|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The precise locations of neurokinin (NK)-1 and NK-2 receptors in human airways, and their role in airway inflammatory diseases, have not been carefully examined. To determine the distribution of NK-1 and NK-2 receptors in human central airways, and to determine whether their distribution was different in smokers, we examined surgical specimens from patients undergoing lung resection for limited lung lesions. We mapped NK-1 and NK-2 receptors in four groups of subjects: four asymptomatic nonsmokers, seven asymptomatic smokers, seven symptomatic smokers with normal lung function, and eight symptomatic smokers with chronic airflow limitation. Tissues were immunostained with anti-NK-1- and anti-NK-2-receptor antibodies. Expression of NK-1 and NK-2 receptors was quantified through light microscopy and image analysis. Both NK-1 and NK-2 receptors were found in bronchial glands, bronchial vessels, and bronchial smooth muscle. Although no receptors were observed in the epithelium, receptors were occasionally found in nerves (NK-1) and in inflammatory cells (NK-2) such as T lymphocytes, macrophages, and mast cells. The distribution of both NK-1 and NK-2 receptors was similar in all the tissues examined in the four groups of subjects. These data show that NK-1 and NK-2 receptors are present in human central airways and that their expression is not modified by cigarette smoking. Mapp CE, Miotto D, Braccioni F, Saetta M, Turato G, Maestrelli P, Krause JE, Karpitskiy V, Boyd N, Geppetti P, Fabbri LM. The distribution of neurokinin-1 and neurokinin-2 receptors in human central airways.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Tachykinins are a family of neuropeptides that share the common carboxyl-terminal sequence Phe-X-Gly-Leu-Met-NH2 (1). This common carboxyl-terminal sequence is essential for the tachykinins' receptor interaction and activation, whereas the distinct amino-terminal sequences of the tachykinins provide their receptor-subtype specificity (2). Three mammalian tachykinin receptor genes have been cloned from several species, including humans. These receptors are members of the superfamily of guanine nucleotide-binding-coupled receptors, and all interact with one or more G-proteins to promote high-affinity binding and signal transduction (3). Although neurokinin (NK)-1 and NK-2 receptors have been characterized in human airways both pharmacologically and by cloning, their precise localization in human airways, and their distribution in airway inflammatory diseases have not been carefully examined. It is known that inflammation has both beneficial and adverse effects, and the balance between host defenses and tissue injury is especially important in the lung, an organ exposed to many noxious agents, such as cigarette smoke. Experimental studies have shown that cigarette smoking releases substance P (SP) from sensory nerves (4), induces adhesion of leukocytes to venules of tracheal mucosa (5), decreases neutral endopeptidase activity (6), and exaggerates neurogenic inflammatory responses. Bai and coworkers (7), in an in situ hybridization study, reported a twofold increase of NK-1 and NK-2 receptor messenger RNA (mRNA) expression over that of nonsmokers in lung samples of smokers without airflow obstruction.
Our goal in the present study was to determine the distribution of NK-1 and NK-2 receptors in human central airways. In view of the potential involvement of tachykinins in the inflammatory response induced by cigarette smoking, we also examined the expression of NK-1 and NK-2 receptors in the airways of smokers. Accordingly, we examined lobar bronchi from 26 patients undergoing lung resection for localized pulmonary lesions. Four were nonsmokers and asymptomatic, seven were smokers and asymptomatic, seven had symptoms of chronic bronchitis with normal lung function, and eight were bronchitic with chronic airflow limitation. Lobar bronchi were examined with immunohistochemical techniques by using anti-NK-1- and anti-NK-2-receptor antibodies.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Subjects
The study population was composed of 26 patients who were undergoing lung resection for a solitary peripheral carcinoma. Twenty-two subjects had a history of cigarette smoking. Of these 22, seven had symptoms of chronic bronchitis with a normal FEV1, eight had symptoms of chronic bronchitis and fixed airway obstruction, and seven were asymptomatic with normal lung function. Four subjects were nonsmokers and asymptomatic. Chronic bronchitis was defined as cough and sputum production occurring on most days of the month for at least 3 mo a year during the 2 yr prior to the study (8). Fixed airway obstruction was defined as FEV1 < 80% predicted, with a reversibility of less than 15% after inhalation of 200 µg of salbutamol. Chronic bronchitic subjects had no exacerbations, which were defined as increased dyspnea associated with a change in the quality and quantity of sputum that led the subjects to seek medical attention (9) during the month preceding the study.
All subjects had been free of acute upper-respiratory-tract infections, and none had received glucocorticoids or antibiotics within the month preceding surgery or bronchodilators within the previous 48 h. The subjects were nonatopic (i.e., they had negative skin test results for common allergen extracts), and had no past history of asthma or allergic rhinitis.
The study conformed to the Declaration of Helsinki, and informed written consent was obtained for each subject undergoing surgery. Each subject underwent an interview, chest radiography, electrocardiogram, routine blood tests, skin tests with common allergen extracts, and pulmonary function tests in the week before surgery.
