Microanatomical Changes in Alveolar Type II Cells in Juvenile Mice Intratracheally Exposed to Stachybotrys chartarum Spores and Toxin

T. G. Rand*,1, M. Mahoney*, K. White* and M. Oulton{dagger}

* Department of Biology, Saint Mary's University, 923 Robie Street, Halifax, Nova Scotia, Canada B3H 3C3; and {dagger} Department of Physiology and Department of Obstetrics and Gynaecology, Dalhousie University, Halifax, Nova Scotia, Canada B3J 3G9

Received August 3, 2000; accepted November 6, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

Key Words: ultrastructure; alveolar type II cells; Stachybotrys chartarum; trichothecenes; intratracheal instillation; fungal conidia; morphometrics.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stachybotrys chartarum is an important environmental fungus. Inhalation exposure of building occupants to mycelial fragments, spores, and building dust, all containing toxins produced by this species, has been linked to a number of health problems. These problems include the onset of a variety of respiratory and nonrespiratory symptoms (Croft et al., 1986Go; Dearborn et al., 1999Go; Hodgson et al., 1998Go; Johanning 1995Go; Johanning et al., 1996Go). Despite the large number of respiratory symptoms associated with exposure to this species in humans, there are only a few in vivo animal studies evaluating the impact of spores and toxins of this species on lung tissues. Moreover, little is known about the specific mechanisms that may lead to these last mentioned symptoms. Several workers have shown that exposure of animals to spores of this species elicit biochemical changes in the bronchoalveolar lavage fluid (BAL) that accord with acute inflammation responses (see Mason et al., 1998Go, 2001Go; McCrae et al., 2001Go; Rao et al., 2000aGo,bGo) but there are few studies employing histology to determine whether these respiratory symptoms accord with anatomical changes in lung tissue. Nikulin et al. (1996, 1997) have described some of the histological changes associated with acute S. chartarum spore exposure in mouse lungs. They showed that intranasal exposure of mice to aerosolized spores of 2 different S. chartarum strains resulted in alveolar and interstitial inflammation with hemorrhagic exudate in the alveoli. Most importantly, they also showed that exposure to spores containing trichothecene toxins produced the most severe inflammation in the lungs, and that severity was dependent on spore toxicity and concentration. Histological work in our laboratory has also revealed that instillation exposure of young mouse lungs to spores of this species initiates an inflammatory response marked by the formation of granulomatous lesions containing a pleomorphic inflammatory cell infiltrate comprising fibroblasts, macrophages, and the occasional polymorphonucleocyte that enclosed sites of spore impaction on lung tissue (Rand et al., manuscript in prepartion).

We have also been studying a mouse model of lung injury induced by exposure to S. chartarum spores and have shown that pulmonary alveolar type II cells are sensitive to exposure to Stachybotrys spores and isosatratoxin-F (see Mason et al., 1998Go; McCrae et al., 2001Go; Sumarah et al., 1999Go). We have shown that alveolar type II sensitivity is manifest at the biochemical level as changes in alveolar surfactant phospholipid composition and concentration (McCrae et al., 2001Go; Sumarah et al., 1999Go), and in surfactant production and homeostasis (Hastings et al., 2001Go; Mason et al., 1998Go, 2001Go). One of the interesting results of some of this work is that exposure of lungs to S. chartarum spores leads to increased release of newly secreted surfactant and accumulation of metabolically used surfactant fractions (Mason et al., 1998Go). Reasons why increased surfactant secretion by alveolar type II cells is a response to exposure to S. chartarum are unclear. However, it may be that increased alveolar surfactant production in mice intratracheally exposed to S. chartarum spores is due to changes in organelle structure and function in alveolar type II cells. It is known that exposure of rats to bacterial lipopolysaccharide (LPS) results in ultrastructural changes in alveolar type II cells in rats, especially in cellular, lamellar body, and mitochondrial profiles (see Fehrenbach et al., 1998Go; Lopez et al., 1987Go; Young and Nicholls, 1996Go). The objectives of this study were to determine whether a single intratracheal exposure of juvenile mice to S. chartarum spores and toxin results in ultrastructural and morphometric changes in alveolar type II cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conidia collection.
S. chartarum conidia as the test fungus, isosatratoxin-F as a positive control toxin, and Cladosporium cladosporioides as the negative-control fungus were employed in this study. The S. chartarum isolate used in this study was recovered from a mold-contaminated Cleveland home and produces trichothecene toxins (Cleveland strain # 58–17). The C. cladosporioides isolate was recovered from an outdoor sampling site in Nova Scotia by T.G.R. Dr. Bruce Jarvis, Department of Chemistry, University of Maryland, College Park, Maryland, provided the purified isosatratoxin-F, isolated from a S. chartarum strain for use in this project.

