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Factors in clinical expression of allergic airways disease

Lopuhaä, C.E.

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Citation for published version (APA):Lopuhaä, C. E. (2003). Factors in clinical expression of allergic airways disease.

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Download date: 30 Apr 2020

CHAPTERR 6

Comparisonn of allergen-induced late inflammatory reactions in the nosee and in the skin in house dust mite allergic patients with or

withoutt asthma

;lChristaa K. Lopuhaa, cMariclle J. dc Ricmcr, l 'Theo A. Out, ' 'Deman H.S. Sjamsoedinf, dRobb C. Aalberse, ; lHenk M. ]ansen and'Jaring S. van der Zee

DepartmentsDepartments ofPalmonology, 'Otorhinoiary neology and' Iixperimental Immunology, /IcademicalMedical Cente,

Amsterdam,Amsterdam, CLB Sanqidn Blood Supply foundation, Amsterdam, 1'be Xefbtriands

submitted submitted

Summary y

Background:Background: It remains to be established which factors contribute to the occurrence of asthma in allergicc individuals. We hypothesised that differences in the late allergic inflammatory reaction to allergenn between asthmatic and non-asthmatic house dust mite allergic individuals might contributee to the difference in clinical presentation of allergy. AimAim of this study: To compare allergen induced changes in parameters for cellular inflammation duringg the phase of the late allergic reaction in the skin and nose, in house dust mite allergic individualss with or without asthma.

MaterialMaterial and methods: Nasal and dermal allergen challenges with house dust mite (D. pteronyssimts)

extractt were performed in 52 house dust mite allergic individuals of whom 26 had mild to moderatee persistent asthma and 26 had perennial rhinitis without current or past asthmatic symptoms.. Serial nasal lavage samples were analysed for the presence of inflammatory cells (eosinophilss and neutrophils) and soluble markers associated with cellular inflammation interleukin-55 (IL-5), interleukin-8 (IL-8), eosinophil cationic protein (ECP) and myeloperoxidase (MPO).. Macroscopic late phase skin reactions were studied after intracutaneous skin tests with housee dust mite extract.

Results:Results: Fixed dose nasal allergen provocation elicited a similar degree of immediate allergic reactionn as judged by plasma protein exudation and histamine concentrations in asthma and non-asthmaticc rhinitis. Subsequently, no differences between groups were found during the phase of thee late allergic reaction (4-24 hours) in inflammatory cell influx, plasma protein leakage, ECP or MPO.. Likewise, there were no differences in levels of chemotactic cytokines IL-5 and IL-8. In agreementt with the results of nasal challenge, the late skin reaction after dermal challenge with a fixedd allergen dose and after an allergen dose 10,000 times above the skin threshold for an early skinn reaction did not differ between the groups.

chapterr 6 121 1

Conclusion:Conclusion: House dust mite allergic patients with or without asthma have very similar late allergic

inflammatoryy reactions in the skin and in the nose after allergen challenge. Hence, the occurrence

off pulmonary7 symptoms in asthma is unlikely explained by a general tendency of asthmatics to

havee an enhanced late allergic cellular inflammatory response. Nasal and dermal allergen

provocationss are adequate models to study allergen-induced inflammation but probably lack the

pivotall link, which is essential for the development of asthma.

122 2 chapterr 6

Introductio n n

Inhalantt allergy plays an important causal role in the development of asthma and rhinitis. Althoughh allergic sensitisation is demonstrable in 30 to 40% of the population in the western industrialisedd countries, only 5 to 10% suffer from allergic asthma and 10 to 20% suffer from allergicc rhinitis. The vast majority of allergic asthmatic patients also experience symptoms of allergicc rhinitis1'2. By contrast, allergic rhino-conjunctivitis is not necessarily accompanied by asthmaticc symptoms although this group may have an increased chance of developing symptoms off the lower airways. Increased non-specific bronchial hyperresponsiveness is reported for the groupp of allergic rhinitis, but this is less pronounced than in asthma3'4. Likewise, inflammatory changess in the bronchi similar to the bronchial inflammation found in asthmatics are demonstrablee in patients with allergic rhinitis without asthma5. Results of studies on quantitative differencess in bronchial inflammation between allergic asthma and non-asthmatic rhinitis remain inconclusive68.. Bronchial allergen challenge is able to induce early bronchoconstriction in rhiniticss as well as in asthmatics9-10. However, it is generally found that a late asthmatic reaction, whichh is associated with an increase in cellular inflammation in the bronchi, occurs more frequentlyy and is more pronounced in asthma. It was our study hypothesis that differences betweenn asthmatic and non-asthmatic allergy might be associated with differences in the late cellularr inflammatory response to allergen. To test this hypothesis we performed a prospective experimentall study to investigate allergen-induced inflammation in the bronchi, in the nose and in thee skin in house dust mite allergic patients with or without asthma. Here, we describe the results off the nasal and dermal allergen provocation tests. Intracutaneous skin provocation was done withh a fixed allergen dose as well as with an allergen dose titrated to a standardised early skin reaction.. Primary outcome of the skin provocation test was the magnitude of the dermal infiltrationn after 7 hours.

