Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.     

Roxithromycin inhibits the effector phase of delayed-type hypersensitivity

In the present paper, the effect of roxithromycin on delayed-type hypersensitivity (DTH) was evaluated. Roxithromycin had no effect on sheep red blood cells (SRBC)-induced food pad swelling when orally administered in induction phase, whereas it suppressed the SRBC-induced DTH reaction and 2,4,6-Trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) significantly when administered to mice in effector phase. For the sustained-CHS model induced by multi-challenge with TNCB, roxithromycin also inhibited the ear swelling when exposed to mice in three effector phases while showed no inhibitory effect on CHS by continuous treatment. Administration of this antibiotic in effector phase also down-regulated the MMP-9 activity and the higher in vitro survival of splenocytes from SRBC-challenged mice. Furthermore, this drug inhibited the gene expression of T-helper type 1 (Th1) cytokines such as IL-2 and IFN-gamma of lymph node cells from mice immuned by TNCB or of Con A-stimulated spleen cells. In addition, roxithromycin administered in vivo decreased the concanavalin A (Con A)-induced splenocyte proliferation without affecting the cell survival in vitro. These results suggest that roxithromycin might alleviate DTH reaction at least by suppressing the function and survival of effector T cells.     

六味地黄多糖体外对正常及衰老小鼠脾细胞免疫功能的影响

目的探讨六味地黄多糖免疫药理活性的物质基础。方法运用现代药理学与植物化学紧密配合的方法,从六味地黄汤中逐步分离获得了六味地黄多糖ＣＡ４ ３Ｂ和Ｐ ３。应用［３Ｈ］ ＴｄＲ参入法和溶血空斑实验观察了其体外对小鼠脾细胞免疫功能的作用。结果ＣＡ４ ３Ｂ、Ｐ ３体外单独使用对正常及快速老化模型ＳＡＭＰ８和ＳＡＭＲ１小鼠脾细胞增殖反应有明显的促进作用,但不能增强有丝分裂原ＣｏｎＡ诱导的脾细胞增殖反应,大剂量时有一定的抑制作用。ＣＡ４ ３Ｂ对绵羊红细胞（ＳＲＢＣ）体外诱导的正常小鼠脾抗体形成细胞生成反应亦有明显的促进作用,使抗体形成细胞（ＰＦＣ）的数目明显增加。结论ＣＡ４ ３Ｂ和Ｐ ３可能为具有免疫调节作用的活性多糖。     

牛膝多糖对T淋巴细胞和天然杀伤细胞功能的影响

牛膝多糖（ＡＢＰ）是从中药牛膝根中分离得到的一种有效成分。ＡＢＰ５０－８００ｍｇ·Ｌ－１在体外增强天然杀伤（ＮＫ）细胞活性和促进伴刀豆球蛋白Ａ（ＣｏｎＡ）诱导的肿瘤坏死因子－β（ＴＮＦ－β）产生；但不能提高ＣｏｎＡ诱导的Ｔ淋已细胞增殖反应和白介素２的产生．ＡＢＰ５０及１００ｍｇ·ｋｇ－１ｉｐ明显提高正常小鼠ＮＫ细胞活性和ＴＮＦ─β生成，增强二硝基氟苯诱导的迟发型超敏反应和对抗环磷酰胺对ＮＫ活性的抑制作用。但对ＣｏｎＡ诱导的Ｔ淋巴细胞增殖反应和白介素２的产生无明显影响。表明ＡＢＰ对Ｔ淋巴细胞功能的影响是有选择性的．ＡＢＰ对ＮＫ细胞的杀伤活性的增强作用是明显的．     

Methionine-enkephalin alteration of mitogenic and mixed lymphocyte culture responses in zinc-deficient mice

