Intranasal Beclomethasone Reduces Allergen-induced ... - ATS Journals

Intranasal Beclomethasone Reduces Allergen-induced Symptoms and Superficial Mucosal Eosinophilia without Affecting Submucosal Inflammation FUAD M. BAROODY, PHILLIP ROUADI, PETER V. DRISCOLL, BRUCE S. BOCHNER, and ROBERT M. NACLERIO Section of Otolaryngology/Head and Neck Surgery, The University of Chicago, Chicago, Illinois; and the Department of Medicine (Division of Allergy and Clinical Immunology) at The Johns Hopkins University School of Medicine, Baltimore, Maryland

Previous investigations have suggested that nasal secretions, obtained by lavage or scraping, and the nasal submucosa, sampled by biopsy, are two distinct compartments. We investigated the effect of intranasal corticosteroids on antigen-induced eosinophil influx into both compartments. We performed a double-blind, placebo-controlled study in 15 patients with seasonal allergic rhinitis. Beclomethasone dipropionate, 84 mg twice a day, was delivered to one nostril while the other nostril received placebo for 1 wk. Subjects were then challenged with grass or ragweed extracts on each inferior turbinate. Nasal scrapings from both inferior turbinates were obtained before and 24 h after challenge, and bilateral inferior turbinate biopsies were obtained 24 h after challenge, with the subjects still receiving treatment. Intranasal steroids led to a significant reduction in sneezes and eosinophil influx in nasal secretions without affecting the number of eosinophils in the submucosa. Furthermore, intranasal steroids had no effect on the numbers of submucosal EG21 (activated eosinophils) or CD251 (IL-2-receptor-bearing) cells, nor did they decrease the endothelial expression of vascular cell adhesion molecule-1 (VCAM-1). These data show that pretreatment with intranasal steroids successfully inhibited the clinical response to allergen and reduced eosinophils in the superficial compartment of the nasal mucosa, but it had no effect on inflammation in the deeper compartment. This might be related to a different distribution of the active medication and antigen into the nasal mucosa or to a specific effect of the active medication on the epithelium resulting in inhibited migration of eosinophils across this layer. Baroody FM, Rouadi P, Driscoll PV, Bochner BS, Naclerio RM. Intranasal beclomethasone reduces allergen-induced symptoms and superficial mucosal eosinophilia without affecting submucosal inflammation. AM J RESPIR CRIT CARE MED 1998;157:899–906.

One of the consistent features of allergic rhinitis is nasal mucosal infiltration by inflammatory cells. These cells secrete mediators and cytokines that are responsible for the symptomatology and regulation of the disease. The allergic inflammatory response leads to an increase in the number of CD41 cells as well as a significant increase in the number of activated helper T-cells (CD251) (1). The helper cells in allergic rhinitis belong predominantly to the Th2 subset as demonstrated by the increased expression of the Th2 cytokines (IL-4, IL-5, and GM-CSF) after antigen challenge (2). In addition to T-cells, eosinophils increase in number in both the nasal secretions (3, 4) and the nasal mucosa after challenge with allergen (5, 6) or during natural exposure (7). Not only are these cells present

(Received in original form July 11, 1997 and in revised form November 12, 1997) Supported in part by grants AI 01236 and DC 02714 from the National Institutes of Health and by a Grant-in-aid from Glaxo Wellcome. Correspondence and requests for reprints should be addressed to Fuad M. Baroody, M.D., Section of Otolaryngology/Head and Neck Surgery, The University of Chicago, 5841 S. Maryland Avenue MC1035, Chicago, IL 60637. E-mail: fbaroody@ surgery.bsd.uchicago.edu Am J Respir Crit Care Med Vol 157. pp 899–906, 1998

but they show clear signs of activation, evidenced by the presence of EG2 on the cell membrane (2, 8), and by an increased amount of specific eosinophil-derived proteins in nasal lavage fluid (9, 10). Previous investigations have shown that nasal secretions, obtained by lavage or scraping, and the nasal submucosa, sampled by biopsy, are two distinct compartments with respect to the patterns of cellular accumulation (1, 11, 12). The superficial compartment contains more eosinophils than lymphocytes, whereas the deeper compartment has a predominance of lymphocytes (1). The mechanisms controlling the number, distribution, and activation of eosinophils and other cells in each compartment is largely unknown. Nevertheless, accumulating evidence supports the importance of vascular endothelial cell adhesion molecules in the selective recruitment of eosinophils into tissue sites during allergic reactions (13). In clinical studies, the expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) on endothelial cells is increased in the nasal mucosa of patients with seasonal (14, 15) and perennial allergic rhinitis (16). Moreover, the influx of eosinophils is inhibited by pretreatment with intranasal steroids (17) and bears a close relationship to the type of allergen stimulation and the technique used to harvest cells

