Department of Molecular and Cellular Physiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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Cyclosporin A (CsA), an inhibitor of protein
phosphatase 2B (calcineurin), has been shown to play a role in
exocytosis and neutrophil mobility. Hyperoxia (>95% oxygen for 72 h)
causes lung injury and reduces lung compliance. This model is
indicative of deficiencies in surfactant and elicits a vigorous immune
response leading to further damage. We examined the effects of CsA on
surfactant-secreting lung alveolar type II cells. CsA enhances
ATP-stimulated increases in whole cell capacitance in the presence of 2 mM extracellular Ca2+. This
measurement corresponds with increases in exocytosis. Because of its
effect on the immune system and exocytosis from type II cells, CsA was
examined for its protective effects against hyperoxia-induced lung
damage in mice. We found that CsA (50 mg · kg1 · day
1)
attenuated hyperoxia-induced reductions in lung compliance when administered before or during 72 h of >95% oxygen
(P < 0.05). CsA (10 mg · kg
1 · day
1)
also had a protective effect against hyperoxia-induced changes in
neutrophil infiltration, capillary congestion, edema, and hyaline membrane formation. Wet lung weight-to-dry lung weight ratios did not
show any significant changes after hyperoxia or hyperoxia plus CsA
(P < 0.05). CsA may be useful to
treat patients undergoing prolonged high-oxygen therapy and possibly
other lung injuries.
lung compliance; capillary congestion; type I cells; type II cells; hyperoxia; whole cell recording
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INTRODUCTION |
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THE ALVEOLAR EPITHELIUM consists of flattened (type I) cells responsible for O2/CO2 exchange and cuboidal (type II) cells that secrete surfactant. Surfactant secretion, which involves exocytosis of surfactant-containing lamellar bodies, reduces the surface tension of the epithelial lining, keeping alveoli open and increasing lung compliance. Exocytosis of surfactant-containing lamellar bodies from alveolar type II cells occurs in response to a variety of secretagogues (28, 29). Stimulation of type II cells with ATP increases lamellar body exocytosis by at least three signal transduction mechanisms (29). Two mechanisms, one involving cAMP-dependent protein kinase A and the other protein kinase C, may both facilitate lamellar body exocytosis by phosphorylation-mediated polymerization of the actin network at the plasma membrane of the cell, facilitating vesicle access to the plasma membrane (39). A third mechanism is via ATP-induced formation of inositol trisphosphate, which mobilizes the intracellular store of Ca2+, an event closely associated with type II cell exocytosis (27).
Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has been shown to both stimulate and reduce secretion depending on the cell type. Inhibition of calcineurin by cyclosporin A (CsA) stimulated glucose-induced insulin secretion in the insulin-secreting cell line MIN6 (11); activation of calcineurin by a neurotransmitter inhibited exocytosis in insulin-secreting cells (25). The systemic therapeutic and toxic effects of the immunosuppressive drugs CsA and FK506 have thus far been shown to be a result of calcineurin inhibition (18). CsA and FK506 disrupt Ca2+-dependent signal transduction pathways that regulate gene activities (33) by first binding to their cognate intracellular receptors termed immunophilins: cyclophilin binds CsA and FK506 binding protein binds FK506 (32). These drug-immunophilin complexes target calcineurin, thereby inhibiting dephosphorylation and subsequent translocation of the cytoplasmic component of the nuclear factor of activated T cells into the nucleus (18).
In vivo, exocytosis of the proteolipid pulmonary surfactant is important for proper function of the alveoli. Neonatal respiratory distress syndrome, for example, is a result of a deficiency in surfactant secretion (17). Alveolar type II cells do not begin secreting surfactant until the last 1-3 mo of gestation. Therefore, many premature infants and some full-term infants are born without the capability of secreting surfactant and show a dramatic reduction in lung compliance. Without surfactant to overcome the surface tension, alveoli collapse, producing respiratory distress.
