Rat Nasal Epithelium Research Articles

Th2 Cytokine Inhibitor Suplatast Tosilate Inhibits Antigen-Induced Mucus Hypersecretion in the Nasal Epithelium of Sensitized Rats

Th2 cytokines such as interleukin (IL) 4 and IL-13 are potential mediators for mucus hypersecretion in allergic inflammation. To elucidate the functions of Th2 cytokines in allergic rhinitis, we examined the in vivo effects of the Th2 cytokine inhibitor suplatast tosilate on mucus hypersecretion and eosinophil infiltration in rat nasal epithelium. We induced hypertrophic and metaplastic changes in goblet cells in the nasal epithelium of ovalbumin-sensitized rats by intranasal challenge with ovalbumin. The effects of orally administered suplatast tosilate on mucus production and eosinophil infiltration were examined. Suplatast tosilate (30 and 100 mg/kg) dose-dependently inhibited ovalbumin-induced mucus production and eosinophil infiltration. These suppressions of mucus production and eosinophil infiltration were only effective when suplatast tosilate was given in the effector phase; administration in the induction phase resulted in no effect. These results indicate that Th2 cytokines are important mediators of mucus hypersecretion and eosinophil infiltration in allergic rhinitis. Suplatast tosilate may be useful for the treatment of allergic rhinitis by attenuating the inflammation of the effector phase.

New Generation of Hybrid Poly/Oligosaccharide Nanoparticles as Carriers for the Nasal Delivery of Macromolecules

We have recently reported a new generation of polysaccharide nanoparticles consisting of chitosan (CS) and cyclodextrin (CD) derivatives, which exhibit a number of advantages when compared to the classical CS nanoparticles. In the present work our goal was to explore the potential of these hybrid CS/CD nanoparticles as carriers for the nasal delivery of macromolecules. First, we evaluated the effect of the amount and type of CD (sulfobutylether-beta-CD or carboximethyl-beta-CD) on the physicochemical properties of the nanocarriers. Second, we investigated the interaction of CS/CD nanoparticles with the nasal epithelium by studying their ability to modulate the tight junctions between epithelial cells (Calu-3 cell model) as well as their capacity to overcome mucosal barriers (nasal epithelium of rats). Finally, we loaded two selected nanocarriers with insulin and studied their potential for enhancing the nasal transport of insulin in rabbits. The results showed that CS/CD nanoparticles caused a reversible reduction in the transepithelial resistance of the cell monolayer, thus increasing the membrane permeability. Moreover, the results obtained following the in vivo administration of fluorescent CS/CD nanoparticles to rats evidenced their capability to overcome the nasal mucosal barrier. Finally, the in vivo evaluation in conscious rabbits revealed that insulin-loaded nanoparticles (association efficiencies > 88%) were able to significantly decrease plasma glucose levels (more than 35% reduction). Overall, these results suggest that these new nanoparticles work as nasal carriers and, therefore, have a potential for enhancing the transport of complex molecules across the nasal barrier.

Comparison of nasal proteins adducted by reactive metabolites of naphthalene (NA) in rat and rhesus macaque using 2 dimensional gel electrophoresis and MALDI TOF/TOF

NA is a volatile pollutant classified by NTP as a ‘reasonably anticipated human carcinogen’ based on concentration‐dependent, rare olfactory epithelial neuroblastomas and respiratory (nasal) epithelial adenomas in rats (NTP 2000). However, epidemiologic studies have not provided a convincing link between exposure and cancer. Reactive metabolites of NA bind covalently to proteins in target cells; good correlations between adducted protein levels and injury have been observed (Drug Metab Rev 34:791, 2002). Accordingly, these studies compared adducted proteins in nasal epithelium of rats and rhesus macaques to develop biomarkers mechanistically associated with toxicity which could be applied to human populations. Rat and monkey nasal tissues were incubated with 14C ‐NA, proteins were resolved by 2D gel electrophoresis, and labeled proteins were imaged and subsequently identified. The adducted protein patterns between rat and rhesus macaque are remarkably similar. 45 adducted proteins identified in rat olfactory incubations include those involved in cell structure, transcription, protein synthesis/folding, ATP synthesis, and cell signaling; similar work is ongoing in monkeys. These studies suggest that several of these adducts may be good biomarker targets for assessing the potential for NA toxicity in humans. Supported by EPRI, NIEHS 04311, 04699 and RR00169.