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(5, 7). The effects of steroids include a reduction in numbers of eosinophils in nasal secretions (17) and the nasal mucosa (18). The aim of this study was to investigate the effect of treatment with intranasal corticosteroids on the symptomatic allergic response as gauged by the number of sneezes and the objective increase in nasal secretion weights after challenge. These effects were related to changes in the number of eosinophils in nasal secretions (sampled by nasal scrapings) and in the nasal mucosa (sampled by nasal biopsies). We also investigated the effects of treatment on the number of lymphocytes in the nasal mucosa as well as the vascular expression of VCAM-1. By monitoring patterns of cell recruitment and endothelial activation, information can be gained regarding the differential action of intranasal glucocorticoids on different compartments of the nasal mucosa and the mechanism by which these agents inhibit allergen-induced cellular influx.

METHODS Study Design To allow each patient to serve as his or her own control, all subjects received beclomethasone dipropionate, 84 mg twice a day, in one nasal cavity and placebo spray in the other, simultaneously, for 1 wk. Patients were randomized as to the side of the nasal cavity receiving active drug or placebo. At the end of 1 wk of treatment, subjects underwent sampling of their surface nasal secretions by scraping the medial aspect of both inferior turbinates. This was followed by a sham challenge with diluent, followed 10 min later by a challenge with 333 PNU of ragweed or grass extract on each inferior turbinate. Sneezes and secretion weights obtained after both sham and antigen challenge were recorded for each nostril. The subjects were then asked to keep taking their medications and to come to the laboratory 24 h later. At that time, nasal scrapings were repeated bilaterally, followed by biopsies of both inferior turbinates at the site of the previous challenges.

Subjects A total of 15 patients, nine women and six men, with seasonal allergic rhinitis caused by ragweed or mixed grass extracts were studied during the allergy season. Their median age was 34 yr; range, 24 to 45 yr. All had allergic rhinitis documented by exacerbation of symptoms during the grass or ragweed pollen seasons and a positive puncture skin test response to either grass or ragweed. All experiments were performed when the subjects had received no oral or inhaled medications for at least 1 mo. The study was approved by the Internal Review Board of The Johns Hopkins Medical Institutions, and all subjects gave informed consent prior to initiation of the experiments.

Nasal Antigen Challenge Stimulation and collection of secretions were performed using filter paper discs made from Shandon filter cards (Shandon Inc., Pittsburgh, PA) with a capacity of 50 ml. Subjects were challenged with either ragweed or mixed grass extracts (timothy, june, meadow fescue, and orchard) as well as the diluent for these extracts (sterile phenol-buffered saline) (Greer Laboratories, Lenoir, NC). During the challenges, discs saturated with 50 ml of control or antigen solutions were applied to the medial surface of the anterior portion of the inferior turbinate for 1 min under direct visualization using a nasal speculum, headlight, and duckbill forceps. Thirty seconds after removal, preweighed collection discs were placed at the same location for 30 s. After removal from the nose, the collection discs were immediately placed in 1.5 ml microtubes (Sarstedt Inc., Newton, NC), which were then sealed. The disc-tube combinations, which had been weighed before collection of secretions, were weighed again using a Mettler AE 240 electronic balance (Mettler Instrument Corp., Hightstown, NJ), and the difference between the two weights yielded the weight of collected secretions. During the challenge visit, the left side of the nose was challenged first followed by the right side 30 min later. The first challenge was done with sham solution, and sneezes were counted during a 10-min interval after the challenge. This was followed by challenge with 333 PNU of allergen extract, and another 10-min observation after that challenge with quan-

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titation of sneezes during that interval. Previous studies using the same disc technique (to collect nasal secretions from both nasal cavities every 2 min for a total of 20 min after challenge of one nostril with 333 PNU of allergen) have shown that the weight of generated secretions approaches that obtained after the diluent challenge on both the ipsilateral and contralateral sides of stimulation 20 min after challenge (19). We therefore chose to wait 30 min between challenges of the two nostrils to allow the immediate secretory response on the contralateral side to resolve. To minimize any secretions left over in the contralateral nostril from the previous challenge, we also asked the subjects to blow their noses to clear any accumulated secretions from both nasal cavities. The same challenge sequence was then performed on the contralateral nostril.