Almost all diffuse lung injury, including hyperoxic injury, is associated with a reduction in lung compliance, which may lead to respiratory distress. Although high oxygen is vital to sustaining life in many critical care situations, prolonged oxygen therapy promotes damage. Oxidative damage occurs in two stages, the acute stage and the proliferative stage. During the acute stage, perivascular, interstitial, and intra-alveolar edema and hemorrhage occur with variably extensive necrosis of the pulmonary endothelial and type I epithelial cells (14). Progressive absorption of exudates and thickening of the alveolar air-blood interface due to hyperplasia and hypertrophy of the type II epithelial cells characterize the proliferative stage. There is collagen deposition and fibrosis in the interstitium of the alveolar walls that contribute to the thickening of the gas-exchange areas of the lung (14). Oxygen toxicity also leads to increased infiltration of immune cells, especially polymorphonuclear leukocytes (34), that increase the damage by releasing oxygen radicals. Because oxygen is a vasodilator in the lung (4, 11), pulmonary capillaries have been found to be engorged with blood (capillary congestion). Oxidative damage can also result in hyaline membrane formation, pleural effusions, and atelectasis. Current treatments for hyperoxic damage are inadequate. Thus modifying the current high-oxygen treatment has the potential to significantly reduce lung damage and improve recovery in critical care patients.
We treated cells with CsA, an inhibitor of calcineurin, to determine its effects on exocytosis. We discovered that CsA enhanced ATP-stimulated exocytosis and therefore may serve as a protective agent against hyperoxic lung damage. To test this hypothesis, we examined the effects of CsA on murine lung function and pathology after hyperoxic injury. Hyperoxia-exposed mice treated with CsA showed a reduction in edema, hyaline membranes, capillary congestion, neutrophil infiltration, and static lung compliance, indicating that CsA protects the lung from hyperoxia-induced damage.
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MATERIALS AND METHODS |
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Animal treatment. Female FVB/n mice
weighing 20-25 g from Harlan (for preliminary studies;
Indianapolis, IN) or Taconic Farms (Germantown, NY) were divided into
four groups: 1) control,
2) CsA treated,
3) hyperoxia exposed, and
4) CsA treated and hyperoxia exposed. The control and hyperoxia groups were injected
intraperitoneally with vehicle consisting of polyethylated castor oil
(Cremaphor EL, Sigma) dissolved in alcohol and diluted in saline. The
mice used in the preliminary studies were preexercised as a means of testing cardiopulmonary fitness. The CsA pretreatment and simultaneous groups were injected once a day for 3-4 days with 10 or 50 mg · kg1 · day
1
of CsA (Sandoz, East Hanover, NJ, or, for preliminary studies, Calbiochem, La Jolla, CA) in Cremaphor EL, alcohol, and saline. After
vehicle (31) or CsA injections were complete, the hyperoxia groups were
placed in Plexiglas chambers flushed with 100% oxygen and maintained
at >95% oxygen for 72 h; oxygen concentrations were measured with a
mini-Ox analyzer (Mine Safety Appliances, Pittsburgh, PA).
The control and CsA-treated groups were maintained in room air. These
studies were repeated in the mice set aside for simultaneous CsA plus
hyperoxia studies, with the time of injections changed to the 3 days of
hyperoxic exposure. The animals were provided water and food ad libitum
and kept in alternating 12-h light-dark cycles.
Lung static compliance measurements.
The mice were injected with a lethal dose of pentobarbital sodium
(Abbott Laboratories, North Chicago, IL) and placed in a container
containing 100% oxygen to ensure complete collapse of alveoli by
oxygen absorption. The trachea was cannulated and connected to a
syringe and pressure transducer (X-ducer, Motorola, Phoenix, AZ) via a
three-way connector. After the diaphragm was opened, the lungs were
inflated in 100-µl increments every 5 s to a maximum inflation
pressure of 30 cmH2O and deflated.
Pressure and volume on inflation and deflation were recorded.
Pressure-volume curves were generated for each animal. Lung compliance
was determined by calculating the slope of the linear portion of the
deflation curve between +10 and 10
cmH2O.
Lung pathology. The lungs were infused via the airway with 1-1.4 ml of 10% Formalin at 25-cm fluid height (the distance between the lungs and the meniscus of the Formalin in reservoir), sectioned (4 µm), and stained with hematoxylin and eosin to examine the pathology. Other lungs were examined for edema in the following manner. Immediately after compliance measurements were made, the lungs were removed and separated from the heart, pulmonary vasculature, trachea, and main stem bronchi. The lungs were then blotted dry, weighed, placed in a 60°C oven for >72 h, and reweighed for dry lung determination.
