- Email: [email protected]
Decreased neuropeptide release may play a role in the pathogenesis of nasal polyps
Decreased neuropeptide release may play a role in the pathogenesis of nasal polyps ANIL GUNGOR, MD, FUAD M. BAROODY, MD, ROBERT M. NACLERIO, MD, STEVEN R. WHITE, MD, and JACQUELYNNE P. COREY, MD, Chicago, Illinois
In this in vivo prospective, controlled study, we have examined the capsaicin-induced levels and secretion patterns of the colocalized neuropeptides substance P, calcitonin gene-related peptide (CGRP), and neurokinin A in nasal secretions of subjects with nasal polyps, and we compared these with secretion patterns from healthy subjects and from subjects with allergic rhinitis. Capsaicin was used to elicit neuropeptide release. The neuropeptide levels were measured by an ELISA technique. For substance P, subjects with nasal polyps responded very poorly to capsaicin stimulation. The atopic group was more reactive to capsaicin stimulation than control subjects. For CGRP the increase was immediate in all groups. Atopic subjects and subjects with polyps had a less pronounced but sustained response to capsaicin stimulation. CGRP levels in atopic subjects and those with polyps were restored rapidly. Atopic subjects had higher neurokinin A levels with an immediate and sustained response to capsaicin. Control subjects had higher levels than those with polyps, but both groups were nonresponsive to capsaicin stimulation. (Otolaryngol Head Neck Surg 1999;121:585-90.)
N
asal polyposis is a common clinical condition. The histologic features of most polyps are similar, but one or multiple causes may lead to this end point. Allergy, bacterial or viral infections, autonomic dysfunction, cystic fibrosis, asthma, and acetylsalicylic acid intolerance are
From the Department of Surgery, Section of Otolaryngology (Drs Gungor, Baroody, Naclerio, and Corey) and the Department of Medicine, Section of Pulmonary Medicine (Dr White), University of Chicago. Supported by the 1995 combined research grant of the American Academy of Otolaryngology–Head and Neck Surgery and the American Academy of Otolaryngic Allergy Foundation. Reprint requests: Anil Gungor, MD, Arkansas Children’s Hospital, Pediatric Otolaryngology, Slot 836, 800 Marshall St, Little Rock, AR 72202. Copyright © 1999 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/99/$8.00 + 0 23/1/98009
often associated with nasal polyps. However, none of these factors is present in most polyp patients. The triggering event for the development of polyps has been a major area of interest, but available tissues represent only end-stage disease. Histologic documentation of the first crucial stages of nasal polyp formation is scarce, mainly because of the lack of predicting factors or animal models. According to a theory by Tos and Mogensen,1 the first stage is localized epithelial rupture, through which fibrous tissue of the lamina propria protrudes. The protruding tissue gradually epithelializes, and eventually a polyp is formed. Several noxious stimuli can lead to epithelial rupture in the nasal mucosa, including allergic and infectious inflammatory processes. Other theories involving contact are equally plausible.2,3 Sensory neuropeptides are potential contributors. These peptides have been identified in the nasal mucosa and are released by sensory nerves during allergic reactions. Neuropeptide release into the nasal mucosa may participate in the genesis of polyps. Neuropeptides may stimulate several processes, leading to hyperproliferation and hypertrophy of the mucosa and leading to polyp formation. Thus examining the pattern of release of the sensory neuropeptides found in the nasal mucosa (substance P [SP], calcitonin gene-related peptide [CGRP], and neurokinin A [NKA]) in patients with nasal polyps as compared with normal and atopic subjects may contribute to our understanding of the pathogenesis of nasal polyps. Neuropeptides are involved in the generation of axon reflexes in the human nasal mucosa. Activation of nasal sensory nerves in disease can lead to vascular congestion by 3 routes: initiation of axon responses, direct effects on the vasculature, or induction of central nervous reflexes involving the sympathetic and parasympathetic motor nerves. These reflexes include sneezing and the sense of irritation as well as vasodilatory effects, causing nasal blockage. Sensory nerves respond to noxious chemical and mechanicothermal stimuli by conveying messages of injury (pain) to the central nervous system and by initiating local vascular inflammatory reactions.4 These nerves can be stimulated by chemicals such as capsaicin, vapor-phase components of cigarette smoke, and mediators of allergenic reac585
586
Otolaryngology– Head and Neck Surgery November 1999
GUNGOR et al
tions such as histamine, bradykinin, prostaglandins, and leukotrienes. Peripheral axon responses lead to release of CGRP, SP, and NKA in the nasal mucosa. These neuropeptides are the product of a peripheral sensory nerve subpopulation that has been termed capsaicin-sensitive primary afferents, consisting mostly of unmyelinated C fibers and some myelinated A fibers.5 SP, CGRP, and NKA are known to cause plasma extravasation, mucus hypersecretion, and activation of mast cells in human and animal respiratory mucosa. An axon reflex induced by SP has been suggested to be involved in allergic reactions. The detectable increase of SP, CGRP, and vasoactive intestinal peptide in nasal secretions after relevant allergen challenge in atopic patients suggests a stimulation of the sensory fibers during the acute allergic reaction.6-11 SP receptors are present on epithelium and on arterial, venous, and sinusoidal vessels and glands. Human nasal glands and epithelium contain binding sites for SP and NKA. SP and NKA stimulate mucous glycoprotein secretion from human nasal mucosa, and SP elicits proliferation of both human fibroblasts and endothelial cells in