Pulmonary Function Tests
Pulmonary function tests were performed as previously described (9). Briefly, they included measurements of FEV1 and FVC in all the subjects examined (Biomedin Spirometer, Padua, Italy). The predicted normal values used were those of the European Coal and Steel Community (10). In order to assess the reversibility of airway obstruction in subjects with a baseline FEV1 < 80% predicted, the FEV1 measurement was repeated 15 min after the inhalation of 200 µg of salbutamol.
Skin Prick Tests
Skin prick tests were performed with a battery of commercially available allergens, including tree pollens, animal danders, molds, house dust mites, and storage mites (Lopharma, Milan, Italy). Tests were considered positive when the wheal area was at least 50% greater than that observed with a positive control (histamine) or a negative control (diluent).
Sample Processing
Bronchial rings were taken from the lobar or segmental bronchus of the
lobe obtained at surgery, away from the tumor site. Bronchial rings were
fixed immediately in freshly prepared 1% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 4 h, washed twice (1 h each)
with PBS containing 15% sucrose, embedded in ornithyl carbamyl transferase compound, snap-frozen in isopentane precooled in liquid nitrogen, and stored at 
70° C to be used later in immunohistochemistry.
Immunohistochemistry
Cryostat sections (10 µm thick) were immunostained with antibodies
to NK-1 and NK-2 receptors through the streptavidin-biotin-complex/peroxidase method, and the peroxidase activity was revealed
with the nickel enhancement method, as previously described (11).
Briefly, endogenous peroxidase activity was blocked by immersing
slides in 0.3% hydrogen peroxide in methanol for 30 min. After washing of the slides in PBS, nonspecific binding was blocked by incubating the slides for 30 min in 3% normal swine serum in PBS containing
0.05% bovine serum albumin and 0.1% sodium azide. The sections
were then incubated overnight at 4° C with the primary antibody. A
rabbit polyclonal antihuman antibody to NK-1 receptor was used
(1:1,500 dilution). The ability of the antibody to selectively recognize
human NK-1 receptor was indicated by its having been generated
against a synthetic peptide corresponding to the last 15 amino acid
residues of the carboxyl terminal of human NK-1 receptor (residues
391 to 407). This sequence is different from the corresponding sequences of the human NK-2 and NK-3 receptors. A description and
characterization of this antibody have recently been reported (12).
Negative controls were produced by preabsorbing the antibody with
the immunogenic peptide diluted at 10
4 or 106 M in a 1:1,000 dilution of the antibody, and incubating for at least 4 h before applying
the antibody to tissue. Further negative controls were produced by
omitting the primary antibody and by substituting the primary antibody with rabbit preimmune serum.
To detect NK-2 receptors, a mouse monoclonal antibody to the
human NK-2 receptor was used (1:50 dilution). It was raised according to the methods reported by Zerari and coworkers (13), using the
human NK-2 receptor (residues 367 to 398) peptide sequence coupled
to keyhole limpet hemocyanin as the immunogen. Specificity of the
antibody was verified with both a solid-phase enzyme-linked immunosorbent assay (ELISA), and a competitive ELISA, and it did not
cross react with synthetic peptides corresponding to the same region
of the human NK-1 or human NK-3 receptors. Further specificity of
the antibody was demonstrated by immunostaining of cells and immunoprecipitation of cell extracts transfected with human NK-2 receptor
complementary DNA (cDNA) but not with human NK-1 or human NK-3 receptor cDNAs (data not shown). Negative controls were produced by preabsorbing the antibody with the immunogenic peptide
diluted at 104 or 105 M in a 1:50 dilution of the antibody, and incubating for at least 4 h before applying the antibody to tissue. Further
negative controls were produced by omitting the primary antibody.
After washing in PBS, the sections stained for NK-1 receptor were incubated for 30 min with biotinylated swine antirabbit IgG antibody
(E431; Dako Ltd., High Wycombe, UK), whereas those stained for
NK-2 receptor were incubated for 30 min with biotinylated rabbit antimouse IgG antibody (E413; Dako). All sections were then washed
and incubated for 60 min with streptavidin-biotin complex reagent
(StreptABComplex/HRP; KO377; Dako). Immunoreactivity was visualized with diaminobenzidine (DAB). Sections were dehydrated and
mounted in a hydrophobic mounting medium (Eukitt; Kindlex GMBH,
Freiburg, Germany).
