INTRODUCTION

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Allergic rhinitis is a common chronic disease of the airways, affecting up to 36 million people in the United States (1) with an estimated direct cost of $3.4 billion in 1993 alone (2). Its counterpart in the lower airways, asthma, affects approximately 14 million people in the U.S., and is the cause of 1.9 million emergency room visits in 1995 alone (3). It is well known that these two conditions share the salient features of inflammation, including mucosal edema, increased vascular permeability, and leukocytic infiltration. In addition, allergic rhinitis and asthma are both characterized by significantly increased responsiveness to various inhalants. Hyperresponsiveness of the upper airways may account for the induction of nasal symptoms upon exposure to environmental irritants such as tobacco smoke or cold dry air (4). Similarly, hyperresponsiveness of the lower airways is believed to be responsible for acute bronchial narrowing upon inhalation of stimuli such as methacholine or hypertonic saline (5, 6).

The nature of airway hyperresponsiveness has been largely unknown. There is recent evidence that, in the upper airways, hyperresponsiveness can be attributed to increased neural reactivity. We have found that subjects with allergic rhinitis, compared with healthy individuals, exhibit significantly enhanced nasal reflexes in response to stimuli such as capsaicin, bradykinin, and hyperosmolar saline, and that these responses can be attenuated by pretreatment with a local anesthetic (7). The role of the sensorineural apparatus in hyperresponsiveness of the lower airways has not been as established, largely due to methodological limitations. Nonetheless, findings that neural stimuli such as histamine, bradykinin, and hyperosmolar saline induce significantly greater bronchoconstriction in asthmatics, compared with control subjects, suggest that it may be significant (6, 11, 12).

Although it is widely believed that inflammation leads to hyperresponsiveness of the airways, the pathophysiologic basis of this relationship has not been elucidated. We hypothesize that the prototypical neurotrophin nerve growth factor (NGF) (13) plays an important role in bridging this gap, by being a mediator of increased sensorineural responsiveness in the airways. NGF is expressed and released by several types of cells that actively participate in the allergic inflammatory process (14), and effects biological changes that can significantly enhance sensorineural responsiveness (21, 22). In the present investigation, we determined whether NGF synthesis and release is dysregulated in the upper airways of patients with allergic rhinitis in comparison with healthy individuals.

	METHODS

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Study Subjects

A total of 30 volunteers participated in these studies. Twenty subjects reported chronic symptoms of rhinitis during the period of investigation, and exhibited sensitivity to multiple aeroallergens including grass and/or ragweed pollen by puncture skin test (rhinitis subjects: eight males and 12 females, mean age 36.6 yr, range 18 to 54 yr.) Ten subjects had no history of chronic nasal symptoms and had a negative skin test using a panel of common aeroallergens (control subjects: four males and six females, mean age 34.3 yr, range 25 to 54 yr). All subjects avoided the use of antihistamines for at least 1 wk and of steroids or cromolyn for at least 1 mo prior to the study. Informed consent was obtained at the start of the investigation, which was approved by the institutional review board of the Johns Hopkins Bayview Medical Center.

Study Design

Baseline nasal lavage fluids were collected from all rhinitis and control subjects. Nasal provocation with allergen was performed on 20 rhinitis and five control subjects. As part of the control experiments, 10 of the rhinitis subjects underwent sham nasal challenge with the vehicle for allergen, and the other 10 were challenged with histamine, 1 wk before allergen provocation. In a separate visit, nasal mucosal scrapings were obtained from five rhinitis and four control subjects.

Nasal Challenges and Collection of Samples

The challenge solutions were administered into both nostrils using metered dose nasal sprays that deliver approximately 75 µl per actuation. Nasal secretions were obtained by lavage technique as described previously (23). Briefly, 5 ml of prewarmed lactated Ringer's solution were instilled into both nostrils with a pipette and, after approximately 10 s, expelled into a collecting basin and transferred into conical tubes. After this baseline lavage, six additional lavages were performed to wash out preexisting biological components in nasal secretions, and the fluids were discarded. After a 5-min pause, prechallenge nasal lavage fluids were collected 5 min before nasal provocation. In a pilot experiment to evaluate the occurrence and time course of NGF release in nasal fluids after allergen exposure, a patient with allergic rhinitis underwent nasal challenges with 10, 100, then 1,000 protein nitrogen units (pnu) of ragweed extract, 15 min apart. Postchallenge nasal lavage fluids were collected 10 min after each dose and 30 min, 1 h, and every hour for 8 h after the last allergen challenge. The samples were kept on ice until centrifugation at 2,500 × g for 15 min at 4° C. Aliquots of the supernatant were then frozen at 80° C for subsequent protein electrophoresis and Western immunoblotting.

