Stress amplifies lung tissue mechanics, inflammation ...

Experimental Lung Research, 38, 344–354, 2012 Copyright © 2012 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2012.704484

Stress amplifies lung tissue mechanics, inflammation and oxidative stress induced by chronic inflammation Fabiana G. Reis,1 Ricardo H. Marques,1,4 Claudia M. Starling,1 Rafael Almeida-Reis,1 Rodolfo P. Vieira,1 Claudia T. Cabido,1 Luiz Fernando F. Silva,2 Tatiana Lanças,2 Marisa Dolhnikoff,2 M´ılton A. Martins,1 Edna A. Leick-Maldonado,1 Carla M. Prado,3 and Iolanda F. L. C. Tibério1 1

Medicine Department LIM 20, School of Medicine, University of São Paulo, São Paulo, Brazil

2

Pathology Department LIM 5, School of Medicine, University of São Paulo, São Paulo, Brazil Department of Biological Sciences, Federal University of São Paulo, Diadema, Brazil

Exp Lung Res Downloaded from informahealthcare.com by UNICAMP on 12/04/12 For personal use only.

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Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil A B STRACT Background: Mechanisms linking behavioral stress and inflammation are poorly understood, mainly in distal lung tissue. Objective: We have investigated whether the forced swim stress (FS) could modulate lung tissue mechanics, iNOS, cytokines, oxidative stress activation, eosinophilic recruitment, and remodeling in guinea pigs (GP) with chronic pulmonary inflammation. Methods: The GP were exposed to ovalbumin or saline aerosols (2×/wk/4wks, OVA, and SAL). Twenty-four hours after the 4th inhalation, the GP were submitted to the FS protocol (5×/wk/2wks, SAL-S, and OVA-S). Seventy-two hours after the 7th inhalation, lung strips were cut and tissue resistance (Rt) and elastance (Et) were obtained (at baseline and after OVA and Ach challenge). Strips were submitted to histopathological evaluation. Results: The adrenals’ weight, the serum cortisol, and the catecholamines were measured. There was an increase in IL-2, IL-5, IL-13, IFN-γ , iNOS, 8-iso-PGF2α, and in %Rt and %Et after Ach challenge in the SAL-S group compared to the SAL one. The OVA-S group has had an increase in %Rt and %Et after the OVA challenge, in %Et after the Ach and in IL-4, 8-iso-PGF2α, and actin compared to the OVA. Adrenal weight and cortisol serum were increased in stressed animals compared to nonstressed ones, and the catecholamines were unaltered. Conclusion & clinical relevance: Repeated stress has increased distal lung constriction, which was associated with an increase of actin, IL-4, and 8-iso-PGF2α levels. Stress has also induced an activation of iNOS, cytokines, and oxidative stress pathways. KEYWORDS asthma, cytokines, iNOS, isoprostane, lung distal tissue mechanics, stress

role of stress in the pathophysiology of asthma remains unclear. Stress seems to modulate this response, both through nerve connections between the immune system and the autonomic nervous system, and through the release of hormones and neuropeptides. Many studies have shown that stress can influence the migration, proliferation, and function of inflammatory cells, as well as the production of cytokines and other inflammatory mediators [3]. These findings could partially contribute to the comprehension of the mechanisms that aggravate asthmatic symptoms induced by stressful situations [2, 3]. The relevance of the distal lung tissue in functional asthma impairment has been recently emphasized by other studies [5–8]. Several authors have

INTRODUCTION Stress threatens homeostasis [1] and is associated with the activation of the hypothalamic-pituitaryadrenal axis (HPA) and the sympathetic and adrenomedullary systems (SAM) [1, 2]. Recent studies have shown that emotional states can also influence the course and treatment of atopic and asthmatic diseases [2–4]. However, the precise Received 29 January 2012; accepted 15 June 2012. This work was supported by FAPESP, CNPq, LIM-20-FMUSP. Address correspondence to Iolanda F.L.C. Tibério, M.D. Phd, Departamento de Cl´ınica Médica, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Sala 1210, CEP 01246-903 São Paulo, SP, Brazil. E-mail: [email protected]