Identification of Phenotype of Inflammatory Cells by Double Immunohistochemistry
To examine the phenotype of inflammatory cells expressing NK-2 receptor, cryostat sections were studied with double immunohistochemistry. Sections were washed in Tris-buffered saline (TBS; 0.05 M Tris base, 0.15 M NaCl, pH 7.6) between each experimental step. After pretreatment with 0.3% hydrogen peroxide in methanol for 30 min and with 3% normal swine serum for 20 min, antibodies for the identification of inflammatory cells were applied to the sections for 30 min. We used a polyclonal anti-tryptase antibody (1:50) for the identification of mast cells (rabbit antihuman tryptase; 9245-5007; Biogenesis Ltd., Poole, UK), a polyclonal anti-CD3 antibody (1:50) for the identification of T lymphocytes (rabbit antihuman CD3; A452; Dako), and a monoclonal antibody (1:50) for the identification of macrophages (mouse antihuman CD68; M718; Dako). To identify mast cells and T lymphocytes, sections were incubated for 30 min with a goat antirabbit Ig conjugated to alkaline phosphatase-labeled dextran polymer (Envision AP; K4017; Dako), whereas for macrophages, sections were incubated for 30 min with rabbit antimouse immunoglobulins (Z0259; Dako) and with a mouse monoclonal antibody to alkaline phosphatase-antialkaline phosphatase (D0651; Dako). Immunoreactivity for all inflammatory cells was visualized with fast red. All sections were incubated overnight at 4° C with monoclonal anti-NK-2 antibody (1:50). The second antibody consisted of a biotinylated rabbit antimouse IgG antibody (E413; Dako) (1:60) applied for 30 min, and the third layer consisted of streptavidin-biotin complex reagent (StrpABCComplex/HRP; K0377; Dako) applied for 30 min. Immunoreactivity for NK-2 receptor was visualized with DAB. Sections were counterstained with hematoxylin and mounted in glycerol gel (Dako). Negative controls were produced by omitting the primary antibodies.
Quantification
Light-microscopic analysis of the vessels of the bronchial submucosa and bronchial glands in the coded slides was performed with a Zeiss microscope (Zeiss, Wetzlaar, Germany). In order to measure the surface area of tissue examined, an eyepiece graticule with a graded scale was superimposed on the microscopic field. Vessels were counted at a magnification of ×312 in the area 500 µm beneath the epithelial basement membrane in nonoverlapping fields until all of the available area was covered. Results were expressed as number of vessels positive for NK-1 or NK-2 receptors per mm2 of bronchial wall examined. Acini of bronchial glands were counted at a magnification of ×500 in alternate, nonoverlapping fields until all the gland area was covered. Results were expressed as number of acini positive for NK-1 or NK-2 receptors per mm2 of bronchial gland area examined.
The area of bronchial smooth muscle positive for NK-1 or NK-2 receptors was evaluated with an image analysis system (Windows, Image version 3.4; CASTI Imaging s.r.l., Venice, Italy). Images of microscopic fields at a magnification of ×100 were captured via a video camera attached to a microscope (Leyca, Wetzlar, Germany). The area of receptor staining in the image was measured by interactive delineation. Using the computer mouse, we delineated the boundaries of the bronchial wall (considered to be the area between the basement membrane and the bronchial cartilage) and the boundaries of bronchial smooth muscle positive for NK-1 or NK-2 receptors. The number of pixels contained between the boundaries, traced with the computer cursor, was converted to the tissue area by using a factor determined by calibrating the image analyzer with a graded scale viewed through the same microscope and objective used for the study. Results were expressed as the percentage of area of smooth muscle positive for NK-1 or NK-2 receptors per mm2 of bronchial wall.
Statistical Analysis
Group data were expressed as mean ± SEM or as median and range for normally and nonnormally distributed data, respectively. Differences between the four study groups were analyzed with the nonparametric Kruskal-Wallis analysis of variance followed by the Mann- Whitney U test for nonnormally distributed data. The unpaired t test was used for normally distributed data. Values of p < 0.05 were accepted as significant. At least three replicate measurements were performed by the same observer on 10 randomly selected slides, and the intraobserver reproducibility was assessed with the coefficient of variation (CV) for repeated measures.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Clinical Findings
The characteristics of the four groups of subjects examined are given in Table 1. The three groups of smokers were similar with regard to age, sex, and smoking history (packs/yr). Nonsmokers were similar with regard to age, but three subjects were females. As expected from the selection criteria, subjects with chronic bronchitis and chronic airflow limitation had a significantly lower value of FEV1 (% predicted) and significantly lower FEV1/FVC (%) than did nonbronchitic subjects or bronchitic subjects with normal FEV1. Subjects with FEV1 less than 80% predicted showed no significant response to bronchodilator (range: 3.8 to 5.4%).
|
Immunohistochemical Findings
Quantification of NK-1 and NK-2 receptors was satisfactory in all the subjects except Subject 1 in the group of nonbronchitic subjects, in whom quantification of both NK-1 and NK-2 receptors in smooth muscle and in bronchial vessels could not be performed. Positive immunostaining for both NK-1 and NK-2 receptors was found in the bronchial smooth-muscle layer (Figure 1), in the myoepithelial cells of bronchial glands (Figure 2), in the smooth-muscle layer of bronchial vessels (Figure 3), and in the endothelium and in the smooth-muscle layer of pulmonary arteries (Figure 4). Positive immunostaining for NK-1 receptors was found in the endothelium of bronchial vessels (Figure 3), and occasionally in nerves (Figure 5), whereas positive immunostaining for NK-2 receptors was found in inflammatory cells, frequently located close to bronchial glands (Figures 2 and 6). On the basis of morphologic criteria, we observed that inflammatory cells positive for NK-2 receptor were mononuclear cells. By using double immunohistochemistry, we found that NK-2 receptor was expressed by CD68+ (macrophages), tryptase-positive (mast cells), and CD3+ (T lymphocytes) cells. The expression occurred in a limited number of inflammatory cells present in the central airways of the subjects examined. No receptors were observed in the epithelium. Positive immunostaining for NK-2 receptors in bronchial glands and bronchial vessels was less extensive than that for NK-1 receptors, and in bronchial smooth muscle it was less uniform than was the staining for NK-1 receptors. Preabsorption of both the anti-NK-1- and anti-NK-2-receptor antibodies with the immunogenic peptide completely abolished the staining.