In the ensuing main study, allergen nasal challenge was administered as a single dose of 1,000 pnu of mixed grass or ragweed extract (Greer Laboratories, Inc., Lenoir, NC), and nasal lavage fluids were collected before and 10 min after challenge. Nasal administration of the vehicle (NaCl 0.9%, phenol 0.4%; Greer Laboratories, Inc.) or of 1 mg histamine (Sigma-Aldrich, St. Louis, MO) served as control challenges. In this protocol, concentrations of NGF in supernatant samples of nasal lavage fluids were measured by enzyme-linked immunosorbent assay (ELISA).

Superficial nasal mucosal cells were harvested by gentle scraping of the inferior turbinate using a nasal mucosal curette (Rhino-Probe; Arlington Scientific, Inc., Arlington, TX). The collected samples were suspended in 200 µl lactated Ringer's solution and kept on ice until microcentrifugation. The resultant cell pellets were frozen immediately at 80° C for quantitative assessment of NGF messenger RNA (mRNA) by reverse transcription-polymerase chain reaction (RT-PCR).

Protein Electrophoresis and Western Immunoblotting

The total protein content of the supernatant samples was measured by the bicinchoninic acid protein assay (Pierce, Rockford, IL), using bovine serum albumin as standard. A 10-µg aliquot from each sample was reduced and denatured with -mercaptoethanol and sodium dodecylsulfate. The samples were then loaded onto a polyacrylamide precast gel (Novex, San Diego, CA) and electrophoresed in parallel with a molecular weight marker (Amersham Life Sciences, Arlington Heights, IL) and 100 ng of human recombinant NGF (a gift from Genentech, San Francisco, CA). After these were transferred to a supported nitrocellulose membrane (Schleicher and Schuell, Keene, NH), Western immunoblotting was performed using 1:2,000 rabbit polyclonal 1° anti-human NGF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 1:2,000 horseradish peroxidase-linked donkey anti-rabbit 2° antibody (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The blot was analyzed using an enhanced chemiluminescence system (Amersham Life Sciences). For absorption control experiments, the membrane was stripped of bound antibody, and human recombinant NGF (100 µg/ml) was incubated with the 1° antibody overnight before reprobing as previously described.

Enzyme-linked Immunosorbent Assays (ELISA)

The concentrations of NGF in supernatant samples of baseline, pre- and post-challenge nasal lavage fluids were measured by sandwich-type ELISA using a modification of previously described methods (24). Sheep and rabbit polyclonal anti-NGF antibodies were purified over affinity column before use as 1° and 2° antibodies, respectively. Briefly, 96-well plates were coated with 1° antibody and incubated overnight. After blocking with 10% goat serum, supernatant samples were added, with high-performance liquid chromatography (HPLC)- purified mouse NGF serving as standard. After overnight incubation with 2° antibody conjugated with -galactosidase (Pierce), the reaction was developed with 4-methylumbelliferyl--galactoside and stopped with the addition of glycine. Samples were then analyzed using a Microfluor ELISA reader (PerSeptive Biosystems, Farmingham, MA) at 360/450 nm. The sensitivity of this assay is approximately 10 pg/well of NGF, with the lowest detectable concentration of NGF being  2× the standard deviation of the background.

The concentrations of human serum albumin (HSA) in nasal lavage fluids were similarly determined by ELISA as described previously (8). Sheep anti-HSA (The Binding Site Ltd., Birmingham, UK) and horseradish peroxidase-conjugated sheep anti-HSA (Dako Corp., Carpinteria, CA) were used as the 1° and 2° antibodies, respectively, and HSA was used as standard. The reaction was developed with o-phenylenediamine dihydrochloride (Sigma Chemical Co., St. Louis, MO), stopped with the addition of sulfuric acid, and read at 490 nm.

