阿托伐他汀诱导BMDCs源性外泌体对EAMG模型大鼠临床症状的缓解及与自然杀伤细胞和自然杀伤T细胞的关系

目的 分析阿托伐他汀诱导骨髓来源树突状细胞(BMDCs)源性外泌体对实验性自身免疫性重症肌无力(EAMG)模型大鼠临床症状的缓解及与自然杀伤细胞和自然杀伤T细胞的关系。方法 分别采用二甲基亚砜、阿托伐他汀与BMDCs进行共培养,分别设置对照组(二甲基亚砜与BMDCs共培养)和实验组(阿托伐他汀与BMDCs共培养),通过流式细胞术检测2组BMDCs表面共刺激分子的表达水平; 通过梯度离心法提取2组BMDCs源性外泌体,将其注射于EAMG大鼠体内,实验组给予阿托伐他汀,对照组未进行任何治疗,记录2组大鼠临床症状评分; 采用流式细胞术测定2组大鼠淋巴结单个核细胞中自然杀伤细胞和自然杀伤T细胞的比例。结果 实验组BMDCs表面共刺激分子CD80和CD86表达水平较对照组显著下降(P<0.01),而2组主要组织相容性复合体Ⅱ(MHC-Ⅱ)分子表达水平无明显差异(P>0.05); 实验组免疫2、4、6周后临床症状评分较对照组均明显降低(P<0.01)。与对照组比较,实验组大鼠淋巴结单个核细胞中自然杀伤细胞的比例明显升高(P<0.01),而2组大鼠淋巴结单个核细胞中自然杀伤T细胞的比例无明显差异(P>0.05)。结论 阿托伐他汀诱导BMDCs分泌源性外泌体可有效减轻EAMG大鼠临床症状,其作用机制可能与提高大鼠淋巴结单个核细胞中自然杀伤细胞的比例有相关     

Circulating natural killer cells but not cytotoxic T lymphocytes are reduced in patients with active relapsing multiple sclerosis and little clinical disability as compared to controls

Triple antibody flow cytometry was used to compare the populations of CD56⁺ effector cells in the peripheral circulation of 29 patients with relapsing multiple sclerosis (MS) and little disability who were exacerbation-free for over 2 months and 29 healthy control subjects. Populations were characterized by two panels of antibodies (CD8, CD16, CD56 and CD3, CD8, CD56), as well as by size or granularity. In the MS patients, mature natural killer (NK) cells (CD3⁻CD8⁻CD56⁺) of small size and low granularity were significantly reduced compared to normals (P 〈 0.0003). The quantities of other effector cells (cytotoxic T lymphocytes, large granular lymphocytes and monocytes) were not different in MS patients compared to the control subjects. Also, we identified a previously unrecognized population of CD56⁺ monocytes (CD3⁻CD14⁺CD56⁺) in both the normal control subjects and the MS patients which would have been misclassified as NK cells using one or two antibody cytometry employed in previous studies.     

The differential expression of natural killer cells in NMOSD and MS

Natural killer (NK) cells are involved in the pathogenesis of inflammatory demyelinating diseases of the central nervous system. However, the differential expressions of NK cells in the peripheral blood of patients with neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS) are unknown. This study aimed to explore the differential expressions of NK cells in NMOSD and MS and evaluate the clinical implications of this difference. We performed a cross-sectional study to investigate the expression of NK cells in the peripheral blood of patients with NMOSD (n = 78) and MS (n = 24) and of healthy controls (HC, n = 27). Furthermore, we investigated the relationship between NK cell level and disease phase in 102 patients with NMOSD and MS through Spearman correlation analysis and receiver operating characteristic (ROC) analysis. Our results showed that the median (interquartile range) NK cell levels in acute-phase NMOSD patients, remission-phase NMOSD patients, acute-phase MS patients, and HC subjects were 114.10 (64.75–153.38) cells/µL, 167.60 (116.35–266.15) cells/µL, 282.55 (140.57–368.20) cells/µL, and 221.00 (170.40–269.55) cells/µL, respectively (p < 0.001). The Spearman correlation coefficient (95%) for the relationship between NK level and disease phase in NMOSD patients was 0.366 (0.150–0.550) (p < 0.001). Furthermore, ROC analysis revealed that patients with NK cell values lower than 172.200 cells/µL were more prone to have acute-phase NMOSD than MS. In conclusion, the expression of NK cells in peripheral blood was lower in patients with NMOSD than in patients with MS in the acute phase, and a low expression of NK cells may suggest having acute-phase NMOSD rather than MS.     