Nasal Scrapings Nasal scrapings were obtained from the medial surface of the inferior turbinates using a plastic curette (Rhinoprobe; Apotex Scientific, Arlington, TX). The contents of the sample were spread over a glass slide, air-dried, fixed, and stained using a modified Wright stain (DiffQuik; Baxter Health Corp., Miami, FL). The percentages of eosinophils, mononuclear cells (including lymphocytes and monocytes), and polymorphonuclear leukocytes in each slide were counted using a light microscope (Zeiss; Carl Zeiss Inc., Thornwood, NY) with oil immersion and a magnification 31,000. When possible, 200 cells were counted, and the percentage of each cell type was determined. When the total number of cells counted was less than 25, the specimen was considered inadequate. Specimens from each nostril during each of the study visits were “blinded” and counted by a single investigator.

Nasal Biopsy and Tissue Preparation Twenty-four hours after allergen challenge, the nasal mucosa at the anterior tips of the inferior turbinates was decongested with oxymetazoline and anesthetized topically with pontocaine. Next, 0.5 to 1.0 ml of 1% lidocaine in 1:100,000 epinephrine (Elkins-Sinn, Inc., Cherry Hill, NJ) was injected. Nasal biopsy specimens, with dimensions averaging 4 3 3 mm, were then obtained from the anterior tips of the inferior turbinates with a punch forceps. All subjects had biopsy specimens taken bilaterally from the same site of challenge. Bleeding after biopsy was controlled by the local application of silver nitrate. Each specimen was bisected with one part fixed in formalin and embedded in paraffin and the other part placed in optimal cutting temperature medium (Tissue-Tek; Miles, Inc., Elkhart, IN), snap-frozen with liquid nitrogen, and kept at 2808 C until cryosectioning. The paraffin-embedded tissue was sectioned into 5-mm sections and stained with OrceinGiemsa for the identification of eosinophils.

Immunohistology A murine monoclonal antibody (mAb) recognizing VCAM-1 (2G7, IgG1 subclass) was generously donated by Dr. Walter Newman (Otsuka America Pharmaceutical, Inc.). The following were purchased: murine mAb against CD25 to identify IL-2-receptor-bearing cells (Becton Dickinson, San Jose, CA); murine mAb against EG2 (Kabi Pharmacia, Uppsala, Sweden) to identify the cleaved secreted form of eosinophil cationic protein, thereby identifying activated eosinophils (20); murine mAb against von Willebrand factor (vWf), which specifically stains vascular endothelial cells (vWf, F8/86, IgG1 subclass) (Dako Corp., Carpenteria, CA); mouse irrelevant IgG1 and IgG2a mAb used as negative controls (Becton Dickinson); biotinylated antimouse IgG, avidin biotin complex-alkaline phosphatase kit (ABC-AP), and substrate chromogen solution, Fast Red-Naphthol AS-MX (Vector Laboratories, Burlingame, CA); normal goat serum, Mayer’s hematoxylin solution, poly-L-lysine, and levamisole (Sigma Diagnostics, St. Louis, MO). Specimens were cryosectioned into 5-mm thicknesses (2800 Frigocut N; Reichert-Jung Co., Germany) and mounted on poly-L-lysinecoated glass slides. Cryostat sections were air-dried and then fixed in acetone (48 C) for 10 min. Immunostaining of specimens was performed using the avidin biotin complex-alkaline phosphatase technique (ABC-AP) as follows: after acetone fixation, the slides were washed with TRIS-buffered saline (TBS), assembled with coverplates, and inserted into a cassette (Shandon Coverplate System; Shandon Inc.) to hold them for the rest of the staining. All reagents and buffer