EP4 Agonist Inhibits Lipopolysaccharide-Induced Mucus Secretion in Airway Epithelial Cells

We examined the in vivo effects of agonists for prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) on mucus hypersecretion. We also examined the in vitro effects of EP agonists on airway epithelial cells. For the in vivo study, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal lipopolysaccharide (LPS) instillation. For the in vitro study, we used NCI-H292 cells and cultured human nasal epithelial cells. Subcutaneous injection of the EP4 agonist (1 to 100 microg/kg) dose-dependently inhibited LPS-induced mucus production and neutrophil infiltration. The EP3 agonist (100 microg/kg) also had some inhibitory effects on mucus production, whereas the EP1 and EP2 agonists showed no effect. The LPS-induced mucus secretion was significantly inhibited by the EP3 and EP4 agonists at 10(-6) mol/L in cultured epithelial cells. The LPS-induced interleukin-8 secretion was also inhibited by the EP3 and EP4 agonists. These results indicate that the EP4 agonist inhibited LPS-induced airway mucus hypersecretion directly or indirectly through the suppression of interleukin-8 secretion and neutrophil infiltration.

Role of prostaglandin E2 as a protective mediator for airway inflammation

SummaryProstaglandin (PG) E2 is a cyclooxygenase metabolite of arachidonic acid that has anti‐inflammatory properties including suppression of lymphocyte proliferation, inhibition of cytokine/chemokine production, and dilation of human bronchi. Recently, four types of PGE2 receptors (EP1, EP2, EP3, and EP4) were identified, and their roles have been extensively examined. We found mRNA expression of all four PGE2 receptors in human airway epithelial cells, and EP3 and EP4 agonists significantly inhibited mucus secretion and inflammatory cell infiltration induced by lipopolysaccharide (LPS) or by allergic inflammation in rat nasal epithelium. These EP agonists significantly inhibited LPS‐induced secretion of IL‐8 and mucus in cultured airway epithelial cells. These results indicate that PGE2 acts as an anti‐inflammatory mediator via EP receptors of airways. EP agonists may serve as a new therapeutic modality for the treatment of airway inflammation.

A Method to Integrate Benchmark Dose Estimates with Genomic Data to Assess the Functional Effects of Chemical Exposure

The use of genomic technology for assessing health risks associated with chemical exposure has significant potential, but its direct application has proven to be challenging for the toxicology and risk assessment communities. In this study, a method was established for analyzing dose-response microarray data using benchmark dose (BMD) calculations and gene ontology (GO) classification. Gene expression changes in the rat nasal epithelium following acute formaldehyde exposure were used as a case study. The gene expression data were first analyzed using a one-way ANOVA to identify genes that showed significant dose-response behavior. These genes were then fit to a series of four statistical models (linear, second-degree polynomial, third-degree polynomial, and power models) and the least complex model that best described the data was selected. The genes were matched to their associated GO categories, and the average BMD and benchmark dose lower confidence limit (BMDL) were calculated for each GO category. The results were used to identify doses at which individual cellular processes were altered. For the formaldehyde exposures, the BMD estimates for the GO categories related to cell proliferation and DNA damage were similar to those measured in previous studies using cell labeling indices and DNA-protein cross-links and consistent with the BMD estimated for rat nasal tumors. The method represents a significant advance in applying genomic information to risk assessment by allowing a comprehensive survey of molecular changes associated with chemical exposure and providing the capability to identify reference doses at which particular cellular processes are altered.

