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2. Author's personal copy
Immunology Letters 135 (2011) 78–87
Contents lists available at ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/
Effects of restraint stress on NALT structure and nasal IgA levels
Rigoberto Oros-Pantoja a , Adriana Jarillo-Luna a,b , Víctor Rivera-Aguilar c , Luvia Enid Sánchez-Torres d ,
Marycarmen Godinez-Victoria a , Rafael Campos-Rodríguez e,∗
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, Mexico
b
Departamento de Morfología, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, CP. 11340, México, DF, Mexico
c
Departamento de Microbiología, UBIPRO, FES-Iztacala, UNAM, Avenida de los Barrios s/n, Tlalnepantla Edo, de Mexico, CP. 54090, Mexico, DF, Mexico
d
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación Carpio s/n, Col. Santo Tomas, CP. 11340, México, DF, Mexico
e
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 a b s t r a c t
Article history: The effects of stress on the mucosal immune responses in inflammatory disorders of the gut, as well as on
Received 27 March 2010 salivary and intestinal IgA levels are well known. However, its effects on the structure and function of the
Received in revised form NALT have not yet been reported, and are examined in the present study. Balb/c mice were submitted to
23 September 2010
restraint stress for 3 h per day during 4 or 8 d. The immunohistochemistry and flow cytometric analysis
Accepted 3 October 2010
revealed that repeated restraint stress (4 and 8 d) decreased the percentage, compared to the control
Available online 16 October 2010
group, 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 NALT
Keywords:
Restraint stress
and nasal IgA levels showed a similar pattern, being significantly lower at 4 d of stress and significantly
NALT higher at 8 d. In summary, repeated restraint stress altered the distribution and number of lymphocytes
IgA and IgA+ cells in nasal mucosa, probably due to changes in norepinephrine and corticosterone levels.
Lymphocytes © 2010 Elsevier B.V. All rights reserved.
Catecholamines
Glucocorticoids
1. Introduction It has been documented that psychological stress alters suscep-
tibility to several different strains of respiratory viruses [14], and
The effects of stress on the mucosal immune responses have numerous reports indicate that exercise stress can increase the risk
been widely analyzed in relation to inflammatory disorders of the for upper respiratory tract infection, particularly in highly trained
gut and the secretion of IgA in saliva. The robust information avail- and elite athletes [15–17]. Although some elite athletes and sub-
able confirms that psychological stress plays a key role in the jects under severe stress produce less IgA in saliva, it has not been
pathophysiology and clinical presentation of inflammatory bowel established that this is the cause of the higher incidence of res-
disease [1–5]. piratory infections in these populations [17–20]. Other causative
There are contradictory reports on the relationship between factors that have been proposed are the presence of infiltrated
secretory IgA (S-IgA) levels in saliva and different conditions of inflammatory cells in mucous membranes and the removal of one
stress, such as exercise, mood states and academic examina- or more immune functions [15,16,21].
tions. Whereas some studies found decreases in S-IgA, others It is unknown whether chronic stress can alter the structure
detected increases or no change [6–11]. We recently reported that and/or function of the nasal-associated lymphoid tissue (NALT), and
stress decreases intestinal IgA levels, and affects the population if so whether such change would contribute to the increased inci-
of intraepithelial lymphocytes in the duodenal mucosa of mice dence of respiratory infections found among elite athletes. Studies
[12,13]. However, the effects of stress on the nasal immune system on animals suggest that stress can affect the immune responses
have not been explored in detail. in the upper respiratory tract. In mice infected intranasally with
influenza virus, restraint stress increases levels of IgM and IgG
antibody-secreting cells, which are virus-specific responses in the
superficial cervical lymph node, the latter being considered part
of the NALT [22]. On the other hand, restraint stress inhibits the
∗ Corresponding author at: Departamento de Bioquímica. Escuela Superior de
production of IgE, IgG1 and IgG2a, specific for an allergen inocu-
Medicina. 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. lated intranasally [23]. Moreover, acute treadmill exercise of mice
E-mail address: citli@prodigy.net.mx (R. Campos-Rodríguez). decreases 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

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R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87 79
nodes, cells which play a critical role in the induction of immune with 2 ml sterile saline solution, which was collected in Eppendorff
responses to antigens in the eye, upper respiratory tract and oral tubes. The tubes were stored at −70 ◦ C until the analysis was done
mucosa [24,25]. of secretory IgA (sIgA) by paired ELISA. Protein concentration and
The NALT is defined as the oropharyngeal lymphoid tissue of quantification curves were constructed by the Bradford method.