Nasall provocation was performed with a fixed dose of house dust mite extract for all individualss and the reaction was monitored by measuring changes in repeated nasal lavages. Primaryy outcomes were levels of IL-5, IL-8, Eosinophil Cationic Protein (ECP), Myeloperoxydase (MPO)) and cell differentials obtained during the phase of the late allergic reaction (4-24 hours).

chapterr 6 123 3

Earlyy protein leakage (alfa-2-macroglobulin) and histamine levels were monitored as an indicator

forr early mast cell degranulation in the nose11-14. The study was designed to detect a 25%

differencee in late skin reactivity- and a 30% difference in log normalised levels of soluble markers

wit hh a power of 0.80 and a 95% confidence level.

Material ss and methods

Patients Patients

Patientss with asthma, diagnosed according to the American Thoracic Society (ATS) criteria, were

recruitedd from the outpatient department of Pulmonology, and had a documented history of

recurrentt episodes of wheezing, chest tightness and dyspnoea and a normal lung function

betweenn asthmatic attacks1-"1. Patients with perennial rhinitis were recruited from the outpatient

departmentt of otorhinolaryngology and were characterised by episodes of sneezing, watery

rhinorrhoea,, pruritus in the nose, eyes and palate and nasal obstruction16. Patients with non-

asthmaticc perennial rhinitis were only included if they did not have a history of current or past

dyspnoeaa or wheezing, use of bronchodilators or a doctors diagnosis of asthma.

Thee following inclusion criteria applied to all participants: a) FEVi > 70% of predicted

value;; b) specific IgE level to house dust mite > 0.5 IU /ml ; c) able to stop short acting J32-

adrenoceptorr agonists for at least 8 hours, oral anti-histamines for 2 weeks and inhaled

corticosteroidss for 6 weeks prior to the start of the study; d) no significant change in

environmentall allergen exposure (e.g. not moved) during the past 12 months; e) whenever co-

sensitisationn against pollen was present, patients were studied outside the relevant season.

Exclusionn criteria for all were: a) history7 of immunotherapy; b) respirator)- tract infection 6

weekss prior to the study; c) immunosuppressive medication; d) smoking.

Thee AM C Medical Ethics Committee approved the study, and all subjects gave written

informedd consent.

124 4 chapterr 6

StudyStudy design

Priorr to the inclusion all subjects were screened by Radio-Allergo-Sorbent-Test (RAST) and skin prickk test (SPT) with a standard panel of aero-allergens and by spirometry.

Corticosteroidss were withheld for 6 weeks and anti-histamines for 2 weeks before the studyy period. On the first day of the study intracutaneous tests were performed and blood sampless were obtained. The next day nasal challenge with diluent was followed by allergen challengee one hour later. Changes induced by diluent challenge were monitored by three nasal lavagess during the first hour. Similarly, allergen challenge was followed by three lavages in the first hourr and subsequendy by lavages every hour for 8 hours. Twenty-four hours after allergen challengee nasal lavage and venepuncture were repeated.