For in vivo analysis, random-bred, pathogen-free Carworth Farms White (CFW) male mice were obtained. The animals were housed in accordance with the standards set forth by the Canadian Council for Animal Care (CCAC, 1993Go), and with the approval of the Saint Mary's University Animal Care Committee.

Intratracheal instillations.
Control and treatment mice were separated into groups of 5–6 mice. Preweighed 21–28-day-old juvenile mice were anesthetized via an intramuscular injection containing ketamine (Ketaleen) and xylazine (Rompun) as previously described by Mason et al. (1998).

With the exception of untreated controls, each mouse was inoculated intratracheally with 50 µl of 1 of the following treatments: 0.9% NaCl, 0.02 µg isosatratoxin-F/ml (= 0.7 ng toxin or 35 ng/kg BW/animal), S. chartarum or C. cladosporioides spores (1.4 x 106 conidia/ml = 70,000 spores/animal), as described by Mason et al. (1998). Based on previous work by Sorenson et al. (1987) who determined that 1 mg S. chartarum spore dust can contain from 7.2 to 12.7 ng satratoxin/mg dust we estimated toxin concentration in the S. chartarum spore loads administered to mice to be about 0.5 ng toxin or 25 ng/kg BW satratoxin equivalents/animal.

The mice were placed back into their cages immediately after instillation. During recovery, mice were continuously monitored for signs of sickness or distress as outlined in the CCAC guidelines (CCAC, 1993Go). If the animals showed distress, they were immediately euthanized using a sodium pentobarbital (65 mg/ml) overdose and excluded from the study.

Fixation.
Mice were killed with a 300 µl ip injection of sodium pentobarbital (65 mg/ml), 24 and 48 h postinoculation. These times were chosen because results of our previous studies (see Mason et al., 1998Go; Sumarah et al., 1999Go) indicated that the most significant changes in surfactant composition and homeostasis were manifest between 24 and 48 h postinoculation. Once euthanized, each mouse was immediately weighed then placed ventral side up. The body cavity was opened and the abdominal artery severed for exsanguation. The chest cavity was then exposed and the lungs degassed by puncturing the pleural sac. The trachea was cannulated using an 18-gauge butterfly needle. The needle was connected by plastic tubing to a 30 ml syringe filled with a fixative mixture of 1.5% glutaraldehyde and 1.5% freshly prepared paraformaldehyde in 0.1 M cacodylate buffer (pH 7.35; Bozzola and Russell, 1992Go), for fixation by positive pressure instillation at 18 cm H2O as described by Davies (1980), for 30 min. After this time, the inflated lungs with instilled fixative were then carefully excised from the thoracic cavity in toto, disconnected from the catheter, and placed into a 250-ml beaker filled with the fixative solution for 12 h before processing for transmission electron microscopy (TEM). After primary fixation, the right cranial lung lobe from each mouse was excised and cut into roughly 1 mm3 sections. Pieces from each sectioned mouse lung were randomly selected, washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide (OsO4) in buffer for 1 h, washed in buffer, dehydrated through an ascending acetone series, and flat-embedded in Epon-Araldite.

Thin sections (gray to silver interference colors) were cut from at least 3 blocks of embedded lung per animal using a Reichert UM 2 Ultramicrotome. Sections were mounted onto #300 mesh copper grids, stained using 5% uranyl acetate and Reynold's lead citrate (see Bozzola and Russell, 1992Go), and examined using a JOEL 100 STEM microscope at 6600 magnifications and operated at 80 kV.

For morphometrics, 10 to 12 alveolar type II cells per animal, for a total of between 50 and 60 cells per group, were identified and photographed using 70 mm Kodak plate film at 6600 magnifications. Care was taken to ensure that each cell photographed included a lumenal surface and nuclear profile. Electron micrographs of alveolar type II cells for analysis were either printed on Ilford Polycontrast 8 x 11-in. paper or scanned using a flat-bed scanner at a final magnification x16,500 and used to determine the volume density of alveolar type II cells, lamellar bodies, mitochondria, and nuclei.

