Stress Enhances Airway Reactivity and Airway Inflammation in an Animal Model of Allergic Bronchial Asthma Ricarpa A. Joscuns, MD, Davin Quarcoo, MD, Peta C. Ack, MD, Upo Herz, Pu, Haratn Renz, MD, axo Buncuarn F. Kua, MD Objective: Despite the long-standing clinical assumption tat stress and asthma morbidity are associcted. convincing experimental evidence on mechanisms has been uravailible. A wide range of immurological, endocrinological, andl neurowal pathways are knowin t9 mediate and modalate a systomie stress response. Intorestingly, most of those mediators piay a crucial role in intating and perpetuating symptoms associated with bronchial asthma, To explore potential mechanisms linking stess to asthma exscer- bation we developed an animal model that combines allensic airway inflammation and exposure to stress. Methods: CBA/J mice ‘were sensitized by intraperitoneal injection of ovalbumin (OVA) and challenged with OVA aerosol via the airways. Additionally, ‘some mice were stressel hy exposure to an ulissonic sressor_ Airway hyperreactivity (AHR) was measured in vit by elestic fleld stimulation (EFS) of tracheal smoeth muscle elements. Bronchoalveolar lavage fluid (BAL) was cbvained and cell numbers setctmined. Cytokine levels of IL-4, IL-5, and IFN-y in BAL were determined by ELISA. Results: Ou findings demonstate that exogenously applied stress drarstically enhances airway reactivity in OVA-sensitized and challenged mice. Further, siress ‘sgmificantly increases allergen-induced airway inflammation identified by increased leukocyte (ie, easinonil) numbers in tronchoulveolar lavage Plaids, Conclusions: We found further evidence that stress can indcod exacctbate airway hypesreactivit sand sirway inflammation in sn animal mode! of allergi bronchial asthma and now introduce a novel marine model to identify ssresstriggered pathways, including mediators as neuroborones, neuropeptides. and markers of inflammation. Key words: sonic ssres, allergic asthma, airway inflammation, airway hyperreactivity, mouse mode! AHIR = airway hyperreactivity; AR = airway reactivity; BAL = bronchoalveolar lavage; EFS ~ electrical field stimulation; ELISA. enzyme Inked immuno sorbent assay: ES., = ffequency of halt ‘muximal tracheal contraction; GM-CSF = granulocyte-macrophage colony-stimuleting factor, IFN ~ Interferon; IL = Interleukin; IP = iraperitoneal; LaGetSi = Landesamt fir Arkeitsschatz, Gesurd- haeischucz und technische Sichecbeit Berlin (aubouty for animal reicarch condvc!); OVA ~ Ovelbumin; PBS ~ phosphate buffered saline: TAV/TH2 = T helper cell type and 2: TNF factor, VCAM-L = vascular cell adhesion molecule INTRODUCTION tress has long been suspested asa possible eause of acute asthma exacerbation in humans, even though conclusive experimental evidence is still lacking. For example, anecdotal reports have oflen associated stressful life events with the onset of reversible airway constriction besides other extiasic stimulant-like allergens, chemical initants, acute airway in fection, cold air, and exercise, Siess has been associated with asthina morbidity by clinical observation and epidemiclogical research (J-3), but litle is known about its underlying ‘mechanisms Stress as a precipitating or provoking factor in men has been implicate in diseases as varied as inflammatory bowel disease (4,5), eanoer (6,7) and atopic dermatitis (8, 9). There is increasing evidence from psychoneuroimmunological st ies in both animals and hurnans that stress results in concom- itart activation of cells fiom the nervous, endocrine, and immune systems and in the release of diverse biologically Tron te Deparinet of Inemil Medicina Chait-Carspue Vicchow, Humboldt University (A.J, PCA. BFR), Beto, Department of Petit: ries, Chante-Campis Virckow, Humboldt Univenity (D.03. Beri: and Deparment of Clivcel Chomstry and Molsulr Disgnoetc, Philippe Uni erst (UH, H.R, Marburg, Germany “Adress reprint request to" Risse Toachim, MD. Chaité-Compus Vie chow, Diotelissichen, Forchunzsceninan, R 20594, Augoterburger Pla, 13353 Beelm, Gemany. Eda: near