Published ahead of print on March 5, 2003, doi:10.1164/rccm.200212-1508OC
© 2003 American Thoracic Society
Neural Expression and Increased Lavage Fluid Levels of Secretoneurin in Seasonal Allergic RhinitisDepartments of Clinical Pharmacology and Otorhinolaryngology, University Hospital, Department of Physiological Sciences, Lund University, Lund, Sweden; Department of Pharmacology, Innsbruck University, Innsbruck, Austria; and School of Biomolecular and Biomedical Science, Griffith University, Brisbane, Australia Correspondence and requests for reprints should be addressed to Dr. Magnus Korsgren, M.D., Department of Clinical Pharmacology, University Hospital, SE-221 85 Lund, Sweden. E-mail: magnus.korsgren{at}klinfarm.lu.se
Secretoneurin is a neuropeptide potentially involved in migration of eosinophils, monocytes, and dendritic cells. Whether secretoneurin is present in the human airway mucosa and whether it is released at ongoing allergic airway inflammation is currently unknown. In patients with allergic rhinitis, we have explored the occurrence of secretoneurin in nasal mucosal biopsies and lavage fluids before and during natural allergen exposure. Immunohistochemical analysis revealed an abundance of nerves displaying secretoneurin immunoreactivity, which were distributed predominantly around blood vessels and submucosal glands. A majority of nerve fibers containing vesicular acetylcholine transporter, tyrosine hydroxylase, calcitonin gene–related peptide, and vasoactive intestinal peptide were also secretoneurin-immunoreactive, indicating a localization of secretoneurin in cholinergic, adrenergic, and sensory nerves. Lavage fluid levels of secretoneurin were increased at allergen exposure (p < 0.01–0.05). Levels of secretoneurin did not correlate with eosinophil cationic protein (  = 0.1, p = 0.7). We conclude that secretoneurin has a widespread occurrence in nasal mucosal nerves of patients with seasonal allergic rhinitis and that increased nasal lavage fluid levels of secretoneurin may characterize ongoing allergen exposure. These data favor a role of secretoneurin in the local traffic of immune cells in human airway mucosa.
Key Words: allergic airway inflammation • neuropeptides • eosinophils Allergic airway inflammation is characterized by recruitment and activation of inflammatory cells as well as by increased activities of mucosal end organs. The result is an exceedingly complex molecular environment rich in cytokines, chemokines, and other mediators, originating from various cells and tissue compartments. In allergic rhinitis, this process is characterized by recruitment and activation of eosinophils (1, 2) and by nerve activities, indicated by symptoms such as itching and sneezing, pathophysiologic features such as (reflex-mediated) secretion, and release of neurotransmitters and neuropeptides (3, 4). It has been suggested that such nerve activities may interact with inflammatory/immune cells and that such interactions may be of proinflammatory importance (5–8). For example, it has been demonstrated that topical substance P may increase the nasal output of eosinophils in seasonal allergic rhinitis (5). Furthermore, influx of inflammatory cells into the nasal mucosa has been reported after challenges with high-dose capsaicin (7). However, to what extent neural activities can induce or modify airway inflammatory processes is still an open question (8, 9). There is a need for a more complete analysis of the airway innervation to understand the role of airway neural activities in health and disease (10). Secretoneurin is a 33-amino acid peptide derived by proteolytic processing from its precursor secretogranin II (chromogranin C) (11, 12). Preliminary observations suggest the occurrence of secretoneurin in rat tracheal tissue and in human bronchial neuroendocrine cells (13, 14). It has been shown that this neuropeptide may have effects on various immune cells. For example, secretoneurin has been demonstrated to attract immature, and arrest mature, blood-derived dendritic cells (15). Moreover, secretoneurin may trigger migration of human monocytes in vitro and in vivo, and combinations of secretoneurin and sensory neuropeptides (substance P, somatostatin) synergistically stimulate this migration (16). Intriguingly, secretoneurin may also act as a potent eosinophil chemoattractant (17, 18). Taken together, these previous studies indicate that secretoneurin may play a role in inflammatory conditions (12). However, whether secretoneurin is present in the human nasal mucosa is unknown. Furthermore, whether secretoneurin is released at ongoing allergic airway inflammation and whether any increased secretoneurin activity is associated with increased eosinophil activity is presently unknown. In the present study, involving patients with allergic rhinitis, we have explored the expression and distribution of secretoneurin immunoreactivity in the nasal mucosa before and during a pollen season. Furthermore, using radioimmunoassays, we have examined levels of secretoneurin and eosinophil cationic protein (ECP) in nasal lavages obtained from these patients. In an attempt to explore whether nerve stimulation affects any nasal output of secretoneurin, we have also performed histamine challenges/lavages and analysis of lavage levels of secretoneurin.