culture.12 CGRP is found in sensory nerve fibers that innervate vessels and glands in human nasal mucosa. CGRP receptors are present on arterial vessels and to a minor extent on other vessels, and CGRP is a potent and longacting arterial vasodilator.13 CGRP induces proliferation of human upper airway epithelial cells in culture, suggesting a role in regulating airway epithelial cell growth and the potential of stimulating the repair of damaged nasal epithelium and hyperproliferation of the epithelium in nasal polyposis.14-16 Capsaicin, the active substance of hot peppers, acts on a nonspecific cation channel and allows the influx of calcium into the cell, leading to depolarization of the neuron and to pain, neuropeptide release, and the axon response. Capsaicin-induced sensory nerve activation is evidenced symptomatically by the immediate burning sensation, rhinorrhea, and lacrimation induced by topical application. Repeated high-dose capsaicin administration in the human nose leads to a prolonged decrease in capsaicin responsiveness, which may last several weeks.17 Neuropeptides secreted in response to noxious stimuli like allergens may contribute to the airway reactivity of allergic subjects.18 Neuropeptides along with other mediators released after allergen exposure of the nasal mucosa may be responsible for the symptoms of allergic patients.7,19 An increase in the level of SP, CGRP, and vasoactive intestinal peptide in nasal secretions after relevant aller-
gen challenge in atopic subjects has been demonstrated, supporting the contribution of these peptides to the sequelae of allergen exposure. Each neuropeptide shows a rapid, brief, and allergen dose-dependent elevation that suggests stimulation of sensory fibers.7 In this study we tested the difference in the nasal mucosal neuropeptide response to capsaicin in an in vivo model, using subjects with polyps, atopic subjects, and healthy control subjects. METHODS AND MATERIAL We recruited adult human subjects between the ages of 18 and 50 years, and using results of their histories, screening tests, and examinations, we assigned each to 1 of 3 study groups: (1) subjects with polyps (n = 8, 5 men and 3 women, age range 30 to 63 years, mean age 42.0 years); (2) subjects with allergic rhinitis but no polyps (n = 13, 9 men and 4 women, age range 20 to 46 years, mean age 28.0 years); and (3) control subjects with no nasal disease (n = 9, 6 men and 3 women, age range 18 to 34 years, mean age 24.4 years). Atopy was determined by a history of perennial or seasonal allergic rhinitis with sensitivity to at least 1 inhalant aeroallergen confirmed by skin test and/or RAST. Control subjects had no history of allergic disease, including their nasal, dermatologic, and pulmonary systems, and had negative skin test or RAST results. Nasal polyposis was confirmed by anterior rhinoscopy, fiberoptic endoscopic nasal examination, or both. Atopic subjects with histories of immunotherapy in the preceding 1-year period or steroid use in the preceding two-week period were excluded. Subjects with nasal polyps having cystic fibrosis or histories of steroid use in the preceding 2-week period were excluded. All subjects were free of any acute infection at the time of the study. Subjects were not permitted to use any antihistamines, decongestants, or antibiotics 1 week before the study day. Pregnancy was ruled out in female subjects with urine pregnancy testing the day of the challenge, and subjects with positive test results were excluded. The study protocol was approved by the institutional review board of the University of Chicago (protocol no. 7700). Informed consent was obtained from each subject. Nasal Stimulation with Capsaicin and Collection of Nasal Secretions Nasal stimulation with capsaicin was performed as follows. Filter-paper disks (no. 190005; Shandon, Pittsburgh, PA) were first saturated with 10–6 mol/L phosphoramidon solution and air dried. Each capsaicin disk was saturated with 30 µL of capsaicin solution (6 mmol/L in 1% EtOH vol/vol in 0.9% saline solution); this volume saturates the disk with 50 µg of capsaicin. The vehicle disk was saturated with 30 µL of the vehicle (1% EtOH in 0.9% saline solution) only. All disks were kept in the refrigerator after being weighed (Mettler Analytical Balance AE 240; Mettler Instruments, Highstown, NJ).
Otolaryngology– Head and Neck Surgery Volume 121 Number 5
GUNGOR et al
587
Fig 1. Neuropeptides in control subjects. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Fig 2. Neuropeptides in atopic subjects. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Before the challenge, 6 preliminary nasal lavages were performed to wash away preexisting secretions. Lavages were performed by instilling 5 to 8 mL 0.9% saline solution at 37° C into the nasal cavity. Before the first lavage, a preweighed dry disk was placed on the anterior nasal septum under direct visualization. This disk was removed after 2 minutes of collection, immediately resealed into a preweighed 1.5-mL microfuge tube containing 10–6 mol/L phosphoramidon in 270 µL 0.9% saline solution, weighed, and placed on ice. This procedure was repeated for each disk. Phosphoramidon prevents the enzymatic degradation of neuropeptides by neutral endopeptidase (NEP) in nasal secretions. The weight of collected secretions was calculated. This initial disk was the first baseline value for the prechallenge neuropeptide levels. After the 6 lavages, another dry disk was used to collect the second baseline. Then we placed the vehicle disk for 2 minutes, followed in 5 minutes by a dry collection disk. The capsaicin disk was placed next on the opposite nasal septum, followed in 5 minutes by a dry collection disk. After completion of the challenge, the tubes were placed in a freezer at –70°C until the day of the Sep-Pak/enzyme immunoassay (EIA) protocol. The assays were performed in batches of 3 or 4 with standard curve and spiked controls.
