Titel
Modulation of pulmonary CD4+, CD8+, γδ T cells, apoptosis and proliferation by stress in a murine asthma bronchiale model

Authors
Isabel Masson1, Ricarda A. Joachim1, Matthias Rose1, Eva M. J. Peters1,2*

Affiliations
1Charite Centre 12 (CC12) for Internal Medicine and Dermatology, Department of Psychosomatic Medicine and Psychotherapy, Universitätsmedizin Charité, Berlin, Germany.
2Psychoneuroimmunology Laboratory, Department of Psychosomatic Medicine and Psychotherapy, Justus-Liebig University, Giessen, Germany.

*Corresponding author at: Psychoneuroimmunology Laboratory, JLU Gießen and Charité Berlin, Postal Address: Ludwigstr. 78; D-35392 Gießen, Germany
Phone: +49 641 99 45642 or +49 30 450 553113; Fax: +49 30 80499632
Email:eva.peters@eva-peters.com 

SUMMARY 
Stress enhances airway inflammation in asthma and worsens disease severity. Activated stress response-systems affect important cellular players in allergic asthma such as CD4+, CD8+ and γδ T cells. Resulting enhanced inflammation, apoptosis and proliferation within bronchoepithelial area are important pathogenic factors. However, interaction kinetics of stress and allergic inflammation is missing. Based on an established murine stress-asthma-model we studied kinetics of pulmonal CD4+, CD8+ and γδ T cells from 6 until 96 h after allergen challenge and termination of 24 h noise-stress exposure. In addition, peribronchial proliferating and apoptotic cells and bronchoalveolar lavage (BAL) leucocytes were examined. Stress significantly reduced peribronchial CD4+ cells at 12 h and 24 h, and CD8+ cells at 96 h without significant effects on γδ T cells. Stress also significantly increased apoptotic cells at 6 h and reduced proliferating cells at 96 h in bronchial epithelia and peribronchial tissue. At the same time, results of BAL examination indicated that stress significantly accelerated leucocyte invasion of the airway lumen 24 h after allergen challenge, including eosinophils and lymphocytes. In summary, we found stress to reduce CD4+ and CD8+ cells within the peribronchial tissue while enhancing leucocytic accumulation in BAL. An accelerated migration towards the airway lumen can be discussed. In addition, via reduced proliferation and increased apoptosis in epithelia, stress can affect bronchoepithelial regeneration and barrier function.
Keywords
Noise stress; airway inflammation kinetics; allergic asthma; mouse model; T cells, apoptosis; proliferation.
1.	Introduction 
Bronchial asthma is a common chronic inflammatory airway disease that leads to airway hyperresponsiveness (AHR) with variable airway obstruction. Genetic predisposition, respiratory infections and environmental factors potently affect the development and course of the disease (German Medical Association, 2009). Stress is an additional, now undisputed component of asthma bronchiale exacerbations (Chen and Miller, 2007). In a longitudinal cohort study for example, association of stress with asthma incidence, allergic rhinitis and atopic dermatitis was shown (Rod et al., 2012). 
In the course of stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis and the sympatho-adrenergic system (SAS), the concentration of corticotropin releasing hormone (CRH) is increased, as are adrenocorticotropic hormone (ACTH), corticosteroids and catecholamines. Besides these, stress leads to release of neurotrophins and neuropeptides, among them Substance P (SP) (Chen and Miller, 2007; Black, 2002). A substantial number of studies employing mostly the mouse model of asthma bronchiale assessed stress-mediated increased airway inflammation and AHR with a focus on these classical stress-mediators (Priftis et al., 2009). These studies revealed for example an increased concentration of SP in sensory neurons of vagal ganglia that innervate the respiratory system, which was accompanied by an increase of inflammatory BAL leukocytes 24 h after stress in mice (Joachim et al., 2006). 

