2011 effects of restraint stress on nalt structure and nasal ig a levels

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Immunology Letters 135 (2011) 78–87

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Immunology Letters

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Effects of restraint stress on NALT structure and nasal IgA levels

Rigoberto Oros-Pantojaa, Adriana Jarillo-Lunaa,b, Víctor Rivera-Aguilarc, Luvia Enid Sánchez-Torresd,Marycarmen Godinez-Victoriaa, Rafael Campos-Rodrígueze,∗

a Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Diaz Miron, CP. 11340, México, DF, Mexicob Departamento de Morfología, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, CP. 11340, México, DF, Mexicoc Departamento de Microbiología, UBIPRO, FES-Iztacala, UNAM, Avenida de los Barrios s/n, Tlalnepantla Edo, de Mexico, CP. 54090, Mexico, DF, Mexicod Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación Carpio s/n, Col. Santo Tomas, CP. 11340, México, DF, Mexicoe Departamento Bioquímica, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, CP. 11340, México, DF, Mexico

a r t i c l e i n f o

Article history:Received 27 March 2010Received in revised form23 September 2010Accepted 3 October 2010Available online 16 October 2010

Keywords:Restraint stressNALTIgALymphocytesCatecholaminesGlucocorticoids

a b s t r a c t

The effects of stress on the mucosal immune responses in inflammatory disorders of the gut, as well as onsalivary and intestinal IgA levels are well known. However, its effects on the structure and function of theNALT have not yet been reported, and are examined in the present study. Balb/c mice were submitted torestraint stress for 3 h per day during 4 or 8 d. The immunohistochemistry and flow cytometric analysisrevealed that repeated restraint stress (4 and 8 d) decreased the percentage, compared to the controlgroup, of CD3+ and CD4+ T cells, without affecting the percentage of CD8+ T cells or B220+ cells (B cells).The numbers of IELs (CD4+ and CD8+ T cells) were lower at 4 d of stress and higher at 8 d. IgA+ cells in NALTand nasal IgA levels showed a similar pattern, being significantly lower at 4 d of stress and significantlyhigher at 8 d. In summary, repeated restraint stress altered the distribution and number of lymphocytesand IgA+ cells in nasal mucosa, probably due to changes in norepinephrine and corticosterone levels.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The effects of stress on the mucosal immune responses havebeen widely analyzed in relation to inflammatory disorders of thegut and the secretion of IgA in saliva. The robust information avail-able confirms that psychological stress plays a key role in thepathophysiology and clinical presentation of inflammatory boweldisease [1–5].

There are contradictory reports on the relationship betweensecretory IgA (S-IgA) levels in saliva and different conditions ofstress, such as exercise, mood states and academic examina-tions. Whereas some studies found decreases in S-IgA, othersdetected increases or no change [6–11]. We recently reported thatstress decreases intestinal IgA levels, and affects the populationof intraepithelial lymphocytes in the duodenal mucosa of mice[12,13]. However, the effects of stress on the nasal immune systemhave not been explored in detail.

∗ Corresponding author at: Departamento de Bioquímica. Escuela Superior deMedicina. Instituto Politécnico Nacional, Plan de San Luis y Diaz Mirón, CP 11340,México, DF, Mexico. Tel.: +52 55 57 48 20 04; fax: +52 55 57 14 54 55.

E-mail address: [email protected] (R. Campos-Rodríguez).

It has been documented that psychological stress alters suscep-tibility to several different strains of respiratory viruses [14], andnumerous reports indicate that exercise stress can increase the riskfor upper respiratory tract infection, particularly in highly trainedand elite athletes [15–17]. Although some elite athletes and sub-jects under severe stress produce less IgA in saliva, it has not beenestablished that this is the cause of the higher incidence of res-piratory infections in these populations [17–20]. Other causativefactors that have been proposed are the presence of infiltratedinflammatory cells in mucous membranes and the removal of oneor more immune functions [15,16,21].

It is unknown whether chronic stress can alter the structureand/or function of the nasal-associated lymphoid tissue (NALT), andif so whether such change would contribute to the increased inci-dence of respiratory infections found among elite athletes. Studieson animals suggest that stress can affect the immune responsesin the upper respiratory tract. In mice infected intranasally withinfluenza virus, restraint stress increases levels of IgM and IgGantibody-secreting cells, which are virus-specific responses in thesuperficial cervical lymph node, the latter being considered partof the NALT [22]. On the other hand, restraint stress inhibits theproduction of IgE, IgG1 and IgG2a, specific for an allergen inocu-lated intranasally [23]. Moreover, acute treadmill exercise of micedecreases the number of CD4+ T cells in the submandibular lymph

0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.imlet.2010.10.001


R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87 79

nodes, cells which play a critical role in the induction of immuneresponses to antigens in the eye, upper respiratory tract and oralmucosa [24,25].

