2011 repeated restraint stress reduces the ig a producing cells in peyers patches
1 REPEATED RESTRAINT STRESS REDUCES THE IgA PRODUCING CELLS IN PEYER’S PATCHESBeatriz 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, EscuelaSuperior de Medicina-IPN, Salvador Díaz Mirón y Plan de San Luis s/n, Colonia SantoTomás, 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, México3 Departamento de Morfología, Escuela Superior de Medicina-IPN, Salvador Díaz Mirón yPlan 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 deMéxico, Avenida de los Barrios s/n, Tlalnepantla Estado. de México. CP. 54090, México,D. F.
26 Laboratorio de Microscopía Electrónica, Facultad de Medicina, Universidad La SalleFuentes 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:email@example.com
3AbstractThe few reports that analyze the effects of stress on the immune cells of the intestinalmucosa or the functions of these cells tend to focus on S-IgA levels in saliva, and thesestudies have shown contradictory results. The principal objective of this study was toanalyze the effects of repeated restraint stress on the number and distribution of immunecells of Peyer’s patches (PPs), and also the effects of glucocorticoid and catecholamineadministration on the same stress-related parameters. Upon analyzing the effect of repeatedrestraint stress on PPs, it was found that there was no modification in the morphologicalstructure of the PPs, but that restraint stress reduced the total number of lymphocytes, thenumber of CD8+ T cells, B cells and plasma cells in PPs. Only in the site of PPs whereIgA-producing plasma cells are most numerous (the dome) was a decrease found in thistype of cell. These effects were due at least in part to the effect of glucocorticoids andcatecholamines. Since IgA produced in the Peyer’s patches is a natural antibody thatimpedes bacterial infections, repeated stress may favor the entrance of pathogens throughthe intestine.Keywords: Repeated restraint stress, Peyer’s patches, IgA, glucocorticoids,catecholamines.
4IntroductionThe neuroendocrine system regulates the immune responses, including those of theintestinal mucosa [1, 2]. Studies done on the neuroendocrine regulation of the mucosalimmune system have focused mainly on the quantity of IgA secreted in saliva [3, 4], thesynthesis and secretion of the secretory component of the tear glands [5, 6], and theregulation 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 immunesystem have focused almost exclusively in relation to inflammatory diseases of the intestineand the secretion of IgA in saliva.The abundant information available confirms that psychological stress plays a fundamentalrole in the physiopathology and clinical symptoms of intestinal inflammatory diseases inhumans [9, 10]. However, in relation to IgA levels in human saliva, both reductions andincreases have been reported, depending on the type of psychological and/or physical stressprotocol employed [4, 11, 12]. These contradictions make it difficult to reach anyconclusion 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 restraintstress reduces IgA levels in mouse intestine as well as the intraepithelial lymphocytepopulation in mouse duodenal mucosa [13, 14].The immune system of the mucosa can be divided into inductor and effector sites. In theformer, 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 Blymphocytes. In the latter, the effector cells perform their action, such as the production of
5S-IgA [15, 17]. PPs, the most studied inductor site, have fundamental importance in thecapture of antigens in the intestinal lumen and in the induction of the humoral immuneresponse, mainly through the production of S-IgA, in the intestine [18, 19]. It is known thatPPs are innervated by nerve fibers containing norepinephrine, vasoactive intestinalpolypeptide (VIP), substance P (SP) and somatostatin (SOM) [20, 21], and that thelymphocytes of PPs express receptors for neuropeptides (e.g., SOM and SP). In vitrostudies have demonstrated that the production of IgA, IgG and IgM by PP lymphocytes isregulated by β-endorphin, ACTH and various peptides, which also regulate the migrationand proliferative response of lymphocytes .Stress-related effects on the structure and functions of PPs have been done in the context ofGALT, with a focus on changes in T lymphocyte populations [22, 23]. Since theneuroendocrine regulation of the function of inductor sites has been little studied, the aimof the current study was to analyze the effects of repeated restraint stress on themorphological structure as well as the number and percentage of lymphocytes, includingthe IgA-producing plasma cells, in the different regions of Peyer´s patches, and to comparethese stress-related effects with those produced by treatment with glucocorticoids andcatecholamines.
