Alteration in Goblet Cell Numbers and Mucin Gene Expression in a Mouse Model of Allergic Conjunctivitis

Abstract

purpose. To determine whether the number of filled conjunctival goblet cells and mucin gene expression are altered in a mouse model of allergic conjunctivitis.

methods. A/J mice were sensitized intraperitoneally with cat dander or the peptide P3-1 from the protein Fel d1. Two weeks later, the mice were challenged for 7 consecutive days with eye drops containing the allergens. Conjunctival tissue was harvested at 0, 6, 24, or 48 hours after final antigen challenge. Control samples were naïve animals and mice sensitized with cat dander and challenged with OVA-peptide or PBS. The mean number of filled goblet cells per square millimeter in three forniceal fields for each group was determined in wholemounts of conjunctiva prepared using rhodamine–phalloidin labeling followed by confocal microscopy. RNA was isolated from conjunctiva of the contralateral eye and taken for relative quantitation of mRNA of the goblet cell mucin Muc5AC and the epithelial membrane-spanning mucin Muc4, by real-time RT-PCR.

results. The number of filled goblet cells was significantly decreased with both cat dander and P3-1, after final ocular challenge (P < 0.001). The most significant decrease over naïve mice was seen at 6 hours after final challenge with both allergens. The number of filled goblet cells was still decreased but was returning toward naïve levels at 24 hours (P < 0.05), and at 48 hours no significant difference was seen compared with naïve, PBS-treated, and OVA-peptide–treated control samples. For both cat dander and P3-1, Muc5AC and Muc4 mRNA was found to be decreased at the time of final ocular challenge. The level of Muc5AC mRNA from goblet cells rebounded from the decrease to show an increase over control by 24 hours after final challenge, and by 48 hours, the mRNA level had returned to naïve control range. In contrast, significant increases in Muc5AC mRNA were evident after final control challenge with PBS or OVA-peptide, indicating a potential irritant effect of drop application. The Muc4 mRNA level was significantly reduced at all time points except 24 hours after the last challenge. By comparison with allergen-challenged eyes, no change in Muc4 message levels was noted at any time point in OVA-peptide– or PBS-treated control eyes.

conclusions. These findings demonstrate that, in the conjunctiva of mice, repetitive application of allergens induces a reduction in the number of filled goblet cells and a decrease in Muc5AC and Muc4 mRNAs. After a period of 24 to 48 hours, the goblet cell number return to naïve levels, and goblet cell mucin mRNA levels return to above or within normal range, indicating a rapid recovery in the mucus secretion system.

Mucins on the surface of the eye are believed to provide a barrier to prevent pathogens and particulate matter from entering the ocular surface epithelium and, through their heavy O-linked glycosylation, maintain hydration of the ocular surface. Of the 13 human mucin genes described to date, two major mucins of the ocular surface are the large secreted mucin MUC5AC, limited to the conjunctival goblet cells that are interspersed among the stratified cells in the conjunctival epithelium, and the membrane-spanning mucin MUC4, expressed by the stratified squamous epithelia of the entire ocular surface in rodents and primarily by the conjunctival epithelium in humans. ^{1 2 3} The rat homologue of MUC4 called sialomucin complex (SMC) also known as ascites sialylglycoprotein (ASGP) can be found in both membrane and soluble forms. SMC can be shed into the tear fluid of the rat, ⁴ suggesting that shed membrane-spanning mucins as well as secreted MUC5AC may be present in the tear fluid. 