The NALT is defined as the oropharyngeal lymphoid tissue ofthe upper respiratory airways of rodents, considered analogousto Waldeyer’s ring in humans [26–28]. It consists of paired lym-phoid structures situated above the soft palate at the entrance tothe bifurcated pharyngeal duct, which are composed in part of sec-ondary lymphoid aggregates characterized by follicular B-cell areasand parafollicular T-cell areas [29], as well as the many lympho-cytes found in and underneath the epithelial lining of the nasalmucosa [28]. The NALT is covered by an epithelium or follicle-associated epithelium (FAE), which consists of ciliated columnarcells, M cells (alone or in clusters), intraepithelial lymphocytesand a few goblet cells. Antigen-presenting cells, including dendriticcells and macrophages, are also found in the NALT. Therefore, thisorgan must have an important role in the induction and regulationof mucosal immune responses to antigens in the upper respiratorytract [27–29].

To the best of our knowledge there have not yet been any reportson the effects of stress on the structure and function of the NALT.Thus, the aim of the present study was to determine whether or notrepeated restraint stress induces a change in the levels of plasmaticglucocorticoids and catecholamines, and/or in the distribution oflymphocytes in the nasal mucosa, and if so, whether such changeshave any correlation with nasal IgA levels in mice. The results showthat repeated restraint stress selectively affects individual compo-nents of the immune system of the nasal mucosa of mice and thebasal production of IgA.

2. Materials and methods

2.1. Animals

Ten week old male Balb/c mice (Harlan, Mexico) were ran-domly placed in three groups (n = 7): two experimental groups thatunderwent restraint stress and a non-stressed control group. Of theexperimental groups, one underwent restraint stress during 4 d andthe other during 8 d. Animals were handled and treated accordingto a protocol approved by the Ethics and Institutional Animal Careand Use Committees.

2.2. Restraint stress protocol

The experimental groups were submitted to 3 h restraint stresssessions daily, always from 8:00 to 11:00 am. Restraint stress wascarried out by placing the mice in cylindrical plastic containers 6 cmlong, 3 cm high and 3.5 cm wide, with many ventilation holes toprevent hyperthermia. At the time of restraint stress for the exper-imental groups, non-restrained mice were left undisturbed in theirhome cages, but without access to food or water. Apart from therestraint stress schedule, the experimental animals were kept incages and all three groups were provided with food and water adlibitum.

To avoid adaptation during the 3 h restraint stress sessions, themice received various stimuli in 30 min cycles, the first cycle con-sisting of (i) the agitation of the containers for 10 s after 10 min, and(ii) the rotation of the containers for 10 s after 20 min, followed byanother 30 min cycle that began in the same way and included theimmersion of the mouse tails in cold water for 10 s after 30 min.

2.3. Obtaining and processing biological material

Control and experimental mice were anaesthetized with ether,bled by direct cardiac puncture, and sacrificed by decapitation. Thenasal wash was done by retrograde infusion through the trachea

with 2 ml sterile saline solution, which was collected in Eppendorfftubes. The tubes were stored at −70 ◦C until the analysis was doneof secretory IgA (sIgA) by paired ELISA. Protein concentration andquantification curves were constructed by the Bradford method.

After the nasal wash procedure, the skin of the head wasremoved, as were the inferior jawbone and soft tissue, accord-ing to the method described by Asanuma et al. and Heritageet al. [30–32]. The extracted palate was placed upside down in1 cm3 aluminum containers embedded in tissue inclusion medium(Tissue-tek, Sakura, 4583). The containers were frozen and storedat −70 ◦C until the embedded tissue was cut in a cryostate. Afterremoving the NALT, the skulls were fixed by immersion in 4%paraformaldehyde for 24 h, washed, and decalcified with 8% EDTA(Baker analyzed) at pH 7.6. The solution was changed and this cyclewas repeated daily for 8 d. The skulls were then included in paraffin.

2.4. Processing

From the samples of frozen NALT, 7 �m thick cuts were madeon the crown portion, and then placed on slides previously treatedwith 1% gel. Some slides were fixed in acetone for 20 min and othersin 4% formaldehyde for the same time. Those cuts fixed in formalde-hyde and the cuts from the samples processed in wax were stainedwith haematoxylin and eosin for a general morphological analysis.

2.5. Immunohistochemistry

Cells were quantified by utilizing immunohistochemical meth-ods. 7 �m crown sections of NALT were fixed in acetone for 20 min.Later, the slides were hydrated with PBS and the endogenous per-oxidase was blocked by incubation with 3% H2O2 and 0.1% NaN3in PBS for 10 min. The samples were washed, incubated with 5%bovine serum for 30 min, and washed again with 0.05% Tween-20in PBS. Plasmatic cells producing IgA were determined by a directimmunohistochemical technique, utilizing goat anti-mouse IgAperoxidase conjugate polyclonal antibodies (HRP-Serotec). Addi-tionally, monoclonal biotin conjugate mouse antibodies were usedfor an indirect immunohistochemical technique. The followinglymphocytes were detected: CD3+ (BD Pharmingen, 553323), CD4+

(BD Pharmingen, 553728), CD8+ (BD Pharmingen, 553029) andCD45-R (B220 BD Pharmingen, 553085). Estreptavidine peroxidaseconjugate (Jackson Immuno Research) was later applied.

