Departments of 1 Pediatrics, 3 Pathology, and 4 Surgery, Washington University School of Medicine, St. Louis, Missouri 63110; and 2 Neurogen Corporation, Branford, Connecticut 06405
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ABSTRACT |
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Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.
parasympathetic nervous system; autonomic denervation; trachea
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INTRODUCTION |
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THE RECENT DEMONSTRATION that gene-targeted disruption of the neurokinin-1 (NK-1) receptor (the main receptor for substance P) protects murine lungs from immune complex injury (5) has bolstered the notion of a tachykinin-based mechanism for neural amplification of immune inflammation in the lungs. The existence of a similar, but smaller-scale, mechanism had been suspected since the discovery that airway sensory fibers contain substance P and other tachykinins encoded by the preprotachykinin (PPT)-A gene and release them in response to nociceptive signals (48).
Sensory fibers, however, may not be the only source of tachykinins in the lungs. Substance P immunoreactivity has been observed in airway ganglia from several species, including humans (7-10, 44, 49). These observations have two intriguing implications. First, airway neurons may function as a tachykinin reservoir available even when the sensory innervation of the airways is disrupted (e.g., after lung transplantation). In addition, because airway ganglia are themselves innervated by peptidergic sensory fibers (42) and undergo membrane depolarization when exposed to substance P (43, 50), ganglion neurons are likely to contain NK-1 receptors and may even coexpress NK-1 receptor and PPT-A genes. Indeed, it is conceivable that the intrinsic neuronal network of the airways contributes to the amplification and propagation of immune-mediated inflammation in the lungs through a system of synaptic and parasynaptic peptidergic interactions, independent of the sensory nerves.
The present study was designed to characterize the expression of PPT-A and NK-1 receptor mRNAs and protein by intrinsic airway neurons in the rat. First, we applied immunohistochemical and mRNA detection techniques to elucidate whether PPT-A and NK-1 receptor gene products are expressed independently or are coexpressed by airway neuronal bodies. Second, we investigated whether the expression of these genes is altered by the removal of vagal and sensory inputs in tracheal xenografts, syngeneic lung grafts, and cultured parasympathetic ganglia. Finally, we took advantage of the requisite role of the NK-1 receptor for the injury produced by antigen-antibody complexes in the lung to determine whether nonsensory sources of substance P can support the development of this type of injury in syngeneic lung grafts.
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METHODS |
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All experiments were performed following protocols approved by the Washington University Animal Studies Committee. Rodents were housed in a climate-controlled room at 22°C with full access to a standard rat chow and water.
Preparation of trachea and tracheal xenografts. Tracheae were removed from 10- to 12-wk-old Sprague-Dawley rats (Charles River, Wilmington, MA) under pentobarbital sodium anesthesia (100 mg/kg ip). After separation from the esophagus, each trachea was divided into two halves, upper and lower, by a transverse coronal section at the level of the thoracic inlet. Each half was randomly assigned for immediate preservation as a control or for implantation as a xenograft into immunodeficient mice. The dissection of the trachea from the neighboring tissue was not designed to preserve the tracheal nerves or the longitudinal trunk parasympathetic ganglia attached to these nerves (2, 52). At least in the ferret, the majority of the neurons in these ganglia are cholinergic and lack substance P expression (7). The tracheal xenografts were prepared with a modification of a technique previously described (13). Immediately after removal, the selected tracheal segments were rinsed and soaked for 4 h at 4°C in antibiotic-antimycotic solution (Sigma, St. Louis, MO) reconstituted in PBS to concentrations of 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The segments were then implanted subcutaneously into male C57BL/6J nu/nu mice (Jackson Laboratory, Bar Harbor, ME) under pentobarbital sodium anesthesia (50 mg/kg ip). The xenografts were removed at 1 (n = 8), 3 (n = 5), 5 (n = 5), 7 (n = 7), 14 (n = 16), or 28 (n = 22) days after implantation and placed in fixative solution (4% paraformaldehyde or 2% paraformaldehyde, 0.1% glutaraldehyde, and 15% picric acid, both in 0.1 M sodium phosphate-buffered saline) for 24 h. Although some mucus accumulated in the tracheal lumen, the xenografts did not appear distended even after 28 days. Therefore, no provisions were made for continuous drainage.
