C3 binding glycoprotein from Cuscuta europea induced different cytokine profiles from human PBMC compared to other plant and bacterial immunomodulators

The immunomodulatory properties of bioactive agents include the ability to induce cytokine production by the activated target cell. The effect of immunomodulatory C3 binding glycoprotein isolated from Cuscuta europea on the induction of human PBMC cytokine synthesis and the cell viability was investigated. Isolated PBMC from healthy donors were cultured for 24 h with C3bgp. We also studied the influence of C3bgp on the cytokine production in LPS, PHA, PWM and Dex treated PBMC. The quantitative determination of TNF-alpha, IL-12, IL-6 and IL-10 was performed in culture supernatant by ELISA. Results obtained demonstrated that C3bgp induced proinflammatory and immunoregulatory cytokine production, in the highest degree IL-12, followed by IL-6 and in lower degree TNF-alpha. IL-12 quantity was significantly increased in C3bgp stimulated cultures in comparison with LPS, PHA and PWM stimulated PBMC. C3bgp also increased IL-12 in PHA or PWM stimulated cultures, but not in LPS stimulated culture. C3bgp significantly increased IL-6 production compared to the PHA and PWM but not to LPS stimulation. On the other side, C3bgp inhibited IL-10 production after LPS, PHA and PWM stimulation. Cell viability in C3bgp stimulated cultures retained on the same level from 72 to 120 h of culturing, in contrast to LPS and PHA stimulated cultures. Based on the results presented, we conclude that the C3bgp exhibited immunomodulatory properties on the human PBMC. The ability of PDTC and Dex to down-regulate the effect of C3bgp on the proinflammatory cytokine production suggests that a part of the mechanism of action of C3bgp is mediated through NF-kB signal transduction pathway.     

重组人胸腺素α原体外对IFN-γ,IFN-α及TNF-α的影响

目的体外观察重组人胸腺素α原(prothymosin α,Pro Tα)对几种重要细胞因子分泌的影响。方法用脾淋巴细胞、脾巨噬细胞及腹腔巨噬细胞,以ELISA法检测Pro Tα对IFN-γ,IFN-α和TNF-α分泌的影响。结果1×10^-7 mol·L^-1 Pro Tα明显促进脾细胞分泌IFN-γ(P<0.05),该浓度的Pro Tα也明显刺激小鼠脾巨噬细胞分泌IFN-α(P<0.01);在小鼠腹腔巨噬细胞中,Pro Tα能明显刺激IFN-α和TNF-α的分泌(P<0.01)。结论Pro Tα对细胞因子IFN-γ,IFN-α和TNF-α的分泌均有促进作用。     

The immune-enhancing effect of the herbal combination Bouum-Myunyuk-Dan

The herbal formulation Bouum-Myunyuk-Dan (BMD) has long been used for various diseases. It has been shown to have antimicrobial and anti viral activity clinically. However, it is still unclear how BMD exerts these effects in experimental models. In this study, we investigated the effect of BMD on the production of cytokines in a human T cell line, MOLT-4 cells, and in mouse peritoneal macrophages. As a result, BMD significantly increased the viability and proliferation of splenocytes (p<0.05) and also significantly increased interleukin (IL)-2 and IL-4 production compared with media control (about 2.7-fold for IL-2 and 6.7-fold for IL-4, p<0.05) after 24 h. BMD increased the interferon (IFN)-gamma production by 3.7-fold but there were no significant differences compared with controls. Maximal effective concentrations of BMD were 1 mg/ml for IL-2 and IL-4 and 0.1 mg/ml for IFN-gamma. In addition, BMD (0.01 mg/ml) increased the production of tumor necrosis factor (TNF)-alpha and IL-12 in mouse peritoneal macrophages (by 2.7-fold for TNF-alpha and 42.5-fold for IL-12, p<0.05). In conclusion, these data indicate that BMD may have an immune-enhancing effect through the production of various cytokines.     

Multiplexed cytokine detection in microliter microdialysis samples obtained from activated cultured macrophages

Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.     

