Prevention of immunosuppression in stressed mice by neurotropin(NSP)

It is well known that the immune function can be compromised by stress. To investigate immune function in mice stressed by experimental restraint or unavoidable and opioid dependent stress, we evaluated the changes in total body weight and in organ weights (liver, spleen and thymus) of these animals, as well as the phagocytic activity of macrophages, the cytotoxicity of T cells and inhibitory effects on tumor growth and changes in T cell subset populations. At the same time we evaluated the effects of Neurotropin (NSP), a substance extracted from the inflamed skin of rabbits inoculated with the vaccinia virus and which appears to possess neuroimmunomodulating activity. The experimentally stressed group exhibited a reduction of phagocytic activity of macrophages, cytotoxicity of T cells and inhibitory effects on tumor growth. In addition there were changes in the population of T cell subsets. In those animals pretreated with NSP, the immunosuppression induced by stress was ameliorated. As compared with several agents which influence phagocytosis, neurotropin exhibited effects similar to that of agents that blocked the adrenaline receptor and an opioid antagonist rather than tranquilizer (diazepam) and a cholinergic receptor blocker. The pharmacologic effects of neurotropin support a relationship between the actions of the central nervous system and the immune system.     

Renewal rates of murine T-lymphocyte subsets

The proliferative dynamics of T-cell subsets in lymphoid organs of BALB/c mice have been evaluated by means of [3H]thymidine injections for up to 3 days, followed by FACS and autoradiography. In the thymus, cells positive for both CD4 and CD8 antigens were more rapidly renewed than single-positive cells. Cell populations showing strong expression of Thy-1 and weak expression of CD5 antigens were more rapidly renewed than populations with the opposite antigen expressions. The results obtained from the spleen and the lymph nodes indicated that the majority of T cells have life spans exceeding a few days, and comparable cell kinetics were found in the different T-cell subpopulations. Results obtained after injections of hydroxyurea, which causes elimination of cycling cells, paralleled the autoradiographic results.     

Restraint stress and anti-tumor immune response in mice

Psychological stress modulates the immune system through the hypothalamic-pituitary-adrenal axis, the sympatho-adrenomedullary axis and the opioid system. According to literature data, restraint stress increases the immune cell apoptosis, decreases the spleen and thymus cell content, the natural killer (NK) activity in the spleen, and it compromises the anti-tumor immune response in mice. We immobilized mice in two consecutive nights, and then determined the cell number, apoptosis, NK cell content, NK activity and the level of cytokine mRNAs (TNF-beta, TNF-alpha, IL-4, IL-5, IL-1alpha, IFN-gamma, IL-2, IL-6, IL-1beta and IL-3) in the thymus and spleen. No consistent changes were detected in any of the immune parameters either in C57Bl/6 or in DBA/2 mice. Stressed or control B6 mice were injected with B16 melanoma cells immediately after the immobilization or one week later. No significant differences were found in the growth of primary tumors and lung metastases in stressed and control animals. Taken together, our mice, kept in a general-purpose non-SPF animal house, seemed to be refractory to the stress-induced immunomodulation. Our interpretation is that stress-induced immunomodulation can occur only in mice isolated from any background stressors, or rather natural stimuli, throughout their life.     

The migration of bone marrow cells to thymic culture supernatants is inhibited by stress

The effect of immobilization stress on precursor cell migration from bone marrow to the thymus was studied in C57BL/6 mice. The in vitro migration assays, using Nuclepore chambers, showed that precursor cell migration to thymus supernatants was strongly inhibited in stressed animals. This inhibition of migration seemed to be cell-associated what can explain the thymic involution observed in mice under stress conditions. The migration of precursor cells from bone marrow may be one of the mechanisms by which the thymus gland is involuted by stress.     

Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.     

Anti-glycoprotein D monoclonal antibody protects against herpes simplex virus type 1-induced diseases in mice functionally depleted of selected T-cell subsets or asialo GM1+ cells.

Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for the functional loss of one but not two T-lymphocyte subpopulations.     

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.     