Seasonal or perennial allergic conjunctivitis is a common ocular problem. It is characterized by a burning, itching sensation and may be accompanied by the presence of mucin threads. ⁵ The degree and severity of the allergic response may be highly variable. ^{6 7} In vernal conjunctivitis, mucus discharge is a common feature, as is hyperplasia of the conjunctiva and goblet cells, during which folds or pegs of conjunctival tissue develop and give a cobblestone appearance. It has been shown by impression cytology that there is a significant increase in the number of goblet cells in persons with vernal conjunctivitis over those in normal control subjects. ⁸ Similarly, study of conjunctival biopsy specimens from patients with atopic keratoconjunctivitis demonstrated a significant increase in both the epithelial mitotic rate and goblet cell frequency. ⁹  

An increase in the number of goblet cells with mucus overproduction is a common feature of allergic asthma. ¹⁰ A more recent study associated this increase with an increased expression of Muc5AC mRNA in a murine model of allergic asthma. ¹¹ In cases of allergic conjunctivitis, however, little information is available on mucin expression or goblet cell number. Two studies of goblet cells in a guinea pig model of allergic conjunctivitis demonstrated a decrease in conjunctival goblet cells, ^{12 13} but there does not appear to be information on mucin gene expression relative to ocular allergic response. Keane-Meyers et al. ¹⁴ have developed a murine model of allergic conjunctivitis that closely mimics the clinical and histologic features of human disease (Keane-Myers et al., manuscript submitted). Sensitized mice were challenged in the eye with either cat hair extract containing known amounts of the major cat allergen Felis domesticus allergen 1 (Fel d1) or a 14-amino-acid peptide (P3-1) from chain I of the immunodominant Fel d1 component of the cat dander protein. An immediate response after ocular challenge with allergens was observed in the mice, including increased photosensitivity, itching, chemosis, and edema. These early clinical symptoms correlated with mast cell degranulation observed 1 hour after challenge. Twenty-four hours after final challenge, there was a significant infiltration of inflammatory cells, including eosinophils. Particularly during the late phase, antigen-specific increases in T-helper (Th)2–associated cytokines (IL-4 and -5) were detected in in vitro cultures of splenocytes and purified T cells from the affected animals. Additionally, increased serum levels of the Th2-dependent antibodies IgE and allergen-reactive IgG1 were noted. We hypothesize that goblet cells and mucin production, perhaps through Th2-associated cytokines, are important components in the epithelial innate immune defense against allergens. The purpose of this study was therefore to evaluate the presence of filled goblet cells as well as Muc5AC and Muc4 mucin gene expression in mice, by using the murine model for allergic conjunctivitis. 

Materials and Methods

Animals

Six- to eight-week-old female A/J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Use of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Schepens Eye Research Institute Animal Care and Use Committee. Three to 11 mice were used for each experimental group at each time point studied. To prevent any potential aerosolization of allergen from affecting PBS-challenged and naïve control mice, all animals were kept in cages with filter-topped lids. 

Antigen-Challenge Protocols

Two different protocols developed by Keane-Myers et al. (manuscript submitted) ¹⁴ were used to elicit an allergic response in A/J mice. In the first protocol, the animals were sensitized systemically by intraperitoneal (IP) injection of cat dander extract (ALK Laboratories Inc., Milford, CT), containing defined amounts of the major cat allergen, a 35-kDa protein Fel d1. Fifty micrograms per mouse was injected with an equal volume of Incomplete Freund’s Adjuvant (IFA; Gibco BRL-Life Technologies, Gaithersburg, MD) prepared as an emulsion (100 μl per mouse). Two weeks later, mice received 7 consecutive days of ocular challenge with topical application of cat dander containing Fel d1 (1 μg/μl) suspended in PBS (5 μl/eye). 

In the second protocol, designed to show antigen specificity, mice were sensitized systemically with IP injection of a 14-amino-acid peptide (P3-1) from chain I of the immunodominant Fel d1 component of the cat dander protein (gift from Julian Bond, ImmuLogic, Waltham, MA). Fifty micrograms per mouse was injected with an equal volume of IFA prepared as an emulsion (100 μl per mouse). Two weeks later, animals were challenged for 7 consecutive days with eye drops containing the P3-1 peptide (1 μg/μl) suspended in PBS (5 μl/eye). 

Control animals for both protocols were mice sensitized with cat dander and challenged with 5 μl PBS and naïve mice without sensitization or challenge. To control for any potential response to protein or for nonspecific irritation due to the deposition of protein on the eye surface, a third group of animals was sensitized with IP injection of cat dander-IFA and challenged with eye drops using the same concentration (5 μg) of an irrelevant protein (OVA-peptide 323-339; Analytical Biotechnology Services, Boston, MA). The animals were killed by cervical dislocation at 0, 6, 24, or 48 hours after final antigen challenge, and tissue was harvested. 