It is in fact believed that stress worsens the pathology in asthma bronchiale primarily by enhancing airway inflammation (Chen and Miller, 2007). In the airways of asthmatics, inflammatory pulmonal infiltrats consist to a large part of CD4+ T cells (Ricciardolo et al., 2003; Lommatzsch et al., 2006), CD8+ T cells (Robinson et al., 1993) and γδ T cells (Hamazoui et al., 2002) after allergen inhalation. CD4+ T cells recognize allergens processed by antigen-presenting cells (APC) and trigger the allergy cascade that ultimately leads to IgE formation, mast cell degranulation, and migration of inflammatory cells into the airways (Holgate, 2008). In addition, CD8+ and γδ T cells are involved in asthma pathology (Robinson, 2010). 
It is well known, that T cells express receptors for mediators of the stress response (Chen and Miller, 2007). Stress, corticosteroids and catecholamines affect the number of T cells bearing CD4 and CD8 for example in murine blood, skin (Dhabhar et al.; 2012) and intestinal tissue, also including  γδ T cells(Jarillo-Luna et al, 2008. In addition, SP can affect immune responses via interaction with the HPA and the SAS (Black; 2001) and modulate T cells via neurokinine (NK) receptors, as demonstrated in vitro for human blood cells (Hood et al; 2000). Thus neuroendocrine-immune interaction may control the described deterioration of asthma bronchiale by stress via T cells. These cells could therefore represent an important general pathophysiological target for stress. 
In chronic airway inflammation, pathological remodeling processes characterize the clinical picture in addition to inflammation and a dysregulation of proliferation and apoptosis is often observed in and around the airway epithelia of asthmatics (Puddicombe et al. 2003; Benayoun et al., 2001).This results in a limited barrier function of the bronchial epithelium and an increased permeability for further defective influences (Holgate, 2008). Negative regulation of tissue regeneration processes by stress is a wellknown phenomen (Choi et al., 2005; Gouin et al., 2011). As epithelial damage with barrier dysfunction contributes to the pathology of asthma (Holgate, 2008; Puddicombe et al, 2003), stress-impaired epidermal barrier function and proliferation is of note (Choi et al., 2005) and possibly changes in apoptosis or proliferation in peribronchial tissue by stress may contribute to the observed stress-induced clinical worsening of asthma bronchiale.
At present, the question whether stress potentially promotes inflammatory airway responses via modulation of T cell kinetics and kinetic of apoptosis and proliferation in pulmonal tissue is open. Here we explore, if stress can modulate kinetics of T cell subsets involved in murine airway inflammation, namely CD4+, CD8+ and γδ T cells (Robinson, 2010), as well as the number of apoptotic and proliferating cells in pulmonal tissue, especially in and around the bronchial epithelia. We chose an immunohistochemical method to assess respective stress effects in situ and investigated the development of CD4+, CD8+, and γδ T cell numbers in alveolar and peribronchial tissue simultaneously with apoptotic and proliferating cell numbers in peribronchial tissue tissue and leukocyte subpopulations in BAL. We chose an observation period of 6 h to 96 h after exposure to noise stress and establishment of allergic airway inflammation, when inflammatory cellular infiltration of murine lung tissue can be expected in a periodic manner (Lommatzsch et al., 2006). Because peribronchial areas are innervated densely by nerval systems (Lundberg et al, 1984; Dinh et al., 2011) known to be involved in stress reactions of the organism, we distinguished between peribronchial and alveolar areas. 

