1. 1
REPEATED RESTRAINT STRESS REDUCES THE IgA PRODUCING CELLS IN
PEYER’S PATCHES
Beatriz Elina Martínez-Carrillo1, Marycarmen Godinez-Victoria1,2, Adriana Jarillo-Luna3,
Rigoberto Oros-Pantoja1, Edgar Abarca-Rojano4, Víctor Rivera-Aguilar5, Judith Pacheco-
Yepez6, Luvia Enid Sánchez-Torres2 and Rafael Campos-Rodríguez1*.
1
Departamento de Bioquímica y Sección de Estudios de Posgrado e Investigación, Escuela
Superior de Medicina-IPN, Salvador Díaz Mirón y Plan de San Luis s/n, Colonia Santo
Tomás, México, D.F., C.P. 11340, México.
2
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas-IPN, Prol.
Carpio y Plan de Ayala s/n, Colonia Santo Tomás, México, D.F., C.P. 11340, México
3
Departamento de Morfología, Escuela Superior de Medicina-IPN, Salvador Díaz Mirón y
Plan de San Luis s/n, Colonia Santo Tomás, México, D.F., C.P. 11340, México.
4
Laboratorio de Inmunobiología del Endotelio, Escuela Superior de Medicina-IPN,
Salvador Díaz Mirón y Plan de San Luis s/n, Colonia Santo Tomás, México, D.F., C.P.
11340, México.
5
Departamento de Microbiología, Unidad de Biología Tecnología y Prototipos (UBIPRO),
Facultad de Estudios Superiores (FES)-Iztacala, Universidad Nacional Autónoma de
México, Avenida de los Barrios s/n, Tlalnepantla Estado. de México. CP. 54090, México,
D. F.

2. 2
6
Laboratorio de Microscopía Electrónica, Facultad de Medicina, Universidad La Salle
Fuentes 17, Tlalpan, CP. 14000, México, D. F.
*Corresponding author: Rafael Campos-Rodríguez PhD. Escuela Superior de Medicina-
IPN, Salvador Díaz Mirón y Plan de San Luis s/n, Colonia Santo Tomás, México, D.F.,
C.P. 11340, México. Tel. +52(55) 57482004; Fax. +52(55) 57145455. E-mail:
citli@prodigy.net.mx

3. 3
Abstract
The few reports that analyze the effects of stress on the immune cells of the intestinal
mucosa or the functions of these cells tend to focus on S-IgA levels in saliva, and these
studies have shown contradictory results. The principal objective of this study was to
analyze the effects of repeated restraint stress on the number and distribution of immune
cells of Peyer’s patches (PPs), and also the effects of glucocorticoid and catecholamine
administration on the same stress-related parameters. Upon analyzing the effect of repeated
restraint stress on PPs, it was found that there was no modification in the morphological
structure of the PPs, but that restraint stress reduced the total number of lymphocytes, the
number of CD8+ T cells, B cells and plasma cells in PPs. Only in the site of PPs where
IgA-producing plasma cells are most numerous (the dome) was a decrease found in this
type of cell. These effects were due at least in part to the effect of glucocorticoids and
catecholamines. Since IgA produced in the Peyer’s patches is a natural antibody that
impedes bacterial infections, repeated stress may favor the entrance of pathogens through
the intestine.
Keywords: Repeated restraint stress, Peyer’s patches, IgA, glucocorticoids,
catecholamines.