Visualization and Quantitation of Filled Goblet Cells

To visualize and quantitate the filled conjunctival goblet cells, a conjunctival wholemount technique was used. ¹⁵ One eye from each animal was fixed for 5 to 10 minutes in situ with 4% paraformaldehyde in PBS. Eyes were enucleated, and wholemounts of superior and inferior conjunctiva were dissected and fixed overnight in 4% paraformaldehyde. Tissues were then rinsed three times (10 minutes per rinse) in PBS and incubated with rhodamine-conjugated phalloidin, which primarily binds to the filamentous actin at the cell periphery (dilution 1:100; Molecular Probes, Eugene, OR). With this technique, only filled goblet cells were visualized. Conjunctival wholemounts were postfixed for 30 minutes in 4% paraformaldehyde and rinsed three times for 10 minutes each in PBS. Tissues were flatmounted on a glass slide in mounting medium (Vectashield; Vector, Burlingame, CA). 

Tissue was viewed by confocal microscopy using a confocal microscope (TCS 4D; Leica, Heidelberg, Germany). Optical sections were taken of 3 forniceal fields using a 40× PL Fluotar oil immersion objective. In this manner, the entire forniceal area was usually captured. 

Images were imported into image management software (Photoshop, ver. 5.0; Adobe, San Jose, CA), and measurement of the entire epithelial area was achieved by tracing the area by using the “lasso” tool in the software program. The total data area, measured in pixels, was acquired through the “image: histogram” command in the program. Two independent counts were recorded for filled, phalloidin-labeled goblet cells. Goblet cells per unit area of pixels were adjusted to real unit area or cells per square millimeter of real epithelial area, based on 28.346 pixels/cm in the software and a calibration factor of 1 mm = 4096 pixels at ×40 magnification on the confocal microscope. Data were recorded as goblet cells per square millimeter, and the results were analyzed on computer with ANOVA (StatView software, ver. 5.0; Abacus Concepts; Berkeley, CA). 

Real-Time RT-PCR to Determine Relative Muc5AC and Muc4 Expression

The contralateral eye was taken for RNA isolation to use for relative quantitation of Muc5AC and Muc4 mRNAs using real-time RT-PCR. Mouse conjunctivas were snap frozen in liquid nitrogen, and total RNA was isolated by a single-step extraction technique with reagent (TRIzol; Gibco-Life Technologies, Grand Island, NY). The first strand of cDNA was synthesized from 1 μg of DNase-treated RNA with random primers, using reverse transcriptase (SuperScript II; Gibco-Life Technologies), followed by RNase H treatment. 

Real-time RT-PCR was performed using a sequence-detection system (TaqMan PCR GeneAmp 5700; (PE Applied Biosystems, Foster City, CA), essentially as previously described. ^{16 17} The Muc5AC-specific primers (based on GenBank accession number L42292; provided in the public domain by the National Center for Biotechnology and available at http://www.ncbi.nlm.nih.gov) ¹⁸ were sense 5′-AAAGACACCAGTAGTCACTCAGCAA-3′ (nucleotides [nt] 745-769) and antisense 5′-CTGGGAAGTCAGTGTCAAACCA-3′ (nt 883-862), and the TaqMan probe sequence was 5′-TCACACACAACCACTCAACCAGTGACCA-3′ (nt 802-829). The Muc4-specific primers (based on GenBank accession number AF218265) were sense 5′-CTCCAAGAAATGTAGTGGCTTTCAG-3′ (nt 2925-2949), antisense 5′-CACGGTCTTGGGCTGGAGTA-3′ (nt 883-862), and TaqMan probe 5′-AACATCCCCAGAAGCGTGTACCCTGG-3′ (nt 802-829). 