AllergenAllergen extract

AA standardized freeze dried house dust mite extract (ALK Diephuis, Houten, The Netherlands) wass dissolved in phosphate buffered saline, pH 7.4 (PBS), 0.03% human serum albumin (HSA), 0.5%% phenol (ALK Diephuis, Houten, The Netherlands) at 1 mg/ml (equivalent to 106 SQ(ALK-Standardd Quality) units/ml and 50,000 BU(Biological Units)/ml) and was kept at -20°C in small aliquots.. This allergen batch was used for all assays, and contained 85 [j.g Der pi and 9.7 ptg Der p22 per mg of protein. Allergen dilutions for tests in vivo and in vitro were made freshly from stock.. For nasal allergen challenge it was diluted in PBS, 0.03% (w/v) HSA, 0.0125% (w/v) benzalkoniumm chloride (ALK Benelux, Houten, The Netherlands).

DermalDermal allergen challenge

Tenfoldd dilutions of the extract and 30 BU/ml were administered intracutaneously (ICT) in the forearmm as described before17. In addition, diluent control and histamine dihydrochloride at 0.1 mg/mll were tested. After 15 minutes, wheal- and flare reactions were determined by marking bothh reactions and copying the marks on a form via an adhesive tape. Results were summarised byy the sum of the largest diameter and its orthogonal diameter. At 7 hours the late skin induration wass quantified by the sum of the largest diameter and the orthogonal diameter. Late phase

chapterr 6 125 5

reactionss were evaluated for 30 BU /ml and for the allergen concentration 10,000 times above the

thresholdd concentration for eliciting an early skin reaction.

NasalNasal allergen challenge

Nasall allergen challenge and repeated nasal lavages were performed according to the method

describedd by Naclerio et al.18 For lavage, both nostrils were instilled with 5 ml of 0.154 M NaCl at

37°CC with the patient sitting with the head in 45° anteflexion. After 10 seconds dwell time the

flui dd was collected in a polypropylene tube and immediately put on ice. The challenge protocol

startedd with 4 baseline nasal washings and subsequent application of oxymetazoline 0.05%,

0.0125%% benzalkoniumchloride dissolved in NaCl 0.9% to limi t mucosal swelling. Subsequently,

diluentt fluid (0.15 ml) was sprayed into both nostrils as a control challenge and nasal lavages were

performedd after 20, 30 and 40 minutes. Then, allergen challenge was performed with 0.15 ml of

10,0000 B U / ml in each nostril. Nasal lavage was repeated 20, 30 and 40 minutes after application

off allergen. Thereafter, nasal washings were done hourly from 3 to 8 hours and again 24 hours

afterr challenge. Dur ing the procedure the subjects were allowed to wipe their noses but asked not

too blow their nose.

SampleSample processing

Thee lavage fluids were centrifuged for 5 minutes at 700 g. Supernatant was separated from the

cellss and stored in aliquots at -20°C. The cell pellet was resuspended in PBS and an equal volume

off dithiotreitol solution (10 mM D TT in 135 mM Tris buffer, pH 8.0) was added followed by

mixingg the sample in a shaking water bath for 15 minutes at 37°C (DTT; Sigma Chemical

Company,, St. Louis, Missouri, USA). After centrifugation for 5 minutes at 700 g, the cell pellet

wass resuspended in 1 ml PBS containing 0.5% HSA and cells were counted in a Bürker counting

chamber.. Cells were than cvto-centrifuged at 550 rpm for 2 minutes in a Shandon Cytocentrifuge

andd stained with Romanovsky (Diff-Quick) and Jenner Giemsa. Al l differential cell counts were

performedd by one investigator blinded for patient identity and sample time point. Epithelial cells

(squamouss and non-squamous), neutrophils, eosinophils, lymphocytes and macrophages were

identified.. A total of 500-1000 cells were counted, depending on the percentage of eosinophils.

126 6 chapterr 6

Duee to low cell numbers in repeated lavages data from samples 4-24 hours after challenge were pooled. .

ProteinProtein assays

Levelss of ECP were determined in the lavage supernatants with an ELISA described previously19. Thee detection limit was 15 pg/ml. Assay reagents (Rabbit-anti-human ECP antiserum and anti-ECPP antibody-biotin) were kindly donated by dr A. Zuurbier (CLB Sanquin, Amsterdam, The Netherlands).. A standard dilution curve of ECP was derived from Pharmacia & Upjohn (Uppsala, Sweden).. MPO20, IL-521, IL-822 and Alpha-2-macroglobulin (A2M)23 were measured by ELISA as describedd earlier. Histamine was measured with an automated fluorometric analysis as described byy Siraganian24. Total serum IgE was quantified by a binding and an inhibition assay, as described before25.. Levels of specific IgE against house dust mite (Dermatophagoides pteronyssinus) were measuredd in duplicate with the Radio-Allergosorbant test (RAST)25. The results were expressed in internationall units per millilitr e using an in house-standard, calibrated against the World Health Organisationn standard26.

