Immunomodulating effect of lysed immunoactive fractions of selected Escherichia coli strains on the macrophage system. An in vitro study

The immunomodulator Uro-Vaxom (an immunoactive fraction of E. coli, FEC) is a therapeutic agent used to control bacterial infections. FEC was applied to macrophages of C57BL/6 mice to investigate the in vitro activation of these cells of the unspecific immune system. Experiments were designed to test the secretory, immuno-regulatory and cytotoxic function of macrophages after application of FEC. As presented here, production of Interleukin-6 and tumor-necrosis-factor were significantly and dose-dependent way increased, whereas it was not possible to induce a secretion of Interleukin-1. In addition, FEC activated macrophages kill Leishmania donovani promastigotes, Candida albicans and Staphylococcus aureus. In comparison to a pressed echinaceae-preparation, FEC activated mouse macrophages secrete Interleukin-6 and tumor-necrosis-factor and kill protozoa, fungi and bacteria, with higher efficiency.     

Effect of acetylsalicylic acid, ascorbate and ibuprofen on the macrophage system.

The influence of ascorbic acid (CAS 50-81-7), acetylsalicylic acid (CAS 50-78-2) and ibuprofen (CAS 15687-27-1) on macrophages of C57BL/6 mice was investigated in vitro. It has been shown that ascorbic acid or acetylsalicylic acid alone did not stimulate or inhibit the production of interleukin-6, whereas a combination of both substances caused a significant stimulation. The viral replication in L929 fibroblasts was not affected by ascorbate and/or acetylsalicylic acid. In addition, the tumor-necrosis factor (TNF) synthesis of peritoneal macrophages was neither stimulated nor inhibited by both substances, alone or in combination. The oxygen radical production, however, was definitely inhibited by ascorbic acid, the effect of acetylsalicylic acid was far less marked, but at the high concentrations the inhibition was clearly discernible. Ibuprofen, a propionic acid derivate, was able to reduce the replication of vesicular stomatitis virus in L929 fibroblast cells. At the highest concentration of ibuprofen, 100 micrograms/ml, 34% of the fibroblast were able to survive. This protective effect declined as the ibuprofen concentration decreased. Ibuprofen could not stimulate peritoneal macrophages to secrete TNF, whereas the oxygen radical production was significantly reduced. In addition, ibuprofen activated mouse macrophages to produce interleukin-6 in a dose dependent way. The results of the in vitro experiments presented clearly show that ascorbic acid, acetylsalicylic acid in ibuprofen influenced the unspecific immune system.     

Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions

3, 5,7-trihydroxy-4'-methoxy-8-(3-hydroxy-3-methylbutyl)-flavone (ICT) is a novel derivative of Icariin (ICA), the major active ingredient of Herba Epimedii, a herb used in traditional Chinese and alternative medicine. We previously demonstrated its anti-inflammatory effect in murine innate immune cells and activated human PBMCs. We report herein that ICA or ICT treatment reduces the expression of MRP8/MRP14 and toll-like receptor 4 (TLR4) on human PBMCs. Administration of ICA or ICT inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC numbers in the spleen of these mice. Further, we saw a restoration of IFN-γ production by CD8+ T cells in tumor bearing mice when treated with ICA or ICT. ICA and ICT significantly decreased the amounts of nitric oxide and reactive oxygen species in MDSC in vivo. When MDSC were treated in vitro with ICT, we saw a significant reduction in the percent of these cells with concomitant differentiation into dendritic cells and macrophages. Concomitant with this cell type conversion was a down-regulation of IL-10, IL-6 and TNF-α production. Decreased expression of S100A8/9 and inhibition of activation of STAT3 and AKT may in part be responsible for the observed results. In conclusion, our results showed that ICA, and more robustly, ICT, directly modulate MDSC signaling and therefore altered the phenotype and function of these cells, in vitro and in vivo.     

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-alpha) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-alpha and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-alpha and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-kappaB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-kappaB activation.     

Stimulation of various functions in murine peritoneal macrophages by high mannuronic acid-containing alginate (HMA) exposure in vivo

High mannuronic acid-containing alginate (HMA) was tested to affect murine peritoneal macrophages. In the present study, we measured various functions of murine peritoneal macrophages that were isolated 20 h after intraperitoneal injection with HMA (25 and 100 mg/kg). HMA increased the number of peritoneal macrophages and phagocytosis. Macrophages from HMA-treated mice significantly inhibited growth of tumor cells compared to macrophages from control mice. In addition, supernatants from macrophages of HMA-treated mice contained nitric oxide (NO), hydrogen peroxide (H2O2) and TNF-alpha. The increased production of these cytotoxic molecules induced by HMA is consistent with tumoricidal activity of activated macrophages. Furthermore, HMA-induced tumoricidal activity was partially abrogated by anti-TNF-alpha, inhibitors of NO and the scavenger of reactive oxygen. Thus, the tumoricidal activity induced by HMA appeared to be mediated by the production of TNF-alpha, NO and H2O2. Taken together, these results suggest that HMA has the immunostimulating effect on macrophages after in vivo exposure of it.     