6Materials and Methods2.1 AnimalsTwo-month old male Balb/c mice (from the animal house of the Escuela Superior deMedicina, ESM), weighing 25 to 30 g, were treated with the appropriate dose ofmebendazole and metronidazole to eliminate parasites. They were housed 6 per cage in aroom with little noise and kept on a 12:12 h light/dark cycle (lights on at 6 am). Allhandling and assays were carried out between 8 am and 12 am to avoid the influence of thecircadian cycles of ACTH and cortisol. The mice were handled in accordance with thenorms of the Institutional Commission for the Care and Use of Lab Animals of the ESM.2.2 Restraint stress protocolMice 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 backand forward freely in the container, but could not turn around. The duration of the restraintcycle was 1 or 4 h for four successive days. Unrestrained mice were left undisturbed intheir home cages without access to food or water during the same period.2.3 Hormonal treatmentGroups of 6 mice were treated subcutaneously with epinephrine at 0.1 or 0.5 mg/kg/day for4 consecutive days. Other groups of 6 mice were treated subcutaneously withdexamethasone 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
7On the fourth day, all animals were sacrificed and the small intestine was extracted andwashed with PBS. The segments that contained Peyer’s patches were separated with finedissection scissors, obtaining 3 to 5 patches, depending on the mouse. One PP from theproximal 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 PPswere used to quantify the number of lymphocytes by flow cytometry.Briefly, PPs that were not fixed or frozen were immediately disaggregated and resuspendedin 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 cellswere washed three times with the same solution, then the button cells were resuspended in1 mL of HBSS to conduct a count. The total number of cells in each patch was counted byemploying a violet crystal solution, while the viability was evaluated by Trypan blueexclusion analysis. Finally, the suspension was adjusted to a concentration of 10x106cells/mL of HBSS.2.5 Reverse Hemolytic Plaque AssayThe reverse hemolytic assay for the detection of IgA was adapted from a previouslydescribed 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 antibodydissolved in 1 mL of PBS, used as a coupler, and 0.5 mL of CrCl3 solution (1 mg/mL in0.15 M NaCl) at 37oC for 1 hr with continuous shaking. One-tenth milliliter of cellsuspension of PPs in HBSS was mixed with 20 μL of 20% v/v suspension in 0.1 5 M NaClof freshly anti-IgA-coupled SRBC, 20 μL of the serum of rabbit anti-mouse IgA
8(“developer”, diluted 1:150), and 20 μL of guinea pig serum (complement source). Themixture was incubated in Cunningham´s chambers at 37oC for 90 min, then the hemolyticplates were counted and the mean of IgA producing cells per million viable cells wascalculated.2.6 Topographic stainingThe PPs fixed in formaldehyde were cut in slices of 7 µm and stained with Hematoxiline-Eosine (H-E) and Gomoris trichomic technique for morphological analysis.2.7 In situ detection of lymphocytes by immunohistochemical techniquesThe PPs frozen in isopentane were cut in slices of 7 µm. The cells were stained using animmunohistochemical technique with monoclonal biotynilated antibodies specific foridentifying T CD4+ and T CD8+ cells, followed by the application of streptavidinconjugated with HPR. To detect the IgA-producing plasma cells in situ, a monoclonalantibody 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 counterstainedwith Harris’ hematoxylin. After dehydration, cells were covered with synthetic resin andcounted by tissue area, each area being measured by an ocular micrometer calibrated with ahemocytometer.2.8 Immunophenotyping by flow cytometryFor cell immunophenotyping, directly labeled monoclonal antibodies were used: anti-CD19-APC or -PE, CD45-PercCP, CD138-APC, IgA-FITC, CD3-FITC, CD8-PE andCD4-PercCP (all from BD Biosciences, San Jose, CA, USA).
