Sage Journals: Discover world-class research

Abstract

Substance P contributes to the physiological homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. The distribution of substance P receptor [neurokinin-1 receptor (NK-1R)] in lungs of normal sheep and sheep with acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica was determined by immunohistochemistry (IHC). Three rabbit polyclonal antibodies generated to the same cytosolic C-terminal portion of NK-1R (residues 393-407) were tested. NK-1R immunoreactivity was traced in digital images and quantified with IPLAB software. There were no significant differences in NK-1R protein density between normal and infected lambs. Antibody 1 had the broadest distribution and intensity, and stained alveolar septae, smooth muscle cells of airways and vessels, epithelial cells of airways and alveoli, and submucosal glands. When all animals from the study were included, there was a trend towards decreased NK-1R immunoreactivity over time. The work suggests that (a) the density of NK-1R does not change during progression of bacterial (M. haemolytica) bronchopneumonia, (b) NK-1R is widely distributed in ovine lung and decreases with age, and (c) antibodies to the same NK-1R cytosolic region can vary in specificity and affinity.

Keywords

substance P NK-1 receptor ovine lung immunohistochemistry

Tachykinins (TKs) belong to a family of neuropeptides that share a common carboxyl-terminal domain Phe-X-Gly-Leu-Met-NH₂, where X is either an aromatic (Tyr or Phe) or a branched aliphatic (Val or Ile) amino acid (Erspamer 1981). This common carboxyl-terminal domain is essential for the agonist activity, while the amino terminal domain provides selectivity for different receptor subtypes (Piedimonte 1995). The mammalian TKs, also called neurokinins (NKs), include substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the N-terminal extended forms of NKA, neuropeptide K (NPK) and neuropeptide γ (NP7) (Piedimonte 1995). In the airways, SP and NKA, but not NKB, are expressed by unmyelinated sensory C-fibers (Barnes 2001) which, in parallel to the efferent autonomic nerves (cholinergic and adrenergic), innervate the upper and lower respiratory tract of several species, including humans (Piedimonte 1995). These sensory nerves can be activated by a variety of stimuli such as the neurotoxin capsaicin, electrical nerve stimulation, changes in osmolarity or temperature, histamine, bradykinin, SO₂, methacholine, prostaglandins, leukotrienes, cigarette smoke, ozone, and other inflammatory mediators released by allergic or non-allergic mechanisms (Piedimonte 1995). In addition to sensory C-fibers, there is a growing body of evidence that certain immune cell types such as monocytes-macrophages, neutrophils, eosinophils, lymphocytes, and dendritic cells also have the ability to produce SP and NKA in inflamed lungs (Maggi 1997; Ramirez-Romero et al. 2001). These studies suggest that TKs may contribute to inflammatory responses of the airways (Barnes 2001).

Among TKs, SP is considered to be the main sensory neurotransmitter in mammalian airways (Corcoran 1996). The distribution of capsaicin-sensitive SP-like immunoreactive (SP-Li) nerve fibers in the respiratory tract has been examined in cattle (Nishi et al. 2000), sheep (Corcoran 1996; Ramirez-Romero et al. 2000,2001), and horses (Sonea et al. 1994a,b). These studies indicate that SP-Li nerve fibers are much more numerous in young animals than in adults and are mainly seen in the epithelium (cattle, horses), in connective tissue beneath the epithelium and around blood vessels (cattle, horses, sheep), submucosal glands (cattle, horses, sheep), airway smooth muscles (neonatal foals, sheep), and bronchial and pulmonary vessels (horses). A similar distribution has been found in the human respiratory tract (Bai et al. 1995). Together with NKA, SP produces a proinflammatory effect, that contributes to the physiological homeostasis of pulmonary airways and vasculature and is called “neurogenic inflammation” (Geppetti and Holzer 1996). It stimulates mucus secretion from submucosal glands and goblet cells (Rogers 2000), mucociliary clearance (Wong et al. 1990), plasma protein extravasation (Gamse and Saria 1985; Keith 2000), vasodilatation (Pedersen et al. 2000), leukocyte adhesion to the vascular endothelium (Matis et al. 1990), alveolar macrophage and monocyte release of inflammatory cytokines, and mast cell degranulation (Barnes 2001). SP also enhances cholinergically mediated broncho-constriction, angiogenesis, and proliferation and chemotaxis of human lung fibroblasts (Barnes 2001; Rice et al. 2001). However, there are some exceptions, such as SP-induced contraction of pulmonary arteries in rabbits (Shirahase et al. 1995) or relaxation of bronchial smooth muscles in rats (Bodelsson et al. 1999). These species differences might be attributed to different pathways (direct and indirect) of neurokinin receptor activation (Bodelsson et al. 1999; Amadesi et al. 2001) and distribution, indicating the precautions needed during interpretation of interspecies diversity.

