Non-specific bronchial hyperresponsiveness in asthma patients with or without allergic rhinitis

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1 237 Non-specific bronchial hyperresponsiveness in asthma patients with or without allergic rhinitis Institute for Biomedical Research, Kaunas University of Medicine, Lithuania Key words: bronchial hyperresponsiveness, methacholine, exercise, eosinophils Summary Bronchial hyperresponsiveness is the main pathophysiological feature of asthma Eosinophilic inflammation of airway is one of main factors influencing bronchial hyperresponsiveness The aim of the study was to evaluate bronchial responsiveness to methacholine and exercise of asthma patients with or without allergic rhinitis and to estimate relations between eosinophil count in nasal secretion and non-specific bronchial hyperresponsiveness Ninety eight patients with mild or moderate asthma were examined Seventy eight patients had mild allergic rhinitis Patients were divided in two groups: asthma with rhinitis group (n=78) and asthma group (n=20) Bronchial responsiveness was tested with methacholine and exercise challenges Allergic status was determined by skin prick tests, mean wheal size and eosinophil count in the blood and nasal secretion Atopy was more frequent (p=0001) and mean wheal size larger (p=0002) in asthma with rhinitis group No difference estimated on blood eosinophil count (p=0125) between both groups Nasal eosinophil count was higher in asthma with rhinitis group comparing with asthma group (p=0001) Methacholine provocative dose (PD 20 ) was lower and the slope of the dose-response curve higher in asthma with rhinitis group, but not statistically significantly: PD ±270 µg and 2121±529 µg, accordingly, p=0179; the slope of the dose-response curve 236±16 and 207±25, p=0219 Exercise-induce bronchoconstriction developed equally: to 46% patients (n=36) from asthma with rhinitis group and 45% patients (n=9) asthma group No significant differences were found between maximal fall of after exercise ( ): 242±23% and 259±40%, accordingly, p=0,744 ; area under the curve (AUC 0- ): 4516 ± 489 % sec and 4840±1110% sec, p=0777 No statistical significant 30 correlations were evaluated between nasal eosinophilia and PD 20, the slope of the doseresponse curve, and AUC 0-30 Conclusions No significant differences were estimated on bronchial responsiveness to methacholine and excercise between asthma patients with or without allergic rhinitis Eosinophil count in nasal secretion did not correlate with non-specific bronchial hyperresponsiveness Introduction Bronchial hyperresponsiveness (BHR) is a key pathophysiological feature of asthma, which presents itself as an exaggerated response to a variety of stimuli that lead to bronchoconstriction The important link of asthma pathogenesis is chronic eosinophilic inflammation of airway, often related to the increased airway responsiveness (1, 2) More than 90% patients with asthma have BHR It is known that persons with allergic rhinitis have nasal hyperreactivity (3) Some of them may present the asymptomatic non-specific BHR It is shown that methacholine, exercise, adenosine-5 monophosphate may cause bronchial obstruction up to 40% of patients with allergic rhinitis (4, 5, 6) Exacerbation of the allergic rhinitis may raise severity of asthma and bronchial responsiveness This is considered to have links with inflammation of bronchial mucosa by release of bronchoconstricting mediators, post-nasal mucous drip, and sinobronchial reflex (7) It is ascertained that natu- Correspondence to K Malakauskas, Laboratory of Pulmonology, Institute for Biomedical Research, Kaunas University of Medicine, Eivenių 4, 3007 Kaunas, Lithuania kestutismalakauskas@kmult