Statisticall analysis

SPSS,, version 10.0 Statistics U.K. (Chicago, IL , USA) was used for statistical analyses. The analysiss of the ECP, MPO, IL-5 and IL-8 during late allergic reaction was done with the average off the values measured at 4,6 and 8 hours after challenge. When applicable data were log-transformedd to obtain normal distribution of data. Within group changes were evaluated with pairedd t-test and Wilcoxon test (IL-5). Between groups comparisons were done with t-test and analysiss of variance controlling for the level of specific IgE to HDM. Mann-Whitney test was donee in the absence of a (log)normal distribution of data (IL-5 and skin reactions). Comparison of groupss with respect to allergen-induced changes in parameters was done with analysis of covariancee with post challenge values as dependent variable, patient group as fixed factor and baselinee values as a covariate. Associations between parameters were determined with the

chapterr 6 127 7

Spearmann rank test. All reported p-values are two-tailed and p-levels of <0.05 are considered significant. .

Results s

AA summary of demographic and clinical data of both patient groups is presented in table 1. There wass no difference in mean age and total IgE between the groups. The amount of IgE directed againstt house dust mite was slightiy lower in the non-asthmatic group. Adjustment for this parameterr was added to the statistical analysis of differences in the primary outcomes between groups.. As expected, there was a significant difference in baseline FEVi and histamine thresholds betweenn asthmatics and non-asthmatic patients.

Tablee 1 Patient characteristics.

Non-asthmaticc rhinitis Asthma a Gender r

Age e

FEVi i

Pc2ohistamine e

Perenniall rhinitis symptoms

Totall IgE

HDMM specific IgE

RASTT results:

Grass pollen3

Treee pollen3

Catt or dog dander3

HDMM mono allergy3

(male/female) )

(mean,, range)

(%% predicted)'

(mg/ml)2 2

(%) ) (IU/ml) 2 2

(IU/ml) 2 2

6/20 0

277 (18-37)

103.2(12.7) )

9.66 (2.7)**

100 0

126.11 (3.8)

13.0(3.4)* *

50 0

46 6

23 3

31 1

6/20 0 277 (18-40) 94.77 (14.6) 1.2(4.0) ) 73 3 243.44 (3.8) 26.66 (2.8)

50 0 31 1 50 0 38 8

*p<0.05,, **p<0.01 j,, mean and standard deviation; 2, geometric mean and standard deviation.3, (% pos).

128 8 chapterr 6

Tablee 2 Analysis of changes in nasal lavage from baseline to 24 hour post-challenge

Eosinophils5 5

(io4/i) ) ECP P

(ng/ml) )

IL- 5 5

(pg/ml) )

Neutrophils* *

(1071) ) IL- 8 8

(Pg/ml) ) MPO O

(ng/ml) )

A2M M

(ng/ml) )

Non-asthmatics s

baseline e

Mean* *

(SE) )

2.2 2

(1.6) ) 2.20 0

(1.3) )

<0.1 1

2.5 5

(1.5) )

136.0 0

(1.2) )

75.5 5

(1.2) )

503.6 6

(1.2) )

(nn = 26)

24hrs s

Mean* *

(SE) )

19.9 9

(1.4) )

4.32 2

(1.4) ) 1.53 3

(0.7) )

8.1 1

(1.4) )

140.0 0

(1.1) )

72.9 9

(1.2) ) 642.7 7

(1.3) )

pp value

24hrs s

versus s

baseline e

0.001 1

0.003 3

0.02 2

0.003 3

>0.2 2

>0.2 2

>0.2 2

baseline e

Mean* *

(SE) )

3.8 8

(1.7) ) 1.6 6

(1.3) )

O .l l

1.4 4

(1.6) ) 95.1 1

(1.2) )

45.9 9

(1.3) ) 404.7 7

(1.3) )

Asthma a

(nn = 26)

24hrs s

Mean* *

(SE) )

14.8 8

(1.5) )

4.0 0

(1.4) )

1.51 1

(0.5) )

7.7 7

(1.5) )

94.9 9

(1.2) )

38.2 2

(1.3) ) 490.1 1

(1.4) )

pp value

24hrs s

versus s

baseline e

0.004 4

0.002 2

0.04 4

0.001 1

>0.2 2

>0.2 2

>0.2 2

Non--asthmaticss vs.