The specificity of the EIA was determined by previous studies to be 0.1% for SP and CGRP and 0.2% for NKA. Every assay run included all neuropeptides. At least 1 sample from each study group was included in each assay. Spike control ensured a reliable detection within the error level of half a log throughout the measurements. All results were expressed as moles of the specific neuropeptide per disk, and then a log transformation was calculated. The level of detection was at the 10–14 level (10 fmol) range, and therefore the log-transformed values were negative. For statistical analysis between groups, analysis of variance (ANOVA) was used. When ANOVA showed a significant difference, an unpaired t test was used for comparison between groups. P < 0.05 was considered significant. Comparison of the capsaicin response between groups was done by focusing on the change in SP levels with capsaicin stimulation. For each subject, the mean vehicle (sham challenge) value was obtained—(V + 5 min after V)/2—and subtracted from the mean of capsaicin stimulation value—(C + 5 min after C)/2.
Determination of Neuropeptide Concentrations by EIA After the specimen was thawed, the supernatant was transferred by pipette to 1.5-mL microfuge tubes. Glacial acetic acid was added to a final concentration of 5%, to block remaining NEP. An equal volume of 0.1% trifluoroacetic acid was added to precipitate proteins, followed by centrifugation. Samples were then purified through C-18 Sep-Pak chromatography cartridges. Elution of neuropeptides was done with 3 mL of 60% acetonitrile/0.1% trifluoroacetic acid. The eluates were desiccated overnight.
RESULTS
Normal subjects exhibited an increase in all 3 neuropeptide levels with capsaicin stimulation, which returned quickly to baseline values. CGRP seems to be the most abundant or the most easily detected neuropeptide (Fig 1). Atopic subjects seemed to have slightly higher baseline levels of all 3 neuropeptides. The response to capsaicin challenge for NKA was insignificant, but SP and CGRP showed increases similar to those in control subjects. The increase in CGRP levels seems to be sustained in atopic subjects because a return to the baseline pattern was not observed in the first 5 minutes after the capsaicin challenge (Fig 2). In subjects with polyps there was an obvious blunt-
588
GUNGOR et al
Fig 3. Neuropeptides in subjects with polyps. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Fig 4. SP. Atopic and control subjects have a significant response to capsaicin stimulation as compared with the polyp group (P < 0.005). Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
ing of the response to capsaicin stimulation for all 3 neuropeptides. There also seemed to be a greater difference between the 3 neuropeptides, which was less apparent in the atopic and control groups. NKA baseline levels were lower, and CGRP baseline levels were higher than those of the control subjects (Fig 3). The levels of SP, as analyzed by ANOVA and subsequent t tests, were found to be significantly different in the polyp group than in the control and the atopic groups (P < 0.05). The postchallenge values of the polyp group were significantly different than the postchallenge values of the atopic and the control groups (P < 0.001, unpaired t test) (Fig 4). The observed difference between the atopic and control subjects failed to reach statistical significance (P > 0.05). Considering only the change from the sham challenge values induced by capsaicin stimulation, we
Otolaryngology– Head and Neck Surgery November 1999
Fig 5. SP deltas. Increase in the SP level induced by capsaicin stimulation has been calculated by subtracting the value 5 minutes after vehicle challenge from the capsaicin challenge value. Result for each subject has been plotted on the y-axis. There is no significant difference between groups.
Fig 6. NKA. For interpretation see text. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
found that the distribution of individual SP values and mean showed that the delta (the change over vehicle stimulation value) for the polyp group was smaller than that of the atopic and control subjects, but ANOVA did not show a statistically significant difference between groups (P > 0.05) (Fig 5). NKA was lowest in the polyp group, and the atopic subjects had the highest levels without a significant response to capsaicin stimulation (Fig 6). However, the groups were not significantly different from each other at the 0.05 level (ANOVA). The change in NKA levels with capsaicin stimulation did not show any difference between groups at the 0.05 level (ANOVA) (Fig 7). For CGRP, the control group showed a clearly visi-
Otolaryngology– Head and Neck Surgery Volume 121 Number 5
Fig 7. NKA deltas. Design same as that of Fig 5, but with NKA data. For interpretation see text.
GUNGOR et al
589
Fig 8. CGRP. For interpretation see text. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
ble response to capsaicin stimulation (Fig 8). This response was not observed in the polyp and atopic groups. These 2 groups also seemed to respond to vehicle stimulation, hence the less prominent response to capsaicin. This observation is verified by comparing the deltas (Fig 9). At the 0.05 level the control group had significantly higher values than both the polyp and the atopic groups (unpaired t test). DISCUSSION
Finding overall lower neuropeptide levels in the polyp group was a surprising discovery. The observed increase in CGRP response to nonspecific stimulus and subsequent blunting of the response to capsaicin in subjects with polyps and atopic subjects was an interesting finding. Both observations were contrary to what was proposed in our hypothesis. An activated neuroinflammatory response system, as we would expect, could be evident by increased levels of the suspected neuropeptides. However, under conditions of chronic stimulation, the response suggests depletion of stores to give an absent or blunted response. In a recent study Perkins et al20 investigated the presence of SP and CGRP in nasal lavage fluid and nasal mucosal specimens in 10 patients with polyps. They were unable to measure SP. Low levels of CGRP were detected in patients with polyps. The authors related this finding to the relatively avascular anatomy of the polyps and to the lack of neural structures in the polyp tissue. However, in view of the design and results of our study, a priming effect on CGRP release in atopy and polyposis may have caused the observed “depletion” of CGRP. Furthermore, we have looked at the nasal mucosal response, not at the response of polyps in our study.
Fig 9. CGRP deltas. Design same as that of Figs 5 and 7, using CGRP data. Atopic subjects and those with polyps are significantly different from the control group (P < 0.05). For interpretation see text.