4. 4
Introduction
The neuroendocrine system regulates the immune responses, including those of the
intestinal mucosa [1, 2]. Studies done on the neuroendocrine regulation of the mucosal
immune system have focused mainly on the quantity of IgA secreted in saliva [3, 4], the
synthesis and secretion of the secretory component of the tear glands [5, 6], and the
regulation of the synthesis of the secretory component in the female reproductive apparatus
[7, 8]. On the other hand, studies done on the effects of stress on the mucosal immune
system have focused almost exclusively in relation to inflammatory diseases of the intestine
and the secretion of IgA in saliva.
The abundant information available confirms that psychological stress plays a fundamental
role in the physiopathology and clinical symptoms of intestinal inflammatory diseases in
humans [9, 10]. However, in relation to IgA levels in human saliva, both reductions and
increases have been reported, depending on the type of psychological and/or physical stress
protocol employed [4, 11, 12]. These contradictions make it difficult to reach any
conclusion about the effects of stress on the humoral immune response of the mucosas,
which is represented by secretory IgA (sIgA) levels. Our recent studies show that restraint
stress reduces IgA levels in mouse intestine as well as the intraepithelial lymphocyte
population in mouse duodenal mucosa [13, 14].
The immune system of the mucosa can be divided into inductor and effector sites. In the
former, consisting of principally of lymphoid tissue in PPs, the appendix and solitary nodes
[15, 16], the antigens captured from the mucosa surface stimulate a response from T and B
lymphocytes. In the latter, the effector cells perform their action, such as the production of

5. 5
S-IgA [15, 17]. PPs, the most studied inductor site, have fundamental importance in the
capture of antigens in the intestinal lumen and in the induction of the humoral immune
response, mainly through the production of S-IgA, in the intestine [18, 19]. It is known that
PPs are innervated by nerve fibers containing norepinephrine, vasoactive intestinal
polypeptide (VIP), substance P (SP) and somatostatin (SOM) [20, 21], and that the
lymphocytes of PPs express receptors for neuropeptides (e.g., SOM and SP). In vitro
studies have demonstrated that the production of IgA, IgG and IgM by PP lymphocytes is
regulated by β-endorphin, ACTH and various peptides, which also regulate the migration
and proliferative response of lymphocytes [21].
Stress-related effects on the structure and functions of PPs have been done in the context of
GALT, with a focus on changes in T lymphocyte populations [22, 23]. Since the
neuroendocrine regulation of the function of inductor sites has been little studied, the aim
of the current study was to analyze the effects of repeated restraint stress on the
morphological structure as well as the number and percentage of lymphocytes, including
the IgA-producing plasma cells, in the different regions of Peyer´s patches, and to compare
these stress-related effects with those produced by treatment with glucocorticoids and
catecholamines.

6. 6
Materials and Methods
2.1 Animals
Two-month old male Balb/c mice (from the animal house of the Escuela Superior de
Medicina, ESM), weighing 25 to 30 g, were treated with the appropriate dose of
mebendazole and metronidazole to eliminate parasites. They were housed 6 per cage in a
room with little noise and kept on a 12:12 h light/dark cycle (lights on at 6 am). All
handling and assays were carried out between 8 am and 12 am to avoid the influence of the
circadian cycles of ACTH and cortisol. The mice were handled in accordance with the
norms of the Institutional Commission for the Care and Use of Lab Animals of the ESM.
2.2 Restraint stress protocol
Mice were placed in cylindrical plexiglass containers 9 cm long and 3.5 cm in diameter,
with many ventilation holes to prevent hyperthermia (n = 6). Animals could move to back
and forward freely in the container, but could not turn around. The duration of the restraint
cycle was 1 or 4 h for four successive days. Unrestrained mice were left undisturbed in
their home cages without access to food or water during the same period.
2.3 Hormonal treatment
Groups of 6 mice were treated subcutaneously with epinephrine at 0.1 or 0.5 mg/kg/day for
4 consecutive days. Other groups of 6 mice were treated subcutaneously with
dexamethasone at 5 or 50 mg/kg/day, also for 4 consecutive days. Control mice (n = 6)
received only the vehicle (NaCl 0.89%).
2.4 Isolation of Peyer’s patches and purification of lymphocytes