For relative quantitation as used in this study, a comparative threshold (C _T) method normalizes the amount of target to an internal standard control—for example, a suitable housekeeping gene. The relative amount of target gene in different samples was determined and compared with the amount in the naïve control specimens (calibrator specimen). The internal standard control gene was amplified using rodent GAPDH (TaqMan Control Reagents; PE Biosystems). To verify the validity of using GAPDH as the internal calibration standard, the efficiencies of the Muc5AC-Muc4 and GAPDH amplifications were compared and found to be equivalent. The identities of the Muc5AC and Muc4 PCR products were verified by sequencing performed by the DNA Sequencing Core Facility of the Massachusetts General Hospital (Boston, MA). 

Each PCR reaction contained equivalent amounts of cDNA. Assays were performed in triplicate using a kit according to the manufacturer’s recommendations (TaqMan PCR Reagent Kit; PE Biosystems). Relative quantitation of amounts of Muc5AC and Muc4 mRNAs at 0, 6, 24, and 48 hours after final antigen challenge was determined as previously described. ¹⁶ Results were analyzed using the unpaired t-test (StatView, ver. 5.0; Abacus Concepts). 

Results

Quantitation of Goblet Cells

Challenge with the two allergens—the cat dander extract or the peptide P3-1 from the major cat allergen Fel d1—resulted in a similar response of goblet cells throughout the 48-hour time course after final ocular challenge. To illustrate the method of goblet cell quantitation, Figure 1 shows representative optical sections of conjunctival wholemount confocal images from naïve (Fig. 1A) and allergen-challenged mice taken 6 hours after final ocular challenge (Fig. 1B) . Goblet cells are labeled with rhodamine-conjugated phalloidin, which penetrates goblet cells, primarily binding to the filamentous actin at the cell periphery. As demonstrated previously, this method visualizes filled goblet cells as indicated by lectin binding. ¹⁵ There was a significant decrease in the number of filled goblet cells after sensitization and ocular challenge with either allergen in comparison with naïve control animals (Fig. 2) . Statistical analysis revealed a significant decrease at the time of final ocular challenge (0 hour; P < 0.001). With both allergens, the most significant decrease compared with naïve mice occurred at 6 hours after final challenge (P < 0.001). At this time point, the filled goblet cell population was diminished by approximately 50% in comparison with the number in naïve control samples. At 24 hours, cell numbers were still significantly decreased compared with that in naïve control samples (P < 0.05 for the cat dander group [Fig. 2A ]; P < 0.001 for the peptide P3-1 group [Fig. 2B ]). However, this decrease was not as dramatic as the decrease 6 hours after final ocular challenge. Goblet cell numbers returned to the naïve control number at 48 hours after final ocular challenge with either cat dander or the peptide P3-1. 

A slight, but not significant, decrease in the number of filled goblet cells was seen at the time of final challenge (0 hour) for either PBS (Fig. 2C) or OVA-peptide control (Fig. 2D) , whereas no difference in the number of goblet cells in both control groups was observed at any later time point. 

Real-Time RT-PCR

Treatment with both allergens—the cat dander extract and the peptide P3-1 from the major cat allergen Fel d1—resulted in a decrease in both Muc5AC and Muc4 mucin gene mRNA levels. mRNA levels of both genes increased to significantly higher levels 24 hours after the last challenge with cat dander (P < 0.08). Down- and upregulation of the two genes occurred in a coordinate fashion in response to allergen challenge. 

For Muc5AC mRNA, there was a significant decrease relative to naïve control samples seen at the time of final ocular challenge (0 hour) in response to the cat dander extract (P < 0.05; Fig. 3A ). The level of Muc5AC mRNA was also reduced after final challenge with the active peptide from Fel d1, but the reduction did not reach significance (Fig. 3B) . Muc5AC mRNA in response to both the cat dander extract and the peptide P3-1 returned to the naïve control range by 6 to 24 hours. In contrast, there was a significant upregulation in Muc5AC mRNA levels noted at the time of final ocular challenge for the PBS and OVA-peptide control samples (Figs. 3C 3D) . 