Asthmatics s

baseline e

pp value

between n

groups s

>0.2 2

>0.2 2

>0.2 2

>0.2 2

>0.1 1

>0.1 1

>0.2 2

24hrs s

pp value adjusted d

forr baseline

(unadjusted) )

>0.2 2

>0.2 2

NA A

>0.2 2

>0.2 2

>0.2 2

>0.2 2

(>0.2) )

(>0.2) )

(>0.2) )

(>0.2) )

(>0.1) )

>0.05) )

(>0.2) )

*,, geometric mean and standard error for ECP, IL-8, MPO, A2M. $,, pooled cell data from baseline lavages and from lavages 4 —24 hr after challenge. NA,, not available;

Withinn group comparison: paired t-test (Wilcoxon signed ranks for IL-5); between groups comparisonn unpaired t-test (baseline and unadjusted 24 hrs) and analysis of covariance (24 hrs adjustedd for baseline) signifying differences in changes from baseline (Mann-Whitneyy test for IL-5).

chapterr 6 129 9

Nasa/Nasa/ challenge

Meann recovery of the 10 ml lavage fluid was similar in asthmatics and non-asthmatics (7.7 [SD

0.9]] vs. 7.6 [SD 0.9] ml, p>0.2).

Thee baseline eosinophil and neutrophil counts and levels of alpha-2-macroglobulin

(A2M) ,, ECP, M P O, IL- 5 and IL- 8 were similar in the nasal washings of asthma and non-

asthmaticc rhinitis (table 2).

Thee repeated nasal lavage procedure caused some washout effect of soluble markers and

cells.. This is shown by a gradual decrease in the concentrations of the soluble markers during the

initiall baseline lavages and after diluent challenge. It generally resulted in levels at 40 minutes after

thee diluent challenge of about 10-15% of the initial baseline lavage. Extended control procedures

duringg 24 hours after diluent challenge in a limited number of patients indicated a slight further

decreasee in soluble markers up to 40 minutes after sham challenge, followed by a gradual return to

pre-challengee values at 6 hours and to baseline values at 24 hours (fig. 1). Therefore, 24 hours

lavagee results, with no preceding lavage for 16 hours, was compared with baseline values, and

lavagess after 4 to 8 hours were compared with post diluent lavages. In none of the patients plasma

proteinn exudation was found during the first hour after diluent challenge. During the first hour

afterr allergen administration A2M levels increased in all padents and to a similar extent in both

groupss (fig. 1). In contrast, house dust mite challenge did not induce an increase in A2M in

patientss without sensitisation to house dust mite, excluding a significant non-immunological effect

off the house dust mite extract on the nasal mucosa.

A2MM expressed as area under the curve (AUC) during the first hour after allergen

challengee was not significantly different between the two groups (fig. 1). This was not changed by

adjustmentt for specific IgE. Similarly, histamine levels during the early allergic reaction did not

differr between asthmatics and non-asthmatics (Mean [SE]: asthma 0.76[0.10], non-asthmatics

0.811 [0.12] ng /ml, p>0.2).

130 0 chapterr 6

ChangesChanges in markers of inflammation during the phase of the late allergic reaction

Plasmaa protein exudation as detected by the level of A2M showed a second increase during the phasee of the late allergic reaction with a maximum 8 hours post-challenge in both groups (fig. 1). Thee latter increases were statistically significant in the non-asthmatics as well as in the asthma groupp (both p<0.001). At 24 hours after the challenge levels had returned to baseline values. The coursee of A2M as assessed by the AUC from 2 to 8 hours, was not different between the two groups.. This was unaffected by adjustment for IgE directed against house dust mite. In addition, theree was no difference between the subgroups of asthmatic patients with or without rhinitis symptoms. .