The effects of methionine-enkephalin (Met-Enk) on mitogenic and mixed lymphocyte culture (MLC) proliferation of splenocytes from Zn-deficient, restricted and control mice were evaluated. The data from this experiment show that Met-Enk can suppress the responses of splenocyte from the 3 groups to concanavalin A (Con A), but less inhibition was observed in the Zn-deficient group. Met-Enk can also enhance the responses to pokeweed mitogen (PWM) and decrease the response to lipopolysaccharide (LPS) in all groups. Alteration of proliferative responses to Con A and PWM were reversible in the presence of naloxone 10 mumol/L indicating that the effect of Met-Enk on cellular proliferation was mediated by the opioid receptor. In the proliferation of MLC, the response of lymphocytes from Zn-deficient mice was increased in the absence of Met-Enk and Met-Enk can suppress this increased response. It is therefore concluded that Met-Enk can modify the pattern of mitogenic responses and the alteration in Con A and MLC responses can be influenced by zinc deficiency.     

褐藻糖胶的免疫调节作用

观察了从海带中提取的褐藻糖胶对小鼠免疫功能的影响，褐藻糖胶在体外可诱导白细胞介素１（ＩＬ－１）和丙型干扰素（ＩＦＮ－γ）产生；体内给药可增强Ｔ细胞，Ｂ细胞，巨噬细胞（Ｍ）和自然杀伤细胞（ＮＫ细胞）功能，促进对绵羊红细胞（ＳＲＢＣ）的初次抗体应答。     

Immunological alterations induced by polyamine derivatives on murine splenocytes and human mononuclear cells

Three polyamine derivatives assigned as bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm) were studied to determine their effects on the proliferation of murine splenocytes and human peripheral blood mononuclear cells (PBMC) induced by the mitogens, Con A, LPS and PHA. All compounds showed a dose dependent inhibitory effect on mouse and human T cell proliferation induced by the mitogens, with BNIPPut exhibiting the most potent antiproliferative activity, followed by BNIPSpd and by BNIPSpm, respectively (Put > Spd > Spm), when considering human T cells. This suppressive activity also affects the capacity of mouse spleen cells to produce Th1 cytokines, namely IL-2 and INF-gamma after in vitro stimulation with Con A. The polyamine-induced inhibition also occurred in the case of LPS-stimulated B cells with a marked decrease of CD69 expression by these cells. Furthermore, the ability for these polyamine derivatives to induce apoptosis on Con A-stimulated splenocytes could be related to their antiproliferative activity.     

Stemucronatoside K, a novel C(21) steroidal glycoside from Stephanotis mucronata, inhibited the cellular and humoral immune response in mice

Stephanotis mucronata (Blanco) Merr. has been used for rheumatoid arthritis in Chinese folk herb medicine. Guided by bioactive test, a novel potent immunosuppressive C(21) steroidal glycoside stemucronatoside K (SMK) was isolated from this plant. Its structure was elucidated on the basis of the chemical evidence and extensive spectroscopic methods. We investigated the immunosuppressive effects of SMK in vitro and in vivo. SMK significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. ICR mice were immunized subcutaneously with OVA on the first day and administered intraperitoneally with SMK at the doses of 2.5, 5, and 10 mg/kg once daily for 10 days. At 24 h after the last administration, mitogen- and OVA-stimulated splenocyte proliferation, the levels of cytokines from splenocytes, and specific antibody titers in serum were measured. SMK significantly inhibited Con A-, LPS- and OVA-induced splenocyte proliferation in the immunized mice in a dose-dependent manner. OVA-specific IgG, IgG1, and IgG2b antibody titers were significantly reduced by SMK compared with the control group. SMK also significantly decreased OVA-induced interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and IL-4 production from splenocytes in the OVA-immunized mice. These results demonstrated that SMK could suppress the cellular and humoral immune response in mice. This study provided evidence to understand the therapeutic effects of S. mucronata and an immunosuppressive natural product compound to further researches to be developed as immunosuppressant.     