2 238 ral exposure to pollen during season provokes an increase in bronchial responsiveness in subjects with seasonal allergic rhinitis and also induces inflammatory cell recruitment and IL-5 expression, leading to bronchial inflammation (8) More that 75% of patients with asthma have symptoms of allergic rhinitis (9) Allergic rhinitis constitutes a risk factor for the occurrence of asthma (10) Patients with allergic rhinitis have clear signs of allergic inflammation in nasal mucosa (11, 12), however, there are evident morphological changes in bronchial mucosa that is typical for asthma: infiltration with eosinophils, T-lymphocytes, damage of epithelium, and submucosal fibrosis (13, 14) Gaga et al (2000) showed that allergic inflammation of nasal mucosa existed in patients with asthma even without clear clinical symptoms of allergic rhinitis Thereby, pathophysiological and morphological similarities and differences exist between the nasal and bronchial mucosa in rhinitis and asthma The aim of the research was to evaluate bronchial responsiveness to methacholine and exercise in asthma patients with or without allergic rhinitis, to determinate the links between the bronchial responsiveness and eosinophil count in nasal secretion Materials and methods Subjects We studied 98 outpatients (aged 17-51) who have been diagnosed as having mild or moderate asthma according to the Global Initiative for Asthma (GINA) (15) Allergic rhinitis was diagnosed on the basis of characteristic history of clinical symptoms (rhinorrhea, nasal itch, sneezing, and nasal obstruction), their duration and frequency, and positive skin prick tests (16) Seventy eight patients were diagnosed as having seasonal or persistent mild allergic rhinitis longer than 12 months All subjects were divided in 2 groups: asthma with rhinitis group (n=78) and asthma group (n=20) A control croup consisted of 8 healthy nonsmokers and non-allergic subjects All subjects neither had acute respiratory infection for 4 weeks before study, nor used inhaled/intranasal steroids for 2 months Before the bronchial provocation tests they also neither took any antihistamine preparations for 3 days, nor used long-acting β 2 agonists for 48 hours, short-acting β 2 agonists for 8 hours The study protocol was approved at the Institute for Biomedical Research, Kaunas University of Medicine Spirometry Spirometric measurements were obtained using a spirometer Vmax229 (SensorMedics, USA) Baseline forced expiratory volume in one second ( ) and forced vital capacity (VC) were recorded as the highest of three reproducible measurements performed according to methodology (17) Methacholine challenge Methacholine challenges were performed using an reservoir method (Provocations Test I, Pari, Germany) (18), starting with 15 mg methacholine dose and doubling till the cumulative 1300 mg delivered dose or when more than 60% fall of had been reached or when intolerable dyspnea had appeared was measured at 30 and 90 seconds after each dose and the largest was used for further analysis Results were expressed as: provocative dose of methacholine in mg needed to decrease by 20% from the baseline value (PD 20 ); slope of the dose-response curve PD 20 was calculated by log-linear interpolation between the data points either side of the 20% fall The slope of the dose-response curve was calculated by linear regression analysis of the data points in the middle part of the curve, including the two data points used in determining the PD 20 and all consecutive data points up to the first point of the plateau, or up to the last data point if no plateau was demonstrated A maximum response plateau was considered to be present if two or more measurements fall within 5% of each other Exercise challenge Subjects exercised on an electrically driven treadmill (Series 2000 Treadmill, Marquette, USA) for 5 min with a fixed workload adjusted to increase the cardiac frequency to 90% of the individuals age-predicted maximum (220-age) Workload was gradually achieved by changing the treadmill speed and inclination according to methodology (20) All subjects breathed unconditioned room air (temperature C and relative humidity less than 50%) Pre-exercise spirometry was performed before exercise and the best of three attempts was considered the pre-exercise Spirometry was repeated after exercise at 1, 3, 5, 7, 10, 15 and 30 minutes The best value of two attempts from each set of measurements was used for analysis The test was considered positive when decreased more than 10% compared with preexercise value The exercise challenge endpoints included: maximal percentage fall in after exercise; area under the curve (AUC) AUC 0-30min area under the curve of the percentage fall in plotted against for 30 min AUC summarizes both the extent and duration of bronchoconstriction after exercise AUC was calculated using trapezoidal integration method and expressed by percentage min