Point counting with an unbiased double square lattice grid was used to determine relative volume density measurements. For this, a transparent, unbiased lattice grid with 112 lines, each 1-cm long, was superimposed over alveolar type II (PII) cell images. Electron micrograph images were identified by number only to ensure that evaluations were unbiased. Volume density (points per structure) was considered for alveolar type II cells (VPII), lamellar bodies (VLB), mitochondria (Vmito), and nuclei (Vnuc) using the appropriate formulae (see Bozzola and Russell, 1992Go; Davies, 1980Go; Weibel and Cruz-Orive, 1997Go). Points for cells or organelles were only considered when intersecting grid lines fell entirely within the structure observed. Fraction of the cell cytoplasm occupied by lamellar body or mitochondria was calculated using the following formula: Vv (fraction of cytoplasm occupied by organelle) = (points counted on organelle (Porganelle) ÷ (total points on alveolar type II cell profile (PT) – points counted on nucleus (Pnuc)). Data were analyzed using normality of variance and 2-way ANOVA, and at 95% (p <= 0.05) confidence level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice exposed to either 50 µl 106 conidia/ml of S. chartarum conidia, C. cladosporioides conidia, or to 50 µl isosatratoxin-F, did not show any apparent clinical signs of respiratory distress or sickness.

Ultrastructure
Alveolar type II cells from untreated controls, saline and C. cladosporioides treated animals were similar (Figs. 1A and 1BGo). Generally, the cytoplasm of cells from these animals was moderately electron dense. The cells supported a centrally to basally located nucleus with peripherally dispersed chromatin and usually a prominent, centrally located nucleolus. The cells contained moderate numbers of elongate mitochondria, and well-defined, separate, membrane bound lamellar bodies. The surface of cells exposed directly to the alveolar space supported numerous, small microvilli. Mild mitochondrial swelling was seen in a few alveolar type II cells of the C. cladosporioides treated animals.



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FIG. 1. Electron micrographs of alveolar walls. These are (A) a wall from a saline treated animal and (B) a wall from a Cladosporium cladosporioides treated animal. Each micrograph shows an alveolar type II cell with prominent nucleus (N), lamellar bodies (L), mitochondria (M), and microvilli (arrows). AS, alveolar space; original magnification x9000.

 
Alveolar type II cells from animals treated with either S. chartarum spores or isosatratoxin-F showed remarkable ultrastructural changes compared to the controls. Alveolar type II cells from these animals often featured mitochondria that were condensed (Figs. 2A and 2BGo), electron dense, and irregularly aligned cristae (Fig. 2AGo). Nuclei of some alveolar type II cells from treatment animals had scattered chromatin (Fig. 2CGo) and a poorly defined nucleolus. These cells were swollen and their cytoplasm often, but not always, showed rarefaction and clustered electron dense granules (Figs. 2C and 2DGo). The plasmalemma of some of the treatment animals showed marked distension and some lacked microvilli (Fig. 2DGo).Lamellar bodies in alveolar type II cells from these animal treatment groups were also often swollen and contained lamellae with irregular profiles (Figs. 2B and 2CGo). Other ultrastructural changes were the vesicles and membranal figures that could also be found in the alveolar space of treatment animals.



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FIG. 2. Electron micrographs of alveolar type II cells. (A) Cell with condensed mitochondria from a Stachybotrys chartarum treated animal. AS, alveolar space; N, = alveolar type II cell nucleus; microvilli (single arrows); lamellar bodies (L); original magnification x8000. (B) Cell from a Stachybotrys chartarum treated animal. Note condensed mitochondria (double arrows); marked distension of lamellar bodies with irregularly arranged lamellae (L); microvilli (single arrows). AS, alveolar space; original magnification x10,500. (C) Cell from an isosatratoxin-F treated animal. Note condensed mitochondria (M); dispersed nuclear chromatin (N2); electron dense aggregates (single arrows); and cytoplasmic rarefaction; original magnification x10,500. (D) Cell from a Stachybotrys chartarum treated animal. Note condensed mitochondria (double arrows) and membranal figures (V) in alveolar space (AS); cytoplasmic rarefaction; electron dense aggregates (single arrows). AS, alveolar space. Note the lack of microvilli along the margin of the alveolar type II cell; original magnification x16,500.