Because of the design of our study, our findings support the idea that the neuropeptide stores were depleted by continuous noxious stimuli, which may have led to the development of polyps. However, our study group is small, and some of the observed differences between groups need to be verified in a larger study. SP was lowest in the polyp group (P < 0.0001) (Fig 4), but the difficulties associated with the detection of this neuropeptide limit the interpretation of our data. It can be speculated that polyp development is possible when neuropeptide-initiated hyperproliferation intended to repair the damaged mucosa becomes deregulated. In some, this may happen because the noxious stimulus is sustained and/or severe, and in others, effective neutralizing mechanisms (eg, NEP production) are
590
GUNGOR et al
insufficient. It is also possible that the interrelationship and/or cooperation between colocalized neuropeptides determines the nature of the mucosal response. A highly speculative mechanism based on our limited observations would suggest that if the neuropeptide system is only partially activated (only CGRP in the atopic group) the mucosal response is limited to edema and congestion, whereas simultaneous activation and subsequent depletion of all 3 neuropeptides (as in the polyp group) leads to polyp development as a result of severe inflammation. Considering the mitogenic effects of CGRP on respiratory epithelium, CGRP is most likely activated to provide the much needed stimulus for hyperproliferation and repair. Blunt responses to capsaicin stimulation and overall low levels of neuropeptides, as compared with those in the control group, are considered to be signs of depleted neuropeptide stores, which in turn suggest an activated system of mucosal repair. Sensitivity to vehicle stimulation was interpreted as the implication of a primed system responding to nonspecific stimuli as well. It is interesting that the polyp group seemed to be depleted of NKA, whereas the atopic subjects showed above-control values. Low levels of SP in the polyp group imply that the nasal mucosa of subjects with polyps does not show a response to capsaicin stimulation with an increase in SP levels. The important role of the NEP subsystem and the effects of tachyphylaxis were not addressed in this study. A different study design is needed to approach this important issue. The potential causes of neuropeptide release in polyp patients also were not addressed in this study. These and other aspects of the neurogenic inflammation deserve further attention, and results of our pilot study need to be verified by future studies. REFERENCES 1. Tos M, Mogensen C. Pathogenesis of nasal polyps. Rhinology 1977;15:87-95. 2. Jankowski R, Bene MC, Moneret-Vautrin AD, et al. Immunohistological characteristics of nasal polyps. Rhinology 1989;8 (Suppl):51-8.
Otolaryngology– Head and Neck Surgery November 1999
3. Larsen K, Tos M. Clinical course of patients with primary nasal polyps. Acta Otolaryngol (Stockh) 1994;114:556-9. 4. Lundblad L, Lundberg JM, Anggard A. Local and systemic capsaicin pretreatment inhibits sneezing and the increase in nasal vascular permeability induced by certain chemical irritants. Naunyn Schmiedebergs Arch Pharmacol 1984;326:254-61. 5. Lundblad L. Protective reflexes and vascular effects in the nasal mucosa elicited by activation of capsaicin-sensitive substance P immunoreactive trigeminal neurons. Acta Physiol Scand 1984; 529(Suppl):1-42. 6. Holtzer P. Local effector functions of capsaicin sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739-63. 7. Mossiman BL, White MV, Hohman RJ, et al. Calcitonin generelated peptide and vasoactive intestinal peptide increase in nasal secretions after allergen challenge in atopic patients. J Allergy Clin Immunol 1993;92:95-104. 8. Ichimura K, Mineda H, Seki A. Vascular effects of neuropeptides on the nasal mucosa. Ann Otol Rhinol Laryngol 1988;97:289-93. 9. Uddman R, Anggard A, Widdicombe JG. Nerves and neurotransmitters in the nose. In: Mygind N, Pipcorn U, editors. Allergic and vasomotor rhinitis: pathophysiological aspects. Copenhagen: Munksgaard; 1987. p. 50-62. 10. Guarnaccia S, Baraniuk JN, Bellanti J, et al. Calcitonin generelated peptide nasal provocation in humans. Ann Allergy 1994; 72:515-9. 11. Holtzer P. Capsaicin: cellular targets, mechanism of action and selectivity for thin sensory neurons. Pharmacol Rev 1991;43: 143-201. 12. Ziche M, Morbidelli L, Pacini M, et al. NK1-receptors mediate the proliferative response of human fibroblasts to tachykinins. Br J Pharmacol 1990;100:11-4. 13. Struthers AD, Brown MJ, McDonald DW, et al. Human calcitonin gene-related peptide; a potent endogenous vasodilator in man. Clin Sci (Lond) 1986;70:389-93. 14. Baraniuk JN, Lundgren JD, Okayama M, et al. SP and NKA in human nasal mucosa. Am Rev Respir Dis 1991;4:228-36. 15. Corey JP, Sigrist KS, White SR. Proliferation of human upper airway epithelial (HUAE) cells stimulated by CGRP but not neurokinins [abstract]. Am J Respir Crit Care Med 1994;149:A997. 16. White SR, Hershenson MB, Sigrist KS, et al. Proliferation of guinea pig tracheal epithelial cells induced by calcitonin generelated peptide. Am J Respir Cell Mol Biol 1993;8:592-6. 17. Stjarne P, Lundblad L, Anggard A, et al. Local capsaicin treatment of the nasal mucosa reduces symptoms in patients with nonallergic nasal hyperreactivity. Am J Rhinol 1991;5:145-51. 18. Nieber K, Baumgarten CR, Rathsack R, et al. SP and beta-endorphin–like immunoreactivity in lavage fluids of subjects with and without allergic asthma. J Allergy Clin Immunol 1992;90:646-52. 19. Naclerio RM, Meier HL, Sobotka AK, et al. Mediator release after nasal airway challenge with allergen. Am Rev Respir Dis 1983;128:597-602. 20. Perkins JA, Moore KH, Canonico DM, et al. Neuropeptide levels in the nasal secretion and nasal mucosa of patients with chronic sinusitis and nasal polyposis. Am J Rhinol 1994;8:117-21.