7. 7
On the fourth day, all animals were sacrificed and the small intestine was extracted and
washed with PBS. The segments that contained Peyer’s patches were separated with fine
dissection scissors, obtaining 3 to 5 patches, depending on the mouse. One PP from the
proximal segment was fixed in 4% formaldehyde and processed for cuts on a wax block,
while another was frozen in isopentane and stored at -70°C until use. The rest of the PPs
were used to quantify the number of lymphocytes by flow cytometry.
Briefly, PPs that were not fixed or frozen were immediately disaggregated and resuspended
in Hank´s balanced salt solution (HBSS). The cell suspension was filled with gauze twice,
with the aim of eliminating epithelial cells and remaining tissue. After that, suspension cells
were washed three times with the same solution, then the button cells were resuspended in
1 mL of HBSS to conduct a count. The total number of cells in each patch was counted by
employing a violet crystal solution, while the viability was evaluated by Trypan blue
exclusion analysis. Finally, the suspension was adjusted to a concentration of 10x106
cells/mL of HBSS.
2.5 Reverse Hemolytic Plaque Assay
The reverse hemolytic assay for the detection of IgA was adapted from a previously
described method [24, 25]. Briefly 0.5 mL of washed and packed sheep erythrocytes
(SRBC) was incubated with 0.1 mL solution of 10 mg rabbit anti-mouse IgA antibody
dissolved in 1 mL of PBS, used as a coupler, and 0.5 mL of CrCl3 solution (1 mg/mL in
0.15 M NaCl) at 37oC for 1 hr with continuous shaking. One-tenth milliliter of cell
suspension of PPs in HBSS was mixed with 20 μL of 20% v/v suspension in 0.1 5 M NaCl
of freshly anti-IgA-coupled SRBC, 20 μL of the serum of rabbit anti-mouse IgA

8. 8
(“developer”, diluted 1:150), and 20 μL of guinea pig serum (complement source). The
mixture was incubated in Cunningham´s chambers at 37oC for 90 min, then the hemolytic
plates were counted and the mean of IgA producing cells per million viable cells was
calculated.
2.6 Topographic staining
The PPs fixed in formaldehyde were cut in slices of 7 µm and stained with Hematoxiline-
Eosine (H-E) and Gomori's trichomic technique for morphological analysis.
2.7 In situ detection of lymphocytes by immunohistochemical techniques
The PPs frozen in isopentane were cut in slices of 7 µm. The cells were stained using an
immunohistochemical technique with monoclonal biotynilated antibodies specific for
identifying T CD4+ and T CD8+ cells, followed by the application of streptavidin
conjugated with HPR. To detect the IgA-producing plasma cells in situ, a monoclonal
antibody to the heavy α-chain of mice and an antibody conjugated to HPR were used.
Finally, the reactions were revealed for 10 min with diaminobenzidine and counterstained
with Harris’ hematoxylin. After dehydration, cells were covered with synthetic resin and
counted by tissue area, each area being measured by an ocular micrometer calibrated with a
hemocytometer.
2.8 Immunophenotyping by flow cytometry
For cell immunophenotyping, directly labeled monoclonal antibodies were used: anti-
CD19-APC or -PE, CD45-PercCP, CD138-APC, IgA-FITC, CD3-FITC, CD8-PE and
CD4-PercCP (all from BD Biosciences, San Jose, CA, USA).

