CpG ODN enhances uptake of bacteria by mouse macrophages

Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages. In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages. Flow cytometric analysis of mouse macrophages (RAW 264.7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei, Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages. The enhancement of bacterial uptake by CpG ODN was concentration-dependent. The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay. There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B. pseudomallei. These observations may provide significant insights for future investigation into host cell-pathogen interaction.     

CpG ODN对rHBsAg免疫小鼠脾细胞周期与凋亡的影响

目的：研究合成含CpG基序的寡脱氧核苷酸（CpGODN）对重组乙型肝炎表面抗原（rHBsAg）免疫小鼠脾淋巴细胞细胞周期及细胞凋亡的效应。方法：采用BALB／c小鼠作为免疫实验动物，经后腿胫骨前肌免疫两次，FACS法检测脾细胞的细胞周期与细胞凋亡。结果：FACS检测结果显示，加CpGODN各组与不加CpGODN相应组比较，脾细胞细胞周期明显由G0／G1期向S期转化；细胞凋亡则呈现降低趋势。结论：CpGODN可以有效刺激免疫小鼠脾淋巴细胞细胞周期由G0／G1期向S期转化，并且有抑制细胞凋亡的效应。     

CpG ODN对ZP~(121-140)表位合成肽诱导的免疫应答和免疫避孕效应影响

目的:探讨CpG ODN对透明带(四)抗原表位ZP~(121-140)合成肽诱导的免疫应答和免疫避孕效应影响.方法:应用人工合成ZP2~(121-140)表位肽,与20μg CpG ODN或等量完全弗氏佐剂(CFA)混悬后左胫前肌免疫雌性BALB/c小鼠,初次免疫后2、4、6周各加强免疫一次,共免疫四次.在每次加强免疫前和末次免疫后两周断尾取血,分离血清,分析特异性lgG抗体及非特异性细胞因子IFN-γ、TNF-Ⅱ,IL-10水平;收集小鼠阴道冲洗液,离心取上清,分析特异性IgA抗体水平.免疫小鼠生育实验结束后,取卵巢组织行病理学分析.结果:CpG ODN组诱导的特异性IgG和IgA水平较CFA组有增高趋势,但无显著性差异(P＞O.05).CpG ODN组诱导的非特异性IFN-γ和TNF-水平明显高于CFA组(P＜0.05);而CpG ODN组诱导的IL-10水平显著低于CFA 组(P＜0.05).两种佐剂对小鼠的受孕率影响没有显著性差异,但CpG ODN组孕鼠每胎产仔数明显低于CFA组(P＜O.05).病理学分析显示实验小鼠卵巢组织无病理性变化.结论:CpG ODN对Zp~(121-140)合成肽诱导的免疫应答和免疫避孕效应略优于CFA,更适合用于避孕疫苗佐剂研究.     

CpG ODN增强乙型肝炎表面抗原免疫小鼠的抗体产生

目的：探讨合成含CpG基序的寡核苷酸（CpG ODN)对重组乙型肝炎表面抗原（rHBsAg)及乙型肝炎疫苗增强小鼠特异性抗体产生的效应。方法：采用非纯系（Km)及纯系（Balb/c)小鼠作为免疫对象，经后腱胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗－HBs)效价。结果：加CpG ODN组，其抗-HBs效价均较单独注射rHBsAg和疫苗组明显增高，持续时间长，且纯系鼠的抗体效价明显高于非纯系鼠。结论：CpG ODN对小鼠抗－HBs产生具有增强作用，具与疫苗中的铝佐剂有协同效应。     

Lipopolysaccharide induces calcitonin gene-related peptide in the RAW264.7 macrophage cell line

Calcitonin gene‐related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up‐regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme‐linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS‐induced inflammatory mediators such as nerve growth factor (NGF), interleukin‐1β (IL‐1β), IL‐6, prostaglandin E₂ (PGE₂) and nuclear factor‐κB (NF‐κB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS‐induced CGRP, which leads to the fine‐tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration‐dependent manner, stimulated, inhibited or had no effect on basal or LPS‐induced release of monocyte chemoattractant protein‐1, IL‐1β, IL‐6, tumour necrosis factor‐α and IL‐10 in RAW macrophages. The ligand‐concentration‐dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation‐induced CGRP has a positive or negative reciprocal effect on the production of other pro‐ and anti‐inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.     

Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响。噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响。结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100μg/ml时促进作用最明显,呈剂量相关,作用72h时细胞因子分泌量达到最大,72h后下降。VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足。VEPS促进G1期细胞增多,提高细胞的增殖能力。VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW264.7细胞有相似的作用规律。以上结果证明VEPS能激活巨噬细胞,也可能最终激活淋巴细胞,达到增强非特异性和特异性免疫的作用。     

CpG ODN对rHBsAg免疫小鼠淋巴细胞增殖反应的影响

目的：进一步探讨以合成含CpG基序的寡核苷酸（CpG ODN）作为免疫增强剂与重组乙型肝炎表面抗原（rHBsAg）联合免疫小鼠,观察其淋巴细胞增殖反应效应.方法：经后腿胫骨前肌初次免疫BALB/c小鼠两周后加强免疫1次;MTT和3H-TdR掺入法分别检测免疫小鼠胸腺及脾脏淋巴细胞增殖反应.结果：加CpG ODN组与单纯注射抗原及疫苗组相比,淋巴细胞增殖反应显著增强.而且,除胸腺特异性增殖中的CpG加疫苗组和疫苗组外,其他各实验组与对照组相比,淋巴细胞增殖反应也都显著增强.结论：CpG ODN能够明显刺激小鼠胸腺及脾脏淋巴细胞增殖反应,显示出强而有效的免疫增强效应,有望成为一种新型的免疫佐剂.     

新型免疫佐剂在淋巴细胞杂交瘤技术中的应用

目的：观察新型免疫佐剂ImmunEasy^TM(Cp GODN)的免疫调节作用，并与弗氏佐剂相比较，探讨其在单克隆抗体制备中的应用前景。方法：将小鼠用新型免疫佐剂ImmunEasy^TM或弗氏佐剂混合原核表达的重组APRIL抗原，分别以常规免疫和少量抗原免疫方案免疫Balb／c小鼠，用间接ELISA法检测不同时相小鼠免疫血清抗APRIL抗体的总效价及各类和亚类的效价。建立一种能在单个细胞水平反映小鼠B细胞分泌特异性抗体亚类的改良ELISPOT方法，并对两种佐剂免疫组中配对的小鼠进行检测。常规方法制备杂交瘤，以间接ELISA法筛选阳性杂交瘤，比较不同佐剂免疫小鼠所获得的杂交瘤上清液的类及其亚类频率。结果：与弗氏佐剂相比，ImmunEasy^TM在单克隆抗体制备过程中可明显减少抗原用量，缩短免疫周期，提高免疫血清效价，并增加IgG2a、IgM及IgA抗体的比例。结论：在细胞融合制备鼠源性mAb中，应用ImmunEasy^TM，可减少免疫原用量，提高效率，还可优先获得某些类及其亚类的mAb。     

含M-CSF条件培养液对RAW264.7细胞产生NO的影响

巨噬细胞集落刺激因子 (M CSF )是一种单核吞噬细胞特异性的生长因子。近年来的研究表明 ,M CSF可以增强巨噬细胞杀伤细菌等病原体及肿瘤细胞的能力 ,对抗感染及抗肿瘤具有重要意义。一氧化氮 (NO )是可由单核吞噬细胞产生的一种自由基 ,对病原体及肿瘤细胞同样具有杀伤作用。为探讨M CSF的抗感染、肿瘤作用是否与其刺激单核吞噬细胞产生NO有关 ,我们以L92 9细胞条件培养液 (L92 9 CM )作为M CSF来源 ,观察了M CSF对巨噬细胞系RAW2 64 7细胞NO产生的影响。结果发现 ,L92 9 CM可以刺激RAW2 64 7细胞NO的产生 ;且RT PCR显示L92 9 CM处理使RAW2 64 7细胞诱导型NO合酶 (iNOS )表达增加。     