RNA Extraction and RT-PCR

Cellular RNA was isolated from nasal mucosal scrapings with TRIzol (GibcoBRL, Gaithersburg, MD), using a modification of the acid guanidinium thiocyanate-phenol-chloroform extraction method described previously (25). First-strand complementary DNA (cDNA) was synthesized using 2 µg of total RNA and oligo(dT) primers with the SuperScript Preamplification System (GibcoBRL, Gaithersburg, MD). AmpliWax gem facilitated hot-start PCR (Perkin Elmer, Branchburg, NJ) was performed with 0.5 µM gene-specific primers, 1.25 U AmpliTaq DNA polymerase, and 1 µl cDNA from the reverse transcription reaction. The primer sequences for NGF were 5'-GTGGTGCTGCCCCCTTCAA-3'(sense) and 5'-CAAGGTGTGAGTCGTGGTA-3' (antisense) generating a fragment of 301 bp. Used as a control for RNA input, the primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense) generating a fragment of 452 bp. Amplification was conducted in the linear range with the following cycling parameters: initial denaturation, 94° C, 5 min; (25 to 30 cycles) denaturation, 94° C, 30 s; annealing, 57° C for NGF, 60° C for GAPDH, 30 s; extension, 72° C, 45 s; and final extension, 72° C, 5 min. The amplified products were resolved on 1.6% agarose gels, stained with ethidium bromide, and visualized with ultraviolet illumination. The agarose gels were then dried and apposed to a Storage Phosphor Screen (Molecular Dynamics, Sunnyvale, CA) and the PCR products were quantified using ImageQuant software (Molecular Dynamics). PCR products were identified by size and restriction digest analysis.

Data Analysis

Nonparametric statistics were applied. Mann-Whitney U test was used for group comparisons between rhinitis and control subjects with respect to concentrations of NGF protein in nasal lavage fluids and to ratios of NGF/GAPDH mRNA in nasal mucosal scrapings (using StatView 5.0 software; Abacus Concepts Inc., Berkeley, CA). Wilcoxon signed ranks paired test was used for comparisons between the prechallenge and postchallenge values, and between the effects of allergen and of histamine on NGF or albumin. A two-tailed p value of < 0.05 was considered significant.

	RESULTS

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The presence of NGF protein in nasal lavage fluids was initially demonstrated by Western immunoblotting (Figure 1). Subsequent evaluation by ELISA of NGF protein in nasal lavage fluids showed significantly higher baseline concentrations among subjects with allergic rhinitis compared with control subjects (p = 0.007; Figure 2). On the other hand, evaluation by RT-PCR of baseline concentrations of NGF mRNA in superficial nasal scrapings showed significantly decreased levels among rhinitis subjects, compared with control subjects (p = 0.01; Figure 3).

View larger version (69K): [in this window] [in a new window]   Figure 1.   Protein electrophoresis and Western blotting for NGF. (Upper panel ) NGF is present in nasal lavage fluids collected 10 min after nasal challenge with ragweed extract in a subject with allergic rhinitis. The first column confirms the migration pattern of NGF detected in nasal lavage fluids with a parallel electrophoresis of human recombinant NGF (hrNGF, 100 ng). Hourly evaluation up to 8 h postchallenge shows dissipation of NGF levels over time. (Lower panel ) NGF bands are abolished after incubation of the 1° antibody with hrNGF (100 µg/ml).

View larger version (12K): [in this window] [in a new window]   Figure 2.   Baseline levels of NGF protein in nasal lavage fluids. Concentrations of NGF in nasal lavage fluids, measured by ELISA, are significantly higher in the 20 subjects with allergic rhinitis compared with the 10 healthy control subjects. Data are presented as means ± SEM.