The immunomodulatory effect(s) of lead and cadmium on the cells of immune system in vitro.

A number of studies documented that the heavy metals are not only toxic for the organisms but they may modulate immune responses. The immunomodulatory activity was proved in several in vivo and in vitro model systems. In the current study, immunomodulatory activities of lead and cadmium are presented. The viability of both lymphocytes and macrophages was affected by heavy metals in a dose- and time-dependent manner. In the case of lead, the depression of N-oxide production closely correlated with increased blast transformation of spleen cells induced by concanavalin A (ConA). On the contrary, cadmium suppressed the production of N-oxides but stimulated significantly the proliferation of spleen cells. The production of cytokines by lymphocytes and macrophages was dependent on the in vitro model used. Generally, the treatment of macrophages with lead results in disregulation of the production of proinflammatory cytokines [tumour necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha) and interleukin 6 (IL-6)] and preferential production of Th1 type of cytokines (IFN-gamma and IL-2). Cadmium seemed to trigger the Th2 cytokine regulatory pathway [interleukin 4 (IL-4), interleukin 10 (IL-10)]. The results suggest the metal-induced changes in immunoregulatory mechanism of host with potentially severe clinical consequences.     

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1)beta, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-alpha mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1beta and IL-12 mRNA were highly induced at 24 h. IL-1beta and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 microg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-alpha activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 microg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-gamma. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 microg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.     

Interleukin-6 production by peritoneal mesothelial cells and its regulation by inflammatory factors in rats administered carbon tetrachloride intraperitoneally

We previously reported that a high level of interleukin-6 (IL-6), which is protective against CCl(4)-induced hepatotoxicity, is produced in the peritoneal cavity in the early period after ip carbon tetrachloride (CCl(4)) administration. The objective of this study was to identify the tissues and cells involved in IL-6 production and clarify the mechanisms underlying its regulation. IL-6 mRNA levels increased significantly in the serous membranes of the mesentery and peritoneum, but not in the parenchymal organs including liver, kidney and spleen, 3 h after ip CCl(4) administration. Peritoneal mesothelial cells (PMCs), a major cell population in serous membranes, were isolated from rat peritoneal walls by trypsin digestion and cultured with peritoneal exudate fluid (PEF) from CCl(4)-administered rats. PMCs produced a high level of IL-6 in the presence of PEF recovered 0.5 h after ip CCl(4) administration. Analyses of PEF revealed that the levels of prostaglandin E(2) (PGE(2)), histamine, IL-1alpha, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased immediately after ip CCl(4) administration. These inflammatory factors, except for histamine, stimulated IL-6 production to varying degrees, in the following order: IL-1alpha>IL-1beta>TNF-alpha>PGE(2). In summary, the present study indicates that the high level of IL-6 observed in the rat peritoneal cavity after ip CCl(4) administration is at least partially produced by PMCs stimulated cooperatively with IL-1alpha, IL-1beta, TNF-alpha and PGE(2). These inflammatory factors may be released from tissues or cells either stimulated or injured directly by CCl(4).     

Modulation of IFN-gamma production by TNF-alpha in macrophages from the tumor environment: significance as an angiogenic switch

BACKGROUND: The role of macrophages in tumor angiogenesis has been known to influence in the production of angiogenic cytokines and growth factors including TNF-alpha. Recently, macrophages were also found to produce INF-gamma, which were found to be involved in angiogenic inhibition. Thus, the importance of macrophages in tumor angiogenesis might be the angiogenic switch. The hypothesis tested here is that TNF-alpha can modulate the INF-gamma production in macrophages in tumor environment as part of the tumor angiogenic switch. METHODS: Macrophages in tumor environment were obtained from peritoneal cavity and s.c. grown tumor of C57BL/6 mice injected with B16F10 melanoma cell line for 6 and 11 days, respectively. Mac1+-macrophages were purified using magnetic beads (MACs; Milteny Biotech, Germany) and cultured with various concentrations of TNF-alpha at various time points at 37 degrees C. The supernatants were analyzed for IFN-gamma or VEGF by ELISA kit. RESULTS: Residential macrophages from peritoneal cavity did not respond to LPS or TNF-alpha to produce INF-gamma. However, the cells from tumor environment produced IFN-gamma as well as VEGF. Upregulation of IFN-gamma production by the addition of LPS or TNF-alpha was observed in macrophages from the tumor bearing peritoneal cavity. RT-PCR analysis revealed external TNF-alpha-induced IFN-gamma gene expression in macrophages from tumor environment. CONCLUSION: The overall data suggest that the macrophages in tumor environment might play an important role not only in angiogenic signal but also in anti-angiogenic signal by producing related cytokines. Moreover, TNF-alpha might be a key cytokine functioning as a tumor angiogenic switch.     