Immunohistochemical detection of surfactant protein B. Adult mouse lung sections (4 µm) were deparaffinized and incubated overnight at 4°C with rabbit polyclonal antibody produced against the mature surfactant protein (SP) B protein. The PBS (2% Triton X-100)-washed sections were incubated with biotinylated goat anti-rabbit antibody. The Vectastain ABC Peroxidase Elite Rabbit IgG Kit (Vector Laboratories, Burlingame, CA) was used to detect antigen-antibody complexes. The enzymatic reaction was enhanced with nickel cobalt to produce a black precipitate. The sections were counterstained with nuclear fast red. The whole lung sections were examined for the presence of SP-B in the bronchiolar (Clara cells) and alveolar (type II cells) epithelia as well as in alveolar macrophages. After low-power examination, randomly selected high-power fields of alveoli were selected to determine the number of alveolar type II cells distinctly SP-B reactive over the total number of alveoli. Type II cells with black staining greater than ~1.5-µm diameter were counted as positive.
Isolation and primary culture of rat type II cells. Type II cells were isolated from the lungs of pathogen-free male (200-220 g) Sprague-Dawley rats (Harlan) as previously described (27). Briefly, the rats were anesthetized with pentobarbital sodium (Abbott Laboratories) injected intraperitoneally, and the lungs were perfused via the pulmonary artery until visually free of blood. Twenty units of elastase (Worthington Biochemicals, Freehold, NJ) were instilled into the lungs via a tracheal catheter, and the type II cells were digested from the basement membrane for 10-15 min. The cells were purified by the rat IgG (Sigma, St. Louis, MO) panning method and plated on 1 × 25-mm circular glass coverslips that were previously coated with rat tail collagen (Upstate Biotechnology, Lake Placid, NY). The cells were cultured on the coverslips overnight in Dulbeccos's modified Eagle's medium (DMEM) with 10% fetal bovine serum at 37°C in 8% CO2.
Whole cell recording. During
exocytosis, vesicles fuse with the plasma membrane and increase the
total surface area of the cell. Because the membrane has capacitance,
changes in its surface area can be detected as changes in whole cell
capacitance (with whole cell voltage-clamp techniques). The method used
for measuring membrane capacitance was developed in one of our
laboratories (R. Pun) and is based on a computational approach. This
approach allows for the determination of changes in membrane
capacitance that are associated with alterations in membrane
resistance. This method (23) allows for the study of agonist-induced
capacitance changes (secretion) without the complications associated
with the accurate determination of the phase angle that is necessary for the correct measurement of capacitance. The method is based on
measuring the current responses of a sinusoidal wave with a digital
phase detector and calculation of the values of the parameters of the
equivalent circuit, i.e., a resistor (the series or access resistance) in series with a capacitor (membrane capacitance) that is
in parallel with a resistor (membrane resistance). Briefly, after
formation of a high-resistance seal between the patch pipette and the
membrane, the pipette potential was clamped at 70 mV, and a
series of sinusoidal waves was sent, via injection, into the type II
cell. The amplitude of the sinusoidal wave was either 30 or 50 mV, with
an angular frequency of 5,234 rad/s, corresponding to a frequency of
833 Hz. Membrane conductance was determined from the response to a
30-mV step pulse applied before the sinusoidal waves. From the
amplitudes of the current responses, the input voltage, the gains used,
the angular frequency, and the various parameters of the equivalent
circuit (see above) were determined.
The time course of changes in the three parameters was measured at 1-s intervals on the cell bathed in HEPES-buffered saline (140 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM NaHCO3, with the osmolarity adjusted to 310-325 mosmol and pH 7.4). The intracellular recording solution was a high-K+, HEPES-buffered medium (140 mM KCl, 2 mM MgCl2, 10 mM glucose, 10 mM HEPES, 5 NaHCO3, and 3 mM EGTA, pH 7.2, 310 mosmol). Two millimolar ATP was added to prevent possible rundown of Ca2+ currents. Rat type II cells were subjected to whole cell recording 18-19 h after isolation from the lung. The cells were stimulated externally for 10 s with 100 µM ATP (Sigma) supplied to the cells via a local perfusion pipette placed 30-50 µm from the cell. CsA (Calbiochem) was included in the pipette solution to evaluate its effect on membrane capacitance changes. In the absence of ATP stimulation, CsA (1 µM) did not induce a change in membrane capacitance. The magnitude of the change in capacitance for each responding cell was obtained by averaging three values measured around the peak of the response and then subtracting the average of three values measured at baseline, i.e., before stimulation with ATP.
