Steroid-sensitive indices of airwayinflammation in children with seasonalallergic rhinitis

As an inflammatory disease of the nasal mucosa,allergic rhinitis is characterized by plasma exu-dation (1–3). Because this mechanism involvestissue distribution and airway luminal entry ofadhesive, leukocyte-activating, growth- factoractive, and other biologically active proteins,pharmacological inhibition of plasma exudationis a potentially important action (4). However, itis only in adults that drug (glucocorticosteroid)treatment has been demonstrated to inhibitplasma exudation (5, 6). In adult seasonal allergicrhinitis, it has further been demonstrated that thenasal mucosa exhibits abnormally great exudative

responses to histamine challenges (7, 8). Whe-ther the exudative hyperresponsiveness is alsoexpressed in the nasal mucosa of allergic childrenand whether it is at all affected by glucocorticos-teroid treatment are not known.The eosinophil granulocyte, a potential effec-

tor cell in allergic airway diseases (9), may be ofspecial importance in allergic rhinitis as in thisdisease the nasal mucosa is particularly rich inhighly degranulated eosinophils (10). Glucocort-icosteroid treatment inhibits the eosinophilia ofallergic rhinitis in adults (11). This action maylargely reflect reduced recruitment of eosinophils

Meyer P, Andersson M, Persson CGA, Greiff L. Steroid-sensitiveindices of airway inflammation in children with seasonal allergicrhinitis.Pediatr Allergy Immunol 2003: 14: 60–65.� 2003 BlackwellMunksgaard

Previous studies involving adults have demonstrated that airwayglucocorticosteroids inhibit plasma exudation and eosinophil activity inallergic rhinitis. This study explores the possibility that plasma exuda-tion, exudative responsiveness, and the occurrence of eosinophil activ-ity-related proteins are glucocorticosteroid-sensitive nasal mucosalindices in allergic children. Using a placebo-controlled, parallel-groupdesign effects of nasal budesonide (64 lg per nasal cavity b.i.d) weredetermined in children with seasonal allergic rhinitis. Nasal lavage fluidlevels of eotaxin, eosinophil cationic protein (ECP), and a2-macro-globulin, indicating plasma exudation, were determined, the latter withand without challenge with topical histamine. Nasal lavage fluid levelsof a2-macroglobulin and ECP increased significantly during the pollenseason, and the acute plasma exudation response to histamine wassignificantly greater during than outside the season. There was a trendtowards a seasonal increase in nasal lavage fluid levels of eotaxin.Budesonide significantly inhibited the seasonal increase in a2-macro-globulin as well as the exudative hyperresponsiveness to histamine. Anytendency of increases in mucosal output of eotaxin and ECP wasabolished by the glucocorticosteroid treatment. We conclude thatmucosal exudation of plasma, as a global sign of active inflammatoryprocesses, is a glucocorticosteroid-sensitive facet of allergic rhinitis inchildren. Exudative hyperresponsiveness, potentially caused by severalweeks of mucosal inflammation, emerges as a significant feature ofallergic rhinitis in children, and its development is prevented by localtreatment with a glucocorticosteroid drug. The seasonal increase in ECPand the trend for an increase in eotaxin were absent in the glucocorti-costeroid-treated subjects.

Peter Meyer1, Morgan Andersson2,Carl G. A. Persson3 and Lennart Greiff2

Departments of 1Pediatrics, 2Otorhinolaryngology,Head & Neck Surgery, and 3Clinical Pharmacology,University Hospital, Lund, Sweden

Key words: airway; inflammation; pediatric; plasmaexudation; rhinitis

Lennart Greiff, Department of Otorhinolaryngology,Head & Neck Surgery, University Hospital, SE-221 85Lund, SwedenTel.: +46 46 171705Fax: +46 46 2110968E-mail: lennart.greiff@skane.se;lennartgreiff@hotmail.com

Accepted 4 May 2002

Pediatr Allergy Immunol 2003: 14: 60–65

Printed in UK. All rights reserved

Copyright � 2003 Blackwell Munksgaard

PEDIATRIC ALLERGY AND

IMMUNOLOGYISSN 0905-6157

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to the nasal mucosa as elimination of the nasaltissue eosinophils, by either apoptosis or luminalentry, may not be affected by glucocorticoster-oids in vivo (12–14). A few previous reports havesuggested that glucocorticosteroids are anti-eosinophilic agents in children suffering fromallergic rhinitis (15), but this possibility has notbeen extensively studied.This study examines children with seasonal

allergic rhinitis during as well as outside theiractive disease period. Inflammatory indicesappearing on the nasal mucosal surface areexamined by use of an efficient sampling tech-nique that can be handled by the childrenthemselves (16). Specifically, the present studyexamines the possibility that nasal mucosaloutputs of plasma (a2-macroglobulin), an eosin-ophil chemoattractant (eotaxin), and an eosino-phil granule protein (eosinophil cationic protein;ECP) are glucocorticosteroid-sensitive indices inthe allergic child. In addition, the possibility thatseasonal hyperresponsiveness develops and thatit is a glucocorticosteroid-sensitive disease vari-able is also explored in these patients.