9Cells were harvested, washed twice with PBS and 0.5% BSA, and then stained for T cellphenotype with a cocktail of anti-CD3, -CD4 and -CD8 mAb, or for B cell phenotype usinganti-B220 and anti-CD19 mAb, for 30 min at room temperature in darkness. The cells werethen 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 stainedaccording to BD Bioscience’s protocol for intracellular staining. The fluorescent signalintensity was recorded and analyzed in a FACSCalibur flow cytometer (Becton Dickinson).For each sample 15,000 events were collected. Data were analyzed using the Summitsoftware v4.3 (Dako, Colorado Inc.). The total number of lymphocytes was calculated fromthe percentage of cells located in the lymphocyte region in the dot-plot of FSC vs SSC, andthe total number of cell/patch according to the following formula: (# total cells/Peyer´spatch) x (% lymphocytes)/100. The absolute number of positive cells (subsets oflymphocytes) was calculated from the total number of lymphocytes, according to thefollowing formula: (# total cells/Peyer´s patch) x (% positive cells)/100. The percentageand number of CD4+ cells and CD8+ cells were calculated from the CD3+ cells.2.9 Statistical AnalysisThe differences between two groups were determined by the Student’s t test. The analysisof data from 3 or more groups was done with one-way ANOVA. All values were presentedas the mean ± SD of at least three independent assays. Statistical analyses were performedby 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.
10Results3.1 Restraint stress did not modify the morphology of PPsIt is well established that repeated stress in the short or long run modifies the number andfunction of immune and inflammatory cells [26, 27]. However, there have been no studieson the effects of repeated stress on PPs of the mouse intestinal mucosa. Since PPs havefundamental importance in the capture of antigens in the intestinal lumen and in theinduction of the humoral immune response, we evaluated the structure as well as thenumber and percentage of lymphocytes in this tissue. We observed that the normal structureof the germinal center, internodal regions and dome remained intact in the PPs of stressedmice (Figure 1). Also, there was no significant difference in this parameter between themice stressed for 1 or 4 h.3.2. Restraint stress modified the cellular composition of PPsThe percentage of B cells was significantly lower in mice stressed for 4 h than in the othertwo groups: animals stressed for 1 h or those non-stressed (Figure 2A, P < 0.001), asdetected by flow cytometry. There were no differences in the percentages of the othersubsets of lymphoid cells, including plasma cells, among the three groups. However, whenthe absolute number of each cellular subset was determined, the total number oflymphocytes in the PPs was found to be lower in both groups of stressed mice than thecontrol animals (Figure 2B, P < 0.001, Bonferroni t-test). The absolute number of CD8+ Tcells, B cells and plasma cells was significantly lower in mice stressed for 4 h than the othertwo groups: animals stressed for 1 h or those non-stressed (Figure 2B, P < 0.001). Amongthese parameters, only plasma cells were found to be lower in mice stressed for 1 h than in
11control animals (P < 0.05). The total number of T cells and CD4+ T cells was not affectedby the restraint stress protocol employed.3.3. Restraint stress reduced the number of IgA-producing plasma cellsConsidering that Peyer’s patches contain antibody-producing effector cells [28-30], weevaluated the effect of repeated restraint stress on the total number of IgA-producingplasma cells in PPs, as well as the number of these cells in each region. Theimmunohistochemical assay demonstrated that repeated restraint stress diminished thenumber of IgA-producing plasma cells in the dome, but not in the corona, germinal centeror intermodal region of PPs (Figure 3A and 3B; P < 0.001, Bonferroni t-test). The flowcytometric 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-producingplasma cells, the only significant difference between the three groups was the lowerpercentage of these cells found in mice stressed for 1h compared to the control animals(Figure 3D).3.4. Effects of dexamethasone and epinephrineIn several studies it has been demonstrated that changes observed in the immune responseinduced by stress are mediated principally through the release of glucocorticoids andcatecholamines in different kinds of tissues. Therefore in the present study the effect ofdexamethasone (a glucocorticoid) and epinephrine (a catecholamine) on the number oflymphocytes and subsets of T and B cells in PPs was evaluated.
12The 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). Thedose 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. Dexamethasoneat doses of 5 and 50 mg/kg/day significantly reduced: (i) the size of the patches, in whichno germinal centers were found (Figure 1C), (ii) the total number of lymphocytes (Figure4A, 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 ofplasma cells (Figure 4A P < 0.001).In relation to epinephrine, the structure of the PPs remained normal (Figure 1D) and therewere no differences in the percentage of T cells, CD4+ T cells and plasma cells among thethree groups of mice (Figure 5A). The dose of 0.5 mg increased the percentage of CD8+ Tcells (**P < 0.05) and both doses reduced the percentage of B cells (*P < 0.001). Therewere 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 oflymphocytes, 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 ofIgA-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
13dexamethasone 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 ofIgA-producing plasma cells (Figure 6E, **P < 0.05), whereas only the 0.1 mg/Kg/day dosereduced the percentage of IgA-producing plasma cells (Figure 6F, P < 0.001).3.5. The reverse hemolytic assayTo confirm the findings of immunohistochemistry and flow cytometry, we performed afunctional 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 stressedanimals (1 or 4 hours) compared to the control group. Also, it confirmed that both doses ofdexamethasone (*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).
