Distribution of Substance P Receptor (Neurokinin-1 Receptor) in Normal Ovine Lung and During the Progression of Bronchopneumonia in Sheep
Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa (BG,JMG,MRA); Facultad de Medicina Veterinaria y Zootecnia, Universidad Autonoma de Nuevo Leon, Monterrey, Nuevo Leon, Mexico (RRR); and College of Liberal Arts and Sciences, Iowa State University, Ames, Iowa (TBB)
Correspondence to: Dr. Branka Grubor, Dept. of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011-1250. E-mail: brankag{at}iastate.edu
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Summary |
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Key Words: substance P NK-1 receptor ovine lung immunohistochemistry
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Introduction |
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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
; RamirezRomero 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 bronchoconstriction, 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 superfamily of guanine nucleotide-binding, protein-coupled receptors, with seven putative transmembrane-spanning
-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 immunoreactivity 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 (RamirezRomero 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 393407) in ovine lung.
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Materials and Methods |
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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 (RamirezRomero 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 hematoxylineosin 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 streptavidinbiotin 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 7.27.4 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
-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 streptavidinHRP 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 ultrapure 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 KTMTESSSFYSNMLA, which corresponds to a 15-amino-acid section (residues 393407) 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.
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 (x20 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.
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Results |
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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 393407.
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)
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Discussion |
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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 (RamirezRomero 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 393407), 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.
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Acknowledgments |
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Footnotes |
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