1 Department of Pathology, University of Vermont, Burlington, Vermont 05405-0068; and 2 Department of Pediatrics, University of Arizona, Tucson, Arizona 85724-5073
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ABSTRACT |
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Recent evidence suggests that neurokinin (NK)-receptor activation may have a protective role in maintaining lung integrity when challenged by airborne toxicants such as sulfur dioxide, ozone, acrolein, or hydrocarbons. To investigate the effect of NK1-receptor activation on hydrocarbon-induced lung injury, B6.A.D. (Ahr d/Nats) mice received subchronic exposures to JP-8 jet fuel (JP-8). Lung injury was assessed by the analysis of pulmonary physiology, bronchoalveolar lavage fluid, and morphology. Hydrocarbon exposure to target JP-8 concentrations of 50 mg/m3, with saline treatment, was characterized by enhanced respiratory permeability to 99mTc-labeled diethylenetriaminepentaacetic acid, alveolar macrophage toxicity, and bronchiolar epithelial damage. Mice administered [Sar9,Met(O2)11]substance P, an NK1-receptor agonist, after each JP-8 exposure had the appearance of normal pulmonary values and tissue morphology. In contrast, endogenous NK1-receptor antagonism by CP-96345 administration exacerbated JP-8-enhanced permeability, alveolar macrophage toxicity, and bronchiolar epithelial injury. These data indicate that NK1-receptor activation may have a protective role in preventing the development of hydrocarbon-induced lung injury, possibly through the modulation of bronchiolar epithelial function.
tachykinin; JP-8 jet fuel; inhalation; pulmonary toxicology
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INTRODUCTION |
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THE PRESENCE OF tachykinin-containing sensory nerve fibers within the airways of mammals has been demonstrated by radioimmunoassay and immunohistochemistry (15). Substance P (SP) is a member of the tachykinin family of neuropeptides, which are widely distributed in the peripheral and central nervous systems (19). Exposure of airways to mechanical or chemical irritants such as capsaicin and Formalin results in protective reflex responses such as bronchoconstriction, increased vascular permeability, vasodilatation, mucus secretion, and enhanced mucociliary activity (19). These actions are mediated through plasma membrane-bound neurokinin receptors (NK1, NK2, and NK3) that have seven transmembrane domains and are G protein coupled (17). SP has the highest affinity for the NK1 receptor. The pulmonary effects of SP have been shown to be inhibited by CP-96345, a nonpeptide NK1-receptor antagonist (29). Furthermore, autoradiographic, immunochemical, and receptor binding studies have demonstrated that NK1 receptors are localized at both the basal and apical sites on airway epithelium from the trachea to the respiratory bronchioles (8, 18).
Although NK1-receptor activation has been shown to enhance vascular permeability, recent studies suggest that NK1-receptor activation may protect against enhanced airway epithelial permeability. Yu and co-workers (36) demonstrated that SP administration had a protective effect on ozone-induced permeability in primary human and canine bronchial epithelial cells, which was predominately mediated through NK1 receptors. De Sanctis and co-workers (4) showed that SP depletion in rats produced a decrease in airway transepithelial electrical potential difference and enhanced permeability to 99mTc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA). These studies were based on Rangachari and McWade's (24) postulate that SP or NK1-receptor activation may modulate epithelial permeability via paracellular anion flux. Yu and co-workers (36) concluded that SP, through NK1-receptor activation, functions to maintain bronchial epithelial barrier integrity under conditions of environmental or toxicant challenge. In a rat model of occupational hydrocarbon exposure, Pfaff and co-workers (20, 21) demonstrated that JP-8 inhalation decreases pulmonary epithelial barrier function, which was mediated by suppression and/or depletion of SP release.
We have recently characterized the acute and subchronic pulmonary responses to hydrocarbon inhalation in B6.A.D. (Ahr d/Nats) mice (25 and unpublished observations). Hydrocarbon exposure appeared to initially target distal airways with the onset of increased respiratory permeability, epithelial necrosis, and perivascular edema. With repeated exposures, tissue injury progressed to include pulmonary edema, capillary endothelial vacuolization, and type II epithelial cell alterations. The present study was carried out to investigate the potential modulatory effects of NK1-receptor activation and antagonism on hydrocarbon-induced lung injury, with an emphasis on bronchiolar airways.