|
|
|
|
|
|
There was no significant difference in the expression of NK-1 and NK-2 receptors among the four groups of subjects in bronchial smooth muscle (Figure 7), glands (Figure 8), or bronchial vessels (Figure 9) (Kruskal-Wallis test, p > 0.05).
|
|
|
The intraobserver CV ranged from 1% to 15% for smooth muscle, from 7% to 14% for glands, and from 3% to 12% for vessels.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
By using immunohistochemical techniques and quantitative methods, we found that both NK-1 and NK-2 receptors are present in several structures of human central airways, including smooth muscle, glands, vessels, and pulmonary arteries. Immunostaining for NK-1 and NK-2 receptors was absent in the epithelium, but sparse immunostaining was found in nerves (NK-1 receptor) and in inflammatory cells (NK-2 receptor).
Moreover, expression of both NK-1 and NK-2 receptors was not found to differ in the central airways of nonsmokers, asymptomatic smokers, smokers with symptoms of chronic bronchitis and those with normal lung function, and chronic bronchitic patients with chronic airflow limitation.
Previous studies of the inflammatory changes in chronic bronchitis have shown that inflammation appears to be present throughout the bronchial tree and the respiratory portion of the lung in chronic obstructive pulmonary disease (COPD) (14). Neurogenic inflammation contributes markedly to the inflammatory process in the airways of different animal species. Thus, stimulation of NK-1 and NK-2 receptors causes bronchospasm, stimulation of gland secretion, increased vascular permeability in postcapillary venules, recruitment and activation of inflammatory cells, and facilitation of cholinergic neurotransmission (15). It has been suggested that the abnormal stimulation of the sensory nerve terminals that can occur in chronic inflammatory diseases of the airways results in the enhanced release of tachykinins in the airway wall (16). The neurogenic component of inflammation might contribute to the overall airway inflammation present in both asthma and COPD.
In rodent airways, NK-2 receptors, and to a lesser extent NK-1 receptors, have been shown to be involved in bronchoconstriction, whereas NK-1 receptors were found to be involved in mucus secretion, microvascular leakage, vasodilation, and most effects on inflammatory cells (17). It would therefore appear that SP is more potent than neurokinin A (NKA) in inducing mucus secretion. We found that NK-1 receptors were localized in the myoepithelial cells surrounding bronchial glands, a finding that confirms those in previous studies (18). In the canine trachea, Coles and associates found that SP appears to stimulate secretion without morphologic effects on secretory cells, suggesting that SP may cause myoepithelial cell contraction and expulsion of presecreted mucus from the lamina of secretory and/or collecting glands, rather than causing direct degranulation of secretory cells of acini. We demonstrated that NK-2 receptors were also localized in the myoepithelial cells surrounding bronchial glands, even if their expression was not as great as the expression of NK-1 receptors. It is possible that the NK-2 receptor plays a role in the normal physiology of glands and also in diseases associated with mucus hypersecretion. In addition to their effects on mucus secretion, tachykinins constrict smooth muscle of human airways in vitro via NK-2 receptors (19). The contractile response to NKA is greater in smaller human bronchi than in more proximal airways, suggesting that tachykinins have a more important constrictor effect in peripheral airways (22). More conflicting data have been obtained with regard to NK-1 receptors and the contractility of isolated human airways. The use of selective NK-1 receptor agonists has shown both constrictor and dilator activity (23). More recently, it has been shown that NK-1 receptors mediate a prostanoid-dependent contraction of human small bronchi (24). NK-1 receptors have also been shown to cause relaxation of human pulmonary arteries (25). In some specimens in the present study, we were able to observe the pulmonary artery adjacent to the bronchus, and in this structure we detected positive immunostaining for both NK-1 and NK-2 receptors.