Three mammalian NK receptors have been cloned from several species, and including NK-1R, NK-2R, and NK-3R (Joos et al. 1994). All belong to the super-family of guanine nucleotide-binding, protein-coupled receptors, with seven putative transmembrane-spanning α-helices, an extracellular amino-terminus, and an intracellular carboxyl tail (Piedimonte 1995; Joos et al. 2000). Although, when present at sufficiently high concentrations, each of the naturally occurring TKs can act as a full agonist on all three receptors (Regoli et al. 1987), SP has the highest binding affinity for NK-1R (Regoli et al. 1988). In human subjects, positive immunostaining for NK-1R is present in the bronchial smooth muscles, myoepithelial cells of bronchial glands, in the endothelium and smooth-muscle layer of bronchial vessels and pulmonary arteries, and occasionally in nerves (Mapp et al. 2000). NK-1R is also present in the apical portion of bronchial epithelium (particularly in goblet cells) and in some inflammatory cells (Chu et al. 2000). In rat lung, NK-1R is present in the apical portion of the airway epithelium, from the trachea to the respiratory bronchioles, but not in alveoli, submucosal glands, or smooth muscle of the airways or most blood vessels, except for the smooth muscle of some intermediate-sized pulmonary veins (Ichikawa et al. 1995). In contrast to rats, guinea pig lung has NK-1R immuno-reactivity in smooth muscle from trachea to small bronchioles and from pulmonary vessels (Carstairs and Barnes 1986). These studies underscore the lack of complete correlation between the anatomic distribution of receptors and SP-like nerve fibers in the lung of various species, indicating the importance of revealing the distribution of NK-1R, particularly in attempting to understand the neuropeptide's mechanisms of action and functions.

Corcoran and Haigh (1992) have suggested that NK-1R predominates in sheep airways compared to other NK receptors. Therefore, the first aim of our study was to assess NK-1R distribution in the lung of healthy weaned lambs and to compare its immunoreactivity with various stages of pneumonia caused by the ovine pathogen M. haemolytica (Ramirez-Romero et al. 2001) during the 45-day period. Our second objective was to test the distribution of immunoreactivity of three rabbit polyclonal antibodies generated against a synthetic NK-1R peptide identical to a 15-amino-acid section of the cytosolic C-terminal portion of NK-1R (residues 393-407) in ovine lung.

Materials and Methods

Experimental Animals

Nineteen sheep of both sexes and mixed breed, 3 months old (weaned), were obtained from Iowa State University Laboratory Animal Resources. The lambs were randomly assigned to two groups, each group being maintained in a separate climate-controlled isolation room until sacrifice. After a 24-hr adaptation period, one group (nine animals) received saline and the other (10 animals) received Mannheimia (Pasteurella) haemolytica. The lambs were subdivided into six groups according to the time of euthanasia at day 1, 15, or 45 after inoculation (Table 1). This protocol was approved by the Iowa State University Animal Care Review Committee.

Inoculation

M. haemolytica serotype A1 ovine strain 82-25 was obtained from a natural case of pneumonic pasteurellosis in sheep. The animals received 5 ml of either bacterial inoculum containing 1.9 × 10⁸ cfu/ml (Brogden et al. 1995) or pyrogen-free saline (PFS). Intrabronchial inoculation was performed using fiberoptic bronchoscopy as previously described (Brogden et al. 1984,1995). Briefly, in non-intubated, sedated lambs, the bronchoscope (model VFS-2; Schott Fiber-optics, Southbridge, MA) was inserted via the nasotracheal route. The inoculum was deposited into the main bronchus of the right cranial lobe.

Table 1

Experimental design

	Number of lambs and days post inoculation
Inoculum a	Day 1	Day 15	Day 45
PFS b	3	3	3
M. haemolytica	3	4	3

^aSee Materials and Methods for inoculation procedure.

^bPFS, pyrogen-free saline.

Tissue Collection and Processing

The animals were sacrificed at day 1, 15, or 45 after inoculation with an IV (intrajugular) overdose of sodium pentobarbital. At necropsy, samples were taken from the pulmonary deposition site (right cranial lobe). One sample was taken proximal to the hilus and included the cranial bronchus. The other one was from the distal part and included the apex of the cranial lobe (Ramirez-Romero et al. 2001). Samples were fixed by immersion in 10% neutral buffered formalin [48 hr at room temperature (RT)] and replicate sections were stained with hematoxylin-eosin to assess the microscopic lesions of the lung samples, or stained by immunohistochemistry (IHC) for localization of the NK-1 receptor in the same areas of lung.