The primary antibodies were incubated for 2 h and estreptavi-dine for 1 h, both at RT in a humidified chamber. Gentle washeswere carried out with PBS at the end of each incubation period.The peroxidase reaction was revealed according to the Karnovskymethod with DAB (Pierce, 34065). The samples were counter-stained with one part of Harris’ haematoxylin diluted in 3 partsof water, then dehydrated and covered with synthetic resin. Withthe control samples for each antibody, stains were conducted by thesame method except that the first antibody primer was substitutedby PBS. Other control samples were incubated with a peroxidatedantibody before staining for anti-mouse IgA antibodies.

2.6. Microscopic analysis and cell quantification

The total area of NALT was measured in �m2 in the cuts stainedwith H–E by using Imagen Pro Plus software, calibrated at 200×magnification. With the same software, using constant areas of2500 �m2 from images magnified 400× (see Fig. 4A), the numberof T CD3+, CD4+, CD8+, B IgA+ and CD45+ lymphocytes were quan-tified in the follicular and parafollicular zones. The software toolsemployed in the count were: adjustment of the minimum and max-imum range of the area of cells to be counted, manual selection ofthe color of cells to be counted, and Watershed-split and Autosplitto separate cells that were very close together. In the lamina propria


80 R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87

the IgA+ cells were counted in the same way. In the quantification ofCD4+ and CD8+ intraepithelial lymphocytes, the average longitudeof the respiratory epithelium in the crown cuts of the NALT, whichturned out to be 800 linear �m, was used as a reference. Finally, thevolume of the NALT was calculated by measuring the total area ofthis tissue from each cut, averaging the various cuts made on eachanimal, then averaging this value for all the animals in each group.Each count was made in duplicate.

2.7. Flow cytometry

Nasal-associated lymphoid tissue (NALT) cell suspensions andthe nasal passage (non-NALT) lymphocytes, which were preparedfrom the portion of the nasal cavity remaining after isolation ofNALT, were obtained according to procedures previously described[30,32]. For cell immunophenotyping, directly labeled antibodieswere used: anti-CD19-PE, IgA-FITC, CD138-APC, CD3-FITC, CD4-PEand CD8-APC (all from BD Biosciences, San Jose, CA, USA). Cells wereharvested, washed twice with PBS and 0.5% BSA and then stainedfor T cell phenotype with a cocktail of anti-CD3, -CD4 and -CD8mAb, for 30 min at room temperature in the dark. The cells werethen washed with PBS and fixed in 2% formaldehyde in PBS. B cellswere fixed, permeabilized and stained according to BD Biosciences’protocol for intracellular staining. Stained cells were acquired witha FACSCalibur flow cytometer (BD Biosciences). Data were analyzedusing the Flow-jo software v7.5 (Tree Star, Inc.).

2.8. Enzyme-linked immunosorbent assay (ELISA) for IgA in thenasal wash

Rabbit anti-mouse IgA immunoglobin (20 �g/ml) was placedin each well, which was incubated for 18 h at 4 ◦C. After wash-ing 3 times with a phosphate-Tween 20 (PBS-T) buffer at pH 7.2,the samples were directly applied and incubated for 2 h at 37 ◦C.The plates were then washed 5 times with PBS-T and 5 timeswith PBS, then incubated for 2 h at 37 ◦C with goat anti-mouseIgA conjugate (BD Pharmingen, 55549) diluted 1:3000 in PBS-T.Finally, the plates were washed 3 times with PBS-T and 3 timeswith PBS before the substrate (Ortophenylendiamina, Sigma-OPD)was added at RT. After 15 min the reaction was stopped with 2.5 Msulfuric acid and the absorbance was determined at a wave lengthof 490 nm. A standard curve was made utilizing purified mouse IgAfrom myeloma (MP-Biomedicals, 64334) at a concentration rangeof 50 �g to 150 ng.

2.9. Determination of plasma corticosterone and epinephrine

A determination of plasma corticosterone was made by a com-mercial kit of ELISA (Cayman Chemical Company, 500651), and thatof plasma epinephrine by a commercial kit of radioimmunoassay(RIA-LDN Labor Diagnostika Nord, BA-0100).

2.10. Statistical analysis

Data are presented as the mean ± SD. The comparison of twogroups was analyzed by using the Student’s unpaired two-tailedt-test. One-way ANOVA was performed to compare more than twogroups, and if a significant main effect or association was identi-fied (P < 0.05), the respective group means were compared usingthe Bonferroni t-test. All analyses were performed using the statis-tical program SigmaStat for Windows Version 2.03 software, andgraphed with Sigma Plot software (SPSS Inc.).

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Corticosterone Epinephrine

Fig. 1. Effect of restraint stress on serum corticosterone and epinephrine levels.Mice were subjected to restraint stress during 3 h for 4 or 8 consecutive days. Later,peripheral blood was collected and subjected to corticosterone and norepinephrineradioimmunoassay. Data are expressed as the mean ± SD (n = 5–8). Corticosteronelevels were significantly lower in mice restrained for 4 d than in the other twogroups: the 8 d restrained group and the unrestrained control (P < 0.001, Bonfer-roni t-test). Norepinephrine levels were significantly higher in mice restrained for4 and 8 d than in the unrestrained control (P < 0.001, Bonferroni t-test).