MethodsStudy design

Children with allergic rhinitis, receiving topicalglucocorticosteroid treatment or placebo, wereexamined during a birch pollen season. Nasallavages with saline were carried out once beforeand at two occasions during the season. Inaddition, nasal challenges and lavages with hista-mine were carried out once during and once afterthe pollen season. This particular design waschosen to avoid any effects of a histamine challengecarried out close to the pollen season on allergen-induced nasal mucosal outputs of eotaxin, ECP,and a2-macroglobulin. The levels of eotaxin andECP were determined in the saline lavages. Fur-thermore, the levels of a2-macroglobulin weredetermined in nasal lavage fluids obtained aftersaline as well as the histamine exposure.

Treatment

Topical glucocorticosteroid treatment (budeso-nide aqueous nasal spray, 64 lg per nasal cavityb.i.d) was given in a double-blind, placebo-controlled, randomized, and parallel groupdesign. Nine patients received budesonide andnine placebo. The treatment started before theexpected start of the pollen season and continuedthroughout the part of the study that wascarried out during the pollen season. No other

drugs were allowed except rescue medication:Loratadine tablets (Clarityn�, Schering-Plough,Brussels, Belgium) and cromoglycate eyedrops(Lomudal�, Aventis Pharma, Cheshire, England)in clinical doses. The patients were instructed touse rescue medication if more than moderatesymptoms occurred.

Patients

Eighteen children (10 boys and 8 girls, 7–13 yearsold, mean age 10.6 years) participated in thestudy. The children had a history of birch pollenallergic rhinitis, which was verified by a positiveskin-prick test. The children had no history ofperennial nasal or bronchial disease, no history ofrecent respiratory tract infection, and nohistory ofrecent drug treatment. The study was approved bythe local ethics committee and informed consentwas obtained from the patients and their parents.

Symptom scores

The children scored nasal symptoms, i.e. rhinor-rhea, blockage, and sneezes, as well as eyesymptoms in a diary once daily during the pol-len season. Score 0: no, 1: mild, 2: moderate, and3: severe symptoms.

Nasal pool challenge and lavage technique

The nasal pool-device was used for saline lavagesand for concomitant histamine challenge andlavage of the nasal mucosa (16). The nasal pooldevice is a compressible plastic container equippedwith a nasal adapter. The adapter is inserted into anostril and the sitting patient, leaning forward in a60�flexedneckposition, compresses the container.The nasal pool-fluid is then instilled in one of thenasal cavities and maintained in contact with alarge and reproducible area of themucosal surfacefor adeterminedperiodof time.When thepressureon the device is released the fluid returns into thecontainer. In the present study, the volume of thenasal pool-fluid was 12 ml. The right nasal cavitywas used for all lavages.

Isotonic saline and histamine lavages of the nasal mucosa

Nasal lavages with isotonic saline, each with aduration of 2 min, were carried out once beforeand at two occasions during the pollen season.Immediately after the second seasonal salinelavage, an additional 5-min saline lavagefollowed by a 5-min histamine (100 lg/ml)combined challenge and lavage was carried out.These three lavages were carried out with 5-min

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intervals. A 2-min saline lavage followed by a5-min saline lavage and a 5-min combined hista-mine (100 lg/ml) challenge and lavage was alsocarried several months after the pollen season hadended (December). These three lavages werecarried out at 5-min intervals.The lavages were carried out on the same days

for all patients and at about the same time pointof the day. The recovered lavage fluid wascentrifuged (G ¼ 105 g, 10 min, 4�C) and aliqu-ots were prepared from the supernatants andfrozen ()20�C) for later analysis of eotaxin, ECP,and a2-macroglobulin.

Analysis of eotaxin, ECP, and a2-macroglobulin

The nasal lavage fluid levels of eotaxin weremeasured using a commercially available radio-immunoassay (Pharmacia-Diagnostics, Uppsala,Sweden). The detection level of the eotaxinassay was <5 pg/ml. ECP were measured byfluoroimmunoassay (Pharmacia-Diagnostics).The detection level of the ECP assay was<2 ng/ml. a2-macroglobulin were determinedusing a radioimmunoassay sensitive to 7.8 ng/ml.Rabbit anti-human a2-macroglobulin (Dakopatts,Copenhagen, Denmark) was used as antiserumand human serum (Behringwerke Diagnostica,Marburg, Germany) as standard. Human

a2-macroglobulin (Cappel-Organon Teknika,Turnhout, Belgium) was iodinated using thelactoperoxidase method (17). Tracer and stand-ard (or sample) was mixed with antiserum beforeadding goat anti-rabbit antiserum (AstraZeneca,Lund, Sweden). The bound fraction was meas-ured using a gamma counter. The intra- andinter-assay coefficients of variation were between3.8–6.0% and 3.1–7.2%, respectively.