Neuroenteric peptides affect natural killer activity by intestinal lamina propria mononuclear cells

The effect of the neuropeptides bombesin and vasoactive intestinal peptide, as well as neurotensin, on natural killer activity by lamina propria mononuclear cells isolated from histologically normal mucosa was assessed. Bombesin and vasoactive intestinal peptide were found to dose-dependently stimulate natural killer activity against Caco-2 colon carcinoma target cells, especially in a short incubation assay, whereas no alterations of cytotoxicity were found against K-562 target cells. Neurotensin, on the contrary, was not found to affect this type of cellular cytotoxicity. Differences in the modulatory effects of these gastrointestinal peptides on natural killer activity by lamina propria mononuclear cells may be related to the distinct localization differences of these peptides within the bowel. The present findings illustrate the existence of neuro-immune interactions at the intestinal mucosa level.     

Natural killer (NK) cells in chronic progressive multiple sclerosis patients treated with lymphoblastoid interferon

Natural killer (NK) cell functional activity, as defined by the lysis of 51Cr-labelled K-562 cells, and number, defined phenotypically by anti-Leu-11, are significantly decreased in chronic progressive multiple sclerosis (MS) when compared to normal controls. When age- and sex-matched populations are compared, NK cell functional activity is again significantly reduced in MS compared to controls but not when compared to a control group of other medical disease (OMD). The MS group could be differentiated from the OMD group, however, when results of NK cell functional activity are combined with NK cell phenotype. With the administration of lymphoblastoid interferon daily for 6 months, NK cell activity increased significantly at 48 h and at 1 week. By 1 month, activity decreased to a level slightly above placebo treatment values. The results likely reflect interferon's enhancement of mature NK cell activity combined with a variable effect on recruitment of pre-NK cells.     

Glucocorticoid dysregulation of natural killer cell function through epigenetic modification

It is well-established that psychological distress reduces natural killer cell activity (NKCA) and dysregulates cytokine balance. This may be mediated by stress-induced release of glucocorticoids, which have broad effects on the immune system, including the suppression of NKCA and alteration of cytokine production. The purpose of this study was to evaluate epigenetic mechanisms that may underlie the effect of glucocorticoids on NK cells, using the human NK cell line, NK92. Treatment of NK92 cells with the synthetic glucocorticoid, dexamethasone, at a concentration of 10⁻⁷ M, produced a significant reduction in NKCA. Glucocorticoid inhibition was a consequence of not only a reduced capacity of the NK cells to bind to tumor targets but also a reduced production of granule constituents (perforin and granzyme B) with no detectable effect on granule exocytosis. Glucocorticoids also reduced the constitutive and the stimulated production of the cytokines, IL-6, TNF alpha and IFN gamma, and reduced the surface expression of LFA-1. Glucocorticoid treatment also reduced global histone acetylation, the acetylation of histone 4 lysine position 8, and the accessibility of the proximal promoters of perforin, interferon gamma and granzyme B. Histone acetylation was recovered by treatment of the NK cells with a histone deacetylase inhibitor, which also restored NKCA and IFN gamma production. These results demonstrate glucocorticoids to dysregulate NK cell function at least in part through an epigenetic mechanism, which reduces promoter accessibility through modification of histone acetylation status. This epigenetic modification decreases the expression of effector proteins necessary to the full functional activity of NK cells.     

Absence of natural killer (NK) cell activity against oligodendrocytes in multiple sclerosis

The cytotoxicity of natural killer (NK) cells directed to enriched cultures of bovine oligodendrocytes was investigated in multiple sclerosis (MS) patients and controls. Macrophage-depleted peripheral blood lymphocytes were used as effector cells in a 4-h 51Cr release assay. No significant cytotoxic activity to oligodendrocytes was identified in either MS or control groups. In contrast, a definite cytotoxic activity directed toward K562 cells was observed in the study populations. No statistically significant difference was observed between chronic progressive or stable MS, other neurological diseases (OND), and normal controls. These results indicate that NK cell activity directed toward intact bovine oligodendrocytes is not significantly different between MS and control groups and question the significance of studies employing K562 cells as the target in NK assays in MS. Furthermore, these observations suggest that NK-mediated cytotoxicity against oligodendrocytes is unlikely to be a specific mechanism mediating demyelination in MS.     