Patients Twenty-four patients (mean age 28, range 21–46) with allergic rhinitis were studied before and during a birch pollen season. Inclusion criteria were a history of strictly seasonal allergic rhinitis and a positive skin prick test to birch pollen allergen. Exclusion criteria were a history of chronic nasal disease, positive skin prick tests to perennial allergens, asthma, chronic obstructive pulmonary disease, and ongoing treatment. The study was approved by the Ethics Committee and informed consent was obtained. Nasal symptoms (rhinorrhea, blockage, and sneezes) were scored every morning during the study. Each symptom was scored on a four-grade scale: score 0: no, 1: mild, 2: moderate, and 3: severe symptoms. The score of each symptom was added to yield a total daily score (range 0–9).
Nasal Lavages and Biopsies Paired nasal biopsies were taken from the inferior turbinate using a forceps with a drilled-out punch. Lidocain (34 mg/ml) and naphazolin (0.17 mg/ml) were applied using a spray device and a cotton swab. Also, carbocain (10 mg/ml) and adrenaline (5 µg/ml) were injected. The specimens were processed as described previously (20), and stored at -80°C until sectioning. Additional details on the sampling methods are provided in the online supplement.
Immunohistochemistry
To reveal the coexistence of two different antigens, simultaneous double immunostaining was used (see Reference 20 and the online supplement). However, simultaneous double immunostaining was not possible using primary antisera raised in the same species. Therefore, to examine the coexistence of VAChT/secretoneurin and secretoneurin/TH, respectively (i.e., antisera raised in rabbits), sequential immunostaining was performed (see Reference 23 and the online supplement).
Radioimmunoassays
Statistical Analysis
Nasal Symptoms and Output of ECP The regional birch pollen counts revealed moderately increased pollen levels during a period of three weeks in late April and early May. Accordingly, nasal symptoms recorded during this period were increased (Figure 1) . Compared with the first study week (March 18–24), the increase in total nasal symptoms reached statistical significance in the third (p < 0.05), fifth (p < 0.05, sixth (p < 0.001), and seventh (p < 0.001) study weeks. The nasal biopsies were obtained on March 18 and on May 9 (i.e., before and late into the pollen season). Nasal lavages were performed on the same dates as well as on April 27 and on May 2. Nasal lavage fluid levels of ECP were significantly increased at the second (p < 0.05) and the third (p < 0.05) of the seasonal observations (cf. before the season) (Figure 2) , indicating that the patients developed a seasonal, eosinophilic airway inflammation.
Distribution of Secretoneurin Immunoreactivity An abundance of nerves displaying secretoneurin immunoreactivity were observed in all biopsies examined. These fibers were distributed mainly around seromucous glands and blood vessels in the subepithelial connective tissue (Figures 3A–3G) : The acini of glands were surrounded by a network of varicose nerve fibers. Secretoneurin-immunoreactive nerves were numerous in blood vessel walls, particularly arteries, forming a plexus in the adventitia or at the junction between the adventitia and media. Positive fibers were also seen in nerve bundles deep in the mucosa. Secretoneurin-positive nerve fibers were scarce beneath and within the epithelium. Secretoneurin immunoreactivity was detected exclusively in nerve fibers. Although no quantitative analysis was made, there was no apparent evidence of an altered occurrence or distribution of secretoneurin immunoreactivity associated with seasonal allergen exposure.