the upper respiratory airways of rodents, considered analogous After the nasal wash procedure, the skin of the head was
to Waldeyer’s ring in humans [26–28]. It consists of paired lym- removed, as were the inferior jawbone and soft tissue, accord-
phoid structures situated above the soft palate at the entrance to ing to the method described by Asanuma et al. and Heritage
the bifurcated pharyngeal duct, which are composed in part of sec- et al. [30–32]. The extracted palate was placed upside down in
ondary lymphoid aggregates characterized by follicular B-cell areas 1 cm3 aluminum containers embedded in tissue inclusion medium
and parafollicular T-cell areas [29], as well as the many lympho- (Tissue-tek, Sakura, 4583). The containers were frozen and stored
cytes found in and underneath the epithelial lining of the nasal at −70 ◦ C until the embedded tissue was cut in a cryostate. After
mucosa [28]. The NALT is covered by an epithelium or follicle- removing the NALT, the skulls were fixed by immersion in 4%
associated epithelium (FAE), which consists of ciliated columnar paraformaldehyde for 24 h, washed, and decalcified with 8% EDTA
cells, M cells (alone or in clusters), intraepithelial lymphocytes (Baker analyzed) at pH 7.6. The solution was changed and this cycle
and a few goblet cells. Antigen-presenting cells, including dendritic was repeated daily for 8 d. The skulls were then included in paraffin.
cells and macrophages, are also found in the NALT. Therefore, this
organ must have an important role in the induction and regulation 2.4. Processing
of mucosal immune responses to antigens in the upper respiratory
tract [27–29]. From the samples of frozen NALT, 7 m thick cuts were made
To the best of our knowledge there have not yet been any reports on the crown portion, and then placed on slides previously treated
on the effects of stress on the structure and function of the NALT. with 1% gel. Some slides were fixed in acetone for 20 min and others
Thus, the aim of the present study was to determine whether or not in 4% formaldehyde for the same time. Those cuts fixed in formalde-
repeated restraint stress induces a change in the levels of plasmatic hyde and the cuts from the samples processed in wax were stained
glucocorticoids and catecholamines, and/or in the distribution of with haematoxylin and eosin for a general morphological analysis.
lymphocytes in the nasal mucosa, and if so, whether such changes
have any correlation with nasal IgA levels in mice. The results show 2.5. Immunohistochemistry
that repeated restraint stress selectively affects individual compo-
nents of the immune system of the nasal mucosa of mice and the Cells were quantified by utilizing immunohistochemical meth-
basal production of IgA. 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-
2. Materials and methods oxidase was blocked by incubation with 3% H2 O2 and 0.1% NaN3
in PBS for 10 min. The samples were washed, incubated with 5%
2.1. Animals bovine serum for 30 min, and washed again with 0.05% Tween-20
in PBS. Plasmatic cells producing IgA were determined by a direct
Ten week old male Balb/c mice (Harlan, Mexico) were ran- immunohistochemical technique, utilizing goat anti-mouse IgA
domly placed in three groups (n = 7): two experimental groups that peroxidase conjugate polyclonal antibodies (HRP-Serotec). Addi-
underwent restraint stress and a non-stressed control group. Of the tionally, monoclonal biotin conjugate mouse antibodies were used
experimental groups, one underwent restraint stress during 4 d and for an indirect immunohistochemical technique. The following
the other during 8 d. Animals were handled and treated according lymphocytes were detected: CD3+ (BD Pharmingen, 553323), CD4+
to a protocol approved by the Ethics and Institutional Animal Care (BD Pharmingen, 553728), CD8+ (BD Pharmingen, 553029) and
and Use Committees. CD45-R (B220 BD Pharmingen, 553085). Estreptavidine peroxidase
conjugate (Jackson Immuno Research) was later applied.