A role for natural killer cells in the immunopathogenesis of multiple sclerosis

Seventeen relapsing–remitting (R/R) multiple sclerosis (MS) patients and age/sex matched controls were studied every 6 weeks for 2 years. Disease activity, determined both clinically and by serial MRI, was correlated with natural killer (NK) cell functional activity (FA) and phenotype. Mean NK cell FA is significantly lower in MS patients, compared to controls (P<0.001), while variability around the means is significantly greater (P<0.01). The spectrum of mean NK cell FA, observed in the patient cohort, along with cyclical nature of the FA and phenotype over time, observed in both patients and controls, may begin to explain the discrepant results reported in previous studies. In R/R MS, there is a significant correlation between reductions (valleys) in NK cell FA and the development of active lesions on MRI, new (P<0.001) or enlarging (P=0.05). More importantly, a significant number of active lesions, new (P=0.01) and enlarging (P=0.02), are preceded by a reduction in NK cell FA. The correlation between the onset of clinical attacks and valleys of NK cell FA is also significant (P=0.002). When taken together, the results suggest that reductions (valleys) in NK cell FA represent periods of susceptibility for the development of active lesions on MRI and clinical attacks. A significant positive correlation is also identified between mean NK cell FA for each R/R MS patient and total number of active MRI lesions developed by that patient over the 2 years (P=0.001). The results would suggest that R/R MS patients with a higher mean NK cell FA are at greater risk for the development of active lesions. These results support the proposal that NK cells may play a role in the immunopathogenesis of R/R MS.     

The biphasic changes in splenic natural killer cell activity following ventromedial hypothalamic lesions in rats

The cytotoxic activity of natural killer (NK) cells in the spleen was measured by a standard 4-h chromium release assay following electrical lesioning of the ventromedial hypothalamic nucleus (VMH) in rats. The splenic NK cell activity of VMH lesioned rats was found to be significantly suppressed at effector: target cell ratios of 100:1 and 50:1 compared with that of sham lesioned rats on day 4 after the lesion. On the other hand, on day 49, the VMH lesioned animals that had become hyperphagic and obese showed an enhancement of splenic NK cell activity compared with sham lesioned animals. The mechanisms of the biphasic change in NK cell activity following the VMH lesions are discussed.     

Coping in an intermittent swim stress paradigm compromises natural killer cell activity in rats

The effects of intermittent swim stress and stressor controllability on natural killer cell activity (NKCA) was examined. Significant decreases in splenic NKCA were observed immediately post-stress, but only when the stress was controllable. Although decreased NKCA was also observed in yoked rats subjected to the same stressor, it failed to attain statistical significance. Previous results suggest these effects are not due to corticosterone. The results suggest a cost of coping on the acute, in vitro immune measure of NKCA.     

Microglial cells are a component of the perivascular glia limitans

The ultrastructural relation between microglial cells and cerebral blood vessels was studied in rat brains by immune electron microscopy using antibodies against the common leukocyte antigen (Ox1), the complement receptor 3 (Ox42), and against class I and class II histocompatibility antigens (MHC antigens; Ox3, Ox6, Ox18, and I1-69). Microglial cell processes were found incorporated between the astrocytic foot processes of the glia limitans in 4-13% of cerebral microvessels. After intravenous injection of gamma-interferon, either alone or in combination with tumor necrosis factor, these microglial cell processes expressed classes I and II MHC antigens. Studies in (Lewis X DA)F1-DA bone marrow chimeras demonstrated that these cell processes belonged to resident microglia. This study suggests that microglial cells may play an important role in antigen recognition at the blood-brain barrier.     

Arecoline a muscarinic cholinergic agent conditions central pathways that modulate natural killer cell activity

The central nervous system plays an active role in the regulation of the immune system. Modulation of immune activities appears to be in part under the control of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the effect of a muscarinic cholinergic agonist, arecoline, which stimulates the secretion of corticotropin-releasing hormone (CRF) and adrenocorticotropic hormone (ACTH) on the immune system. In this report we demonstrate that peripherally administered arecoline or ACTH can increase activity of pre-activated NK cells. Second, we show that central administration of arecoline at a dose too low to alter peripheral events is sufficient to induce a significant increase in the activity of pre-activated natural killer (NK) cells. Finally, we demonstrate by using a Pavlovian conditioning paradigm that the pairing of a novel odor (camphor) with administration of arecoline can be used to alter NK cell activity. Subsequent to the conditioning trial, exposure to the odor alone is sufficient to raise NK cell activity. From these observations, we infer that the pathway(s) that are conditioned reside in sites located within the CNS and the conditioned response is evoked in the peripheral compartment (NK cell activity).     