Type II cell hyperoxia studies. Murine type II cells were isolated (7) and allowed to adhere to collagen-coated coverslips for ~2 h. The medium was changed to L-15 medium with 10% fetal bovine serum, 1% penicillin-streptomycin, and either vehicle (methanol) or 500 nM CsA (Calbiochem) diluted in methanol. The type II cells were placed in a sealed, humidified chamber at 37°C and exposed to either 100% oxygen or room air for the remainder of the 24 h (9, 21) postplating (22 h). The cells were immediately fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in PBS-saponin and immunostained for SP-B as described in Immunohistochemical detection of surfactant protein B.
Statistical analysis. All statistical comparisons were made by one-way analysis of variance (ANOVA). P values < 0.05 were considered significant. These analyses were followed by Student-Newman-Keuls tests in which P values < 0.05 were considered significant.
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RESULTS |
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CsA pretreatment and hyperoxia.
Because CsA is a powerful immunosuppressant and stimulated an increase
in exocytosis in isolated alveolar type II cells, we evaluated whether
CsA would have a protective effect against hyperoxia-induced lung
damage. Changes in static lung compliance of hyperoxia-exposed mice
treated with CsA were compared with those in hyperoxia-exposed mice not
treated with CsA. Pretreatment of mice with CsA (50 mg · kg1 · day
1
for 3 days) showed a significant protection against hyperoxia-induced reductions in lung compliance. Hyperoxia (>95% oxygen for 72 h)-treated mice showed a significant reduction in the mean compliance
value [45.86 ± 3.63 (SE)
µl/cmH2O] compared with
that in room air control mice (67.76 ± 2.43 µl/cmH2O;
P < 0.05). Mice given CsA before hyperoxic exposure showed a significant increase in lung compliance to
near-normal levels (55.98 ± 2.57 µl/cmH2O;
P < 0.05). Room air plus CsA mice
had a mean compliance of 77.52 ± 3.48 µl/cmH2O (Fig.
1). Representative lung
compliance curves from each experimental group are presented in Fig.
2.
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We also analyzed whether CsA pretreatment inhibited hyperoxia-induced
lung damage as determined by pathology. Representative photographs
showing neutrophil infiltration, edema, capillary congestion, hyaline
membrane formation, and increased alveolar wall thickness for the four
experimental groups are presented in Fig.
3. Hyperoxia-exposed lungs pretreated with
CsA (10 mg · kg1 · day
1)
had significantly less edema and congestion than hyperoxia-exposed lungs pretreated with vehicle. Room air lungs showed no significant edema and congestion. There was also a reduction in neutrophil infiltration in CsA-pretreated hyperoxia-damaged lungs compared with
vehicle-pretreated hyperoxia-damaged lungs. There was significant and
extensive hyaline membrane formation and increased alveolar wall
thickness in the lungs of hyperoxia-exposed mice that was absent in the
other three groups (Fig. 3).
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Wet lung weight-to-dry lung weight ratios were analyzed to determine
whether CsA protected against hyperoxia-induced increases in lung water
(edema). Although the difference between the mean (±SE) wet lung
weight-to-dry lung weight ratios (Fig. 4)
for the hyperoxia and hyperoxia plus CsA (4.79 ± 0.18) groups was
not significant (P > 0.05), the
difference between the control (4.13 ± 0.04) and hyperoxic (4.54 ± 0.03) group ratios was also not significant
(P > 0.05). The CsA-treated group
ratio (4.15 ± 0.08) was significantly lower than the hyperoxia
group ratio (P < 0.05).
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The effects of CsA on the amount of SP-B in alveolar type II cells as
well as the distribution of SP-B-positive type II cells were examined.
SP-B was found in the bronchiolar epithelium, alveolar epithelium, and
alveolar macrophages. Positive-staining alveolar type II cells were
found throughout the alveoli of all four experimental groups, with the
highest density of positive-staining cells near the airways and the
highest density of negative-staining cells near the outer edges of the
lung lobes. Hyperoxia-exposed lungs had a less homogeneous distribution
of positive-staining alveolar type II cells and larger areas of
negative-staining alveolar type II cells than room air-exposed lungs.
The areas of negative-staining alveolar type II cells were smaller in
the hyperoxia plus CsA group compared with the hyperoxia group.
Furthermore, the SP-B content of the positive-staining type II cells in
the hyperoxia plus CsA groups was increased (Fig.