So far, few studies have described the distribution of NK-1 and NK-2 receptors in airways of asthmatic subjects through the use of immunohistochemical methods, and to the best of our knowledge, no other quantitative immunohistochemical studies of surgical bronchial specimens for tachykinin expression in nonsmokers and smokers have been reported. Chu and colleagues (26) examined NK-1 and SP expression in endobronchial biopsies from asthmatic subjects, using immunohistochemical techniques and semiquantitative analysis of NK-1 expression. The asthmatic subjects showed greater NK-1 expression in the bronchial epithelium than did normal controls. The expression decreased in both epithelium and submucosa upon treatment with clarithromycin, a drug used in chronic asthma when it is associated with the presence of mycoplasma in the airways. In situ hybridization studies indicated that NK-1 mRNA was localized predominantly in airway epithelium and in vascular endothelium, and that NK-1 receptor gene expression was increased in asthmatic lungs (27). A recent study (7) showed that NK-1 and NK-2 receptor expression was twofold greater in smokers without airflow obstruction than in nonsmokers, whereas NK-1 receptor mRNA expression was significantly lower in patients with COPD than in smoking controls. The investigators explained this unexpected finding as a consequence of the process leading to chronic airflow limitation in susceptible smokers, or to a contributing factor leading to obstruction. They postulated that in COPD, an increased release of tachykinins leads to downregulation of NK-1 receptor gene transcription, or that the low level of NK-1 receptors may be associated with a less efficient protective mucus barrier, thus predisposing to increased injury and airflow obstruction.
Radioligand binding assays and autoradiographic studies have also been used as tools for studying receptors. Through the use of autoradiographic techniques with labeled tachykinins, such as SP and NKA (28, 29), it has been found that in guinea-pig lung, NK-1 receptors are localized to bronchial vessels, epithelial cells, and submucosal glands, whereas NK-2 receptors are predominantly localized to airway smooth muscle. Autoradiographic techniques identify only the receptors available for binding, and do not quantify receptor sites masked by other factors or by changes in receptor affinity. Moreover, it is difficult to correlate receptor density with function, since the ability of a specific tissue to respond to tachykinins may depend more on the intracellular processes that couple receptor binding to response than on the total number of receptors available. Our experiments did not show immunostaining of airway epithelium for either NK-1 or NK-2 receptors. In the guinea pig lung, specific labeling for NK-1 receptor in the epithelium increased gradually with diminishing size of airways (30). Therefore, absence of the expression of NK-1 receptors in the epithelium of large airways does not exclude their localization to the epithelium of small airways. Our failure to detect tachykinin receptors in the epithelium is unlikely to have been due to low specificity of the antibodies we used, since these antibodies were effective in detecting NK-1 and NK-2 receptors in other airway structures. An alternative explanation could be a technical defect (i.e., an artifact caused by cutting). NK-1 receptor gene expression has been demonstrated in human airway epithelium, particularly in goblet cells (7), and our cryostat sections could have been less than ideal for preserving the integrity of these cells. Low levels of protein expression could also explain the lack of immunostaining for NK-1 and NK-2 receptors in the airway epithelium in our study.
An interesting finding in our study was the expression of NK-2 receptor by macrophages (CD68+ cells), mast cells (tryptase-positive cells) and T lymphocytes (CD3+ cells). Since we performed double immunohistochemistry for CD68 and for NK-2, using two monoclonal antibodies, we cannot exclude the possibility that doubly immunostained cells were the result of a cross reaction between the secondary antibodies with the two primary antibodies. However, the morphology of these cells was the typical morphology of macrophages. These results are in keeping with the studies by Saetta and coworkers (31) and Pesci and associates (32), which showed that T lymphocytes, macrophages, and mast cells are key cells in the airways of subjects with chronic mucus hypersecretion.
The immunohistochemical technique used in our study appears to identify the total pool of NK-1 and NK-2 receptors, including the intracellular pool, and allows quantification of the airway structures that express the receptors but not of the exact number of peptide molecules expressed in cells bearing the receptors. The absence of differences in the expression of tachykinin receptors in chronic bronchitic and COPD patients is in accord with the findings in previous studies (33). We examined the difference in nerve density (SP, calcitonin gene- related peptide [CGRP], and vasointestinal peptide [VIP]) in central airways of smokers with and without symptoms of chronic bronchitis, and found no differences in SP- and CGRP-positive nerve density in the two groups. However, the density of VIP-positive nerves was increased in the bronchial glands of bronchitic as compared with asymptomatic smokers, suggesting a role for VIP in chronic mucus hypersecretion. Of interest is the observation of an increased concentration of SP in the induced sputum of bronchitic subjects, suggesting an increased release of tachykinins from airway sensory nerves that may downregulate pulmonary tachykinin receptors (34).
The present study demonstrates the presence of both NK-1 and NK-2 receptors in several structures of human central airways, and a similar expression of these receptors on glands, vessels, and airway smooth muscle in bronchitic subjects with normal lung function or with chronic airflow limitation, in nonbronchitic subjects, and in nonsmokers. It could therefore be hypothesized that changes in tachykinin receptor expression do not occur in the airways of bronchitic individuals, or that they occur only in the small airways or during exacerbations. The bronchitic subjects in our study were selected to be in stable condition with their disease far from clinical exacerbation. No subjects took glucocorticoids in the month prior to the study, excluding the possibility of a decrease in tachykinin NK-2 receptor gene transcription as a result of such treatment.