14DiscussionIn some lymphoid organs, such as the thymus, spleen, and nodes, diverse types of stresscause 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 hrestraint stress did not cause atrophy of the PPs (Figure 1B), although it did indeed result ina decrease in the total number of lymphocytes (Figure 2B).Similarly, both doses ofepinephrine administered in the current contribution caused a decrease of the total numberof 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 moresignificant decrease in the total number of lymphocytes (Figure 4A) than that found withepinephrine.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 micerestrained for 1 h, a reduction was found in the total lymphocytes and the number of plasmacells and IgA-producing plasma cells. In the mice restrained for 4 h, these same reductionswere observed along with decreases in CD8+ T cells and B cells (Figure 2). Similar resultswere reported from another study, where 12 h of restraint stress caused a decrease in thenumber of B cells (B220+ cells), CD8+ T cells and total T cells in PPs of mice . In thatstudy, plasma cells were not evaluated. The fact that a decrease in total T cells wasobserved was likely due to the greater time of restraint stress.
15Even 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 wereevaluated in the present study. The flow cytometric analysis showed that compared to thenon-stressed animals, the total number of IgA-producing plasma cells in PPs was lower inboth groups of restraint-stressed mice. The immunohistochemical study confirmed theseresults, and also revealed that of the regions of the PPs, only in the dome was there adecrease in the number of these cells in stressed mice. In others sites of the PPs, such as thecorona, germinal center and internodal region, there were no significant differences in thenumber of IgA-producing plasma cells between stressed and non-stressed groups (Figure3). The fact that the only change in the number of IgA-producing plasma cells was found inthe dome is to be expected, as it is known that IgA-producing plasma cells arepredominantly located in this region of PPs.The reduction in the response of IgA-producing plasma cells was not significantly greaterin the mice stressed for 4 hours than those stressed for 1 hour, which could be due to theeffect 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 responseto mitogens in the spleen (but not in the blood), whereas such effect caused by subsequentexposures 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]. Thefunction of the latter antibodies is not clear, but they could be important for reducing thefrequency of allergies, as well as inflammatory and autoimmune diseases in the intestine
16. Furthermore, these innate secretory antibodies may protect against some infections,such as Salmonella typhimurium . Since repeated restraint stress reduced the number ofIgA-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 principallythrough the release of glucocorticoids and catecholamines in different kinds of tissues, thatadministration of high doses of glucocorticoids notably suppresses the humoral immuneresponse [36, 37], and that dexamethasone and epinephrine at least partially mediate someof the effects of stress on the systemic immune response [38-40]. Therefore, an evaluationof the effects of these hormones was included in the present study. The doses ofdexamethasone (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 thenumber of IgA-producing plasma cells in PPs, as determined by flow cytometry (Figure 6).The immunohistochemical analysis confirmed these results, with the exception of findingno significant difference in this parameter with the lower dose of epinephrine.Compared to control animals, dexamethasone and epinephrine at both doses induced adecrease in the number of all other evaluated sub-populations of lymphoid cells (Figure 4and Figure 5). The only exception was with dexamethasone at the lower dose, whichproduced no significant reduction in total plasma cells. Interestingly, the effects on thepopulations of lymphocytes were progressively greater, considering the 1 hr restraint stressgroup, 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 theeffect of glucocorticoids and catecholamines, and it is possible that the doses of these
17hormones administered represent the effect of a more intense stress than that provoked bythe restraint stress protocol of the current contribution.In previous studies on rats, dexamethasone significantly reduced the S-IgA levels in the bileand the number of IgA-producing plasma cells in the ileum , and favored the adherenceof bacteria to the epithelium as well as the invasion of the mucosa . There have notbeen any previous reports, to the best of our knowledge, regarding the effect ofdexamethasone on the IgA-producing plasma cells in PPs of mice. However, it has beenreported that a single injection of dexamethasone reduces the number of Ig-producingplasma cells (IgM, IgG and IgA) in the spleen and mesenteric lymph nodes of mice ,and that a treatment with a dose between 