3 Non-specific bronchial hyperresponsiveness in asthma patients 239 (21) Only the area below the pre-exercise baseline was counted in calculating the AUC 0-30min Allergen skin prick tests Skin prick tests were performed with 7 standard glycerin-preserved allergens: house dust, house dust mite Dermatophagoides pteronyssinus, dog hair-dander, mould mix, mixed trees and mixed grasses (Bayer Corp, Pharmaceutical Div, USA) The reaction was measured after 20 min, subtracting that of the positive histamine and the negative glycerin control The mean wheal size was the arithmetic mean of the reactions to the seven allergens assessed at least one allergen yielded a wheal diameter of 3 mm and more than 50% of histamine wheal size Eosinophil count in nasal secretion and blood Nasal secretion smears were stained with ethanol and Giemsa-Romanovsky dye and examined under microscope Results were expressed by eosinophil count in percents of all granulocytes (22) Blood eosinophils were counted with a hematology system (ADVIA 120, Bayer, Ireland) and expressed as a total number of cells (x10 9 /l) Blood for measurements was taken before the methacholine challenge on the same day Statistical analysis Statistical analysis was performed using standard statistics program (SPSS Windows 80) PD 20 values were log-transformed for correlation analysis All summary statistics were expressed as means ± SEM To evaluate normality of distributions the Kolmogorov-Smirnov test was used To estimate reliability of mean differences between groups it was used non-parametric Mann-Whitney U-test The correlation was investigated with the Spearman or Pearson correlation coefficient tests Statistical significance was assumed for p<005 Results Basic characteristics of the subjects are summarized in Table 1 Asthmatics with rhinitis were younger than the ones from the asthma group (p=0031) Positive skin prick was found more frequently in asthma with rhinitis group than in asthma group (p=0001) The mean wheal size in response to the seven most common allergens was higher in the asthmatics with rhinitis (p=0002) All subjects had normal pulmonary function and baseline did not differ significantly Eosinophilia in nasal secretion was statistical significantly higher in asthma with rhinitis group than in asthma group (214 ± 37% vs 37 ± 13%, p=0001) (Figure) However, eosinophil count in nasal secretion was lower in control group than in other groups (p<002) Though blood eosinophil count was higher in asthma with rhinitis group, it was not statistical significantly different comparing with asthma group (p=0125) Peripheral blood eosinophil count was lower in control group than in other groups (p<005) BHR to methacholine was slightly higher in subjects group with asthma and rhinitis, though PD 20 and the slope of the dose-response curve were not statistically different from the asthma group (Table 2) Exercise-induced bronchoconstriction was observed equally in both groups: exercise provoked bronchial obstruction for 45% subjects from the Table 1 Characteristics of study subjects Data Asthma with rhinitis Asthma group Control group group (n=78) (n=20) (n=8) Sex (male/female) 52/28 12/8 6/2 Age (years) 230±07* 283±22 221±30 Smoking (non-smokers/ex-smokers/smokers) (n) 60/12/6 13/4/3 7/1/0 Baseline (l) 422± ± ±034 Baseline (%) 1060±16 994± ±53 Atopy (%) 100** 29 0 Mean wheal size (mm) 22±01*** 09±02 0 Blood eosinophils ( 10 9 /l) 054± ± ±007**** forced expiratory volume in one second * p=0031 compared with asthma group ** p=00001 compared with asthma group *** p=0002 compared with asthma group **** p<005 compared with asthma group and asthma with rhinitis group