Statistics

Differences in lavage fluid levels of eotaxin, ECP,and a2-macroglobulin were examined usingFriedman’s test. If statistical significance’s emer-ged, further analyses were performed usingWilcoxon’s signed rank test. Differences in nasalsymptoms within each treatment group weresimilarly examined. Differences between budeso-nide and placebo treatment were examined usingthe Mann–Whitney U-test. p-values of less than0.05 were considered significant. Data are pre-sented as mean ± SEM.

Results

The regional birch pollen counts demonstrated amild pollen season. Accordingly, there were onlysmall differences in symptom scores (Fig. 1): In

Fig. 1. Nasal symptom scores(mean ± SEM) during thestudy period. The scoresdemonstrated mild symptoms.Yet, in patients receiving place-bo, the nasal symptom scoreswere significantly increased onstudy days 3–6, 9, 11, 15, 21, 29and 32–35 (p-values<0.05).

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patients receiving placebo, nasal symptom scoresincreased very mildly but significantly on 13 ofthe study days (p-values<0.05, cf. preseasonlevels). In patients receiving budesonide, nasalsymptom scores increased significantly on 11 ofthe study days (p-values<0.05, cf. preseasonlevels). Focusing on the cumulative seasonalsymptoms, budesonide failed to reduce symp-toms of allergic rhinitis. Loratadine tablets andcromoglycate eyedrops were used very infre-quently during the study period.In patients receiving placebo, nasal lavage fluid

levels of eotaxin increased during the pollenseason, but this increase failed to reach statisticalsignificance (Fig. 2). At the observations duringthe pollen season, the levels of eotaxin were lowerin patients receiving budesonide (cf. placebo), butthis effect failed to reach statistical significance.In patients receiving placebo, nasal lavage fluid

levels of ECP increased during the pollen season,and this increase reached statistical significanceat the first as well as the second seasonal obser-vation (p<0.05, cf. before the pollen season)(Fig. 3). This observation was not seen inpatients receiving budesonide. At the observa-tions during the pollen season, the levels of ECPwere lower in patients receiving budesonide (cf.placebo), but this effect failed to reach statisticalsignificance.In patients receiving placebo, nasal lavage fluid

levels of a2-macroglobulin increased during thepollen season, and this increase reached statisti-cal significance at the first (p<0.01) as well asthe second (p<0.05) seasonal observation cf.

before the pollen season) (Fig. 4). This effect wasnot seen in patients receiving budesonide. Atboth points of observation during the pollenseason, the levels of a2-macroglobulin wereattenuated in patients receiving budesonide(p<0.05, cf. placebo).Histamine produced significant plasma exu-

dation, i.e. increased nasal lavage fluid levels of a2-macroglobulin (p<0.001) (Fig. 5). In patientsreceiving placebo, the exudative responsiveness tohistamine was significantly increased during thepollen season (p<0.05, cf. histamine challenges

Fig. 2. Levels of eotaxin in nasal saline lavages obtainedbefore (April) and at two occasions (May 10 and 20) duringthe birch pollen season. In patients receiving placebo,eotaxin levels tended to be increased by the seasonal allergenexposure, but this effect failed to reach statistical signifi-cance. Budesonide prevented the seasonal increase ineotaxin levels, but again this effect failed to reach statisticalsignificance.

Fig. 3. Levels of ECP in nasal saline lavages obtained be-fore (April) and at two occasions (May 10 and 20) duringthe birch pollen season. In patients receiving placebo, ECPlevels were significantly increased at seasonal allergenexposure (significance levels are given elsewhere). Budeso-nide reduced the seasonally increased ECP levels, but thiseffect failed to reach statistical significance.

Fig. 4. Levels of a2-macroglobulin in nasal saline lavagesobtained before (April) and at two occasions (May 10 and20) during the birch pollen season. In patients receiv-ing placebo, a2-macroglobulin levels were significantlyincreased by the seasonal allergen exposure (significancelevels are given elsewhere). Budesonide attenuated the sea-sonal increase in a2-macroglobulin levels (*p<0.05).

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carried out well after the pollen season). Budeso-nide attenuated the seasonal exudative hyperre-sponsiveness (p<0.05, cf. placebo).