Syngeneic lung transplantation. Lung transplantation was performed in 12-wk-old inbred Fischer rats (n = 16; Charles River). The trachea of the donor rat was cannulated, and the lungs were ventilated with a rodent ventilator (Harvard Apparatus, S. Natick, MA) under pentobarbital sodium anesthesia (50 mg/kg ip). The thoracic and abdominal organs were exposed through a midline incision. Heparin (1,000 U/kg) was injected into the inferior vena cava, which was then sectioned and allowed to bleed until exsanguination. The pulmonary vessels were flushed with cold saline through a pulmonary artery cannula, and the lungs were removed into a cold saline bath. The recipient rat was anesthetized with halothane (0.5-3%) piped into an induction box or, after cannulation of the trachea, into the inspiratory limb of the rodent ventilator. After a lateral thoracotomy and left pneumonectomy, the pulmonary artery and vein were attached to the corresponding vessels of the donor rat's lung by use of polyethylene cuffs (41, 47). The left main stem bronchus was anastomosed with an 8-0 Prolene running suture. The thoracotomy was closed by layers, with care taken to remove all air remaining in the pleural cavity through a silicone rubber catheter. On recovery from anesthesia, the pleural catheter was removed, and after a short period of observation, the rat was transferred to the rodent holding facility without supplemental oxygen.
Immune complex injury. The objective of the experiments was to determine whether the destruction of peptidergic sensory nerve fibers during syngeneic lung transplantation prevents the development of an immune complex injury. In mice, it is known that this kind of injury is attenuated by specific pharmacological NK-1 receptor antagonists (24) and prevented by disruption of the NK-1 receptor gene (5). Lacking similar information in other species, we investigated first whether the NK-1 receptor plays a similar conditional role in the rat. To this effect, we compared the responses of five rats pretreated with the NK-1 receptor antagonist LY-306740 (38) (a gift of Lilly Research Laboratories, Indianapolis, IN; 30 mg/kg ip in 2 ml of normal saline) and six rats pretreated with saline vehicle alone to the simultaneous injection of chicken ovalbumin intravenously (20 mg/kg in 2 ml of normal saline; Sigma) and a rabbit polyclonal antibody against ovalbumin intratracheally (0.9 mg/kg in 1 ml of normal saline; Chemicon, Temecula, CA). The rats were injected 2.5 h later with FITC-labeled albumin (5 mg/kg iv; Sigma), and after an additional 1.5 h, the lungs were exposed through a median sternotomy under pentobarbital sodium anesthesia. A clip was placed on the left main stem bronchus, and the left lung was removed for analysis. The right lung was then lavaged three times, each with 2 ml of normal saline. Disruption of the permeability of the alveolar-capillary membrane was quantified as the ratio of lavage fluid to serum FITC albumin concentrations measured spectrofluorometrically (excitation = 495 nm, emission = 520 nm; model SPF 500, American Instruments, Silver Spring, MD).
Once the necessary participation of the NK-1 receptor in the immune complex injury was established, additional experiments were performed in 15 rats that had undergone syngeneic lung transplantation 2 wk earlier and in 16 control rats (6 of which had undergone a left thoracotomy 2 wk earlier). Each rat was injected with chicken ovalbumin intravenously and either antibody against ovalbumin or an equivalent volume of normal saline intratracheally as described above. After 4 h or at death, the lungs were exposed through a median sternotomy, and each lung was lavaged separately in three passes of 2 ml of normal saline through a tapered cannula that was wedged alternatively into the right and left main stem bronchi. The correct position of the cannula was corroborated by selective inflation of the desired lung as the lavage liquid was injected. The lavage sample was rejected if the lavage fluid leaked into the chest cavity or if <50% of the injectate was recovered. Finally, except for five transplant recipients and eight control rats whose lungs were frozen for RNA extraction, the pulmonary artery was perfused with normal saline and then with 4% buffered paraformaldehyde for morphological studies. In three of the graft recipients and five control rats, the NK-1 receptor antagonist LY-306740 (30 mg/kg ip) was injected before the administration of ovalbumin and anti-ovalbumin antibodies at doses similar to those reported previously in rats (38) to verify once again the participation of the NK-1 receptor in the immune complex-induced injury.Primary culture of airway neurons.