Immunomodulatory and cytotoxic effects of Nigella sativa and thymoquinone on rat splenocytes

Three different concentrations of Nigella sativa (N. sativa) ethanolic extract, thymoquinone (TQ), dexamethasone, and saline were examined to see whether they had any effects on cell viability, proliferation, and interleukin 4 (IL-4) and interferon-γ (IFN-γ) secretion in non-stimulated, phytohemagglutinin (PHA) and concavaline A (Con A)-stimulated splenocytes. In PHA and Con A-stimulated splenocytes, cell viability and proliferation were increased and Con A shifted cytokine profile towards Th2 balance. Dexamethasone treatment showed a suppression in viability, IFNγ and IL-4 secretion in non-stimulated and stimulated splenocytes. Extract and TQ reduced the viability and inhibited the proliferation of stimulated and non-stimulated splenocytes concentration-dependently. Higher concentrations of N. sativa (1000 mg/ml) and TQ (5 and 10 mg/ml) reduced the secretion of IL-4 in stimulated cells. Two higher concentrations of N. sativa had decreased IFNγ secretion in both stimulated and non-stimulated cells. In non-stimulated cells, only the highest and in Con A-stimulated cells, all TQ concentrations had inhibited IFNγ secretion. The highest concentration of N. sativa increased IFNγ/IL-4 ratio in both stimulated and non-stimulated cells while higher concentrations of TQ only had the same effect on stimulated cells. N. sativa and TQ showed cytotoxic inhibitory effect on rat splenocytes and on Th1/Th2 cytokines concentration-dependently. Higher concentrations of extract and TQ increased cytokines balance in Th1/Th2.     

康乐霉素C对T—和B—淋巴细胞活化的抑制作用

目的：阐明康乐霉素Ｃ对脾细胞增殖和Ｔ－细胞亚型的作用。方法：氚掺入法或噻唑蓝比色法测定细胞增殖；用荧光激活细胞分选仪测定细胞亚群；曲利苯蓝排斥法测定细胞存活率。结果：Ｋａｎ８，４０，８０和４００ｎｍｏｌ．Ｌ＾－１除抑制丝 同种异型抗原刺激的小鼠脾细胞增殖外；与Ｃｉｃ不同，抑制ＬＰＳ刺激的脾细胞增殖；使Ｌ３Ｔ４＾｜Ｌ－细胞亚型比倒置；     

An in vitro study of liposomal curcumin: stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells

Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.     

Enhancement of T lymphocyte proliferation and suppression of antibody producing cell formation by methionine-enkephalin

Methionine-enkephalin (met-enk) 0.1-100 nmol/L significantly enhanced lymphocyte proliferation induced by T cell mitogens. On the other hand, the peptide markedly inhibited splenocyte blastogenesis induced by B cell mitogen lipopolysaccharides (LPS) and sheep red blood cell (SRBC)-driven plaque-forming cell (PFC) formation in vitro. Met-enk alone had no effect on immune responses. However, naloxone 50 nmol/L had also a stimulating effect on Con A-induced splenocyte proliferation. The similar results were also observed in vivo. The results also indicated that the enhancement of T cell function by met-enk was stronger in immunosuppressed mice than in the normal mice.     

人参皂苷-Ro促进小鼠脾细胞增殖及调节小鼠脾细胞Th1/Th2细胞因子的产生

目的研究人参皂苷-Ro对小鼠脾细胞增殖及细胞因子产生的影响。方法［³H］ TdR参入法检测人参皂苷-Ro对小鼠脾淋巴细胞增殖的影响；酶联免疫吸附法检测人参皂苷-Ro对小鼠脾淋巴细胞产生细胞因子白介素-2、干扰素-γ和白介素-4的影响；逆转录聚合酶链式反应分析法研究人参皂苷-Ro对小鼠脾淋巴细胞中干扰素-γ、白介素-4 mRNA表达的影响。结果人参皂苷-Ro在1-10 μmol·L^-1显著促进Con A诱导的小鼠脾淋巴细胞增殖及小鼠脾淋巴细胞白介素-2的产生；在2-10 μmol·L^-1促进Con A诱导的小鼠脾淋巴细胞产生和表达Th2细胞因子白介素-4, 而降低Con A诱导的小鼠脾淋巴细胞产生和表达Th1细胞因子干扰素-γ。结论人参皂苷-Ro通过调节脾细胞内Th1型和Th2型细胞因子的转录和表达发挥免疫调节作用。     