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solutions were then applied to the slides by pipetting into the coverplate-slide assembly. Nonspecific binding sites were blocked with normal goat serum (1:20) for 20 min at room temperature. One hundred microliters of primary Ab or negative control solutions (diluted appropriately with TBS) were then applied and allowed to incubate for 60 min at 378 C. Primary antibodies were then washed with TBS, and 100 ml of biotinylated goat antimouse secondary Ab (1:100 diluted in TBS) were applied and allowed to incubate for 45 min at 378 C. After washing with TBS, 100 ml of avidin-biotin-alkaline phosphatase complex were added to the slides and allowed to incubate for 30 min at room temperature. The slides were then washed with TBS, and 100 ml of the red chromogen solution were added and allowed to incubate in the dark for 20 min at room temperature. The chromogen was then washed with 2 ml of TBS and the slides were placed in staining dishes. They were rinsed briefly in distilled water and counterstained with hematoxylin for 2 min and 15 s. Hematoxylin was washed off the slides by running water for 5 min. The specimens were then dehydrated by brief sequential immersions in 95% alcohol, 100% alcohol and xylene, dried, and coverslips mounted on the specimens using aqueous mounting medium (Baxter Healthcare Corp., McGaw Park, IL). Appropriate dilutions of the monoclonal antibodies provided maximal red staining of the positive cells or vessels with little or no background staining. Optimal concentrations of the antibodies were as follows: 10 mg/ml for 2G7 (VCAM-1), 30 ng/ml for F8/86 (vWf), 100 ng/ml for EG2, and 1 mg/ ml for CD25. All specimens were stained with each of the monoclonal antibodies in the same assay to eliminate interassay variability. Available nasal specimens with positivity to each of the monoclonal antibodies, as determined by previous staining, were run with each assay and served as positive controls, whereas negative controls consisted of tissue specimens stained with irrelevant antibodies (IgG1 or IgG2a). For evaluation of the percentage of vessels staining for VCAM-1, we always stained consecutive tissue sections with vWf and VCAM-1.

examined under magnification 3100 for vascular expression of VCAM-1 and vWf. Consecutive sections had been stained with either VCAM-1 or vWf. Vessels were considered to be positively stained if the endothelial cells lining their lumen were stained red. Comparisons were always made between study specimens and positive and negative controls stained simultaneously. Qualitative evaluation of the intensity of staining was deemed to be too subjective and was not done. We counted only stained vessels with a distinct lumen to avoid counting nonvascular expression of adhesion molecules. This conservative approach eliminated capillaries without a distinct lumen. The percentage of total vessels (as stained by vWf) that showed positive staining for VCAM-1 was used to report the results.

Microscopic Assessment of Biopsy Specimens

RESULTS

All stained sections were examined blind in coded random order using a light microscope with an eyepiece reticule. With the use of the reticule, the central 0.5 mm2 of the lamina propria immediately underneath the basement membrane was examined under magnification 3400 for eosinophils and CD25 and EG2-positive cells, and the final counts were expressed in cells/mm2. All of the biopsy specimen was

Sneezes and Secretion Weights

Statistical Analysis Because the data were not normally distributed, they were analyzed using nonparametric statistics (21). When analyzing the results of the allergen challenge, the responses after antigen were compared with those after diluent on the two treatments using Wilcoxon’s Signed Ranks test. This allowed determination of whether sneezes, secretion weights, and eosinophils in nasal scrapings were significantly increased by allergen challenge when the nasal cavities were premedicated with either placebo or steroid. To compare the effect of premedication with steroid to placebo, we compared the net change from diluent challenge (value after allergen challenge minus value after diluent challenge) for the above parameters after the two treatments using Wilcoxon’s Signed Ranks test. Cell counts and the percentage of vessels positive for VCAM-1 in the nasal biopsies were also compared using Wilcoxon’s Signed Ranks test. The data are presented in RESULTS as median values with (range), and the individual data are graphed to allow the reader to appreciate the differences between patients.

Compared with diluent challenge, there was a significant increase in sneezes after antigen challenge on the side treated with placebo [0 (0–2) versus 6 (0–13), p 5 0.001] (Figure 1). There was a similar, albeit less pronounced, increase in sneezes

Figure 1. Effect of premedication with intranasal steroids on number of sneezes after allergen challenge. The X axis indicates diluent or allergen challenge. Horizontal bars represent median values. The panel to the right shows the net change from diluent in the number of sneezes after each of the treatments. There was a significant increase in sneezes after allergen challenge after premedication with both placebo and intranasal steroids; the increase was less marked after steroid treatment. The net change in the number of sneezes was significantly reduced by steroid premedication. Data of 15 subjects are presented with *p , 0.05 allergen versus diluent in the two panels on the left and steroid versus placebo in the panel on the right.