雷公藤氯内酯醇对小鼠肺泡巨噬细胞体外炎性反应的影响(英文)

目的:探讨雷公藤单体雷公藤氯内酯醇(tripcholorolide,T4)对肺泡巨噬细胞(AM)炎症反应的影响及机制.方法:小鼠AM受脂多糖(LPS)10mg/L刺激的同时,加入T4 500 μg/L或地塞米松 100μmol/L;ELISA法测定上清液中TNFα、IL-1β、IL-6及IL-10浓度:RT-PCR检测上述因子及iNOS基因mRNA的表达.结果:AM受10 mg/L LPS刺激24小时后,上清液中TNFα、IL-1β、IL-6、IL-10及NO释放均明显增加.T4 500 μg/L及地塞米松100μmol/L对上述介质均有不同程度的抑制作用.LPS刺激5小时后,AM中TNFα、IL-6、IL-10和iNOS的mRNA表达均明显增加.T4和地塞米松对上述介质的mRNA表达均有明显抑制作用.另外,T4对TNFα、IL-6、IL-10 mRNA的稳定性无明显影响.结论:T4具有抑制AM中促炎介质和抗炎介质表达的作用.     

Ursodeoxycholic acid protects concanavalin A-induced mouse liver injury through inhibition of intrahepatic tumor necrosis factor-alpha and macrophage inflammatory protein-2 production

Ursodeoxycholic acid (UDCA) is widely used for the therapy of liver dysfunction. In this study, we investigated the protective effect of UDCA in concanavalin A-induced mouse liver injury. The treatment with UDCA at oral doses of 50 and 150 mg/kg at 2 h before concanavalin A injection significantly reduced the elevated plasma levels of aminotransferases and the incidence of liver necrosis compared with concanavalin A-injected control group without affecting the concentrations of liver hydrophobic bile acids. UDCA significantly inhibited elevated levels of tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), and interleukin 6 (IL-6) in blood of concanavalin A-injected mice. To clarify the influence of UDCA on production of cytokines, we examined intrahepatic mRNA expressions and the protein levels of TNF-alpha, MIP-2, interferon-gamma (IFN-gamma), IL-4, IL-6, and IL-10 at 1 h after concanavalin A injection. The treatment with UDCA significantly decreased the intrahepatic levels of TNF- alpha and MIP-2, whereas this compound showed no clear effect on IFN-gamma, IL-4, IL-6, or IL-10. Furthermore, UDCA significantly decreased myeloperoxidase activity as well as MIP-2 level in the liver and histological examination of liver tissue revealed that intrasinusoidal accumulation of neutrophils was decreased markedly by UDCA. In addition, UDCA significantly inhibited the production of TNF-alpha and MIP-2 when cultured with nonparenchymal and lymph node cells. In conclusion, these findings suggest that UDCA protects concanavalin A-induced liver injury in mice by inhibiting intrahepatic productions of TNF-alpha and MIP-2, and the infiltration of neutrophils into the liver.     