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METHODS |
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Animals. Twelve 25- to 35-g male B6.A.D. (Ahr d/Nats) mice were randomly assigned to each exposure group, with all 12 mice being subjected to pulmonary function and respiratory permeability measurements. Within each exposure group, nine mice were then randomly assigned for bronchoalveolar lavage analysis, and three mice were randomly assigned for morphological evaluation. The B6.A.D. (Ahr d/Nats) mice were derived from stock maintained at the University of Arizona and have been described previously (13). Briefly, the mice are genetically the same as the C57BL/6 mice except that they are double congenic for nonresponsiveness to aryl hydrocarbon hydroxylase induction and slow N-acetylation. These mice were originally chosen to investigate the possible role of aryl hydrocarbon hydroxylase activity in the susceptibility to JP-8 jet fuel (JP-8)-induced pulmonary toxicity and immunotoxicity (7, 25). Both studies found no significant differences in toxicity between B6.A.D. (Ahr d/Nats) and C57Bl/6 strains that could be attributed to aryl hydrocarbon hydroxylase activity or N-acetyltransferase activity. All mice were housed in an American Association for Accreditation of Laboratory Animal Care-approved animal facility of the Department of Animal Resources at the University of Arizona Health Sciences Center; the mice were housed in groups of four mice per pan in a level 4 microisolation room and were fed ad libitum.
Exposure system and animal treatments.
Hydrocarbon exposures were performed as previously described (25 and
unpublished observations). Briefly, JP-8 (Wright-Patterson Air Force
Base, Dayton, OH) aerosol was generated with an Ultra-Neb 99 nebulizer
(model 099 HD, DeVilbiss, Somerset, PA) and was allowed to mix with
ambient air. The hydrocarbon-air mixture was drawn through a nose-only
inhalation exposure chamber (IN-TOX, Albuquerque, NM) using a constant
vacuum. JP-8 concentration and aerosol characterization were determined
with a seven-stage cascade impactor (IN-TOX), and the results are
summarized in Table 1. Mice were exposed
for 1 h/day over a period of 7 days to an average target concentration
of 50 mg/m3. Previous studies have
shown that exposures as low as 50 mg/m3 induce consistent pulmonary
toxicity (25 and unpublished observations). Control animals were
exposed to ambient air (0 mg/m3). Nose-only exposures were
used to minimize oral ingestion of JP-8 during grooming and to more
closely simulate occupational exposure.
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To test the effect of NK1-receptor activation on JP-8-induced pulmonary toxicity, the selective NK1-receptor agonist [Sar9,Met(O2)11]SP (Sigma, St. Louis, MO) was chosen. Mice receiving agonist treatment were administered a 1 µM aerosol (to limit proteolytic digestion) of [Sar9,Met(O2)11]SP in saline for 15 min immediately after each JP-8 exposure. The concentration of SP was based on the work of Joos and co-workers (9). This was the highest concentration of NK1-receptor agonist that did not cause bronchoconstriction in normal or asthmatic subjects. Negative control animals were exposed to ambient air followed by saline vehicle or SP treatment. Positive control animals were exposed to an average concentration of 53 mg/m3 JP-8, which was immediately followed by 15 min of aerosolized saline. To obtain insight on the potential role of NK1-receptor activation in protecting against hydrocarbon-induced lung injury, it was decided to antagonize endogenous activation with CP-96345 (Pfizer, Groton, CT). CP-96345 (NK1-receptor antagonist)-pretreated mice were administered a 2.5-mg/kg (saline vehicle) intraperitoneal injection 15 min before (3) each JP-8 exposure. Control mice were pretreated with CP-96345 as described and were exposed to ambient air. Mice exposed to ambient air alone or to JP-8 alone have been previously described and were omitted from this study to avoid redundancy (25 and unpublished observations).