2.2. Restraint stress protocol The primary antibodies were incubated for 2 h and estreptavi-
dine for 1 h, both at RT in a humidified chamber. Gentle washes
The experimental groups were submitted to 3 h restraint stress were carried out with PBS at the end of each incubation period.
sessions daily, always from 8:00 to 11:00 am. Restraint stress was The peroxidase reaction was revealed according to the Karnovsky
carried out by placing the mice in cylindrical plastic containers 6 cm method with DAB (Pierce, 34065). The samples were counter-
long, 3 cm high and 3.5 cm wide, with many ventilation holes to stained with one part of Harris’ haematoxylin diluted in 3 parts
prevent hyperthermia. At the time of restraint stress for the exper- of water, then dehydrated and covered with synthetic resin. With
imental groups, non-restrained mice were left undisturbed in their the control samples for each antibody, stains were conducted by the
home cages, but without access to food or water. Apart from the same method except that the first antibody primer was substituted
restraint stress schedule, the experimental animals were kept in by PBS. Other control samples were incubated with a peroxidated
cages and all three groups were provided with food and water ad antibody before staining for anti-mouse IgA antibodies.
libitum.
To avoid adaptation during the 3 h restraint stress sessions, the 2.6. Microscopic analysis and cell quantification
mice 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 The total area of NALT was measured in m2 in the cuts stained
(ii) the rotation of the containers for 10 s after 20 min, followed by with H–E by using Imagen Pro Plus software, calibrated at 200×
another 30 min cycle that began in the same way and included the magnification. With the same software, using constant areas of
immersion of the mouse tails in cold water for 10 s after 30 min. 2500 m2 from images magnified 400× (see Fig. 4A), the number
of T CD3+ , CD4+ , CD8+ , B IgA+ and CD45+ lymphocytes were quan-
2.3. Obtaining and processing biological material tified in the follicular and parafollicular zones. The software tools
employed in the count were: adjustment of the minimum and max-
Control and experimental mice were anaesthetized with ether, imum range of the area of cells to be counted, manual selection of
bled by direct cardiac puncture, and sacrificed by decapitation. The the color of cells to be counted, and Watershed-split and Autosplit
nasal wash was done by retrograde infusion through the trachea to separate cells that were very close together. In the lamina propria

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80 R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87
the IgA+ cells were counted in the same way. In the quantification of 12000 250
CD4+ and CD8+ intraepithelial lymphocytes, the average longitude P < 0.001
Control
of the respiratory epithelium in the crown cuts of the NALT, which 10000 Stress 4 d *
* Stress 8 d 200
turned out to be 800 linear m, was used as a reference. Finally, the *
volume of the NALT was calculated by measuring the total area of 8000
this tissue from each cut, averaging the various cuts made on each 150
ng / ml
pg / ml
animal, then averaging this value for all the animals in each group. 6000
Each count was made in duplicate.
100
4000
2.7. Flow cytometry 2000
50
*
Nasal-associated lymphoid tissue (NALT) cell suspensions and
0 0
the nasal passage (non-NALT) lymphocytes, which were prepared Corticosterone Epinephrine
from the portion of the nasal cavity remaining after isolation of
NALT, were obtained according to procedures previously described Fig. 1. Effect of restraint stress on serum corticosterone and epinephrine levels.