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demonstrated that the peripheral lung parenchyma of asthmatic patients presents eosinophilic inflammation, an over expression of IL-5 and extracellular matrix remodeling, which has also been reproduced in animal models [5, 7–9]. The above data support the hypothesis that repeated stress should be conceptualized as a factor that disrupts physiological pathways, resulting in enhanced asthmatic responses [2, 4]. The importance of the distal lung responses to the global pulmonary alterations enhancing asthma symptoms has been recently assessed emphasized by other studies [5, 9, 10]. Apart from these evidences discussed, the relevance of induced stress effects on the distal lung has not been previously analyzed. In this study, we have attempted to investigate whether repeated forced swim stress influence the lung tissue mechanics and to analyze the possible morphological determinants of these functional alterations. In order to perform this task, we have assessed eosinophilic inflammation, oxidative stress, cytokines

expression, and extracellular matrix remodeling levels in the distal lung tissue of guinea pigs with chronic allergic pulmonary inflammation.

MATERIALS AND METHODS Animals were handled in compliance with the Guide for care and use of laboratory animals [10]. The protocols were approved by Ethics Committee of University of São Paulo. Thirty-two male Hartley guinea pigs were divided in four different groups (n = 8 animals each): nonsensitized (SAL); sensitized (OVA); nonsensitized and stressed (SAL-S); sensitized and stressed (OVA-S). As it has been previously described [10], seven ovalbumin aerosols were performed [Figure 1] during 15 minutes each, over a 4-week period, while the controls have received saline aerosols. Twenty-four hours after the 4th inhalation, animals were placed in a box filled with water (25◦ C) and were forced to swim

FIGURE 1. Timeline of the experimental protocol. The guinea pigs were submitted to seven

inhalations (2 per week- with 2 to 3-days intervals, during 4 weeks) with aerosols of saline or ovalbumin solution with increasing doses of antigen. From the 1st to the 4th inhalations, the dose used was 1 mg/mL of ovalbumin (2 weeks). In the 5th and 6th inhalations (3rd week), animals were submitted to 2.5 mg/mL of ovalbumin and in the 7th inhalation (beginning of the 4th week) the dose of 5 mg/mL of antigen was used. The solution of ovalbumin or saline was continuously aerosolized for 15 minutes or until respiratory distress occurred (sneezing, coryza, cough or retraction of the thoracic wall). The forced swim stressor (FSS) starts 24 hours after the 4th inhalation during 5 days a week for 2 weeks. Seventy-two hours after 7th inhalation, all guinea pigs were anesthetized, exsanguinated, and lungs were removed and submitted to the experimental protocol of oscillatory mechanics. C