Differences in thymus dependency among the alloreactive T-cell subpopulations in their development

Effects of thymectomy at various times after birth (Tx-1, Tx-3, Tx-7, Tx-14) on mixed lymphocyte reaction (MLR), cell-mediated cytotoxicity (CMC), and graft-versus-host reaction (GVHR) were examined by experiments with spleen cells obtained at 8 weeks of age. All of MLR, CMC, and GVHR were detected in spleen cells of Tx-14 mice. However, the ability of spleen cells to induce GVHR was abolished by thymectomy at 7 days after birth. On the other hand, MLR and CMC were not affected in such mice. In Tx-1 or Tx-3 mice, only MLR was detected in spleen cells. These results suggest that thymus dependency of T cells responsible for MLR are lower in their maturation than those for CMC and that thymus dependency of T cells responsible for GVHR are the highest among these T-cell subpopulations.     

番石榴多糖对糖尿病小鼠的血糖及胸腺、脾指数的影响

研究采用两种不同方法提取的番石榴多糖对四氧嘧啶致糖尿病的小鼠血糖值及胸腺、脾指数的影响。通过给小鼠腹腔注射四氧嘧啶(200 mg/kg BW)建立糖尿病小鼠模型,尾端取血,采用血糖仪分别在给小鼠灌胃多糖三天和十天后检测小鼠血糖值,第十天解剖小鼠,分别对小鼠胸腺和脾脏称重。结果表明:与糖尿病对照组比较,两组灌喂番石榴多糖的小鼠的生存质量提高,血糖值显著降低,同时胸腺指数显著增加,提示番石榴多糖具有降血糖作用,是一种潜在的糖尿病治疗药物。     

大豆异黄酮对电离辐射小鼠T淋巴细胞亚群的影响

目的:研究大豆异黄酮对辐射小鼠T淋巴细胞亚群的影响。方法:32只雄性昆明小鼠,随机分为正常对照组、辐射对照组和辐射补充0.5%大豆异黄酮组,喂养两周后,4.0Gyγ射线照射;于照射后两周杀死小鼠取外周血、胸腺和脾脏,流式细胞仪测定T淋巴细胞亚群。结果:辐射使小鼠外周血CD3、CD4和CD8百分比降低,并且CD8的变化有统计学意义;使胸腺和脾脏的CD3和CD4百分比升高、CD8百分比降低,其中CD4的升高显著(P<0.05)。补充大豆异黄酮,可使外周血、胸腺和脾脏CD3以及血CD4比例升高,但对辐射引起的CD8变化无明显作用。结论:大豆异黄酮可对辐射小鼠的T淋巴细胞亚群起到辐射防护的作用。     

Protective effect of a Potentilla anserine polysaccharide on oxidative damages in mice

Potentilla anserine polysaccharide (PAP) was studied in vivo to investigate its antioxidant activity using the model of dexamethasone-induced oxidative stress in mice. The investigation demonstrated that PAP at 50, 100 or 200 mg/kg body weight for 7 days respectively increased thymus index and spleen index, glutathione level, superoxidase dismutase activity and total antioxidant capacity in both thymus and spleen and decreased the content of H₂O₂ in spleen and NO in both thymus and spleen of mice. The results revealed that PAP was able to overcome dexamethasone-induced oxidative stress and might play an important role in repairs of oxidative damage in immunological system.     

Pituitary Adenylate Cyclase Activating Peptide Deficient Mice Exhibit Impaired Thymic and Extrathymic Regulatory T Cell Proliferation during EAE

We have shown that mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) manifest enhanced sensitivity to experimental autoimmune encephalomyelitis (EAE), supporting the anti-inflammatory actions described for this neuropeptide. In addition to an increased proinflammatory cytokine response in these mice, a reduction in regulatory T cell (Treg) abundance in the lymph nodes (LN) was observed, suggesting altered Treg kinetics. In the present study, we compared in PACAP deficient (KO) vs. wild type mice the abundances and rates of proliferation FoxP3⁺ Tregs in three sites, the LN, central nervous system (CNS) and thymus and the relative proportions of Th1, Th2, and Th17 effector subsets in the LN and CNS. Flow cytometry analyses revealed a decrease in Treg proliferation and an increased T effector/Tregs ratio in the LN and CNS of PACAP KO mice. In the thymus, the primary site of do novo natural Treg production, the total numbers and proliferative rates of FoxP3⁺ Tregs were significantly reduced. Moreover, the expression of IL-7, a cytokine implicated in thymic Treg expansion during EAE, failed to increase at the peak of the disease in the thymus and LN of PACAP KO mice. In addition to these Treg alterations, a specific reduction of Th2 cells (about 4-fold) was observed in the lymph nodes in PACAP KO mice, with no effects on Th1 and Th17 subsets, whereas in the CNS, Th1 and Th17 cells were increased and Th2 decreased. Our results suggest that endogenous production of the neuropeptide PACAP protects against EAE by modulating Treg expansion and Th subsets at multiple sites.     

