喘可治注射液对人外周血单个核细胞Th1/Th2细胞因子谱的影响

目的：通过研究喘可治注射液对人外周血单个核细胞（PBMCs）Th1／Th2细胞因子谱的影响，探讨喘可治注射液的免疫调节作用机制。方法：以流式微球分析（CBA）法检测不同处理情况下，人外周血单个核细胞分泌Th1（IFN-γTNF-α、IL-2）和Th2（IL4、IL-6、IL-10）细胞因子水平。结果：健康人PBMCs体外培养12小时后，上清中细胞因子主要为TNF-α和IL-6，喘可治使Th1和Th2细胞因子全面升高；喘可治对PDB加离子霉素诱导的PBMCs分泌Th1和Th2细胞因子具有抑制作用，并能抑制流感病人异常升高的INF-γ、TNF-α、IL-6和IL-10分泌。结论：喘可治注射液上调健康人PBMCs分泌TH1和Th2细胞因子，对异常活化的PBMCs分泌的Th1和Th2细胞因子则具有下调作用。     

Lactobacillus isolates from healthy volunteers exert immunomodulatory effects on activated peripheral blood mononuclear cells

As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immuno-modulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti-CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases.     

Modulation of immunoglobulin production and cytokine mRNA expression in peripheral blood mononuclear cells by intravenous immunoglobulin

Intravenous immunoglobulin (IVIG) has the potential to regulate Ig production, but the mechanism(s) responsible for this effect is unknown. In experiments reported here, we examined the ability of IVIG to regulate Ig production in human peripheral blood mononuclear cells (PBMCs) stimulated with pokeweed mitogen (PWM). IVIG (2–10 mg/ml) showed a potent (80–85%) inhibition of PWM-stimulated IgG, IgM, and IgA production. To determine more precisely how IVIG mediated the inhibition of Ig production, we studied Ig promoting cytokine gene expression after PWM stimulation with or without IVIG (2 and 10 mg/ml) using dot-blot techniques. RNA was isolated from PBMCs at predetermined time points and probed with cDNAs specific for human cytokines (IL-1-, IL-2, IL-2R, IL-4, IL-5, IL-6, -IFN, and TNF-). IL-6 mRNA accumulation was maximal at 4.5 hr post-PWM stimulation and was inhibited 64–75% when IVIG (10 mg/ml) was present. -IFN mRNA levels peaked at 72 hr poststimulation and were also 68–75% inhibited by IVIG. IL-2 mRNA levels peaked at 4.5 hr and were 23–46% inhibited by IVIG. The inhibitory effect of IVIG on production of these cytokines (IL-6 and -IFN) was also observed at the protein level in sonicated PBMCs after incubation with PWM and IVIG. The mRNA levels for other cytokines were not or only minimally inhibited by IVIG. Addition of IL-6, -IFN, or IL-2 partially restored Ig production in IVIG-treated PWM-stimulated cultures, suggesting that inhibition of other cytokines or another mechanism(s) independent of cytokine inhibition might also be involved, although inhibition of IL-6, -IFN, and IL-2 may be one of the critical factors in the suppression of Ig production by IVIG.     

Increased interleukin-4-positive lymphocytes in patients with Hashimoto's thyroiditis and concurrent non-endocrine autoimmune disorders

A prevalent T helper type 1 (Th1) subset of lymphocytes has been described in Hashimoto's thyroiditis (HT), but whether a similar polarization may characterize HT when associated with non-endocrine autoimmune disorders (NEAD) is not known. The aim of the present study was to analyse the intracellular Th1 and Th2 distinctive cytokines in patients with isolated HT or associated with non-endocrine autoimmune disorders. Intracellular cytokine expression was assessed in peripheral blood lymphocytes (PBL) of 68 out-patients (females = 55; males = 13; median age = 6 years) with HT : 33 had isolated HT and 35 had a concurrent NEAD. The percentage of interferon (IFN)-γ and interleukin (IL)-2 Th1- and IL-4 Th2-positive cells was measured by flow cytometric analysis. We found an increased percentage of IL-2-positive cells in all patients, without differences between patients with isolated HT or associated with NEAD. IFN-γ(+) cells were also increased in both groups, but the median percentage of those with isolated HT was lower than in patients with HT+NEAD (19·0 versus 29·9%; P = 0·0082). An increased number of IL-4-positive cells was observed in three of 33 (9·1%) patients with isolated HT and in 25 of 35 patients with NEAD [71%; P < 0·0001; relative risk (RR) = 3·18]. The median values of IL-4(+) cells (HT = 5·0% versus HT + NEAD = 16·8%) confirmed this large difference (P < 0·0001). A clear-cut increase of IL-4(+) lymphocytes characterizes patients with autoimmune thyroiditis who have associated non-endocrine autoimmune disorders. These findings may represent an initial tool to detect patients with autoimmune thyroiditis in which additional non-endocrine autoimmune disorders may be awaited.     

Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in prmary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-γ, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-α. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-γ. IL-2, IL-3, TNF-α and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-γ, IL-2 and TNF-α. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.     

Kinetics of mononuclear cell infiltration and cytokine expression in iodine-induced thyroiditis in the NOD-H2h4 mouse

Mononuclear cell infiltration of the thyroid gland is a common histologic feature of chronic lymphocytic thyroiditis. Although the infiltrating mononuclear cells have been implicated in the destruction of the thyroid, information concerning the progression of infiltration into the thyroid is limited. In this report, we examine the composition and kinetics of mononuclear cell infiltration in the thyroid and the expression of major histocompatibility complex class II (I-Ak), IL-12, and IFN-gamma in the thyroid of the NOD-H2h4 mouse, a model of spontaneous autoimmune thyroiditis accelerated by the administration of excess dietary iodine. Mice were given a low dose of 0.015% NaI in their drinking water for 2, 4, 6, 8, and 16 weeks, and thyroids were removed, serially sectioned, and stained in an avidin-biotin peroxidase assay. The thyroid infiltrate included CD4+ and CD8+ T cells, F4/80+ macrophages, and B220+ B cells. After 2 weeks of iodine treatment, CD4+ T cells were the first seen in the thyroid, followed by CD8+ T cells and F4/80+ macrophages. B220+ B cells entered the thyroid after 4 weeks of iodine treatment. IL-12 and IFN-gamma positive cells were located in the thyroid early in disease and were up-regulated in the focal accumulations of infiltrating cells. Thyrocytes clearly expressed I-Ak after 4 weeks of iodine treatment near the location of mononuclear cell infiltration.     

Effect of glutamine on Th1 and Th2 cytokine responses of human peripheral blood mononuclear cells

Decreased glutamine concentrations are found in patients with catabolic stress and are related to susceptibility to infections. In this study, we evaluated the role of glutamine in Th1/Th2 cytokine responses. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA), live attenuated bacillus Calmette-Guérin (BCG), or measles virus in the presence of different glutamine concentrations. We found that glutamine at an optimal concentration (0.6 mM) significantly enhanced PHA-stimulated lymphocyte proliferation as well as Th1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] and Th2 cytokine (IL-4 and IL-10) production. In the absence of glutamine, BCG and measles virus elicited minimal lymphocyte proliferation, whereas BCG enhanced Th1 cytokine response and measles virus promoted Th2 cytokine response. Interestingly, addition of glutamine promoted the BCG-elicited Th1 cytokine response (IFN-gamma), but suppressed the measles-induced Th2 cytokine response (IL-10). These results suggest that appropriate glutamine levels may influence host responses to different antigens and microorganisms. Furthermore, predominately Th1, but not Th2, cytokine responses required the presence of optimal concentrations of glutamine.     

帕金森病人外周血单个核细胞分泌IL-2的实验研究

目的 探讨帕金森病病人中外周血单个核细胞(PBMC)分泌IL-2的能力.方法 采用酶联免疫吸附法(ELISA)分别检测体外培养的PBMC上清液、及PBMC接受刀豆蛋白(ConA)刺激后上清液中IL-2含量.结果 PD Ⅰ期患者中未受刺激的PBMC分泌的IL-2显著高于PDⅡ-Ⅲ期患者(P＜0.05),接受ConA刺激后,以PD Ⅱ-Ⅲ期患者的PBMC生成IL-2最低,显著低于正常对照组(P＜0.05),刺激后同刺激前相比各组IL-2都有升高,但仅健康对照组刺激前后有显著差异(P＜0.05).结论 帕金森病患者的外周血单个核细胞的分泌IL-2能力存在异常.     

Graves病患者外周血单个核细胞TIM-3、TIM-1及其相关基因mRNA的表达与临床意义

目的 研究TIM-3、TIM-1、T-bet、GATA-3、IFN-γ、IL-4和Galectin-9基因在Graves病(GD)患者外周血的表达及其临床意义.方法 通过实时定量RT-PCR分别检测70例Graves病患者及22例健康正常人外周血单个核细胞(PBMC)中TIM-3、TIM-1及其相关基因mRNA的表达情况,并分析TIM-3、TIM-1与其之间的关系.结果 GD患者PBMC中TIM-3、TIM-1 mRNA的表达异常增高,其突眼GD组TIM-3 mRNA的表达要高于无突眼GD组,而TIM-1 mRNA的表达却无明显差异.GD初诊患者,TIM-3 mRNA的表达要显著高于复发组,但TIM-1 mRNA的表达却恰恰相反.GD缓解患者,TIM-3 mRNA的表达与健康对照组差异无统计意义,TIM-1 mRNA的表达虽显著下降,但仍高于正常对照组.结论 TIM-3、TIM-1可能参与了GD的发生、发展和晚期转归,TIM-3或(和)TIM-1有可能为治疗GD提供一个新的靶点.     