[30,32]. For cell immunophenotyping, directly labeled antibodies Mice were subjected to restraint stress during 3 h for 4 or 8 consecutive days. Later,
were used: anti-CD19-PE, IgA-FITC, CD138-APC, CD3-FITC, CD4-PE peripheral blood was collected and subjected to corticosterone and norepinephrine
radioimmunoassay. Data are expressed as the mean ± SD (n = 5–8). Corticosterone
and CD8-APC (all from BD Biosciences, San Jose, CA, USA). Cells were
levels were significantly lower in mice restrained for 4 d than in the other two
harvested, washed twice with PBS and 0.5% BSA and then stained groups: the 8 d restrained group and the unrestrained control (P < 0.001, Bonfer-
for T cell phenotype with a cocktail of anti-CD3, -CD4 and -CD8 roni t-test). Norepinephrine levels were significantly higher in mice restrained for
mAb, for 30 min at room temperature in the dark. The cells were 4 and 8 d than in the unrestrained control (P < 0.001, Bonferroni t-test).
then washed with PBS and fixed in 2% formaldehyde in PBS. B cells
were fixed, permeabilized and stained according to BD Biosciences’
protocol for intracellular staining. Stained cells were acquired with 3. Results
a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed
using the Flow-jo software v7.5 (Tree Star, Inc.). 3.1. Effect of restraint stress on serum corticosterone and
norepinephrine
2.8. Enzyme-linked immunosorbent assay (ELISA) for IgA in the Given that the changes in the immune response induced by
nasal wash stress are mediated principally through the release of glucocor-
ticoids and catecholamines, we determined plasma corticosterone
Rabbit anti-mouse IgA immunoglobin (20 g/ml) was placed and norepinephrine concentrations in restrained and unrestrained
in each well, which was incubated for 18 h at 4 ◦ C. After wash- mice (Fig. 1). Compared to control animals, restraint stress signif-
ing 3 times with a phosphate-Tween 20 (PBS-T) buffer at pH 7.2, icantly modified serum levels of corticosterone (one-way ANOVA;
the samples were directly applied and incubated for 2 h at 37 ◦ C. F(2,15) = 80; P < 0.001) and norepinephrine (F(2,15) = 32; P < 0.001).
The plates were then washed 5 times with PBS-T and 5 times Corticosterone levels were significantly lower in mice at 4 d of
with PBS, then incubated for 2 h at 37 ◦ C with goat anti-mouse restraint stress than in the other two groups; at 8 d restraint stress
IgA conjugate (BD Pharmingen, 55549) diluted 1:3000 in PBS-T. or in control animals (P < 0.001, Bonferroni t-test). On the contrary,
Finally, the plates were washed 3 times with PBS-T and 3 times the norepinephrine concentrations in both groups of restrained
with PBS before the substrate (Ortophenylendiamina, Sigma-OPD) mice (4 and 8 d) were significantly higher than in control mice
was added at RT. After 15 min the reaction was stopped with 2.5 M (P < 0.001, Bonferroni t-test).
sulfuric acid and the absorbance was determined at a wave length
of 490 nm. A standard curve was made utilizing purified mouse IgA 3.2. Effect of restraint stress on the microscopic structure and
from myeloma (MP-Biomedicals, 64334) at a concentration range volume of the NALT
of 50 g to 150 ng.
We analyzed the morphology of the NALT after staining this
tissue with H&E. In unstressed mice we observed one area with
2.9. Determination of plasma corticosterone and epinephrine two ovoid masses, one on each side of the midline of the nasal
face of the palate next to the sidewall of the nose (Fig. 2A). In the
A determination of plasma corticosterone was made by a com- structure of the NALT it was difficult to identify typical lymphoid
mercial kit of ELISA (Cayman Chemical Company, 500651), and that nodules. Regarding irrigation, both conventional and high endothe-
of plasma epinephrine by a commercial kit of radioimmunoassay lial venules (HEV) and arterioles were identified in the NALT.