Immunohistochemistry

Sections were stained with antibodies to the NK-1 receptor using the streptavidin-biotin complex peroxidase method, and the metal-enhanced diaminobenzidine (DAB) method was used to reveal the peroxidase activity as previously described (Shu et al. 1988). Briefly, the slides were heated in the oven (58C) for 30 min, deparaffinized in xylene, immersed through a succession of graded alcohols, and then fully hydrated. Next they were immersed in 1% H₂O₂ in ultrapure water for 40 min to block endogenous peroxidase activity. After several rinses in BioGenex PBS at pH 7.2-7.4 containing 0.1% Tween-20 (Wash Buffer; BioGenex, San Ramon, CA), nonspecific binding was blocked by incubation in 10% normal goat serum (NGS; Sigma, St Louis, MO) and 3% bovine serum albumin (essentially γ-globulin-free BSA; Sigma A-7030) in BioGenex PBS for 20 min. The sections were then placed in a humidified container and incubated with primary antibodies for 18 hr at 4C. The primary antibodies used in this study were polyclonal rabbit anti-NK-1R peptide, in the form of hyperimmune serum, diluted 1:4000 (antibody 1 from Oncogene rabbit #653), 1:3000 (antibody 2 from Oncogene rabbit #652), and 1:3000 (antibody 3 from Oncogene rabbit #654) in antibody diluent (Common Antibody Diluent; BioGenex). Then the slides were placed on the OptiMax Plus automated cell-staining machine (BioGenex) to complete the remaining procedures. The machine washed the slides with BioGenex PBS, applied 2% H₂O₂ in BioGenex PBS for 20 min, and then washed with BioGenex PBS again. The sections were then preincubated briefly (30 sec) with a biotinylated goat anti-rabbit secondary antibody and then, with no rinse in between, the same reagent was applied for 50 min (ready-to-use human serum-adsorbed biotinylated goat anti-rabbit already at 2 μg biotinylated IgG/ml; Kirkegaard & Perry Laboratories, Gaithersburg, MD). After several rinses in BioGenex PBS, the sections were incubated for 50 min at RT with a streptavidin-HRP complex (Super-Sensitive peroxidase-conjugated streptavidin; BioGenex). Finally, the slides were washed with BioGenex PBS and exposed for 12 min to a metal (cobalt chloride and nickel chloride)-enhanced DAB substrate (ImmunePure Metal Enhanced DAB Substrate Kit; Pierce Chemical, Rockford, IL) to visualize immunoreactivity (IR). The slides were then washed thoroughly with ultra-pure water to stop the DAB reaction, counterstained for 2 min with quarter-strength Shandon's acidified Harris haematoxylin, subjected to Scott's tapwater (10 g MgSO₄ and 2 g NaHCO₃ per liter of water) for 1 min, dehydrated through graded alcohols, cleared in xylene, and coverslipped with Permount (Sigma). Dark brown precipitate was accepted as a positive reaction. Control sections lacked staining and included (a) no primary antibody, (b) normal rabbit serum, (c) normal rabbit IgG (Upstate Biotechnology; Lake Placid, NY), and (d) NK-1R antibody preincubated with synthesized NK-1R peptide.

Primary Antibodies

Antisera tested in this study were purchased from Oncogene (Oncogene Research Products, a division of CN Bioscience; San Diego, CA). They were raised in three rabbits (#652, #653, and #654 at Oncogene) in response to the peptide KT-MTESSSFYSNMLA, which corresponds to a 15-amino-acid section (residues 393-407) of the cytosolic carboxy-terminal region of the NK-1 receptor. The peptide was crosslinked via glutaraldehyde to bovine thyroglobulin. These hyperimmune rabbit sera were tested for specificity to NK-1R by preincubating them for 45 min with different concentrations of a synthesized 15-mer peptide (KTMTESSSFYSNMLA), which was identical to the peptide used by Oncogene (now Calbiochem) to immunize the rabbits. This peptide was synthesized by automated solid-phase peptide synthesis and purified by reversed-phase high-pressure liquid chromatography (Joel Nott; Protein Facility, ISU, Ames, IA). A minimum of 33.93 μg/ml (20 μM) concentration of this peptide completely inhibited IHC staining by all three rabbit anti-NK-1R IgGs.