Theree was an increase in ECP in nasal washings during the late phase, which was statisticallyy significant in both groups (fig. 2). At 24 hours post-challenge levels were still elevated comparedd to baseline values (table 2). Again, increases at 4 to 24 hours were statistically indistinguishablee for asthma and non-asthmatic rhinitis.

chapterr 6 131 1

allergen n

1000--

300 0

ÖJO O

5! !

100--

30--

10--

non-asthmatics s

asthmatics s

/ / --baselinee 20 min 30 min 40 min 3 hr 4hr r 5hr r 6hr r 7hr r 8hr r

Timee after allergen challenge

Figur ee 1 Level of oc-2-macroglobulin (A2M) in nasal lavages after nasal allergen challenge.

Baselinee was level after diluent challenge. The broken line indicates geometric mean (SE) of A2M

valuess after diluent challenge in 3 patients.

Theree was a significant increase in A2M during the early allergic reaction (20-40 minutes, both

groupss p<0.0001) and during the phase of the late allergic reaction (4-8 hours) in both group

(bothh groups p<0.001), relative to baseline as well as to controls after sham challenge. There were

noo significant differences between asthmatics and non-asthmatics during early phase (p = 0.3)

andd late phase (p = 0.5).

1322 chapter 6

e e

U U w w

3--

1--

0.3J J

allergen n

non-asthmatics s

asthmatics s

11 1 1 1 I —

baselinee 30 min 4hr 6hr 8hr

Timee after allergen challenge

Figuree 2 Concentrations of eosinophil cationic protein (ECP) in nasal washings after nasal

allergenn challenge. Baseline was level after diluent challenge. There was a significant increase in

bothh groups at 4-8 hours (p<0.0001) but there was no significant difference between asthmatics

andd non-asthmatics (p = 0.8).

chapterr 6 133 3

OH H

100--

10--

1--

0.05--

non-asthmatics

asthmatics

allergen n

(37) )

baseline e 300 min 44 hrs 66 hrs 88 hrs 244 hrs

T imee after allergen challenge

Figur ee 3 Levels of U-5 in nasal washings following nasal allergen challenge. Baseline was level

afterr diluent challenge. The number of samples below detection level are indicated at each time

point.. There was a significant increase in IL- 5 in both groups at 4-8 hours (Wilcoxon p<0.05).

Theree were no significant differences between asthmatics and non-asthmatics (Mann-Whitney p

== 0.7).

1344 chapter 6

Theree was also a significant rise in MPO levels at 4 to 8 hours compared to levels after control challengee in both groups (geometric mean[SE] asthma 7.9 [1.4] to 20.9 [1.3] ng/ml and non-asthmatics:: 10.5 [1.3] to 28.8 [1.3] ng/ml, both p<0.0001). In contrast to ECP, MPO levels returnedd to baseline levels after 24 hours (table 2).

IL-55 levels significantly increased in both groups during the late allergic reaction and were stilll elevated after 24 hours (both p<0.05, Wilcoxon) (fig. 3). Again, no difference was found betweenn the groups at any of the time points studied.

IL-88 showed a similar but modest increase in both groups after 4 to 8 hours compared to levelss after diluent challenge in the asthma as well as in the non-asthmatic group (geometric mean [SE]] asthma 24.0 [1.3] to 45.6 [1.2] ng/ml, non-asthmatics 28.1 [1.2] to 45.9 [1.2] ng/ml, p<0.00011 and p = 0.005, respectively). After 24 hours levels were similar to baseline levels (tablee 2).

Duee to low cell numbers in the repeated nasal washings data for influx of inflammatory cellss were pooled for 4-24 hours. There was a significant increase in both groups for eosinophil andd neutrophil numbers compared to baseline numbers, but no significant difference between groupss (table 2). Results were not affected by controlling for the level of specific IgE to HDM.

