lamellar body
and shows a uranium peak at the UO4,5 edge at 96 eV, which is due to
the bloc staining with uranyl acetate, and a phosphorus peak at the PL2,3
edge at 132 eV. The spectrum in the center was recorded over a lipid
body and shows no clear signals. The left-hand spectrum was recorded
over a pure resin region and served as control.
TABLE 1. Stereological data of lipid bodies in canine type II pneumocytes during
cold hypothermic ischemia†
Experimental Group
20-min ischemia at 4°C
4-hr ischemia at 4°C
8-hr ischemia at 4°C
12-hr ischemia at 4°C
Vv (lb, cell)a
V/S (lb)a
Vv (li, cell)
V/S (li)
Vv (lilb, cell)
V/S (lilb)
21.3 (2.3)
16.6 (1.6)
15.6 (1.0)
17.3 (1.3)
18.0 (1.3)
191.0 (13.8)
172.3 (5.4)
153.1 (5.0)
167.2 (13.3)
165.5 (8.8)
0.2 (0.1)
0.9 (0.2)*
1.1 (0.2)*
1.3 (0.4)*
1.0 (0.2)*
105.0 (27.1)
126.7 (14.0)
196.4 (38.5)
207.5 (46.2)
201.9 (52.5)
3.2 (0.4)
2.7 (0.4)
2.6 (0.4)
2.6 (0.6)
3.2 (0.7)
203.8 (17.4)
171.7 (19.4)
184.7 (20.8)
162.9 (33.5)
229.1 (29.2)
Volume densities (Vv) and volume-to-surface ratios (V/S) of type II pneumocyte components. Values for lamellar bodies (lb),
lipid bodies (li), and intermediate forms (lilb) are shown with reference to the cell volume (, cell). Data are given as mean
Data from Ochs et al. (2001) for comparison.
*P ⬍ 0.05 vs. control.
PAF is an important proinflammatory mediator involved
in the pathogenesis of both ALI/ARDS and I/R injury
(Lewis et al., 1997; Nagase et al., 1999; de Perrot et al.,
2003). Beside being in alveolar macrophages, PAF-acetylhydrolase is also present in type II pneumocytes and is
secreted into the alveolar space in association with surfactant (Jehle et al., 2001). PAF has as well been shown to
induce the formation of lipid bodies in leukocytes (Bozza et
al., 1996; Weller et al., 1999; de Assis et al., 2003).
In a previous study, we demonstrated that cold ischemic
canine lung storage of up to 12 hr does not lead to ultrastructural alterations of lamellar bodies in type II pneumocytes (Ochs et al., 2001), supporting the idea that successful surfactant preservation is possible during
prolonged ischemic storage (Lewis et al., 1997). However,
the present results demonstrate an increased occurrence
of lipid bodies under the same conditions. Therefore, intracellular lipid metabolism and/or trafficking might be
altered as an early response to ischemic stress before
intracellular surfactant alterations become visible. The
concept that the formation of lipid bodies in type II pneumocytes can be induced during ischemia is supported by
observations in a case of human single lung transplantation where type II pneumocytes of the nontransplanted
donor single lung showed an accumulation of lipid bodies
together with increased amounts of intracellular surfactant at the end of ischemic storage. The patient receiving
the contralateral single lung developed severe I/R injury,
which finally made retransplantation necessary (Fehrenbach et al., 1998).
A specific role of lipid bodies in type II pneumocytes
might be to provide fatty acid substrates for surfactant
synthesis under certain conditions. This has already been
suggested for lipid bodies in pulmonary lipofibroblasts
located in the alveolar interstitium (McGowan and Torday, 1997). The connection between lipid bodies and the
surfactant system could be regarded as a specialized version of the general functions that lipid bodies have as
intermediates in the pathways of membrane lipid formation, trafficking, and turnover. Impairment of these pathways is often manifested by a pathological accumulation of
lipid bodies (Murphy, 2001). Type II pneumocytes are
directly involved in pulmonary host defense by secreting
surfactant components with immunomodulatory functions
(Wright, 1997). Furthermore, a direct role for surfactant
proteins A and D in the protection against oxidative damage has been demonstrated (Bridges et al., 2000). Previous
in vitro studies on type II pneumocytes indicate that they
also actively contribute to inflammatory processes by generating a variety of proinflammatory mediators, including
a broad profile of eicosanoid metabolites, cytokines, and
chemokines (Grimminger et al., 1992; Vlahakis et al.,
1999; Rose et al., 2002; Sato et al., 2002; Vanderbilt et al.,
2003). In this respect, the formation of lipid bodies might
be regarded as a morphological feature of type II pneumocytes that indicates activation of this cell type in the
inflammatory response.