2.	Methods
2.1.	Animals
Balb/c mice were purchased from the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV, Berlin, Germany) and were kept in the animal barrier facility at the Charité, Virchow Hospital, University Medicine Berlin, Germany under pathogen free conditions with a 12 h light/dark cycle and food and water ad libitum. Animal care and experimental procedures were followed according to institutional guidelines and conformed to the requirements of the state authority for animal research conduct (LaGetSi, Berlin).
2.2.	Allergen sensitization and challenge
All animals (n=60) underwent sensitization procedure. After sensitization by intraperitoneal (i.p.) injection of 10 µg chicken ovalbumin (OVA, Grad VI, Sigma Chemie, Deisenhofen, Germany) emulsified in 1.5 mg Al(OH)3 (Alum Imject Pierce, Rockford, IL, USA) on days 0, 14, and 21, Balb/c mice were challenged with OVA aerosol (1% OVA diluted in phosphate buffered saline (PBS) via the airways on days 26 and 27 for 20 min (Joachim et al., 2006).
2.3.	Stress exposure
Sensitized mice were randomized into two experimental groups. Starting with the first OVA challenge, mice of the stress group were exposed to noise for a single 24 h period in a separate room (n=30), while control mice remained undisturbed (n=30). Noise stress was generated by a rodent repellant device (Conrad Electronics, Berlin, Germany) placed within the cage, which emitted a 300 Hz tone in irregular intervals 4 x per min (Joachim et al., 2006).
2.4.	Tissue collection
Mice were sacrificed 6 h, 12 h, 24 h, 48 h and 96 h after the second airway challenge by cervical dislocation and the lungs and trachea were dissected. After BAL, lungs were intrabronchially injected with 800 μl of tissue freezing medium (Tissue Tek, Sakura Finetek, Torrance, CA, USA) diluted with PBS (1:5) and immediately snap frozen in liquid nitrogen. Lungs were stored at -80°C until cryo-sectioning (8 μm) at -20 to -25°C (Microm, Walldorf, Germany). Sections were fixed, air-dried and stored at -20°C. Sections for terminal deoxynucleotidyl transferase (TdT) mediated nick end labeling (TUNEL) - and Ki67-immunofluorescence staining were mounted on microscope slides treated with 3‑aminopropyltriethoxysilane.
2.5.	Immunohistochemistry for T-cell surface markers
Defrosted acetone-fixed cryosections were treated with avidin and biotin blocking solution (Vector laboratories, Burlingame, CA, USA), and protein block (DAKO, Hamburg, Germany) to prevent unspecific bindings. Incubation with primary antibodies dissolved in tris buffered saline (TBS) and fetal calf serum (FCS) 1% was performed for 1h (BD Pharmingen, Heidelberg; CD4-, CD8-rat-anti-mouse 1:100, γδ TZR-hamster-anti-mouse 1:70). Secondary antibodies were dissolved in TBS, mice normal serum (MNS) 4%, and (goat normal serum) GNS 2% (Jackson Immunoresearch, West Grove, PA, USA; CD4-, CD8-goat-anti-rat: 1:200, γδ-TCR-goat-anti-hamster 1:70), and were incubated for 30 min. Avidin-biotin-peroxidase complex with components ABC-A and ABC-B (Vector laboratories, Burlingame, CA, USA) was dissolved in TBS (1:100) for 30 min and sections were incubated for 30 min. After this, sections were treated with alkaline phosphatase developer. Sections were counter-stained with haemalaun. Negative control was performed using rat normal serum (RNS) in TBS instead of the primary CD4- and CD8-antibodies, and using hamster normal serum (HNS) in TBS instead of the primary γδ-antibody. 
2.6.	Immunofluorescence double staining for TUNEL and Ki67