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instinctual up to 10 minutes or until there was danger of drowning, during 5 days a week for 2 weeks. Controls were left in their cages. The forced swim stress has been used as an animal model of behavioral despair/depression and it is a type of unavoidable stress that induces an effort for survival with an escape deficit [11]. In contact with water, animals instinctively start to swim in an effort to survive, as it has been previously shown by other authors [11, 12]. Seventy-two hours after the 7th inhalation, animals received an overdose of thiopental barbitone. Blood samples were taken by cardiac puncture. Lungs were excised and infused with Krebs physiological solution. Strips from the left lower lobe (10 mm × 2 mm × 2 mm) were cut. The lung oscillatory mechanics protocol has been previously described [5]. Briefly, resting tension (T) was set by movement of a screw thumb wheel system. Strips were fixed at 1 g and maintained in Krebs solution aerated with 95% O2 and 5% CO2 (frequency of oscillation: 1 Hz; amplitude: 2.5% Lr) [5]. Tissue resistance (Rt) and elastance (Et) were obtained at baseline and after OVA (0.1%). Ach (10−3 ) administration into the organ bath chamber was assessed by the equation of motion and standardized for the strip size. The unstressed cross-sectional area (A0) of the strip was obtained through the formula: A0(cm2 ) = W0/(p × Lr), [5], where p is the mass density of the tissue taken as 1.06 g/cm3 , W0 represents the wet weight (gr), and Lr the resting length (cm). Values of Rt and Et were multiplied by Lr/A0. The percent increases of Rt and Et were obtained. The body and the adrenal weight ratios were expressed relative to the animal weight (ReAW). The blood in the samples was centrifuged and the supernatant was assayed using a Cortisol kit [13]. Catecholamines were measured through the use of a high-performance liquid chromatography [14]. Strips were fixed and submitted to histological and immunohistochemical analysis and were then stained with H & E, Picro-Sirius, and LUNA staining [9]. Immunohistochemistry was performed with anti-iNOS [10]; anti-IL-2, anti-IL-4, anti-IL-5, antiIFN-γ , and anti-IL-13 [15]; and anti-α-smooth muscle actin and anti-8-iso-PGF2α antibodies [9], by the biotin-streptavidin-peroxidase method. Using a 100point grid (area: 104 µm2 at ×1000 magnification), the number of points hitting alveolar tissue and the number of eosinophils and positive cells for IFN-γ , IL-2, IL-4, IL-5, IL-13, and iNOS within the alveolar septa were counted in 10 fields. Results were expressed as cells/104 µm2 [16]. The fractional area of tissue constituents, and the collagen and actin content were evaluated under ×400 magnification, as previously described by Star-

ling et al. [9]. Results were expressed as percentages. Lung tissue 8-iso-PGF2α content was evaluated under ×400 magnification by the Image-Pro Plus 4.5 v image analysis system [16] and the results were obtained dividing the positive area of 8-iso-PGF2α by the tissue area and then expressed as percentages. All morphometric analysis described in this section were performed by individuals blinded to the protocol design. Statistical analysis was performed with the SigmaStat-10.0 Software (Jandel, CA). Comparison among groups was performed by a Two-Way Analysis of Variance, followed by the Holm-Sidak method. A P value of < .05 was considered significant.

RESULTS The ReAW of the animals submitted to the stress protocol was greater than that observed in the nonstressed groups (P < .001). There was an increase in the mean cortisol values between stressed and nonstressed animals (P < .004). There were no differences in the mean values of catecholamines among the experimental groups. We have observed an increase in the baseline values of Rt and Et between saline and ovalbumin exposed animals after the ovalbumin challenge [Rt: (SAL: 575.297 ± 77.35; OVA: 1014.52 ± 86.48; SAL-E: 645.37 ± 86.48; OVA-E: 1000.96 ± 81.53, N−s /m2 ), Et: (SAL 35151.77 ± 5740.56; OVA: 60390.95 ± 4971.47; SAL-E: 39420.34 ± 4971.47; OVA-E: 56090.21 ± 4687.15 - N/m2 ) P < .001]. Figure 2A and B demonstrates an increase in the %Rt and the %Et on the OVA and the OVA-S groups after the ovalbumin challenge when in comparison to the saline groups (P < .05). Animals of the OVA-S group (quantos OVA-S grupos existem?) have had an increase in %Rt and %Et in comparison to the OVA group (P < .05). Figure 2C and D shows that there was an increase in %Rt and %Et for the OVA, the OVA-S and the SAL-S groups after the Ach challenge in comparison to the saline group (P < .05). We have also observed an increase in the %Et in the OVA-S group in comparison to the OVA group (P < .05). There were no differences in hysteresivity among the four experimental groups. Figure 3A shows an increase in the 8-iso-PGF2α positive area of the OVA group in comparison to the SAL group (P < .05). Animals submitted to the forced swim stress had an increase in the 8-isoPGF2α positive area in comparison to nonstressed animals (P < .001). Figure 3B shows that there was an increase in the iNOS-positive cells in the OVA, the Experimental Lung Research