In conclusion, the present study has shown that cold
ischemic lung storage without further preservation measures induces the formation of lipid bodies in canine type
II pneumocytes. This finding is consistent with the concept of lipid bodies as specialized intracellular domains in
a variety of cell types involved in the inflammatory response. The connection between the impairment of lipid
metabolism and trafficking leading to the formation of
lipid bodies in type II pneumocytes and the surfactant
alterations associated with I/R injury of the lung remains
to be further investigated. Since type II pneumocytes are
considered as a major target in I/R injury (Novick et al.,
1996), lipid body formation in type II pneumocytes should
also be considered as an ultrastructural criterion for the
assessment of lung preservation quality in studies evaluating new lung preservation strategies.
We thank S. Freese, A. Gerken, and H. Hühn for excellent technical assistance and C. Maelicke for checking the
Ambrosio G, Oriente A, Napoli C, Palumbo G, Chiariello P, Marone G,
Condorelli M, Chiariello M, Triggiani M. 1994. Oxygen radicals
inhibit human plasma acetylhydrolase, the enzyme that catabolizes
platelet-activating factor. J Clin Invest 93:2408 –2416.
Atwal OS, Sweeny PR. 1971. Ultrastructure of the interalveolar septum of the lung of the goat. Am J Vet Res 32:1999 –2010.
Barkett VM, Coalson JJ, Greenfield LJ. 1969. Early effects of hemorrhagic shock on surface tension properties and ultrastructure of
canine lungs. Bull Johns Hopkins Hosp 124:87–94.
Bozza PT, Payne JL, Goulet JL, Weller PF. 1996. Mechanisms of
platelet-activating factor-induced lipid body formation: requisite
roles for 5-lipoxygenase and de novo protein synthesis in the compartimentalization of neutrophil lipids. J Exp Med 183:1515–1525.
Bozza PT, Yu W, Penrose JF, Morgan ES, Dvorak AM, Weller PF.
1997. Eosinophil lipid bodies: specific, inducible intracellular sites
for enhanced eicosanoid formation. J Exp Med 186:909 –920.
Bridges JP, Davis HW, Damodarasamy M, Kuroki Y, Howles G, Hui
DY, McCormack FX. 2000. Pulmonary surfactant proteins A and D
are potent endogenous inhibitors of lipid peroxidation and oxidative
cellular injury. J Biol Chem 275:38848 –38855.
Chevalier G, Collet AJ. 1972. In vivo incorporation of choline-3H,
leucine-3H and galactose-3H in alveolar type II pneumocytes in
relation to surfactant synthesis: a quantitative radioautographic
study in mouse by electron microscopy. Anat Rec 174:289 –310.
Chilton FH, Fonteh AN, Surette ME, Triggiani M, Winkler JD. 1996.
Control of arachidonate levels within inflammatory cells. Biochim
Biophys Acta 1299:1–15.
Crouch EC. 2000. Surfactant protein-D and pulmonary host defense.
Respir Res 1:93–108.
de Assis EF, Silva AR, Caiado LF, Marathe GK, Zimmerman GA,
Prescott SM, McIntyre TM, Bozza PT, de Castro-Faria-Neto HC.
2003. Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists
generated during low density lipoprotein oxidation that induces
lipid body formation in leukocytes. J Immunol 171:2090 –2098.
de Perrot M, Liu M, Waddell TK, Keshavjee S. 2003. Ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med 167:490 –
Dietl P, Haller T, Mair N, Frick M. 2001. Mechanisms of surfactant
exocytosis in alveolar type II cells in vitro and in vivo. News Physiol
Sci 16:239 –243.