In this in vivo prospective, controlled study, we have examined the capsaicin-induced levels and secretion patterns of the colocalized neuropeptides substance P, calcitonin gene-related peptide (CGRP), and neurokinin A in nasal secretions of subjects with nasal polyps, and we compared these with secretion patterns from healthy subjects and from subjects with allergic rhinitis. Capsaicin was used to elicit neuropeptide release. The neuropeptide levels were measured by an ELISA technique. For substance P, subjects with nasal polyps responded very poorly to capsaicin stimulation. The atopic group was more reactive to capsaicin stimulation than control subjects. For CGRP the increase was immediate in all groups. Atopic subjects and subjects with polyps had a less pronounced but sustained response to capsaicin stimulation. CGRP levels in atopic subjects and those with polyps were restored rapidly. Atopic subjects had higher neurokinin A levels with an immediate and sustained response to capsaicin. Control subjects had higher levels than those with polyps, but both groups were nonresponsive to capsaicin stimulation. (Otolaryngol Head Neck Surg 1999;121:585-90.)
N
asal polyposis is a common clinical condition. The histologic features of most polyps are similar, but one or multiple causes may lead to this end point. Allergy, bacterial or viral infections, autonomic dysfunction, cystic fibrosis, asthma, and acetylsalicylic acid intolerance are
From the Department of Surgery, Section of Otolaryngology (Drs Gungor, Baroody, Naclerio, and Corey) and the Department of Medicine, Section of Pulmonary Medicine (Dr White), University of Chicago. Supported by the 1995 combined research grant of the American Academy of Otolaryngology–Head and Neck Surgery and the American Academy of Otolaryngic Allergy Foundation. Reprint requests: Anil Gungor, MD, Arkansas Children’s Hospital, Pediatric Otolaryngology, Slot 836, 800 Marshall St, Little Rock, AR 72202. Copyright © 1999 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/99/$8.00 + 0 23/1/98009
often associated with nasal polyps. However, none of these factors is present in most polyp patients. The triggering event for the development of polyps has been a major area of interest, but available tissues represent only end-stage disease. Histologic documentation of the first crucial stages of nasal polyp formation is scarce, mainly because of the lack of predicting factors or animal models. According to a theory by Tos and Mogensen,1 the first stage is localized epithelial rupture, through which fibrous tissue of the lamina propria protrudes. The protruding tissue gradually epithelializes, and eventually a polyp is formed. Several noxious stimuli can lead to epithelial rupture in the nasal mucosa, including allergic and infectious inflammatory processes. Other theories involving contact are equally plausible.2,3 Sensory neuropeptides are potential contributors. These peptides have been identified in the nasal mucosa and are released by sensory nerves during allergic reactions. Neuropeptide release into the nasal mucosa may participate in the genesis of polyps. Neuropeptides may stimulate several processes, leading to hyperproliferation and hypertrophy of the mucosa and leading to polyp formation. Thus examining the pattern of release of the sensory neuropeptides found in the nasal mucosa (substance P [SP], calcitonin gene-related peptide [CGRP], and neurokinin A [NKA]) in patients with nasal polyps as compared with normal and atopic subjects may contribute to our understanding of the pathogenesis of nasal polyps. Neuropeptides are involved in the generation of axon reflexes in the human nasal mucosa. Activation of nasal sensory nerves in disease can lead to vascular congestion by 3 routes: initiation of axon responses, direct effects on the vasculature, or induction of central nervous reflexes involving the sympathetic and parasympathetic motor nerves. These reflexes include sneezing and the sense of irritation as well as vasodilatory effects, causing nasal blockage. Sensory nerves respond to noxious chemical and mechanicothermal stimuli by conveying messages of injury (pain) to the central nervous system and by initiating local vascular inflammatory reactions.4 These nerves can be stimulated by chemicals such as capsaicin, vapor-phase components of cigarette smoke, and mediators of allergenic reac585
586
Otolaryngology– Head and Neck Surgery November 1999
GUNGOR et al
tions such as histamine, bradykinin, prostaglandins, and leukotrienes. Peripheral axon responses lead to release of CGRP, SP, and NKA in the nasal mucosa. These neuropeptides are the product of a peripheral sensory nerve subpopulation that has been termed capsaicin-sensitive primary afferents, consisting mostly of unmyelinated C fibers and some myelinated A fibers.5 SP, CGRP, and NKA are known to cause plasma extravasation, mucus hypersecretion, and activation of mast cells in human and animal respiratory mucosa. An axon reflex induced by SP has been suggested to be involved in allergic reactions. The detectable increase of SP, CGRP, and vasoactive intestinal peptide in nasal secretions after relevant allergen challenge in atopic patients suggests a stimulation of the sensory fibers during the acute allergic reaction.6-11 SP receptors are present on epithelium and on arterial, venous, and sinusoidal vessels and glands. Human nasal glands and epithelium contain binding sites for SP and NKA. SP and NKA stimulate mucous glycoprotein secretion from human nasal mucosa, and SP elicits proliferation of both human fibroblasts and endothelial cells in culture.12 CGRP is found in sensory nerve fibers that innervate vessels and glands in human nasal mucosa. CGRP receptors are present on arterial vessels and to a minor extent on other vessels, and CGRP is a potent and longacting arterial vasodilator.13 CGRP induces proliferation of human upper airway epithelial cells in culture, suggesting a role in regulating airway epithelial cell growth and the potential of stimulating the repair of damaged nasal epithelium and hyperproliferation of the epithelium in nasal polyposis.14-16 Capsaicin, the active substance of hot peppers, acts on a nonspecific cation channel and allows the influx of calcium into the cell, leading to depolarization of the neuron and to pain, neuropeptide release, and the axon response. Capsaicin-induced sensory nerve activation is evidenced symptomatically by the immediate burning sensation, rhinorrhea, and lacrimation induced by topical application. Repeated high-dose capsaicin administration in the human nose leads to a prolonged decrease in capsaicin responsiveness, which may last several weeks.17 Neuropeptides secreted in response to noxious stimuli like allergens may contribute to the airway reactivity of allergic subjects.18 Neuropeptides along with other mediators released after allergen exposure of the nasal mucosa may be responsible for the symptoms of allergic patients.7,19 An increase in the level of SP, CGRP, and vasoactive intestinal peptide in nasal secretions after relevant aller-