5).
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An evaluation of the number of cells staining positive for SP-B per alveoli yielded a significant decrease in the mean ratio of positive-staining type II cells per alveoli in the hyperoxia group (0.372 ± 0.010) compared with that in the control group (0.584 ± 0.057; P < 0.05). Although quantitative analysis showed an insignificant increase in positive-staining type II cells per alveoli in the hyperoxia plus CsA group (0.457 ± 0.045) compared with that in the hyperoxia group (0.372 ± 0.010; P > 0.05), CsA also increased the actual amount of SP-B in the positive-staining alveolar type II cells. The CsA group had a ratio of 0.4896 ± 0.076. The control and hyperoxia groups were the only groups that were significantly different. The lung sections without primary antibody exposure yielded absolutely no black staining.
CsA administration during hyperoxia.
We wished to determine whether CsA would have a protective effect when
administered at the same time as high-oxygen treatment to simulate the
clinical situation when there is no time for CsA pretreatment before
oxygen administration. Two different doses of CsA were used to
determine which concentration would be most effective. Studies using 10 mg · kg1 · day
1
of CsA showed no significant difference between the hyperoxia plus CsA
and the hyperoxia plus vehicle groups of mice. However, 50 mg · kg
1 · day
1
of CsA, administered during hyperoxic exposure, had a protective effect
against hyperoxia-induced reductions in lung compliance (Fig.
6). Mean (±SE) lung
compliance was significantly reduced in the hyperoxic (49.53 ± 4.34 µl/cmH2O) mice compared with
that in the room air control mice (83.68 ± 5.85 µl/cmH2O;
P < 0.05). Hyperoxia plus CsA mice
had significantly increased lung compliance (75.98 ± 8.63 µl/cmH2O) compared with that in
hyperoxia plus vehicle mice (P < 0.05). Representative lung compliance curves from each experimental
group are presented in Fig. 7.
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To compare the effects on lung edema of CsA (50 mg · kg1 · day
1)
administered during hyperoxia with the effects of CsA administered before hyperoxia, wet lung weight-to-dry lung weight ratios (Fig. 8) were measured. There is no significant
difference between the mean wet lung weight-to-dry lung weight ratios
for the control (4.25 ± 0.05) and hyperoxia plus vehicle (4.58 ± 0.08) groups (P > 0.05). The
difference between the ratios of the hyperoxia plus vehicle and
hyperoxia plus CsA groups (4.39 ± 0.12) was also not significant
(P > 0.05). The CsA group ratio
(4.30 ± 0.11) was significantly different from the hyperoxia group
ratio (P < 0.05).
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Effect of CsA on isolated alveolar type II cell
secretion. Type II cells stimulated with 100 µM ATP
for 10 s in a bath medium that contained 2 mM
Ca2+ responded with a single
transient exocytotic response (see example in Fig.
9A)
detected as a rapid capacitance increase (mean 2.43 ± 0.33 pF,
range 0.5-5.78 pF; n = 27 cells) immediately after the stimulation. Whole cell
capacitance decreased to near prestimulated levels within 25-50 s
of reaching peak capacitance, indicating a similar amount of membrane
retrieval after the exocytotic response. No further changes were
observed for the duration of the experiments. When type II cells were
stimulated with both 100 µM ATP and 1 µM CsA in a 2 mM
Ca2+ bath medium, multiple cycles
of capacitance changes were observed relative to those in the control
cells (Fig. 9B). The patterns of the
extended ATP and/or CsA exocytotic responses were variable, but the
general trend in exocytotic response to ATP and/or CsA stimulation was
a transient rise and fall in capacitance (mean 2.25 ± 0.39 pF,
range 0.6-5.48 pF; n = 17 cells)
followed by multiple secondary undulations in capacitance over the
following 4-6 min.
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In addition to increasing the duration of the exocytotic response, CsA also increased the percentage of cells that responded to ATP. With Ca2+ present in the extracellular medium, the percentage of cells that responded to ATP was 36% (27 of 75), which increased to 57% (17 of 30 cells) in the presence of CsA. CsA did not induce spontaneous increases in capacitance nor did it affect the basal membrane capacitance of the responding cells (with 2 mM Ca2+).