Neurons were cultured from tracheae of 10- to 12-wk-old Sprague-Dawley
rats as previously described (14, 17). Each culture combined the
tracheae from four rats. The posterior quadrant of each trachea was
separated through bilateral longitudinal incisions at the level of the
trachealis muscle's attachments to the tracheal cartilage. The
resultant tissue strips were denuded of epithelium and digested for
30-45 min by placement in two consecutive 37°C baths of
sterile low-calcium Krebs-Henseleit solution with 2.5 mg/ml BSA
(Sigma), 3 mg/ml (1st bath) or 1.5 mg/ml (2nd bath) collagenase I
(Sigma), and 0.15 mg/ml (1st bath) or 0.50 mg/ml (2nd bath) elastase IV
(Sigma). The digestion product was washed with a solution containing
10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Sigma) in DMEM-F-12 (equal volumes of DMEM and
Ham's F-12 medium) and centrifuged twice (1,000 rpm for 10 min at
4°C). After cell clumps were broken with a heat-smoothed glass
pipette, the cells were seeded onto Falcon Primaria plates (Becton
Dickinson, Lincoln Park, NJ) and incubated overnight at 37°C in 5%
CO2. On the following day, the plates were rinsed with serum-free medium to remove nonadhered cells.
The cell suspension was washed twice in fresh serum-free medium and
centrifuged. The cell pellet was then resuspended in DMEM-F-12
containing antibiotics (see above for concentrations), 100 ng/ml
-nerve growth factor (Harlan Bioproducts for Science, Indianapolis,
IN), 1 µg/ml bovine insulin (Sigma), 20 µg/ml human transferrin
(Jackson ImmunoResearch Laboratories, West Grove, PA), 20 µg/ml
glutamine (Sigma), and 30 nM sodium selenite (Sigma) and seeded on
Matrigel-coated round coverslips (Becton Dickinson, Bedford, MA). (Some
of the cultures were performed in the absence of
-nerve growth
factor.) Cytosine arabinoside (1 µM; Sigma) was added 24 h later. The
medium was replenished every 48 h for the duration of the culture
(7-14 days). Neuronal cultures were preserved by 30 min of
fixation in solutions containing 4% paraformaldehyde and 0.25%
glutaraldehyde (immunohistochemistry) or 2% paraformaldehyde, 0.1%
glutaraldehyde, and 15% picric acid (in situ hybridization).
Immunohistochemical analysis.
Paraffin-embedded tissue sections (5 µm) were deparaffinized with
xylene and hydrated in incremental concentrations of ethanol. Tissue
sections and neuronal cultures were permeabilized with 0.3% Triton
X-100 for 15 min and treated with 0.1% hyaluronidase in 0.1 M sodium
acetate buffer for 30 min at 37°C. Tissue sections and cultures
were then exposed to donkey serum (Sigma) diluted 1:50 in Tris-buffered
saline (TBS) for 30 min and incubated overnight at 4°C
with the specific primary antisera (Table
1). The samples were then washed thoroughly
and placed in a 1:50 solution of the specific fluorescent-labeled
(FITC, tetramethylrhodamine isothiocyanate, or aminomethylcoumarin
acetate) or biotinylated anti-IgG antiserum for 3-4 h at room
temperature. All anti-IgG antisera were raised in donkey and purchased
from Jackson ImmunoResearch Laboratories. After a final wash, each
sample was mounted on a glass slide and protected with a sealed
coverslip. The specificity of substance P and NK-1 receptor antibodies
was confirmed by showing no staining in samples chosen at random for
each processing batch when 10 µM immunizing peptide was added to the
primary antiserum solution (1). The specificity of the macrophage
metalloelastase antisera was corroborated by the absence of staining
when the primary antiserum was replaced with preimmune serum (46).