gen challenge in atopic subjects has been demonstrated, supporting the contribution of these peptides to the sequelae of allergen exposure. Each neuropeptide shows a rapid, brief, and allergen dose-dependent elevation that suggests stimulation of sensory fibers.7 In this study we tested the difference in the nasal mucosal neuropeptide response to capsaicin in an in vivo model, using subjects with polyps, atopic subjects, and healthy control subjects. METHODS AND MATERIAL We recruited adult human subjects between the ages of 18 and 50 years, and using results of their histories, screening tests, and examinations, we assigned each to 1 of 3 study groups: (1) subjects with polyps (n = 8, 5 men and 3 women, age range 30 to 63 years, mean age 42.0 years); (2) subjects with allergic rhinitis but no polyps (n = 13, 9 men and 4 women, age range 20 to 46 years, mean age 28.0 years); and (3) control subjects with no nasal disease (n = 9, 6 men and 3 women, age range 18 to 34 years, mean age 24.4 years). Atopy was determined by a history of perennial or seasonal allergic rhinitis with sensitivity to at least 1 inhalant aeroallergen confirmed by skin test and/or RAST. Control subjects had no history of allergic disease, including their nasal, dermatologic, and pulmonary systems, and had negative skin test or RAST results. Nasal polyposis was confirmed by anterior rhinoscopy, fiberoptic endoscopic nasal examination, or both. Atopic subjects with histories of immunotherapy in the preceding 1-year period or steroid use in the preceding two-week period were excluded. Subjects with nasal polyps having cystic fibrosis or histories of steroid use in the preceding 2-week period were excluded. All subjects were free of any acute infection at the time of the study. Subjects were not permitted to use any antihistamines, decongestants, or antibiotics 1 week before the study day. Pregnancy was ruled out in female subjects with urine pregnancy testing the day of the challenge, and subjects with positive test results were excluded. The study protocol was approved by the institutional review board of the University of Chicago (protocol no. 7700). Informed consent was obtained from each subject. Nasal Stimulation with Capsaicin and Collection of Nasal Secretions Nasal stimulation with capsaicin was performed as follows. Filter-paper disks (no. 190005; Shandon, Pittsburgh, PA) were first saturated with 10–6 mol/L phosphoramidon solution and air dried. Each capsaicin disk was saturated with 30 µL of capsaicin solution (6 mmol/L in 1% EtOH vol/vol in 0.9% saline solution); this volume saturates the disk with 50 µg of capsaicin. The vehicle disk was saturated with 30 µL of the vehicle (1% EtOH in 0.9% saline solution) only. All disks were kept in the refrigerator after being weighed (Mettler Analytical Balance AE 240; Mettler Instruments, Highstown, NJ).
Otolaryngology– Head and Neck Surgery Volume 121 Number 5
GUNGOR et al
587
Fig 1. Neuropeptides in control subjects. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Fig 2. Neuropeptides in atopic subjects. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Before the challenge, 6 preliminary nasal lavages were performed to wash away preexisting secretions. Lavages were performed by instilling 5 to 8 mL 0.9% saline solution at 37° C into the nasal cavity. Before the first lavage, a preweighed dry disk was placed on the anterior nasal septum under direct visualization. This disk was removed after 2 minutes of collection, immediately resealed into a preweighed 1.5-mL microfuge tube containing 10–6 mol/L phosphoramidon in 270 µL 0.9% saline solution, weighed, and placed on ice. This procedure was repeated for each disk. Phosphoramidon prevents the enzymatic degradation of neuropeptides by neutral endopeptidase (NEP) in nasal secretions. The weight of collected secretions was calculated. This initial disk was the first baseline value for the prechallenge neuropeptide levels. After the 6 lavages, another dry disk was used to collect the second baseline. Then we placed the vehicle disk for 2 minutes, followed in 5 minutes by a dry collection disk. The capsaicin disk was placed next on the opposite nasal septum, followed in 5 minutes by a dry collection disk. After completion of the challenge, the tubes were placed in a freezer at –70°C until the day of the Sep-Pak/enzyme immunoassay (EIA) protocol. The assays were performed in batches of 3 or 4 with standard curve and spiked controls.
The specificity of the EIA was determined by previous studies to be 0.1% for SP and CGRP and 0.2% for NKA. Every assay run included all neuropeptides. At least 1 sample from each study group was included in each assay. Spike control ensured a reliable detection within the error level of half a log throughout the measurements. All results were expressed as moles of the specific neuropeptide per disk, and then a log transformation was calculated. The level of detection was at the 10–14 level (10 fmol) range, and therefore the log-transformed values were negative. For statistical analysis between groups, analysis of variance (ANOVA) was used. When ANOVA showed a significant difference, an unpaired t test was used for comparison between groups. P < 0.05 was considered significant. Comparison of the capsaicin response between groups was done by focusing on the change in SP levels with capsaicin stimulation. For each subject, the mean vehicle (sham challenge) value was obtained—(V + 5 min after V)/2—and subtracted from the mean of capsaicin stimulation value—(C + 5 min after C)/2.