Table 2

Mean values for the area of neurokinin 1 receptor (NK-1R) protein expression in the lung of sheep inoculated with PFS or M. haemolytica at 1, 15, and 45 days post inoculation (p.i.)

	Mean value ± SEM (% area)
Days p.i.	PFS a	M. haemolytica
Day 1	14.47 ± 2.30	13.13 ± 2.30
Day 15	13.02 ± 2.30	13.50 ± 1.99
Day 45	10.70 ± 2.30	6.07 ± 2.30

^aPFS, pyrogen-free saline.

Table 3

Mean values for distribution of neurokinin 1 receptor (NK-1R) protein in sheep lung during the period of 45 days; values for all three primary antibodies were pooled

	Mean value ± SEM (% area) a
Structures	Day 1	Day 15	Day 45
Alveoli	32.63 ± 2.72 b	23.37 ± 2.52 b	3.39 ± 2.72
Bronchioles	13.26 ± 2.72 b	9.86 ± 2.52	4.07 ± 2.72
Bronchi	16.90 ± 3.15 b	15.91 ± 2.85	7.89 ± 2.72
Sub. glands	23.23 ± 3.33	22.39 ± 2.85	16.97 ± 2.72
Vessels	10.50 ± 2.72	10.50 ± 2.52	5.22 ± 2.72

^aDays 1, 15, and 45 are time points when animals were sacrificed.

^bSignificantly different from day 45.

Image Analysis for NK-1R Immunoreactivity

The structures examined in the lung samples included alveoli, bronchi, bronchioles, submucosal glands, and vessels. Specific compartments observed within bronchi and bronchioles included lamina epithelialis mucosae, lamina propria mucosae, and lamina muscularis mucosae, while vessels included tunica intima and media as one compartment and adventitia as the other. Alveoli and submucosal glands were assessed as single compartments. Three fields of each structure, chosen randomly from every slide, were examined with a Zeiss Axioskop fitted with Neoflur objectives (×20 objectives), a 100-W light source, and a Sony DXC-3000A camera. The images were then captured with IPLAB Software (Scanalytics; Fairfax, VA) on a Macintosh G3 (Apple; Cupertino, CA). Images were edited on a Micron workstation with Photoshop (Adobe Photoshop 6.0; Mountain View, CA), thresholded for analysis, and quantified on the G3 with the IPLAB software.

Statistical Analysis

Results represent percentages of stained areas over total areas of randomly picked lung structures/compartments. Data are expressed as means ± SEM of three randomly chosen fields from each structure/compartment for each of the assessed animals. Differences in means between three tested antibodies, day, treatment, and structure/compartment effects were analyzed using two-factor analysis of variance (ANOVA) test. All analyses were performed using SAS software. Values were considered to be significant at p<0.05.

Table 4

Mean values for total area of neurokinin 1 receptor (NK-1R) protein expression in sheep lung during the period of 45 days; three primary antibodies were compared

	Mean value ± SEM (% area) b
Days p.i. a	Ab1	Ab2 and Ab3
Day 1	33.91 ± 1.86 c, d	4.70 ± 1.90
Day 15	28.15 ± 1.67 c, d	4.66 ± 1.73
Day 45	11.98 ± 1.75 a	3.04 ± 1.75

^aDays 1, 15, and 45 are time points when animals were sacrificed.

^bAb1, Ab2, and Ab3 denote antibodies 1, 2, and 3, respectively (see Materials and Methods).

^cSignificantly different from Ab2 and Ab3 on the same day.

^dSignificantly different from Ab1 on day 45 p.i.

Results

Microscopic Lesions

Animals inoculated with M. haemolytica developed severe lesions at the site of inoculum deposition and have been described previously (Ramirez-Romero et al. 2001). Briefly, in animals sacrificed at 1 day after inoculation with bacteria, lungs had extensive areas of seroproteinaceous fluid, neutrophil infiltration, hemorrhage, and necrosis. Lungs of the animals at 15 days after inoculation were characterized by fibrosis of alveolar and interlobular septa and pleura, marked proliferation of type II alveolar cells, many alveolar macrophages within the alveoli, and occasional foci of pyogranulomatous inflammation. Finally, at 45 days after inoculation, lungs had epithelial cell proliferation in alveoli and bronchioles as well as marked pleural and interlobular fibrosis. In addition, there was increased tortuosity of the small airway lumens that were partially obliterated by fibrous connective tissue and occasional areas of alveolar atelectasis or emphysema. All control animals, inoculated with saline, lacked lesions.