A confounding factor in any study done on surgical specimens obtained from patients with lung cancer is that cancer itself may influence the results. Surgical specimens allow analysis of the entire bronchial wall and a better quantification of events occurring at the level of the bronchial-gland compartment. Because we examined only tissue away from tumors and included nonsmoking subjects with lung cancer in our control group, we feel confident that our findings of a similar expression of both NK-1 and NK-2 receptors in the pulmonary structures of subjects with and without symptoms of chronic bronchitis and with and without chronic airflow limitation are valid. However, we cannot exclude the possibility that the absence of significant differences in the distribution of both NK-1 and NK-2 receptors in our four study groups was due to our inability to include a group of control subjects without lung cancer in our study.
In conclusion, by using immunohistochemical techniques, we mapped the expression of NK-1 and NK-2 receptors in human central airways. We found positive immunostaining for both NK-1 and NK-2 receptors in the bronchial smooth-muscle layer, in the myoepithelial cells of the bronchial glands, in the smooth-muscle layer of bronchial vessels, and in the endothelium and smooth-muscle layer of pulmonary arteries. NK-2 receptor was also expressed by macrophages, mast cells, and T lymphocytes. We observed no differences in the expression of NK-1 and NK-2 receptors in the central airways of nonsmokers, asymptomatic smokers, patients with chronic bronchitis and normal lung function, and bronchitic subjects with chronic airflow limitation.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Cristina E. Mapp, M.D., Istituto di Medicina del Lavoro, Universita' degli Studi di Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail: mapp{at}ux1.unipd.it
(Received in original form March 29, 1999 and in revised form June 21, 1999).
Acknowledgments: The authors thank Drs. S. Chiarelli, G. Cavallesco, and G. Azzena for their expert collaboration; E. Nardo for revising the manuscript; and G. Fulgeri for typing the manuscript.
Supported by MURST, Boehringer Ingelheim Italia, Ferrara Ricerche, Associazione Ricerca Asma, Associazione Studio Tumori Malattie Polmonari, Arcispedale S. Anna, Ferrara.
| |
References |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1. Uddman, R., R. Hakanson, A. Luts, and F. Sundler. 1997. Distribution of neuropeptides in airways. In P. J. Barnes, editor. Autonomic Control of the Respiratory System. Harvard Academic, London. 21.
2. Nakanishi, S.. 1991. Mammalian tachykinin receptors. Annu. Rev. Neurosci. 14: 123-136 [Medline].
3.
Hershey, A. D., and
J. E. Krause.
1990.
Molecular characterization of functional cDNA encoding the rat substance P receptor.
Science
247:
958-962
4. Lundberg, J. M., and A. Saria. 1983. Capsaicin-induced desensitization of the airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 302: 251-253 [Medline].
5. Baluk, P., C. Bertrand, P. Geppetti, D. M. McDonald, and J. A. Nadel. 1996. NK1 receptor antagonist CP-99,994 inhibits cigarette smoke- induced neutrophil and eosinophil adhesion in rat tracheal venules. Exp. Lung Res. 22: 409-418 [Medline].
6. Dusser, D. J., T. D. Djokic, D. B. Borson, and J. A. Nadel. 1989. Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea-pig: possible role of free radicals. J. Clin. Invest. 84: 900-906 .
7. Bai, T.-R., D. Zhou, T. Weir, B. Walker, R. Hegele, S. Hayashi, K. McKay, G. P. Bondy, and T. Fong. 1995. Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases. Am. J. Physiol. 269: 309-317 .
8. American Thoracic Society. 1995. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 152: S77-S120 .
9. Saetta, M., A. Di Stefano, P. Maestrelli, G. Turato, M. P. Ruggieri, A. Roggeri, P. Calcagni, C. E. Mapp, A. Ciaccia, and L. M. Fabbri. 1994. Airway eosinophilia in chronic bronchitis during exacerbations. Am. J. Respir. Crit. Care Med. 150: 1646-1652 [Abstract].
10. European Coal and Steel Community. 1971. Aide-memoire of Spirographic Practice for Examining Ventilatory Function, 2nd ed. Industrial Health and Medicine, Luxemburg.
11. Shu, S., G. Ju, and L. Faw. 1988. The glucose-oxidase DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85: 169-171 [Medline].
12. McDonald, D., S. C. Silberman, J. A. Lowe, S. E. Drozda, S. E. Leeman, and N. D. Boyd. 1996. Photoaffinity labeling of the human substance P (neurokinin-1) receptor with (H-3(2)) azido-CP-96,345, a photoreactive derivative of a nonpeptide antagonist. Mol. Pharmacol. 49: 808-813 [Abstract].
13. Zerari, F., V. Karpitskiy, J. Krause, L. Descarries, and R. Couture. 1998. Astroglial distribution of neurokinin-2 receptor immunoreactivity in the rat spinal cord. Neuroscience 84: 1233-1246 [Medline].
14. Jeffery, P. K.. 1998. Structural and inflammatory changes in COPD: a comparison with asthma. Thorax 53: 129-136 [Medline].