Pulmonary function and respiratory permeability. Pulmonary function and respiratory permeability measurements were performed as previously described (25 and unpublished observations). Briefly, 24-30 h after JP-8 exposure, the mice were anesthetized with an intramuscular injection mixture of ketamine hydrochloride (80 mg/kg), xylazine (10 mg/kg), and acepromazine maleate (3 mg/kg). A tracheostomy was performed, with the insertion of a Teflon intravenous catheter (20 gauge; Critikon, Tampa Bay, FL) serving as an endotracheal tube. The mice were placed under pressure-controlled respiration (Kent Scientific, Litchfield, CT) and were given an intraperitoneal injection of gallamine triethiodide (8 mg/kg) to suppress spontaneous breathing. Airflow was measured with a pneumotachograph (Fleisch no. 0000, Instrumentation Associates) that was coupled to a differential pressure transducer (Validyne, Northridge, CA). Airflow and pressure signals were used to measure dynamic compliance, and pulmonary resistance was measured with a modified PEDS-LAB (Medical Associated Services, Hatfield, PA) pulmonary function system by the method of Rodarte (27). Pulmonary function measurements were normalized to individual animal weight. After pulmonary function recording, respiratory permeability was determined by measuring the pulmonary clearance of intratracheally instilled 99mTc-DTPA.
Bronchoalveolar lavage. Immediately
after respiratory permeability testing, nine animals per exposure group
were randomly assigned for bronchoalveolar lavage. Bronchoalveolar
lavage analyses were also performed as described previously (25 and
unpublished observations). Briefly, anesthetized animals were
euthanized by exsanguination of the abdominal aorta, and the lungs were
removed and cannulated. The lungs were lavaged six times with sterile isotonic saline at a volume of 1 ml via the cannula. Bronchoalveolar lavage fluid (BALF) cell number and differentials were determined from
a 0.2-ml sample by hemocytometer counting and cytocentrifuge preparation, respectively. Cytocentrifuge preparations were stained with Diff-Quik (Dade Diagnostics, Aguada, Puerto Rico). The remaining BALF from each mouse was centrifuged to pellet cells. The cell-free BALF was analyzed for total protein (Coomassie Plus, Pierce, Rockford, IL), lactate dehydrogenase (LDH; Sigma), and
N-acetyl--D-glucosaminidase (NAG; Boehringer Mannheim, Mannheim, Germany) activities with colorimetric reagent kits.
Lung morphology. Immediately after alveolar permeability testing, three animals per exposure group were randomly assigned for pulmonary morphological evaluation. Animals were euthanized, and lungs were removed as described in Bronchoalveolar lavage. Lungs were fixed by intratracheal instillation of half-strength Karnovsky's fixative (2% paraformaldehyde, 2% glutaraldehyde, and 0.01% picric acid in 0.1 M HEPES; pH 7.4; 550 mosmol) at a constant pressure of 20 cmH2O for 1 h. The lungs were then immersed in fixative for 24 h at 4°C. Sections (2-3 mm) from the fixed lungs were taken from the midportion of the left and right lung lobes for routine qualitative assessment of distal airway and alveolar morphology. Tissue sections were randomly chosen for light microscopy or minced into 1-mm3 pieces for electron microscopy. Light microscopy sections (5 µm) were embedded in paraffin and stained with hematoxylin and eosin. Electron microscopy sections (silver to gold interference colors) were prepared by osmication, sectioning, and staining with lead citrate and uranyl acetate and were examined with a Philips (Mahwah, NJ) CM-12 transmission electron microscope.
Statistical analysis. All data are presented as means ± SD. Differences between means for group data were tested for significance by a factorial ANOVA. Fisher's protected least significant difference multiple t-test was used to identify significant differences between groups. Statistical analyses were carried out with StatView 4.5 statistical software (Abacus Concepts, Berkeley, CA), and P values < 0.05 were considered significant.