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Figure 2. Effect of premedication with intranasal steroids on nasal secretion weights (mg) after allergen challenge. The panel on the right shows the net change from diluent in secretion weights after each of the treatments. There was a significant increase in nasal secretions after allergen challenge after premedication with both placebo and intranasal steroids, with the increase being less marked after steroid treatment. The net change in secretion weights was significantly reduced by steroid premedication. Data of 15 subjects are presented with *p < 0.05 allergen versus diluent in the two panels on the left and †p 5 0.056 steroid versus placebo in the panel on the right.

after antigen challenge on the side treated with intranasal steroids [0 (0–0) versus 3 (0–13), p 5 0.008] (Figure 1). When the antigen-induced net change over diluent was compared for both treatments, there was a significant inhibition of sneezes after intranasal steroids [6 (0–13) with placebo versus 3 (0–13) with steroids, p 5 0.03] (Figure 1). There was a significant increase in secretion weights after antigen challenge compared with diluent in both placebo [9.9 (4.2–47.4) mg versus 42.4 (13.9– 70.3) mg, p 5 0.0007] and steroid-treated nostrils [7.1 (2.1–43.6)

mg versus 38.9 (3.4–51.5) mg, p 5 0.002] (Figure 2). When the responses after diluent and allergen challenges were compared for both treatments, there were significantly less secretions after both diluent (p 5 0.01) and allergen challenges (p 5 0.01) in the nostrils treated with steroids. When the net change from diluent for secretion weights was examined, intranasal steroids resulted in a decrease compared with placebo [26.5 (5.1–50.6) mg with placebo versus 13.0 (1.0–44.2) mg], which almost reached statistical significance (p 5 0.056) (Figure 2).

Figure 3. Effect of premedication with intranasal steroids on the percentage of eosinophils in nasal secretions 24 h after allergen challenge. The panel on the right shows the net change from diluent in the percentage of eosinophils after each of the treatments. There was a significant increase in nasal secretion eosinophils after allergen challenge after premedication with both placebo and intranasal steroids, with the increase being less marked after steroid treatment. The net change in eosinophil percentage was significantly reduced by steroid premedication. Data of 14 subjects are presented with *p , 0.05 24 h postchallenge versus prechallenge in the two panels on the left and steroid versus placebo in the panel on the right.

Baroody, Rouadi, Driscoll, et al.: Effect of Intranasal Beclomethasone on Eosinophil Influx Nasal Scrapings

The percentage of eosinophils in nasal scrapings was assessed before and 24 h after antigen challenge in the placebo and steroid-treated nostrils. The slide from one of the patients in the placebo group had too few cells to be adequately interpreted, so the data from only 14 subjects are presented. There was a significant increase in the percentage of eosinophils in nasal scrapings 24 h after antigen challenge with the patients receiving placebo [0.8% (0–41) versus 31.3% (0–60.5), p 5 0.008] (Figure 3). A similar, but less pronounced, increase in eosinophils was noted when the patients were treated with steroids [0.0% (0–56) versus 12.3% (0–48), p 5 0.05] (Figure 3). The median number of eosinophils at baseline was not significantly different in the nostrils treated with either placebo or steroid (p 5 0.84). Comparing the net increase in eosinophils 24 h after antigen challenge for both treatments, a significant inhibition of eosinophil influx into nasal secretions was noted after intranasal steroids [26.8% (240–56.5) with placebo versus 4.0% (217–25) with steroids, p 5 0.01] (Figure 3). There were no statistically significant effects of steroid treatment on the influx of neutrophils or mononuclear cells 24 h after antigen challenge (data not shown). Nasal Biopsies

The Orcein-Giemsa-stained specimens of only 13 subjects were suitable for evaluation. There was a reduction in the number of eosinophils in the nasal biopsies obtained from the nostrils medicated with beclomethasone compared with placebo, but this did not reach statistical significance [214 (10– 642) cells/mm2 with placebo versus 94 (16–884) cells/mm2 with steroid, p 5 0.35, n 5 13] (Figure 4). Similarly, there were no

