• BMB Reports
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• 제55권2호
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• pp.81-86
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• 2022

Macrophages are a major cellular component of innate immunity and are mainly known to have phagocytic activity. In the tumor microenvironment (TME), they can be differentiated into tumor-associated macrophages (TAMs). As the most abundant immune cells in the TME, TAMs promote tumor progression by enhancing angiogenesis, suppressing T cells and increasing immunosuppressive cytokine production. N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene, whose expression is down-regulated in various cancers. However, the effect of NDRG2 on the differentiation of macrophages into TAMs in breast cancer remains elusive. In this study, we investigated the effect of NDRG2 expression in breast cancer cells on the differentiation of macrophages into TAMs. Compared to tumor cell-conditioned medium (TCCM) from 4T1-mock cells, TCCM from NDRG2-over-expressing 4T1 mouse breast cancer cells did not significantly change the morphology of RAW 264.7 cells. However, TCCM from 4T1-NDRG2 cells reduced the mRNA levels of TAM-related genes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7 cells. In addition, TCCM from 4T1-NDRG2 cells reduced the expression of TAM-related surface markers, such as CD206, in peritoneal macrophages (PEM). The mRNA expression of TAM-related genes, including IL-10, YM1, FIZZ1, MR1, ARG1 and iNOS, was also downregulated by TCCM from 4T1-NDRG2 cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced the expression of PD-L1 and Fra-1 as well as the production of GM-CSF, IL-10 and ROS, leading to the attenuation of T cell-inhibitory activity of PEM. These data showed that compared with TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cells suppressed the TAM differentiation and activation. Collectively, these results suggest that NDRG2 expression in breast cancer may reduce the differentiation of macrophages into TAMs in the TME.

• 대한한방내과학회지
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• 제25권1호
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• pp.16-27
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• 2004

목적 : 골쇄보(骨碎補) (Drynariae Rhizoma)는 한의학에서 골격계 질환의 치유력을 강화시키는 것으로 알려져 있는데, 항 virus, 항 박테리아, 항 염증 작용이 있는 것으로 보고되고 있다. 질병을 치료하기 위하여 면역 반응을 조절하는 기전에 관하여 오랫 동안 많은 관심을 기울여왔는데, 식물에서 추출한 약재들이 면역기능을 조절할 수 있는 가능성에 대하여 광범위하게 연구되었다. 이에 저자는 골쇄보(骨碎補)의 ethanol 추출물을 가지고 항 세포성과 변역 조절 기능에 대하여 연구하였다. 방법 : 사람의 혈액단핵구 (PBMC)의 배양은 thymidine 법으로 검정하고 nitric oxide (NO) 생성은 mouse macrophage RAW 264.7 세포주를 이용하였으며 IL-2, IFN 와 TNF-a 생성은 ELISA 기술로 검정하였다. 세포 증식은 FACScan으로 측정하고 세포 표면항원 CD16, CD25 및 HLA-DR 은 FITC/PE 항체로 측정하였다. 결과 : 골쇄보(骨碎補)는 mitogen (phytohaemagglutinin; PHA) 과 antigen (purified protein derivative; PPD) 에 의해 자극받은 human peripheral blood mononuclear cells (PBMCs) 의 증식을 억제하였다. 더욱이, 골쇄보(骨碎補)는 mouse 와 인간에 기원한 여러 세포들의 성장을 억제하였다. 또한, nitric oxide (NO), interleukin-2 (IL-2)와 tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ 의 생성을 억제하였다. 한편, human PBMCs 에서 intracytoplasmic $interferon-{\gamma}\;(IFN-{\gamma})$와 cell surface markers 인 CD16, HLA-DR 의 expression은 골쇄보(骨碎補)에 의하여 영향을 받지 않았으나, CD25 expression 은 현저히 통제되었다. 결론 : 골쇄보(骨碎補) ethanol 추출물이 in vitro 에서 항 증식성과 변역 억제작용을 가지고 있다는 가능성을 의미하는 것으로 사료된다.