(RIA-LDN Labor Diagnostika Nord, BA-0100). Upon comparing the analysis of the NALT from stressed and
unstressed animals, the morphology of the NALT was found to be
similar. The total volume of the NALT was estimated by graph-
2.10. Statistical analysis ing the volume of 35 serialized sections taken from the front to
the back portion. When the points were joined, the organ showed
Data are presented as the mean ± SD. The comparison of two a cylindrical shape with a greater volume in the central portion
groups was analyzed by using the Student’s unpaired two-tailed than at the extremes. This form was similar in control and stressed
t-test. One-way ANOVA was performed to compare more than two groups (Fig. 2B). The mean volume was 2.95 ± 0.56 mm3 in the
groups, and if a significant main effect or association was identi- control group, 2.68 ± 0.42 mm3 in the group stressed for 4 d, and
fied (P < 0.05), the respective group means were compared using 3.27 ± 0.65 mm3 in the group stressed for 8 d. Analysis with one-
the Bonferroni t-test. All analyses were performed using the statis- way ANOVA revealed that there were not any significant differences
tical program SigmaStat for Windows Version 2.03 software, and between the control and the experimental groups (F(2,15) = 1.4,
graphed with Sigma Plot software (SPSS Inc.). P = 0.27), although there was a tendency towards the atrophy of

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R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87 81
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 lateral
face (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 analysis
did 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 NALT In the follicular area (Fig. 3C), the ANOVA showed a statis-
volume in the 8 d stressed group. tically significant difference in CD3+ cells between the animal
groups (F(2,15) = 24; P < 0.001). The number of such cells in the
restrained groups (4 and 8 d) was significantly lower than that in
3.3. Effect of restraint stress on the distribution of NALT the control animals (P < 0.001, Bonferroni t-test). However, there
lymphocytes was not any significant difference between the 4 and 8 d stressed
mice (P = 0.766). Hence, the stress protocol of the present study
The immunohistochemical analysis of the NALT from control decreased the population of CD3+ lymphocytes in both the follicular
animals showed several patterns of cell distribution, which were and parafollicular areas.
determined 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 no 3.4.2. CD4+ and CD8+ T cells
germinative center, surrounded by a parafollicular area largely In the parafollicular area (Fig. 3B) a statistically significant
composed of T lymphocytes (CD3+ , CD4+ and CD8+ cells) (Fig. 3A). decrease was found in CD4+ T cells at 4 or 8 d of restraint stress com-
However, there were no well-defined boundaries between the pared to control mice (F(2,15) = 64; P < 0.001). Also, the number of
two areas, as part of the parafollicular area was found mixed these cells was significantly lower in the 8 d than 4 d restrained
with follicular area. CD4+ T cells existed in both the follicular and group (P = 0.04). In the follicular area (Fig. 3C), there were not
parafollicular areas, while CD8+ T cells showed a pattern of dis- any significant differences between the three groups (F(2,15) = 23;
tribution more circumscribed to the parafollicular area (Fig. 3A). P = 0.7). Therefore, the results show a progressive decrease in the
The immunohistochemical analysis of the NALT from stressed number of CD4+ cells in the parafollicular area with restraint stress.
mice (both the 4-day and 8-day groups) did not show any alter- Regarding the CD8+ T cells in the parafollicular area, there
ation in the distribution patterns of B or T lymphocytes or their were no statistically significant differences between the groups
subpopulations. (F(2,15) = 0.09; P = 0.9; Fig. 3B). In the follicular area the CD8+ T cells
were absent or very scarce in all groups (Fig. 3C). Therefore, this
subpopulation was resistant to activation by the sympathetic ner-
3.4. Effect of stress on the T cell subpopulations of the NALT vous system and hypothalamic–pituitary–adrenal axis during the
restraint stress protocol of the present study.
3.4.1. CD3+ T cells
To determine if our stress protocol affected the number of CD3+
cells, we compared the number of these cells in both the parafollicu- 3.5. Intraepithelial lymphocytes
lar and follicular areas of the NALT for stressed and control animals.
In the parafollicular area, the analysis with ANOVA showed a sta- There was a statistically significant difference between the
tistically significant difference in CD3+ cells between the animal groups in the number of intraepithelial lymphocytes in the mid-
groups (F(2,15) = 10; P = 0.002; Fig. 3B). Further analysis with Bon- dle part of the respiratory epithelium of the NALT (F(2,15) = 35;
ferroni’s pairwise comparison procedure revealed that the number P < 0.001; Fig. 4). The number of CD4+ IEL cells (Fig. 4A) was signifi-
of CD3+ cells in mice at 8 d of stress (223 ± 10.6) was significantly cantly lower at 4 d of restraint stress than in the other two groups:
lower (P = 0.006) than that at 4 d of stress (249 ± 7.5) or in control the 8 d restrained group (P = 0.021, Bonferroni t-test) and control
animals (237 ± 12.3). On the other hand, the number of CD3+ cells animals (P < 0.001, Fig. 4C). The number of these cells was also sig-
was not statistically different between control animals and those nificantly lower in the 8 d restrained group than in control animals
stressed for 4 d. (P < 0.05).

