Effect of topical corticosteroids on seasonal increases in epithelial eosinophils and mast cells in...

Effect of topical corticosteroids on seasonal increases in

epithelial eosinophils and mast cells in allergic rhinitis: a

comparison of nasal brush and biopsy methods

M. R. JACOBSON, S. JULIUSSON*, O. LOÈ WHAGEN², B. BALDER², A. B. KAY³and S. R. DURHAM

Upper Respiratory Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London, UK,

*Department of Lung Medicine, University Hospital of Iceland, Reykjavik, Iceland, ²Sahlgrenska Hospital, GoÈtheburg,

Sweden and ³Allergy and Clinical Immunology, Imperial College School of Medicine at National Heart and Lung Institute,

London, UK.

Summary

Background Nasal brushing and nasal biopsy are well-tolerated sampling techniques.

Seasonal grass pollen-induced rhinitis is characterized by epithelial mast cell in®ltration and

seasonal increases in both epithelial and sub-mucosal eosinophils.

Objective To compare the ability of the nasal brush and nasal biopsy techniques to detect

natural seasonal increases in eosinophils and mast cells, and to assess the in¯uence of

topical corticosteroid.

Methods Nasal brush samples and nasal biopsies were collected from 46 grass pollen-

sensitive seasonal rhinitis patients before the grass pollen season and at the peak of the

pollen season following 6 weeks' treatment with either ¯uticasone propionate aqueous nasal

spray (200 mg, twice daily) or placebo nasal spray.

Results Placebo patients showed seasonal increases in epithelial eosinophils both with

nasal brushing (P < 0.0001) and biopsy (P < 0.001). Epithelial mast cell numbers also

increased during the pollen season as detectable by brushing (P < 0.0001) and biopsy

(P < 0.03). Changes in cell numbers measured by nasal brushing correlated with those

observed with nasal biopsy, both for eosinophils and mast cells (P< 0.05). Sub-mucosal

eosinophils but not mast cells also increased during the pollen season (P< 0.002). Nasal

brushing and biopsy revealed that ¯uticasone treatment inhibited seasonal increases in

epithelial eosinophils (P < 0.00001) and epithelial in®ltration by mast cells (nasal brushing

P < 0.00001 and nasal biopsy P < 0.01). Fluticasone also inhibited seasonal increases in sub-

mucosal eosinophils (P < 0.001) and signi®cantly reduced nasal symptoms (P < 0.001).

Conclusion Nasal brushing harvests suf®cient in¯ammatory cells from the surface of the

nasal mucosa to be used in lieu of nasal biopsies in observation of the effect of drugs on the

nasal epithelium.

Keywords: biopsy, corticosteroids, eosinophils, mast cells, nasal brushing, seasonal

allergic rhinitis

Clinical and Experimental Allergy, Vol. 29, pp. 1347±1355. Submitted 21 August 1998;

revised 9 December 1998; accepted 30 January 1999.

Introduction

Seasonal allergic rhinitis due to grass pollen is characterized

by nasal discharge, blockage, sneezing and itching. One of

Clinical and Experimental Allergy, 1999, Volume 29, pages 1347±1355

1347q 1999 Blackwell Science Ltd

Correspondence: S. R. Durham, Upper Respiratory Medicine, Imperial

College School of Medicine at National Heart and Lung Institute,

Dovehouse Street, London SW3 6LY, UK.

the prominent features of allergic rhinitis is the in®ltration

of the nasal mucosa by in¯ammatory cells. These cells

secrete mediators and cytokines which give rise to the

symptoms and regulation of the disease. We previously

used the biopsy technique to demonstrate that seasonal

allergic rhinitis is accompanied by local tissue eosinophilia

and epithelial migration of tryptase-only (MCT) mast cells

[1]. These cellular increases are thought to be regulated by

the local production and release of cytokines such as IL-4,

IL-5 and IL-6. Immunoreactivity for these particular cyto-

kines has been localized to mast cells and in addition, to

eosinophils in the case of IL-5, in the nasal mucosa of

patients with seasonal allergic rhinitis [2]. We have shown a

seasonal increase in IL-5 mRNA expression in the nasal

mucosa of patients with seasonal allergic rhinitis [3].