cachimachartede ‘Resi for pblison July 29, 20D; revision resned Dacor 1, 2002 DOE 1010970 .FS¥ DO0DOSES2 SiON. AS Peychoromatic Medicine 65:811-815 (2003) DossL 740865054811 active compounds, including glucocorticoids, catecholamines, neuropeptides, und cytokines (10). Many of these have been shown to play a critical role in the pathogenesis of asthma and ‘might be the key for understanding the complex regulation processes that are involved. An animal model was developed fo stidy the psychoneuroimmunological network in a con- trolled environment. For this purpose we combined two es tablished models (11, 12)}-a model of allergic airway inflam- mation and a stress mouse model, The murine model of allergic airway inflammation reflects major pathophysiologi cal findings of allergic bronchial asthma, namely airway re- activity and airway inflammation (II, 13, 14) In asthmatie patients, enhanced zirway reactivity (AHR) to ‘unspecific stimuli and eosinophilic airway inflammation cor- relate well with the severity of the disease (13, 16). Ithas been suggested that both in asthmatic patients as i animals sub- jected to allergen-induced airway inflammation the develop: ‘ment of AHR is based on abnormalities of the neural system of the airways (17-20), OF the different methods to assess airway reactivity in mice, the ex vivo technique of electrical field stimulation of tracheal smooth muscle segments used in this study is best suited 10 dissect neuronal dysregulation of the airways (21), In patients with bronchial asthma, increased total leukocyte and eosinophil numbers in bronchial biopsies and in bron- choalveolar lavage ‘uid are proof of an increased airway inflammation (22-24). Similarly, in mice allergic airway in- flammation can be assessed by measuring these parameters A wealth of data demonstrates an important role for T helper type 2 cell (Th2) in the allergic inflammation. These activated cells release the cytokines interleukin (IL)4 (25) and IL-13 (26), which lead to the production of allergen. specific IgE antibodies by plasma-cells, and IL-S, a major regulating factor for growth, differentiation, and activation of eosinophils (27-30). Stress has been proposed to enbance the allergic inflammatory response by altering the Thl/Th2 bal- ance (31), Copyright96¥_tppinootbWitheine'& Wilkins. Unauthorized reproduction of this article is prohibited.Sound stress as an effective inductor of stress reactions has been used in various animal models (12, 32-34). The appara- tus and the stress protocol used in this study have been adopted fiom a murine model of stress-induced abortion. Here, a 24-hour period of sound stress induces maximal abor- tion rates in pregnant CBA/I mice (12), Data from pit studies in the model of allergen-induced airway inflammation combined with exogenously applicd stress suggested that a 24-hour period of sound stress induces the maximal reaetion in this model, The aim of this study was to investigate whether and how exogenously applied stress influences the airway reactivity ‘and to test the hypothesis that siress acts through increased airway inflammation in OVA-sensitized and provoked mice, METHODS Animals CBA mee wee purchased trom Charles River (Suzie, Germary) and :nainiined in an animal facility with a 12-hoe ligh/aek cle. Animal care ‘and experinenal procedures were followed according 19 institutional guide lines and conformed to the requirements of the state authority for animal research conduct (LaGetS, Bein) Protocol of Sensitization BAI mice were sensitized by inrapertoneal injecten (IP) of 10 us cickes ovalbumin (OVA, Grad VI. Signa Chom, Desentoin, Gennany) ccnulifed in 15 mg AVM), (Alum Inject Pvc, Rackfor IL, USA) on day 0, 14, and 21. To induce a strong local iflammavery response with increased leeeyte nme inthe bronchoalveslar invnge (BAL) mice were challenged twice with OVA aerosol (1% OVA dled in PBS) via the airways ‘on dey 26 and 27, as described previously (11\Fig. 1). Stress Treatment Afor IP teastization, mice were randomized iato wo diffont experi ‘mental groups. Coincidng with