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RESULTS |
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Pulmonary function and respiratory
permeability. At 24-30 h after the final exposure,
there were no significant changes in dynamic compliance or total
pulmonary resistance (Table 2). However, at
24-30 h after the last exposure, there was a significant increase in respiratory permeability (115%) in mice exposed to an average concentration of 53 mg/m3 JP-8 and
receiving aerosolized saline administration (JP-8 + Sal) compared
with mice exposed to ambient air and receiving aerosolized saline (0 + Sal) or aerosolized
[Sar9,Met(O2)11]SP
(0 + SP) and mice pretreated with CP-96345 and exposed to ambient air
(CP + 0; Table 2). The increase in respiratory permeability to
99mTc-DTPA was prevented
in mice exposed to an average JP-8 concentration of 52 mg/m3 and receiving aerosolized
[Sar9,Met(O2)11]SP
administration (JP-8 + SP). Conversely, mice pretreated with CP-96345
and exposed to JP-8 (CP + JP-8) were observed to have an exacerbated
increase in respiratory permeability (
50%) compared with the JP-8 + Sal group.
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BALF analysis. There were significant
increases in both BALF total protein (110%) and LDH (
180%)
levels for the JP-8 + Sal group, which are suggestive of increased
plasma extravasation and cellular death compared with 0 + Sal, 0 + SP,
and CP + 0 control groups (Table 3). As
with respiratory permeability,
[Sar9,Met(O2)11]SP
administration after each JP-8 exposure prevented an increase in BALF
levels of both total protein and LDH. The increase in BALF total
protein was not exacerbated in the CP + JP-8 group, but LDH was
exacerbated by approximately another 110% compared with the JP-8 + Sal
group. There appeared to be a significant reduction in alveolar
macrophage activity as indicated by the decrease in BALF NAG activity
(
45%) in JP-8 + Sal mice (Table 3). The decrease in NAG activity
was not exacerbated in the CP + JP-8 group, but the decrease was
prevented in the JP-8 + SP group.
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BALF total cell counts significantly decreased (20%) in the JP-8 + Sal group compared with the control groups (Table
4). The BALF decrease in total cell
number was exacerbated by approximately another 30% in the CP + JP-8 group compared with that in JP-8 + Sal mice. Once again,
the administration of
[Sar9,Met(O2)11]SP
after JP-8 exposures prevented the decrease in BALF cell numbers. There
were no changes in BALF neutrophil or lymphoctye populations for all
treatment groups. The alveolar macrophage population was significantly
decreased in the JP-8 + Sal group and was further decreased in the CP + JP-8 group (Table 4). The decrease in BALF alveolar macrophage
population was prevented in the JP-8 + SP group.
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Morphological evaluation. The
morphological observations were based on the examination of all tissue
sections, and qualitative results were representative of all animals
examined for each treatment group. Light- microscopic examination of 0 + SP mice revealed lung tissues within normal limits (Figs.
1B and
2C)
compared with previous work (25). Evaluations of 0 + Sal and CP + 0 control groups were also within normal limits. Lung morphology for 0 + Sal and CP + 0 mice was not different from that for 0 + SP mice. Therefore, representative micrographs for 0 + Sal and CP + 0 mice were
omitted to avoid redundancy. Examination of JP-8 + Sal mice revealed
marked focal areas of alveolar septal thickening and collapsed air
spaces (Fig. 1A). Evaluation of
distal airways was characterized by the appearance of swollen and
exfoliated bronchiolar epithelial cells (Fig.
2A). Areas of vacuolization between
bronchioles and venules, which suggest the formation of moderate
perivascular and mild subendothelial edema, were also observed (Fig.
2B). Histological evidence of lung
injury was absent in JP-8 + SP mice (Fig. 1, C and
D). Examination of the alveolar
region and distal airways of JP-8 + SP mice revealed tissues that were
similar to 0 + SP mice (Figs. 1 and 2). In contrast, CP + JP-8 mice had
morphological evidence of enhanced lung injuries compared with JP-8 + Sal mice. Microscopic evaluation of the alveolar region of CP + JP-8
mice revealed dense areas of alveolar septal thickening associated with
the intra-alveolar hemorrhage and collapse (Fig.
1D). Swelling and exfoliation of
bronchiolar epithelial cells were also apparent (Fig.
2E). In contrast to JP-8 + Sal mice,
there appeared to be sporadic collapsing of terminal airways adjacent
to uncollapsed airways in CP + JP-8 mice (Fig.