15. Barnes, P. J., J. N. Baraniuk, and M. G. Belvisi. 1991. Neuropeptides in the respiratory tract: part II. Am. Rev. Respir. Dis. 144: 1187-1198 [Medline].
16. Barnes, P. J.. 1986. Asthma as an axon reflex. Lancet 1: 242-245 [Medline].
17. Martling, C. R., E. Theodorsson-Norheim, I. Norheim, and J. M. Lundberg. 1987. Bronchoconstrictor and hypotensive effects in relation to pharmacokinetics of tachykinins in the guinea-pig-evidence for extraneuronal cleavage of neuropeptide K to neurokinin A. Naunyn-Schmiedebergs Arch. Pharmacol. 336: 183-189 [Medline].
18.
Coles, S. J.,
K. G. Neill, and
L. Reid.
1984.
Potent stimulation of glycoprotein secretion by substance P.
J. Appl. Physiol.
57:
1323-1327
19. Naline, E., P. Devillier, G. Drapeau, L. Toty, H. Bakdach, D. Regoli, and C. Advenier. 1989. Characterization of neurokinin effects and receptor selectivity in human isolated bronchi. Am. Rev. Respir. Dis. 140: 679-686 [Medline].
20. Advenier, C., E. Naline, L. Toty, H. Bakdach, X. Emors-Alt, P. Vilain, J. Breliere, and G. Le Fur. 1992. Effects on the isolated human bronchus of SR48968, a potent and selective nonpeptide antagonist of the neurokinin A (NK2) receptors. Am. Rev. Respir. Dis. 146: 1177-1181 [Medline].
21. Sheldrick, R. L., K. F. Rabe, A. Fischer, H. Magnussen, and R. A. Coleman. 1995. Further evidence that tachykinin-induced contraction of human isolated bronchus is mediated only by NK2-receptors. Neuropeptides 29: 281-292 [Medline].
22. Frossard, N., and P. J. Barnes. 1991. Effect of tachykinins on small human airways. Neuropeptides 19: 157-162 [Medline].
23. Chitano, P., P. Di Blasi, R. E. Lucchini, F. Calabro', M. Saetta, P. Maestrelli, L. M. Fabbri, and C. E. Mapp. 1994. The effects of toluene diisocyanate and of capsaicin on human bronchial smooth muscle in vitro. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 270: 167-173 .
24.
Naline, E.,
M. Molimard,
D. Regoli,
X. Elmonds-Alt,
J. F. Bellamy, and
C. Advenier.
1996.
Evidence for functional tachykinin NK1 receptors
on human isolated small bronchi.
Am. J. Physiol.
271:
L763-L767
25. Corboz, M. R., M. A. Rivelli, S. I. Ramos, M. A. Rivelli, and J. A. Hey. 1998. Tachykinin NK1 receptor-mediated vasorelaxation in human pulmonary arteries. Eur. J. Pharmacol. 350: R1-R3 [Medline].
26. Chu, H. W., M. Kraft, J. E. Krause, D. N. Cortright, and R. J. Martin. 1998. Neurokinin-1 (NK-1) and substance P (SP) expression in asthmatic airways: downregulation with clarithromycin (abstract). Am. J. Respir. Crit. Care Med. 157: A24 .
27.
Adcock, I. M.,
M. Peters,
C. M. Gelder,
H. Shirasaki,
C. R. Brown, and
P. J. Barnes.
1993.
Increased tachykinin receptor gene expression in
asthmatic lung and its modulation by steroids.
J. Mol. Endocrinol.
11:
1-7
28. Strigas, J., and E. Burcher. 1996. Autoradiographic localization of tachykinin NK2 and NK1 receptors in the guinea-pig lung, using selective radioligands. Eur. J. Pharmacol. 311: 177-186 [Medline].
29. Castairs, J. R., and P. J. Barnes. 1986. Autoradiographic mapping of substance P receptors in lung. Eur. J. Pharmacol. 127: 295-296 [Medline].
30. Zhang, X.-L., J. C. W. Mak, and P. J. Barnes. 1995. Characterization and autoradiographic mapping of (3H)CP96,345, a nonpeptide selective NK1 receptor antagonist in guinea pig lung. Peptides 16: 867-872 [Medline].
31. Saetta, M., A. Di Stefano, P. Maestrelli, A. Ferraresso, R. Drigo, A. Potena, A. Ciaccia, and L. M. Fabbri. 1993. Activated T-lymphocytes and macrophages in bronchial mucosa of subjects with chronic bronchitis. Am. Rev. Respir. Dis. 147: 301-306 [Medline].
32. Pesci, A., G. A. Rossi, G. Bertorelli, A. Aufiero, P. Zanon, and D. Olivieri. 1994. Mast cells in the airway lumen and bronchial mucosa of patients with chronic bronchitis. Am. J. Respir. Crit. Care Med. 149: 1311-1316 [Abstract].
33.