Discussion

This study demonstrates that treatment with anasal glucocorticosteroid inhibits plasma exuda-tion and exudative hyperresponsiveness, and mayreduce eosinophil activity, in children who sufferfrom seasonal allergic rhinitis. These data indi-cate that allergic rhinitis, irrespective of age,is characterized by glucocorticosteroid-sensitiveinflammatory processes.The nasal pool-device, as confirmed in this

study, is readily handled by 7 year olds. We havepreviously demonstrated that children using thisdevice regularly manage to recover over 85% ofthe lavage fluids (16), which is far better thanobtained with other methods of nasal lavageemployed in children (16). Because only 18children were recruited and a parallel-groupdesign was used the power of the present studywas low. Yet, the present methodology may havecontributed to data consistency and, hence, to thedetection of differences between small groups ofpatients in this study. We could thus demonstratestatistically significant increases in nasal lavagefluid levels of ECP and a2-macroglobulin as wellas development of exudative hyperresponsivenessduring the season. Furthermore, significant inhi-bition of the exudative indices was produced byglucocorticosteroid treatment in this study. It is

also possible that this treatment, in agreementwith previous observations (11, 18, 19), wouldhave reduced nasal lavage fluid levels of ECP andeotaxin more clearly than in this study had agreater number of patients been included.Given the low power and the mild seasonal

allergen exposure, the exudative and anti-exuda-tive effects that turned out significant in thisstudy may be considered quite characteristicfeatures of allergic rhinitis in children. In chal-lenge experiments involving adults as well aschildren, we have previously demonstrated thatluminal entry of plasma extends to thresholdinflammatory stimulation, that it occurs inairways with a maintained epithelial integrity,that it is largely a non-sieved process and, hence,that lavage fluid levels of the large plasmaprotein a2-macroglobulin is a useful index ofthis response (20). Plasma exudation may there-fore be viewed as a global measure of the overallinflammatory process, especially reflecting theextent to which the airway mucosal tissue itself isaffected by the inflammation (20). The presentdata indicate that the exudative response is welldeveloped in children. This feature is importantin creating a bioreactive proteinaceous milieu forin vivo inflammatory processes. For example, as aconsequence of its non-sieved nature, the kininand coagulation systems (20) as well as thecomplement proteins will also be exuded (21, 22).As shown by the saline lavage levels in this

study, and as corroborated by the present effic-acy of glucocorticosteroids, the exudativeresponse provides a means of monitoring diseaseintensity and treatment effects in the allergicchild. Because luminal entry of plasma, incontrast to luminal entry of cells, directly reflectsthe magnitude of subepithelial events, plasmaexudation indices may provide more usefulquantitative data on disease activity than thestudy of inflammatory cells (20). Interestingly,Benson et al. (23), in a study involving 60schoolchildren with allergic rhinitis, could dem-onstrate glucocorticosteroid-sensitive increases inboth eosinophil numbers and in interleukin-5levels during seasonal allergic rhinitis. While theeosinophil count seems well established as a grossindex of allergic airway disease, the interleukin-5levels may too frequently be below detectionlimit to be of regular use (23). Thus, if the presentpromising findings regarding consistency ofplasma exudation are confirmed in larger groupsof children with allergic rhinitis, a2-macroglobu-lin may become of great importance as an indexfor monitoring of this disease. In addition, thepresent study demonstrated that the exudativeresponse to histamine challenge was greater

Fig. 5. Levels of a2-macroglobulin in saline and histaminelavages obtained during (May 20) and well after (December)the birch pollen season. In patients receiving placebo, asignificant exudative hyperresponsiveness developed duringthe pollen season (significance levels are given elsewhere).Budesonide attenuated the seasonal exudative hyperre-sponsiveness (*p<0.05).

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during than outside the pollen season. Hence,during several weeks of allergic inflammationthere is development of an exudative hyperre-sponsiveness in children similar to what has beenobserved in adults (7). Moreover, the presenthyperresponsiveness was inhibited by glucocort-icosteroid treatment. Further studies are warran-ted to examine whether this latter originalobservation in allergic children carries over toadult patients with seasonal allergic rhinitis.In conclusion, the present data indicate that

mucosal exudation of plasma and the exudativehyperresponsiveness are both characteristic featuresand glucocorticosteroid-sensitive indices of seasonalallergic rhinitis in children. Inhibition of plasmaexudation and reducing hyperresponsiveness likelyreflect clinically important anti-inflammatory effic-acy of drug treatment. Hence, monitoring ofexudative properties of the nasal mucosa is poten-tially of value for assessment of treatment effects inchildren suffering from allergic rhinitis.

AcknowledgmentsThe present study is supported by the Swedish ResearchCouncil, the Vardal Foundation, the Swedish Associationagainst Asthma and Allergy, the Medical Faculty of LundUniversity, the Konsul Th. C. Berghs Foundation, andAstraZeneca. We thank Lena Glanz-Larsson, Eva Anders-son, and Berit Holmskov for expert laboratory assistance.

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