4 240 Eosinophil count (%)in nasal secretion Asthma gr Asthma with rhinitis gr Control gr Figure Eosinophil count (%) in nasal secretion * p=0001 compared with asthma group; ** p=002 compared with asthma with rhinitis and asthma groups Table 2 Characteristics of bronchial hyperresponsiveness to methacholine and exercise Parameters Asthma with rhinitis Asthma group p group (n=78) (n=20) PD 20 ( µg ) 1730± ± Slope of the dose-response curve 236±16 207± (%) 242±23 259± AUC 0-30 (% min) 4516± ± PD 20 provocative dose of methacholine needed to decrease by 20 % from the baseline; maximum fall in after exercise; AUC 0 30 under the curve of the percentage fall in plotted against for 30 min asthma group (n=9) and 46% (n=36) - from the asthma with rhinitis group There were no statistically significant differences between D and AUC 0-30min comparing asthmatics with or without rhinitis (Table 2) All the subjects in control group had negative provocative tests with methacholine and exercise No statistically significant correlation was determined between eosinophils in nasal secretion and PD 20, the slope of the dose-response curve, D, and AUC 0-30min in asthma with rhinitis group Discussion Eosinophilic bronchial inflammation has been reported in sputum and bronchial biopsies from allergic rhinitis patients (13, 14) Almost half of patients with allergic rhinitis have increased non-specific bronchial responsiveness There are two ways to measure nonspecific BHR: using pharmacological and non-pharmacological agents Directly acting pharmacological agents such as methacholine and histamine act on specific airway receptors and cause the contraction of bronchial smooth muscles Physical stimuli as exer-

5 Non-specific bronchial hyperresponsiveness in asthma patients 241 cise, cold air, hypertonic or distilled water give an indirect challenge because they effect the osmotic or thermal properties of the airway surface while also releasing mediator from cells in the bronchial airway epithelium and muscle Airway cooling and drying during exercise, affect airway resulting in local inflammatory mediators histamine and leukotrienes release Bronchial obstruction develops as mediators, possible along with rewarming, cause bronchoconstriction, vascular engorgement and leakage, and increase mucus production (23) Bronchial response to methacholine is different in allergic rhinitis and asthma patients In asthma patients the dose-response curve of methacholine is shifted to the left (lower PD 20 ), the curve has steeper slope, it often does not reach the maximum response plateau However, allergic rhinitis patients have the dose-response curve more shifted to the right (higher PD 20 ), lower slope and the plateau is usually reached (24) All subjects with asthma with or without rhinitis in our study had increased bronchial responsiveness to methacholine Asthmatics with rhinitis had more shifted to the left dose-response curve, i e lower PD 20, steeper slope of dose-response curve; however, these differences were not significant comparing them with asthmatics without rhinitis Nowadays it is discussed that atopy may be associated with exercise-induced bronchoconstriction in asthmatics (25) Though positive skin prick tests were found more frequent and mean wheal size was higher in the asthmatics with rhinitis, but exercise-induced bronchoconstriction observed equally in asthma with rhinitis and asthma groups Comparing D and AUC values there was no statistically significant differences between asthma and asthma with rhinitis groups In our study a higher eosinophil count in nasal secretion was found in asthma with rhinitis group It is proved, that there is minimal persistent allergic inflammation of nasal mucosa during the period without a contact with allergen of patients having allergic rhinitis (26) We found higher eosinophil count in nasal secretion of patients with asthma compared with the healthy persons This does not contradict opinion about the possible asymptomatic eosinophilic inflammation of the nasal mucosa asthmatics without rhinitis (27) It is known, that increased intensity of eosinophilic inflammation in nasal mucosa is correlated with the severity of symptoms and bronchial responsiveness (28) In our study there was no significant relation between nasal eosinophilia and non-specific BHR to methacholine and exercise of patients with asthma and allergic rhinitis Chanez et al (29) had shown that extent of eosinophilic infiltration, the thickness of regular basement membrane and the epithelium shedding is greater in bronchial then in nasal mucosa of the same untreated patients suffering from asthma and rhinitis We did not find any difference between bronchial responsiveness to methacholine and exercise in asthma patients with or without allergic rhinitis It was estimated a higher