Lucchini, R. E.,
F. Facchini,
G. Turato,
M. Saetta,
G. C. Caramori,
A. Ciaccia,
D. R. Springall,
J. M. Polak,
L. M. Fabbri, and
C. E. Mapp.
1997.
Increased VIP-positive nerve fibers in the mucous glands of subjects with chronic bronchitis.
Am. J. Respir. Crit. Care Med.
156:
1963-1968
34. Tomaki, M., M. Ichinose, M. Miura, Y. Hirayama, H. Yamauchi, N. Nakajima, and K. Shirato. 1995. Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am. J. Respir. Crit. Care Med. 151: 613-617 [Abstract].
This article has been cited by other articles:
 |
|
 |
|
  K. Mizuta, G. Gallos, D. Zhu, F. Mizuta, F. Goubaeva, D. Xu, R. A. Panettieri Jr., J. Yang, and C. W. Emala Sr. Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L523 - L534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Zee, S. Schomberg, and T. C. Carpenter Hypoxia upregulates lung microvascular neurokinin-1 receptor expression Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L102 - L110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Barnes Distribution of Receptor Targets in the Lung Proceedings of the ATS, December 1, 2004; 1(4): 345 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Barnes Mediators of Chronic Obstructive Pulmonary Disease Pharmacol. Rev., December 1, 2004; 56(4): 515 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Joachim, V. Sagach, D. Quarcoo, Q. T. Dinh, P. C. Arck, and B. F. Klapp Neurokinin-1 Receptor Mediates Stress-Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness in Mice Psychosom Med, July 1, 2004; 66(4): 564 - 571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.F. Joos, W. Vincken, R. Louis, V.J. Schelfhout, J.H. Wang, M.J. Shaw, G.D. Cioppa, and R.A. Pauwels Dual tachykinin NK1/NK2 antagonist DNK333 inhibits neurokinin A-induced bronchoconstriction in asthma patients Eur. Respir. J., January 1, 2004; 23(1): 76 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Grubor, R. Ramirez-Romero, J. M. Gallup, T. B. Bailey, and M. R. Ackermann Distribution of Substance P Receptor (Neurokinin-1 Receptor) in Normal Ovine Lung and During the Progression of Bronchopneumonia in Sheep J. Histochem. Cytochem., January 1, 2004; 52(1): 123 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Freed, S. McCulloch, T. Meyers, and R. Suzuki Neurokinins Modulate Hyperventilation-induced Bronchoconstriction in Canine Peripheral Airways Am. J. Respir. Crit. Care Med., April 15, 2003; 167(8): 1102 - 1108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.G. Tournoy, K.O. De Swert, P.G. Leclere, R.A. Lefebvre, R.A. Pauwels, and G.F. Joos Modulatory role of tachykinin NK1 receptor in cholinergic contraction of mouse trachea Eur. Respir. J., January 1, 2003; 21(1): 3 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Maghni, M.-C. Michoud, M. Alles, A. Rubin, V. Govindaraju, C. Meloche, and J. G. Martin Airway Smooth Muscle Cells Express Functional Neurokinin-1 Receptors and the Nerve-Derived Preprotachykinin-A Gene: Regulation by Passive Sensitization Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 103 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S Wong, N. N Sun, R C. Lantz, and M. L Witten Tissue-specific patterns of neurokinin-1 receptor (NK-1R) gene expression in mice exposed to sidestream cigarette smoke Toxicology and Industrial Health, October 1, 2002; 18(9-10): 435 - 444. [Abstract] [PDF]
|
|
|
|
|
|
J. Van Schoor, G.F. Joos, and R.A. Pauwels Effect of inhaled fluticasone on bronchial responsiveness to neurokinin A in asthma Eur. Respir. J., June 1, 2002; 19(6): 997 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. J. Van Rensen, P. S. Hiemstra, K. F. Rabe, and P. J. Sterk Assessment of Microvascular Leakage via Sputum Induction: The Role of Substance P and Neurokinin A in Patients with Asthma Am. J. Respir. Crit. Care Med., May 1, 2002; 165(9): 1275 - 1279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. TOBIN Chronic Obstructive Pulmonary Disease, Pollution, Pulmonary Vascular Disease, Transplantation, Pleural Disease, and Lung Cancer in AJRCCM 2000 Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1789 - 1804. [Full Text] [PDF]
|
|
|
|
|
|
F. SCHMIDLIN, S. AMADESI, R. VIDIL, M. TREVISANI, N. MARTINET, G. CAUGHEY, M. TOGNETTO, G. CAVALLESCO, C. MAPP, P. GEPPETTI, et al. Expression and Function of Proteinase-activated Receptor 2 in Human Bronchial Smooth Muscle Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1276 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. AMADESI, J. MOREAU, M. TOGNETTO, J. SPRINGER, M. TREVISANI, E. NALINE, C. ADVENIER, A. FISHER, D. VINCI, C. MAPP, et al. NK1 Receptor Stimulation Causes Contraction and Inositol Phosphate Increase in Medium-size Human Isolated Bronchi Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1206 - 1211. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Cell Mol. Biol. |