9. 9
Cells were harvested, washed twice with PBS and 0.5% BSA, and then stained for T cell
phenotype with a cocktail of anti-CD3, -CD4 and -CD8 mAb, or for B cell phenotype using
anti-B220 and anti-CD19 mAb, for 30 min at room temperature in darkness. The cells were
then washed with PBS and fixed in 1 % formaldehyde in PBS. IgA-producing plasma cells
(CD138+ cells) and B cells (CD19+/B220+ cells) were fixed, permeabilized and stained
according to BD Bioscience’s protocol for intracellular staining. The fluorescent signal
intensity was recorded and analyzed in a FACSCalibur flow cytometer (Becton Dickinson).
For each sample 15,000 events were collected. Data were analyzed using the Summit
software v4.3 (Dako, Colorado Inc.). The total number of lymphocytes was calculated from
the percentage of cells located in the lymphocyte region in the dot-plot of FSC vs SSC, and
the total number of cell/patch according to the following formula: (# total cells/Peyer´s
patch) x (% lymphocytes)/100. The absolute number of positive cells (subsets of
lymphocytes) was calculated from the total number of lymphocytes, according to the
following formula: (# total cells/Peyer´s patch) x (% positive cells)/100. The percentage
and number of CD4+ cells and CD8+ cells were calculated from the CD3+ cells.
2.9 Statistical Analysis
The differences between two groups were determined by the Student’s t test. The analysis
of data from 3 or more groups was done with one-way ANOVA. All values were presented
as the mean ± SD of at least three independent assays. Statistical analyses were performed
by using the statistical program Sigma Stat for Windows Version 2.03 software (SPSS Inc).
A P-value equal or less than 0.05 was considered statistically significant.

10. 10
Results
3.1 Restraint stress did not modify the morphology of PPs
It is well established that repeated stress in the short or long run modifies the number and
function of immune and inflammatory cells [26, 27]. However, there have been no studies
on the effects of repeated stress on PPs of the mouse intestinal mucosa. Since PPs have
fundamental importance in the capture of antigens in the intestinal lumen and in the
induction of the humoral immune response, we evaluated the structure as well as the
number and percentage of lymphocytes in this tissue. We observed that the normal structure
of the germinal center, internodal regions and dome remained intact in the PPs of stressed
mice (Figure 1). Also, there was no significant difference in this parameter between the
mice stressed for 1 or 4 h.
3.2. Restraint stress modified the cellular composition of PPs
The percentage of B cells was significantly lower in mice stressed for 4 h than in the other
two groups: animals stressed for 1 h or those non-stressed (Figure 2A, P < 0.001), as
detected by flow cytometry. There were no differences in the percentages of the other
subsets of lymphoid cells, including plasma cells, among the three groups. However, when
the absolute number of each cellular subset was determined, the total number of
lymphocytes in the PPs was found to be lower in both groups of stressed mice than the
control animals (Figure 2B, P < 0.001, Bonferroni t-test). The absolute number of CD8+ T
cells, B cells and plasma cells was significantly lower in mice stressed for 4 h than the other
two groups: animals stressed for 1 h or those non-stressed (Figure 2B, P < 0.001). Among
these parameters, only plasma cells were found to be lower in mice stressed for 1 h than in

11. 11
control animals (P < 0.05). The total number of T cells and CD4+ T cells was not affected
by the restraint stress protocol employed.
3.3. Restraint stress reduced the number of IgA-producing plasma cells
Considering that Peyer’s patches contain antibody-producing effector cells [28-30], we
evaluated the effect of repeated restraint stress on the total number of IgA-producing
plasma cells in PPs, as well as the number of these cells in each region. The
immunohistochemical assay demonstrated that repeated restraint stress diminished the
number of IgA-producing plasma cells in the dome, but not in the corona, germinal center
or intermodal region of PPs (Figure 3A and 3B; P < 0.001, Bonferroni t-test). The flow
cytometric analysis confirmed that the total number of IgA-producing plasma cells
(CD138+/IgA+) in PPs was lower in mice stressed for 1 and 4 h than in the control animals
(Figure 3C, P < 0.001). However, when we determined the percentage of IgA-producing
plasma cells, the only significant difference between the three groups was the lower
percentage of these cells found in mice stressed for 1h compared to the control animals
(Figure 3D).
3.4. Effects of dexamethasone and epinephrine
In several studies it has been demonstrated that changes observed in the immune response
induced by stress are mediated principally through the release of glucocorticoids and
catecholamines in different kinds of tissues. Therefore in the present study the effect of
dexamethasone (a glucocorticoid) and epinephrine (a catecholamine) on the number of
lymphocytes and subsets of T and B cells in PPs was evaluated.