Biotinylated antibody labeling was demonstrated by incubation with an
avidin-horseradish peroxidase complex (Vectastain ABC kit, Vector
Laboratories, Burlingame, CA) followed by staining with 0.05%
diaminobenzidine tetrahydrochloride and gold intensification.
Macrophage metalloelastase-immunoreactive cells were counted at
×200 magnification after lung tissue sections were divided into
eight equal sectors, and two of them were selected randomly. Cell
density was calculated by dividing the total number of immunoreactive
cells in each sector by the sector's area measured with a 10 × 10 40-µm-square reticle.
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In situ hybridization.
Samples (tissue slides or culture coverslips) were prepared for
hybridization as previously described (6). Digoxigenin-labeled cRNA
probes were prepared for the detection of PPT-A and NK-1 receptor gene
transcripts with a commercial digoxigenin-U labeling kit (Roche
Molecular Biochemicals, Indianapolis, IN). PPT-A probes were derived
from a cDNA cloned into the BamH I
site of pGEM1 [pG1-b-PPT (36)]. The antisense probe was
transcribed with T7 RNA polymerase (Promega Biotec, Madison, WI) after
the plasmid was linearized with Hind
III (Promega Biotec). This resulted in a cRNA complementary to a 508-bp
fragment of the -PPT mRNA, including the 75-bp
5'-untranslated region, the entire protein-coding region, and a 43-bp fragment of the 3'-untranslated region (36). A sense
-PPT probe was transcribed with SP6 RNA polymerase (Promega Biotec) to provide a control for the specificity of the antisense probe. NK-1
receptor probes were derived from cDNAs inserted into the Hinc II site of pBluescript (21).
Antisense probes complementary of the 5' (nt
1 to
642)- and 3'-coding regions (nt
637 to
1224) of the NK-1 receptor mRNA were transcribed with T3 RNA polymerase (Promega Biotec) after the plasmid was linearized with
Xho I (Promega Biotec) and
Xba I (GIBCO BRL, Life Technologies,
Gaithersburg, MD), respectively. Sense NK-1 receptor probes were
transcribed with T7 RNA polymerase after the plasmid was linearized
with Xba I and
Xho I. Hybridization was carried out
overnight by exposing the samples to a solution containing 2.5 µg of
probe per milliliter at 42°C. Each sample was then washed with
saline-sodium citrate buffer for 15 min, incubated with 20 µg/ml
RNase A for 30 min at 37°C to remove nonhybridized probe, and
washed again in saline-sodium citrate buffer. Digoxigenin-labeled probe
was detected by enzyme-linked immunoassay followed by an overnight
color reaction (BCIP, Genius 3 kit, Roche Molecular Biochemicals)
according to the recommendations of the manufacturer. The samples were
counterstained with eosin and sealed with coverslips.
RT-PCR amplification of PPT transcripts.
Total RNA was extracted from whole lung, tracheal tissue, or neuronal
cultures, as appropriate, with RNAzol B (Tel-Test, Friendswood, TX). Single-stranded DNA was synthesized from 2 µg of RNA
with use of dT priming and Moloney murine leukemia virus RT (GIBCO BRL). PCR reactions contained 6 µl of 10× reaction buffer, 30 pmol of each primer in 1 µl of solution, 2 µl of 5 mM
deoxynucleotide triphosphate mixture, 1 U of
Taq polymerase, and 66 µl of sterile water. Amplification was conducted in a programmable thermal controller (model PTC-100, MJ Research, Watertown, MA) and involved denaturation at 96 and 94°C for 3 min and 30 s, respectively, annealing at 57°C for 1 min, and extension at 72°C for 4 min for a total of 25 cycles. Primers for RT-PCR of PPT-A transcripts were as follows: sense 5'-ATGCCCGAGCCCTTTGAGC-3' (exon 2) and antisense
5'-ATTGCGCTTTCATAAGCC-3' (exon 7). These primers
allowed discrimination of the three alternatively spliced transcripts
of the PPT-A gene by the size of the PCR products: 181-bp -PPT,
235-bp
-PPT, and 190-bp
-PPT. As a control for the stability of
the neuronal populations present in the tracheal xenografts, we
performed RT-PCR of the microtubule-associated protein 2 (MAP-2), a
constitutive protein expressed exclusively in the axon, dendrites, and
cell bodies of neurons (23). The primers were as follows: sense
5'-ACGAAGGAAAGGCACCACACT-3' and antisense
5'-GTATCTGAATAGGTGCCCTGT-3'. The 148-bp
product was sequenced to confirm its identity with the
corresponding segment of the rat MAP-2 gene. All PCR products were
electrophoresed on 1.5% agarose gels and stained with ethidium
bromide. Southern blotting was performed with
32P-labeled antisense probes:
pG1-
-PPT and a probe generated by cloning the MAP-2 PCR product into
a pCR-TOPO vector (Invitrogen, San Diego, CA).