2E). This observation may be
consistent with the pathogenesis of atelectasis. The severity of
perivascular and subendothelial edema also appeared to increase (Fig.
2F), but these and all morphological
observations were not quantified by morphometry.
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Electron microscopy focused on alveolar type II epithelial cells and
terminal airway epithelium for all treatment groups. As with light
microscopy, ultrastructural evaluation of lung tissues from 0 + SP and
CP + 0 exposure groups revealed no indication of toxicity (Figs.
3B and
4C).
There were no differences between 0 + SP and CP + 0 control groups, so
once again photomicrographs for CP + 0 mice were omitted to prevent
redundancy. Examination of the JP-8 + Sal exposure group revealed an
apparent increase in the number and size of surfactant-producing
lamellar bodies within alveolar type II epithelial cells (Fig.
3A). Under higher magnification,
there was also the appearance of lamellar inclusion bodies that
appeared to be secondary lysosomes containing intracellular debris (not
shown). Affected type II epithelial cells were also characterized by
dilation and/or vacuolization of endoplasmic reticulum. As with
previous work, there were also scattered areas of diffuse vacuolization
of adjacent endothelium with subendothelial edema (Fig.
3A). In contrast, ultrastructural
evaluations of the JP-8 + SP-exposed mice were absent of toxicological
changes to alveolar type II epithelium and capillary endothelium (Fig.
3C). The only indication of JP-8
exposure was the appearance of an increase in the size but not in the
number of lamellar bodies. Endothelial and alveolar type II epithelial
alterations in CP + JP-8-exposed mice were observed to be slightly more
severe than those in JP-8 + Sal mice. However, alveolar type II
epithelial injury progressed to include intranuclear inclusion bodies
and cells absent of lamellar bodies with organelle vacuolization (Fig. 3D). The occasional intranuclear
inclusion bodies were of an unknown osmiophilic granular-like
structure.
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As with light microscopy and ultrastructural evaluation of alveoli, terminal bronchiolar epithelium remained within the normal limits for 0 + SP and CP + 0 control tissues (Fig. 4C). Examination of the JP-8 + Sal group revealed terminal airway changes that were moderate with respect to incidence and severity. Bronchiolar alterations were characterized by the dilation of intercellular junctions and nuclear membranes (Fig. 4A). Also apparent were areas of isolated cytoplasmic vacuolization of rough endoplasmic reticulum within nonciliated epithelial cells (Clara cells), leading to the appearance of necrosis and lamellar-like inclusion bodies (Fig. 4, A and B). Ciliated epithelial cells appeared relatively unaffected except for changes to intercellular junctions and nuclear membranes. In contrast, examination of bronchiolar epithelium from JP-8 + SP-treated mice revealed the absence of toxicological alterations, consistent with control groups (Fig. 4D). As opposed to both JP-8 + SP and JP-8 + Sal treatment groups, CP + JP-8-exposed mice had more progressive alterations within bronchiolar lining cells. Intercellular cytoplasmic vacuolization of endoplasmic reticulum was clearly more diffuse within Clara cells (Fig. 4, E and F) but also within ciliated epithelial cells. For the first time, there were indications of necrotic ciliated epithelial cells (Fig. 4E). Nuclear alterations within Clara cells caused the appearance of less dense chromatin staining, which could be indicative of the onset of necrosis.
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DISCUSSION |
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The present study demonstrates that subchronic administration of saline, [Sar9,Met(O2)11]SP, or CP96345 alone had no direct effect on B6.A.D. (Ahr d/Nats) murine lung physiology or morphology compared with a previous subchronic study (unpublished observations). Subchronic hydrocarbon inhalation of JP-8 produced lung injury consistent with previous mouse work (unpublished observations), and a previous study in rats observed that subchronic JP-8 inhalation results in decreased BALF SP levels that correlate with lung injury (20). A regimen of NK1-receptor activation by [Sar9,Met(O2)11]SP after JP-8 inhalation exposures prevented the development of lung injury. In contrast, antagonism of endogenous NK1-receptor activation by CP-96345 pretreatment exacerbated the hydrocarbon-induced lung injury.