Determination of Neuropeptide Concentrations by EIA After the specimen was thawed, the supernatant was transferred by pipette to 1.5-mL microfuge tubes. Glacial acetic acid was added to a final concentration of 5%, to block remaining NEP. An equal volume of 0.1% trifluoroacetic acid was added to precipitate proteins, followed by centrifugation. Samples were then purified through C-18 Sep-Pak chromatography cartridges. Elution of neuropeptides was done with 3 mL of 60% acetonitrile/0.1% trifluoroacetic acid. The eluates were desiccated overnight.
RESULTS
Normal subjects exhibited an increase in all 3 neuropeptide levels with capsaicin stimulation, which returned quickly to baseline values. CGRP seems to be the most abundant or the most easily detected neuropeptide (Fig 1). Atopic subjects seemed to have slightly higher baseline levels of all 3 neuropeptides. The response to capsaicin challenge for NKA was insignificant, but SP and CGRP showed increases similar to those in control subjects. The increase in CGRP levels seems to be sustained in atopic subjects because a return to the baseline pattern was not observed in the first 5 minutes after the capsaicin challenge (Fig 2). In subjects with polyps there was an obvious blunt-
588
GUNGOR et al
Fig 3. Neuropeptides in subjects with polyps. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
Fig 4. SP. Atopic and control subjects have a significant response to capsaicin stimulation as compared with the polyp group (P < 0.005). Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
ing of the response to capsaicin stimulation for all 3 neuropeptides. There also seemed to be a greater difference between the 3 neuropeptides, which was less apparent in the atopic and control groups. NKA baseline levels were lower, and CGRP baseline levels were higher than those of the control subjects (Fig 3). The levels of SP, as analyzed by ANOVA and subsequent t tests, were found to be significantly different in the polyp group than in the control and the atopic groups (P < 0.05). The postchallenge values of the polyp group were significantly different than the postchallenge values of the atopic and the control groups (P < 0.001, unpaired t test) (Fig 4). The observed difference between the atopic and control subjects failed to reach statistical significance (P > 0.05). Considering only the change from the sham challenge values induced by capsaicin stimulation, we
Otolaryngology– Head and Neck Surgery November 1999
Fig 5. SP deltas. Increase in the SP level induced by capsaicin stimulation has been calculated by subtracting the value 5 minutes after vehicle challenge from the capsaicin challenge value. Result for each subject has been plotted on the y-axis. There is no significant difference between groups.
Fig 6. NKA. For interpretation see text. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
found that the distribution of individual SP values and mean showed that the delta (the change over vehicle stimulation value) for the polyp group was smaller than that of the atopic and control subjects, but ANOVA did not show a statistically significant difference between groups (P > 0.05) (Fig 5). NKA was lowest in the polyp group, and the atopic subjects had the highest levels without a significant response to capsaicin stimulation (Fig 6). However, the groups were not significantly different from each other at the 0.05 level (ANOVA). The change in NKA levels with capsaicin stimulation did not show any difference between groups at the 0.05 level (ANOVA) (Fig 7). For CGRP, the control group showed a clearly visi-
Otolaryngology– Head and Neck Surgery Volume 121 Number 5
Fig 7. NKA deltas. Design same as that of Fig 5, but with NKA data. For interpretation see text.
GUNGOR et al
589
Fig 8. CGRP. For interpretation see text. Baseline 1, Measurement before intervention; Baseline 2, measurement after nasal lavage; EtOH (Vehicle), measurement with ethyl alcohol stimulation.
ble response to capsaicin stimulation (Fig 8). This response was not observed in the polyp and atopic groups. These 2 groups also seemed to respond to vehicle stimulation, hence the less prominent response to capsaicin. This observation is verified by comparing the deltas (Fig 9). At the 0.05 level the control group had significantly higher values than both the polyp and the atopic groups (unpaired t test). DISCUSSION
Finding overall lower neuropeptide levels in the polyp group was a surprising discovery. The observed increase in CGRP response to nonspecific stimulus and subsequent blunting of the response to capsaicin in subjects with polyps and atopic subjects was an interesting finding. Both observations were contrary to what was proposed in our hypothesis. An activated neuroinflammatory response system, as we would expect, could be evident by increased levels of the suspected neuropeptides. However, under conditions of chronic stimulation, the response suggests depletion of stores to give an absent or blunted response. In a recent study Perkins et al20 investigated the presence of SP and CGRP in nasal lavage fluid and nasal mucosal specimens in 10 patients with polyps. They were unable to measure SP. Low levels of CGRP were detected in patients with polyps. The authors related this finding to the relatively avascular anatomy of the polyps and to the lack of neural structures in the polyp tissue. However, in view of the design and results of our study, a priming effect on CGRP release in atopy and polyposis may have caused the observed “depletion” of CGRP. Furthermore, we have looked at the nasal mucosal response, not at the response of polyps in our study.
Fig 9. CGRP deltas. Design same as that of Figs 5 and 7, using CGRP data. Atopic subjects and those with polyps are significantly different from the control group (P < 0.05). For interpretation see text.