To visually identify surfactant secretion, isolated type II cells were
exposed to hyperoxia plus CsA. The type II cell SP-B content of the
cells was identified with immunocytochemistry. Murine type II cells
were exposed to CsA (500 nM) during 22 h of 100% oxygen. Under
normoxic conditions, CsA-treated cells had more SP-B at the surface
than vehicle-treated cells. Because SP-B is released from type II cells
through lamellar bodies (36), it can be concluded that these cells have
more lamellar body-associated SP-B at the plasma membrane surface than
the control cells. When comparing control with hyperoxia-exposed type
II cells, there was also more SP-B at the plasma membrane surface (Fig.
10). Type II cells in the hyperoxia plus
CsA group looked similar to both hyperoxia-exposed and room air plus
CsA cells. Therefore, it appears that hyperoxic insult and CsA
treatment separately stimulate secretion in type II cells. CsA and
hyperoxic actions did not appear to be synergistic.
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DISCUSSION |
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In an effort to identify treatment strategies to reduce lung injury and improve recovery from injury, the effects of CsA in hyperoxia-damaged lungs of rodents was evaluated. Because of the immunosuppressive and exocytosis-modulating actions of CsA in various cell types, we wished to determine whether CsA treatment could reduce lung damage due to hyperoxia. CsA protects against hyperoxia-induced reductions in lung compliance, edema, hyaline membranes, capillary congestion, and neutrophil infiltration. CsA also enhances exocytosis from isolated alveolar type II cells.
The effects of CsA in the entire lung after injury induced by high
oxygen was also evaluated. Static lung compliance is a well-established
measure of lung function and has been determined to be a very sensitive
measure of hyperoxic lung damage (1). Decreases in lung compliance have
been shown to be caused by changes in the mechanical properties of the
surface lining and its phospholipid content (3). In addition to reduced
lung compliance, hyperoxic lungs have been shown to have impaired
surfactant phospholipid synthesis (16) as well as decreased SPs and
surfactant mRNA (22). Abnormal pressure-volume curves are, in part, a
result of decreases in the absorption and spreading of SPs and damage from the inflammatory response associated with hyperoxia (22). There
may be direct damage to surfactant from oxygen radicals. Proteins in
the edema fluid damage or inactivate surfactant. Our results show that
both pretreatment and simultaneous treatment with CsA at 50 mg · kg1 · day
1
have a significant protective effect against hyperoxia-induced reductions in lung compliance. We expect CsA to modify this increase in
static lung compliance through multiple synergistic pathways including
protection of the alveolar epithelium and endothelium. Protection of
alveolar endothelial and epithelial cells will prevent edema and the
consequent inactivation of surfactant by proteins in the edema fluid.
It appears that protection of surfactant function is a significant
pathway by which normal lung compliance values are maintained by CsA in
hyperoxia-exposed lungs. Without this mechanism, enhancement of
surfactant secretion will not contribute significantly to the
maintenance of normal lung function.
Furthermore, injury to alveolar type II cells that has been observed after hyperoxic exposure (19) may directly reduce surfactant availablity. Oxidative injury has been shown to damage receptors on alveolar type II cells specific for secretagogues such as ATP (38). Interestingly, hyperoxia reduces bronchiolar lavage levels of ATP in a dose-dependent manner (26). ATP levels in bronchiolar lavage have been shown to be sufficient to elicit secretion (26). Our single-cell studies demonstrate that CsA enhances ATP-stimulated exocytosis from surfactant-secreting alveolar epithelial cells. In the injured lung, increased surfactant secretion alone may not be sufficient to improve lung function but includes reductions in type II destruction and damage to surfactant by serum proteins that will prevent surfactant from carrying out its function.
Wet lung weight-to-dry lung weight ratios were used as an index of
edema in both the pretreatment and simultaneous treatment groups with
50 mg · kg1 · day
1
of CsA. Because wet lung weight-to-dry lung weight ratios were not
increased after 72 h of high-oxygen treatment, this may not be a
sensitive early indicator of edema. Arkovitz et al. (1) found that
although mice exposed to 95% oxygen for 5 days did show significant
increases in wet lung weight-to-dry lung weight ratios, mice exposed to
95% oxygen for 72 h did not show increases in wet lung weight-to-dry
lung weight ratios. Therefore, we cannot determine whether CsA had a
protective effect against edema after 72 h of hyperoxia using wet lung
weight-to-dry lung weight ratios. However, pathology results
demonstrate that pretreatment with 10 mg · kg
1 · day
1
of CsA reduces edema as well as capillary congestion and neutrophil infiltration.