Human natural killer cell activity is reduced by treatment of anti-myelin-associated glycoprotein (MAG) monoclonal mouse IgM antibody and complement

Human mononuclear cells could be stained by anti-myelin-associated glycoprotein (MAG) monoclonal mouse IgM antibody. The remaining human natural killer (NK) cell activity examined by using K-562 cells at 20:1 as effector:target ratio after treatment of anti-MAG monoclonal mouse or anti-Leu-7 (HNK-1) antibody and complement revealed 13.4% and 15.1%, respectively (untreated NK activity was 40.8%). However, human NK activity could be abrogated by anti-Leu-11 and complement. The remaining NK activity shown as lytic units after treatment with anti-MAG, anti-Leu-7 or anti-Leu-11 and complement was 6.1, 5.3 and below 1.0, respectively (untreated NK cells showed 15.4). When NK activity was examined in another target cell, MOLT-4, the remaining activity shown as lytic units was also decreased with anti-MAG antibody (4.3) or with anti-Leu-7 (3.0) (untreated NK activity was 8.3). Our findings suggest that NK cells may be influenced by anti-MAG antibody if it is found in the sera as anti-lymphocytotoxic antibody.     

Interferon production and natural killer (NK) activity in leukocyte cultures from multiple sclerosis patients

Peripheral blood leukocyte (PBL) cultures from only 37% of MS patients produced detectable HuIFN-gamma in response to ConA as opposed to 85% of the cultures derived from normal blood donors. However, the yields in patient-derived cultures that were responsive, were not lower than those in cultures from controls. Production of HuIFN-alpha after stimulation with Sendai virus was not aberrant in cells taken from MS patients. The difference in HuIFN-gamma response rate between MS and normal donor-derived cells was more pronounced when DR2+ carriers were compared amongst each other than when DR2-k carriers were compared. Among the MS patients, the failure of PBLs to produce HuIFN-gamma in response to ConA was not correlated with age, sex, disease duration and type of disease. However, positive correlations were found with current disability indices and past disease progression rates. Unstimulated NK-activities of MS patient-derived PBLs were not different from those of normal donor-derived cells. the degree of augmentation of the activity by stimulation with ConA and interferon-alpha was also normal. Within the MS patients group, but not in the control group, there was a trend for DR2+ carriers to have lower spontaneous and stimulated NK-activities than DR2- individuals.     

Mononuclear cell infiltrate and HLA-DR expression in low grade astrocytomas

Summary Frozen samples from 23 low grade (grade I and II) astrocytomas were studied by means of a panel of monoclonal antibodies to macrophages, lymphocytes (and their subsets) and HLA-DR antigens. Macrophages were present in low to moderate numbers in 38%–86% of cases, the variance in figures depending on the antibody used. T lymphocytes, the majority of CD8 phenotype, were detected in low numbers in 78% of tumours. B lymphocytes were scanty in 22% (5/22) and totally absent in the remaining cases. HLA-DR antigen was expressed by tumour cells in 35% (6/17) of cases. These findings indicate that in some low grade astrocytomas there is a mononuclear cell infiltrate with macrophages and secondarily CD8+lymphocytes playing the major role. The significance of these findings remains speculative at present.Supported in part by a grant from the Oxford District Health Authority and the A. von Humboldt Foundation     

Recognition of major histocompatibility complexantigens on murine glial cells

Recognition of autologous major histocompatibility complex (MHC) antigens by T cells is an essential step in the induction of an immunologic reaction to either endogenous or exogenous antigens. We investigated the ability of murine glial cells of different ages to stimulate clones of allospecific T lymphocytes. We also investigated the effects of supernatants from cultures of activated T cells on the immunologic recognition of MHC antigens on murine glial cells. Lymphocyte clones specific for Class I, Class II and non-MHC, background antigens were obtained from C57B1/6J-anti-DBA/2 mixed lymphocyte cultures. Glial cell cultures were prepared from newborn syngeneic (C57B1/6J) and allogeneic (DBA/2) mouse brains. Glial cultures 1-4 weeks of age were able to stimulate alpha-Class I-specific clones. No stimulation of alpha-Class II or alpha-background clones was noted. Incubation of glial cells with supernatants from cultures of alloantigen-activated spleen cells (C57B1/6J-anti-DBA/2) resulted in a decreased ability of glial cells to stimulate alpha-Class I responses. In contrast supernatant-treated cultures acquired the capacity to stimulate alpha-Class II-specific clones. No responses were noted in clones responsive to non-MHC antigens. The ability to stimulate alpha-Class II-specific clones was most prominent with one-week-old glial cultures and was lost by four weeks of culture. The increased susceptibility of younger glial cultures to the modulatory effects of lymphokines from activated T cells may be a factor in the increased susceptibility of the immature central nervous system to persistent viral infections and the development of autoimmune phenomena.     