View larger version (36K): [in this window] [in a new window]   Figure 3.   NGF mRNA in nasal scrapings. RNA was extracted from superficial nasal mucosal cells, obtained from the inferior turbinate with a curette, in five rhinitis and four control subjects. RT-PCR using primer sequences for NGF generated 301 bp fragments, which were resolved on agarose gels and stained with ethidium bromide. The panel demonstrates NGF mRNA in all the nasal scrapings examined, with decreased concentrations in subjects with allergic rhinitis compared with healthy control subjects.

Nasal provocation with allergen induced immediate symptoms of nasal irritation, rhinorrhea, congestion, and sneezing in all subjects with allergic rhinitis and in none of the control subjects. Western immunoblotting demonstrated a dose-dependent increase in concentrations of NGF protein in nasal lavage fluids, as early as 10 min after allergen challenge, that dissipated over several hours. Immunoblotting specificity was established by the control experiment showing that NGF bands were eliminated when incubation in the 1° antibody was done in the presence of an excess of human recombinant NGF (Figure 1). These results provided the basis for the 10-min postchallenge time point used in the main study. Nasal provocation with allergen induced a mean 12-fold increase in NGF protein concentrations of nasal lavage fluids in rhinitis subjects, from pre- to 10 min postchallenge (p = 0.01; Figure 4). In contrast, neither diluent nor histamine affected NGF protein levels in these subjects (p = 0.4 and p = 0.5, respectively). Allergen provocation likewise did not affect NGF protein concentrations of nasal lavage fluids in control subjects (p = 0.9; Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Concentrations of NGF in nasal lavage fluids before and after nasal challenge with allergen or vehicle. Nasal provocation with 1,000 pnu of ragweed or grass extract significantly increased nasal lavage NGF concentrations in the 20 subjects with allergic rhinitis. On the other hand, NGF levels were not affected by allergen in five healthy control subjects nor by the vehicle in 10 subjects with allergic rhinitis. Open bars = control subjects; solid bars = rhinitis subjects.

Nasal provocation with histamine was performed as a control measure to determine whether any allergen-induced increase in NGF concentrations could be simply attributable to plasma extravasation. Increased vascular permeability is reflected by a significant elevation of albumin in nasal lavage fluids (10). Both 1,000 pnu of allergen extract and 1 mg of histamine caused an increase in the concentrations of albumin in nasal lavage fluids (Figure 5). There was no difference between allergen and histamine provocation in terms of albumin concentrations in nasal lavage fluids, both before and after challenge. There was similarly no difference between these two protocols in terms of NGF concentrations in nasal lavage fluids before challenge. However, the levels of NGF in nasal lavage fluids after provocation with allergen were significantly greater than those with histamine (p = 0.04).

View larger version (26K): [in this window] [in a new window]   Figure 5.   Comparison between allergen and histamine nasal challenge. There is no difference between 1,000 pnu of allergen and 1 mg of histamine in their ability to induce plasma extravasation in 10 subjects with allergic rhinitis, manifested by comparable increases in the albumin content of nasal lavage fluids. Allergen, however, is distinguished from histamine in its ability to induce significantly greater concentrations of NGF in nasal lavage fluids collected 10 min postchallenge. Hatched bars = histamine challenge; dark bars = allergen challenge.

	DISCUSSION

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Our data provide the first evidence that NGF is constitutively expressed and secreted in the human upper airways in vivo, and is released acutely in the course of an allergic reaction. More importantly, we found significant differences between patients with allergic rhinitis and healthy individuals, demonstrating a specific abnormality in the nasal mucosa that may provide a neurological explanation for the hyperresponsiveness that characterizes this disease.

Patients with allergic rhinitis report increased symptomatic responsiveness to environmental irritants such as tobacco smoke compared with nonatopic individuals (26). Experimentally, we and others have found that such patients exhibit significantly exaggerated nasal reflexes upon provocation with capsaicin, bradykinin, cold dry air, or hyperosmolar saline compared with those with nonallergic rhinitis or with healthy control subjects (7). The reflex response induced by nasal provocation can be attenuated by pretreatment with a local anesthetic (9, 10, 27), neural desensitization by repetitive application of capsaicin (9), or more definitively by vidian nerve resection (28). These observations collectively indicate that the presence of allergic inflammatory disease predisposes the nasal mucosa to hyperresponsiveness that is nerve-mediated. Indeed, previous studies have shown that allergen exposure in sensitized animals causes increased responsiveness of the airways to nerve stimulation (29, 30).