For Muc4 mRNA, there was a significant decrease at the time of final ocular challenge (0 hour) in response to both allergens—the cat dander (P < 0.05) or the peptide P3-1 (P < 0.08; Figs. 4A 4B ). In response to the cat dander challenge, Muc4 mRNA increased to greater than the naïve control level by 24 hours (P < 0.08). At 48 hours after final ocular challenge, both responses had decreased to significantly less than the naïve control level (P < 0.05). There was no significant change in Muc4 mRNA levels seen at any of the time points for the PBS or OVA-peptide control samples (Figs. 4C 4D) . 

Discussion

The role of mucus in defense of the ocular surface from allergens is not established. Mouse models of allergic conjunctivitis have been developed recently and have shown that mice sensitized and challenged with allergens show characteristics similar to those seen in human allergic conjunctivitis (Keane-Myers et al., manuscript submitted). ^{14 19} Clinical, cellular, and humoral parameters have been described in these models, but to our knowledge data on the goblet cell number and mucin gene expression have not been recorded. In the current study, the number of filled goblet cells was reduced, and mucin gene expression was downregulated at the time of final challenge in response to ocular challenge with a specific allergen—cat dander and peptide P3-1 from the Fel d1 component of the cat dander protein. The fact that, within 24 hours after challenge, the number of goblet cells was still significantly decreased but mucin mRNA levels exceeded naïve levels suggests that conjunctival epithelial cells mount a local epithelial defense against allergens by increasing goblet cell differentiation and mucus secretion to remove the offending agent. 

A significant decrease in the number of filled goblet cells was noted at the time of final ocular challenge with either allergen. This decrease continued over 6 hours, and at 24 hours, the number was still decreased but was higher than at 6 hours. The goblet cell number returned to control level within 48 hours. These data correlate well with the significant decrease in goblet cell Muc5AC mRNA at the time of final challenge and the return to the naïve level by 48 hours. Similar to these findings, Toda et al. ¹² reported a reduction in goblet cell density in a guinea pig model of allergic conjunctivitis. In contrast to our study, studies conducted on human vernal and atopic keratoconjunctivitis (VKC and AKC, respectively) revealed an increase in conjunctival goblet cell frequency when compared with normal control samples. ^{8 9} A possible explanation for this observed difference in goblet cell numbers between the animal models and humans may be that human VKC and AKC represent more chronic disease states in comparison with the relatively intense, acute 7-day allergen exposure in these rodent models of allergic conjunctivitis. It should also be noted that the VKC and AKC disease states are far more severe than that of the animal model, in that they exhibit chronic inflammation and various epithelial abnormalities, along with increased goblet cells. Epithelial disease severity may be a major reason for differences between VKC, AKC, and the mouse model. The clinical implications for the mouse model in relation to severe allergic conjunctivitis are uncertain. 

The effectors of ocular mucin gene expression are not known, but it is evident that specific allergens downregulated mucin genes in this mouse model of allergic conjunctivitis, because Muc5AC and Muc4 mRNA levels were significantly decreased at the time of final ocular challenge. After 24 hours, the situation reverted to within normal range, thus showing the specificity of this event. By comparison, the Muc5AC levels in PBS and OVA control groups were significantly upregulated at the time of final challenge. These data suggest that the eye drops of PBS and of PBS with OVA-peptide may act as an irritant as opposed to the specific allergen effect. A similar increase in mucin production has been demonstrated in respiratory tract epithelia in response to various irritants. In the airway of rat and human, exposure to irritants, infections, and tobacco smoke have been shown to increase goblet cells in the epithelium. ^{20 21} In fact, cigarette smoke has been reported to induce upregulation of MUC5AC mRNA in an airway epithelial cell line, through activation of epidermal growth factor receptors. ²² Furthermore, environmental irritant stimuli such as SO₂ can induce an increase in Muc2 mRNA in rat airway. ²⁰ As in airway epithelia, nonallergenic irritants may increase mucin gene expression in goblet cells of the conjunctiva. 

Airway inflammation, hyperactivity, and increases in goblet cells with mucus overproduction are common features of asthma. ¹⁰ A more recent study associated increases in goblet cells with an increase in Muc5AC mRNA in a murine model of allergic asthma. ¹¹ Marked elevation of Muc5AC mRNA was observed in the lungs of OVA- and IL-13–treated compared with control animals 24 hours after the last challenge. 

