Occurence of lipid bodies in canine type II pneumocytes during hypothermic lung ischemia.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 277A:287–297 (2004) Occurence of Lipid Bodies in Canine Type II Pneumocytes During Hypothermic Lung Ischemia MATTHIAS OCHS,1* HEINZ FEHRENBACH,2 AND JOACHIM RICHTER1 Department of Anatomy, Division of Electron Microscopy, Georg August University, Göttingen, Germany 2 Clinical Research Group Chronic Airway Diseases, Department of Internal Medicine (Respiratory Medicine), Philipps University, Marburg, Germany 1 ABSTRACT 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, 2001). 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 © 2004 WILEY-LISS, INC. 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: email@example.com Received 9 May 2003; Accepted 14 December 2003 DOI 10.1002/ar.a.20013 288 OCHS ET AL. 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. MATERIALS AND METHODS 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). Fixation 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. Sampling 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. Processing 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. CTEM For CTEM analysis, ultrathin sections of 70 nm thickness were examined using an EM 10 transmission elec- 289 LIPID BODIES IN TYPE II PNEUMOCYTES tron microscope (Zeiss, Oberkochen, Germany). Sections were counterstained with uranyl acetate and lead citrate. EFTEM 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 study. Statistics 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). RESULTS CTEM 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 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). Stereology 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- 290 OCHS ET AL. 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. DISCUSSION The general morphological appearance of lipid bodies has been well described (Fawcett, 1981; Ghadially, 1997). They are usually present within the cytoplasm as spheri- LIPID BODIES IN TYPE II PNEUMOCYTES 291 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- 292 OCHS ET AL. 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. LIPID BODIES IN TYPE II PNEUMOCYTES 293 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 294 OCHS ET AL. 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. 295 LIPID BODIES IN TYPE II PNEUMOCYTES TABLE 1. Stereological data of lipid bodies in canine type II pneumocytes during cold hypothermic ischemia† Experimental Group Control 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 [nm] Vv (li, cell) [%] V/S (li) [nm] Vv (lilb, cell) [%] V/S (lilb) [nm] 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 (SEM). a 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. ACKNOWLEDGMENTS We thank S. Freese, A. Gerken, and H. Hühn for excellent technical assistance and C. Maelicke for checking the article. LITERATURE CITED 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 296 OCHS ET AL. 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 – 511. 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 58:152–171. 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 – L1156. 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: 381–396. 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 79:85–91. 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. LIPID BODIES IN TYPE II PNEUMOCYTES 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 – H301. 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. 297 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.