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FIGURE 2. (A and B). Mean ± SEM post-ovalbumin challenge values (percentage of increase) of

tissue resistance (A) and elastance (B) in guinea pigs previously exposed to seven inhalations with ovalbumin or saline, ∗ P < .05 comparing ovalbumin exposed animals (OVA and OVA-S groups) to saline exposed ones (SAL and SAL-S groups); ∗∗ P < .05 comparing ovalbumin exposed animals submitted to repeated swim stressor (OVA-S group) to nonstressed ones (OVA group). (C and D). Mean ± SEM post-Ach challenge values (percentage of increase) of tissue resistance (A) and elastance (B) in guinea pigs previously exposed to seven inhalations with ovalbumin or saline, ∗ P < .05 comparing ovalbumin group, ovalbumin stressed group and saline stressed group (OVA, OVA-S, and SAL-S) to saline animals (SAL), ∗∗ P < .05 comparing ovalbumin stressed group to ovalbumin group.

OVA-S, and the SAL-S groups compared to the SAL one (P < .001). Figure 4A demonstrates an increase in the collagen content in the alveolar septa of the OVA and the OVA-S groups subjects compared to the SAL and the SAL-S groups ones (P < .01). There were no differences between the OVA and the OVA-S groups. Figure 4B shows an increase in the eosinophil density for the OVA and the OVA-S animals compared to controls (P < .05) and no differences between the OVA and the OVA-S groups. Figure 4C shows that there was an increase in actin content in the OVA and the OVA-S animals compared to controls (P < .001). The OVA-S group has had an increase in the actin content compared to the OVA one (P < .05). Figure 5A and E shows that there was an increase in the IL-2 and IFN-γ positive cells only in animals of the SAL-S group in comparison to other groups (P < .05). Figure 5B shows an increase in the IL-4 positive cells in the OVA and OVA-S groups C


compared to the saline-exposed ones (P < .05). The OVA-S group had an increase in the IL-4 positive cells compared to the OVA one (P < .05). Figure 5C shows that there was an increase in the IL-13 positive cells in the OVA, OVA-S, and SAL-S groups compared to the SAL groups (P < .05). Figure 5D shows that there was an increase in the IL-5 positive cells of OVA and OVA-S subjects, when compared to controls. The SAL-S group had an increase in IL-5 positive cells compared to the SAL group (P < .05). We have observed that the lung strips were mainly represented by alveolar septa as described in Table 1. Figures 6 and 7 show representative photomicrographs of the lung tissue. The guinea pigs aerosolized with saline and submitted to the forced swim stress have presented an increase in the number of 8-isoPGF2α, iNOS, IL-2, IL-5 IL-13 and INF-γ positive cells when compared to the SAL group. The lung tissue of the sensitized animals that were submitted to the forced swim stress have presented a prominent

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TABLE 1 Percentage of Alveolar Septa, Blood Vessels, and Airways in the Four Experimental Groups—Guinea Pigs Previously Exposed to Even Inhalations with Saline (SAL) or Ovalbumin (OVA) and Guinea Pigs Previously Exposed to Seven Inhalations with Saline or Ovalbumin and Also Submited to Repeated Swim Stressor (SAL-S and OVA-S) Groups

Alveolar septa (%)

Blood vessels (%)

Airways (%)

SAL OVA SAL-S OVA-S

87.45 ± 1.28 87.41 ± 2.46 91.40 ± 1.60 89.38 ± 0.55

9.05 ± 0.78 9.98 ± 1.41 6.56 ± 1.27 8.76 ± 0.65

3.48 ± 0.68 2.60 ± 1.14 2.03 ± 0.70 1.84 ± 0.38

8-iso-PGF2α expression

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Note. Lung strips were mainly represented by alveolar septa (p < 0.001 for all comparisons).

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FIGURE 3. (A). Mean values ± SEM of iNOS positive cells density in lung tissue of guinea pigs previously exposed to seven inhalations with ovalbumin or saline, either or not submitted to repeated forced swim stress; ∗ P < .001 comparing to SAL group. (B). 8-iso-PGF2α content (mean ± SEM) in the alveolar septa of guinea pigs exposed to seven inhalations with ovalbumin or saline, either or not submitted to repeated forced swim stress, ∗ P < .001 comparing ovalbumin-exposed guinea pigs (OVA group) to saline-exposed one (SAL group) and ∗∗ P < .001 comparing animals submitted to forced swim stress (OVA-S and SAL-S) to nonstressed groups (OVA and SAL groups).

increase in IL-4 positive cells, 8-iso-PGF2α, and actin content in comparison to sensitized animals which were not submitted to that test.