Apoptotic cells were stained by direct immunofluorescence using the TUNEL assay, proliferating cells were stained by indirect immunofluorescence labeling of Ki67. Cryosections were fixed with formaline and postfixed in ethanol-acetic acid at -20°C. Tissue was incubated with equilibration buffer for 5 min, incubated with a solution of TdT-digoxine and reaction buffer for 60 min at 37°C and afterwards sections were treated with stop buffer as described by the manufacturer (TUNEL kit; Intergen, New York, USA) for 30 min at 37°C. After incubation with blocking solution (ZNS 10% in PBS) for 20 min, sections were incubated over night with primary Ki67 rat-anti-mouse antibody (Monotec, Hamburg; 1:100 in PBS and ZNS 2%). Apoptotic cells were visualized by treatment with anti-digoxigenin-linked fluoresceine iso-thiocyanate (FITC) (TUNEL kit, Intergen, New York, USA) for 30 min. Proliferating cells were visualized by incubation with secondary tetramethyl rhodamine iso-thiocyanate (TRITC)-linked goat-anti-rat-antibody (Jackson Immunoresearch, West Grove, PA, USA, 1:200 in PBS, MNS 4%, ZNS 2%) for 30 min in the dark. Nuclei were counterstained by incubation with 4’, 6 diamidino-2-phenylindole-dihydrochloride (DAPI) (Roche, Mannheim, Germany) for 5 min. Negative control was performed by leaving out TdT in the procedure for TUNEL, and by replacing Ki67 antibody with RNS in PBS. Sections were mounted on microscopy slides using a fluorescence protective medium (Vecta Shield, Vector Laboratories, Burlingame, CA, USA) and stored at -80°C. All light sensitive procedures were performed in the dark. 
2.7.	Bronchoalveolar lavage
6 h, 12 h, 24 h, 48 h and 96 h after the second airway challenge on day 28 BAL fluid was obtained after sacrification of the animals. Tracheal cannulation was done and airways were lavaged twice with 0.8 ml icecold PBS, and the total number of cells in BAL fluid was determined. Cytospins were prepared by centrifugation of 50 µl BAL fluid for each sample. After fixation, cytospins were stained with Diff Quik (Dade Behring, Marburg, Germany) according to the manufacturer’s data and differential cell counts were performed. Standard morphological criteria were used to classify cells as eosinophils, lymphocytes, neutrophils or macrophages. Not less than 100 cells per cytospin were counted. Airway inflammation was evaluated by inflammatory cell counts in BAL (Joachim et al., 2006).
2.8.	Data analysis
At each point in time one control group and one stress group (n=6) were sacrificed. Assignment to groups and microscopic cell counts were performed by two independent investigators. Cells in alveoles, peribronchial tissue and BAL were counted in 10 consecutive microscopic fields per mouse, using a grit (1 mm2) at a magnification of 1:400. CD4+, CD8+ and γδ T cells were counted using a light microscope (Zeiss, Jena, Germany). Immunfluorscence microscopy of apoptotic and proliferating cells was performed within the next two weeks after staining under protection of light (Axioplan, Zeiss, Jena, Germany). Photo documentation was performed using a digital imaging system (AxioVision; Zeiss, Göttingen, Germany; Spot advanced software, Visitron, Puchheim, Germany). Statistic calculations were done by SPSS software based on means per animal. Data are presented in boxplots, showing means of medians per group +- standard error of the mean (SEM). Univariate analysis of variance (ANOVA) was performed to detect general influences of points in time after allergen challenge or stress (n=30). For further evaluation concerning the factor time (n=12), posthoc dunnett test was applied. Significant differences between stressed and control mice (n=6) were determined using the nonparametric Mann-Whitney U-test. Pearsons correlation test was applied to assess correlations between numbers of CD4+, CD8+ and γδ T cells with numbers of apoptotic, proliferating and BAL cells. P values of <0,05 were regarded as significant concerning all statistic calculations in this experiment. 