A single exposure of rats to water-immersion restraint stress induces oxidative stress more severely in the thymus than in the spleen

Abstract

Objectives

We examined whether a single exposure of rats to water-immersion restraint stress (WIRS) induces oxidative stress in the thymus and spleen.

Methods

Vitamin E, ascorbic acid, reduced glutathione (GSH), and lipid peroxide (LPO) were assayed in the thymus and spleen of rats with and without 6 hours of WIRS.

Results

In unstressed rats, vitamin E, ascorbic acid, GSH, and LPO levels were higher in the thymus than in the spleen. Thymic ascorbic acid level was lower in stressed rats than in unstressed rats. Splenic ascorbic acid level was similar in both groups. Thymic and splenic GSH levels were lower in stressed rats than in unstressed rats but the reduced amount of GSH was lower in the spleen than in the thymus. Thymic vitamin E level was lower in stressed than in unstressed rats. Splenic vitamin E level was higher in stressed rats than in unstressed rats. Thymic and splenic LPO levels were higher in stressed rats than in unstressed rats but the increased amount of LPO was higher in the thymus than in the spleen.

Conclusion

It is indicated that a single expose of rats to WIRS induces oxidative stress more severely in the thymus than in the spleen.     

Dependence of organism stability on chronic stress from thyroid status

In experiences on 108 male rats, the effect of acute (immobilization during 3 hrs) and chronic (immobilization for 3 hrs during 5 days) stresses on the organism general stability was studied as evaluated with changes of body weight, adrenal glands, spleen, thymus relative mass, gastric mucosa state, animals physical endurance. Chronic stress evoked more obvious decreasing of spleen and thymus relative mass than the acute one, as well as lesion of gastric mucosa accompanied with decrease of the rat resistance to physical loading. Thyroid function suppression by merkazolil (1.2 mg/100 g body weight during 14 days) promotes further the reduction of the organism stability in acute and, especially, in chronic stress, while physiological doses of thyroid hormones (5.0-8.0 mcg of thyroxin on kg of body weight during 28 days), on the contrary, increased it in both stress conditions. Existence of the organism stability dependence on thyroid status both in acute and chronic stress proves iodothyronine's important role in the organism antistress-system.     

Evidence for interaction between T cell populations of tumor-bearing and normal mice in immune suppression

Spleen cells of DBA/2 mice bearing subcutaneous implants of the syngeneic tumor L5178Y induce suppression of the in vitro antibody response of normal spleen cells to sheep erythrocytes (SRBC). Cells mediating suppression are detected in the spleens of tumor-bearing mice as early as 24 hr post-implantation but are no longer detected there 15 days post-implantation. These spleen cells are nylon wool nonadherent, sensitive to anti-Thy 1.2 + C and anti-Lyt 1.1 + C, and insensitive to anti-Lyt 2.1 + C treatment. The anti-SRBC response of the unfractionated spleen cells from the tumor-bearing mice is not itself suppressed at the cell numbers used. This along with the finding that suppression occurs in the presence of spleen cells from normal mice suggest that a cell population from the normal mouse spleen is also involved in the suppression. Spleen cells from mice inoculated with irradiated (nonproliferating) L5178Y cells are similarly capable of mediating nonspecific suppression for the same limited period of time after the inoculation. In addition, spleen cells from mice stimulated with several nontumorigenic cellular antigens interact with normal spleen cells to produce suppression. These findings suggest that suppression observed in vitro with spleen cells from these tumor-bearing mice may be the result of antigen-activated cells triggering normal immunoregulatory cells.     