• 한국식품과학회지
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• 제46권1호
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• pp.68-72
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• 2014

본 연구는 동충하초(Cordyces militaris) 유래의 기능성 물질인 코디세핀의 면역활성을 검증하기 위하여 C57BL6 마우스 복강 대식세포를 이용하여 코디세핀이 대식세포의 활성화에 미치는 영향에 대하여 시험하였다. 그 결과 LPS에 의해 유도된 마우스 복강세포는 코디세핀의 작용에 의해 IL-$1{\beta}$, IL-12, TNF-${\alpha}$의 염증성 사이토카인의 생성이 증대되어 초기 염증매개 반응을 유도하여 선천면역반응의 활성화와 그리고 면역작용에 있어 후기 적응면역의 전환으로의 T 림프구의 활성화가 예상된다. 또한 IL-6의 생성증대로 활성화된 T 림프구에 의해 B 림프구의 항체생성반응을 매개하는 면역반응도 상승할 것으로 사료된다. 그리고 대식세포에 의한 염증반응에서 염증매개인자인 NO와 $H_2O_2$의 생성을 증대시킴에 따라 대식세포의 독성작용을 활성화시켜 염증반응을 효과적으로 유도할 것으로 보이며, 또한 $H_2O_2$의 후기 생성을 저해하였는데 이는 염증반응에 유도될 수 있는 세포의 손상으로부터 세포를 보호할 수 있을 것으로 사료된다. 따라서 코디세핀은 외부인자로부터 염증매개성 면역반응의 증강작용을 나타내는 것으로 사료된다.

• BMB Reports
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• 제51권2호
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• pp.92-97
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• 2018

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the $NF-{\kappa}B$ family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity.

• IMMUNE NETWORK
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• 제11권5호
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• pp.281-287
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• 2011

Background: Biodegradable polymers have increasingly been recognized for various biological applications in recent years. Here we examined the immunostimulatory activities of the novel poly(aspartic acid) conjugated with L-lysine (PLA). Methods: PLA was synthesized by conjugating L-lysine to aspartic acid polymer. PLA-nanoliposomes (PLA-NLs) were prepared from PLA using a microfluidizer. The immunostimulatory activities of PLA-NLs were examined in mouse bone marrow-derived dendritic cells (BM-DCs). Results: PLA-NLs increased the number of BM-DCs when added to cultures of GM-CSF-induced DC generation on day 4 after the initiation of cultures. Examination of the phenotypic properties showed that BM-DCs generated in the presence of PLA-NLs are more mature in terms of the expression of MHC class II molecules and major co-stimulatory molecules than BM-DCs generated in the absence of PLA-NLs. In addition, the BM-DCs exhibited enhanced capability to produce cytokines, such as IL-6, IL-12, TNF-${\alpha}$ and IL-$1{\beta}$. Allogeneic mixed lymphocyte reactions also confirmed that the BMDCs were more stimulatory on allogeneic T cells. PLA- NL also induced further growth of immature BM-DCs that were harvested on day 8. Conclusion: These results show that PLA-NLs induce the generation and functional activities of BM-DCs, and suggest that PLA-NLs could be immunostimulating agents that target DCs.

• IMMUNE NETWORK
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• 제11권3호
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• pp.163-168
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• 2011

Background: Nanoparticles (NPs) prepared from biodegradable polymers, such as poly (D,L-lactic acid-co-glycolic acid) (PLGA), have been studied as vehicles for the delivery of antigens to phagocytes. This paper describes the preparation of antigen-loaded PLGA-NPs for efficient cross-priming. Methods: NPs containing a similar amount of ovalbumin (OVA) but different sizes were produced using a micromixer-based W/O/W solvent evaporation procedure, and the efficiency of the NPs to induce the cross-presentation of OVA peptides were examined in dendritic cells (DCs). Cellular uptake and biodistribution studies were performed using fluorescein isothiocyanate (FITC)-loaded NPs in mice. Results: The NPs in the range of $1.1{\sim}1.4{\mu}m$ in size were the most and almost equally efficient in inducing the cross-presentation of OVA peptides via $H-2K^b$ molecules. Cellular uptake and biodistribution studies showed that opsonization of the NPs with mouse IgG greatly increased the percentage of FITC-positive cells in the spleen and lymph nodes. The major cell type of FITC-positive cells in the spleen was macrophages, whereas that of lymph nodes was DCs. Conclusion: These results show that IgG-opsonized PLGA-NPs with a mean size of $1.1{\mu}m$ would be the choice of biodegradable carriers for the targeted-delivery of protein antigens for cross-priming in vivo.