Bisucaberin, a new siderophore, sensitizing tumor cells to macrophage-mediated cytolysis. I. Taxonomy of the producing organism, isolation and biological properties

Alteromonas haloplanktis strain SB-1123 isolated from deep-sea mud produced a new siderophore, bisucaberin. Bisucaberin rendered tumor cells susceptible to cytolysis mediated by murine peritoneal macrophages which were elicited by Proteose peptone and not yet activated by lymphokine. Bisucaberin exerted its sensitizing activity by both the preincubation with tumor cells and the addition to co-culture of macrophages and tumor cells. The activity of bisucaberin was specifically inhibited by ferric ion. Bisucaberin showed direct cytostasis for tumor cells but did not cause cytolysis in the absence of macrophages. Cytostasis by bisucaberin was attributable to the specific inhibition of DNA synthesis in tumor cells.     

Macrophage involvement in the antitumoral effect of Nocardia-delipidated cell mitogen (NDCM).

Several immunomodulatory fractions derived from Nocardia have been found to inhibit the growth of several experimental tumors, including Lewis lung carcinoma (3LL). An involvement of both macrophages and lymphocytes in the antitumoral effect of Nocardia fractions has been suggested. The mechanism of the Nocardia delipidated Cell Mitogen (NDCM)-induced tumor-inhibiting effect was investigated further in the present study. Macrophages activated by NDCM exerted a cytotoxic effect on 3LL cells in vitro, indicating a direct influence of macrophages on the tumor cells. These results correlate with previous findings which showed a local accumulation of macrophages (but also lymphocytes) at the tumor site in NDCM-treated mice. In tumor-bearing mice--both treated and non-treated with NDCM--a splenomegaly due to a pronounced extramedullary hematopoiesis was seen. Concomitant with the gradual evolution of the extramedullary hematopoiesis in the red pulp, a depletion in white pulp component was observed, more pronounced in the control 3LL-bearing mice than in the 3LL-inoculated NDCM-treated animals. The disappearance of the lymphatic follicles in 3LL-bearing mice may be responsible for the failure to cope with the tumor. It is therefore possible that by delaying white pulp depletion, NDCM favors a better host defense against the tumor. Examination of lungs in 3LL-bearing mice treated by NDCM showed a rich infiltration of macrophages in the vicinity of isolated tumor cells, probably indicating a defensive role of NDCM-activated macrophages against metastatic spread of the tumor. Although the macrophage appears to be of major importance in the NDCM-induced host response against the tumor, other components of the immune system are probably system are probably also activated by the Nocardia fraction in defense against the neoplasm.     

Protection of human keratinocytes from UVB-induced inflammation using root extract of Lithospermum erythrorhizon

UVB irradiation is an important inducer of biological changes in skin and can activate inflammatory reactions and apoptotic pathways, leading to skin damage. A root extract of Lithospermum erythrorhizon (SK), which has naphthoquinone pigments containing shikonin and shikonin derivatives, is known for its anti-inflammatory, anti-bacterial, and anti-tumor activity, and for its scavenging of reactive oxygen species. However, the effect of SK against UV damage is not clear. The aim of this study was to evaluate the efficacy of SK against UVB induced damage in normal human epidermal keratinocytes (NHEK). UVB-irradiated NHEK showed decreased cell viability, increased production of interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor-alpha, and induced apoptosis. In an apoptosis pathway assay, UVB-irradiated NHEK showed increased caspase-3 activity, p53 and its phosphorylation at serine 15 compared with non-irradiated cells. All these effects induced by UVB irradiation were clearly inhibited by treatment with SK before and after UVB irradiation for 24 h. It is suggested that SK can protect epidermal cells against harmful effects of UVB irradiation and that SK treatment is probably beneficial for photoprotection of the skin.     

Combined anticancer effects of sphingosine kinase inhibitors and sorafenib

The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.     