気道炎症とアラキドン酸代謝物の新しい展開

Arachidonic acid metabolites, prostanoids and leukotrienes, are important mediators in airway inflammation. Over the last decade, selective receptors of these mediators have been identified, and the biological functions of these receptors have been clarified in detail. Prostaglandin (PG)D2, thromboxane A2, and cysteinyl leukotrienes may augment allergic inflammation. On the contrary, PGE2, PGI2, and lipoxins may be potent anti-inflammatory mediators involved in airway inflammation.PGE2 receptors are characterized into four subtypes: EP1, EP2, EP3, and EP4. In our study, EP agonists inhibited mucus secretion and IL-8 release from cultured human airway epithelial cells in vitro. An in vivo study revealed that mucus production and neutrophil or eosinophil infiltration caused by LPS stimulation or allergic inflammation were inhibited by the subcutaneous injection of EP agonists in rat nasal epithelium. These results indicate that PGE2 acts as an anti-inflammatory mediator via EP receptors, and that EP agonists may constitute a new therapeutic strategy.In this article, recent knowledge of prostanoid and leukotriene functions in the pathogenesis of airway inflammation is reviewed. Their selective receptors and synthetic enzymes may provide novel targets for future drugs.

Basal Gene Expression in Male and Female Sprague-Dawley Rat Nasal Respiratory and Olfactory Epithelium

The nasal epithelium is an important target site for chemically induced toxicity and carcinogenicity. Experimental studies show that site-specific lesions can arise within the nasal respiratory or olfactory epithelium following the inhalation of certain chemicals. Moreover, gender differences in epithelial response are also reported. To better understand and predict gender differences in response of the nasal epithelium to inhaled xenobiotics, gene expression profiles from naive male and female Sprague-Dawley rats were constructed. Epithelial cells were manually collected from the nasal septum, naso- and maxillo-turbinates, and ethmoid turbinates of nine male and nine female rats. Gene expression analysis was performed using the Affymetrix Rat Genome 430 2.0 microarray. Surprisingly, there were few gender differences in gene expression. Gene ontology enrichment analysis identified several functional categories, including xenobiotic metabolism, cell cycle, apoptosis, and ion channel/transport, with significantly different expression between tissue types. These baseline data will contribute to our understanding of the normal physiology and selectivity of the nasal epithelial cells' response to inhaled environmental toxicants.

Physiological Significance of Resistin and Resistin-Like Molecules in the Inflammatory Process and Insulin Resistance

Resistin was initially identified as a protein, secreted by adipocytes, which inhibits insulin action and adipose differentiation. The three proteins homologous to resistin were termed resistin-like molecules (RELM) alpha, beta and gamma. Resistin and RELMalpha are abundantly expressed in adipose, but RELMbeta and RELMgamma are secreted mainly from the gut. Recently, resistin and RELMs were reported to be associated with inflammation. For example, RELMalpha, viewed as an inflammation-related protein, was originally identified in broncho-alveolar lavage fluid obtained from animals with experimentally induced pulmonary inflammation. RELMbeta is also related to bacterial colonization, but RELMbeta injection or hepatic overexpression of RELMbeta induced insulin resistance. RELMgamma isolated from rat nasal respiratory epithelium was found to be altered by cigarette smoke. Thus, resistin and RELMs could be useful for assessing the inflammatory condition in vivo. On the other hand, whether the serum resistin or RELM concentration is strongly related to insulin resistance remains unclear. However, taking recent studies showing a close relationship between inflammation and insulin resistance in diabetes into consideration, these proteins may have interactive roles linking inflammation and insulin resistance, both of which major involvement in the progression of atherosclerosis. If so, the serum resistin or RELM concentration may be a good marker of atherosclerotic risk. In addition, these proteins or unidentified receptors are potential therapeutic targets for the treatment of diabetes and prevention of atherosclerosis. These possibilities merit further study.