CorrelationCorrelation between outcomes

U-55 early during the late phase (4-6 hours) was associated with late phase levels of ECP (Rss = 0.46, p = 0.001), A2M (0.40, p = 0.003) and eosinophil numbers (Rs = 0.47, p = 0.001), but wass not associated with neutrophils (p = 0.6) and MPO (p = 0.16). Early rise in 11-8 (4-6 hours) wass associated with late phase levels of MPO (Rs = 0.72, p<0.0001), A2M (Rs = 0.42, p = 0.002), ECPP (Rs = 0.47, p = 0.001) and showed a trend for an association with post challenge neutrophil numberss (Rs = 0.26, p = 0.07), but no association with eosinophil numbers (p = 0.9).

chapterr 6 135 5

DermalDermal challenge

Thee allergen threshold concentration for a positive early skin reaction was significantly lower in

asthmaa than in non-asthmatic rhinitis (geometric mean [SE] asthma 0.09[1.4] BU/ml, non-

asthmaticss 0.21 [1.3] B U / m l , p = 0.04). However, after correction for the difference in the level of

H D MM specific IgE, the difference between groups disappeared (p = 0.3). Skin reactions with

histaminee were very similar in both groups (median[IQR] flare reaction, asthma: 8.0[2.3] cm, non-

asthmatics:: 7.5[1.5]cm; wheal reaction, asthma: 2.3[0.5]cm, non-asthmatics: 2.0[0.5]cm, p>0.2).

Earlyy skin reaction with H DM showed no differences in size of the wheal and flare reactions

betweenn the asthmatics and non-asthmatics, neither after a challenge with 30 BU/ml

(median[lQR]] flare reaction, asthma: 10.3[3.6]cm, non-asthmatics 9.6[2.8]cm; wheal reaction,

asthmaa 2.7[ l . l ]cm, non-asthmatics 2.6[0.5]cm, both p>0.2) nor at a concentration of 10,000 times

abovee the skin allergen threshold (median[IQR] flare reaction, asthma 10.4[2.0] cm, non-

asthmaticss 9.9[2.4]cm; wheal reaction, asthma: 2.9[1.0]cm, non-asthmatics: 2.9[0.7] cm, both

p>0.2). .

Obviouss late skin reactions were observed in both groups after challenge with 30 BU

(median[IQR]] skin induration, asthma 9.3[3.9]cm, non-asthmatics 8.1[3.7]cm, p>0.2), as well as

afterr challenge with a concentration 10,000 times above the threshold (median[IQR] skin

induration,, asthma 10.4[2.0]cm, non-asthmatics l l . l [6.0]cm, p>0.2) with no significant difference

betweenn groups.

AssociationsAssociations between nasal and dermal late phase reactions

Theree were significant associations between the late phase skin reaction with fixed dose of

allergenn and late phase nasal eosinophils (Rs = 0.30, p = 0.05), ECP (Rs = 0.45, p = 0.001), IL- 5

(Rss = 0.38, p = 0.006) and A2M (Rs = 0.45, p = 0.001). Significant associations with late phase

nasall neutrophils, M PO and IL- 8 were lacking (P>0.1). In a stepwise multiple linear regression

analysiss with late phase skin reaction as variable to be explained and nasal eosinophils, E.CP, 11-5,

A2MM and specific IgE to H DM as explaining variables, IgE to H DM turned out to be the only

significantt independent predictor.

136 6 chapterr 6

Discussion n

Inn this study of allergen induced late allergic inflammation in nose and skin we were not able to detectt differences between house dust mite allergic individuals with or without asthma.

Validationn of the classification as being asthmatic or non-asthmatic in this study was supportedd by a significantly lower baseline FEVi and histamine threshold in asthma. Both parameterss had not been used for inclusion into either group.

Thee majority of the patients with asthma also reported symptoms of perennial allergic rhinitiss which is in keeping with the literature27'28. No significant differences in nasal and dermal outcomess were found between asthmatics who indicated to have perennial nasal symptoms and thosee who did not. In concordance, comparison of primary outcomes between non-asthmatic rhinitiss and the subgroups of asthmatics with and without symptoms of perennial rhinitis did not reveall differences. Moreover, at baseline nasal inflammation was very similar in non-asthmatic rhinitiss and asthma. So, it seems unlikely that any difference in allergen-induced inflammation wouldd have been obscured by differences in the occurrence of nasal symptoms or initial nasal mucosall inflammation.

Becausee challenge and lavage procedures were identical and recovery of lavage fluids was veryy similar in non-asthmatics and asthmatics comparison of both groups is not hampered by the washoutt phenomenon of repeated nasal washings. However, estimation of the kinetics of the nasall inflammation during the first hours after challenge may be slightly distorted by the phenomenon. .