Self-regulatory cognition and immune reactivity: idiographic success and failure feedback effects on the natural killer cell

Inducing depressed and anxious individuals to write about their personal goals decreases natural killer (NK) cell activity, revealing a psychobiological pathway whereby experiences of failure can influence health (Strauman et al., 1993). However, it is unclear whether similar effects also occur in non-distressed individuals. This study used the same writing task to examine the acute physiological effects of presenting idiographic success and failure feedback by priming self-congruencies or self-discrepancies on three occasions (including a control condition). Blood samples were collected after each writing session to determine NK activity, and the number and type of lymphocytes in circulation were enumerated to help explain the cytolytic changes. The two self-relevant priming conditions were associated with significant alterations in immunity, and the high self-discrepant participants were more responsive. Both self-congruent (success) and self-discrepant (failure) priming induced significant shifts in mood, which partially mediated immune alterations but did not account for them completely. If repeated and sustained over time, incidental activation of self-discrepancies and self-congruencies could account for individual variation in immune responses.     

癫痫患儿外周血CD19+B、 CD20+B淋巴细胞和自然杀伤细胞的检测及意义

目的探讨儿童癫痫患者外周血CD 19+13、CD20+B淋巴细胞和自然杀伤(NK)细胞的表达及其意义。方法选择中山大学孙逸仙纪念医院自2008年1月至2010年12月收治的癫痫患儿458例为病例组,另设同期该院52例健康对照者为对照组。应用流式细胞仪对2组成员外周血CD19+B细胞、CD20+B细胞和NK细胞的表达进行检测及比较,同时分析92例应用静脉注射免疫球蛋白(IVIG)治疗的癫痫患儿治疗前后细胞表达的改变。 结果癫痫患儿CD19+B细胞和CD20+B细胞比例分别为(22.35％±6.54％)、(21.50％±8.41％),明显高于正常对照组(16.86％±4.02％)、(16.13％±4.19％),差异有统计学意义(P≤0.05); NK细胞比例为(9.11％±4.90％),明显低于正常对照组(14.72％±4.15％),差异有统计学意义(P≤0.05)。IVIG治疗6个月后癫痫患儿CD19+B细胞和CD20+B细胞比例分别为(18.26％±5.03％)、(16.74％±5.12％),较治疗前(22.74％±6.25％)、(21.61％±8.03％)明显降低,差异有统计学意义(P＜0.05);而NK细胞为(14.65％±4.58％),较治疗前(9.07％±4.76％)明显升高,差异亦有统计学意义(P＜0.05)。92例IVIG治疗患儿中,22例无效,70例有效,有效与无效患儿外周血CD19+B细胞和CD20+B细胞比例治疗前后差值差异具有统计学意义(P＜0.05),但外周血NK细胞比例治疗前后差值差异无统计学意义（P＞0.05）。 结论癫痫患儿存在B淋巴细胞和NK细胞功能异常,IVIG治疗能改善癫痫患儿的免疫功能紊乱;外周血CD19+B细胞、CD20+B细胞可做为癫痫IVIG治疗疗效的监测指标。     

Effect of cerebral hemisphere decortication on the cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse

A comparison was made of the effects of left and right cerebral decortication on cytotoxic activity of natural killer and natural cytotoxic lymphocytes in the mouse. Natural killer cytotoxic activity was significantly reduced after right decortication, whereas left decortication led to a less pronounced, though still significant fall. The cytotoxic activity of natural cytotoxic cells, on the other hand, was significantly i increased, particularly 15 days after left decortication. These findings mirror the results of previously published personal findings following electrothermocoagulation of the hypothalamus. The suggestion is made that the cortex and the hypothalamus form an integrated system for the control of certain aspects of natural immunity.     

The expression of MHC antigens on oligodendrocytes: Induction of polymorphic H-2 expression by lymphokines

Neither class I nor class II major histocompatibility complex (MHC) antigen has been demonstrated in native oligodendrocytes, the possible target of viral or immune damage in multiple sclerosis (MS). In this report, we show that H-2, but not Ia, antigen expression is induced on isolated mouse oligodendrocytes in vitro by crude supernatant from lectin-activated spleen cells, lectin-free interleukin 2, and cloned gamma-interferon. This induction of H-2 expression is not accompanied by proliferation of oligodendrocytes, whereas MHC induction in spleen cells is highly related to their proliferation, or blastoid transformation. Oligodendrocytes as well as other brain cells are probably isolated from these lymphokines by the blood-brain barrier (BBB). However, it is possible that oligodendrocytes express MHC class I antigen as a consequence of impairment of the BBB, or in the presence of activated T-cells which have been demonstrated in active MS lesions. This activation then renders oligodendrocytes possible target cells for MHC-restricted cytotoxic T-cells.