Because of the design of our study, our findings support the idea that the neuropeptide stores were depleted by continuous noxious stimuli, which may have led to the development of polyps. However, our study group is small, and some of the observed differences between groups need to be verified in a larger study. SP was lowest in the polyp group (P < 0.0001) (Fig 4), but the difficulties associated with the detection of this neuropeptide limit the interpretation of our data. It can be speculated that polyp development is possible when neuropeptide-initiated hyperproliferation intended to repair the damaged mucosa becomes deregulated. In some, this may happen because the noxious stimulus is sustained and/or severe, and in others, effective neutralizing mechanisms (eg, NEP production) are
590
GUNGOR et al
insufficient. It is also possible that the interrelationship and/or cooperation between colocalized neuropeptides determines the nature of the mucosal response. A highly speculative mechanism based on our limited observations would suggest that if the neuropeptide system is only partially activated (only CGRP in the atopic group) the mucosal response is limited to edema and congestion, whereas simultaneous activation and subsequent depletion of all 3 neuropeptides (as in the polyp group) leads to polyp development as a result of severe inflammation. Considering the mitogenic effects of CGRP on respiratory epithelium, CGRP is most likely activated to provide the much needed stimulus for hyperproliferation and repair. Blunt responses to capsaicin stimulation and overall low levels of neuropeptides, as compared with those in the control group, are considered to be signs of depleted neuropeptide stores, which in turn suggest an activated system of mucosal repair. Sensitivity to vehicle stimulation was interpreted as the implication of a primed system responding to nonspecific stimuli as well. It is interesting that the polyp group seemed to be depleted of NKA, whereas the atopic subjects showed above-control values. Low levels of SP in the polyp group imply that the nasal mucosa of subjects with polyps does not show a response to capsaicin stimulation with an increase in SP levels. The important role of the NEP subsystem and the effects of tachyphylaxis were not addressed in this study. A different study design is needed to approach this important issue. The potential causes of neuropeptide release in polyp patients also were not addressed in this study. These and other aspects of the neurogenic inflammation deserve further attention, and results of our pilot study need to be verified by future studies. REFERENCES 1. Tos M, Mogensen C. Pathogenesis of nasal polyps. Rhinology 1977;15:87-95. 2. Jankowski R, Bene MC, Moneret-Vautrin AD, et al. Immunohistological characteristics of nasal polyps. Rhinology 1989;8 (Suppl):51-8.
Otolaryngology– Head and Neck Surgery November 1999
3. Larsen K, Tos M. Clinical course of patients with primary nasal polyps. Acta Otolaryngol (Stockh) 1994;114:556-9. 4. Lundblad L, Lundberg JM, Anggard A. Local and systemic capsaicin pretreatment inhibits sneezing and the increase in nasal vascular permeability induced by certain chemical irritants. Naunyn Schmiedebergs Arch Pharmacol 1984;326:254-61. 5. Lundblad L. Protective reflexes and vascular effects in the nasal mucosa elicited by activation of capsaicin-sensitive substance P immunoreactive trigeminal neurons. Acta Physiol Scand 1984; 529(Suppl):1-42. 6. Holtzer P. Local effector functions of capsaicin sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739-63. 7. Mossiman BL, White MV, Hohman RJ, et al. Calcitonin generelated peptide and vasoactive intestinal peptide increase in nasal secretions after allergen challenge in atopic patients. J Allergy Clin Immunol 1993;92:95-104. 8. Ichimura K, Mineda H, Seki A. Vascular effects of neuropeptides on the nasal mucosa. Ann Otol Rhinol Laryngol 1988;97:289-93. 9. Uddman R, Anggard A, Widdicombe JG. Nerves and neurotransmitters in the nose. In: Mygind N, Pipcorn U, editors. Allergic and vasomotor rhinitis: pathophysiological aspects. Copenhagen: Munksgaard; 1987. p. 50-62. 10. Guarnaccia S, Baraniuk JN, Bellanti J, et al. Calcitonin generelated peptide nasal provocation in humans. Ann Allergy 1994; 72:515-9. 11. Holtzer P. Capsaicin: cellular targets, mechanism of action and selectivity for thin sensory neurons. Pharmacol Rev 1991;43: 143-201. 12. Ziche M, Morbidelli L, Pacini M, et al. NK1-receptors mediate the proliferative response of human fibroblasts to tachykinins. Br J Pharmacol 1990;100:11-4. 13. Struthers AD, Brown MJ, McDonald DW, et al. Human calcitonin gene-related peptide; a potent endogenous vasodilator in man. Clin Sci (Lond) 1986;70:389-93. 14. Baraniuk JN, Lundgren JD, Okayama M, et al. SP and NKA in human nasal mucosa. Am Rev Respir Dis 1991;4:228-36. 15. Corey JP, Sigrist KS, White SR. Proliferation of human upper airway epithelial (HUAE) cells stimulated by CGRP but not neurokinins [abstract]. Am J Respir Crit Care Med 1994;149:A997. 16. White SR, Hershenson MB, Sigrist KS, et al. Proliferation of guinea pig tracheal epithelial cells induced by calcitonin generelated peptide. Am J Respir Cell Mol Biol 1993;8:592-6. 17. Stjarne P, Lundblad L, Anggard A, et al. Local capsaicin treatment of the nasal mucosa reduces symptoms in patients with nonallergic nasal hyperreactivity. Am J Rhinol 1991;5:145-51. 18. Nieber K, Baumgarten CR, Rathsack R, et al. SP and beta-endorphin–like immunoreactivity in lavage fluids of subjects with and without allergic asthma. J Allergy Clin Immunol 1992;90:646-52. 19. Naclerio RM, Meier HL, Sobotka AK, et al. Mediator release after nasal airway challenge with allergen. Am Rev Respir Dis 1983;128:597-602. 20. Perkins JA, Moore KH, Canonico DM, et al. Neuropeptide levels in the nasal secretion and nasal mucosa of patients with chronic sinusitis and nasal polyposis. Am J Rhinol 1994;8:117-21.