Immunoreactivity of Primary Antibodies

IHC for NK-1R was repeated on the same set of lung tissues using three different polyclonal antibodies, each of which was produced in a different rabbit but to the same region of NK-1R. Control sections lacked staining and included no primary antibody, normal rabbit serum from Sigma, normal rabbit IgG, and NK-1R antibody preincubated with a synthetic 15-aa peptide of NK-1R residues 393-407.

Figure 1

NK-1R immunoreactivity in lung tissues stained with Ab1 on day 1 (A1-5), 15 (B1-5), and 45 (C1-5) after inoculation progressively decreased over time. Structures examined included alveoli (A-C1), bronchioles (A-C2), bronchi (A-C3), submucosal glands (A-C4), and blood vessels (A-C5).

Figure 2

(A) Negative control that lacks immunoreactivity. (B) Lung tissues stained with Ab2, in which there is immunoreactivity within the smooth muscle of the bronchiole (B1; M.h., day 45 p.i.), bronchus (B2; PFS, day 15 p.i.), and within the vessel (B3; PFS, day 15 p.i.). (C) Lung tissues stained with Ab1, where there is staining within the alveolar septae (C1; PFS, day 1 p.i.), within the bronchial epithelial cells (C2; PFS, day 15 p.i.), within the submucosal glands (C3; M.h., day 1 p.i.), and in the smooth muscle of the vessel (C4; PFS, day 1 p.i.). (D) Lung tissues stained with Ab3, where staining is present within the bronchiolar epithelial cells (D1; M.h., day 45 p.i.), within the submucosal glands (D2; M.h., day 15 p.i.), and in the smooth muscle of the vessel (D3; M.h., day 45 p.i.).

Quantitative Image Analysis of NK-1R

The NK-1R immunoreactivity was widely distributed in ovine lung. However, we did not see any difference in the amount of NK-1R protein expression when control (PFS) and infected (M. haemolytica) animals were compared at day 1, 15, and 45 p.i. (Table 2). Because of this finding, all the values from control and infected animals were pulled together and further analyzed. The area of NK-1R immunoreactivity were greatest at 1 day after inoculation and progressively decreased at 15 and 45 days after inoculation (Table 3). However, this progressive decrease in immunoreactivity was significant only for Ab1 (p<0.05) (Table 4; Figure 1).

When immunoreactivity for three primary antibodies was compared, the area of immunoreactivity for Ab1 was significantly greater (p<0.05) than for Ab2 and Ab3 in all the assessed structures (alveoli, bronchioles, bronchi, submucosal glands, and vessels) (Table 5; Figure 2). The area of immunoreactivity for Ab1 was significantly greater in alveoli and submucosal glands of bronchi (p<0.05) than in bronchioles, bronchi, and blood vessels. In contrast, the areas of immunoreactivity for Ab2 and Ab3 were significantly greater in bronchi, submucosal glands of bronchi, and in blood vessels (p<0.05) than in alveoli and bronchioles. Within bronchi and bronchioles, the area of immunoreactivity was greatest in the lamina muscularis and lowest in the lamina propria for all three antibodies (p<0.05) (Table 6). The area of immunoreactivity was significantly greater in lamina muscularis as well as the bronchial and bronchiolar epithelium for Ab1 (p<0.05) compared to Ab2 and Ab3 (Table 6). Within the vessels, the area of immunoreactivity was significantly greater in the tunica media than in the tunica adventitia for all antibodies (p<0.05) (Table 7).

Table 5

Mean values for the area of neurokinin 1 receptor (NK-1R) protein expression in various regions of lung

	Mean value ± SEM (% area) a
Region	Ab1	Ab2 and Ab3
Alveoli	37.84 ± 2.15 b , c	1.75 ± 2.15
Bronchioles	16.80 ± 2.15 b	1.32 ± 2.15
Bronchi	19.91 ± 2.28 b	7.22 ± 2.44
Submucosal glands	35.82 ± 2.37 b , c	5.90 ± 2.44
Vessels	13.00 ± 2.15 b	4.48 ± 2.15

^aAb1, Ab2, and Ab3 denote antibodies 1, 2, and 3, respectively (see Materials and Methods).

^bSignificantly different from Ab2 and Ab3.

^cSignificantly different from bronchioles, bronchi, and vessels.

Table 6

Mean values for the area of neurokinin-1 receptor (NK-1R) protein expression in the bronchial and bronchiolar compartments

	Mean value (%) a
	Ab1	Ab2	Ab3
Lamina epithelialis	17.04 b , c	0.18	1.46
Lamina propria	2.97 b	0.30	0.04
Lamina muscularis	30.71 b , c	18.96 d	5.15 e

^aSEM for each of the values is 1.67. Ab1, Ab2, and Ab3 denote antibodies 1, 2, and 3, respectively (see Materials and Methods).