3.	Results
3.1.	Dynamics of pulmonal CD4+, CD8+ and γδ T cells infiltrate development after noise stress
CD4+ cells represented the predominating cell population in alveolar and pereibronchial areas compared to CD8+ and γδ T cells (Fig. 1A) at all investigated points in time, both in stressed and control mice. In both groups we observed an increase of CD4+ and CD8+ cells 24 h after allergen challenge, which is when onset of infiltration is commonly reported in comparable murine models of asthma bronchiale (Stock et al., 2004; Lommatzsch et al., 2006). γδ T cells were numerous at early points in time but considerably sparse from 24 h after allergen challenge in both stress and control mice. 
Compared to control, stress exposure significantly reduced the number of peribronchial CD4+ cells 12 h and 24 h after allergen challenge (p=0,025; Mann-Whitney test). Peribronchial CD8+ cell numbers were reduced 96 h after allergen challenge (p=0,010; Mann-Whitney test) (Fig. 2A-B). Stress had no significant effect on the number of γδ T cells throughout the investigation period (Fig. 2C), albeit there was a tendency towards a decreased number of γδ T cells in peribronchial tissue 12 h after allergen challenge (p=0,063; Mann-Whitney test). 
In the alveolar tissue stress had no significant effects on cell counts of any population examined in this study (data not shown). 
3.2.	Eosinophils, lymphocytes and neutrophils are increased in BAL after stress
BAL of sensitized mice after allergen challenge revealed high numbers of leukocytes, including eosinophils. These features indicate airway inflammation (Fig. 3A-B), as established for this model of murine allergic airway inflammation (Joachim et al., 2006). Eosinophils were the most abundant leukocyte population all over the examination period in stressed and control mice (Fig. 3B). 
Stress affected the kinetics of BAL cells. In BAL of stressed mice the increase of leukocytes preceded the increase of leukocytes in control mice, already detectable 24 h after allergen challenge. Leukocyte counts were significantly increased in stressed mice compared to control mice at 24 h after allergen challenge (Fig. 3A). These dynamics included eosinophils (p=0,025; Mann-Whitney test), lymphocytes (p=0,045; Mann-Whitney test) and neutrophils (p=0,020; Mann-Whitney test) which were all boosted at 24 h after allergen challenge in the stressed mice (Fig. 3B, 3D, 3E). Concerning BAL-lymphocytes of stressed mice, the increased counts 24 h after allergen challenge were followed by decreased counts at 48 h after allergen challenge (p=0,047; Mann-Whitney test) (Fig. 3D), indicating faster but not generally more intense inflammation in the airway lumen. Neutrophils were furthermore boosted by stress at 6 h after allergen challenge (p=0,018; Mann-Whitney test) (Fig. 3E), while macrophages and monocytes remained generally unaffected by stress (Fig. 3C). 
3.3.	Apoptotic and proliferating cells in peribronchial tissue
Apoptotic and proliferating cells were generally sparse and were seen preferentially in the peribronchial and epithelial tissue (Fig. 4A). 
In stressed and control mice the number of apoptotic cells in and around the bronchial epithelia was not significantly changed by time. Comparison between stress and control mice revealed that stress had early effects on the number of apoptotic cells by reducing them significantly at 6 h after allergen challenge (p=0,006; Mann-Whitney test) (Fig. 5A).
Proliferating cells in stressed and control mice were more numerous at earlier stages and more sparse at 96 h after allergen challenge. Comparison between stress and control mice revealed that stress decreased the number of proliferating cells significantly at 96 h after allergen challenge (p=0,006; Mann-Whitney test) (Fig. 5B). 
No correlation was found between development of CD4+, CD8+ and γδ T cell numbers with apoptotic and proliferating cell numbers (analysis with Pearsons’s correlation test, data not shown).