3. Results

3.1. Effect of restraint stress on serum corticosterone andnorepinephrine

Given that the changes in the immune response induced bystress are mediated principally through the release of glucocor-ticoids and catecholamines, we determined plasma corticosteroneand norepinephrine concentrations in restrained and unrestrainedmice (Fig. 1). Compared to control animals, restraint stress signif-icantly modified serum levels of corticosterone (one-way ANOVA;F(2,15) = 80; P < 0.001) and norepinephrine (F(2,15) = 32; P < 0.001).Corticosterone levels were significantly lower in mice at 4 d ofrestraint stress than in the other two groups; at 8 d restraint stressor in control animals (P < 0.001, Bonferroni t-test). On the contrary,the norepinephrine concentrations in both groups of restrainedmice (4 and 8 d) were significantly higher than in control mice(P < 0.001, Bonferroni t-test).

3.2. Effect of restraint stress on the microscopic structure andvolume of the NALT

We analyzed the morphology of the NALT after staining thistissue with H&E. In unstressed mice we observed one area withtwo ovoid masses, one on each side of the midline of the nasalface of the palate next to the sidewall of the nose (Fig. 2A). In thestructure of the NALT it was difficult to identify typical lymphoidnodules. Regarding irrigation, both conventional and high endothe-lial venules (HEV) and arterioles were identified in the NALT.

Upon comparing the analysis of the NALT from stressed andunstressed animals, the morphology of the NALT was found to besimilar. The total volume of the NALT was estimated by graph-ing the volume of 35 serialized sections taken from the front tothe back portion. When the points were joined, the organ showeda cylindrical shape with a greater volume in the central portionthan at the extremes. This form was similar in control and stressedgroups (Fig. 2B). The mean volume was 2.95 ± 0.56 mm3 in thecontrol group, 2.68 ± 0.42 mm3 in the group stressed for 4 d, and3.27 ± 0.65 mm3 in the group stressed for 8 d. Analysis with one-way ANOVA revealed that there were not any significant differencesbetween the control and the experimental groups (F(2,15) = 1.4,P = 0.27), although there was a tendency towards the atrophy of



Fig. 2. Morphology and stress effect on NALT volume. (A) Two lymphoid nodes can be observed on the floor of the nose in the crown section of the middle NALT segment.In the portion photographed, these nodes are covered by respiratory epithelium (RE). Next to the middle portion is the epithelium of the sectioned nasal septum. The lateralface (LF) is followed by lateral walls, then by the basal face (BF), all forming part of the lamina propria (LP). The epithelial palate (EP) can be seen in the lower part. H&E. 4×.(B) In comparison to the control group, there was a decrease in the average total volume (measured in mm3) at 4 d of stress, but an increase at 8 d of stress. Statistical analysisdid not show any significant differences between the three groups (F(2,15) = 1.44; P = 0.27).

tissue in the 4 d stressed group and toward an increase in NALTvolume in the 8 d stressed group.

3.3. Effect of restraint stress on the distribution of NALTlymphocytes

The immunohistochemical analysis of the NALT from controlanimals showed several patterns of cell distribution, which weredetermined mainly by the organization of B and T lymphocytes.In the center of the NALT there were predominantly B lympho-cytes (IgA+ and CD45+ cells) forming a lymphoid follicle with nogerminative center, surrounded by a parafollicular area largelycomposed of T lymphocytes (CD3+, CD4+ and CD8+ cells) (Fig. 3A).However, there were no well-defined boundaries between thetwo areas, as part of the parafollicular area was found mixedwith follicular area. CD4+ T cells existed in both the follicular andparafollicular areas, while CD8+ T cells showed a pattern of dis-tribution more circumscribed to the parafollicular area (Fig. 3A).The immunohistochemical analysis of the NALT from stressedmice (both the 4-day and 8-day groups) did not show any alter-ation in the distribution patterns of B or T lymphocytes or theirsubpopulations.

3.4. Effect of stress on the T cell subpopulations of the NALT

3.4.1. CD3+ T cellsTo determine if our stress protocol affected the number of CD3+

cells, we compared the number of these cells in both the parafollicu-lar and follicular areas of the NALT for stressed and control animals.In the parafollicular area, the analysis with ANOVA showed a sta-tistically significant difference in CD3+ cells between the animalgroups (F(2,15) = 10; P = 0.002; Fig. 3B). Further analysis with Bon-ferroni’s pairwise comparison procedure revealed that the numberof CD3+ cells in mice at 8 d of stress (223 ± 10.6) was significantlylower (P = 0.006) than that at 4 d of stress (249 ± 7.5) or in controlanimals (237 ± 12.3). On the other hand, the number of CD3+ cellswas not statistically different between control animals and thosestressed for 4 d.