Various techniques are available to sample the cellular

component of the nasal mucosa. Nasal lavage, mucosal

scraping, mucosal imprints, blown secretions, nasal

smears and nasal brushings all sample the mucosal surface

and secretions [4]. Nasal brushing is thought to harvest a

proportion of the cells within the nasal epithelial lining and

thus provide more information than nasal lavage [5]. Nasal

brushing is a simple, safe, noninvasive, well-tolerated

technique which is useful for repeated harvesting of cells

from the nasal mucosa [6] and also for studying the nasal

surface of children [7,8]. Nasal brushing yields cell samples

with well-preserved cytological detail, easy to identify on

morphological criteria [5,9]. Nasal biopsy, although more

invasive, is also a well-tolerated technique [10]. Nasal

biopsy is advantageous in that it enables studies of both

the nasal epithelium and sub-mucosa providing a broader

picture of the pathogenesis of allergic rhinitis. Nasal biopsy

and brushing have previously only been compared in an

allergen -challenge study assessing the dynamics of nasal

eosinophilia during a period of prolonged allergen exposure

[6]. Nasal mucosal scraping has been used to assess the

effect of intranasal ¯uticasone propionate [11] but this paper

is novel in its use of nasal brushings to assess the effect of

corticosteroids on the in¯ammatory cells of the nasal

epithelium.

Topical steroids are highly effective in the treatment of

allergic rhinitis [12,13]. Pretreatment with intranasal ster-

oids inhibits the seasonal in¯ux of eosinophils and mast

cells into the nasal secretions and mucosa of patients with

seasonal allergic rhinitis [2,14,15]. Fluticasone propionate

aqueous nasal spray is a topically active corticosteroid

molecule of known ef®cacy [16,17] and minimal systemic

adverse effects.

This study compares the nasal brush and nasal biopsy

techniques with regards to their ability to detect natural

seasonal changes in both eosinophils and mast cells. In

addition we tested the ability of these techniques to allow

observation of drug-induced changes.

Materials and methods

Patients

Forty-six non-smoking timothy grass pollen-sensitive

patients were recruited from the outpatient clinic of the

Asthma and Allergy Centre, Department of Medicine,

Sahlgrenska Hospital, GoÈtheburg, Sweden. They were

selected on the basis of (1) a clinical history of moderate

to severe hay fever for at least 2 years, and (2) a positive skin

prick test (weal diameter greater than 5 mm) to grass pollen

extract (Phleum pratense Soluprick; ALK, Horsholm, Den-

mark). Venous blood was taken for measurement of total

and allergen-speci®c IgE concentrations by ELISA. Patients

were excluded if they had a history of birch pollen allergy,

perennial allergy, had received immunotherapy in the last

5 years or had taken oral or topical corticosteroids in the

previous 6 months.

Study design

The study was performed with the approval of the hospital

Ethics Committee and the written informed consent of all

participants. The 46 patients were divided into two groups

(moderate and severe) on the basis of their disease severity

as determined by clinical history and their skin prick test.

`Severe' � history of symptoms on most days for at least 10

weeks during the pollen season and a skin prick test

> 10 mm; `moderate' � patients with seasonal symptoms

who did not ful®l all of the above criteria. Patients in each

group were then randomized separately to receive either

¯uticasone propionate aqueous nasal spray 200 mg twice

daily (Flixonase, Glaxo, Sweden) or matched placebo spray

(containing the aqueous diluent) to ensure an even distribu-

tion of disease severity in the active and placebo groups.

Baseline nasal brushing followed by nasal biopsy of the

opposite nostril was performed out of season at a time when

all patients were asymptomatic. There was a two week run-

in period during which patients recorded symptoms on a

diary card. Repeat nasal sampling took place 6 weeks after

treatment with ¯uticasone or matched placebo spray timed

to correspond with the week following the peak pollen

count. The side of biopsy/brush was reversed relative to

the baseline samples.

Diary cards

Patients were provided with 2-week diary cards throughout

the study period and were asked to keep a daily record of all

their symptoms of rhinitis namely: nasal blockage; sneez-

ing; rhinorrhoea (nasal discharge) and nasal itching. Symp-

toms were individually assessed using a four-point rating

scale from 0 (no symptoms) to 4 (breathing through nose

very dif®cult, more than 10 episodes a day of sneezing,

1348 M. R. Jacobson et al.

q 1999 Blackwell Science Ltd, Clinical and Experimental Allergy, 29, 1347±1355

persistent distraction of discharge and itching). The diary

card symptom scores were summarized by the percentage of

symptom-free days for each symptom separately.

Nasal brushing

A commercially available 5.5 mm diameter nylon brush

(Doft AB, OÈ sthammar, Sweden) was used for cell sampling.