The acute pulmonary response to inhaled JP-8 in B6.A.D. (Ahr d/Nats) mice was previously characterized by a targeting of bronchiolar epithelium (25). Bronchiolar epithelial injury included the vacuolization of smooth endoplasmic reticulum, degranulation, and necrosis of Clara cells. In a similar subchronic study, the severity of bronchiolar epithelial injury increased with progression of the injury to include deterioration within the alveolar-capillary barrier (unpublished observations). Nonciliated airway epithelial (Clara) cells appear to be initial targets for hydrocarbon-induced lung injury in this and previous studies (25 and unpublished observation). This suggests that hydrocarbon-induced lung injury may be mediated through Clara cell metabolic activity. These findings suggest that NK1-receptor activation may prevent hydrocarbon-induced lung injury by either decreasing access or modulating metabolism of JP-8 within Clara cells.
NK1 receptors could decrease the access of toxicants to Clara cells by modulating the composition or movement of the epithelial lining fluid. NK1-receptor activation is known to stimulate bronchial mucus secretion (28) and mucociliary activity (31). Lindberg (14) has also shown that toxicant exposure, such as cigarette smoke, can increase mucociliary beat as measured photoelectrically. The observed increase in mucociliary beat was determined to be partially mediated through the activation of NK1 receptors by SP. The mucous layer of the epithelial lining fluid is responsible for trapping particulate matter, and the ciliary beat within the aqueous layer is responsible for the movement of the mucous layer and subsequent pulmonary clearance of foreign compounds. Increased mucus secretion could increase the solubility of hydrocarbons within the epithelial lining fluid and effectively dilute the concentration of hydrocarbons, which can gain access to airway epithelia. A concomitant increase in ciliary beat frequency would also enhance the movement of the hydrocarbon-containing mucous layer in larger airways, which appear to be less susceptible to hydrocarbon-induced injury (30). Stimulated ciliary beat frequency could also prevent hydrocarbon-induced injury by enhancing hydrocarbon clearance.
The metabolism of xenobiotics within the lung often leads to toxicity, although certain pulmonary cells are more readily damaged than others. This differential susceptibility can result from cell-specific differences in xenobiotic activation and detoxification. Clara cells are known to be the primary site for cytochrome P-450-mediated xenobiotic biotransformation in the lung and are most readily damaged as a consequence of the bioactivation of many pulmonary toxicants (1). In vitro experiments have shown that straight-chain hydrocarbons can inhibit cytochrome P-450-mediated detoxification pathways (23). To our knowledge, the effect of NK1- receptor activation on metabolic activities within Clara cells has not been studied. However, SP has been shown to modulate metabolic activity in other cellular systems. Ekstrom and co-workers (5) have shown that SP influences polyamine metabolism in salivary glands by increasing ornithine decarboxylase activity, and others have shown that SP can stimulate arachidonic acid metabolism and NADPH oxidase activity in neutrophils or alveolar macrophages (16, 32). The mechanism by which SP modulates cellular metabolism for these studies is not completely understood, but it is believed to be NK1 receptor mediated. NK1-receptor activation could modulate Clara cell metabolism by stimulating oxidation of JP-8, which could lead to the formation of metabolites that are more readily cleared from the lung and less toxic. Conversely, CP-96345 pretreatment could be exacerbating lung injury by depleting endogenous NK1-receptor activation of detoxification pathways.
The most consistent hypothetical mechanism by which SP and NK1-receptor activation prevents toxicant-induced lung injury is through the maintenance of airway epithelial barrier function. Capsaicin pretreatment to deplete SP has been shown to exacerbate acrolein- and ozone-induced airway epithelial denudation, necrosis, and inflammation (33, 34). Yu and co-workers (36) have shown that NK1-receptor activation protects and maintains epithelial barrier function from ozone in primary human and canine bronchial epithelial cells. Furthermore, Killingsworth and co-workers (10) observed a protective effect by SP in a rat model of sulfur dioxide-induced chronic bronchitis. NK1-receptor activation may protect against hydrocarbon and other toxicant-induced lung injuries through a common mechanism for maintaining bronchiolar epithelial barrier function. In support of this hypothesis, we (unpublished observations) have found that hydrocarbon-induced decreases in epithelial barrier function by enhancing [3H]mannitol permeability in a human bronchial epithelial cell line. However, the mechanism of tachykinin protection remains to be determined and may end up being a combination of pulmonary actions elicited by NK1-receptor activation.