 
Stereology
Point count stereological analyses revealed that Pt volume-density estimates for alveolar type II cells from all the animal groups were not significantly different (p > 0.05; Table 1Go). Lamellar body abundance was increased in animals exposed to S. chartarum for 48 h but this was not significant (p > 0.05). However, PiLB and Vvlam estimates for this group and in animals exposed to isosatratoxin-F for 48 h were significantly elevated (p < 0.05), compared to the other treatment groups (Table 1Go). Mitochondria abundance was not significantly different among treatment groups, but Pi and Vvmito estimates for mitochondria were significantly lower (p < 0.05) in the isosatratoxin-F (PIH 48 h) and S. chartarum treated animals (PIH 24 and 48 h) compared to the other treatment groups (Table 1Go). Nucleus Pi and Vvnuc estimates were the same for all treatment groups. Volume-density outcome was not influenced by exposure time in any of the treatments (p > 0.05).


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TABLE 1 Volume Density of Lamellar Bodies and Mitochondria in Alveolar Type II Cells from Untreated and Treated Mouse Groups
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar type II cells comprise some 15–60% of the cells of the pulmonary epithelium (see Burkitt et al., 1993Go; Mason and Shannon, 1997Go). These cells synthesize and store major components of pulmonary surfactant, a phospholipid-rich substance (Haagsman and van Golde, 1991Go; Hawgood and Shiffer, 1991Go; Oulton et al., 1993Go; Possmayer, 1984Go) that lines the alveolar surface. Surfactant promotes lung stability by reducing the surface tension of the air-alveolar interface (Guyton et al., 1984Go; Notter, 1984Go), and it performs important functions in alveolar defense (Curti and Genghini, 1989Go; Jarstrand, 1984Go; Schurch et al., 1990Go). Ultrastructural features of alveolar type II cells have been described for many animals and, in healthy tissues, have been found to exhibit remarkable similarity amongst species (Adamson, 1990Go; Mason and Shannon, 1997Go). Results of the present study have revealed that alveolar type II cells from untreated and saline treated animals generally exhibited ultrastructural features similar to those reported for cells from normal, healthy alveolar tissue (see Burkitt et al., 1993Go).

This study has also revealed that instillation exposure to mold spores and toxin will elicit ultrastructural changes in alveolar type II cells, although the nature and degree of the cellular response toward spores from different species appears to vary considerably. Exposure to Cladosporium cladosporioides spores resulted in some ultrastructural changes, which was evidenced as modest mitochondrial swelling in alveolar type II cells. Mitochondrial swelling is a commonly observed, early manifestation of cell injury and has been reported in cases of reversible lung damage resulting from acute oxygen toxicosis (Adamson, 1990Go), and exposure to bacterial endotoxin (Fehrenbach et al., 1998Go; Lopez et al., 1987Go; Young and Nicholls, 1996Go). However, this response in the C. cladosporioides treated animals was mild compared to the response of alveolar type II cells from animals exposed to S. chartarum and isosatratoxin-F.

Alveolar type II cells from the S. chartarum and isosatratoxin-F treated animals clearly showed evidence of cytological damage, manifest at both the ultrastructural and morphometric levels. Differences in the degree of cellular response associated with exposure to C. cladosporioides spores and to S. chartarum spores and isosatratoxin-F supports the position that the nature and degree of the alveolar type II cell response toward pollutants can vary considerably. More importantly, it also supports the position that exposure to S. chartarum spores and toxin results in cellular responses in vivo different from those associated with exposure to spores of nontoxigenic mold species.