In the follicular area (Fig. 3C), the ANOVA showed a statis-tically significant difference in CD3+ cells between the animalgroups (F(2,15) = 24; P < 0.001). The number of such cells in therestrained groups (4 and 8 d) was significantly lower than that inthe control animals (P < 0.001, Bonferroni t-test). However, therewas not any significant difference between the 4 and 8 d stressedmice (P = 0.766). Hence, the stress protocol of the present studydecreased the population of CD3+ lymphocytes in both the follicularand parafollicular areas.

3.4.2. CD4+ and CD8+ T cellsIn the parafollicular area (Fig. 3B) a statistically significant

decrease was found in CD4+ T cells at 4 or 8 d of restraint stress com-pared to control mice (F(2,15) = 64; P < 0.001). Also, the number ofthese cells was significantly lower in the 8 d than 4 d restrainedgroup (P = 0.04). In the follicular area (Fig. 3C), there were notany significant differences between the three groups (F(2,15) = 23;P = 0.7). Therefore, the results show a progressive decrease in thenumber of CD4+ cells in the parafollicular area with restraint stress.

Regarding the CD8+ T cells in the parafollicular area, therewere no statistically significant differences between the groups(F(2,15) = 0.09; P = 0.9; Fig. 3B). In the follicular area the CD8+ T cellswere absent or very scarce in all groups (Fig. 3C). Therefore, thissubpopulation was resistant to activation by the sympathetic ner-vous system and hypothalamic–pituitary–adrenal axis during therestraint stress protocol of the present study.

3.5. Intraepithelial lymphocytes

There was a statistically significant difference between thegroups in the number of intraepithelial lymphocytes in the mid-dle part of the respiratory epithelium of the NALT (F(2,15) = 35;P < 0.001; Fig. 4). The number of CD4+ IEL cells (Fig. 4A) was signifi-cantly lower at 4 d of restraint stress than in the other two groups:the 8 d restrained group (P = 0.021, Bonferroni t-test) and controlanimals (P < 0.001, Fig. 4C). The number of these cells was also sig-nificantly lower in the 8 d restrained group than in control animals(P < 0.05).



Fig. 3. Distribution of T lymphocytes in the NALT (A). Great amounts of lymphocytes can be observed in the parafollicular area (PA) and a lesser number in the follicular area(FA), marked with monoclonal anti-CD3 (a), anti-CD4 (b), and anti-CD8 (c) antibodies (100×). The scheme (S) shows their pattern of distribution. Effect of restraint stresson the parafollicular (B) and follicular areas (C). Mice restrained for 4 or 8 d, or unrestrained. Immediately after the last stress session, the mice were sacrificed and nasaltissue samples were obtained. The number of CD3+, CD4+ and CD8+ T cells (analyzed as the number of cells per �m2) was determined by immunohistochemistry. Data wereobtained from 7 mice/group and are presented as the mean ± SD. The statistical analysis was performed by using ANOVA, followed by the Bonferroni’s multiple comparisontest. In the parafollicular area (B), compared with the unrestrained control group, restraint stress of 8 d reduced the number of CD3+ T cells (**P < 0.05, Bonferroni t-test), andrestraint stress of 4 and 8 d reduced the number of CD4+ T cells (*P < 0.001), but did not affect CD8+ T cells (P < 0.05). In the follicular area (C), compared with the unrestrainedcontrol group, restraint stress reduced the number of CD3+ T cells (*P < 0.001), but the number of CD4+ and CD8+ T cells was not affected. Similar results were obtained intwo independent experiments.

Although the number of CD8+ IEL cells was scarce (Fig. 4B),we found a statistically significant difference between the groups(F(2,15) = 48; P = 0.007; Fig. 4C). Like CD4+ IEL cells, the number ofCD8+ IEL cells was significantly lower at 4 d of restraint stress thanin the other two groups: the 8 d restrained group (P < 0.001) andcontrol animals (P < 0.01). However, unlike CD4+ IEL cells, the num-ber of CD8+ IEL cells was significantly higher in the 8 d restrainedgroup than in control animals (P < 0.001).

3.6. Effect of stress on the B cell subpopulations of the NALT

The number of cells that expressed the B220 marker (Fig. 5A)in the follicular area was not significantly different in control andexperimental groups (F(2,15) = 1.04; P = 0.4; Fig. 5B). Our results

suggest that B cells were resistant to the hormonal response to therestraint stress protocol used.