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significant effects of steroid treatment on the number of EG21 cells in the nasal mucosa after allergen challenge [106 (14– 292) cells/mm2 with placebo versus 112 (0–210) cells/mm2 with steroid, p 5 0.2, n 5 15] (Figure 4). There was a decrease in the number of CD251 cells in the nasal mucosa 24 h after antigen challenge in the steroid-treated nostril, but this did not reach statistical significance [24 (0–166) cells/mm2 in the placebo group versus 12 (0–56) cells/mm2 in the steroid group, p 5 0.16, n 5 15] (Figure 5). When examining the percentage of VCAM-1-positive vessels in the nasal biopsies, there was no significant difference between the expression of VCAM-1 24 h after allergen challenge in the nostrils treated with steroid versus placebo [33.6% (2–100) with placebo versus 27.9% (16.5– 91.7) with steroid, p 5 0.65, n 5 15] (Figure 5).

DISCUSSION The design of this study enabled us to use each patient as his or her own control and subsequently decrease the variability in responses to antigen challenges between patients. Treating both nostrils simultaneously also prevented potential variability induced by environmental exposure. Our data show that treatment with intranasal steroids for 1 wk significantly inhibited sneezes and secretion weights after nasal provocation with grass or ragweed in patients with seasonal allergic rhinitis. These findings further support the inhibitory role of intranasal steroids on the symptoms of the early response to allergen challenge (22, 23). The significant inhibition of secretion weight production in the response to diluent challenges in the nostrils medicated with intranasal steroids suggests that, in addition to their inhibitory effects on the allergen-induced secre-

Figure 4. Effect of premedication with intranasal steroids on the number of eosinophils in nasal biopsies 24 h after allergen challenge. The left panel shows the number of eosinophils/mm2 enumerated by examining Orcein-Giemsa-stained specimens (n 5 13), and the right panel shows the number of EG21 cells/ mm2 representing activated eosinophils (n 5 15). The X axis indicates the treatment, and median values are depicted by the horizontal bars. Pretreatment with intranasal steroids had no significant effect on the number of these cells in the nasal mucosa. NS 5 not significant.

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Figure 5. Effect of premedication with intranasal steroids on the number of CD25 1 cells (left panel) and the percentage of VCAM-1 positive vessels (right panel) in nasal biopsies 24 h after allergen challenge. Data of 15 subjects are shown, with horizontal bars depicting median values. Pretreatment with intranasal steroids had no significant effect on either parameters in the nasal mucosa. NS 5 not significant.

tory response, these agents also inhibit nonspecific reactivity of the nasal mucosa to the control challenge. We also evaluated the influx of eosinophils in the superficial compartment of the nasal mucosa by examining the percentage of eosinophils in nasal scrapings before and 24 h after allergen provocation. As demonstrated in other studies, allergen challenge led to a significant increase in the percentage of eosinophils in nasal secretions when the patients were receiving placebo (7, 11, 17). This increase was also noted, albeit to a much lesser extent, when the patients were pretreated with intranasal steroids. However, when the net change in eosinophils was compared between the two treatments, there was a significant inhibition by intranasal steroids, which has also been documented in previous studies (17). Contrary to its effect on nasal secretions, intranasal steroid therapy did not significantly alter the number of total eosinophils or that of activated eosinophils (EG21) in the nasal mucosa 24 h after allergen challenge. Lozewicz and colleagues (24) investigated the effect of a 2-wk pretreatment with fluticasone propionate nasal spray on eosinophils in nasal biopsies 24 h after allergen challenge in subjects with seasonal allergic rhinitis. Their data showed that intranasal steroids did not inhibit the increase in total eosinophils but resulted in a significant reduction in the number of activated eosinophils (EG21) in the nasal mucosa as well as a significant reduction in levels of eosinophil cationic protein hours after allergen challenge. A similar study was conducted by Rak and colleagues (18), who premedicated subjects with grass allergies out of season with 6 wk of intranasal fluticasone propionate or placebo and compared baseline nasal biopsies with those obtained 24 h after allergen challenge. The total number of submucosal eosinophils (MBP1) was not affected by fluticasone pretreatment, but the number of activated eosinophils (EG21) was significantly reduced in the subjects receiving fluticasone compared with placebo. In the epithelium, the effect of fluticasone pre-