DISCUSSION In the present study, we have evaluated the importance of repeated exposure to the forced swim stress, which represents an animal model of behavioral despair/depression, in an experimental model of chronic allergic pulmonary inflammation, particularly showing the influences on mechanics, inflammation, remodeling, and oxidative stress pathway activation of the distal lung tissue. We had previously found that chronic allergic inflammation is associated with a hyper responsiveness of the distal lung tissue [5, 9, 10] and this is also replicated in the present study. The stress protocol was able to promote an increase in resistance and elastance of distal lung tissue after acetylcholine challenge in nonsensitized animals. Evaluating the effects of stress in this model of chronic allergic inflammation, we have observed an enhancement in resistance and elastance after ovalbumin challenge and in elastance after acetylcholine challenge. There are several mechanisms that may be involved in control of the distal lung mechanical alterations, many of which were evaluated in this study. Tissue actin content was increased in the ovalbumin exposed guinea pigs and we have observed a synergistic effect of the stress with actin, probably present in the myofibroblasts. These cells are able to synthesize several extracellular matrix proteins as well as contracting the lung parenchyma [17]. Another aspect was related to the production of nitric oxide. Several studies have shown that NOderived isoenzymes from constitutive acts on smooth muscle cell tone and NO-derived isoenzymes mainly from iNOS regulate inflammatory responses [10, 16]. Blockage of only iNOS attenuates airway and distal lung parenchyma constriction and inflammatory cell recruitment induced by chronic allergic inflammation [9, 10]. Moreover, Chi et al. [18] have demonstrated that iNOS derived from NO production induced by LPS had been further enhanced by epinephrine and noradrenalin. We have observed a great number of iNOS positive cells in animals submitted to the stress protocol (SAL-S group) as well as in animals submitted to the sensitized protocol (OVA group). We believe that the increase in the iNOS positive cells has contributed to the increase of the contractile response. However, this is not likely to be the only mechanism involved, especially in the sensitized-stressed animals. Experimental Lung Research


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FIGURE 4. Mean ± SEM values of collagen content (A), volume proportion of smooth

muscle-specific actin (B), and eosinophil density (C) in guinea pigs previously exposed to seven inhalations with ovalbumin or saline, either or not submitted to repeated forced swim stress; (A) ∗ P < .01 comparing ovalbumin exposed animals (OVA and OVA-S groups) to saline exposed ones (SAL and SAL -S groups). (B), ∗ P < .001 comparing ovalbumin exposed animals (OVA and OVA-S groups) to saline exposed ones (SAL and SAL -S groups) and ∗∗ P < .03 comparing ovalbumin exposed animals submitted to repeated forced swim stress (OVA-S group) to OVA group. (C), ∗ P < .001 comparing ovalbumin exposed animals (OVA and OVA-S groups) to saline exposed ones (SAL and SA -S groups).

One aspect that may be involved in the mechanic responses is the increase in the 8-iso-PGF2α content in sensitized animals, and this effect was empowered by stress. Moreover, animals that were submitted only to the stress protocol have also presented this effect. Oxidative stress pathways play an important role in the regulation of inflammatory mediators that may be involved in atopy [2, 19]. The manner by which the iNOS-derived NO production leads to the potentiation of lung tissue mechanics may be related to the generation of isoprostanes (8-iso-PGF2α). These lipid-derived substances contribute to the contraction of smooth muscle actin via Rho and Rho kinase [20]. It has been demonstrated that exposure to stressful situations can lead to an increase of free radicals [19, 21]. Torres et al. [19] showed that the chronic stress variable increases pulmonary lipid peroxidation. Sivonová et al. [21] showed that stress induces oxidative damage to DNA and lipid oxidation as well as a decrease in the plasma antioxidant activity. C