^bSignificantly different from Ab2 and Ab3.

^cSignificantly different from lamina propria for Ab1.

^dSignificantly different from lamina epithelialis and lamina propria for Ab2.

^eSignificantly different from lamina epithelialis and lamina propria for Ab3.

Discussion

The findings regarding the amount of NK-1R mRNA and protein expression in the lung vary greatly. One study shows that, in asthmatics and patients with COPD, NK-1R mRNA levels are unchanged compared with non-smoking controls but increase twofold in smokers (Bai et al. 1995). Conversely, Chu et al. (2000) demonstrated that asthmatic subjects have increased expression of SP and NK-1R protein in their airway epithelium but not in the submucosa, compared with normal control subjects. In our study, no differences in NK-1R protein densities were note on comparing control to infected lambs. The reason for that may be that the extent of SP production does not change during the M. haemolytica infection. Even though the density of SP-Li nerve fibers tends to decrease during the initiation and at 45 days of M. haemolytica infection, SP production appears increased in macrophages and neuronal ganglion cells during infection (Ramirez-Romero et al. 2001). Therefore, up-regulation of NK-1R appears to be dependent on SP, which is in agreement with studies performed on rat intestine (Pothoulakis et al. 1998). However, we cannot exclude the possibility that the overall level of NK-1R remains unchanged, i.e., any increase in SP binding could be due to an increased presence of NK-1R during pulmonary immune responses resulting from the influx of receptor-bearing leukocytes which, as of yet, have not been assessed by IHC.

Table 7

Mean values for the area of neurokinin-1 receptor (NK-1R) protein expression in the vascular compartments

	Mean value (%) a
	Ab1	Ab2	Ab3
Tunica media	38.04 b , c	23.71 b , c	7.31 b
Adventitia	1.31	0.42	0.10

^aSEM for each of the values is 1.97. Ab1, Ab2, and Ab3 denote antibodies 1, 2, and 3, respectively (see Materials and Methods).

^bSignificantly different from adventitia.

^cSignificantly different from Ab3.

Our data suggest that NK-1R is widely distributed in healthy ovine lung. These results were expected because it was demonstrated that, when injected IV into sheep, SP is a more potent bronchoconstrictor than NKA (Corcoran and Haigh 1992; Rice et al. 2001). It is also likely that the density of pulmonary NK-1R decreases with age (at least for Ab1). Progressive loss of SP-Li nerve fibers during postnatal maturation of the respiratory tract was found in the foal lung (Sonea et al. 1994b), human bronchi (Hislop et al. 1990), and the nasal septum of lambs (Ramirez-Romero et al. 2001). Furthermore, McLeod et al. (1998) demonstrated that the anatomic distribution of the SP-containing fibers and NK-1R in the rat spinal cord correlate well. When combined, these results favor our hypothesis that the density of both the SP-Li nerve fibers and NK-1R decreases with aging in the sheep lung. It has also been shown that NK-1R -/- mice lack generalized immune complex-mediated lung injury compared to wild-type NK-1R +/+ animals (Bozic et al. 1996). Higher numbers of NK-1R in young animals could increase their susceptibility to inflammatory responses.

Localization and characterization of tachykinin receptors have been extensively studied using various morphological and pharmacological approaches (Joos et al. 2000). In the present study, applying a standard IHC procedure, we have evaluated three “different” polyclonal antibodies generated to the same antigen (NK-1R cytosolic region, residues 393-407), whose density and expression have been shown to be related to the NK-1R protein expression (Vigna et al. 1994). After testing these antibodies for their specificity and affinity, we thought that Ab1 was the most specific in recognizing the antigen of interest, for two reasons. First, Ab2 and Ab3 recognized only some of the targets stained by Ab1 with a lower intensity than stained by Ab1. Second, there was no background staining or staining of the structures labeled by Ab1, Ab2, or Ab3 when normal rabbit serum or NRS IgG was used instead of the polyclonal antibodies for the IHC procedure as negative controls. These findings are not unusual because it is well known that antisera produced in response to immunization are a mixture of a heterogeneous population of antibody molecules (Janeway et al. 1999). This heterogeneity can be due to production of antibodies to different epitopes on the immunizing agent (bovine thyroglobulin) as well as to different spots on the single antigenic determinant, such as hapten (peptide in our study).