treatment was more marked and led to significant reductions in the numbers of both total and activated eosinophils (18). These studies, unlike ours, support the efficacy of the intranasal steroid, fluticasone, in reducing allergen-induced eosinophil activation in the nasal mucosa. This might be due to several differences between the studies, including the dose of allergen used in the challenges, the potency and dosage of the administered steroid preparation (fluticasone propionate 200 mg once daily [24] and fluticasone propionate 200 mg twice a day [18] versus beclomethasone dipropionate 84 mg twice a day), the length of the pretreatment period (6 and 2 wk versus 1 wk) or the study design. In the current study, intranasal steroid treatment had no effect on the number of CD251 cells or the endothelial expression of VCAM-1 in the lamina propria. Rak and colleagues (18) have shown a significant decrease in allergen-induced CD251 cells in both the nasal submucosa and the epithelium after prolonged treatment with fluticasone propionate. In a study of patients with dust-mite-induced perennial rhinitis, Holm and colleagues (25) compared baseline nasal biopsies with biopsies obtained after 3 mo of treatment with fluticasone propionate aqueous nasal spray. Although they showed a significant reduction in Langerhans’ cells (CD11) and HLADR1 cells in the lamina propria after treatment, the numbers of CD31, CD41, CD81, and CD251 cells were not significantly affected. Because their tissue allowed the examination of the epithelial layer, they also showed that fluticasone pretreatment resulted in a significant decrease in intraepithelial CD11 and HLA-DR1 cells, a trend toward a significant reduction in CD31 (p 5 0.06) and CD81 (p 5 0.06) cells, and no significant change in intraepithelial CD41 and CD251 cells. We were unable to quantitate the number of inflammatory cells in the epithelial layer in our study because immunohistochemistry was performed on frozen tissue, and freezing results in poor epithelial preservation. Furthermore, our biop-

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sies were obtained from the most anterior portion of the inferior turbinate, where the epithelium is squamous and very thin. This is in contrast to other investigators, who obtained biopsies from the inferior portion of the inferior turbinate farther back into the nasal cavity where pseudostratified columnar epithelium predominates (18, 25). Because epithelial counts represent the superficial portion of the nasal mucosa, we sampled that compartment by obtaining nasal scrapings using a plastic curette. We examined the percentage of eosinophils in the scrapings and found that premedication with intranasal steroids led to a significant reduction in these percentages 24 h after allergen challenge compared with placebo. This agrees with the results of Rak and colleagues (18), which show a reduction of total and activated eosinophils in the nasal epithelium after challenge in the patients receiving steroids. Differences in methodology could account for the difference in effects on CD251 cells, which were inhibited in the study of Rak and colleagues but not in ours. On the other hand, in a study performed in a slightly different population of patients with rhinitis, namely, those with perennial symptoms caused by dust-mite sensitivity, Holm and colleagues (25) did not show a reduction in CD41 or CD251 cells in the nasal mucosa after 3 mo of treatment with fluticasone. Conceptually, the antigen challenge in a perennial population is ongoing but less vigorous, therefore leading to a less prominent inflammatory infiltrate, which, theoretically, would be easier to inhibit by intranasal steroids. Despite that, the number of lymphocytes in the lamina propria was not inhibited by steroid pretreatment. Alternatively, the number of lymphocytes devoted to allergen may constitute only a small portion of total lymphocytes, hence, the lack of inhibition by steroids. Comparing the effects of intranasal steroids on the lamina propria and the epithelium in the study of Holm and colleagues, it seemed that the active medication was a little more effective in inhibiting CD81 and CD31 cells in the epithelium than in the lamina propria, lending credibility to the hypothesis that intranasal steroids might be more active on the superficial compartment of the nasal mucosa than on deeper compartments. This was also seen in our study with respect to eosinophils as pretreatment with beclomethasone reduced eosinophils in nasal secretions but not in the lamina propria. Commensurate with the lack of efficacy of the steroid pretreatment in inhibiting total and activated eosinophils in the lamina propria is the lack of reduction in the percentage of VCAM-1-positive vessels. Because VCAM-1 is important in the selective recruitment of eosinophils and lymphocytes (13), these results support a lack of effect of the steroids in the deeper compartments of the nasal mucosa. This selective inhibition of secretion eosinophils without significant effects on submucosal inflammation was associated with a significant reduction of symptoms of sneezing and rhinorrhea after allergen provocation, an indicator of the clinical efficacy of the steroids used in this trial. This suggests that inhibition of the inflammatory response in the lamina propria is not a prerequisite for clinical efficacy of intranasal agents. Limitations of our study design might be partly responsible for the discrepancy between our results and those in the literature. As challenges and subsequent nasal biopsies were performed in both nasal cavities at very close time intervals, the possible effect of the nasonasal reflex has to be entertained. It has been well established that challenging one nasal cavity with allergen or histamine will result in a reflex increase in secretions in the contralateral nasal cavity (26, 27). This secretory response is mostly under the control of the parasympathetic nervous system as evidenced by almost complete inhibition by atropine premedication (27). The methods sec-