4.	Discussion 
The present study reveals dynamic and adverse effects of stress on cell numbers in peribronchial tissue and BAL. Our study shows that after induction of allergic inflammation, stress can reduce CD4+ and CD8+ cells in peribronchial tissue. Here CD4+ cells seem to react faster to stress than CD8+ cells, as indicated by reduction of CD4+ cells at 12h and 24h, while peribronchial CD8+ cells were reduced later at 96h after allergen challenge by stress. At the same time, in our study BAL examination demonstrates accelerating effects of stress on the inflammatory cell influx kinetics into the bronchoalveolar lumen with most pronounced effects observed for eosinophils, lymphocytes and neutrophils at 24h after allergen challenge. Stress also compromises peribronchial regeneration as evidenced by enhanced apoptosis at 6h and reduced proliferation at 96h post allergen challenge in peribronchial and epithelial areas. These tissue destructive stress-effects are likely contributing to the reported enhanced airway inflammation and clinical worsening in asthma bronchiale affected mammals exposed to noise stress and other perceivable stressors (Priftis et al, 2009; Chen and Miller, 2007).
In murine lung tissue and blood of mice without pretreatment short-term stress has been reported to decrease CD4+ and CD8+ cells simultaneously within the first 4 hours after stress, leaving open when recovery happens (Kanemi et al., 2005). In several desease models stress-induced changes of CD4+ and CD8+ cell numbers have been reported and connected to dysfunctional immune responses. Induction of malignant skin tumors led to a more rapid tumor progression in association with decreased numbers of cutaneous CD4+ cells when mice were exposed to chronic immobilization stress (Saul et al., 2005). Also, chronic immobilization stress was reported to impair immune defense against herpes simplex virus related to decreased CD8+ T cell numbers in murine infected neuronal ganglia (Freeman et al., 2007) and in the course of murine choriomeningitis, social stress reduced numbers of virus specific CD8+ splenocytes (Sommershof et al., 2011). Stress can also enhance immune function. In skin of mice with cutaneous squamosal cancer short term stress enhanced numbers of CD4+  and CD8+ cells in skin while preventing tumor progression (Dhabhar et al., 2010). 
In our study the stress-induced peribronchial tissue cell decrease is guided by transiently enhanced airway inflammation after stress, as indicated by BAL results in our murine stress asthma model. Several murine studies reveal enhancing effects of stress on allergen-induced leukocyte accumulation in BAL and also in sputum of asthmatic children eosinophils were increased by stress (Priftis et al., 2009; Liu et al., 2002). Discrepant development of leukocytes in lung tissue and BAL in the murine asthma model after 24 h noise stress was reported before. Leukocytes in stressed compared to control mice were increased in BAL at 24 h, while they were reduced in pulmonal tissue at 6 h (Joachim et al., 2007).(  Decrease of pulmonal tissue CD8+ cells has been demonstrated for CBA/J mice, which were examined only at 24 h after noise stress and allergen challengeand also showed enhanced inflammation in BAL by stress (Brünahl, 2005)). However, these studies were missing further kinetics. 
In the course of airway inflammation migration of inflammatory cells out of the lung tissue into the bronchoalveolar lumen is a common phenomen and can explain discrepant cell counts in BAL and bronchial tissue (Persson and Uller, 2010). Stress is known to affect migration of lymphocytes into different compartments and can thereby instantly enhance immune function (Dhabhar et al., 2012). Here we show, that stress-induced CD4+ cell reduction in peribronchial tissue precedes stress-induced enhancement of BAL-leukocytes, including lymphocytes. According to the transiently increased number of BAL-lymphocytes after stress exposure in our study, we assume that stress increases migration of lymphocytes towards the airway lumen, where the immediate contact with the allergen takes place. We don’t suppose proliferation or apoptosis to be relevant for stress effects on peribronchial cell counts, as there is no statistical correlation between the peribronchial development of CD4+ or CD8+ cell counts and apoptotic or proliferating cells (analysis with Pearsons’s correlation test, data not shown). 
In the murine asthma model it is established that a large part of CD4-expressing cells corresponds to activated T cells in the airways (Garlisi et al., 1997). There is supporting evidence that lymphocytes in murine BAL after allergen challenge consist for the most part of CD4+ cells, at least in Balb/c mice after treatment with Ovalbumin (Lommatzsch et al., 2006). Antigen-specific activated pulmonal CD4+ cells have a high migratory capacity towards the airway lumen (Hasegawa et al., 2010) and specialized T effector cells with high migratory capacity are more affected by stress-induced mobilization into different compartments than are naive cells (Dhabhar et al., 2012; Atanackovic et al., 2006). We assume, that stress triggered invasion of the bronchoalveolar lumen by inflammatory cells, including CD4+ cells, can lead at least to a transient deteoration of asthma bronchiale. Simultaneous increase of AHR and inflammatory BAL leukocytes after stress was demonstrated in some murine asthma models and could reflect this idea (Priftis et al; 2009). At the same time passage of the bronchial epithelia is an important mechanism for resolution of pulmonal inflammation (Persson and Uller, 2010). With respect to decreased CD4+ cells in peribronchial tissue and accelerated lymphocyte accumulation in BAL after stress in our study an accelerated resolution of airway tissue inflammation by stress in the further course can be discussed.