the fist OVA aeresel challenge, one gr04p of ance was exposed 1 seund stress fora single M4-hour perio, whi conta animals remsineé unlisturted (Fig 1) The scund sess was emit by a ‘eden sepellint device (Coarad Electonics, Helin, Germany) a «frequency ‘of 300 Hz for {second each time in intervals of 13 seconds, The stess device ‘was placed in the rouse cage ao that the ne could not excape poreapion of the sound, While tesed animals expenieeced exposure othe stesso in their 2h Sound ‘Stress 262728 Analysis. vA. Aerosol ‘llemen exposure Fig |. Experimental protocol After systemiesersiization on days 0, 14, ant 21, OVA IP mice ware clalleged twice wih OVA aeresal on ‘ays 26 and 27. Coineidng wit the first challenge, the sess group was exposed to sound sess for 24 hours while controls remained undisturbed ai R. A. JOACHIM et al. home cage, they were Kept in adiffret room rom consol animals so that he te groups wore sepaazed, Determination of Allergen-Specific Antibodies Blood was sampled from the lteral caudal veins on day 0 and 25 of sensitization (e, befoe sues appicalion). Bloed was eleted at oom te perature and centifagsd. Total jg ard allergen-specitie IaF antibody thers were detemnined in sera by enzyme liked immunosorbent assay (ELISA) 1s described previously (11). Ant-mouse IgE antibodies were obtained from Pharmingen, Hamburg, Germany. The anii-OVA IgE antbody ters of the samples were related to pooleé standard sera that were generated in the Inberatry. Detection limi were 1.95 ngiml for toa IgE ad 1S EU/ml for antiOVA IRE Determination of Airway Responsiveness. Airway reactivity (AR) was evaluated 24 hours afier the last OVA stalenge (ay 28). AR as well airway inflammation reflected by minders ‘oF infiltrating imine eels ni the irsays have Been show to be maximal between 24 co 48 hours afer the his allergen chlenge (pers communi~ cation FHamelman), AR was assessed in vir by cleric field simlaion (EFS) of vacheal snooh muscle wement aspreviusly described (LT, 21)-10 tele, animale were suri” and tracheal smooth muscle segments were prepared an placed in oan bus containing Krebs-HenseetbutTer (Sia, Germany). and suspended by tongular supports transicing the force of comractons. EFS was delivered by a Grass S48 simular using 12V, 2 ms ple duration, and 5 to 30 He frequencies. Each simelaton was ainained ‘unt peak contacile responses were obtained. The corractin in response to EFS stimalas was measured and the frequency that caused 50% of the ‘maximal coataction vas calculated ftom lozaithmic plots oF the contractile response versus the frequency of EFS and expressed 8 ES. Moan ES, from honstesscd animals was se es 100% and compared with mean ES, from strosed animale Bronchoalveolar Lavage Bronchealvoohr lnage (RAL) was obtained 24 hourt afer the second aionay ellen (day 28). ‘Animale were aserificed and trcheas cannulated, Airways were Lavaged twig with 0.8m cp-cold PSS andthe frat umber of ces was determined as previously dered (11). Cytogpine were prepared for cock sample by cenfugation of 30 ul BAL Mai. Aer fixation, etospis were stated with Diff Quik (Dads Baling, Marburg, Germany) and eiferental call courts wore performed. Cells were clasiied #8 newropals, eosinophils, Iympho- ste, or macrophages using standard porphologicl entra, Calle lage Huds were stored a -20°C unt futher analysis Determination of Cytokin Lavage Fluids Invrleukin (IL) 4, 11-5, nd feteferon (IFN) yeontent of BAL Muds was determined by FLISA as described previously (11) Anitodies were obsained fiom Pharmingen, Hamburg, Germany. Detstion limits were 16 petal for IL-4, 27 pel for IL-S, and 40 ppm! for IFNy Histological Evaluation For asiessmenr of airway inflammation, lungs were tke insta with 7% formaldshyde via the tmaches, then removed and stored in formaldehyde, Profinembedded sections were stained with hematoxylin and eosin (H 8 5. Statistics Significance of diforences between groups was determinad usieg the nonparametric Mant-Whitney U-test. Significance was set atp = (5. Ress ago prosoefed as moun ales, Psychosomatic Medicine 