Casein zymograms and Western blotting.
Immediately after collection, lung lavage fluid was separated from
suspended cells by centrifugation at 4°C and frozen at 70°C.
Casein zymograms were performed as described previously (12). For
Western blot analysis, 30 µl of lavage fluid were loaded on a 10%
SDS-polyacrylamide gel and electrophoresed. The gel was then
transferred to a polyvinylidene difluoride membrane, which was placed
in a blocking solution of 3% powdered milk in TBS-Tween for 1 h. The
membrane was incubated in a solution of primary antibody (Table 1) in
equal parts of TBS-Tween and 3% powdered milk for 1 h at room
temperature and washed thoroughly. Antibody binding was detected by
incubation for 45 min with a horseradish peroxidase-conjugated
secondary antibody (donkey anti-rabbit, 1:2,000) at room temperature
and development by chemiluminescence (Amersham Pharmacia Biotech,
Piscataway, NJ).
RIA. Substance P RIA was performed as previously described (37). Bolton-Hunter-iodinated substance P (2,000 Ci/mmol) was purchased from NEN Life Sciences (Boston, MA). The anti-substance P antiserum R5 (30) was used at a dilution of 1:400,000. Samples were assayed in duplicate at multiple dilutions and compared with synthetic substance P standards. The linear range of the standard curve was 25-300 pg/ml.
Data analysis. Southern blots of the RT-PCR products were processed in a Storm phosphor screen and analyzed for pixel intensity with ImageQuant (Molecular Dynamics, Sunnyvale, CA). Pixel intensities or, when appropriate, their ratios were compared by Student's t-test or Wilcoxon signed rank test (depending on whether the data were or were not normally distributed) for single comparisons or by ANOVA with replication for multiple comparisons (effects of time on mRNA levels in tracheal xenografts).
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RESULTS |
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Baseline expression of PPT-A and NK-1 receptor mRNA and protein by
airway ganglia.
Both -PPT and NK-1 receptor mRNAs were detected by in situ
hybridization in tracheal superficial muscular plexus neurons, peribronchial neurons, and occasionally in longitudinal trunk ganglia
(Fig. 1). PPT-A and NK-1 receptor proteins
were also demonstrated by immunohistochemistry, coexisting in neuronal
bodies identified by neurofilament M or protein gene product 9.5 immunoreactivity in the same locations (Fig.
2).
-PPT mRNA staining was similar between rats and, within each rat, among all the stained neurons. In
contrast, the intensity of immunostaining for PPT-A protein (and
presumably the individual cellular levels of the peptides) varied
substantially. In some rats, virtually all the neurofilament M-stained
cells in the area of the muscular superficial plexus were
immunoreactive for PPT-A protein (Fig. 2). In other rats, only a
portion of the cells contained detectable PPT-A protein. We found no
discrepancies in the immunofluorescent patterns generated in
adjacent slides by three antisera of different specificity against
PPT-A-encoded peptides (Table 1). We confirmed the specificity of
double-fluorescent staining for
-PPT and NK-1 receptor proteins by
the disappearance of the appropriate fluorescent signal in the samples
pretreated with immunizing antigens. We used these controls and the
differences in the intracellular distribution of the fluorescent
signals to distinguish specific antibody labeling from bleed-through
and autofluorescence artifacts.