Migration of pulmonal T cells can be facilitated by several stress-sensitive mechanisms. Upregulation of mRNA for intercellular-cell-adhesion-molecule (ICAM-1) in pulmonal tissue of untreated mice after stress treatment was observed (Curry et al., 2010) and elevation of BAL-eotaxin levels was detected in murine airway inflammation after noise stress (Joachim et al., 2007). Possibly these observations are transferable to our experimental set up and explain the pro-inflammatory nature of involved CD4+ T cells and the mechanisms behind their relocation after stress exposure. 
We show peribronchial CD8+ cell decrease by stress to be a relatively late event, at 96 h after allergen challenge. Expression of CD8 is found almost exclusively on T cells in the context of ovalbumin-provoked pulmonal inflammation in Balb/c mice (Stock et al., 2004). On the one hand, murine pulmonal CD8+ T cells can enhance airway inflammation by synthesis of IL-4 and IL-5 (Stock et al., 2004). Accordingly, in human asthmatic airways association of leukotriene B4 receptor positive IL-13-secreting CD8+ cells with airway obstruction was demonstrated (Dakhama et al., 2013). On the other hand they can regulate it by synthesis of IL-10 (Stock et al., 2004), which can limit inflammatory processes, including reduction of TH2-cytokines and impaired T cell proliferation (Ng et al., 2013). Against this background peribronchial CD8+ cell reduction after stress in our study may have divergent effects on airway inflammation.
We found no significant difference in pulmonal γδ T cell counts between stressed and control mice. Also, in both stressed and control mice, pulmonal γδ T cells were reduced from 24 h until 96h after allergen challenge. Since in our study, allergen challenge via the airways was done twice, the intensity of inflammation generated may not have been sufficient to increase γδ T cell numbers. However, there was a tendency of decreased peribronchial γδ T cell numbers after stress at 12 h after allergen challenge (p=0,063; Mann-Whitney test). γδ T cells consist of several subtypes with differing functions and presence of γδ T cells in airways is based predominantly on their local proliferation (Kirby et al., 2007). Under defined conditions as in murine asthma models, a high percentage of Vγ4+ (Hahn et al., 2003) and Vy6+ cells (Wands et al., 2005) can be expected. These cells can reduce pulmonal inflammation (Carding and Egan, 2002; O’Brien and Born, 2010). Against this background the peribronchial γδ T cell reduction after stress in or study can be interpreted as noxious impact of stress on asthma bronchiale within the peribronchial tissue.
Apoptotic cells in epithelial and peribronchial areas were increased only as early as 6 h after allergen challenge by stress in our study, hence preceded inflammatory effects. Increased apoptosis in bronchial epithelia has been demonstrated in murine asthma models and for asthmatics and linked to epithelial damage (White, 2011). Epithelial damage contributes to pathological remodeling processes of the airways and is even regarded as causing itself airway inflammation (White, 2011; Holgate, 2008). In the murine hair follicle noise stress as well as SP increased apoptosis (Arck et al., 2001). Glucocorticoids can promote apoptosis in peribronchial tissue of asthmatics (O'Sullivan et al., 2004), in human airway epithelial cells (White, 2011) and murine intestinal epithelial cells (Jarillo-Luna et al., 2008). According to the increased number of apoptotic cells in and around the bronchial epithelia after stress in our study, stress could cause airway epithelia damage already at early points in time after allergen contact and thereby contribute to worsening of airway inflammation.
Proliferating cells were reduced by stress in and around the bronchial epithelia only at 96 h after allergen challenge in our study. Decreased proliferation of epithelial cells from asthmatics has been reported, resulting in epithelial damage which is linked to epithelial leakage and reduced barrier function (Holgate, 2008; Puddicombe et al., 2003). In the murine model of cutaneous inflammation stress compromises epidermal proliferation and epidermal barrier function (Choi et al., 2005). Among the stress-mediators responsible for this effect, glucocorticoids have a high potential to decrease epithelial proliferation (Choi et al., 2006). Inhibition of proliferation has been demonstrated also for SP and noise stress in the murine hair follicle (Arck et al., 2001). As in our study impairment of proliferation by stress was a relatively late event, when tissue regeneration in allergic airway inflammation plays a major role (Ricciardolo et al., 2003), we suppose a negative impact of stress on airway epithelia tissue protecting processes, thereby increasing airway inflammation.
The present study reveals certain time windows for the respective stress effects in allergic airway inflammation. Further studies are necessary to clarify the precise mechanisms of the stress-induced decrease in peribronchial CD4+ and CD8+ cell numbers. It also remains open if the change in proliferation and apoptosis after stress is restricted to certain cell types in and around the peribronchial epithelia. In conclusion, stress exposure leads to reduction of CD4+ and CD8+ cell infiltrates in peribronchial tissue while it accelerates mobilization of some leukocyte subsets into the airway lumen and compromises peribronchial regeneration in a timed and disease relevant fashion. 