The brush was placed between the nasal septum and the

inferior turbinate and removed gently whilst being rotated

90 8 [5]. No anaesthesia was used. After sampling the brush

was placed immediately in a 3-mL plastic tube containing

2 mL phosphate-buffered saline. The brush was then shaken

vigorously in the solution and carefully brushed off against

the side of the wall. Aliquots (100 mL) of the cell suspension

were loaded into a cytocentrifuge (Cytocentrifuge II, Shan-

don Southern Products Ltd, Cheshire, UK), spun for 7 min at

750 r.p.m., and air dried prior to storing at ÿ 80 8C wrapped

in foil.

Nasal biopsy

Local anaesthesia of the inferior nasal turbinate was

achieved using 3% cocaine and 0.025% adrenaline. After

10 minutes a 2.5-mm biopsy was taken using Gerritsma

forceps [10]. Biopsies were snap frozen and stored at

ÿ 80 8C for immunohistology.

Immunohistology

Immunohistology was performed on 6-mm cryostat sections

(®xed for 7 min in 60 : 40; acetone : methanol) using the

alkaline phosphatase-anti-alkaline phosphatase method as

described previously [1]. Anti-major basic protein (MBP)

antibody, BMK13 (IgG1) was kindly provided by Miss J.

Barkans and Dr R. Moqbel to quantify the total number of

eosinophils in samples. Monoclonal antibody recognizing

mast cell tryptase (IgG1) labelled with alkaline phosphatase

was obtained from Chemicon International, London, UK.

Cytospin slides were brought to room temperature then

®xed in methanol (5 min) prior to incubation with BMK13

or were ®xed in Carnoys ¯uid (60% ethanol: 30% chloro-

form: 10% glacial acetic acid) for 15 min prior to incubation

with antitryptase. Human allergen-challenged nasal biopsies

were used as positive controls and mouse IgG1 (anti-

Aspergillus niger glucose oxidase) was used as a negative

control (Dako Ltd, High Wycombe, UK).

Quanti®cation

Slides were counted blind using an Olympus BH2 micro-

scope (Olympus Optical company Ltd, Tokyo, Japan) with

an eye piece grid (0.202 mm2 at 200 ´ magni®cation). At

least two tissue sections (giving a minimum of six micro-

scope ®elds) were counted per patient. For epithelial counts

the cross-sectional area of the nasal epithelium was quanti-

®ed by use of a computer-assisted graphics tablet. Counts

were expressed per square millimetre of epithelium. Sub-

mucosal counts were made one grid depth (0.45 mm)

beneath the basement membrane and were expressed as

mean counts per square millimetre of sub-mucosa. Cytospin

eosinophil counts were made in the four ®elds of view

labelled `1' in Fig. 1. A total of 300 cells were counted at

each position. If there were less than 300 cells in ®eld 1 then

counts continued in ®elds 2 and 3 as necessary. The number

of eosinophils within this 300 was recorded. The results for

the four areas were summed and the number of eosinophils

was recorded as a percentage of the total cell count.

Cytospin mast cell counts were made by counting all the

mast cells within the cytospin. The total cell count for the

entire cytospin was then estimated by counting the number

of cells in the four ®elds labelled `1' (Fig. 1) and multi-

plying up. Mast cells were expressed as a percentage of total

cells.

Statistical analysis

Wilcoxon matched-pairs signed rank test was used for

within-group comparisons (peak season counts minus pre-

season counts). Between group comparisons were made

using the Mann Whitney U-test. Correlations were per-

formed using Spearman's rank method. P-values less than

Topical corticosteroid use in rhinitis 1349


3

1 1

332

1 2

2 1

3 2

Fig. 1. Diagrammatic representation of cytospin demonstrating the

position of microscope ®elds of view at ´ 20 magni®cation.

0.05 were considered statistically signi®cant. Statistical

tests were performed using a standard computer package

(MiniTab Inc., PA, USA).

Results

All 46 patients completed the study and underwent two

nasal biopsies and two nasal brushings. The ¯uticasone

group (n� 23) and placebo group (n� 23) were well

matched for age, gender, disease severity and for their

degree of grass pollen sensitivity as determined by skin

prick tests and allergen-speci®c IgE antibody concentrations

(Table 1). With the exception of two patients all patients had

suffered from seasonal rhinitis for at least 5 years.