• IMMUNE NETWORK
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• 제3권4호
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• pp.295-301
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• 2003

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard $^{51}Cr$-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with $mIFN-{\gamma}$ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of $IFN-{\gamma}$ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

• IMMUNE NETWORK
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• 제2권4호
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• pp.195-201
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• 2002

Background: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. Methods: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the ${\beta}$-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semiquantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. Results: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at $56^{\circ}C$ illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. Conclusion: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.

• Archives of Pharmacal Research
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• 제29권10호
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• pp.890-897
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• 2006

In inflammatory responses, induction of cytokines and other immune regulator genes in macrophages by pathogen-associated signal such as lipopolysaccharide (LPS) plays a crucial role. In this study, the gene expression profile changes by LPS treatment in the macrophage/monocyte lineage cell line RAW264.7 was investigated. A 60-mer oligonucleotide microarray of which probes target 32381 mouse genes was used. A reverse transcription-in vitro translation labeling protocol and a chemileuminescence detection system were employed. The mRNA expression levels in RAW264.7 cells treated for 6 h with LPS and the control vehicle were compared. 747 genes were up-regulated and 523 genes were down-regulated by more than 2 folds. 320 genes showing more than 4-fold change by LPS treatment were further classified for the biological process, molecular function, and signaling pathway. The biological process categories that showed high number of increased genes include the immunity and defense, the nucleic acid metabolism, the protein metabolism and modification, and the signal transduction process. The chemokine-cytokine signaling, interleukin signaling, Toll receptor signaling, and apoptosis signaling pathways involved high number of genes differentially expressed in response to LPS. These expression profile data provide more comprehensive information on LPS-target genes in RAW264.7 cells, which will be useful in comparing gene expression changes induced by extracts and compounds from anti-inflammatory medicinal herbs.

• 생약학회지
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• 제42권3호
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• pp.253-259
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• 2011

Macrophages play a vital role in the innate immune system involving defensive cytokines such as TNF (tumor necrosis factor)-${\alpha}$ and nitric oxide (NO). Therefore, we try to elucidate the anti-inflammatory activity of Chaga mushroom (Inonotus Obliquus, IO) in murine macrophages. Raw 264.7 cells and peritoneal macrophages of mice were cultured with or without LPS/LPS + IFN-${\gamma}$ in the presence of IO aqueous extracts (IOE 0.2, 2, 20, 100 ${\mu}g$/mL) for 24 hr and 48 hr, respectively. Exposure of IOE caused the decrease of NO production and increase of TNF-${\alpha}$ production in dose-dependent manner in activated peritoneal macrophage in vitro. To further investigate anti-inflammatory effects of IO ex vivo, we orally administrated capsaicin (PC, 3 mg/kg/day) and IOE (100, 200, 400 mg/kg/day) for 4 consecutive days to C57BL/6 mice (7~9 weeks old, female), then observed the NO secretion and cytokine (TNF-${\alpha}$) production of LPS/LPS + INF-${\gamma}$-stimulated peritoneal macrophages. IOE inhibits NO secretion in dose-dependent manner both ex vivo and in vitro and increases the production of TNF-${\alpha}$ in vitro. In addition, we found that IOE possessed suppressive effects of LPS-stimulated TNF-${\alpha}$, IL-$1{\beta}$, COX-2, as well as iNOS expressions in Raw 264.7 cells. These findings indicate that IOE suppress not only the LPS-induced NO overproduction of murine peritoneal macrophages, but also iNOS, COX-2, TNF-${\alpha}$, and IL-$1{\beta}$ overexpression of LPS-induced Raw 264.7 cells. Consequently, our results suggest that IO may have the anti-inflammatory effects via suppression of the inflammatory cytokines and mediators, and be useful for the treatment of inflammatory diseases.