30 µg/kg and 2.5 mg/kg of BW reduces thenumber 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 thisglucocorticoid could owe itself to three mechanisms: a) a decrease in the number oflymphoid cells, principally by apoptosis [42, 48], b) an inhibitory effect on macrophagesand helper T cells, as these are responsible for the induction of the immune response ,and c) changes in the distribution and migration of lymphocytes, since these are able toalter 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 productionof antibodies in PPs. Contradictory results have been reported regarding the effect ofcatecholamines on the systemic immune response. Whereas some studies report aninhibition of the proliferation of B cells and the production of antibodies, others show the
18opposite effect [51-53]. In one study the effect varied according to the moment of theadministration of the immunogen . Although the precise mechanism by which the dosesof epinephrine employed in the present study inhibited the production of antibodies in thePPs is unclear, it may be through direct action on mature cells . It seems relevant thatPPs are innervated by fibers that contain epinephrine [20, 56, 57], which in turn modulatethe internalization of pathogenic bacteria .ConclusionIn summary, whereas repeated restraint stress of different intensities did not have anynotable effect on the morphological structure of the PPs, it did change the number oflymphocytes in this lymphoid tissue. Usually when a stress response causes such effects, itis 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 decreasesas the 1-hour group, plus a decrease in B cells and CD8+ T cells), and the mice treated withdexamethasone and epinephrine (expressing the same decreases as the 4-hour group, plus adecrease in CD4+ T cells and total T cells). Therefore, these effects were due at least in partto the effect of glucocorticoids and catecholamines, and it seems likely that the effects ofepinephrine and dexamethasone represent the equivalent of an even greater stress than thatproduced by the 4 h restraint stress protocol.
19AcknowledgementsWe thank Bruce Allan Larsen for reviewing the use of English in this manuscript. Thisresearch was financially supported by SIP-IPN and CONACYT, Mexico.Conflict of Interest StatementThe authors declare that they have no conflicts of interest regarding any of the products ortechniques employed in this study.
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24Figure LegendsFigure 1: Effect of restraint stress and treatment with dexamethasone andepinephrine 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 formaldehydeand processed for paraffin embedding and stained with H&E and Gomoris trichomictechniques. Germinative center (GC), mantle zone (MZ), and dome (Do). Restraint stress orepinephrine treatment did not cause atrophy of the Peyer’s patches (B and D). However, thehigher 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 inPeyer’s patches determined by flow cytometry. The percentage and the number of CD4+ Tcells and CD8+ T cells were calculated from T cells. Results are the media± SD at least 3independent experiments. *P < 0.001, **P < 0.05.
25Figure 3. Effects of repeated restraint stress on the number of IgA-producing plasmacells 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 immunohistochemicaltechnique; 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 micedetected by immunohistochemistry. (C) Absolute number and (D) percentage of IgA-producing plasma cells in PPs in stressed and unstressed mice determined by flowcytometry. 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 totallymphocytes and subsets of T cells (CD3+ cells), B cells and plasma cells in Peyer´sPatches analyzed by flow cytometry. The percentage and the number of CD4+ T cells andCD8+ T cells were calculated from T cells. Data represent the media ± SD of threeexperiments. *P < 0.001, **P < 0.05.
26Figure 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. Thepercentage 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 inPPs. Mice were treated with dexamethasone (5 or 50 mg/kg) or epinephrine (0.1 or 0.5mg/kg) daily for 4 days. Control animals were treated with the vehicle. Data were obtainedfrom 6 to 12 mice/group and are presented as the mean ± SD. (A) Number of IgA+ cells inPPs of treated and untreated mice with dexamethasone, determined byimmunohistochemistry. (B) Number and (C) percentage of IgA-producing plasma cells inPPs 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 byimmunohistochemistry. (E) Number and (F) percentage of IgA-producing plasma cells inPPs of treated and untreated mice with ephinephrine, determined by flow cytometry. *P <0.001, ** P < 0.01, Bonferroni’s t-test.
27Figure 7.- Effects of chronic restraint stress on the number of IgA-producing cells inPeyer’s patches detected by an plaque hemolytic assay. Restraint stress, both doses ofdexamethasone (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 ofPeyers patch lymphocytes. Similar results were obtained in four independent experiments.