血小板激活因子拮抗剂WEB2086对小鼠腹腔巨噬细胞释放肿瘤坏死因子的影响(英文)

小鼠ip3%TG 4天后取其腹腔巨噬细胞,血小板激活因子拮抗剂WEB 2086对LPS诱导的巨噬细胞释放肿瘤坏死因子(TNF)有显著的抑制作用。时效研究表明,TNF的产生和WEB 2086的抑制作用从LPS刺激后4 h开始,持续到22 h,在16 h时达到高峰。本文还首次采用一种用放线菌素D和NaF处理的L-929细胞测定TNF的新方法,此法与另外3种生物测定方法进行比较的结果显示此法具有更敏感、方便的特点。     

血小板激活因子拮抗剂WEB2086对小鼠腹腔巨噬细胞释放肿瘤坏死因子的影响(英文)

小鼠ip3%TG 4天后取其腹腔巨噬细胞,血小板激活因子拮抗剂WEB 2086对LPS诱导的巨噬细胞释放肿瘤坏死因子(TNF)有显著的抑制作用。时效研究表明,TNF的产生和WEB 2086的抑制作用从LPS刺激后4 h开始,持续到22 h,在16 h时达到高峰。本文还首次采用一种用放线菌素D和NaF处理的L-929细胞测定TNF的新方法,此法与另外3种生物测定方法进行比较的结果显示此法具有更敏感、方便的特点。     

Inhibition of tumor growth by endohedral metallofullerenol nanoparticles optimized as reactive oxygen species scavenger

Intraperitoneal injection of [Gd@C82(OH)22]n nanoparticles decreased activities of enzymes associated with the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice. Several physiologically relevant ROS were directly scavenged by nanoparticles, and lipid peroxidation was inhibited in this study. [Gd@C82(OH)22]n nanoparticles significantly reduced the electron spin resonance (ESR) signal of the stable 2,2-diphenyl-1-picryhydrazyl radical measured by ESR spectroscopy. Like-wise, studies using ESR with spin-trapping demonstrated efficient scavenging of superoxide radical anion, hydroxyl radical, and singlet oxygen (1O2) by [Gd@C82(OH)22]n nanoparticles. In vitro studies using liposomes prepared from bovine liver phosphatidylcholine revealed that nanoparticles also had a strong inhibitory effect on lipid peroxidation. Consistent with their ability to scavenge ROS and inhibit lipid peroxidation, we determined that [Gd@C82(OH)22]n nanoparticles also protected cells subjected in vitro to oxidative stress. Studies using human lung adenocarcinoma cells or rat brain capillary endothelial cells demonstrated that [Gd@C82(OH)22]n nanoparticles reduced H2O2-induced ROS formation and mitochondrial damage. [Gd@C82(OH)22]n nanoparticles efficiently inhibited the growth of malignant tumors in vivo. In summary, the results obtained in this study reveal antitumor activities of [Gd@C82(OH)22]n nanoparticles in vitro and in vivo. Because ROS are known to be implicated in the etiology of a wide range of human diseases, including cancer, the present findings demonstrate that the potent inhibition of [Gd@C82(OH)22]n nanoparticles on tumor growth likely relates with typical capacity of scavenging reactive oxygen species.     

A synthesized cationic tetradecapeptide from hornet venom kills bacteria and neutralizes lipopolysaccharide in vivo and in vitro

Sepsis is a complex clinical syndrome that results from a harmful host response to infection, in which foreign bacteria and lipopolysaccharide (LPS) are potent activators of different immune cells, including monocytes and macrophages. To date, there are currently few effective adjuvant therapies in clinical use except activated protein C focusing on the coagulation system. Mastoparans (MPs) are wasp venom cationic amphiphilic tetradecapeptides; these are capable of modulating various cellular activities, including stimulation of GTP-binding protein, phospholipase C and can bind to a phospholipid bilayer. Masroparan-1 (MP-1, INLKAIAALAKKLL-NH2), a tetradecapeptide toxin isolated from hornet venom, was synthesized chemically. In this study, Escherichia coli 25922 (E. coli 25922) and LPS were used to induce sepsis in an animal model. We found that MP-1 treatment at 3 mg/kg protected mice from otherwise lethal bacteria and LPS challenges. MP-1 has antibacterial capabilities against Gram-negative and Gram-positive bacteria. Its antibacterial action against E. coli may result from the destruction of bacterial membrane structures. In addition, treatment of murine peritoneal macrophages with MP-1 potently inhibited the respiratory burst. This effect maybe related to an inhibition of NADPH oxidase in the membrane. Furthermore, MP-1, bound with high-affinity to LPS and lipid A with dissociation equilibrium constants of 484 and 456 nM, respectively, and neutralized LPS in a dose-dependent manner. MP-1 also significantly reduced the expression of TLR4, TNF-alpha and IL-6 mRNA and the release of cytokines in LPS-stimulated murine peritoneal macrophages. Our results shows that the MP-1-mediated protection of mice from lethal challenge by live bacteria and LPS was associated with its bactericidal action and inhibition of inflammatory responses by macrophages to both bacteria and LPS (the release of cytokines and reactive oxygen species).     