Rapid-onset intranasal delivery of metoclopramide hydrochloride: Part II: Safety of various absorption enhancers and pharmacokinetic evaluation

In the present study, several nasal absorption enhancers, used in metoclopramide hydrochloride (MCP HCl) nasal solutions, have been screened for their possible damaging effect in the in vitro human erythrocytes lysis experiment. Moreover, the in vivo leaching of biological markers from the rat nasal epithelium was used as a quantitative assessment for possible nasal mucosal irritation whereby the extent of release of total protein and lactate dehydrogenase (LDH) in the nasal lavage fluid was determined. Results showed that insignificant hemolysis from normal saline ( P < 0.05) occurred with the enhancer protamine sulphate while poly- l-arginine and sodium cholate demonstrated very low (<15%) hemolysis and caused insignificant protein and LDH release from the rat nasal mucosa. Conversely, sodium deoxycholate and chitosan polymers (either of low or high molecular weight) showed high (>60%) hemolysis in vitro and the release of the biological markers in vivo was significantly higher ( P < 0.05) than the control solution (no enhancer). A significant correlation ( P < 0.05) existed between the enhancement effect of MCP HCl nasal absorption and the amounts of protein ( r = 0.85) and LDH ( r = 0.88). Furthermore, the pharmacokinetics of MCP HCl was determined after intravenous (IV), per-oral and intranasal administration of 10 mg drug dose in rabbits. The application of a nasal spray (NS) solution containing 0.5% sodium cholate resulted in a significant improvement ( P < 0.05) in both the rate and extent of absorption of MCP HCl where the T max achieved was 23.3 min as compared to 50 min in case of the oral solution while the area under the serum concentration–time curve (AUC 0–∞) were 506.1, 434.9 and 278.7 μg/ml min for IV, NS and oral solutions, respectively. These values corresponded to absolute bioavailabilities of 87.21 and 55.61% for the NS and oral solutions, respectively. It could thus be concluded that NS of MCP HCl represents a viable approach to achieving rapid and high systemic drug absorption during the emergency treatment of severe emesis.

Transcriptomic Analysis of F344 Rat Nasal Epithelium Suggests That the Lack of Carcinogenic Response to Glutaraldehyde is Due to its Greater Toxicity Compared to Formaldehyde

Formaldehyde is cytotoxic and carcinogenic to the rat nasal respiratory epithelium inducing tumors after 12 months. Glutaraldehyde is also cytotoxic but is not carcinogenic to nasal epithelium even after 24 months. Both aldehydes induce similar acute and subchronic histopathology that is characterized by inflammation, hyperplasia, and squamous metaplasia. Because early aldehyde-induced lesions are microscopically similar, we investigated whether transcriptional patterns using cDNA technology could explain the different cancer outcomes. Treatments included 1-, 5-, or 28-day exposure by nasal instillation of formaldehyde solution (400 mM) or glutaraldehyde solution (20 mM). Animals were euthanized and the nasal respiratory epithelium removed for gene expression analysis and a subset of rats treated for 28 days was processed for microscopic examination. RNA was isolated and processed for expression assessment using Clontech Atlas Toxicology II Arrays. Both aldehydes induced hyperplasia, squamous metaplasia, and inflammatory infiltrates with scattered apoptotic bodies in the epithelium covering luminal surfaces of the nasoturbinate, maxilloturbinate, and nasal septum. A subset of 80 genes that were the most variant between the treated and control included the functional categories of DNA repair and apoptosis. Hierarchical clustering discriminated chemical treatment effects after 5 days of exposure, with 6 clusters of genes distinguishing formaldehyde from glutaraldehyde. These data suggest that although both aldehydes induced similar short-term cellular phenotypes, gene expression could distinguish glutaraldehyde from formaldehyde. The gene expression patterns suggest that glutaraldehyde's lack of carcinogenicity may be due to its greater toxicity from lack of DNA-repair, greater mitochondrial damage, and increased apoptosis.