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82 R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87
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 stress
on 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 nasal
tissue 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 were
obtained 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 comparison
test. 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), and
restraint 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 unrestrained
control 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 in
two independent experiments.
Although the number of CD8+ IEL cells was scarce (Fig. 4B), suggest that B cells were resistant to the hormonal response to the
we found a statistically significant difference between the groups restraint stress protocol used.
(F(2,15) = 48; P = 0.007; Fig. 4C). Like CD4+ IEL cells, the number of
CD8+ IEL cells was significantly lower at 4 d of restraint stress than
in the other two groups: the 8 d restrained group (P < 0.001) and 3.7. Effect of stress on IgA+ cells in the NALT and non-NALT
control animals (P < 0.01). However, unlike CD4+ IEL cells, the num-
ber of CD8+ IEL cells was significantly higher in the 8 d restrained IgA+ cells were distributed throughout the NALT (Fig. 6A). In
group than in control animals (P < 0.001). the lamina propria (Fig. 6B), IgA+ cells were observed among
the mucoserous acinus of the mucosa lining of the respiratory
epithelium, predominantly at the cornets. These cells were scarce
3.6. Effect of stress on the B cell subpopulations of the NALT in the mucosa of the septum and in the anterior region of the
nose. In the NALT, there was a statistically significant difference
The number of cells that expressed the B220 marker (Fig. 5A) in the number of IgA+ cells between the groups (F(2,15) = 10;
in the follicular area was not significantly different in control and P = 0.013; Fig. 6C). The number of IgA+ cells was significantly lower
experimental groups (F(2,15) = 1.04; P = 0.4; Fig. 5B). Our results at 4 d of restraint stress than in the other two groups: the 8 d

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R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87 83
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, and
is 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+ T
cells (**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). There decrease followed by a recovery to the basal level. In contrast,
was no significant difference between the 8 d restrained group in the lamina propria there was not any significant difference
and control animals (P = 0.15). Therefore, IgA+ cells located in the in the number of IgA+ cells between the groups (F(2,15) = 0.5;
NALT were susceptible to the stress protocol used, showing a 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×) can
be 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 follicular
area 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).

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84 R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87
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 in
the 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 propria
of the respiratory mucosa (arrow, panel B, 400×). Number of IgA+ cells (C). Compared to the unrestrained control group, the number of lymphocytes diminished significantly
in 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 propria
showed 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 a
statistically significant difference between the groups (F(2,24) = 9;
P < 0.01; Fig. 7). The nasal IgA concentration was significantly lower
at 4 d of restraint stress than in the other two groups: the 8 d
restrained group (P < 0.001) and control animals (P = 0.003). On the
other hand, the IgA concentration was similar in the 8 d restrained
group and control animals (P = 0.451).
3.9. Flow cytometric analysis
T- and B-cell composition of the NALT. We determined the cellular
composition by flow cytometry of isolated lymphocytes from the
NALT of non-stressed and stressed mice. The percentages of T cells
(CD3+ , CD4+ , and CD8+ ) and B cells (B220+ ) were similar to those
reported 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% vs
7%), and the ratio CD4+ /CD8+ T cells was three.
The percentage of CD3+ T cells in the lymphocytes of the NALT
was significantly lower in mice stressed for 4 d and 8 d compared
with the non-stressed group (Fig. 8A, P < 0.001). Lymphocytes of
the 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 number
of CD3+ T cells in the parafollicular area of mice stressed for 8 d, and
in the same cell population in the follicular area of mice stressed
for 4 and 8 d (Fig. 3).