We have found, in the present study, an increase in the collagen content in the alveolar septa of the ovalbumin-exposed animals compared to salineexposed ones. However, there was no significant difference between the OVA and the OVA-S groups. Although stress is associated with inflammation, it can activate collagen fiber deposition; due to an eosinophilic inflammatory process and a Th2 cytokine profile release, it can also modulate the degradation of collagen fibers of the extracellular matrix via activation of metalloproteinases and TGF-β1 [22–24]. In this regard, Yang et al. [22] showed that a norepinephrine treatment increased MMP-2, MMP9, and VEGF levels. Briest et al. [23] showed that noradrenalin increases MMP-2 activity in ventricles. Miller et al. [24] showed that corticosteroids inhibit the expression of TGF-β1 in eosinophils and macrophages. However, other studies do not corroborate with the role of corticosteroids in reducing

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FIGURE 5. Mean values ± SEM of IL-2 (A), IL-4 (B), IL-5 (C), IL-13 (D), and IFN-γ (E)

positive cells lung tissue of guinea pigs previously exposed to seven inhalations with OVA or SAL, either or not submitted to repeated forced swim stress; (A), ∗ P < .05 comparing to SAL, OVA, and OVA-S groups; (B), ∗ P < .05 comparing to SAL, SAL-S, and OVA-S groups, and ∗∗ P < .05 comparing to OVA, SAL, and SAL-S groups; (C) ∗ P < .05 comparing to SAL group and ∗∗ P < .05 comparing to OVA and OVA-S groups; (D), ∗ P < .05 comparing to SAL group; (E), ∗ P < .05 comparing to SAL, OVA, and OVA-S groups.

extracellular matrix remodeling in asthma [25]. The above data emphasize the idea that the extracellular matrix alterations induced by stress responses need further studies. In the present study, the ovalbumin sensitization has induced an increase in the eosinophil density. However, the repeated forced swim stress did not modify the eosinophilic response in ovalbuminstressed animals. The effect of stress in eosinophil recruitment remains a matter of controversy. Portela et al. [26] observed that stressed animals have a significant increase in the amount of edema and lymphomononu-

cleated cells but not in the number of eosinophils in the airways. Machida et al. [27] showed that incubation of human eosinophils with β2-adrenoceptor agonists, inhibits spontaneous eosinophil apoptosis and the apoptosis induced by Fas receptor activation. Capelozzi et al. [11] showed an increase in the eosinophil number in the lung parenchyma of guinea pigs 72 hours after the last stress exposure. Systemic levels of adrenal steroids, catecholamines, and neuropeptides may have different effects on eosinophil recruitment and apoptosis [28]. Glucocorticorticoids induce eosinophil apoptosis in the presence of survival-enhancing asthmatic Experimental Lung Research



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FIGURE 6. Photomicrographs of lung tissue stained for eosinophils (A, B, C,

and D), iNOS positive cells (E, F, G, and H), 8-iso-PGF2α (I, J, K, and L), actin (M, N, O, and P), and collagen (Q, R, S and T) from guinea pigs inhaled with saline (A, E, I, M, and Q) or those chronically exposed to ovalbumin (B, F, J, N, and R). The guinea pigs submitted to repeated exposure to forced swim stress and aerosolized with saline (C, G, K, O, and S) or ovalbumin (D, H, L, P, and T) are also represented. The lung tissue of the OVA-S group presented a prominent increase in 8-iso-PGF2α expression and actin content compared to OVA animals. In addition, animals from SAL-S group, presented an increase in the number of iNOS positive cells as well as in the 8-iso-PGF2α expression compared to SAL. Figures at x400 magnification.