The protective effects of CsA against hyperoxia-induced edema, hyaline membrane formation, capillary congestion, and neutrophil infiltration is likely the result of several synergistic mechanisms interacting to prevent lung damage. The reduction in neutrophil infiltration in the CsA-treated lungs was consistent with a previous study (24) on the inhibitory effect of CsA on isolated neutrophil mobility. Activated neutrophils release partially reduced oxygen intermediates (hydrogen peroxide, hydroxyl radicals, and hypochlorous acid) and hydrolases (8) that cause extensive lung damage, including in pulmonary membranes. Increased permeability of the membranes leads to edema. As edema progresses from the perivascular and interstitial regions to the intra-alveolar regions, edema fluid, serum proteins, fibrin, and necrotic cells form a thick lining along the alveoli termed a "hyaline membrane" (2). The beneficial effect of CsA against edema and hyaline membrane formation is likely to be mediated, at least in part, through inhibition of neutrophil infiltration and other anti-inflammatory and/or antioxidant actions that protect the alveolar capillary membranes from injury and resultant increased permeability. Inhibition of edema and hyaline membrane formation increases the volume of aerated lung, improving lung compliance and gas exchange. Attenuation of edema and hyaline membrane formation leads to further improvements in lung function through protection of surfactant function (12).
The beneficial effect of CsA against hyperoxia-induced capillary congestion may be a result of inhibition of neutrophil infiltration and enhanced secretion of the oxygen radical scavenger pulmonary surfactant as well as a protective action against damage to the pulmonary vasculature. Destruction of the pulmonary vasculature is thought to be the major cause of death in animals exposed to hyperoxia (8, 20). High oxygen damages the alveolar endothelial cells, which subsequently swell. Platelets rapidly adhere, aggregate at the sites of injury, and attract polymorphonuclear leukocytes, which release additional oxygen radicals, perpetuating the damage. In addition to the release of oxygen metabolites by neutrophils, the endothelial cells themselves also release oxygen radicals. Oxygen is an established vasodilator in the lung (4, 13) and is therefore responsible for the dilation of capillaries resulting in congestion. Oxygen radicals released from neutrophils and the damaged endothelium contribute to the dilation of nearby vessels. CsA may be exerting its protective actions against capillary congestion by preventing endothelial injury. Surfactant has been demonstrated to be a scavenger of oxygen radicals (15). CsA-stimulated enhancement of exocytosis from surfactant-secreting cells and inhibition of neutrophil infiltration likely play a role in inhibiting capillary congestion by reducing the amount of oxygen radicals available to dilate capillaries.
Previous studies have indicated that hyperoxic lungs have impaired surfactant phospholipid synthesis (16) as well as decreased SPs and surfactant mRNA (22). In the whole lungs of treated mice, we observed that CsA attenuates hyperoxia-induced reductions in alveolar type II cell SP-B reactivity. These results indicate that the protective effects of CsA against hyperoxic damage are mediated, in part, at the type II cell level to influence surfactant dynamics.
To begin an exploration of the specific actions of CsA and hyperoxia on isolated alveolar type II cells, secretory events in type II cells were examined. Without studying isolated type II cells, the direct effects of CsA on type II cells and surfactant dynamics cannot be distinguished from the indirect effects through other cells types. To study rapidly occurring exocytotic responses in type II cells, the highly sensitive whole cell capacitance measurements were used as an indicator of vesicle fusion with the plasma membrane after brief purinoreceptor agonist (ATP) stimulation was used. This novel approach to the study of secretion in type II cells merited the following important observations. First, in the presence of extracellular Ca2+, isolated type II cells responded to ATP stimulation with a single transient burst of exocytotic activity lasting <1 min. The exocytosis transient was immediately followed by a similar period of endocytosis. Second, CsA, a potent and specific inhibitor of calcineurin (protein phosphatase 2B), elicited multiple cycles of rapid exo- and endocytosis after ATP stimulation. This enhancement of exocytosis may be a result of an increased influx of extracellular Ca2+. Moreover, measurements of outward current showed an increase in the amplitude of the outward current when CsA was present in the recording pipette (Pun, unpublished observations). Current-voltage curves indicate that these outward currents show properties similar to calcium-activated potassium currents (Pun, unpublished observations).
