Are adhesion molecules involved in stress-induced changes in lymphocyte distribution?

Acute psychological stress is associated with important changes in circulating cell populations and reductions in cell-mediated immune responses. However, the mechanisms underlying these phenomena are poorly understood. In this study, we investigated (i) acute and chronic restraint stress effects in Sprague-Dawley rats on peripheral lymphocyte subsets and (ii) adhesion molecule (beta2 integrins) expression and (iii) also determined whether glucocorticoids could underlie stress-related changes in cellular redistribution. We observed time-dependent changes in lymphocyte distribution including decreased (-21%) percentages of peripheral T helper cells and increased (88%) NK cell numbers following acute brief restraint. Acute stress was also found to overall upregulate beta2-integrin (CD11a and CD11b) expression on T cells and to raise (1049%) plasma corticosterone levels. However, this stress response was found habituated (-75% vs. acute) in the animals previously exposed to chronic restraint stress. Stress effects on circulating lymphocytes were not observed in animals previously exposed to chronic intermittent restraint stress or chronically stressed animals re-exposed to the same stressor. Our results indicate that 1) stress alters lymphocyte distribution, 2) that adhesion molecules may be involved in stress-induced alterations of T-cell distribution and 3) that these changes may be related to circulating glucocorticoids and subjected to adaptation with repeated stress exposure.     

应激致免疫功能降低动物模型的建立

目的为探讨应激对免疫系统损伤的作用机制,建立了两种不同的应激导致机体免疫功能降低的动物模型。方法将小鼠分为三组,束缚应激组、热应激组和正常对照组,分别用束缚和高温刺激使小鼠处于应激状态,应激结束后将各组小鼠均分为两部分,一部分感染细菌,比较两种应激状态下小鼠与正常小鼠的死亡率,另一部分摘取脾脏,计算脾脏重量指数,制作脾脏病理切片,观察其组织病理学变化。结果注射大肠埃希菌后束缚应激组和热应激组小鼠死亡率显著高于正常对照组小鼠(P<0.005),束缚应激组和热应激组小鼠死亡率无明显差异(P>0.05)。束缚应激组小鼠和热应激组小鼠脾脏重量指数均较正常对照组明显降低(P<0.05),组织病理学观察显示束缚应激组和热应激组小鼠脾脏切片病理学改变相似,白髓比例减小,脾小体有不同程度的缩小、分布及结构不规则。结论本实验建立的应激导致小鼠免疫功能降低的动物模型,其免疫功能降低与脾脏变化间存在构效关系,与应激的类型关系不大。     

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 radiation modifying properties of carnosine

The influence of carnosine (beta-alanyl-l-histidine) on the survival rate of albino mice subjected to whole-body X-irradiation has been investigated. Carnosine (50-200 mg/kg/day) administered per os during a period of 20 days before irradiation (5.0 Gy) increased the survival rate by 45-65%, whereas the administration of carnosine within 30 days after irradiation (5.5 Gy) produced an insignificant protective effect and caused inhibition of the postirradiation histamine accumulation in the spleen.     

Carnosine attenuates mast cell degranulation and histamine release induced by oxygen-glucose deprivation

Carnosine (beta-alanyl-histidine) is a naturally occurring dipeptide that has been characterized as a putative hydrophilic antioxidant. The protective function of carnosine has been demonstrated in neuronal cells under ischemic injury. The purpose of this study was to investigate the effects of carnosine on oxygen-glucose deprivation (OGD)-induced degranulation and histamine release from mast cells. Cultured mast cells were exposed to OGD for 4 h, and then the degranulation was observed immediately by microscopy. Histamine release was analyzed by high-performance liquid chromatography (HPLC). OGD caused degranulation of mast cells, and increased histamine and lactate dehydrogenase (LDH) release. Carnosine (at a concentration of 5 mM) alone did not produce any appreciable effect on degranulation, histamine, and LDH release from mast cells under normal condition, but significantly inhibited the degranulation, histamine, and LDH release of mast cells induced by OGD. These results indicate that carnosine can protect mast cells from degranulation and histamine release and it may be an endogenous mast cell stabilizer in the pathological processes induced by ischemia.     