Burkholderia pseudomallei RpoS regulates multinucleated giant cell formation and inducible nitric oxide synthase expression in mouse macrophage cell line (RAW 264.7)

Burkholderia pseudomallei is the causative agent of melioidosis. This bacterium can invade and survive inside the phagocytic and nonphagocytic cells. After internalization, the bacteria can escape from the membrane-bound phagosome into the cytoplasm. Internalised B. pseudomallei can also induce a cell-to-cell fusion, resulting in a multinucleated giant cell (MNGC) formation. In the present study, we demonstrated that B. pseudomallei rpoS null mutant was similar to its wild type parent in its ability to survive and multiply inside the mouse macrophages, but it failed to stimulate MNGC formation. The rpoS mutant was also unable to activate inducible Nitric Oxide Synthase (iNOS) in resting mouse macrophages but in gamma interferon (IFN-gamma)-activated macrophages, the mutant was able to induce significantly higher levels of iNOS and NO when compared with its wild-type counterpart, resulting in a significantly lower number of bacteria inside the infected host cells.     

Pam2CSK4 and Pam3CSK4 induce iNOS expression via TBK1 and MyD88 molecules in mouse macrophage cell line RAW264.7

Objective

The aim of this study was to investigate the involvement of TLR adaptor molecules, such as TRIF, MyD88, and TBK1 in the induction of iNOS and nitric oxide (NO) production in Pam2CSK4 and Pam3CSK4-treated mouse macrophages.

Method

Mouse macrophage cell line (RAW264.7) was transfected with trif, myd88, and tbk1 siRNAs before stimulated with Pam2CSK4 and Pam3CSK4. The iNOS gene and protein expression were determined by RT-PCR and immunoblotting, respectively. The NO production was determined by Griess reaction assay.

Results

The results showed that the induction of iNOS expression and NO production by Pam2CSK4 and Pam3CSK4 were diminished in tbk1 and myd88-depleted mouse macrophages but not trif-depleted cells.

Conclusion

These results suggested that the TBK1 and MyD88 molecules were essential for the induction of iNOS expression and NO production by both Pam2CSK4 and Pam3CSK4 via TLR2 signaling.

     

Therapeutic injection of C-class CpG ODN in draining lymph node area induces potent activation of immune cells and rejection of established breast cancer in mice

In order to develop novel CpG ODNs for the treatment of breast cancer, we have designed a series of CpG ODNs and evaluated their anti-tumor activity in a breast cancer mouse model. Interestingly, a C-class CpG ODN, designated as YW002, showed a vigorous activity on the inhibition of tumor growth in mice and completely cured some of the tumor-bearing mice through injection at tumor draining lymph node (TDLN) area. The expansion of immune cells in the TDLN and tumor and the generation of tumor specific immune memory were found associated with YW002-induced anti-tumor activity in mice. These results indicate that C-class CpG ODN could be developed into a medicament in a monotherapeutic regimen for the treatment of breast cancer through injection at TDLN area in clinic.     

In vitro anti-inflammatory activity of the Artemisia montana leaf ethanol extract in macrophage RAW 264.7 cells

Artemisia is one of the largest genera of the family Asteraceae or Compositae, consisting of 500 species. Some Artemisia species, such as Artemisia afra, A. sacrorum, and A. annua, have been widely used as traditional medicine to treat inflammatory and malarial diseases. However, the biological activity of A. montana has not been broadly studied. Therefore, in this study, we investigated the anti-inflammatory activity of A. montana leaf extract (ALE) and its molecular mechanisms in lipopolysaccharide-activated RAW 264.7 cells. Non-cytotoxic concentrations of ALE significantly reduced the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2, resulting in the decrease in NO and prostaglandin E₂. Moreover, ALE inhibited the production of tumour necrosis factor-α and interleukin-6. We also observed that ALE treatment repressed mitogen-activated kinase pathways by inhibiting the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, suggesting that ALE is a therapeutic candidate to treat inflammatory diseases.     