The data obtained in our ocular allergy study seem to be opposite to results in the airway study, in that, immediately after the last challenge, the goblet cells and mucin mRNA levels were significantly decreased. However, there are many differences between the two models. In naïve airway epithelia, there are few if any goblet cells, but by comparison, goblet cells are a major cell type in mouse conjunctival epithelium. In the airway study, the challenge protocol included five intraperitoneal injections of antigen at 3- to 4-day intervals followed 2 weeks later by three daily airway challenges. Assays of goblet cells and mucin mRNA were performed on airway tissue taken only at 24 hours after the last challenge. In our protocol, a single intraperitoneal sensitization injection was followed 2 weeks later by 7 daily ocular surface challenges. Tissue was harvested at 0, 6, 24, and 48 hours after the last challenge. When the 24-hour data points of the two studies are compared, an increase in mucin mRNA is seen in both studies in response to allergen challenge. Thus, there may in fact be similarities in upregulation of mucin production at 24 hours after challenge. 

Mice sensitized and challenged with cat dander or peptide P3-1 showed clinical signs consistent with human allergic conjunctivitis (Keane-Myers et al., manuscript submitted). ¹⁴ These signs correlate with histologic changes in the conjunctival epithelium and stroma: mast cell degranulation in the early phase, and significant infiltration of inflammatory cells, including eosinophils, 24 hours after challenge. Particularly during the late phase, antigen-specific increases in Th2-associated cytokines in spleen cell supernatants and draining lymph nodes were noted as well (Keane-Myers et al., manuscript submitted). ¹⁴ Increasing evidence supports the concept that mucus production is directly affected by cytokines that regulate immune functions in animals. Mucus production appears to be independent of B cells and immunoglobulins ²³ but dependent on Th2-associated cytokines. Initially, IL-4 was shown to cause goblet cell metaplasia and upregulation of MUC2 and MUC5AC mucin gene expression in vitro and in vivo (airway). ^{24 25} Recently, the Th2-associated cytokines IL-9 and IL-13 were demonstrated to be central mediators of allergic asthma and goblet cell metaplasia. ^{11 26 27} Another study indicates that transgenic mice that overexpress the Th2-associated cytokine IL-5 in their lung have increased mucus in their airway epithelia. ²⁸ Thus, several potential Th2-associated cytokines may be involved in mucin gene upregulation in airway epithelia. It remains to be determined whether these cytokines effect mucin gene expression in ocular surface epithelia. 

It also remains to be determined how mucin gene regulation occurs in human ocular surface epithelia. In patients with AKC and VKC, IL-4 and -5 are shown to be increased in tears. ²⁹ Increased expression of IL-4 and -5 was also demonstrated in VKC conjunctival tissues by in situ hybridization. ³⁰ In addition, high levels of IL-4 were found by ELISA from conjunctival brush cytology samples of patients with seasonal allergic conjunctivitis. ³¹ The level of mucin gene expression in human ocular surface allergy has not been studied, and it is not clear whether the initial murine response of the downregulation of mucin genes occurs in humans. Even though the majority of available publications related to regulation of mucin gene expression in allergy concentrate on Th2-associated cytokines, a combination of factors may be responsible for the downregulation of mucin gene expression in the mouse model described herein. Factors associated with mast cells and eosinophils, as major inflammatory components, should be considered and evaluated in regard to mucin gene regulation. Mast cells, for example, release TNF-α, tryptase, and histamine to recruit eosinophils to the site of inflammation. ^{32 33 34} Furthermore, eosinophil products such as eosinophil cationic protein and major basic protein are increased in patients with allergic conjunctivitis and may affect goblet cell function and mucin production. ^{35 36 37}  

The goblet cell Muc5AC and the epithelial cell Muc4 mucin genes apparently respond in a coordinate fashion in response to allergen challenge. This suggests that mucin gene expression is altered not only in goblet cells, affecting the gel-forming mucin Muc5AC, but in the stratified epithelium of the conjunctiva, affecting the membrane-spanning mucin Muc4 as well. The finding of coordinate regulation may indicate a common mechanism of gene regulation for these two types of mucin genes. Further studies are necessary to understand the regulation of mucin gene expression at a molecular and protein level. Such studies will give insight into the underlying mechanisms of allergic conjunctivitis and the role of mucins in epithelial defense against allergens. 