Role of leptin as an immunomodulator of blood mononuclear cells: mechanisms of action

Leptin is a an adipocyte-secreted hormone that regulates weight centrally. However, the leptin receptor is expressed not only in the central nervous system, but also in peripheral tissues, such as haematopoietic and immune systems. Therefore, the physiological role of leptin should not be limited to the regulation of food intake and energy expenditure. Moreover, the leptin receptor bears homology to members of the class I cytokine family, and recent data have demonstrated that leptin is able to modulate the immune response. Thus, the leptin receptor is expressed in human peripheral blood mononuclear cells, mediating the leptin effect on proliferation and activation. In vitro activation and HIV infection in vivo induce the expression of the long isoform of the leptin receptor in mononuclear cells. Also, leptin stimulates the production of proinflammatory cytokines from cultured monocytes and enhances the production of Th1 type cytokines from stimulated lymphocytes. Moreover, leptin has a trophic effect on monocytes, preventing apoptosis induced by serum deprivation. Leptin stimulation activates JAK-STAT, IRS-1-PI3K and MAPK signalling pathways. Leptin also stimulates Tyr-phosphorylation of the RNA-binding protein Sam68 mediating the dissociation from RNA. In this way, leptin signalling could modulate RNA metabolism. These signal transduction pathways provide possible mechanisms whereby leptin may modulate activation of peripheral blood mononuclear cells. Therefore, these data support the hypothesis regarding leptin as a proinflammatory cytokine with a possible role as a link between the nutritional status and the immune response. Moreover, these immunoregulatory functions of leptin could have some relevance in the pathophysiology of obesity.     

脱氧雪腐镰刀菌烯醇对人外周血单个核细胞TAP-1表达的抑制作用

目的: 探讨脱氧雪腐镰刀菌烯醇 (DON)对人外周血单个核细胞 (PBMC)中TAP- 1表达的影响。方法: 采用流式细胞仪(FCM)和半定量RT- PCR方法, 从蛋白和mRNA水平上分析不同浓度的DON对体外培养的人PBMC中TAP- 1分子表达的影响及量 效关系。结果: FCM定量检测表明, 用不同浓度的DON处理, 均可一定程度地抑制人PBMC中TAP -1蛋白的表达, 且二者呈显著的负相关 (r= -0. 865,P<0. 01)。半定量RT -PCR检测显示, 不同浓度的DON处理均可抑制人PBMC中TAP -1mRNA的表达。结论: DON可剂量依赖地抑制体外培养的人PBMC中TAP- 1蛋白和其mRNA的表达, 对阐明食管癌高发区被DON污染的粮食与食管癌发生的关系具有重要的意义。     

Asthma-predictive genetic markers in gene expression profiling of peripheral blood mononuclear cells

Purpose

We sought to identify asthma-related genes and to examine the potential of these genes to predict asthma, based on expression levels.

Methods

The subjects were 42 asthmatics and 10 normal healthy controls. PBMC RNA was subjected to microarray analysis using a 35K array; t-tests were used to identify genes that were expressed differentially between the two groups. A multiple logistic regression analysis was applied to the differentially expressed genes, and area under the curve (AUC) values from receiver operating characteristic (ROC) curves were obtained.

Results

In total, 170 genes were selected using the following criteria: P≤0.001 and ≥2-fold change. Among these genes, 57 were up-regulated and 113 were down-regulated in asthmatics versus normal controls. A multiple logistic regression analysis was done using more stringent criteria (P≤0.001 and ≥5-fold change), and eight genes were selected as candidate asthma biomarkers. Using these genes, 255 models (2⁸-1) were generated. Among them, only 85 showed P≤0.05 by multiple logistic regression analysis. Based on the AUCs from ROC curves for the 85 models, we found that the best model consisted of the genes MEPE, MLSTD1, and TRIM37. The model showed 0.9928 of the AUC with 98% sensitivity and 80% specificity.

Conclusions

MEPE, MLSTD1, and TRIM37 may be useful biomarkers for asthma.     