A T-cell-dependent humoral immune response is preserved during the administration of the nerve agent pre-treatment pyridostigmine bromide in a murine model

Immune regulation, either via the autonomic nervous system or by a proposed "non-neuronal" cholinergic system, suggests that the immune system may be susceptible to perturbation by compounds affecting cholinergic function. Here, the current UK and US nerve agent pre-treatment, pyridostigmine bromide (PB) and the related anti-acetylcholinesterase (AChE) compounds physostigmine (PHY) and BW284c51 were tested for their ability to affect mouse splenocyte function in vitro. In addition, PB, at a dose equivalent to that received during pre-treatment for nerve agent poisoning, was tested for its effect on a T-cell-dependent humoral response to antigen in vivo in the mouse. None of the anti-AChEs tested affected concanavalin A (Con A)-, anti-CD3- or lipopolysaccharide LPS-driven splenocyte proliferation, in vitro, at concentrations expected to give effective nerve agent pre-treatment. However, higher concentrations (>100 microM) particularly of PHY caused some inhibition of the proliferative responses. In vivo, PB or saline was administered via 28-day mini-osmotic pumps to give a 25-40% inhibition of whole blood AChE in the PB-treated animals. During PB or saline administration, primary and secondary doses (i.p.) of sheep red blood cells (SRBC) were given and the humoral response determined by monitoring anti-SRBC IgM and IgG levels. Splenocytes isolated from the experimental animals were also examined for their proliferative and cytokine responses to stimulation. No remarkable effects of PB were seen during the period of AChE inhibition on the humoral immune response. However, a modest elevation in IL-2 and IFN(gamma) in Con A-stimulated lymphocytes was seen in PB-treated animals following pump removal. Overall these data suggest that, in vivo, the SRBC stimulated T-cell-dependent immune response is unaffected by the administration of PB at pre-treatment doses.     

Elucidation of cellular targets responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of antibody responses: II. The role of the T-lymphocyte

Various immunological assays have been applied by our laboratory in an attempt to assess the role of the T-cell in the TCDD-induced suppression of the antibody response by murine B6C3F1 splenic lymphocytes. Animals were treated in vivo (via gavage) with 1.0 microgram/kg TCDD in corn oil for 5 days before in vitro analysis of splenocyte immunocompetence and T-cell function. To study the effects on T-helper cell function, alterations in the proliferative responses of T-cells following TCDD exposure were investigated. Results show no significant difference in [3H]thymidine uptake between vehicle- and TCDD-treated whole splenocytes 24 h after in vitro stimulation with the T-cell mitogen Con A. This is consistent with the finding that IL-2 production at either 24 or 48 h after Con A stimulation of TCDD-treated lymphocytes was not significantly different from that of vehicle-treated controls. The possibility of the induction of a suppressor T-cell by TCDD was also investigated. Titration of T-cells from TCDD-treated mice into naive splenocyte cultures did not suppress the humoral response to either a T-dependent (SRBC) or a T-independent (DNP-Ficoll) antigen. In contrast, titration of cells stimulated in vitro with Con A for 48 h (a positive control for the induction of a suppressor T-cell) inhibited humoral responses of naive cells to both types of antigen. Likewise, T-cells plus macrophages from TCDD-treated mice did not suppress the in vitro humoral responsiveness of naive B-cells plus macrophages to a T-independent antigen (DNP-Ficoll). These results would indicate that an alteration in T-cell function following TCDD exposure does not play a role in the suppression of the antibody response elicited by antigen stimulation of murine B6C3F1 splenocytes.     