The influence of gellan gum on the transfer of fluorescein dextran across rat nasal epithelium in vivo

The nasal uptake of a 3000 Da fluorescein dextran (FD3) was investigated in rats, using fluorescence microscopy. The uptake from a formulation containing deacetylated gellan gum, an in situ gelling agent, was compared to that from a mannitol solution. Additionally, the rheological behavior of the gellan gum in water and saline was studied. It was shown that the gellan gum solution was easily administered owing to its low viscosity, and upon contact with the mucosa, a gel was formed. The epithelial uptake and transfer of FD3 appeared to be increased and prolonged using the gellan gum formulation. This increase was not accompanied by qualitative changes of the epithelial FD3 distribution or any visible harmful effects.

In vivo and in vitro effects of macrolide antibiotics on mucus secretion in airway epithelial cells.

To examine the in vivo effects of macrolide antibiotics on mucus hypersecretion, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats and by intranasal LPS instillation. Oral administration of clarithromycin (CAM) (5-10 mg/kg) significantly inhibited OVA- and LPS-induced mucus production and neutrophil infiltration, whereas josamycin and ampicillin showed no effect. In vitro effects of macrolide antibiotics on airway epithelial cells were examined using NCI-H292 cells and human nasal epithelial cells cultured in air-liquid interface. Mucus secretion was evaluated by ELISA using anti-mucin monoclonal antibodies (anti-MUC5AC and HCS18). CAM and erythromycin significantly inhibited spontaneous and tumor necrosis factor-alpha (20 ng/ml)-induced mucus secretion from NCI-H292 cells at 10-6 to 10-7 M and from human nasal epithelial cells at 10-4 to 10-5 M. MUC5AC messenger RNA expression was also significantly inhibited. These results indicate that the 14-member macrolide antibiotics, CAM and erythromycin, exert direct inhibitory effects on mucus secretion from airway epithelial cells and that they may be useful for the treatment of mucus hypersecretion caused by allergic inflammation and LPS stimulation.

Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium

Formaldehyde (FA), an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carcinomas in the nasal turbinates of rats and mice and squamous metaplasia in monkey noses. Tissue responses to FA include a dose dependent epithelial degeneration, respiratory cell hypertrophy, and squamous metaplasia. The primary target for FA-induced toxicity in both rodents and monkeys is the respiratory nasal epithelium. FA increases nasal epithelial cell proliferation and DNA–protein crosslinks (DPX) that are associated with subsequent nasal cancer development. To address the acute effects of FA exposure that might contribute to known pathological changes, cDNA gene expression analysis was used. Two groups of male F344 rats received either 40 ul of distilled water or FA (400 mM) instilled into each nostril. Twenty-four hours following treatment, nasal epithelium was recovered from which total RNA was used to generate cDNA probes. Significance analysis of microarrays (SAM) hybridization data using Clontech™ Rat Atlas 1.2 arrays revealed that 24 of the 1185 genes queried were significantly up-regulated and 22 genes were significantly downregulated. Results for ten of the differentially expressed genes were confirmed by quantitative real time RT PCR. The identified genes with FA-induced change in expression belong to the functional gene categories xenobiotic metabolism, cell cycle, apoptosis, and DNA repair. These data suggest that multiple pathways are dysregulated by FA exposure, including those involved in DNA synthesis/repair and regulation of cell proliferation. Differential gene expression profiles may provide clues that could be used to define mechanisms involved in FA-induced nasal cancer.

Plunc, a Member of the Secretory Gland Protein Family, Is Up-regulated in Nasal Respiratory Epithelium after Olfactory Bulbectomy

Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy. We isolated the rat homologue of plunc, a murine gene highly expressed in lung and nasopharyngeal regions, by this method. Rat plunc encodes a 270-amino acid protein containing a putative signal peptide. plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization. plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis. Antibodies against Plunc detected a 31-kDa protein in lung, heart, and spleen. Rat nasal epithelium displayed robust immunoreactivity that was highly localized to the microvilli layer of respiratory epithelium. The expression of plunc was up-regulated after bulbectomy in respiratory epithelium. We also detected secreted plunc in rat and human mucus. Sequence and homology analyses suggest that Plunc is a member of the secretory gland protein family with putative bactericidal/bacteriostatic function. This is the first protein found in respiratory epithelium whose expression is regulated by olfactory neuronal injury and may provide protection against infection subsequent to injury.