12. 12
The dose of 5 mg dexamethasone reduced the percentage of CD4+ T cells (Figure 4A, P <
0.001), while increasing that of CD8+ T cells (P < 0.001) and plasma cells (P < 0.05). The
dose of 50 mg increased the percentage of CD8+ T cells (P < 0.001) and plasma cells (P <
0.05). Not only the percentage but also the number of cells was evaluated. Dexamethasone
at doses of 5 and 50 mg/kg/day significantly reduced: (i) the size of the patches, in which
no germinal centers were found (Figure 1C), (ii) the total number of lymphocytes (Figure
4A, P < 0.001), (iii) the number of all subsets of T cells (Figure 4A, CD3+/CD4+, *P <
0.001; and CD3+/CD8+ cells, **P < 0.05), and (iv) the number of B cells (*P < 0.001;
Bonferroni t-test). However, only the higher dose of dexamethasone reduced the number of
plasma cells (Figure 4A P < 0.001).
In relation to epinephrine, the structure of the PPs remained normal (Figure 1D) and there
were no differences in the percentage of T cells, CD4+ T cells and plasma cells among the
three groups of mice (Figure 5A). The dose of 0.5 mg increased the percentage of CD8+ T
cells (**P < 0.05) and both doses reduced the percentage of B cells (*P < 0.001). There
were no significant changes in the percentages of the other cell subpopulations.
Regarding the absolute number of cells, both doses of epinephrine (0.1 and 0.5 mg/kg/day)
significantly reduced the total number of lymphocytes and the different subpopulations of
lymphocytes, including T cells and subsets of T cells, B cells, and plasma cells (Figure 5B,
*P < 0.001, **P < 0.05, Bonferroni t-test).
Dexamethasone at doses of 5 mg and 50 mg significantly reduced the absolute number of
IgA-producing plasma cells in PPs, as determined by immunohistochemistry (Figure 6A;
*P < 0.01). In agreement with these results, flow cytometry also showed that both doses of

13. 13
dexamethasone decreased the number of IgA-producing plasma cells (Figure 6B, **P <
0.05) as well as their percentage, compared to the control animals (Figure 6C, *P < 0.001).
Epinephrine at the higher dose (0.5 mg/Kg/day) significantly reduced the number of IgA-
producing plasma cells in PPs, as detected by immunohistochemistry (Figure 6D; *P <
0.01). However, no significant change was found with the lower dose (0.1 mg/Kg/day).
When using flow cytometry, both doses of epinephrine were found to reduce the number of
IgA-producing plasma cells (Figure 6E, **P < 0.05), whereas only the 0.1 mg/Kg/day dose
reduced the percentage of IgA-producing plasma cells (Figure 6F, P < 0.001).
3.5. The reverse hemolytic assay
To confirm the findings of immunohistochemistry and flow cytometry, we performed a
functional assay, the reverse hemolytic assay, to quantify IgA antibody secreting cells (IgA-
SCs). This test confirmed that the number of IgA-SCs was significantly lower in stressed
animals (1 or 4 hours) compared to the control group. Also, it confirmed that both doses of
dexamethasone (*P < 0.001) and the higher dose of epinephrine (0.5 mg/Kg/day)
significantly reduced the number of IgA-SCs in PPs (Figure 7; *P < 0.001, **P < 0.05).