Redistribution of Lyt-bearing T cells in acute murine experimental allergic encephalomyelitis: selective migration of Lyt-1 cells to the central nervous system is associated with a transient depletion of Lyt-1 cells in peripheral blood

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by using two injections of spinal cord homogenate in incomplete Freund's adjuvant supplemented with mycobacteria. Analysis of circulating Lyt-bearing subsets by indirect immunofluorescence during the course of acute EAE revealed the following: 1) during the pre-clinical phase of EAE (1 to 2 days before the onset of paralysis), there was a decrease in the percentage of Lyt-1- but not of Lyt-2-bearing cells in peripheral blood, and of both Lyt-1- and Lyt-2-bearing cells in spleen; 2) with the onset of clinically evident EAE, there was a decrease in both Lyt-1 and Lyt-2 cells in peripheral blood and an increase in the percentage of Lyt-1-bearing cells in pooled inguinal and axillary lymph node; and 3) after these early changes, there was a rapid reconstitution of the percentages of total Lyt-bearing cells and of both Lyt-1- and Lyt-2-bearing cells in peripheral blood. Immunohistochemical analysis of the central nervous system infiltrate revealed that the earliest lesions consisted predominantly of Lyt-1 T lymphocytes, with few Lyt-2 cells present. These results demonstrate that the influx of cells of the Lyt-1 inducer subset to the central nervous system in acute EAE is accompanied by a transient decrease in Lyt-1 cells in peripheral blood.     

Lyt phenotype analysis of the effector cells responsible for evoking lymphocyte-induced angiogenesis (LIA)

Lymphocyte-induced angiogenesis (LIA) is a vascular response observed when allogeneic or semiallogeneic immunocompetent lymphocytes are inoculated intradermally into immunosuppressed or irradiated host mice. The reported experiments were carried out to characterize the effector cell population(s) responsible for causing LIA. Lyt 1.2, Lyt 2.2, and monoclonal Thy 1.2 antisera were used for negative selection with complement (C′) to investigate the ability of selected subsets of lymphocytes to evoke angiogenesis. Treatment of C57BL/6 spleen cells with either anti-Lyt 1.2 or anti-Thy 1.2 and C′ resulted in an almost complete abrogation of the LIA reaction. In contrast, depletion of Lyt 2+ cells, under conditions which fully abrogated their ability to generate cell-mediated cytotoxicity in allogeneic mixed leukocyte cultures, resulted only in a partial (45%) reduction in the induced vascular response. Synergistic interaction between cell preparations treated separately with either anti-Lyt 1.2 or anti-Lyt 2.2 serum was not observed. We conclude that (i) Lyt 1 + 2?T lymphocytes can induce a significant LIA reaction; (ii) lymphocytes resistant to negative selection with anti-Lyt-1.2 serum are incapable of inducing such a reaction; and (iii) Lyt 1 + 2+ cells directly or indirectly play an additional role in generating a maximal LIA response.     

Effect of chronic psychoemotional stress on subpopulation spectrum of T-lymphocytes in immunocompetent organs in male mice

Subpopulation spectrum of T lymphocytes (CD3+, CD4+, CD8+, CD25+, and CD3(+)25+) in thymus, spleen and inguinal lymphatic nodes have been studied in male mice after 20 days of psychoemotional stress produced by social defeats in daily agonistic confrontations. A reduction of total number of cells, of absolute numbers of all researched subpopulations of lymphocytes and % CD3+ cells in thymus of submissive mice was shown in comparison with intact animals. Reduction of total number of splenocytes and absolute numbers of CD4+ and CD8+ lymphocytes has been observed in a spleen of submissive mice. Besides, % CD3+, CD25+, CD4+ and CD25+ cells were increased in these animals in comparison with intact mice. The absolute number of cells with CD8 phenotype was increased in inguinal lymphatic nodes. The data obtained suggest that the chronic psychoemotional stress is accompanied by serious changes of the cellular link of immunity. The effect of chronic emotional social stress on mutual interaction of the central and peripheral links of immunity has been discussed.