3.7. Effect of stress on IgA+ cells in the NALT and non-NALT

IgA+ cells were distributed throughout the NALT (Fig. 6A). Inthe lamina propria (Fig. 6B), IgA+ cells were observed amongthe mucoserous acinus of the mucosa lining of the respiratoryepithelium, predominantly at the cornets. These cells were scarcein the mucosa of the septum and in the anterior region of thenose. In the NALT, there was a statistically significant differencein the number of IgA+ cells between the groups (F(2,15) = 10;P = 0.013; Fig. 6C). The number of IgA+ cells was significantly lowerat 4 d of restraint stress than in the other two groups: the 8 d



Fig. 4. Effect of restraint stress on the nasal intraepithelial lymphocytes. Immunohistochemical staining of intraepithelial lymphocytes. There are few intraepithelial lym-phocytes (arrows) labeled with anti-CD4 (A, 400×) and anti CD8 (B, 200×). The number of IEL was determined by immunohistochemistry with monoclonal antibodies, andis expressed as the number of cells per linear epithelium (C). Compared with the unrestrained control group, restraint stress of 4 d reduced the number of CD4+ T cells(*P < 0.001). Restraint stress of 8 d also reduced the number of lymphocytes, but to a lesser extent (**P < 0.05). Whereas restraint stress of 4 d reduced the number of CD8+ Tcells (**P < 0.05), restraint stress of 8 d increased their number (*P < 0.001). Similar results were obtained in two independent experiments.

restrained group (P = 0.002) and control animals (P = 0.013). Therewas no significant difference between the 8 d restrained groupand control animals (P = 0.15). Therefore, IgA+ cells located in theNALT were susceptible to the stress protocol used, showing a

decrease followed by a recovery to the basal level. In contrast,in the lamina propria there was not any significant differencein the number of IgA+ cells between the groups (F(2,15) = 0.5;P > 0.6).

Fig. 5. B Lymphocytes in the NALT. The scheme (S) shows the distribution pattern. (A) The B lymphocytes marked with monoclonal anti-CD45 (B220) antibodies (100×) canbe seen predominantly in the follicular area (FA), and in a lesser number in the parafollicular area (PA). (B) The graph compares the number of lymphocytes in a folliculararea in the three groups under study. According to the one way ANOVA, there were no significant differences between the three groups (F(2,15) = 1.044; P = 0.4).



Fig. 6. Effect of restraint stress on nasal IgA cells. Distribution of IgA+ cells in the NALT and lamina propria (A and B). The scheme (s) shows the diffuse distribution pattern inthe follicular area as well parafollicular areas. In the follicular area (FA) of the NALT numerous IgA+ cell can be observed (A, 200×). IgA+ cells are scarce in the lamina propriaof the respiratory mucosa (arrow, panel B, 400×). Number of IgA+ cells (C). Compared to the unrestrained control group, the number of lymphocytes diminished significantlyin the 4 d stress group (F(2,15) = 9.667; P = 0.013) and increased significantly in the 8 d stress group (F(2,15) = 3.238; P = 0.002). The quantity of cells in the lamina propriashowed a similar tendency. However the stress protocol in this study did not affect the number of IgA+ cells (F(2,15) = 0.536; P > 0.596).

3.8. Effects of stress on nasal IgA levels

In terms of the IgA concentration in the nasal lumen, there was astatistically significant difference between the groups (F(2,24) = 9;P < 0.01; Fig. 7). The nasal IgA concentration was significantly lowerat 4 d of restraint stress than in the other two groups: the 8 drestrained group (P < 0.001) and control animals (P = 0.003). On theother hand, the IgA concentration was similar in the 8 d restrainedgroup and control animals (P = 0.451).

3.9. Flow cytometric analysis

T- and B-cell composition of the NALT. We determined the cellularcomposition by flow cytometry of isolated lymphocytes from theNALT of non-stressed and stressed mice. The percentages of T cells(CD3+, CD4+, and CD8+) and B cells (B220+) were similar to thosereported by others in the same strain of mice [30–32]. B cells (56%)were more abundant than T cells (35%), the T-cell population con-tained about 3 times as many CD4+ T cells as CD8+ T cells (22% vs7%), and the ratio CD4+/CD8+ T cells was three.

The percentage of CD3+ T cells in the lymphocytes of the NALTwas significantly lower in mice stressed for 4 d and 8 d comparedwith the non-stressed group (Fig. 8A, P < 0.001). Lymphocytes ofthe NALT include those of the parafollicular and follicular areas.This information corroborates our data obtained by immunohisto-chemistry, according to which a reduction occurred in the numberof CD3+ T cells in the parafollicular area of mice stressed for 8 d, andin the same cell population in the follicular area of mice stressedfor 4 and 8 d (Fig. 3).

The percentage of CD4+ T cells was significantly lower in micestressed for 4 d and 8 d compared with the non-stressed group(Fig. 8A, *P < 0.05). Logically, in the T cell rich parafollicular area,the number of CD4+ T cells detected by immunohistochemistry wasalso lower in mice stressed for 4 and 8 d (Fig. 3) compared to controlanimals.

There were not any differences in the percentage of B cells in theNALT of stressed and non-stressed mice (Fig. 8A, P > 0.1), a resultthat confirms the data obtained by immunohistochemistry.

Fig. 7. Effect of restraint stress on nasal IgA. Immediately after the last stress session,mice were sacrificed and the nasal fluid was obtained. The IgA concentration wasdetermined by ELISA and is expressed as mg/ml for each group. Data were obtainedfrom 10 mice/group and are presented as the mean ± SD. Restraint stress modifiedthe concentration of IgA (F(2,27) = 24.2; P < 0.001), which was significantly lower inmice restrained for 4 d than in the other two groups: the 8 d restrained group andthe unrestrained control animals (*P < 0.001, Bonferroni t-test). Similar results wereobtained in two independent experiments.