紫草素通过TLR4/NF-κB信号通路抑制由脂多糖诱导的巨噬细胞炎症研究

目的 研究紫草素抑制由脂多糖(LPS)诱导的巨噬细胞炎症的机制.方法 以淀粉培养基注射BALB/c小鼠腹腔,诱导并分离巨噬细胞,以APC标记F4/80染色,并用流式细胞仪检测分离巨噬细胞情况;用1 mg·L-1的LPS刺激上述分离的巨噬细胞,分组如下:DMSO溶剂对照组,LPS对照组,LPS+低剂量紫草素组(0.1 μmol·L-1);LPS+中剂量紫草素组(1 μmol·L-1);LPS+高剂量紫草素组(10 μmol·L-1);通过ELISA和实时定量PCR(qRT-PCR)方法分别测定各处理组培养上清液以及细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)的表达情况;Western blot分别测定Toll样受体4(TLR4)和核因子-κB(NF-κB)的p65亚基磷酸化表达情况.结果 流式细胞检测可知,APC标记的F4/80染色巨噬细胞的纯度可达97.8%;ELISA和实时定量PCR结果显示,紫草素处理后可以抑制由LPS刺激细胞因子TNF-α、IL-1β和IL-6的表达(P<0.05),并增加IL-10的表达(P<0.05),且呈现出一定的浓度依赖性;Western blot结果显示,紫草素能下调由LPS刺激的TLR4的表达水平和NF-κB的p65亚基磷酸化表达.结论 紫草素的抗炎机制可能是通过TLR4介导的信号通路活化NF-κB,抑制IL-1β、IL-6和TNF-α的分泌,并促进IL-10的分泌.     

Stable prostaglandin I1 analog SM-10906 modulates productions of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 in mouse macrophages

The present study investigated the effects of stable agonist for prostaglandin (PG) I2 receptor with PGI1 skeleton, SM-10906, on pro-inflammatory cytokine production by mouse peritoneal macrophages (PEMs) in comparison with PGE1 and PGI2. In mouse PEMs, SM-10906 and PGE1 slightly enhanced interleukin (IL)-6 secretion, but had no effects on tumor necrosis factor-alpha (TNF-alpha) or IL-1 production. SM-10906 concentration-dependently inhibited TNF-alpha, IL-1 and IL-6 releases from lipopolysaccharide-activated mouse PEMs, as with PGE1, PGI2 and cAMP analog. Additionally, SM-10906, PGE1 and PGI2 caused concentration-dependent accumulation of cAMP contents in mouse PEMs. It is concluded that PGI1 analog SM-10906 exerts anti-inflammatory effects on stimulated mouse PEMs by increasing in cAMP levels, as with E-series of PG.     

Concanavalin A induced expression of Toll-like receptors in murine peritoneal macrophages in vitro

The differential expression of Toll-like receptors (TLRs 1-9) and their associated proteins in murine peritoneal macrophages in vitro, on treatment with plant lectin concanavalin A (Con A) has been investigated. It is observed that there is enhanced expression of TLRs 2-9 and downstream molecules--MyD88, IRAK1, TRAF6 and IRF3 in murine peritoneal macrophages on in vitro treatment with Con A. Pretreatment of macrophages with inhibitors of JNK, p38, p42/44 and NF-kappaB, significantly decreased the Con A induced expression of TLRs. When cells are pre-treated with Con A and subsequently treated with TLR ligands--Zymosan A, PolyI:C, LPS, CpG DNA, there is enhanced production of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-12 and IFN-gamma,), nitric oxide and iNOS expression in murine peritoneal macrophages. The results suggest that treatment of macrophages with Con A renders them more susceptible to subsequent activation and induction of proinflammatory cytokines and nitric oxide production by different TLR ligands.     

Alleviation of experimental septic shock in mice by acidic polysaccharide isolated from the medicinal mushroom Phellinus linteus