65:811-815 (2003) Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.STRESS AND ASTHMA RESULTS Airway Reactivity ‘The AR was measured by electric field stimulation (EFS) ‘of tracheal smooth muscle elements. It was previously shown that the base-line ESxp value (electrical stimulation [Hz] re ‘quired to induce half-maximal constriction of tracheal smooth muscle segments) of untreated mice is around 4 Hz (11, 21), ‘Twenty-four hours after the second allergen challenge both stressed and nonstressed sensitized mice showed airway reac- tivity, The ESsq was 2.1 Iz in the nonsiressed group (V = 8) ‘and set as 100% AR was further increased by 40% in the stressed group, as ES) was reached at a mean of 1.3 Hz (N = 8p <5) (Fig. 2) Airway Inflammation To determine the cellular constituents of leukocytes infil ‘rating the animals’ airways, lung tissues and bronchoalveolar Iavage (BAL) fluids from stressed and nonstressed OVA- sensitized animals were analyzed. Morphologically, all sensi- tized animals developed signs of airway inflammation with infiltrating mononuclear cells around small blood vessels and lower conducting airways after allergen challenge: In the BAL fluid a significantly higher number of total leukocytes was found in sensitized and siressed animals (mean 276 X 10m, N= 12) compared with sensitized, nonstressed control mice (199 x 10°/ml, = 9, p < 05), Cell differen tiation by Diff Quik staining revealed that the difference between the two groups was predominantly based on an increased number of eosinophils (169 X 10%/ml vs. 101 x. 10%!) (Fig. 3) To characterize inflammation markers, the cytokines Inter- Jeukin (IL}-4, IL-5, and Interferon (IFN)-y were measured in cps Pe | 7 La == Leaks Lympes Eos Neuros Monotac Fig. 3. Total leukocyte musnber and cll differentiation in BAL Aid of OVA-serstized and airway-shallenged CBA mice. Inca are ream and SD fiom stresed (W = 12) and nonstessed (V = 9) 1 sivas (Leen ~ leukocyte, Lymapos ~ Irmphesten, os = cosmopnil granulocytes, Neuros = netephilgratloeytes, Mono/Mae=Mloroeytev/Mscrophager).* p = 08 BAL fluids by ELISA. There was no difference between nonstressed and stressed animals with respect to [L-5 (15 + 28 pwml vs. 60 = 60 pa/mil), whereas the levels of IL-4 and IFN-y remained below detection limits. Ovalbumin Sensitization and Immunoglobulin Synthesis CBAVJ mice sensitized to OVA by intraperitoneal injec tions developed high anti-OVA IgE antibody titers and in~ creased total IgE as evaluated by ELISA before stress appli- cation (Table 1). DISCUSSION In the present study we demonstrate that exogenously ap- plied stress dramatically enhances airway reactivity in OVA sensitized and challenged mice. Additionally, the application 120 . of stress aggravates the allergen-induced airway inflamma- tion, The increased eosinophil counts in BAL fluid of stressed 100 L mice point to a possible mechanism linking stress to the 2 exacerbation of asthma symptoms. B80. Stross and Airway Reactivity 3 Assessing the airway reactivity by electrical field stimula & tion (EFS), we show for the first time that exogenously ap- 3 OO plied stress increases hyperreactivity of the airways (AHR) in a ‘TABLE 1, Assessment of Immunoslobatin Production in CBA/) 40 Mice before and after OVA Sensitiat » Immunoglobulin CBM no stress stress Production’ Day 0 Day 28 OVA ipJ GVA aerosol Total IgE (ng/ml) WS +44 4133 + 1486 ant-OVA Igé U/m) <15 1034» 590 fect of stro on AHIR measured in OVA sensitized and sirwsy” ‘nllenged CBA'F mics. Ics ae mean and SEN fom sessed In’ =") and nonstesced (V = 8) senstized CRALT tice. The Frequency thar eased 509% of maximal contaction (ES) was ‘aleulated. Away contactily i expressed a percent of non strossed cont (21 Ha}. * p = 05. Psychosomatic Medicine 65:811-815 (2003) * Tata and allergen-specie serum anibedy production was determi by ELISA. Blood was deawn fom animals before sensitization and one day bofore aress application t prove sensitization, Presented are means and = SD from 12to 24 animals ia each group. 813 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.