Clinical ef®cacy

The placebo-treated group showed a marked increase in

nasal symptoms during the pollen season. Six weeks' treat-

ment with ¯uticasone during the grass pollen season

resulted in a signi®cant reduction in all symptoms as

demonstrated by signi®cant increases in symptom-free

days. Signi®cantly more symptom-free days for sneezing

were observed with ¯uticasone than with placebo (median

82%, interquartile range 38±95% vs median 17%, inter-

quartile range 15±27%, P< 0.001) (Fig. 2). This marked

increase in symptom-free days was also observed for the

symptoms of nasal discharge 95% (83±100) after ¯utica-

sone treatment and 35% (20±45) following placebo

(P < 0.001), nasal blockage 97% (35±100) with ¯uticasone

and 25% (0±40) with placebo (P < 0.001) and nasal itching

94% (47±100) with steroids vs 35% (25±47) with placebo

(P < 0.001).



Table 1. Clinical details of study patients

Treatment

Placebo Fluticasone

Number of patients 23 23

Male : female ratio 16 : 7 14 : 9

Age (years; mean 6 SD) 33.1 6 10.7 32 6 8.9

RAST* (Timothy grass; score 0±5) 3.2 6 0.8 2.9 6 0.8

(mean 6 SD)

RAST, radioallergosorbant test

100

90

80

70

60

50

40

30

20

10

0

Per

cen

tag

e o

f sy

mp

ton

-fre

e d

ays

Sneezing

P < 0.001

FP PI

Discharge

P < 0.001

FP PI

Blockage

P < 0.001

FP PI

Itching

P < 0.001

FP PI

Fig. 2. Effect of 6 weeks' treatment with ¯uticasone propionate (closed bars, FP) or placebo (open bars, Pl) on the percentage of symptom-

free days for symptoms of rhinitis during natural exposure to grass pollen. Results expressed as median and interquartile ranges. P-values

refer to differences between groups (Mann Whitney U-test).

Immunohistology of nasal brushing samples and biopsy

epithelium

The total cell counts for cytospins prepared from the nasal

brushings were of a similar magnitude pre- and peak pollen

season regardless of treatment. The majority of cells present

were epithelial cells. BMK13-positive eosinophils were

absent from all brushings taken outside the pollen season

(Fig. 3a); this result mirrored the ®ndings in the nasal

epithelium of biopsies (Fig. 3b). During the pollen season

the placebo group showed a signi®cant increase in eosino-

phil numbers both within nasal brush samples (median cell

count from 0 to 2.34%, Wilcoxon test P< 0.0001) and the

nasal epithelium of biopsies (increase in median cell count

from 0 to 21.7 cells/mm2, P < 0.001). In marked contrast this

seasonal increase was abolished by ¯uticasone treatment

whether nasal brushings or nasal biopsies were observed

(between group comparison P< 0.00001 in both cases).

Tryptase-positive mast cells were absent from all bar two

of the nasal brush and nasal epithelial biopsies prepollen

season. At the peak of the pollen season there was a small

but signi®cant increase in mast cell numbers observed in the

nasal brushings of the placebo group (median preseason 0,

median peak season 0.013%, P< 0.0001) which was not

observed in the ¯uticasone group (median mast cell count

zero throughout). The difference between the two groups

was highly signi®cant (P< 0.0001) (Fig. 4a). Analysis of the

epithelium of the biopsies from placebo patients revealed a

similar seasonal increase in mast cells (median cell count

increasing from 0 to 13 cells/mm2, P < 0.03), and likewise

abolition of this increase by treatment with ¯uticasone (Fig.

4b). The difference between the two groups was signi®cant,

P < 0.01.

Correlations between nasal brush cell counts and nasal

epithelial biopsy cell counts

Signi®cant associations were observed between the change

in eosinophil numbers pre- to peak season in nasal brush

samples and the change in eosinophil numbers pre- to peak

season in the nasal biopsy epithelium (r� 0.444, P < 0.05).

A similar correlation was observed with changes in mast

cell numbers (r� 0.5, P < 0.05) (Fig. 5).

Immunohistology of nasal sub-mucosal tissue

Before the pollen season low numbers of eosinophils were

present in the nasal sub-mucosa of both the placebo and

¯uticasone group (2.5 and 1.5 cells/mm2 respectively) (Fig.

6). In the placebo group the number of BMK13� eosinophils

signi®cantly increased during the pollen season to 20 cells/

mm2, P< 0.002. In contrast, the number of sub-mucosal

eosinophils did not increase in the ¯uticasone group. The

seasonal increase in BMK13 eosinophils was signi®cantly

inhibited compared with the placebo group (P< 0.001).