Role of protein kinase C in hydroxyapatite- induced phagocytosis by a murine macrophage cell line (RAW264.7)

The role of protein kinase C (PKC) in hydroxyapatite (HA)-induced phagocytosis by RAW 264.7 cells was investigated. The cells were incubated with HA particles at various incubation time and the levels of PKC activity were determined from the cell lysate. To determine the role of PKC, particles were incubated with the cells pretreated with the various concentrations of bisindolylmaleimide, a PKC inhibitor, and phagocytosis was then assessed at 60 min. Latex beads were used as a control. Our results showed that following incubation with HA particles, the levels of PKC activity in RAW264.7 cells was highest at 7 min and then decreased to reach the baseline levels of the controls at 30 min. Pretreatment of the cells with bisindolylmaleimide significantly reduced phagocytosis of HA particles in a dose-dependent pattern. The results of our present study suggest therefore that ingestion of HA by RAW264.7 cells may depend on PKC activity that may act in the early stages of phagocytosis.     

醛固酮降低新生大鼠心肌成纤维细胞iNOS表达和NO含量

目的为探讨醛固酮诱导心肌成纤维细胞(CFs)增殖及其对诱导型一氧化氮合酶 一氧化氮(iNOS NO)系统活性的影响。方法用胰酶消化法分离、培养新生SD大鼠CFs,采用MTT比色法、硝酸还原酶法、分光光度法和半定量RT PCR技术,观察不同浓度醛固酮对CFs的增殖、NO含量、iNOS活性和iNOSmRNA表达的影响。结果醛固酮(10 -11～10 -7mol/L)能剂量依赖性促进CFs的增殖,降低CFs的NO含量、iNOS活性及iNOSmRNA的表达(均P <0 .0 5 ) ,且螺内酯能逆转醛固酮的上述作用。醛固酮干预下CFs增殖数目与NO含量和iNOS活性呈显著负相关(r分别为- 0 . 94 2、- 0 . 978,均P <0 . 0 1)。结论醛固酮能剂量依赖性降低CFs的iNOS -NO系统活性,促进CFs增殖;螺内酯能逆转此作用。     

Contribution of tumor necrosis factor α and interleukin-1 α on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in a murine macrophage cell line,RAW264.7

The production of several inflammatory cytokines, such as murine macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor (TNF), and interleukin (IL)-1, was investigated in response to respiratory syncytial virus (RSV) infection in a murine macrophage cell line, RAW264.7, with special reference to mutual relation of their productions. The kinetics of MIP-2 production showed a trend for a biphasic pattern, that is, MIP-2 levels became detectable from 2 h postinfection (p.i.) and increased markedly until 8 h p.i. Thereafter, this level fell to the same level until 16 h p.i. and then increased again. TNF α was also detectable at 2 h p.i. and then increased sharply until 8 h p.i., when the peak level attained. Compared with the levels of MIP-2 and TNF α, that of IL-1 α/β, especially IL-1 β, was lower (ng versus pg/ml order). The presence of anti-TNF α or anti-IL-1 α antibody did not influence the early phase of MIP-2 production but significantly inhibited the late phase, suggesting that MIP-2 is induced by the combined effects of RSV infection via direct induction and indirectly after initial induction of TNF α and IL-1 α productions. Although RSV-infected RAW264.7 cells had no alteration inviability compared with mock-infected control, these data demonstrate that RSV is a potent inducer of inflammatory cytokines by direct induction and indirectly via the initial production of other cytokines. J. Med. Virol. 53:145–149, 1997. © 1997 Wiley-Liss, Inc.