Searching for molecular signals that can link allergic inflammation and neural hyperresponsiveness that are both present in allergic rhinitis, we hypothesized that NGF plays a critical role as a mediator in this disease. This prototypical neurotrophin (13) is essential for the development of small-diameter sensory (nociceptive) fibers in the peripheral nervous system (31) and exerts potent biochemical and structural effects on these nerves (32) that can lead to exaggerated responsiveness. For example, transgenic mice overexpressing NGF show bronchial hyperinnervation (21). NGF also upregulates neuropeptides such as substance P that can be antidromically released upon nerve activation (22). As predicted, mice overexpressing NGF in the airways show exaggerated bronchospastic responses to inhalational challenge with capsaicin (21). Similarly, guinea pigs administered exogenous NGF develop airway hyperresponsiveness to histamine (33). These observations resemble previous findings that subjects with allergic rhinitis exhibit hyperresponsiveness to nasal provocation with capsaicin (34) or histamine (35). Airway hyperresponsiveness may be analogous to other conditions that have been associated with NGF, such as skin and muscle hyperalgesia (36, 37) and bladder hyperactivity (38).

While NGF is linked to the development of hyperresponsiveness, it is also associated with allergic inflammation, thus bridging the gap between these two features of allergic rhinitis and asthma. NGF is synthesized, stored, and released by cells that play key roles in the allergic inflammatory disease of the airways, including mast cells, eosinophils, CD4⁺ T cells, B cells, and respiratory epithelial cells (14). The time course of NGF release in allergic rhinitis implicates mast cells, which release mediators within minutes of nasal provocation with allergen (23, 39). Mast cells express and secrete NGF (14, 15), and they have been shown to be in close apposition with tachykinin-containing nerve endings in peripheral tissue (40). Other cells involved in the later phases of inflammation may also contribute to NGF release in the human airways, as suggested by findings of NGF elevation in bronchoalveolar lavage fluids 18 h after segmental allergen challenge in asthmatics (41).

NGF may be increased in the serum of subjects with various allergic disorders (42). We thus applied nasal provocation with histamine as a control to ascertain that allergen-induced increases in concentrations of NGF in nasal lavage fluids could not be solely attributable to plasma leakage. As expected, both histamine and allergen caused extravasation of plasma as shown by comparable increases in the concentrations of albumin in nasal lavage fluids. Unlike histamine, however, allergen challenge caused significant increases in nasal lavage NGF levels in the group of rhinitis subjects. These results indicate that the acute increase in NGF after allergen challenge is not an artifact of plasma leakage but the result of active release of preformed NGF from cells in the nasal mucosa. The negative results of vehicle sham challenge in subjects with allergic rhinitis and of allergen challenge in control subjects show that this is a specific phenomenon related to allergen-sensitivity reactions.

The presence of NGF mRNA in the nasal scrapings of both rhinitis and control subjects is consistent with a constitutive expression of this factor at the epithelial layer of the nasal mucosa. Interestingly, the concentrations of NGF mRNA in these samples were found to be decreased in the group of subjects with allergic rhinitis. Incongruent trends in concentrations of NGF mRNA and protein have been previously observed in the brain (43, 44) and bladder (38) of rats, and may also occur in the skin of patients with diabetic neuropathy (45, 46). Our present findings suggest a steady state of dysregulation in the expression and release of NGF involving at least two types of cells in the nasal mucosa of individuals with allergic rhinitis. Mast cells in the subepithelial layer, for example, may chronically release high concentrations of NGF protein into fluids lining the mucosal surface, with resultant downregulation of NGF mRNA expression in the superficial epithelial cells. The expression, storage, and release of NGF need to be further evaluated through colocalization studies and functional analysis using isolated nasal tissue.

In summary, our data provide the first evidence of dysregulation in NGF expression and release in the upper airways of patients with symptomatic allergic rhinitis. These findings support a role of NGF, released in significantly greater amounts in the context of allergic inflammation, in mediating hyperresponsiveness that characterizes this disease.