In vivo destruction of canine lymphoma mediated by murine monoclonal antibodies

The effect of murine anti-canine lymphoma monoclonal antibodies (MAbs) on tumor cell lysis by thioglycolate activated murine macrophages in vitro and tumor growth inhibition in athymic mice was studied. All IgG1 and IgG2a MAbs tested were able to promote specific destruction of canine lymphoma 17-71 cell line by activated macrophages. A correlation between higher ADCC activity and MAb isotype was not clearly evident. In vivo IgG2a and IgG1 MAbs inhibited the growth of canine lymphoma. These results suggest that MAbs of IgG type have potential in immunotherapy of dogs with lymphoma since they have high tumoricidal activity in vivo.     

Antitumor activity ofBifidobacterium spp. isolated from a healthy Korean

The antitumor activity of Bifidobacterium breve K-110, and K-111, and B. infantis K-525 was investigated. These Bifidobacterial cells and their cell wall preparations (WPG) significantly increased the survival rate of mice who had been intraperitoneally implanted with sarcoma 180 cells. Solid tumor growth was inhibited even when the sarcoma 180 cells were implanted into the groins of the mice. However, the Bifidobacterial cells did not show in vitro cytotoxicity against tumor cell lines. Cell kinetic studies revealed that these WPGs induced neutrophils, which were followed by macrophages, at the site of peritoneal injection. The WPGs directly activated these cells to inhibit the growth of tumor cells in in vitro assays. Our results suggest that Bifidobacterial WPGs induce and activate nonspecific phagocytes in situ to reject growing tumor cells in the mouse peritoneal cavity.     

Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-receptors

Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at l0(-5) M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate (PMA) stimulated macrophages and histamine at a concentration range of 10(-7) to 10(-5) M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFalpha and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-histamine receptors. reserved.     

Delivery method, target gene structure, and growth properties of target cells impact mutagenic responses to reactive nitrogen and oxygen species

Dysregulated production of nitric oxide (NO?) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO? and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO?, a cumulative dose of 620 μM min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 × 10?? (p < 0.05) and 24.8 × 10?? (p < 0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 μM min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 × 10?? and 190 × 10??, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO?. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MF(exp)/MF(control))/cumulative NO? dose] were 5.16 × 10?3 and 4.97 × 10?3 μM?1 min?1 for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO? to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO? or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30-40%) outnumbering transitions (10-20%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10-20% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0-10%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO? and reactive species produced by activated macrophages.     

IL-6在巨噬细胞向M2型极化过程中的诱导作用

目的 将小鼠巨噬细胞系RAW264.7细胞和骨髓瘤细胞系KM3细胞共培养,探讨IL-6在巨噬细胞向M2型极化的过程的作用.方法 取对数生长期细胞,分为3组:A组(KM3细胞组),B组(RAW264.7细胞组),C组(RAW264.7细胞+KM3细胞组).分别予以ACTA(IL-6特异性抑制剂激活素A)或rIL-6(重组人IL-6)处理后,检测肿瘤相关M2型巨噬细胞表达标志F4/80+ CD206+的比例.RT-PCR和Westernblot方法检测各组细胞因子CCL22、IL-10、IL-12、TNF-αmRNA和蛋白表达量.ELISA法检测各组细胞培养上清中IL-6的含量.结果 RAW264.7和KM3细胞共培养24 h后,与对照组相比,M2 型巨噬细胞比例显著增加(P<0.05);予以ACTA处理后M2型巨噬细胞表达明显下降(P<0.05);而予以rIL-6处理后M2型巨噬细胞表达明显上调(P<0.05).与B组(RAW264.7细胞组)相比,C组(RAW264.7细胞+KM3细胞)M2型巨噬细胞相关细胞因子CCL22,IL-10的mRNA和蛋白表达水平明显上调(P<0.05),而M1型巨噬细胞相关因子IL-12,TNF-α的mRNA和蛋白表达水平明显下调(P<0.05).与A组、B组相比,C组细胞上清液中IL-6含量在24 h、48 h、72 h时间点均明显上调(P<0.05).通过浓度梯度改变RAW264.7细胞或KM3细胞的数量,发现RAW264.7细胞数量的改变显著影响了IL-6的表达水平.结论 将RAW264.7细胞和KM3细胞共培养后,后者能诱导RAW264.7细胞向肿瘤相关M2型巨噬细胞转化, IL-6在上述转化过程中发挥重要作用.