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Fig. 8. Flow cytometric analysis of the effect of stress on NALT and non-NALT popu-lations. The animals were stressed 3 h for 4 or 8 d (n = 7 in each case) or not stressed(n = 7). The percentages of lymphocytes isolated from NALT and non-NALT (lam-ina propria) were determined by flow cytometry. Results are expressed as themean ± SD (*P < 0.001, **P < 0.05, vs control). (A) T-cell subsets and B cells in theNALT. (B) IgA + plasma cells in NALT and non-NALT (lamina propria). The data shownare representative of two experiments.

IgA+ cells. The flow cytometric analysis showed that, comparedwith the control animals, the percentage of IgA+ cells in NALTwas lower in the 4 d but higher in the 8 d stressed group (Fig. 8B,*P < 0.001, **P < 0.05, respectively). However, the percentage of IgA+

cells in lamina propria (non-NALT) was not modified by the stressprotocol used (P > 0.1). This data is in agreement with that obtainedby immunohistochemistry.

We were unable to get enough intraepithelial lymphocytes fromthe nasal mucosa to perform a flow cytometry analysis. In fact,there is not any report in the literature about the characterizationof intraepithelial lymphocytes from NALT by flow cytometry.

4. Discussion

There are no reports regarding the effect in the nasal mucosaof mice of restraint stress on the distribution and number of var-ious immune cell populations, or on the basal production of IgA.The results of the present study clearly demonstrate that the stressprotocol used selectively affected individual components of theimmune system of the mouse nasal mucosa.

Regarding corticosterone levels, compared to the control ani-mals there was a marked reduction at 4 d and a notable increaseat 8 d of restraint stress. Contrarily, the plasma norepinephrineresponse showed an increase at both 4 and 8 d of restraint stress.Our results suggest that restraint stress for 4 d inhibited the HPA

axis, and that with restraint stress for 8 d this axis was again nor-mally activated. Several studies have reported habituation andadaptation of the corticosterone response to the same (homotypic)stressor, resulting in a decrease in corticosterone levels in the shortrun followed by a recovery of the same in the long run [33–40].Contrarily, other studies have reported the pattern of continu-ous suppression of the corticosterone response to repeated stress[41–44]. In rats subjected to restraint stress for 3, 7 and 10 d, thecorticosterone response to a 10 min session considerably decreasedafter 3 d and moderately decreased after 7 and 10 d. It is possiblethat the difference is related to the distinct stress protocols.

The significant increase in plasma levels of adrenaline with therestraint stress in the present study is in agreement with otherreports [12,45]. The combination of high levels of adrenaline andcorticosterone in mice restrained for 8 d, along with high levels ofadrenaline and low levels of corticosterone in mice restrained for4 d found in the current contribution is also in agreement with aprevious study by our workgroup [12].

Restraint-stress for 4 or 8 d did not have any effect on the struc-ture and volume of the NALT. Although there are no previousreports of stress on these parameters of the NALT, other studieshave found a reduction in the size of primary lymphoid organs(e.g., the thymus) and secondary nodes and spleen (e.g., the mesen-teric lymphoid) [46–50]. Therefore, it is possible that the NALT ofBalb/c mice is more resistant to the effects of stress (e.g., apoptosis)than other lymphoid organs, which are modified significantly withshort-term and long-term restraint stress protocols [51,52].

In control animals, the CD4+ T cells in the parafollicular areawere three times more abundant per area of tissue than CD8+ Tcells, which is consistent with several studies [27,30,31]. Comparedwith the control animals, the number of CD4+ T cells was signifi-cantly lower in the parafollicular area of 4 and 8 d stressed animals.This was not the case with CD8+ T cells, as no differences werefound between the groups. The flow cytometric analysis revealedthat repeated restraint stress (4 and 8 d) decreased the percentageof CD4+ and CD3+ T cells, without affecting the percentage of CD8+

T cells. These results suggest that there was greater susceptibilityof CD4+ than CD8+ T cells to the effects of the stress protocol used.Our results are in agreement with a previous study in which acutetreadmill exercise decreased the number of CD4+ T cells in the sub-mandibular lymph nodes [24,25], and with still another study inwhich acute restraint stress caused a decrease in the total numberof circulating CD4+, but caused no significant effect on CD8+ T cellsduring either acute or chronic stress [53]. In other studies acuterestraint stress decreased the number of both CD4+ and CD8+ Tcells in Peyer’s patches, thymus and spleen of mice [49,54–56]. Wesuppose that the 3 h restraint stress sessions in the present studydid not reach the threshold of acute stress that could affect CD8+

cells.Upon analyzing the amount of nasal intraepithelial lympho-

cytes (nIEL) in the epithelium covering the middle part of theNALT, we found that they were less abundant than the intestinalintraepithelial lymphocytes (iIEL). Similarly, two studies reportedless abundant nIEL in the respiratory epithelium than iIEL in theintestinal epithelium [57,58].