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tion details the nasal challenge and how we took precautions to eliminate interference of the reflex secretory response with our measurements. This was achieved by waiting 30 min between nostril challenges and asking the subjects to blow their noses and clear secretions between those challenges. We still have to consider that a reflex inflammatory reaction may have occurred in both nostrils as a result of the allergen challenge of the opposite nasal cavity. We have previously reported a reflex increase in CD251 cells in the opposite nasal cavity 24 h after allergen provocation, but we could not show a contralateral influx of eosinophils in the same setting (1, 28). This might have interfered with our results relating to CD251 cells. The efficacy of intranasal steroids in inhibiting the symptomatic reaction to allergen challenge and the influx of eosinophils into nasal secretions is probably related to multiple effects of these agents. The symptomatic improvement is probably due to inhibition of mast cell release during the acute reaction, with a subsequent decrease in sneezing and rhinorrhea. This has been documented in previous studies using a similar challenge model (22, 29). The reduction of eosinophils in nasal secretions is likely to be associated with inhibition of epithelial cytokine and/or chemokine production. It has now been shown that epithelial cells in the airway can secrete a variety of proinflammatory cytokines (IL-1, IL-6, and granulocyte macrophagecolony stimulating factor) and chemokines (RANTES [regulated upon activation, normal T-cell expressed and secreted], IL-8, and monocyte chemotactic protein-4), each of which plays a different role in the growth, differentiation, migration, and activation of eosinophils (30–35). Studies on the effects of corticosteroids show that these can reduce the release of cytokines from airway epithelial cells in vivo and in vitro, and this property might result in an inhibition of eosinophil recruitment into nasal secretions after allergen provocation (31, 35– 37). The lack of a similar effect of steroids in the deeper compartment of the nasal submucosa might be related to lack of penetration of the steroid to the deeper layer in concentrations great enough to affect the inflammatory changes. Whether pretreatment with a more potent steroid for a longer period of time might change the rate of penetration and subsequently the efficacy of the active agent in altering the inflammatory cellular composition of the submucosal compartments needs further investigation. In summary, in a double-blind, placebo-controlled trial, we have shown that a 1-wk treatment with topical corticosteroids inhibits the symptomatic response as well as eosinophil migration into nasal secretions 24 h after allergen provocation. The influx of activated inflammatory cells into the submucosa was not altered by steroid therapy, which may reflect poor absorption of the drug into the deeper nasal compartment, an overtly aggressive antigen stimulation, or diminished potency of the steroid preparation used. References 1. Lim-Mombay, M., F. Baroody, R. Taylor, and R. Naclerio. 1992. Mucosal cellular changes after nasal antigen challenge (abstract). J. Allergy Clin. Immunol. 89:A205. 2. Durham, S. R., S. Ying, V. A. Varney, M. R. Jacobson, R. M. Sudderick, I. S. Mackay, A. B. Kay, and Q. Hamid. 1992. Cytokine messenger RNA expression for IL-3, IL-4, IL-5, and granulocyte/macrophagecolony-stimulating factor in the nasal mucosa after local allergen provocation: relationship to tissue eosinophilia. J. Immunol. 148:2390–2394. 3. Igarashi, Y., M. S. Goldrich, M. A. Kaliner, A. M. Irani, L. B. Schwartz, and M. V. White. 1995. Quantitation of inflammatory cells in the nasal mucosa of patients with allergic rhinitis and normal subjects. J. Allergy Clin. Immunol. 95:716–725. 4. Naclerio, R. M., F. M. Baroody, A. Kagey-Sobotka, and L. M. Lichtenstein. 1994. Basophils and eosinophils in allergic rhinitis. J. Allergy

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