Itt cannot be excluded that there are small differences in allergen induced late cellular inflammationn between asthmatics and non-asthmatic rhinitics beyond the detection limit of this study.. However, the concordance between dermal and nasal challenge results may argue against this. .

Nasall challenge with a fixed dose of allergen resulted in a similar degree of early mast cell activationn in both groups as inferred from the similar increase in plasma protein exudation and histaminee levels. This is in agreement with literature reporting similar early allergic reactions as

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reflectedd by histamine release and plasma protein leakage between allergic patients with or

withoutt asthmadc symptoms9'29.

Thee lack of a difference in the late phase inflammadon in the nose and the skin in this

studyy seems to be in contrast with the general notion that allergen induced bronchial

inflammationn is increased in asthmatics. The evidence for this is mainly indirect and based on the

correlationn between bronchial inflammation and non-specific bronchial hyperresponsiveness on

thee one hand and the finding that allergen-induced late bronchoconstriction occurs more

frequentlyy and is more pronounced in asthmatics than in non-asthmatics on the other hand.

Baselinee non-specific bronchial hyperresponsiveness, which is more increased in asthma, is the

mainn determinant of the late phase bronchoconstriction30. Therefore, it is impossible to conclude

thatt there wil l be differences in late phase cellular inflammation at the bronchial level between

asthmaticss and non-asthmatics just based on differences in allergen-induced late phase

bronchoconstr ict ion.. In fact, the few studies in which allergen induced bronchial inflammation

wass directly compared between allergic asthmatics and non-asthmatics produced conflicting

results6,7.. Direct comparison of allergen induced bronchial inflammation may be hampered by the

differencess in baseline non-specific bronchial hyperresponsiveness and possibly also by

differencess in baseline bronchial inflammation. For this purpose the nasal mucosa is probably a

moree appropriate target because of the similarity in baseline inflammation and in nasal symptoms

inn both groups. Moreover, in nasal challenge the allergen dose is not limited by the degree of

airwayy narrowing, as it is in bronchial challenge, offering the opportunity to apply fixed allergen

dosee regimens, which facilitate direct comparison of allergen, induced inflammation.

Thee increase in nasal IL- 5 and IL-8, the subsequent influx of inflammatory cells

accompaniedd by the increase in the soluble cell markers ECP and MPO as found in this study is in

agreementt with the results of other studies11'13'22-3134. The association between increases in the

chemo-attractantss IL- 5 and IL- 8 and the nasal influx of eosinophils and neutrophils, respectively,

duringg the late phase, are in agreement with their reported role for recruitment of these

inflammatoryy cells.

Thee late phase nasal cellular inflammation closely resembles allergen induced bronchial

inflammation.. It is generally accepted that allergen induced bronchial inflammation contributes to

138 8 chapterr 6

asthmaticc symptoms particularly in those individuals with a, possibly genetically determined, increasedd propensity to develop non-specific bronchial hyperresponsiveness. The high prevalence off asthmatic symptoms in occupational allergy and the ability to induce asthmatic symptoms in non-asthmaticc atopics by artificial high-dose bronchial allergen challenge may demonstrate that highh levels of exposure to allergen, which is associated with an exaggerated allergic inflammatory response,, may overcome the absence of an inherited propensity to develop bronchial hyperresponsiveness.. Natural HDM exposure in our study population was very similar for non-asthmaticc rhinitics and asthmatics as judged from the Der p 1 and Der f 1 levels measured in mattresss dust samples in all participants (results not shown). This finding renders an environmentall role for the occurrence of asthmatic symptoms in this study less likely.

Inn conclusion, we did not find any differences in late phase cellular inflammation followingg nasal and dermal allergen challenge between house dust mite allergic patients with or withoutt asthma. Our results confirm that nasal challenge is an adequate model for studying allergenn induced mucosal inflammation. However, the similarity between asthmatic and non-asthmaticc patients in allergen induced nasal and dermal inflammation suggests that ultimately local factorss in the bronchial tree are decisive for the development of asthma symptoms. Our results indicatee that these factors most likely cannot be targeted in allergic models in other organs other thann the lower airways.

Acknowledgements s

Thiss study was financially supported by the Netherlands Asthma Foundation (grant 32.95.57).

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