The percentage of CD4+ T cells was significantly lower in mice
Fig. 7. Effect of restraint stress on nasal IgA. Immediately after the last stress session,
stressed for 4 d and 8 d compared with the non-stressed group
mice were sacrificed and the nasal fluid was obtained. The IgA concentration was
(Fig. 8A, *P < 0.05). Logically, in the T cell rich parafollicular area, determined by ELISA and is expressed as mg/ml for each group. Data were obtained
the number of CD4+ T cells detected by immunohistochemistry was from 10 mice/group and are presented as the mean ± SD. Restraint stress modified
also lower in mice stressed for 4 and 8 d (Fig. 3) compared to control the concentration of IgA (F(2,27) = 24.2; P < 0.001), which was significantly lower in
animals. mice restrained for 4 d than in the other two groups: the 8 d restrained group and
the unrestrained control animals (*P < 0.001, Bonferroni t-test). Similar results were
There were not any differences in the percentage of B cells in the
obtained in two independent experiments.
NALT of stressed and non-stressed mice (Fig. 8A, P > 0.1), a result
that confirms the data obtained by immunohistochemistry.

9. Author's personal copy
R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87 85
A 70 Control axis, and that with restraint stress for 8 d this axis was again nor-
Stress 4 d mally activated. Several studies have reported habituation and
Stress 8 d
60 adaptation of the corticosterone response to the same (homotypic)
stressor, resulting in a decrease in corticosterone levels in the short
50 run 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
Cells (%)
40
[41–44]. In rats subjected to restraint stress for 3, 7 and 10 d, the
30 * * corticosterone response to a 10 min session considerably decreased
after 3 d and moderately decreased after 7 and 10 d. It is possible
** **
20 that the difference is related to the distinct stress protocols.
The significant increase in plasma levels of adrenaline with the
10 restraint stress in the present study is in agreement with other
reports [12,45]. The combination of high levels of adrenaline and
0 corticosterone in mice restrained for 8 d, along with high levels of
T cells CD4 CD8 B cells adrenaline and low levels of corticosterone in mice restrained for
4 d found in the current contribution is also in agreement with a
40
B Control previous study by our workgroup [12].
Stress 4 d Restraint-stress for 4 or 8 d did not have any effect on the struc-
35 Stress 8 d P < 0.001
ture and volume of the NALT. Although there are no previous
30 reports of stress on these parameters of the NALT, other studies
* have found a reduction in the size of primary lymphoid organs
25 (e.g., the thymus) and secondary nodes and spleen (e.g., the mesen-
IgA+ cells (%)
teric lymphoid) [46–50]. Therefore, it is possible that the NALT of
20 Balb/c mice is more resistant to the effects of stress (e.g., apoptosis)
**
than other lymphoid organs, which are modified significantly with
15
short-term and long-term restraint stress protocols [51,52].
10 In control animals, the CD4+ T cells in the parafollicular area
were three times more abundant per area of tissue than CD8+ T
5 cells, which is consistent with several studies [27,30,31]. Compared
with the control animals, the number of CD4+ T cells was signifi-
0
NALT Lamina propria 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 were
Fig. 8. Flow cytometric analysis of the effect of stress on NALT and non-NALT popu- found between the groups. The flow cytometric analysis revealed
lations. The animals were stressed 3 h for 4 or 8 d (n = 7 in each case) or not stressed that repeated restraint stress (4 and 8 d) decreased the percentage
(n = 7). The percentages of lymphocytes isolated from NALT and non-NALT (lam-
of CD4+ and CD3+ T cells, without affecting the percentage of CD8+
ina propria) were determined by flow cytometry. Results are expressed as the
mean ± SD (*P < 0.001, **P < 0.05, vs control). (A) T-cell subsets and B cells in the
T cells. These results suggest that there was greater susceptibility
NALT. (B) IgA + plasma cells in NALT and non-NALT (lamina propria). The data shown of CD4+ than CD8+ T cells to the effects of the stress protocol used.