eosinophil count in nasal secretion of patients with asthma and rhinitis, but there was no significant correlation between eosinophilia and non-specific BHR The results show, that nasal secretion eosinophils do not influence on non-specific bronchial responsiveness in patients with asthma and allergic rhinitis during non-acute period Nespecifinis bronchų hiperreaktyvumas sergant astma ir alerginiu rinitu arba tik astma Kauno medicinos universiteto Biomedicininių tyrimų institutas Raktažodžiai: bronchų hiperreaktyvumas, metacholinas, fizinis krūvis, eozinofilai Santrauka Bronchų hiperreaktyvumas yra pagrindinis patofiziologinis astmos pokytis, pasireiškiantis neadekvačia bronchų kontrikcija į daugelį dirgiklių Svarbi astmos patogenezės grandis yra lėtinis kvėpavimo takų eozinofilinis uždegimas, dažnai susijęs su padidėjusiu jų reaktyvumu Šio tyrimo tikslas nustatyti bronchų reaktyvumą metacholinui ir fiziniam krūviui sergant astma ir (be) alerginiu rinitu, taip pat nustatyti sąsajas tarp eozinofilų kiekio nosies sekrete ir nespecifinio bronchų hiperreaktyvumo Ištirta 98 pacientų, sergančių lengvos ar vidutinio sunkumo eigos astma 78 tiriamiesiems nustatytas lengvos eigos alerginis rinitas, šie tiriamieji sudarė sergančiųjų astma ir rinitu grupę Astmos grupę sudarė 20 tiriamųjų

6 242 Bronchų reaktyvumas tirtas metacholinu ir fiziniu krūviu Alerginė būklė vertinta atlikus alerginius odos mėginius, nustačius vidutinį pūkšlių dydį, eozinofilų kiekį kraujyje ir nosies sekrete Astmos ir rinito grupėje tiriamieji pasižymėjo dažnesne atopija (p=0,001), didesniu vidutiniu pūkšlių dydžiu (p=0,002) palyginti su astmos grupe Eozinofilų skaičius kraujyje abiejų grupių tiriamųjų nesiskyrė (p=0,125), tačiau jų kiekis nosies sekrete buvo statistiškai didesnis astmos ir rinito tiriamųjų grupėje (p=0,001) Bronchų reaktyvumas metacholinui astmos ir rinito tiriamųjų grupėje buvo kiek didesnis, tačiau jis statistiškai nesiskyrė nuo astmos grupės: provokacinė dozė (PD 20 ) buvo 173,0±27,0 µg ir 212,1±52,9 µg, atitinkamai, p=0,179, dozėsatsako kreivės nuolydis 23,6±1,6 ir 20,7±2,5, atitinkamai, p=0,219 Teigiamas fizinio krūvio provokacinis mėginys abiejų grupių tiriamiesiems nustatytas vienodai dažnai: 46 proc (n=36) astmos ir rinito ir 45 proc (n=9) astmos grupės tiriamiesiems Nerasta reikšmingų skirtumų tarp šių grupių tiriamųjų, kuriems fizinis krūvis sukėlė bronchų obstrukciją: maksimalus sumažėjimas po fizinio krūvio ( ) buvo 24,2±2,3 proc ir 25,9±4,0 proc, atitinkamai, p=0,744, plotas po kreive (AUC 0-30 ) 451,6±48,9 ir 484,0±111,0 proc sek, atitinkamai, p=0,777 Nenustatyta reikšmingų sąsajų tarp eozinofilų skaičiaus nosies sekrete ir PD 20, dozės-atsako kreivės nuolydžio, ir AUC 0-30 Taigi pacientų, sergančių astma ir alerginiu rinitu, bronchų reaktyvumas metacholinui ir fiziniam krūviui nesiskyrė nuo sergančiųjų tik astma, o nespecifinis bronchų hiperreaktyvumas nepriklausė nuo eozinofilų skaičiaus nosies sekrete Adresas susirašinėjimui: K Malakauskas, KMU BMTI Pulmonologijos laboratorija, Eivenių 4, 3007 Kaunas El paštas: kestutismalakauskas@kmult References 1 Bradley BL, Azzawai M, Jacobson M, et al Eosinophils, T- lymphocytes, mast cells, neutrophils and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subject and relationship with bronchial hyperresponsiveness J Allergy Clin Immunol 1991;88: Bousquet J, Chanez P, Campbell AM, Souques F, Lebel B, Enander I, et al Airway inflammation in mild intermittent and in persistent asthma Am J Respir Crit Care Med 1998; 157: De Graaf-in t Veld C, Garrelds IM, Koenders S, Gehrt van Wijk R Relationship between nasal hyperreactivity, mediators and eosinophils in patients with perennial allergic rhinitis and controls Clin Exp Allergy 1996;26: Ramsdale EH, Morris MM, Roberts RS, Tech M, Hargreave FE Asymptomatic bronchial hyperresponsiveness in rhinitis J Allergy Clin Immunol 1985;75: Milgrom H, Taussig LM Keeping children with exercise-induced asthma active Pediatrics 1999;104: Polosa R, Ciamarra I, Mangano G, Prosperini G, Pistorio MP, Vancheri C, et al Bronchial hyperresponsiveness and airway inflammation markers in nonasthmatics with allergic rhinitis Eur Respir J 2000;15: Corren J Allergic rhinitis and asthma: how important is the link? 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