Dvorak AM, Morgan E, Schleimer RP, Ryeom SW, Lichtenstein LM,
Weller PF. 1992. Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells,
alveolar macrophages, type II pneumocytes, and neutrophils. J Histochem Cytochem 40:759 –769.
Eckenhoff RG, Somlyo AP. 1988. Rat lung type II cell and lamellar
body: elemental composition in situ. Am J Physiol 254:C614 –C620.
Fawcett DW. 1981. The cell. Philadelphia: W.B. Saunders.
Fehrenbach H, Richter J, Schnabel PA. 1991. Improved preservation
of phospholipid-rich multilamellar bodies in conventionally embedded mammalian lung tissue: an electron spectroscopic study. J
Microsc 162:91–104.
Fehrenbach H, Ochs M. 1998. Studying lung ultrastructure. In: Uhlig
S, Taylor AE, editors. Methods in pulmonary research. Basel:
Birkhäuser. p 429 – 454.
Fehrenbach H, Wahlers T, Ochs M, Brasch F, Schmiedl A, Hirt SW,
Haverich A, Richter J. 1998. Ultrastructural pathology of the alveolar type II pneumocytes of human donor lungs: electron microscopy, stereology, and microanalysis. Virchows Arch 432:229 –239.
Fehrenbach H. 2001. Alveolar epithelial type II cell: defender of the
alveolus revisited. Respir Res 2:33– 46.
Fisher AB, Dodia C, Ayene I, Al-Mehdi A. 1994. Ischemia-reperfusion
injury to the lung. Ann NY Acad Sci 723:197–207.
Galli SJ, Dvorak AM, Peters SP, Schulman ES, MacGlashan DW Jr,
Isomura T, Pyne K, Harvey VS, Hammel I, Lichtenstein LM,
Dvorak HF. 1985. Lipid bodies. Widely distributed cytoplasmic
structures that represent preferential nonmembrane repositories of
exogenous [3H]arachidonic acid incorporated by mast cells, macrophages, and other cell types. In: Bailey JM, editor. Prostaglandins,
leukotriens, and lipoxins. New York: Plenum Press. p 221–239.
Ghadially FN. 1997. Ultrastructural pathology of the cell and matrix,
4th ed. Boston: Butterworth-Heinemann.
Gil J, Reiss OK. 1973. Isolation and characterization of lamellar
bodies and tubular myelin from rat lung homogenates. J Cell Biol
Gilliard N, Heldt GP, Loredo J, Gasser H, Redl H, Merritt TA, Spragg
RG. 1994. Exposure of the hydrophobic components of porcine lung
surfactant to oxidant stress alters surface tension properties. J Clin
Invest 93:2608 –2615.
Grimminger F, von Kürten I, Walmrath D, Seeger W. 1992. Type II
alveolar epithelial eicosanoid metabolism: predominance of cyclooxygenase pathways and transcellular lipoxygenase metabolism in
co-culture with neutrophils. Am J Respir Cell Mol Biol 6:9 –16.
Günther A, Ruppert C, Schmidt R, Markart P, Grimminger F,
Walmrath D, Seeger W. 2001. Surfactant alteration and replacement in acute respiratory distress syndrome. Respir Res 2:353–364.
Hawgood S. 1997. Surfactant: composition, structure, and metabolism. In: Crystal RG, West JB, Weibel ER, Barnes PJ, editors. The
lung: scientific foundations, 2nd ed. Philadelphia: LippincottRaven. p 557–571.
Hawgood S, Akiyama J, Brown C, Allen L, Li G, Poulain FR. 2001.
GM-CSF mediates alveolar macrophage proliferation and type II
hypertrophy in SP-D gene-targeted mice. Am J Physiol 280:L1148 –
Heffner JE. 1998. Mechanisms underlying ischemia/reperfusion injury of the lung. In: Matthay MA, Ingbar DH, editors. Pulmonary
edema: lung biology in health and disease, vol. 116. New York:
Marcel Dekker. p 379 – 412.
Jehle R, Schlame M, Büttner C, Frey B, Sinha P, Rüstow B. 2001.
Platelet-activating factor (PAF)-acetylhydrolase and PAF-like compounds in the lung: effects of hyperoxia. Biochim Biophys Acta
1532:60 – 66.