 Supported by National Eye Institute Grant R37-EY03306 (IKG), Allergan, Inc. (IKG), and a research fellowship granted by Deutsche Forschungsgemeinschaft (KSK).

 Submitted for publication January 10, 2001; revised May 29, 2001; accepted June 13, 2001.

 Commercial relationships policy: N.

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Figure 1.

 

Goblet cell clusters in wholemounts of conjunctiva from (A) naïve mice and (B) allergen-challenged mice (6 hours after challenge). Filled goblet cells were visualized by phalloidin labeling of the tissue. ¹⁵ The optical section was taken parallel to the plane of the epithelium approximately halfway through the epithelial layer. The decrease in the number of goblet cells in allergen-challenged conjunctiva is shown in (B). Bar, 100μ m.

Figure 2.

 

(A, B) Number of filled goblet cells per square millimeter of conjunctiva after topical application of allergens (n = at least 3 eyes for each group and time point). (A) Goblet cell number at specified time points after 7 days’ challenge with eye drops containing 5 μg cat dander. (B) Number of goblet cells at specified time points after 7 days’ challenge with eye drops containing 5 μg P3-1, a 14-amino-acid peptide from the immunodominant region of the cat dander protein Fel d1. Note the significant decrease in goblet cells compared with naïve control animals at 0, 6, and 24 hours after final ocular challenge (± SEM). (C, D) Number of filled goblet cells per millimeter of conjunctiva after topical application of either PBS or OVA-peptide (n = at least 2 eyes for each group and time point). These two groups served as control samples for nonspecific irritation due to simple drop application or potential response to protein. Number of goblet cells at specified time points after 7 days of challenge with eye drops containing (C) 5μ l PBS and (D) 5 μg OVA-peptide. There was a slight, but not significant, decrease in goblet cells at the time of final challenge (0 hour) for both PBS- and OVA-peptide–treated control samples. However, there was no difference observed compared with naïve control at 6, 24, and 48 hours after final challenge.** P < 0.001; *P < 0.05.

Figure 3.

 

Muc5AC mRNA levels in conjunctival epithelium after topical application of allergens. Real-time RT-PCR was used for the relative quantitation of Muc5AC mRNA, with mRNA concentrations in naïve animals providing the baseline data point. Data points represent the mean ± SEM of all the samples (n = at least 4 eyes for each group). Muc5AC mRNA was decreased in epithelium at the time of final ocular challenge (0 hour) with the cat dander (A) and the peptide P3-1 (B). At 24 hours after the last challenge, message had increased to levels greater than naïve control with both allergens. In contrast, significant upregulation in Muc5AC expression was seen at the time of final challenge in the animals receiving PBS (C) or OVA-peptide (D).* P < 0.05; **P < 0.08.

Figure 4.

 

Muc4 mRNA levels in conjunctival epithelium after topical application of allergens in a mouse model of allergic conjunctivitis. Real-time RT-PCR was used for the relative quantitation of Muc4 mRNA, with mRNA concentrations in naïve animals providing the baseline data point. Data points represent the mean (± SEM) of all the samples (n = at least 4 eyes for each group). Muc4 mRNA was significantly decreased in the epithelium at the time of final ocular challenge (0 hour) and 6 hours after final challenge with both cat dander (A) and the peptide P3-1 (B). At 24 hours after the last challenge, message had increased to levels above or in the range of the naïve control with both allergens. Curiously, mRNA levels were again significantly decreased at 48 hours. In contrast, no significant change in Muc4 mRNA was seen in the animals receiving PBS (C) or OVA-peptide (D) challenge.* P < 0.05; **P < 0.08.