香菇多糖的免疫调节作用

检测香菇多糖（ＬＮＴ）对环磷酰胺（Ｃｙ）诱导的免疫功能低下的小鼠脾细胞溶血素抗体（ＩｇＭ）生成的影响和对ＣｏｎＡ诱导小鼠脾淋巴细胞增殖反应和ＩＬ－２生成的影响。结果表明，国产ＬＮＴ的３种剂量（０．５，１，２ｍｇ·ｋｇ－１·ｄ－１×５ｄ，ｉｐ）显著促进ＩｇＭ抗体的生成，且以１ｍｇ·ｋｇ－１·ｄ－１作用最佳。ＬＮＴ（１～１２５ｍｇ·Ｌ－１）可明显促进ＣｏｎＡ诱导的脾淋巴细胞增殖反应和ＩＬ－２的生成。量效曲线呈钟罩形。提示有浓度依赖性的双向免疫调节作用。     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress both cellular and humoral immunity. Effector T cell-derived type-2 cytokines, including IL-4 and IL-5, play pivotal roles in humoral immunity. Herein, we studied whether TCDD affects type-2 cytokine productions during the immune response. C57Bl/6 mice were intraperitoneally immunized with ovalbumin (OVA) and orally administered 5 or 20 microg TCDD/kg on Day 0, and then challenged with OVA on Day 21. Seven days later (Day 28), antigen-specific antibodies in plasma, and T cell-derived cytokines produced by splenocytes and proliferation of splenocytes upon ex vivo re-stimulation with OVA were investigated. The quantities of IgM class and IgG1 class OVA-specific antibodies in plasma were reduced by 5 or 20 microg TCDD/kg and by 20 microg TCDD/kg, respectively. While thymus weight and cellularity were reduced by 20 microg TCDD/kg, spleen weight and cellularity were not changed by either 5 or 20 microg TCDD/kg. The proportions of B and T cells in the spleen were not affected by TCDD exposure. On the other hand, splenocytes from mice treated with 5 or 20 microg TCDD/kg were shown to produce less IL-4 or IL-5 upon ex vivo re-stimulation with OVA. Production of the T cell growth factor IL-2 was also decreased in splenocytes from TCDD-treated mice. In contrast, the type-1 cytokine IFN-gamma was increased by TCDD. Twenty micrograms of TCDD/kg suppressed OVA- or T cell mitogen (Con A)-stimulated proliferation of splenocytes, but did not affect B cell mitogen (LPS)-stimulated proliferation. These results suggested compromised T cell activation and suppressed type-2 cytokine production by T cells to be involved in the impaired humoral immunity associated with TCDD exposure.     

Immunomodulatory activity of protein-bound polysaccharide extracted from chelidonium majus

In the course of searching immunomodulators from natural sources, the protein-bound polysaccharide, CM-Ala, has been isolated from the water extract of Chelidonium majus L. (Papaveraceae). The immunostimulatory characteristics have been investigated in several experiments such as generation of activated killer (AK) cells, proliferation of splenocytes, activation of macrophages and granulocyte macrophage-colony forming cell (GM-CFC) assay. Of the fractions obtained using Sephacryl S200 column chromatography, CM-Ala was the most effective fraction that augmented the cytotoxicity against Yac-1 tumor cells from 0.88% to 34.18% by culturing with splenocytes for 5 days. CM-Ala also enhanced nitric oxide production by two fold in peritoneal macrophages and exhibited antitumor activity. It showed mitogenic activity on both spleen cells and bone marrow cells. CM-Ala induced proliferation of splenocytes by 84 fold and increased GM-CFC numbers by 1.48 fold over than the non-treated. On the contrary, CM-Ala had cytotoxic activity to a diverse group of tumor cells. From the above results, we proposed that CM-Ala has a possibility of an effective antitumor immunostimulator.