It has been postulated that sensory neuropeptides are involved in the repair of damaged airway epithelium. NK1- and NK2-receptor activation have both been shown to elicit migration of primary cultured airway epithelial cells, with NK1-receptor activation having a greater effect (12). However, only NK1-receptor activation was observed to elicit proliferation of the airway epithelial cells. Subsequent in vivo studies demonstrated that sensory neuropeptide depletion by capsaicin attenuates both epithelial cell proliferation and repair after injury to the trachea (11). However, the capsaicin treatment employed by the aforementioned study depletes sensory neurons of neuropeptides, and the specific role of NK1-receptor agonists in epithelial repair remains undetermined. Furthermore, neuropeptide depletion only attenuated epithelial proliferation and repair in the first 72 h after injury. Tracheal injury was at near-complete repair at 1 wk for both neuropeptide-depleted and nondepleted animals.
NK1-receptor activation could possibly facilitate bronchiolar epithelial cell proliferation within the first 72 h after the initial hydrocarbon exposure. However, based on the observations by Kim et al. (11), CP-96345 should not have had an effect on normal airway epithelial repair after 72 h, whereas, CP-96345 pretreatment was observed to exacerbate lung injury after 7 days of repeated hydrocarbon exposure. In addition, SP or specific NK1-receptor agonists have not been shown to facilitate all three phases of airway epithelial repair (migration, proliferation, and differentiation). Therefore, the present observations that NK1-receptor activation prevents the formation of hydrocarbon-induced lung injury appear to be largely independent of repair mechanisms.
There are limitations to the present study. A major limitation may be the use of CP-96345 as a specific NK1-receptor antagonist. CP-96345 has been shown to act as an antagonist of L-type calcium channels in addition to its NK1-receptor antagonist activity in rat and guinea pig cerebral cortex (6) and mouse spinal cord (35). However, comparable doses of CP-96345 have been shown not to interact with L-type calcium channels in lung tissue (2, 6, 37). The available literature suggests that CP-96345 calcium-channel activity may be tissue and/or dose specific, with little or no activity in the lung. Therefore, the exacerbation of hydrocarbon-induced lung injury by CP-96345 pretreatment appears to be mediated through NK1-receptor antagonism in this study.
A second limitation in this study is the lack of morphometric evaluation of the hydrocarbon-induced lung injuries. The use of morphometry would have enabled the quantification of changes in lung structure, the ranking of changes in lung structure, and the quantification of changes within specific lung compartments like lamellar bodies. However, the goal of this study was to obtain descriptive and qualitative data regarding the effects of NK1-receptor activation on hydrocarbon-induced lung injury. Once these effects have been qualitatively characterized, studies may be designed to quantitatively assess specific lung alterations and mechanisms of injury prevention using morphometry. The design of this study would not have provided optimal sample size or density for an appropriate lung morphometry study (22).
In summary, these data provide further evidence that tachykinins and NK1-receptor activation may be involved in pathophysiological processes that protect the lung from injury induced by environmental or occupational toxicants. The actions of NK1-receptor activation appear to predominantly protect airway epithelial integrity, particularly the Clara cell. However, further experiments with an emphasis on in vitro systems need to be designed to determine the mechanism of protection.
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ACKNOWLEDGEMENTS |
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We thank Veronica Breceda and Shengjun Wang for technical assistance, Susan E. Leeman for the gift of CP-96345, and R. Clark Lantz for help in morphological evaluation and manuscript preparation.
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FOOTNOTES |
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This research was supported by Air Force Office of Scientific Research Grant F49620-94-1-0297 and by the Augmentation Awards for Science and Engineering Research Training Program.
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: M. L. Witten, Dept. of Pediatrics, Univ. of Arizona, PO Box 245073, Tucson, AZ 85724-5073.
Received 13 February 1998; accepted in final form 9 October 1998.
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