Kahwa CKB, Atwal OS, Purton M. 1997. Transmission electron microscopy of the epithelium of distal airways and pulmonary parenchyma of the goat lung. Res Vet Sci 63:49 –56.
Kehrer G, Blech M, Kallerhoff M, Langheinrich M, Bretschneider HJ.
1989. Contribution of amino acids in protective solutions to postischemic functional recovery of canine kidneys. Res Exp Med 189:
Kelly RF. 2000. Current strategies in lung preservation. J Lab Clin
Med 136:427– 440.
King RC, Binns OAR, Rodriguez F, Kanithanon RC, Daniel TM,
Spotnitz WD, Tribble CG, Kron IL. 2000. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation.
Ann Thorac Surg 69:1681–1685.
Kliewer M, Fram EK, Brody AR, Young SL. 1985. Secretion of surfactant by rat alveolar type II cells: morphometric analysis and
three-dimensional reconstruction. Exp Lung Res 9:351–361.
Krause WJ, Cutts JH, Leeson CR. 1976. Type II pulmonary epithelial
cells of the newborn opossum lung. Am J Anat 146:181–188.
Lewis JF, Novick RJ, Veldhuizen RAW. 1997. Surfactant in lung
injury and lung transplantation. New York: Springer.
Mason RJ, Williams MC. 1977. Type II alveolar cell: defender of the
alveolus. Am Rev Respir Dis 115:81–91.
Mason RJ, Shannon JM. 1997. Alveolar type II cells. In: Crystal RG,
West JB, Weibel ER, Barnes PJ, editors. The lung: scientific foundations, 2nd ed. Philadelphia: Lippincott-Raven. p 543–555.
Maurer JR. 1997. The lung following transplantation. In: Crystal RG,
West JB, Weibel ER, Barnes PJ, editors. The lung: scientific foundations, 2nd ed. Philadelphia: Lippincott-Raven. p 2771–2785.
McGowan SE, Torday JS. 1997. The pulmonary lipofibroblast (lipid
interstitial cell) and its contributions to alveolar development.
Annu Rev Physiol 59:43– 62.
Miller ML, Andringa A, Vinegar A. 1982. Ultrastructure and morphometry of the alveolar type II cell of the ferret. J Ultrastruct Res
Modry DL, Chiu RCJ. 1979. Pulmonary reperfusion syndrome. Ann
Thorac Surg 27:206 –215.
Murphy DJ. 2001. The biogenesis and functions of lipid bodies in
animals, plants and microorganisms. Prog Lipid Res 40:325– 438.
Nagase T, Ishii S, Kume K, Uozumi N, Izumi T, Ouchi Y, Shimizu T.
1999. Platelet-activating factor mediates acid-induced lung injury
in genetically engineered mice. J Clin Invest 104:1071–1076.
Notter RH. 2000. Lung surfactants: basic science and clinical applications. New York: Marcel Dekker.
Novick RJ, Gehman KE, Ali IS, Lee J. 1996. Lung preservation: the
importance of endothelial and alveolar type II cell integrity. Ann
Thorac Surg 62:302–314.
Ochs M, Fehrenbach H, Richter J. 1994. Electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) of multilamellar bodies and multilamellar body-like structures in tannic acid
treated alveolar septal cells. J Histochem Cytochem 42:805– 809.
Ochs M. 2001. Pulmonary surfactant preservation in experimental
and clinical lung transplantation. In: Pandalai SG, editor. Recent
research developments in respiratory and critical care medicine.
Trivandrum: Research Signpost. p 59 – 81.
Ochs M, Fehrenbach H, Richter J. 2001. Ultrastructure of canine type
II pneumocytes during hypothermic ischemia of the lung: a study by
means of conventional and energy filtering transmission electron
microscopy and stereology. Anat Rec 263:118 –126.
Pacheco P, Bozza FA, Gomes RN, Bozza M, Weller PF, Castro-FariaNeto HC, Bozza PT. 2002. Lipopolysaccharide-induced leukocyte
lipid body formation in vivo: innate immunity elicited intracellular
loci involved in eicosanoid metabolism. J Immunol 169:6498 – 6506.