Colocalization of Secretoneurin and Other Neuronal Markers The staining pattern of secretoneurin was compared with the distribution of CGRP, a neuropeptide mainly localized to sensory nerves (8, 20, 24, 25). Immunoreactive CGRP was found in nerve fibers that densely innervated the walls of blood vessels (Figure 3B). Occasionally, single delicate fibers were also found between glandular acini. In addition, immunoreactive nerve fibers were seen in the connective tissue beneath the basement membrane and, occasionally, between surface epithelial cells, which confirm previous observations (20, 24, 25). The majority of CGRP-containing nerve fibers seemed also to contain secretoneurin (Figures 3A and 3B). On the other hand, most of the secretoneurin-immunoreactive fibers lacked CGRP. A neuropeptide mainly localized to cholinergic nerves is VIP (20, 26–28). In accordance with previous studies (20, 26–28), a moderate number of VIP-containing nerves were observed around submucosal glands and in the walls of arterial and venous vessels. Occasionally, VIP-positive fibers were seen also just beneath the surface epithelium, but no fibers were detected within the epithelium. Double immunostaining for secretoneurin and VIP revealed that a vast majority of VIP-containing fibers also contained secretoneurin (Figures 3C and 3D), whereas many secretoneurin-immunoreactive fibers lacked VIP. VAChT-immunoreactive (cholinergic) and TH-immunoreactive (adrenergic) nerve fibers were numerous around blood vessels and glands. In the connective tissue just beneath the basement membrane, these two types of nerve fibers were scarce. Neither VAChT-immunoreactive nor TH-immunoreactive fibers were detected within the surface epithelium. Secretoneurin immunoreactivity was found in a population of VAChT-containing (cholinergic) fibers (Figures 3E and 3F) as well as in a population of TH-containing (adrenergic) fibers (Figures 3G and 3H).
Nasal Lavage Fluid Levels of Secretoneurin
The present study, involving patients with seasonal allergic rhinitis, demonstrates a rich occurrence and a widespread distribution of secretoneurin-immunoreactive nerves in the nasal mucosa. Furthermore, it demonstrates, for the first time, secretoneurin in nasal lavage fluids obtained from these patients and increased levels of secretoneurin at seasonal allergen exposure (cf. observations before the pollen season). These data, together with previous in vitro observations on this neuropeptide (15–18), suggest the possibility that secretoneurin may have a role in the traffic of immune cells in the human airway mucosa (29). The present study has originally revealed an extensive occurrence and a widespread distribution of secretoneurin-immunoreactive nerve fibers in the human nasal mucosa. This staining pattern was first compared with the pattern of CGRP, a neuropeptide localized mainly in afferent sensory nerves (8, 20, 24, 25). A vast majority of CGRP-containing nerves were secretoneurin-immunoreactive, potentially identifying secretoneurin as a sensory neuropeptide. In agreement, previous studies in rats have shown that afferent nerve fibers may contain secretoneurin. Secretoneurin thus has a similar distribution as the sensory neuropeptides substance P and CGRP in the nervous system of the rat (13, 30–32). Furthermore, studies in rats have demonstrated that levels of secretoneurin are reduced in peripheral organs, including the trachea, after neonatal capsaicin treatment, although the reduction may be less pronounced than that of substance P and CGRP (13). In the present study, most of the secretoneurin-immunoreactive fibers lacked CGRP, indicating a more widespread distribution of secretoneurin than merely to sensory nerves. Accordingly, the present study revealed secretoneurin immunoreactivity also in a population of TH-containing (adrenergic) fibers as well as in a population of VAChT- and VIP-containing (cholinergic) fibers. Taken together, the present data suggest that secretoneurin is widely distributed in airway mucosal innervation in patients with allergic nasal disease. In the present study, nasal lavage fluid levels of secretoneurin increased significantly at seasonal allergen exposure. The mechanism behind this effect is unclear, but it is possible that allergen exposure stimulates production and/or release of secretoneurin. Indeed, increased secretion of other neuropeptides (substance P, CGRP, and VIP) has been demonstrated previously in allergic rhinitis after experimental allergen challenges (33, 34). Also, significantly greater tissue concentrations of substance P as well as VIP have been reported in patients with allergic rhinitis compared with healthy control subjects (27). Interestingly, in vitro studies using cultured cells have demonstrated