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Expression of PPT-A and NK-1 receptor after denervation.
PPT-A and NK-1 receptor expression persisted after the airway ganglia
were separated from their motor and sensory inputs, independent of
whether this separation was accomplished by grafting tracheal segments
into nu/nu mice, by syngeneic lung
transplantation (Fig. 3), or by isolation
of the ganglion neurons in primary culture (Fig.
4). Substance P-immunoreactive fibers,
which could be found easily at high-power magnification in the
subepithelial region of control tracheae and bronchi, were absent from
the tracheal xenografts and the bronchi contained in the syngeneic lung
grafts. Substance P immunoreactivity was apparent, however, in the
projections from cultured neurons, often colocalizing with
synaptophysin immunoreactivity (Fig. 4). Neuronal NK-1 receptors were
located predominantly in perikarya in tissues and culture. Similar to
Dey et al. (7), we found that only a minority of the ganglia in the
superficial muscular plexus of the trachea were immunoreactive for
choline acetyltransferase. These neurons appeared to retain their
cholinergic phenotype in the tracheal xenografts.
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Cell populations expressing PPT-A and NK-1 receptor genes.
PPT-A gene-encoded mRNA and peptides were detected only in
neurofilament M or protein gene product 9.5-immunoreactive cells. NK-1
receptor immunoreactivity was present frequently in macrophages located
in the wall of the tracheal xenografts or in the alveolar spaces of the
lung (Fig. 5).
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Analysis of PPT mRNAs by RT-PCR.
Tracheal xenografts and lung syngeneic grafts contained greater amounts
of - and
-PPT mRNA than matching tracheal segments and
contralateral lungs, respectively. In the tracheal xenografts, the
increase in PPT mRNAs reached its peak at ~3 days and became attenuated thereafter (Fig. 6). MAP-2 mRNA
levels decreased uniformly after implantation, suggesting neuronal
loss. In the lung grafts, the increase in PPT mRNA was still detectable
2 wk after transplantation (Fig. 7). PPT
mRNA levels were similar after intratracheal instillation of
anti-ovalbumin antibodies (10 ± 2% of brain control) or normal saline alone (21 ± 4%; P = 0.40 by ANOVA) into rats previously injected intravenously with ovalbumin.
This finding suggests that, at least during the 4-h duration of the
experiments, immune complex inflammation does not upregulate local
substance P production or attract foreign substance P-producing cells
into the lungs. There was no difference in PPT mRNA levels between the
left and right lungs of the six control rats subjected to a left
thoracotomy. MAP-2 mRNA was not affected by lung transplantation or
thoracotomy alone. RT-PCR products from
-PPT were ~2.5 times more
abundant than those from
-PPT in trachea and lung. This ratio was
not altered in the lung grafts.
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Effects of NK-1 receptor antagonist on immune complex-mediated
injury in rat lungs.
Intravenous injection of ovalbumin followed by intratracheal
instillation of anti-ovalbumin antibodies caused a diffuse lung injury
characterized by edema, patchy hemorrhage, and infiltration with
polymorphonuclear leukocytes and macrophages. Casein zymograms of the
lavage fluid revealed a band of casein lysis immediately above the
20-kDa marker, which was identified by Western blotting analysis as
corresponding to the activated forms of macrophage metalloelastase and
matrilysin. Pretreatment with LY-306740 inhibited the activation of
both proteases but had only a limited effect on the increase in
alveolar-capillary permeability observed after the immune complex
injury (Fig. 8).
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Immune complex-mediated injury in syngeneic lung grafts.
There were no differences in respiratory rate, activity, or food
consumption between syngeneic lung recipients and control rats before
the administration of ovalbumin and anti-ovalbumin antibodies. After
immune complex formation, however, the transplanted rats developed
greater respiratory difficulty than the control rats. Five of 12 lung
graft recipients untreated with LY-306740 died after developing chest
wall retractions and a gasping pattern of breathing. None died in the
control group during the same period (2 = 5.9;
P = 0.02). At postmortem examination,
none of the lung grafts had signs of vascular or bronchial obstruction.