14. 14
Discussion
In some lymphoid organs, such as the thymus, spleen, and nodes, diverse types of stress
cause atrophy due to a notable reduction in the number of lymphocytes [26, 27, 31].
Contrarily, in relation to the protocol of the present study, whether applied for 1 or 4 h
restraint stress did not cause atrophy of the PPs (Figure 1B), although it did indeed result in
a decrease in the total number of lymphocytes (Figure 2B).Similarly, both doses of
epinephrine administered in the current contribution caused a decrease of the total number
of lymphocytes in the PPs (Figure 5B) without producing atrophy in this tissue (Figure 1D).
However, both doses of dexamethasone caused atrophy of PPs (Figure 1C) due to a more
significant decrease in the total number of lymphocytes (Figure 4A) than that found with
epinephrine.
In the present study, the number and percentage of T cells (CD3+ cells) and their subsets
(CD4+ T cells and CD8+ T cells), B cells (CD19+/B220+ cells) and plasma cells (CD138+
cells) were evaluated in the PPs of mice. Compared to the control group, in the mice
restrained for 1 h, a reduction was found in the total lymphocytes and the number of plasma
cells and IgA-producing plasma cells. In the mice restrained for 4 h, these same reductions
were observed along with decreases in CD8+ T cells and B cells (Figure 2). Similar results
were reported from another study, where 12 h of restraint stress caused a decrease in the
number of B cells (B220+ cells), CD8+ T cells and total T cells in PPs of mice [22]. In that
study, plasma cells were not evaluated. The fact that a decrease in total T cells was
observed was likely due to the greater time of restraint stress.

15. 15
Even though PPs are an inductor site, they also have antibody-producing effector cells [28-
30]. Consequently, the number and percentage of IgA-producing plasma cells were
evaluated in the present study. The flow cytometric analysis showed that compared to the
non-stressed animals, the total number of IgA-producing plasma cells in PPs was lower in
both groups of restraint-stressed mice. The immunohistochemical study confirmed these
results, and also revealed that of the regions of the PPs, only in the dome was there a
decrease in the number of these cells in stressed mice. In others sites of the PPs, such as the
corona, germinal center and internodal region, there were no significant differences in the
number of IgA-producing plasma cells between stressed and non-stressed groups (Figure
3). The fact that the only change in the number of IgA-producing plasma cells was found in
the dome is to be expected, as it is known that IgA-producing plasma cells are
predominantly located in this region of PPs.
The reduction in the response of IgA-producing plasma cells was not significantly greater
in the mice stressed for 4 hours than those stressed for 1 hour, which could be due to the
effect of habituation. In previous studies, rats were exposed to stress by electric shock [32,
33], finding that the initial exposure significantly reduced the T-cell proliferative response
to mitogens in the spleen (but not in the blood), whereas such effect caused by subsequent
exposures was only minor.
Whereas some IgA antibodies are directed against endogenous antigens (e.g., DNA),
others, such as natural polyreactive IgA, also react with exogenous antigens [29, 30]. The
function of the latter antibodies is not clear, but they could be important for reducing the
frequency of allergies, as well as inflammatory and autoimmune diseases in the intestine

16. 16
[34]. Furthermore, these innate secretory antibodies may protect against some infections,
such as Salmonella typhimurium [35]. Since repeated restraint stress reduced the number of
IgA-producing plasma cells, it may facilitate an invasion by pathogenic micro-organisms.
It is known that stress-induced changes in the immune response are mediated principally
through the release of glucocorticoids and catecholamines in different kinds of tissues, that
administration of high doses of glucocorticoids notably suppresses the humoral immune
response [36, 37], and that dexamethasone and epinephrine at least partially mediate some
of the effects of stress on the systemic immune response [38-40]. Therefore, an evaluation
of the effects of these hormones was included in the present study. The doses of
dexamethasone (5 and 50 mg/kg) were much higher than those normally used [41, 42]
Compared to control animals, both doses of dexamethasone and epinephrine reduced the
number of IgA-producing plasma cells in PPs, as determined by flow cytometry (Figure 6).
The immunohistochemical analysis confirmed these results, with the exception of finding
no significant difference in this parameter with the lower dose of epinephrine.
Compared to control animals, dexamethasone and epinephrine at both doses induced a
decrease in the number of all other evaluated sub-populations of lymphoid cells (Figure 4
and Figure 5). The only exception was with dexamethasone at the lower dose, which
produced no significant reduction in total plasma cells. Interestingly, the effects on the
populations of lymphocytes were progressively greater, considering the 1 hr restraint stress
group, the 4 h restraint stress group, and the application of dexamethasone or epinephrine.
Hence, the effects of restraint stress in the present study were due at least in part to the
effect of glucocorticoids and catecholamines, and it is possible that the doses of these