This study reports that acidic polysaccharide (PL) isolated from Phellinus linteus alleviated the septic shock induced by high dose lipopolysaccharide (LPS) injection in mice. To examine the origin of this effect, we investigated cytokine production in serum and the expression of MHC II in B cells and macrophages in areas of inflammation. Pretreatment with PL 24 h before LPS administration resulted in a significant inhibition of up to 68% of circulating tumor necrosis factor (TNF)-alpha, a moderate reduction of 45% of interleukine (IL)-12 and 23% of IL-1beta, but no significant reduction in IL-6. In addition, the expression of MHC II in B cells and macrophages was examined. Our results show that LPS-stimulated cytokine release and the level of MHC II can be modulated by in vivo administration of soluble PL in mice. The decrease of IL-1beta, IL-12 and TNF-alpha in sera and the down-modulation of MHC II during septic shock may contribute to the long survival of mice by PL. Administration of PL in vivo decreases IL-2, IFN-gamma and TNF-alpha production in splencotyes and enhances spontaneous cell apoptosis in macrophages and lymphocytes stimulated with LPS in vitro. Thus, part of the anti-inflammatory effects of PL treatment in vivo may result from the enhanced apoptosis of a portion of the activated macrophages and lymphocytes. The ability of PL to significantly reduce the TNF-alpha production indicates the potential of the polysaccharides in possible therapeutic strategies that are based on down-regulation of TNF-alpha.     

Immunomodulating activity of CVT-E002, a proprietary extract from North American ginseng (Panax quinquefolium).

The activity of CVT-E002, an aqueous extract containing mainly oligosaccharides and polysaccharides from North American ginseng (Panax quinquefolium), as an immunobooster on murine spleen cells and peritoneal macrophages, was studied in-vitro. CVT-E002 stimulated the proliferation of normal mouse spleen cells, of which the major responding subpopulation was identified as B lymphocytes. CVT-E002 also activated peritoneal exudate macrophages leading to enhanced interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production. In addition, CVT-E002 stimulated in-vivo immunoglobulin G (IgG) production in treated mice. These results identify some of the immunomodulating activities of CVT-E002 and suggest its use clinically for the modulation of immune responses.     

The negative immunoregulatory effects of fluoxetine in relation to the cAMP-dependent PKA pathway

Recently, we have shown that various types of antidepressants, including selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, have negative immunoregulatory effects. These antidepressants suppress the interferon-gamma (IFN-gamma)/interleukin-10 (IL-10) production ratio, which is of critical importance for the determination of the capacity of immunocytes to inhibit or activate monocytic/lymphocytic functions. Since cyclic adenosine monophosphate (cAMP) production is stimulated by some antidepressants, and since cAMP inhibits IFN-gamma and stimulates IL-10 production, we postulate that the negative immunoregulatory effects of antidepressants result from their effects on the cAMP-dependent protein kinase A (PKA) pathway. The aim of the present study was to determine whether the negative immunoregulatory effects of fluoxetine may be blocked by antagonists of the cAMP-dependent PKA pathway, such as, e.g., SQ 22536, an adenylate cyclase inhibitor, and Rp-8-Br-cAMPs (Rp-isomer of 8-bromo-adenosine-3',5'-monophosphorothioate), a PKA antagonist. To this end, diluted whole blood collected from 17 normal volunteers was incubated with fluoxetine (10(-6) and 10(-5) M), with or without SQ 22536 (10(-6) and 10(-4) M) and Rp-8-Br-cAMPs (10(-6) and 10(-4) M), afterwards, IFN-gamma, IL-10 and the tumor necrosis factor alpha (TNF-alpha) were determined. Fluoxetine, 10(-6) and 10(-5) M, significantly reduced the production of IFN-gamma and TNF-alpha, and significantly decreased the IFN-gamma/IL-10 production ratio. SQ 22536 and Rp-8-Br-cAMPs were unable to block the suppressant effects of fluoxetine on the IFN-gamma/IL-10 ratio. Rp-8-Br-cAMPs, 10(-4), but not 10(-6) M, normalized the fluoxetine-induced suppression of TNF-alpha production. It is concluded that the suppressant effect of fluoxetine on the IFN-gamma/IL-10 production ratio is probably not related to the induction of the cAMP-dependent PKA pathway, whereas the suppressant effect on TNF-alpha may be related to the induction of PKA. The obtained results suggest that increased activation of the PKA-dependent pathway may constitute an important molecular basis for some (suppression of TNF-alpha production), but not all (suppression of IFN-gamma production), negative immunoregulatory effects of fluoxetine.