cytokines such as IL-13 and IL-5 [27, 29]. On the other hand, higher concentrations of these pro-inflammatory cytokines inhibit the pro-apoptotic effects of glucocorticoids [29]. The complexity of these interactions indicates that the effects of stress responses on eosinophilic recruitment remain a matter of controversy. Cytokines are chemical messengers that stimulate the HPA axis when the body is under stress or experiencing an infection or an inflammatory response [30]. It is noteworthy to emphasize that there is a paucity of C


studies concerning the effects of stress on the expression of these inflammatory cytokines on distal lung parenchyma with alterations related to chronic allergic stimuli. We have observed an increase in Th2 cytokines represented by IL-4, IL-5, and IL-13 positive cells in the ovalbumin-exposed animals. Considering the saline-stressed animals, there was a significant increase in the IFN-γ , IL-2, IL-5, and IL-13 positive cells, compared to the saline animals. Although we did not find an increase in the IL-4 positive cells


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FIGURE 7. Photomicrographs of lung tissue stained for IL-2 (A, B, C, and D),

IL-4 (E, F, G, and H) IL-5 (I, J, K, and L), IL-13 (M, N, O, and P), and INF-γ (Q, R, S, and T) positive cells from guinea pigs aerosolized with SAL (A, E, I, M, and Q) or those chronically exposed to OVA (B, F, J, N, and R). The guinea pigs submitted to repeated exposure to forced swim stressor and aerosolized with saline (C, G, K, O, and S) or ovalbumin (D, H, L, P, and T) are also illustrated. Animals of SAL-S group showed an enhancement in the number of IL-2, IL-5, IL-13, and IFN-γ positive cells compared to SAL group. Furthermore, OVA-S group presented a prominent increase in the number of IL-4 positive cells compared to only OVA ones. Figure at X400 magnification.

in saline stressed animals, we noted that ovalbuminstressed animals had an increase in IL-4 positive cells compared to the ones sensitized only with ovalbumin. The precise role of different cytokines on the modulation of stress responses in animal models of chronic pulmonary inflammation has not yet been fully determined [1, 2, 31]. Okuyama et al. [31] suggest that chronic stress induced an increased IL-4 and IL-5 in bronchoalveolar lavage fluid (BAL). Forsythe et al. [1] showed that sensitized mice stressed for 7 consecutive days had a significant increase in the

inflammatory cells with no change in IL-6, IL-9, and IL-13 expression. Viveros-Paredes et al. [32] analyzed a model of stress in mice using electric shock applied on 7 consecutive days and obtained an increase in cortisol plasma, IFN-γ , IL-4, and IL-10 conventration in the supernatants of splenocytes. Summarizing the above studies, these results suggest that pathogenic mechanisms in atopics may be more strongly affected by stress than others, and that different cytokine profiles, mainly of the Th2

Experimental Lung Research



phenotype, are involved. However, we consider that the differences in the experimental protocols of stress induction used, the animal species evaluated, the methods as well as the materials used for cytokines detection could lead to some of the different entail expression of cytokines. One interesting point was related to the fact that glucocorticoids can also reduce iNOS, cytokines, and other inflammatory mediators expression [25]. We observed an increase in the relative adrenal weight and serum cortisol values in stressed animals compared to nonstressed groups. There would be expected a reduction in iNOS expression via blockage of NF-κB activation induced by treatment with glucocorticoids. However, we observed an increase in the iNOS –positive cells. In conjunction, these findings suggest that repeated exposure to forced swim may induce a state of glucocorticoid resistance, perhaps associated with down-regulation of the expression or the function of glucocorticoid receptors [2, 4, 33]. We did not observe an increase in the mean levels of catecholamines. In fact, several authors had suggested that adrenergic activation was associated with acute stress responses [1, 3]. Taken together, the catecholamine responses and the suspected state of glucocorticoids resistance reinforce the idea that in this animal model the alterations observed were related to a chronic stress response. Our study has some limitations. In this regard, the evaluation of metalloproteinases, glucocorticoid, and neurokinin receptor expression could contribute to a better comprehension of the mechanisms involved. In conclusion, repeated forced swim stress increased lung distal constriction in animals with chronic pulmonary inflammation, which was associated with an increase in actin content, IL-4, and 8-iso-PGF2α expression. These results suggest that activation of inflammatory and oxidative stress pathways were involved in the modulation of stress responses in this animal model of chronic lung inflammation. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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