In the present study the ratio of CD4+ IEL to CD8+ IEL cellswas nearly 2:1. Although one study in human nasal mucosa foundthat CD4+ T cells were the predominant IEL population [59], thereare several other reports of a predominant CD8+ T cell population[60–63]. However, it is difficult to compare our results to any ofthese studies because they were done in human nasal mucosa.

In the present study, compared to the control group the num-ber of intraepithelial CD4+ T cells in the nasal epithelium decreasedin the 4 d stressed group, but recovered (moderately but not com-pletely) in the 8 d stressed group. The intraepithelial CD8+ T cells inthe nasal epithelium also decreased in the 4 d stressed group, but



increased beyond the basal level of the control animals in the 8 dstressed group. The causes of these differences are not known.

Whereas there was no significant change in the percentage andnumber of IgA+ cells in the lamina propria of non-NALT areas, thepercentage and number of IgA+ cells in the NALT and the level ofnasal IgA were lower at 4 d of restraint stress than in the other twogroups: the 8 d restrained group and control animals. The reportsin the literature about stress and the number of IgA+ cells are all inrelation to intestinal and respiratory mucosa. One report showedthat restraint stress does not reduce the number of IgA-producingcells in intestinal lamina propria of mice [12]. The other studiescomparing the number of IgA+ cells in intestinal and respiratorymucosa used stress protocols related to exercise or sickness, and aretherefore difficult to compare to the current contribution [64,65].

The mechanism of reduction of IgA+ cells is unknown. However,in the present study this mechanism could not have been relatedto B cell population changes, since no significant differences werenoted in either absolute numbers or in the percentage of B220+

cells in the NALT between stressed and non-stressed mice. Becausethe level of corticosterone was lower and that of adrenaline washigher in mice stressed for 4 d, it is possible that the lack of signifi-cant change in the population of B cells owes itself to the effects ofcatecholamines, or to the ratio between catecholamines and gluco-corticoids, on the maturation and differentiation of B cells.

Adrenaline modulates B-cell function directly and indirectlyvia changes in Th1-cell cytokine production [66–68]. Althoughconflicting findings exist about the role of adrenaline in the pro-liferation and maturation of B cells into IgA-secreting cells [69,70],it is possible that this hormone inhibits the number of IgA-secretingcells, as well as the IgA produced per cell, by acting on plasmablastand plasma cells in the lamina propria [67,68]. Catecholamines alsomay cause apoptosis of B cells [71,72], and/or modulate lymphocytedistribution [73–75].

In the present study the level of total IgA in the nasal lumenof mice was lower at 4 d of restraint stress than in the other twogroups: the 8 d restrained group and control animals. Similarly, butin mouse intestine, we reported that repeated restraint stress (4 hfor 4 d) reduces IgA levels [12]. We suppose that in the current studythe high levels of norepinephrine greatly influenced the reductionof nasal IgA concentration at 4 d of restraint stress, reflecting thehormonal effects on the number of cells producing IgA as well as theamount of IgA secreted per cell. Catecholamines can have at leastthree effects related to IgA levels: (i) a reduction in the populationof IgA producing cells, (ii) a possible inhibition of the production ofIgA per cell, and (iii) a reduction of blood flow, which has the effectof inhibiting IgA production [66,76,77]. In the present study, witha high level of catecholamines and a low level of glucocorticoids(at 4 d of restraint stress), it is possible that the former acted toinhibit IgA production and the population of IgA+ cells. On the otherhand, when the levels of catecholamines and glucocorticoids wereboth high (at 8 d of restraint stress), perhaps the high levels of thelatter blocked the influence of the former on the production and/orsecretion of IgA.

In summary, repeated restraint stress altered the distributionand number of lymphocytes and IgA+ cells in nasal mucosa. Wedetected significant effects on the mouse NALT with the restraintstress protocol employed, such as the susceptibility of CD4+, CD8+

T cells, and IgA+ cells. Also notable was the lack of susceptibilityof B cells. Furthermore, the effects of restraint stress on CD4+ cells,CD8+ T cells, IgA+ cells and IgA levels were significantly different at4 d and 8 d. The number of IELs (CD4+ and CD8+ T cells) was lowerat 4 d and higher at 8 d of stress compared to the control group.Mice restrained for 4 d had high levels of adrenaline and low levelsof corticosterone, whereas mice restrained for 8 d had high levelsof both adrenaline and corticosterone. The difference in the ratioof these two hormones was probably responsible for the decrease

in (at 4 d of stress) and recovery of (at 8 d of stress) the number ofIgA+ cells and the levels of IgA in the mouse NALT.

Conflict of interest

None of the authors has any conflict of interest in relation to thetechniques used or the subjects mentioned in this manuscript.

Acknowledgements

We thank Bruce Allan Larsen for reviewing the use of English inthis manuscript. This work was supported by SIP-IPN, COFAA-IPN,and CONACYT (Grant 33993).

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