Pharmacological studies on the anxiolytic effect of standardized Schisandra lignans extract on restraint-stressed mice

Fruits of Fructus Schisandrae were used as sedatives and hypnotics in traditional Chinese medicine for a long history. In this study, we investigated the effects of schisandra lignans extract (SLE) on anxiety disorder in restraint-stressed mice using light-dark (L-D) test. The influences of restraint stress on the levels of monoamines: noradrenaline (NE), dopamine (DA) and serotonin (5-HT) in cerebral cortex, as well as plasma corticosterone (CORT) were studied in mice. The HPLC fingerprint of SLE was recorded and the percentage composition of Schisandra lignans was determined as 82.63%. In L-D test, it was found out that 18 h of restraint stress significantly decreased the anxiolytic parameters (explorative behaviors, e.g. number of entries, time spent) in light area indicating high state of anxiety in stressed mice. In addition, restraint stress elevated NE, DA, and 5-HT levels in cerebral cortex of anxiety mice. Plasma CORT level was also increased. Oral administration of SLE (100 and 200 mg/kg/day, 8 days) emolliating the level of stress-induced anxiety by significantly increasing the anxiolytic parameters mentioned above. We also observed decreases in cerebral cortex monoamines levels, as well as plasma CORT level in stressed mice. These results suggested that SLE reversed stress-induced anxiety level, changes of cortex monoamine transmitters and plasma CORT. The anxiolytic effects of SLE might be related to its anti-stress activity by modulation of hyperactive HPA axis.     

Enhanced resistance of restraint-stressed mice to sepsis

Sepsis remains a major health concern across the world. The effects of stress on host resistance to sepsis are still not very clear. To explore the effects of chronic stress on sepsis(') we examined the impact of restraint stress on the resistance of mice to sepsis. Interestingly, it was found that restraint stress enhanced the antisepsis resistance of mice and the concentrations of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNF-alpha in the blood of stressed mice were dramatically reduced post Escherichia coli infection or LPS treatment as compared with that of controls (p < 0.05). In addition, the mRNA expressions of glucocorticoid-induced leucine zipper (GILZ) were up-regulated in the spleen and peritoneal macrophages of mice receiving restraint stress or dexamethasone treatment. These results demonstrate that restraint stress enhances the resistance of mice to sepsis, supporting corticotherapy for sepsis and proposing restraint-stressed mouse as an animal model to elucidate mechanisms of stress-associated, antisepsis resistance.     

Increase in liver-associated natural killer activity by polyribonucleotides

Several polyribonucleotides are currently in clinical trials for the treatment of cancer or viral diseases. The present report in mice demonstrates that polyinosinic-polycytidylic acid and poly-L-lysine which has been stabilized in carboxymethylcellulose (poly (ICLC) as well as polyadenosinic-polyuridylic acid (poly AU), both potently augment natural killer (NK) activity in the liver, which is often a target organ for the formation of metastases during the progression of human cancer. Following the administration of poly ICLC (10 micrograms/mouse), greater NK activity as measured by lytic units (LU), was observed in the liver (445 LU) than in blood (63 LU) or spleen (20 LU). The high level of NK activity in the liver was in contrast to the low levels observed in untreated mice, and was maintained for at least 9 days post injection. NK activity in the blood and spleen returned to normal levels by day 6. Similar results were obtained with poly AU except that approximately 10-fold more poly AU (100 micrograms/mouse) was required to induce optimal augmentation of NK activity. Further studies demonstrated that the increase in liver-associated NK activity induced by poly ICLC was associated with a 10- to 20-fold increase in liver-associated leukocytes, termed nonparenchymal cells (NPC). Fractionation of the NPC on discontinuous density gradients of Percoll demonstrated that the NK activity mediated by NPC was associated with cells morphologically characterized as large granular lymphocytes (LGL). Further studies demonstrated that the repeated administration of poly ICLC resulted in significantly higher levels of liver-associated NK activity and total liver-associated LGL as compared to a single injection.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100?mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.     

Mu-opioid receptor mediates chronic restraint stress-induced lymphocyte apoptosis

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, mu-opioid receptor knockout (MORKO) mice were used to investigate whether the mu-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-gamma production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the mu-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Binding of histamine- and other ligand-conjugated macromolecules to lymphocytes

The presence of histamine receptors on lymphocyte membranes was investigated using conjugates of histamine and macromolecules tritiated or iodinated with I-125. Histamine-RSA conjugate binds to lymphocytes and causes patching and capping of the bound conjugate. It was found, however, that free histamine did not inhibit the binding of histamine-rabbit serum albumin to mouse lymphocytes, nor did His-RSA interfere with the binding of free histamine. In addition conjugates between RSA and other small molecules, such as ethylamine, ethanolamine, tyramine and glycine, were found to bind to the same sites on lymphocyte membrane as did His-RSA. Ethylamine-RSA like His-RSA when coupled to Sepharose, was capable of removing antibody producing cells from spleen cells of mice immunized against sheep red blood cells. In addition, when spleen cells from such immunized mice were passed through ethylamine or histamine-RSA-Sepharose and the unbound cells were subsequently injected into X-irradiated mice, a 1.8 fold increase in the immunological response was noted. We conclude that the selective binding to lymphocytes of the various ligand-macromolecular conjugates may be due to some general properties of the cell membrane and not to any specific receptors. Nevertheless, these conjugates can be used as a tool to remove selectively antibody producing cells as well as some regulatory cells.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Hypothalamic control of the generation of mature natural killer lymphocytes in bone marrow and spleen of the mouse