Conflict of interest
All authors declare that they have no conflicts of interest.

Acknowledgement
The author is greatful to P.C. Arck for   , E. Hagen, P. Moschanski, P. Busse….. for technical advice
This study was supported by a grant from the German Research Association to R.A.J. (DFG jo 356/2-1) and to E.M.J.P (DFG pe 890/4-1).

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6.	Figure captions
 Fig. 1A-C: Immunhistochemical staining of CD4+, CD8+ and γδ T cells in pulmonal tissue. CD4+ (A), CD8+ (B) and γδ T cells (C) in control mice (no stress) and stressed mice (stress) 24 h after allergen challenge (50x). Br: bronchus. Marked sections correspond with the detailed view displayed. 

Fig. 2 A-C: Peribronchial tissue infiltrats of CD4+, CD8+ and γδ T-cells at different points in time after allergen challenge. CD4+ cells (A), CD8+ cells (B), γδ T-cells (C). White bars: control mice, grey bars: stressed mice. Data are shown as median ± standard deviation. *: p<0,05 (Mann-Whitney test) stress vs. control, #: p<0,05 (ANOVA with post hoc Dunnett test: stress and control mice together at each point in time vs. 6 h after allergen challenge), °: outliers.

Fig. 3 A-E: Cells in bronchoalveolar lavage fluid at different points in time after allergen challenge. Leukocytes (Leu) (A), lymphocytes (Lym) (B), neutrophilic granulocytes (Neu) (C), eosinophilic granulocytes (Eos) (D), macrophages and monocytes (Mac) (E). White bars: control mice, grey bars: stressed mice. Data are shown as median ± standard deviation. *: p<0,05 (Mann-Whitney test) stress vs. control, #: p<0,05 (ANOVA with post hoc Dunnett test: stress and control mice together at each point in time vs. 6 h after allergen challenge), °: outliers.

Fig. 4 A-B: Immunfluorescence staining of apoptotic and proliferating cells in pulmonal tissue. Apoptotic cells (Apo) of control mice (no stress) and stressed mice (stress) 6 h after allergen challenge (A) and proliferating cells (Pro) of control mice and stressed mice 96 h after allergen challenge (B). Apoptotic cells were visualized via TUNEL method (green), proliferating cells were visualized by detecting Ki67 with antibody-conjugated rhodamine (red). Nuclei were stained by DAPI (blue). By microscopic examination with different filters in identical sections, cells positive in TUNEL and DAPI staining were considered as apoptotic cells. FITC (TUNEL) and DAPI filters were combined at a magnification of 400x (A). Cells positive in Ki67 and DAPI staining were considered as proliferating cells. TRITC (Ki67) and DAPI filters were combined at a magnification of 400x (B). Apoptotic and proliferating cells were localized especially in peribronchial (p) and epithelial tissues (e). 

Fig. 5A-B: Apoptotic and proliferating cells in peribronchial tissue and epithelia at different points in time after allergen challenge. Apoptotic cells (Apo) (A), proliferating cells (Pro) (B). White bars: control mice, grey bars: stressed mice. Data are shown as median ± standard deviation. *: p<0,05 (Mann-Whitney test) stress vs. control, #: p<0,05 (ANOVA with post hoc Dunnett test: stress and control mice together at each point in time 6 h after allergen challenge), °: outliers.