Tryptase-positive mast cells were constitutively present

in the nasal sub-mucosa of both study groups out of the

pollen season. In the placebo-treated group, the number of



10

8

6

4

2

0

% B

MK

13-

po

siti

ve c

ells

/cyt

osp

in

(a)

(b)

BMK13Placebo Fluticasone

P < 0.00001

(14.7)

100

120

140

80

60

40

20

0

% B

MK

13-p

osi

tive

cel

ls/m

m2

epit

hel

ium

P < 0.00001

(206.3)

Pre Peak Pre PeakFig. 3. BMK13-positive eosinophils in (a) nasal brushings and (b)

nasal epithelium of grass pollen-sensitive rhinitics prepollen season

and peak pollen season following 6 weeks' treatment with ¯utica-

sone propionate (closed circles) or placebo (open circles). Median

values are represented by the horizontal bars. Median differences

were compared using the Mann Whitney U-test.

sub-mucosal mast cells did not change during the pollen

season. Furthermore, in contrast with the epithelial ®ndings,

¯uticasone treatment had no effect on sub-mucosal mast cell

numbers.

Discussion

We have shown that nasal brushing and nasal biopsy both

detect an increase in epithelial eosinophils and mast cells

during seasonal grass pollen-induced rhinitis. Nasal brush-

ing and nasal biopsy both revealed total inhibition of the

seasonal increases in epithelial eosinophils and mast cells

following ¯uticasone therapy. Seasonal sub-mucosal eosi-

nophilia was observed in the nasal biopsies and this was also

inhibited by ¯uticasone treatment whereas sub-mucosal

mast cells were unaffected by treatment. This anti-in¯am-

matory effect of ¯uticasone propionate was paralleled by

signi®cant reductions in nasal symptoms.

Our study has shown that nasal brushing is a useful

alternative to nasal biopsy for sampling the nasal epithe-

lium. Eosinophil and mast cell numbers within the brush-

ings were low but compared favourably with those obtained

by other groups [6,18]. The counting technique used

ensured unbiased counts in which small but highly signi®-

cant changes in cell numbers could be detected. Within the

placebo group the change in eosinophil numbers (peak

season minus preseason) measured using cell counts from



0.075

0.05

0.025

0

%T

ryp

tase

po

siti

ve c

ells

/cyt

osp

in

(a)

(b)

Mast cellsPlacebo Fluticasone

P < 0.00001

(0.34)

(0.16)

100

120

140

80

60

40

20

0

Try

pta

se p

osi

tive

cel

ls/m

m2

epit

hel

ium

P < 0.01

(171.6)

Pre Peak Pre PeakFig. 4. Tryptase-positive mast cells in (a) nasal brushings and (b)

nasal epithelium of grass pollen-sensitive rhinitics prepollen

season and peak pollen season following 6 weeks' treatment with

¯uticasone propionate (closed circles) or placebo (open circles).

Median values are represented by the horizontal bars. Median

differences were compared using the Mann Whitney U-test.

220200

120100

80604020

0

∆ B

MK

13 c

ells

/m

m2

epit

hel

ium

∆ % BMK 13 cells/cytospin

(a)

10 150 5

r = 0.444P < 0.05n = 21

200150100

500

−50−100

∆ M

ast

cell

s/m

m2

epit

hel

ium

∆ % Mast cells/cytospin

(b)

0.25 0.30 0.350 0.05 0.10 0.200.15

r = 0.5P < 0.05n = 19(4)

Fig. 5. Comparison of changes in the number of (a) BMK13-

positive eosinophils and (b) tryptase-positive mast cells recorded

in nasal brushings and the nasal epithelium (peak season cell count

minus preseason cell count). Spearman's rank correlation was

performed.

brush specimens correlated with those obtained from biopsy

sections as did changes in mast cell numbers. Both tech-

niques demonstrated clearly the effectiveness of ¯uticasone

propionate in inhibiting epithelial in®ltration of eosinophils

and mast cells. Nasal brushing is a simple technique which

does not require anaesthetic, it has the advantage of being

more economical than nasal biopsy both in terms of time

and money. Nasal brush specimens require less processing

prior to staining than biopsies but do take longer to assess

under the microscope. Nasal brushing like nasal scraping

(another minimally invasive nasal epithelial sampling tech-

nique) would be particularly well-suited to studies looking

at the effect of treatment on in¯ammatory cells within the

epithelium or on epithelial cells themselves especially

studies involving large numbers of patients or studies

involving children.