This work demonstrates the following. (a) The density of NK-1R is unchanged during progression of bacterial (M. haemolytica) bronchopneumonia. (b) NK-1R is widely distributed in ovine lung and decreases with age. (c) Antibodies to the same NK-1R cytosolic region can vary in specificity and affinity. By revealing the precise location and distribution of NK-1R in the lung, selective NK-1R antagonists can be applied more effectively as potential inhibitors of mucus hypersecretion (MEN 11467; Khan et al. 2001) and plasma extravasation (CP 99,994; Ball et al. 1993), FK 888 (Murai et al. 1993). Non-selective antagonists such as FK 224, which are active on both NK-1 and NK-2 receptor sites and can block both plasma extravasation and bronchoconstriction (Regoli et al. 1994), may also be useful for respiratory diseases such as asthma or COPD.

Footnotes

Acknowledgements

Supported by a grant no. 1999-02323 from the USDA (Ames, IA).

References

Amadesi

Moreau

Tognetto

Springer

Trevisani

Naline

Advenier

. (2001) NK1 receptor stimulation causes contraction and inositol phosphate increase in medium-size human isolated bronchi. Am J Respir Crit Care Med 163:1206–1211

Bai

Zhou

Weir

Walker

Hegele

Hayashi

McKay

. (1995) Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases. Am J Physiol 269:L309–317

Ball

Wren

GRA

Pendry

Smith

Piggott

Sheldrick

RLG

(1993) Effect on the non peptide tachykinin receptor antagonists, (±) CP 99994 and (±) SR 48968, on guinea pig airways. Neuropeptides 24:190

Barnes

(2001) Neurogenic inflammation in the airways. Respir Physiol 125:145–154

Bodelsson

Blomquist

Caverius

Tornebrandt

(1999) Substance P relaxes rat bronchial smooth muscle via epithelial prostanoid synthesis. Respiration 66:355–359

Bozic

Hopken

Gerard

(1996) Neurogenic amplification of immune complex inflammation. Science 273:1722–1725

Brogden

Ackermann

Debey

(1995) Pasteurella haemolytica lipopolysaccharide-associated protein induces pulmonary inflammation after bronchoscopic deposition in calves and sheep. Infect Immun 63:3595–3598

Brogden

Cutlip

Lehmkuhl

(1984) Response of sheep after localized deposition of lipopolysaccharide in the lung. Exp Lung Res 7:123–132

Carstairs

Barnes

(1986) Autoradiographic mapping of substance P receptors in lung. Eur J Pharmacol 127:295–296

10.

Chu

Kraft

Krause

Rex

Martin

(2000) Substance P and its receptor neurokinin 1 expression in asthmatic airways. J Allergy Clin Immunol 106:713–722

11.

Corcoran

(1996) Distribution of calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, substance P and dopamine β-hydroxylase immunoreactive nerve fibers in the trachea of sheep. Res Vet Sci 60:69–75

12.

Corcoran

Haigh

(1992) The effect of tachykinins on sheep bronchomotor tone. Exp Physiol 77:471–479

13.

Erspamer

(1981) The tachykinin peptide family. Trends Neurosci 4:267–270

14.

Gamse

Saria

(1985) Potentiation of tachykinin-induced plasma protein extravasation by calcitonin gene-related peptide. Eur J Pharmacol 114:61–66

15.

Geppetti

Holzer

(1996) Neurogenic Inflammation. Boca Raton, FL, CRC Press

16.

Hislop

Wharton

Allen

Polak

Haworth

(1990) Immunohistochemical localization of peptide-containing nerves in human airways: age-related changes. Am J Respir Cell Mol Biol 3:191–198

17.

Ichikawa

Sreedharan

Owen

Goetzl

(1995) Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung. Am J Physiol 12:L584–588

18.

Janeway

Travers

Walport

(1999) Immunobiology: the Immune System in Health and Disease. 4th ed.

Elsevier Science Ltd/Garland Publishing

19.

Joos

Germonpre

Kips

Peleman

Pauwels

(1994) Sensory neuropeptides and the human lower airways: present state and future directions. Eur Respir J 7:1161–1171

20.

Joos

Germonpre

Pauwels

(2000) Role of tachykinins in asthma. Allergy 55:321–337

21.

Keith

(2000) The role of endogenous lung neuropeptides in regulation of the pulmonary circulation. Physiol Res 49:519–537

22.

Khan

Liu

Khawaja

Manzini

Rogers

(2001) Effect of the long-acting tachykinin NK(1) receptor antagonist MEN 11467 on tracheal mucus secretion in allergic ferrets. Br J Pharmacol 132:189–196

23.