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Substance P concentrations in lung lavage fluid. Equivalent amounts of substance P (measured by RIA) were recovered in the lavage fluid from lung grafts (median substance P recovered: 350 pg; 10th and 90th percentiles: 231 and 471 pg, respectively) and control lungs (median: 360 pg; 10th and 90th percentiles: 251 and 1,350 pg) after immune complex formation. Substance P was usually undetectable in lavage fluid from rats for which the intratracheal antibody was replaced with normal saline.
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DISCUSSION |
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Overwhelming evidence has accumulated in recent years placing the tachykinins and their receptors at the center of a mechanism by which the nervous system can influence the course of inflammation in the lungs (3, 5). The prevailing understanding of this mechanism is that substance P and other products of the PPT-A gene [which are often referred to as "sensory neuropeptides" (48)] are released exclusively by lung sensory fibers in response to inflammatory mediators, mechanical and chemical irritation, and antidromic stimulation of the vagus nerve. Here, we demonstrate that airway intrinsic neurons not only contain PPT mRNA and mature substance P but also express the NK-1 receptor. In addition, we present evidence that, lacking other apparent tachykinin sources, peptidergic airway neurons can support the development of an injury that requires activation of the NK-1 receptor. These observations challenge the concept that sensory fibers are the only source of PPT-A-derived peptides in the airways. Moreover, they suggest that tachykinins act as messengers in a neuronal-based system for the amplification of inflammation in the lungs.
PPT-A and NK-1 receptor expression by parasympathetic ganglia. The presence of substance P in airway ganglia has been refuted (32, 35) and affirmed (8, 9, 44) in multiple studies involving several species. The controversy was clarified substantially after a detailed quantitative study by Dey et al. (7) demonstrated that a large proportion of the neurons in the superficial muscular plexus of the ferret's trachea are immunoreactive for substance P. The present results confirm the findings of these investigators by showing the presence of PPT-A mRNA and protein products in the airway ganglia of the same plexus in the rat.
The coexistence of substance P and NK-1 receptors in the same nerve cells is a novel finding. Its implications include the possibility that ganglia-released tachykinins act in a paracrine or autocrine fashion to regulate ganglionic excitability (43) or even to inhibit their own secretion in the presence of sustained noxious stimuli to the airway. Although presumed from observations that ganglion cells receive inputs from substance P-containing sensory fibers (33, 42) and respond to exogenous application or endogenous release of substance P (18, 50), the existence of NK-1 receptors in airway ganglia has remained under debate. Watson et al. (50), for instance, showed that the selective NK-1 receptor antagonist GR-71251 inhibits the potentiating effect of phosphoramidon on the airway smooth muscle contraction produced by preganglionic electrical stimulation in the guinea pig. Myers and Undem (43), on the other hand, demonstrated, also in the guinea pig, that another NK-1 receptor antagonist, CP-96345, has no influence on the membrane depolarization induced by capsaicin in airway ganglia. The key to understanding these discrepancies may lie with the growing realization that airway ganglia are a heterogeneous population of neurons. The differences that have been described in their anatomic organization (2, 52), morphological characteristics (52), and peptidergic or neurotransmitter phenotype (7) probably denote adaptations to specialized functions. The coexistence of PPT-A and NK-1 receptor gene products in neurons from the superficial muscular plexus, for instance, can be interpreted as an indication that substance P acts as a neurotransmitter within this plexus. Colocalization of substance P and synaptophysin in the projections from cultured ganglia demonstrates that mature peptide can be transported into ganglionic fibers, where it is likely to be released synaptically. On the other hand, the disappearance of most tachykinin-immunoreactive nerve fibers from the tracheal and lung grafts suggests that, rather than forming an extensive network, the short substance P-containing projections seen emerging from the ganglia (Fig. 2) release their peptide products onto nearby neurons or directly into the tissues (smooth muscle or blood vessels). An interganglionic function is concordant with observations made in sympathetic ganglia (26), where substance P is circumscribed to neuronal perikarya and intraganglionic processes. Release into neighboring tissues, on the other hand, is consistent with the recent finding that approximately one-third of the substance P-containing fibers in the trachealis muscle (but no substance P-containing subepithelial fibers) survive in ferret tracheal segments maintained in culture for 7 days (10). Ganglion neurons may also secrete substance P parasynaptically. Calcium-regulated exocytosis of substance P-containing vesicles from cell bodies of dorsal root ganglia neurons has been shown in culture (22).Upregulation of PPT-A gene expression after denervation.