Monocytes are primed to produce the Th1 type cytokine IL-12 in normal human pregnancy: an intracellular flow cytometric analysis of peripheral blood mononuclear cells

This paper considers both monocytes and peripheral blood lymphocytes as potential targets for maternal immunological modulation in pregnancy. Peripheral blood mononuclear cells (PBMCs) from non-pregnant and normal pregnant donors were stimulated in vitro, and cytokine production detected intracellularly by flow cytometry. It was found that monocyte production of TNF-alpha was unaltered in pregnancy, while production of IL-12 was significantly enhanced. In contrast, production of the Th1 type cytokine IFN-gamma was suppressed in the lymphocyte subsets: CD4+ T helper cells and CD56+ NK cells. Production of the Th2 type cytokine IL-4 in CD4+ cells was not significantly altered in pregnancy. These data suggest that the concept that pregnancy is a 'Th2 phenomenon' cannot be generalized to the function of all aspects of maternal cellular immunity as, paradoxically, circulating monocytes are 'primed' to produce the Th1 cytokine IL-12. Furthermore, these data support the hypothesis that components of maternal innate immunity are activated in normal pregnancy.     

Intravenous immunoglobulin treatment in women with recurrent abortions: increased cytokine levels and reduced Th1/Th2 lymphocyte ratio in peripheral blood

PROBLEM: The aim of this study was to investigate changes in peripheral blood Th1/Th2 cytokine levels and lymphocyte ratios after massive intravenous immunoglobulin (MIVIg) treatment for women with recurrent spontaneous abortion (RSA) of unexplained etiology. METHOD OF STUDY: Serum Th1 (IFN-gamma, TNF-alpha) and Th2 cytokine (IL-4, IL-10) levels were assessed by ELISA methods (n = 9) and peripheral blood Th1/Th2 lymphocyte ratios (n = 4) by flow cytometry before and after MIVIg treatments in women with four or more consecutive RSA. RESULTS: Pre-treatment serum IFN-gamma (0.06 +/- 0.09 pg/mL, mean +/- SD), TNF-alpha (0.21 +/- 0.45 pg/mL), IL-4 (0.70 +/- 1.16 pg/mL), and IL-10 (1.12 +/- 1.67 pg/mL) increased to 0.17 +/- 0.16 pg/mL, 0.77 +/- 0.28 pg/mL, 1.82 +/- 0.89 pg/mL, and 3.44 +/- 0.48 pg/mL, respectively, after MIVIg treatments (P < 0.05). CD4-positive IFN-gamma/IL-4 lymphocyte ratios (17.3 +/- 9.1) were reduced to 11.5 +/- 7.1 after treatment (P < 0.05). CONCLUSIONS: Massive intravenous immunoglobulin treatments increased peripheral blood cytokine levels and decreased Th1/Th2 lymphocyte ratios; thus, MIVIg treatments modify the peripheral Th1/Th2 balance.     

Increased expression of allergen-induced in vitro interleukin-10 and interleukin-18 mRNA in peripheral blood mononuclear cells of allergic rhinitis patients after specific immunotherapy

BACKGROUND: During specific pollen immunotherapy (SIT) there is a local mucosal shift from Th2- to Th1- type cytokine predominance, with IL-12 having a major role in this shift. IL-10-induced tolerance is supposed to be a key phenomenon in venom immunotherapy (VIT). However, the role of Th1-promoting cytokines, on the one hand, and the role of regulatory cytokines, on the other hand, have not been studied in parallel during SIT. OBJECTIVE: This study was undertaken to analyse the allergen-induced in vitro mRNA expression of Th1-type effector cytokine IL-18 and regulatory cytokines IL-10 and TGF-beta during SIT in peripheral blood mononuclear cells (PBMC) of allergic rhinitis (AR) patients. METHODS: Thirty patients with AR undergoing pollen SIT and 10 patients with AR who were not treated with SIT were included in the study. The symptoms and medications were registered post-seasonally before the beginning of SIT and after 1 year of therapy. PBMC samples were collected and stimulated with pollen allergen extract prior to the treatment, at the maintenance phase in 12 patients and after 1 year of the treatment. The cytokine mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan). RESULTS: There was a clear increase in the treated AR patients, in comparison with untreated AR patients, in the expression of both IL-10 (mean change from baseline (SEM): 3.1 (0.8) vs. -0.3 (0.3), P<0.002, Mann-Whitney U-test) and IL-18 (2.7 (0.9) vs. -0.2 (0.6), P<0.03) mRNA after 1 year. The clearest increase in IL-10 mRNA expression was seen in patients who did not benefit at all (6.0 (2.3), P<0.001 vs. untreated) and the least increase in patients that had the greatest reduction of symptoms (0.8 (0.6), n.s. vs. untreated) at 1 year. The clearest increase in IL-18 mRNA expression was seen in patients with moderate outcome (3.4 (1.6), P<0.04 vs. untreated). In intermediate samples, taken when the maintenance dose was reached, the peak expression of allergen-induced IL-10 mRNA was associated with the most favourable outcome of SIT (P=0.01, Fisher exact test). A similar trend was seen in IL-18 mRNA expression. CONCLUSIONS: The results suggest that an early and transient increase in allergen-specific IL-10 and IL-18 mRNA expression in PBMC is essential for the therapeutic outcome after 1 year of SIT.     

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.