Maggi

(1997) The effects of tachykinins on inflammatory and immune cells. Respir Peptides 70:75–90

24.

Mapp

Miotto

Braccioni

Saetta

Turato

Maestrelli

Krause

. (2000) The distribution of neurokinin-1 and neurokinin-2 receptors in human central airways. Am J Respir Crit Care Med 161:207–215

25.

Matis

Lavker

Murphy

(1990) Substance P induces the expression of an endothelial-leukocyte adhesion molecule by microvascular epithelium. J Invest Dermatol 94:492–495

26.

McLeod

Krause

Cuello

Ribeiro-da-Silva

(1998) Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord. Proc Natl Acad Sci USA 95:15775–15780

27.

Murai

Maeda

Hagiwara

Miyake

Ikari

Matsuo

Fujii

(1993) Effects of an NK-1 receptor antagonist, FK 888, on constriction and plasma extravasation induced in guinea pig airway by neurokinins and capsaicin. Eur J Pharmacol 236:7–13

28.

Nishi

Kitamura

Otani

Hondo

Taguchi

Yamada

(2000) Distribution of capsaicin-sensitive substance P- and calcitonin gene-related peptide-immunoreactive nerves in bovine respiratory tract. Anat Anz 182:319–326

29.

Pedersen

Buckner

Meeker

Undem

(2000) Pharmacological examination of the neurokinin-1 receptor mediating relaxation of human intralobar pulmonary artery. J Pharmacol Exp Ther 292:319–325

30.

Piedimonte

(1995) Tachykinin peptides, receptors, and peptidases in airway diseases. Exp Lung Res 21:809–834

31.

Pothoulakis

Castagliuolo

Leeman

Wang

Hoffman

Mezey

(1998) Substance P receptor expression in intestinal epithelium in Clostridium difficile toxin A enteritis in rats. Am J Physiol 275(1 pt 1):G68–75

32.

Ramirez–Romero

Brogden

Gallup

Sonea

Ackermann

(2001) Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia. Microb Pathog 30:325–335

33.

Ramirez–Romero

Gallup

Sonea

Ackermann

(2000) Dihydrocapsaicin treatment depletes peptidergic nerve fibers of substance P and alters mast cell density in the respiratory tract of neonatal sheep. Regul Pept 91(1–3):97–106

34.

Regoli

Boudon

Fauchere

(1994) Receptors and antagonists for substance P and related peptides. Pharmacol Rev 46:551–599

35.

Regoli

Drapeau

Dion

Couture

(1988) New selective agonists for neurokinin receptors: pharmacological tools for receptor characterization. Trends Pharmacol Sci 9:290–295

36.

Regoli

Drapeau

Dion

D'Orleans–Juste

(1987) Pharmacological receptors for substance P and neurokinins. Life Sci 40:109–117

37.

Rice

Reynolds

Holmes

Scicchitano

(2001) Tachykinin-induced bronchoconstriction in sheep is NK-1 receptor mediated and exhibits tachyphylaxis. Respirology 6:113–123

38.

Rogers

(2000) Motor control of airway goblet cells and glands. Respir Physiol 125:129–144

39.

Shirahase

Kanda

Kurahashi

Nakamura

Usui

Shimizu

(1995) Endothelium-dependent contraction in intra-pulmonary arteries: mediation by endothelial NK1 receptors and TXA2. Br J Pharmacol 115:1215–1220

40.

Shu

Fan

(1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169–171

41.

Sonea

Bowker

Robinson

Broadstone

(1994a) Substance P and calcitonin gene-related peptide-like immunore-active nerve fibers in lungs from adult equids. Am J Vet Res 55:1066–1074

42.

Sonea

Bowker

Robinson

Holland

(1994b) Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development. Anat Embryol 190:469–477

43.

Vigna

Bowden

McDonald

Fisher

Okamoto

McVey

Payan

. (1994) Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells. J Neurosci 15:834–845

44.

Wong

Miller

Yeates

(1990) Stimulation of tracheal ciliary beat frequency by capsaicin. J Appl Physiol 68:2574–2580

Distribution of Substance P Receptor (Neurokinin-1 Receptor) in Normal Ovine Lung and During the Progression of Bronchopneumonia in Sheep

Abstract

Keywords

Materials and Methods

Experimental Animals

Inoculation

Tissue Collection and Processing

Immunohistochemistry

Primary Antibodies

Image Analysis for NK-1R Immunoreactivity

Statistical Analysis

Results

Microscopic Lesions

Immunoreactivity of Primary Antibodies

Quantitative Image Analysis of NK-1R

Discussion

Footnotes

Acknowledgements

References