Peters SP, Dvorak AM, Schulman ES. 1989. Mast cells. In: Massaro
D, editor. Lung cell biology. New York: Marcel Dekker. p 345–399.
Robinson JM, Karnovsky ML, Karnovsky MJ. 1982. Glycogen accumulation in polymorphonuclear leukocytes, and other intracellular
alterations that occur during inflammation. J Cell Biol 95:933–942.
Rose F, Dahlem G, Guthmann B, Grimminger F, Maus U, Hänze J,
Duemmer N, Grandel U, Seeger W, Ghofrani HA. 2002. Mediator
generation and signaling events in alveolar epithelial cells attacked
by S. aureus ␣-toxin. Am J Physiol 282:L207–L214.
Ryan US, Ryan JW, Smith DS. 1975. Alveolar type II cells: studies on
mode of release of lamellar bodies. Tissue Cell 7:587–599.
Sato K, Tomioka H, Shimizu T, Gonda T, Ota F, Sano C. 2002. Type
II alveolar cells play roles in macrophage-mediated host innate
resistance to pulmonary mycobacterial infections by producing
proinflammatory cytokines. J Infect Dis 185:1139 –1147.
Scarfo LM, Weller PF, Farber HW. 2001. Induction of endothelial cell
cytoplasmic lipid bodies during hypoxia. Am J Physiol 280:H294 –
Schmitz G, Müller G. 1991. Structure and function of lamellar bodies,
lipid-protein complexes involved in storage and secretion of cellular
lipids. J Lipid Res 32:1539 –1570.
Sterio DC. 1984. The unbiased estimation of number and sizes of
arbitrary particles using the disector. J Microsc 134:127–136.
Triggiani M, Oriente A, Seeds MC, Bass DA, Marone G, Chilton FH.
1995. Migration of human inflammatory cells into the lung results
in the remodeling of arachidonic acid into a triglyceride pool. J Exp
Med 182:1181–1190.
Triggiani M, De Marino V, Sofia M, Faraone S, Ambrosio G, Carratu
L, Marrone G. 1997. Characterization of platelet-activating factor
acetylhydrolase in human bronchoalveolar lavage. Am J Respir Crit
Care Med 156:94 –100.
Trulock EP. 1997. Lung transplantation. Am J Respir Crit Care Med
155:789 – 818.
Valdivia E, Sonnad J, D’Amato J. 1966. Fatty change of the granular
pneumocyte. Science 151:213–214.
Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J,
Gonzales R, Dobbs LG. 2003. CXC chemokines and their receptors
are expressed in type II cells and upregulated following lung injury.
Am J Respir Cell Mol Biol 29:661– 668.
Vlahakis NE, Schroeder MA, Limper AH, Hubmayr RD. 1999. Stretch
induces cytokine release by alveolar epithelial cells in vitro. Am J
Physiol 277:L167–L173.
Voorhout WF, Weaver TE, Haagsman HP, Geuze HJ, Van Golde
LMG. 1993. Biosynthetic routing of pulmonary surfactant proteins
in alveolar type II cells. Microsc Res Tech 26:366 –373.
Waever TE, Na CL, Stahlman M. 2002. Biogenesis of lamellar bodies,
lysosome-related organelles involved in storage and secretion of
pulmonary surfactant. Sem Cell Dev Biol 13:263–270.
Weibel ER. 1979. Stereological methods: practical methods for biological morphometry. New York: Academic Press.
Weller PF, Dvorak AM. 1994. Lipid bodies: intracellular sites for
eicosanoid formation. J Allergy Clin Immunol 94:1151–1156.
Weller PF, Bozza PT, Yu W, Dvorak AM. 1999. Cytoplasmic lipid
bodies in eosinophils: central roles in eicosanoid generation. Int
Arch Allergy Immunol 118:450 – 452.
Williams MC. 1982. Ultrastructure of tubular myelin and lamellar
bodies in fast-frozen adult rat lung. Exp Lung Res 4:37– 46.
Wright JR. 1997. Immunomodulatory functions of surfactant. Physiol
Rev 77:931–962.
Без категории
Размер файла
994 Кб
ischemia, pneumocytes, hypothermic, lung, bodies, typed, occurence, canine, lipid
Пожаловаться на содержимое документа