are representative of two experiments. Our results are in agreement with a previous study in which acute
treadmill exercise decreased the number of CD4+ T cells in the sub-
mandibular lymph nodes [24,25], and with still another study in
IgA+ cells. The flow cytometric analysis showed that, compared
which acute restraint stress caused a decrease in the total number
with the control animals, the percentage of IgA+ cells in NALT
of circulating CD4+ , but caused no significant effect on CD8+ T cells
was lower in the 4 d but higher in the 8 d stressed group (Fig. 8B,
during either acute or chronic stress [53]. In other studies acute
*P < 0.001, **P < 0.05, respectively). However, the percentage of IgA+
restraint stress decreased the number of both CD4+ and CD8+ T
cells in lamina propria (non-NALT) was not modified by the stress
cells in Peyer’s patches, thymus and spleen of mice [49,54–56]. We
protocol used (P > 0.1). This data is in agreement with that obtained
suppose that the 3 h restraint stress sessions in the present study
by immunohistochemistry.
did not reach the threshold of acute stress that could affect CD8+
We were unable to get enough intraepithelial lymphocytes from
cells.
the nasal mucosa to perform a flow cytometry analysis. In fact,
Upon analyzing the amount of nasal intraepithelial lympho-
there is not any report in the literature about the characterization
cytes (nIEL) in the epithelium covering the middle part of the
of intraepithelial lymphocytes from NALT by flow cytometry.
NALT, we found that they were less abundant than the intestinal
intraepithelial lymphocytes (iIEL). Similarly, two studies reported
4. Discussion less abundant nIEL in the respiratory epithelium than iIEL in the
intestinal epithelium [57,58].
There are no reports regarding the effect in the nasal mucosa In the present study the ratio of CD4+ IEL to CD8+ IEL cells
of mice of restraint stress on the distribution and number of var- was nearly 2:1. Although one study in human nasal mucosa found
ious immune cell populations, or on the basal production of IgA. that CD4+ T cells were the predominant IEL population [59], there
The results of the present study clearly demonstrate that the stress are several other reports of a predominant CD8+ T cell population
protocol used selectively affected individual components of the [60–63]. However, it is difficult to compare our results to any of
immune system of the mouse nasal mucosa. these studies because they were done in human nasal mucosa.
Regarding corticosterone levels, compared to the control ani- In the present study, compared to the control group the num-
mals there was a marked reduction at 4 d and a notable increase ber of intraepithelial CD4+ T cells in the nasal epithelium decreased
at 8 d of restraint stress. Contrarily, the plasma norepinephrine in the 4 d stressed group, but recovered (moderately but not com-
response showed an increase at both 4 and 8 d of restraint stress. pletely) in the 8 d stressed group. The intraepithelial CD8+ T cells in
Our results suggest that restraint stress for 4 d inhibited the HPA the nasal epithelium also decreased in the 4 d stressed group, but

10. Author's personal copy
86 R. Oros-Pantoja et al. / Immunology Letters 135 (2011) 78–87
increased beyond the basal level of the control animals in the 8 d in (at 4 d of stress) and recovery of (at 8 d of stress) the number of
stressed group. The causes of these differences are not known. IgA+ cells and the levels of IgA in the mouse NALT.
Whereas there was no significant change in the percentage and
number of IgA+ cells in the lamina propria of non-NALT areas, the Conflict of interest
percentage and number of IgA+ cells in the NALT and the level of
nasal IgA were lower at 4 d of restraint stress than in the other two None of the authors has any conflict of interest in relation to the
groups: the 8 d restrained group and control animals. The reports techniques used or the subjects mentioned in this manuscript.
in the literature about stress and the number of IgA+ cells are all in
relation to intestinal and respiratory mucosa. One report showed Acknowledgements
that restraint stress does not reduce the number of IgA-producing
cells in intestinal lamina propria of mice [12]. The other studies We thank Bruce Allan Larsen for reviewing the use of English in
comparing the number of IgA+ cells in intestinal and respiratory this manuscript. This work was supported by SIP-IPN, COFAA-IPN,
mucosa used stress protocols related to exercise or sickness, and are and CONACYT (Grant 33993).
therefore difficult to compare to the current contribution [64,65].
The mechanism of reduction of IgA+ cells is unknown. However,
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