that histamine and nerve growth factor, two mediators relevant to airway inflammation, specifically induce secretogranin II messenger RNA, the precursor of secretoneurin (35, 36). In the present study, histamine challenges, producing neural activity as evidenced by symptomatic responses involving sneezes (not shown), were associated with acutely increased nasal output of secretoneurin before the pollen season but not at seasonal allergen exposure. It may be speculated that histamine, in this context, may stimulate H1-receptors on the slow-conducting nerves that mediate itch and that the release of secretoneurin may reflect an axon-reflex. To explain the difference between observations before and during the pollen season, it may further be speculated that enhanced secretoneurin release is already triggered by allergen exposure during the season and that subsequent histamine challenge during this time period does not further activate the nerves. Further studies are warranted to elucidate whether secretoneurin can be released differently in health and disease. An increase in the numbers of nerve fibers during the season may also affect the levels of secretoneurin on seasonal allergen exposure. In support of this hypothesis, it has been reported previously that the nasal mucosa of patients with rhinitis and ongoing symptoms feature increased numbers of protein gene product 9.5 and VIP-containing fibers, especially around blood vessels, compared with healthy subjects (27, 37, 38). Our present investigation, involving paired biopsies from five patients, did neither quantify nerve fibers in the mucosa nor include healthy control subjects. Therefore, it remains to be elucidated whether the observed increase of secretoneurin in the nasal lavage fluids at seasonal allergen exposure is a consequence of increased production/release from preexisting nerve fibers and/or due to an increased density of nerve fibers in the inflamed mucosa. Secretoneurin has been implicated in various inflammatory responses, as it has been shown to be involved in leukocyte and dendritic cell functions in vitro (15–18). For example, secretoneurin acts as a potent chemoattractant for human eosinophils and monocytes (16–18). Moreover, recent data show that secretoneurin may induce chemotaxis of immature dendritic cells, comparable in its potency to regulated upon activation, normal T cell expressed and secreted (RANTES) (15). However, on maturation of the dendritic cells, secretoneurin inhibits their migration (15). Thus, secretoneurin might fasten dendritic cells at sites of inflammation and once there, keep them arrested (15). These in vitro data, together with the present demonstration of a rich occurrence of secretoneurin in the nasal mucosa of patients with allergic rhinitis, suggest the possibility of a role of secretoneurin in local traffic of immune cells and in various neuroimmune interactions. For example, it may be speculated that secretoneurin, released from perivascular nerves in the nasal mucosa, may promote the transendothelial migration of various leukocytes in vivo, as has been demonstrated previously in vitro (39, 40). Tentatively, secretoneurin may also be involved in allergen-induced clustering of eosinophils around airway nerves (41, 42). However, in the present study there was a lack of correlation between nasal lavage fluid levels of secretoneurin and a marker of eosinophil activity (ECP). Further studies in this field, focusing on the relative importance of secretoneurin, are warranted. In conclusion, the present study has demonstrated a rich occurrence of secretoneurin-immunoreactive nerves in the nasal mucosa of patients with seasonal allergic rhinitis before the pollen season as well as at seasonal allergen exposure. Furthermore, it has demonstrated secretoneurin in nasal lavage fluids obtained from these patients and increased levels of secretoneurin at seasonal allergen exposure. The present data raise the hypothesis that secretoneurin may have a role in the local traffic of immune cells in the human nasal mucosa.
The authors thank Doris Persson, Gertrud Persson, Lena Glantz, and Charlotte Cervin-Hoberg for their expert technical assistance.
Supported by grants from the Swedish Medical Research Council, the Swedish Medical Association, the Swedish Foundation for Strategic Research, the Swedish Otolaryngological Association, the Swedish Heart and Lung Foundation, and the Medical Faculty of Lund University. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form December 19, 2002; accepted in final form March 5, 2003
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2-Macroglobulin and eosinophil cationic protein in the allergic airway mucosa in seasonal allergic rhinitis. Eur Respir J 1999;13:633–637.