Normal gene expression in male F344 rat nasal transitional and respiratory epithelium

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity in rodents. Gene expression profiles were determined in order to provide normal baseline data for nasal transitional/respiratory epithelium from healthy rats. Cells lining the rat nasal passages were collected and gene expression analysis was performed using Clontech™ cDNA Rat Atlas 1.2 arrays (1185 genes). The percentages of genes within specific average expression ranges were 4.2% at 45,000–1000, 14.8% at 1000–200, 25.0% at 200–68, and 56.0% below 68. Nine out of a subset of ten genes were confirmed for relative signal intensity using quantitative real-time RT-PCR. The most highly expressed genes included those involved in phase I (e.g. cytochrome P450s) and phase II (e.g. glutathione S-transferases) xenobiotic metabolism, bioenergetics (e.g. cytochrome oxidase), osmotic balance (e.g. Na +/K + ATPase) and epithelial ionic homeostasis (e.g. ion channels). Such baseline data will contribute to further understanding the normal physiology of these cells and facilitate the interpretation of responses by the nasal epithelial cells to xenobiotic treatment or disease.

Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation: correlation with tissue sulfide concentrations in the rat brain, liver, lung, and nasal epithelium.

Hydrogen sulfide (H2S) is an important brain, lung, and nose toxicant. Inhibition of cytochrome oxidase is the primary biochemical effect associated with lethal H2S exposure. The objective of this study was to evaluate the relationship between the concentration of sulfide and cytochrome oxidase activity in target tissues following acute exposure to sublethal concentrations of inhaled H2S. Hindbrain, lung, liver, and nasal (olfactory and respiratory epithelial) cytochrome oxidase activity and sulfide concentrations were determined in adult male CD rats immediately after a 3-h exposure to H2S (10, 30, 80, 200, and 400 ppm). We also determined lung sulfide and sulfide metabolite concentrations at 0, 1.5, 3, 3.25, 3.5, 4, 5, and 7 h after the start of a 3-h H2S exposure to 400 ppm. Lung sulfide concentrations increased during H2S exposure and rapidly returned to endogenous levels within 15 min after the cessation of the 400-ppm exposure. Lung sulfide metabolite concentrations were transiently increased immediately after the end of the 3-h H2S exposure. Decreased cytochrome oxidase activity was observed in the olfactory epithelium following exposure to > or = 30 ppm H2S. Increased olfactory epithelial sulfide concentrations were observed following exposure to 400 ppm H2S. Hindbrain and nasal respiratory epithelial sulfide concentrations were unaffected by acute H2S exposure. Nasal respiratory epithelial cytochrome oxidase activity was reduced following acute exposure to > or = 30 ppm H2S. Liver sulfide concentrations were increased following exposure to > or = 200 ppm H2S and cytochrome oxidase activity was increased following inhalation exposure to > or = 10 ppm H2S. Our results suggest that cytochrome oxidase inhibition is a sensitive biomarker of H2S exposure in target tissues, and sulfide concentrations are unlikely to increase postexposure in the brain, lung, or nose following a single 3-h exposure to < or = 30 ppm H2S.