The increases observed in membrane capacitance in alveolar type II cells may or may not reflect secretion. Studies in bovine adrenal chromaffin cells demonstrate a significant correlation between capacitance changes and catecholamine secretion (5, 40) and between capacitance changes and fusion of exocytotic vesicles in murine mast cells (41). Furthermore, the increase in capacitance in alveolar type II cells evoked by phorbol ester was about twice that evoked by cAMP (Pun, unpublished observations). These findings agree with measurements of the release of radiolabeled phosphatidylcholine surfactant from type II cells stimulated with phorbol ester and cAMP (10, 26). Whole cell capacitance measurements alone offer no insight into what is being secreted, only the magnitude and duration of the membrane secretory event. Nevertheless, SP-B immunostaining of isolated type II cells indicates that the cellular response is likely lamellar body secretion of SP-B and other surfactant components. Although hyperoxia itself appears to be acting through this pathway, future studies are required to elucidate the molecular mechanisms.
In summary, we have shown that CsA enhances exocytosis from
surfactant-secreting cells. In the injured lung, this effect alone may
not improve lung function because of surfactant inactivation during
injury. CsA has also been demonstrated to have a protective effect
against hyperoxia-induced reductions in lung compliance during
pretreatment and simultaneous treatment with 50 mg · kg1 · day
1
of CsA. CsA reduces capillary congestion, edema, hyaline membrane formation, and neutrophil infiltration during pretreatment with 10 mg · kg
1 · day
1
of CsA. Together these synergistic actions indicate that CsA preserves
both the integrity of the alveolar capillary membranes and pulmonary
surfactant function. CsA would be anticipated to be effective in
reducing lung damage in patients undergoing short-term high-oxygen
therapy for acute lung injury. Because many other forms of lung injury
are associated with a vigorous immune response and decreased lung
compliance, CsA may be beneficial in the treatment of other forms of
lung injury.
Our systemic and cellular examination of the effects of CsA in the lung
parenchyma indicate that CsA acts on numerous cell types such as
alveolar type II cells, endothelial cells, and neutrophils. The precise
cellular and molecular targets of CsA on the overall protective effect
in the lung will be pursued in future studies. Due to the fact that CsA
acts synergistically, inhibits multiple cellular processes, and can
elicit severe side effects (30), it will be valuable to identify the
specific cells involved in promoting the CsA protective effect. The
mouse may prove to be a manipulable model for developing treatment
strategies for lung injury. The SP-B(+/) mouse produces
approximately one-half as much SP-B as wild-type littermates (6). These
animals display severe lung injury after exposure to hyperoxia (35),
demonstrating the importance of surfactant in protecting the lung
epithelium. Additional transgenic mice will provide insight into
designing agents that are targeted to a specific cell type. We have
designed a gene that will neutralize calcineurin only in type II cells. This approach (37) can also be used to selectively inhibit endothelial cells, neutrophils, type I cells, and macrophages. Once we dissect out
the actions of calcineurin inhibition on specific cell types, more
effective strategies for lung protection through calcineurin inhibition
can be designed for patients with specific types of lung damage such as
those found during prolonged hyperoxic exposure.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Christopher Fortner and Dr. John Lorenz for administration of high oxygen in early studies. C. Fortner performed preliminary measurements of static lung compliance (data not shown). Dr. David Millhorn was very helpful in discussing the experimental design. We thank Glenn Doerman for arranging many of the figures, Dr. Paul Succop for help with statistical analysis, Dr. Karen King for suggestions on the manuscript, and Dr. Brenda Wynn for technical assistance (compliance apparatus). We thank Dr. Susan Wert and Sherri Profitt (Molecular Morphology Core, Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH) for generous assistance with the immunohistochemistry.
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FOOTNOTES |
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This work was supported by National Institute of Child Health and Human Development Institutional National Research Service Award (NRSA) Postdoctoral Training Grant 5T-32-HD-07463-03; National Heart, Lung, and Blood Institute Individual NRSA Grant 1-F32-HL-09679-01 (to M. T. Simonich); and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46433 (to J. R. Dedman).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Dedman, Dept. of Molecular and Cellular Physiology, 231 Bethesda Ave., Univ. of Cincinnati Medical Center, PO Box 670576, Cincinnati, OH 45267-0576 (E-mail: john.dedman{at}uc.edu).
Received 13 July 1998; accepted in final form 25 January 1999.
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