One of the most striking results of the study was the significant increase in lamellar body volume density estimates following exposure to S. chartarum spores and isosatratoxin-F. Because these changes were not observed in the C. cladosporioides treated animals, this result suggests that exposure to fungal spore-wall bound (1->3)-ß-D-glucans is not contributing to this response, but that metabolites, including toxins, produced by S. chartarum may be responsible. It is well understood that trichothecenes interact with cellular membranes and cause their disfunction (Riley and Norred, 1996Go). Possibly, exposure to S. chartarum toxins sequestered in and liberated from spores results in lamellar body membrane deregulation resulting in lamellar body swelling and lamellae rearrangement. However, because similar changes in lamellar body volume density to those observed in this study have been reported for alveolar type II cells from rats exposed in vitro and ex vivo to Salmonella minnesota lipopolysaccharide (LPS; Fehrenbach et al., 1998Go; Young and Nicholls, 1996Go), this effect does not appear to be a specific sign of S. chartarum spore or toxin induced lung injury. Nevertheless, that exposure to S. chartarum spores and toxin induces lamellar body fusion helps to explain some of the results reported by Mason et al. (1998). They observed that exposure of mouse lungs to S. chartarum spores resulted in an increase in the production of newly secreted surfactant compared to the untreated, saline and C. cladosporioides controls. Mason et al. (1998) suggested that the increase in newly secreted pulmonary surfactant might reflect a defense mechanism to rid alveolar surfaces of S. chartarum spores. This study provides an alternative explanation that this accumulation could reflect a cellular trauma resulting in lamellar body membrane fusion and possibly, in changes in the amount of pulmonary surfactant exported via lamellar body exocytosis into the alveolar space. Obviously more work, employing pulse-chase radioactive tracer studies, is required to address this issue.

S. chartarum spore and isosatratoxin-F exposure also had a significant impact on alveolar cell mitochondria. It is well known that trichothecene exposure can result in mitochondrial function alterations in vitro (Riley and Norred, 1996Go). It is also recognized that these inhalation exposure to these toxins can cause cell damage and kill animals if exposure concentration is sufficiently high (Creasia et al., 1987Go, 1990Go). However, the effect of fungal spores and mycotoxins on mitochondrial structure is poorly documented. Pertola et al. (1999) reported mitochondrial swelling as a cytological lesion in boar spermatozoa exposed to S. chartarum spores and T-2 toxin. However, these investigators did not evaluate sperm mitochondria objectively using morphometrics. We also noted some swollen mitochondria in alveolar type II cells from S. chartarum and isosatratoxin-F treated animals examined during this study. However, mitochondrial condensation was the overall consequence of exposure. This result supports work of Okumura et al. (1999), who also found that exposure of a mouse cell line to T-2 toxin caused mitochondrial condensation. Okumura et al. (1999) suggested that this change in mitochondrial profile might be due to increased mitochondrial trans-membrane potential due to a rise in the intracellular concentration of calcium, which would result in organelle condensation, although unequivocal evidence for this is lacking. As far as we are aware, work has not been done to evaluate the impact of S. chartarum spores on mitochondrial function.

Okumura et al. (1999) also suggested that cellular changes they observed in mouse cells exposed to T-2 toxin may be linked to preprogrammed cell death, apoptosis. This may be a relatively common cellular response due to exposure to these toxins as Yang et al. (2000) have also found that other trichothecenes, derived from S. chartarum isolates, induce apoptosis in cells exposed to these toxins, in vitro. However, results of our study do not support the position that alveolar type II cells exposed to S. chartarum and isosatratoxin-F are undergoing apoptosis. Instead, we believe that the alveolar type II cells were exhibiting pre-lethal cytological changes more consistent with oncosis onset (see Trump and Berezesky, 1998Go). Oncosis is a form of accidental cell death (Majno and Joris, 1995Go), which differs from the first mentioned remarkably in structure and function (Trump and Berezesky, 1998Go). Ultrastructural features of apoptosis include rapid condensation of the cytoplasm and nuclear chromatin (Zakeri, 1998Go), which we did not observe in this study. However, important diagnostic signs of end-stage oncosis are swollen cells with cytoplasmic paling (rarefaction), mitochondrial condensation, nucleolus fragmentation, and the redistribution of euchromatin to the periphery of the nuclear membrane (see Trump and Berezesky, 1998Go). These features are similar to those we observed in some of the alveolar type II cells from S. chartarum and isosatratoxin-F treated animals. Oncosis often follows a variety of injuries brought about by exposure to toxins, among others, that interrupt ATP synthesis and alter membrane integrity (Trump and Berezesky, 1998Go). Clearly, subsequent studies should be conducted to evaluate the impact of S. chartarum spores and toxin on cell membrane structure and functions.


    ACKNOWLEDGMENTS
 
We thank C. Leggiadro and D. O'Neil, NRC Institute of Marine Biosciences for their assistance and excellent technical support. We would also like to thank Drs. B. Jarvis for the gift of isosatratoxin-F and M. Wiles for use of his stereological templates. This study was supported by a Natural Sciences and Research Council operating grant to T.G.R.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (902) 475-1982. E-mail: thomas.rand{at}stmarys.ca. Back


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 DISCUSSION
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