Effect of the degree of quaternisation of N-trimethyl chitosan chloride on absorption enhancement: in vivo evaluation in rat nasal epithelia

Five TMC polymers with different degrees of quaternisation (12–59%) were synthesised and administered together with [ 14C]-mannitol in the nasal route of rats at a pH of 6.20 and 7.40, respectively. All the TMC polymers increased the nasal absorption of [ 14C]-mannitol significantly at pH 6.20, but only TMC polymers with higher degrees of quaternisation (>36%) were able to increase the absorption of this hydrophilic model compound at pH 7.40. The absorption of [ 14C]-mannitol at pH 7.40 increased with an increase in the degree of quaternisation of TMC until a maximum absorption value was reached with TMC with a degree of quaternisation of 48%. The absorption of [ 14C]-mannitol did not increase further, even when TMC with a higher degree of quaternisation (59%) was used. This can probably be explained by steric effects caused by the attached methyl groups and changes in the flexibility of the TMC molecules with an increase in the degree of quaternisation above an optimum value for absorption enhancement in a neutral environment. It was concluded that the degree of quaternisation of TMC plays an important role in the absorption enhancement properties of this polymer across nasal epithelia in a neutral environment.

Differential Properties of Mucous Glycoproteins in Rat Nasal Epithelium

To examine the differential properties of mucous glycoproteins, we produced hypertrophic and metaplastic changes in goblet cells of rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats, and by intranasal lipopolysaccharide (LPS) instillation. The epithelial mucosubstance was quantitatively examined by alcian blue-periodic acid-Schiff (AB-PAS) and lectin histochemistry. The newly produced mucin after OVA challenge or LPS instillation contained a high amount of sulfomucin and a low amount of neutral glycoprotein: LPS-induced mucin contained more sulfomucin (70.1% of total) and less neutral glycoprotein (8.6%) than OVA-induced mucin (sulfomucin, 33.6%; neutral glycoprotein, 41.8%; p < 0.01). Four of the lectins stained some of the mucosubstance, indicating the presence of galactose-N-acetylgalactosamine, alpha2,3- and alpha2,6-linked sialic acid-galactose, and fucose residues. After LPS instillation, the reactivity was higher for galactose-N-acetylgalactosamine (64.8% of total) and alpha2,3-linked sialic acid-galactose (75.8%) than after saline instillation (3.5 and 19.1%, respectively) or OVA challenge (5.8 and 32.3%; p < 0.05). OVA challenge did not induce the alteration of terminal sugar residues. A 2-fold increase in mucin mRNA (rat Muc5ac) expression was induced after LPS instillation or OVA challenge, compared with animals treated with saline instillation (p < 0.05). These results indicate that mucin mRNA expression (for peptide backbone) increases similarly after LPS instillation or OVA challenge; however, carbohydrate compositions of newly produced mucin are different between the two groups.

Epidermal growth factor receptor signaling mediates regranulation of rat nasal goblet cells

Background: Mucus hypersecretion is a common response to inflammation in the lower airways and is a hallmark of chronic rhinitis. Objective: The purpose of this study was to elucidate the mechanisms of regranulation (mucus production) of goblet cells in nasal epithelium. Methods: Because neutrophils induce an epidermal growth factor (EGFR) cascade, we induced degranulation of goblet cells in rat nasal respiratory epithelium by means of intranasal inhalation of N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we examined regranulation of the goblet cells and the role of EGFR inhibitors and neutrophils in the regranulation process. Results: In the control state Alcian blue/periodic acid–Schiff and mucin MUC5AC staining was present. Degranulation was induced in the nasal septal epithelium 4 hours after intranasal inhalation of fMLP (10–7 mol/L); 48 hours later, goblet-cell regranulation was complete. In the control state EGFR protein staining was absent in the epithelium, but after fMLP-induced degranulation, EGFR protein was expressed. After pretreatment with BIBX1522, a selective EGFR tyrosine kinase inhibitor, fMLP-induced degranulation was unaffected, but goblet-cell regranulation was prevented completely. Conclusion: These data suggest a role for the EGFR cascade in neutrophil-dependent production of goblet-cell mucins. Proving this theory will require the use of selective EGFR inhibitors in clinical studies of nasal hypersecretory states. (J Allergy Clin Immunol 2001;107:1046-50.)