17. 17
hormones administered represent the effect of a more intense stress than that provoked by
the restraint stress protocol of the current contribution.
In previous studies on rats, dexamethasone significantly reduced the S-IgA levels in the bile
and the number of IgA-producing plasma cells in the ileum [43], and favored the adherence
of bacteria to the epithelium as well as the invasion of the mucosa [44]. There have not
been any previous reports, to the best of our knowledge, regarding the effect of
dexamethasone on the IgA-producing plasma cells in PPs of mice. However, it has been
reported that a single injection of dexamethasone reduces the number of Ig-producing
plasma cells (IgM, IgG and IgA) in the spleen and mesenteric lymph nodes of mice [45],
and that a treatment with a dose between 30 µg/kg and 2.5 mg/kg of BW reduces the
number of T and B lymphocytes in PPs in animals other than mice (pigs, neonatal calves,
sheep and rabbits) [41, 42, 46, 47].
The reduction in the number of IgA-producing plasma cells caused by both doses of this
glucocorticoid could owe itself to three mechanisms: a) a decrease in the number of
lymphoid cells, principally by apoptosis [42, 48], b) an inhibitory effect on macrophages
and helper T cells, as these are responsible for the induction of the immune response [49],
and c) changes in the distribution and migration of lymphocytes, since these are able to
alter the capacity of an organ or tissue to mount a specific immune response [22, 50].
No report was found in the literature about the effect of a catecholamine on the production
of antibodies in PPs. Contradictory results have been reported regarding the effect of
catecholamines on the systemic immune response. Whereas some studies report an
inhibition of the proliferation of B cells and the production of antibodies, others show the

18. 18
opposite effect [51-53]. In one study the effect varied according to the moment of the
administration of the immunogen [54]. Although the precise mechanism by which the doses
of epinephrine employed in the present study inhibited the production of antibodies in the
PPs is unclear, it may be through direct action on mature cells [55]. It seems relevant that
PPs are innervated by fibers that contain epinephrine [20, 56, 57], which in turn modulate
the internalization of pathogenic bacteria [57].
Conclusion
In summary, whereas repeated restraint stress of different intensities did not have any
notable effect on the morphological structure of the PPs, it did change the number of
lymphocytes in this lymphoid tissue. Usually when a stress response causes such effects, it
is at least partially through an increased production of glucocorticoids and catecholamines.
There was a progressive scale of stress-related effects for the 1-hour restraint stress group
(expressing a decrease in total lymphocytes and the number of plasma cells and IgA-
producing plasma cells), the 4-hour restraint-stressed mice (expressing the same decreases
as the 1-hour group, plus a decrease in B cells and CD8+ T cells), and the mice treated with
dexamethasone and epinephrine (expressing the same decreases as the 4-hour group, plus a
decrease in CD4+ T cells and total T cells). Therefore, these effects were due at least in part
to the effect of glucocorticoids and catecholamines, and it seems likely that the effects of
epinephrine and dexamethasone represent the equivalent of an even greater stress than that
produced by the 4 h restraint stress protocol.

19. 19
Acknowledgements
We thank Bruce Allan Larsen for reviewing the use of English in this manuscript. This
research was financially supported by SIP-IPN and CONACYT, Mexico.
Conflict of Interest Statement
The authors declare that they have no conflicts of interest regarding any of the products or
techniques employed in this study.