The in®ltration of eosinophils into the nasal mucosa

following natural or laboratory allergen exposure is well

known [1,19,20]. Products from eosinophils such as leuco-

triene C4, platelet-activating factor and eosinophil granule

proteins may account for the prolonged nasal congestion

and mucus secretion observed during natural seasonal

exposure to pollen. Fluticasone propionate has been

shown to inhibit production of chemokines [21] and cyto-

kines [2,22] which may contribute to the inhibition of

eosinophil in®ltration and as a result the improvement of

symptoms observed in this study. The effect of glucocorti-

costeroids on eosinophilia in the nasal epithelium and sub-

mucosa is varied. In an allergen challenge study we have

shown that 6 weeks' pretreatment with ¯uticasone pro-

pionate results in a signi®cant reduction in activated

(EG2�) eosinophils in both the epithelium and the sub-

mucosa whereas total (BMK13) eosinophils were only

reduced in the epithelium [23]. This data supported the

®ndings of Lozewicz and colleagues [24] who investigated

the effect of a 2-week pretreatment with ¯uticasone pro-

pionate on eosinophils in the sub-mucosa of nasal biopsies

24 h postallergen challenge. Their data showed a reduction

in the number of EG2� eosinophils but no change in total

(EG1�) numbers. In contrast, 24 h postallergen challenge

Baroody et al. [25] found that although total eosinophils

(Wright stain) in nasal scrapings were reduced, neither total

(Giemsa stain) nor EG2� eosinophils in the nasal sub-

mucosa were affected following 1 week's treatment with



Fig. 6. Effect of ¯uticasone propionate on the number of BMK13-positive eosinophils and tryptase-positive mast cells. Data are shown for

preseason and peak season following 6 weeks' treatment with ¯uticasone propionate (closed circles) and placebo (open circles). Horizontal

bars represent median values. The Mann Whitney U-test was used to compare median differences.

beclomethasone propionate. In a natural allergen exposure

study Bradding et al. [2] found that 6 weeks' treatment with

once-daily ¯uticasone propionate resulted in inhibition of

seasonal increases in activated (EG2�) eosinophils. We

con®rmed this ®nding following 6 weeks' treatment with

twice-daily ¯uticasone propionate [3]. In this current study

we observed dramatic inhibition of both epithelial and sub-

mucosal BMK13 eosinophils demonstrating that intranasal

steroids can be highly effective in in¯ammatory cell reduc-

tion both in the super®cial and deeper compartments of the

nasal mucosa.

Mast cells are found constitutively in the nasal sub-

mucosa but are not generally found in the nasal epithelium

outside of the pollen season [1,2,23,26,27]. The seasonal

increase in epithelial mast cells is well documented

[1,2,26,27]. These cells are known to be in an activated

state as shown by degranulation evident by electron micro-

scopy [28] and increased levels of the mast cell mediators

histamine and tryptase in nasal lavage ¯uid [29]. Mast cell

degranulation results in histamine, tryptase, prostaglandin

(PG)D2, PGF2 and bradykinin release with the subsequent

induction of nasal symptoms of sneezing, nasal discharge

and transient nasal blockage. Mast cell degranulation con-

tributes to the eosinophilic mucosal in¯ammation seen in

rhinitis since mast cells contain preformed pro-eosinophilic

cytokines and mRNA for IL-4, IL-5, IL-6 and TNF-a [2,30].

As seen in our current and previous studies [1,26] sub-

mucosal mast cell numbers do not change during the pollen

season. The epithelial increase in mast cells is thought to

result from redistribution of mast cells [26,31]. Mast cell

proliferation may also contribute to the number of mast cells

in the nasal epithelium and subepithelial layer of allergic

patients [32]. Since glucocorticosteroids do not prevent

mast cell degranulation [33,34] the improvement in symp-

toms observed in this study must result in part from the

inhibition of mast cell occupation of the nasal epithelium. In

vitro and in vivo evidence suggests that glucocorticoids

decrease tissue mast cell numbers by downregulating

tissue stem cell factor production which is required for the

survival of local mast cells [35,36].

Seasonal grass pollen-induced rhinitis is characterized by

epithelial in®ltration of eosinophils and mast cells, and since

topical corticosteroids have a marked effect on the in¯am-

matory cells of the nasal epithelium nasal brushing is an

appropriate cell-harvesting method to use in studies looking

at treatment effects. The nasal biopsy technique allows more

detailed studies of the nasal mucosa.

Acknowledgements

This study was supported by grants from the Medical

Research Council, UK and ®nancial assistance from Glaxo

Wellcome, UK.

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