Following previous work showing that electrothermocoagulation of the median region of the hypothalamus (MH) caused a marked and permanent decrease in the cytotoxicity of natural killer (NK) cells and in the number of large granular lymphocytes, a study was made of the effect of such lesions on the generation of NK cells in the bone marrow (BM) and spleen of C57BL/6 mice. Fresh spleen and BM cells from MH-lesioned and sham-operated mice were cultured with 40 U of recombinant interleukin-2 (rIL-2). NK activity was significantly higher in BM of lesioned mice, whereas spleen NK activity was greater in the sham-operated controls. NK cells matured by culture with rIL-2 were characterized by assay with fluorescent monoclonal antibodies and found to display the typical NK phenotype. These results show that the number of NK precursors is greater in BM of MH-lesioned mice and that their migration into other organs is probably partially impeded. It can also be concluded that intactness of both BM and the hypothalamus is essential for the physiological generation of NK cells.     

运动员剧烈运动后血中应激免疫抑制蛋白的产生

我们曾经报道,大鼠或小鼠在束缚应激后血中产生了一种能抑制免疫功能的应激免疫抑制蛋白,（又称Ｎｅｕ－ｒｏｉｍｍｕｎｅｐｒｏｔｅｉｎ,ＮＩＰ,神经免疫蛋白）。本工作证明,运动员在大运动量的训练后血清中也产生一种能抑制淋巴细胞转化的物质,它的生化特性及分子量与前述大鼠和小鼠中的应激免疫抑制蛋白相同。在体外实验中,应激大鼠的血清培养人淋巴结细胞,获得了与大鼠实验相同的结果,即人淋巴结细胞也能产生应激免疫抑制蛋白。同时小鼠束缚应激的血清和大运动量的人类血清可以分别抑制人正常淋巴细胞和正常小鼠由ＣｏｎＡ诱导的淋巴细胞转化,以上结果表明,这种应激免疫抑制蛋白的种属特异性不强。     

Ultradian Rhythms of Tumor Resistance System Activity

Ultradian time-dependency of the carcinogenic effect was studied in mice; both the model of transplanted (leukemia EL-4) and that of chemically-induced (urethane-induced lung adenomas) tumor were used. As the possible mechanisms which are attributable to this phenomenon, ultradian variations in cellularity of the lymphoid organs (thymus, lymph nodes, spleen, bone marrow), in natural killer-cell (NK) activity, level of the serum factors modifying lymphocyte migration, and in activity of the liver carcinogen-metabolizing enzyme system were investigated. All these characteristics were determined hourly in intact mice. Both the life span of mice inoculated with tumor cells, and the yield of carcinogen-induced tumors were found to be ultradian dependent (periods 3-8 hr). Ultradian dependency was found also for the number of lymphocytes in the lymphoid organs, the number of karyocytes in bone marrow, the NK activity against tumor cells, the migration index of normal spleen cells in the presence of tumor cells, as well as for the activity of the carcinogen-metabolizing enzyme system. It is suggested that variations in the natural resistance against transplanted or chemically-induced tumors may be due to the variations in these parameters.     

The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.     

Contribution of the antioxidative property of astaxanthin to its protective effect on the promotion of cancer metastasis in mice treated with restraint stress

We investigated the effects of astaxanthin on the antitumor effector activity of natural killer (NK) cells suppressed by stress in mice in order to define the immunological significance of astaxanthin (ASX) when combined with restraint stress treatment. When the mice were treated with restraint stress alone, the total number of spleen cells, and the level NK cell activity per spleen were reduced to a nadir on day 3. The stress also caused a significant increase in the lipid peroxidation of liver tissue. ASX (100 mg/kg/day, p.o., 4 days) improved the immunological dysfunction induced by restraint stress. On the other hand, metastatic nodules were observed in the livers of syngenic DBA/2 mice on day 12 after inoculation of P815 mastocytoma cells. Hepatic metastasis was promoted further by restraint stress when applied on day 3 before the inoculation of P815. Daily oral administration of ASX (1 mg/kg/day, p.o., 14 days) markedly attenuated the promotion of hepatic metastasis induced by restraint stress. These results suggested that astaxanthin improves antitumor immune responses by inhibiting of lipid peroxidation induced by stress.