20. 20
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24. 24
Figure Legends
Figure 1: Effect of restraint stress and treatment with dexamethasone and
epinephrine on the morphology of Peyer’s patches. Mice were unrestrained (A),
restrained for 4 h (B), or were treated with epinephrine (0.5 mg/kg, C) o dexamethasone
(50 mg/kg, D) daily for 4 d. The Peyer’s patches were removed and fixed in formaldehyde
and processed for paraffin embedding and stained with H&E and Gomori's trichomic
techniques. Germinative center (GC), mantle zone (MZ), and dome (Do). Restraint stress or
epinephrine treatment did not cause atrophy of the Peyer’s patches (B and D). However, the
higher dose of dexamethasone caused atrophy and morphological alterations of the patch
(C). (H-E) 100x.
Figure 2. Effects of repeated restraint stress on the on the percentage (A) and number (B)
of total lymphocytes, total and sub-sets of T cells (CD3+ cells), B cells and plasma cells in
Peyer’s patches determined by flow cytometry. The percentage and the number of CD4+ T
cells and CD8+ T cells were calculated from T cells. Results are the media± SD at least 3
independent experiments. *P < 0.001, **P < 0.05.

25. 25
Figure 3. Effects of repeated restraint stress on the number of IgA-producing plasma
cells in Peyer’s patches. Mice were restrained for 1h or 4 h, or unrestrained. (A)
morphological structure of PPs of stressed mice for 4 h by immunohistochemical
technique; observe that the majority of the IgA+ cells are located in the dome (a: 100x, b:
200x). (B) Number of IgA+ cells according to region of PPs of stressed and unstressed mice
detected by immunohistochemistry. (C) Absolute number and (D) percentage of IgA-
producing plasma cells in PPs in stressed and unstressed mice determined by flow
cytometry. Data represent the media ± SD of three experiments. *P < 0.001, **P < 0.05.
Figure 4 .Effect of dexamethasone on the percentage (A) and number (B) of total
lymphocytes and subsets of T cells (CD3+ cells), B cells and plasma cells in Peyer´s
Patches analyzed by flow cytometry. The percentage and the number of CD4+ T cells and
CD8+ T cells were calculated from T cells. Data represent the media ± SD of three
experiments. *P < 0.001, **P < 0.05.

26. 26
Figure 5. Effect of epinephrine on the percentage (A) and number (B) of total lymphocytes,
subsets of T (CD3+ cells) and B cells in Peyer´s Patches analyzed by flow cytometry. The
percentage and the number of CD4+ T cells and CD8+ T cells were calculated from T cells.
Data represent the media ± DS of three experiments. *P < 0.001, **P < 0.05.
Figure 6. Effect of dexamethasone and epinephrine on IgA-producing plasma cells in
PPs. Mice were treated with dexamethasone (5 or 50 mg/kg) or epinephrine (0.1 or 0.5
mg/kg) daily for 4 days. Control animals were treated with the vehicle. Data were obtained
from 6 to 12 mice/group and are presented as the mean ± SD. (A) Number of IgA+ cells in
PPs of treated and untreated mice with dexamethasone, determined by
immunohistochemistry. (B) Number and (C) percentage of IgA-producing plasma cells in
PPs of treated and untreated mice with dexamethasone, determined by flow cytometry. (D)
Number of IgA+ cells in PPs of treated and untreated mice with epinephrine, determined by
immunohistochemistry. (E) Number and (F) percentage of IgA-producing plasma cells in
PPs of treated and untreated mice with ephinephrine, determined by flow cytometry. *P <
0.001, ** P < 0.01, Bonferroni’s t-test.

27. 27
Figure 7.- Effects of chronic restraint stress on the number of IgA-producing cells in
Peyer’s patches detected by an plaque hemolytic assay. Restraint stress, both doses of
dexamethasone (5 and 50 mg) and epinephrine (0.5 mg) significantly reduced (* P <
0.001) the number of IgA-APC detected by a plaque hemolytic assay in a suspension of
Peyer's patch lymphocytes. Similar results were obtained in four independent experiments.