Tracheal and pulmonary PPT mRNA content increased after these tissues
were implanted into nu/nu mice and
syngeneic rats, respectively. Although alveolar macrophages and
intestinal eosinophils can produce substance P (29, 39), we did not
detect -PPT mRNA or substance P in the cellular infiltrates found in
tracheal xenografts and injured lungs. Furthermore, there was no rise
in PPT mRNA after the intense infiltration of control (nongrafted)
lungs by inflammatory cells after immune complex injury. Thus we
ascribe the increase in PPT transcripts to a neuron-specific response
to the grafting process.
Substance P-dependent inflammation in syngeneic lung grafts. There is evidence that activation of the NK-1 receptors is necessary for the inflammatory response produced by the deposition of complexes between a soluble antigen and preformed antibodies in the airways (Arthus reaction). Gene-targeted disruption of the receptor protects mice from this response (5). Pretreatment with NK-1 receptor antagonists reduces the presence of inflammatory cells at the peak of inflammation in murine lungs by 18-64%, depending on cell type (24). In our study, LY-306740 markedly reduced the induction of lung metalloproteases after immune complex injury but had only a limited (30-40%) protective effect against the increase in alveolar-capillary permeability produced by the same injury. These findings confirm our hypothesis that NK-1 receptor stimulation by tachykinins contributes to the Arthus reaction in the rat's lungs. They also suggest the existence of alternative pathways for the immune complex-induced disruption of alveolar-capillary permeability in this species, perhaps involving other tachykinin receptors.
The characteristics of the immune complex injury in the lung grafts provide some new insights into the sources of tachykinin secretion in the lungs. For instance, the extent of the alveolar injury and the concentrations of substance P found in the lavage fluid indicate that this peptide reaches distal portions of the airway tree. Although small airways are well innervated by the parasympathetic system (45), they are also devoid of ganglion neurons. How the tachykinins released by these neurons can participate in a diffuse inflammatory process involving distant portions of the lung remains unresolved. Transbronchial transport from the relatively dense neuronal populations found around large- and medium-size airways provides a possible explanation, especially in small mammals like mice or rats, for which the distances involved are very small. Diffusion into the interstitium or even the lumen of peribronchial air spaces offers an alternative explanation, especially after epithelial permeability becomes disrupted during the early phases of inflammation. Finally, axonal transport of tachykinins to the vicinity of bronchial or pulmonary vessels may place tachykinins near their receptor targets in inflammatory cells as these cells are being recruited into the lungs.Summary. In this study, we demonstrate the existence of an intrinsic population of airway neurons that contain PPT-A-derived tachykinins and NK-1 receptors. In addition, we provide evidence suggesting that these neurons participate in the amplification of immune complex-mediated inflammation in the lungs. It is tempting to speculate that, by a similar mechanism, denervated neurons contribute to local airway inflammation in the course of disease processes such as bronchiolitis obliterans after lung transplantation.
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ACKNOWLEDGEMENTS |
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We thank Prof. G. Alexander Patterson for advice and help with rat syngeneic lung grafts. We also acknowledge the technical assistance provided by Charlene Pearman.
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FOOTNOTES |
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These studies were supported by National Heart, Lung, and Blood Institute Grant HL-57998.
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. J. Pérez Fontán, Dept. of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, One Children's Place, St. Louis, MO 63110 (E-mail: FONTAN{at}KIDS.WUSTL.EDU).
Received 21 July 1999; accepted in final form 24 September 1999.
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