이질아메바에 의한 인체 대장상피세포주 HT-29에서의 interleukin-8 유전자의 발현

김정목,정현채,임경일,조양자,김정룡 INSTITUTE OF TROPICAL MEDICINE YONSEI UNIVERSITY 1995 YONSEI REPORTS ON TROPICAL MEDICINE Vol.26 No.1

이질아메바에 의한 장염 환자의 조직 또는 이질아메바를 실험적으로 감염시킨 동물의 조직 검사에서 호중구의 침윤이 특징적으로 관찰된다. 그러나 이와같은 호중구의 침윤을 설명할 수 있는 기전에 대한 연구는 매우 미흡하다. 따라서 본 연구자들은 아메바 감염 초기에 인체 대장상피세포에서 interleukin-8(IL-8)이 유도되어 호중구 침윤과 같은 염증반응이 유발될 것이라는 가설을 설정하였다. 이를 위하여 인체 대장상피세포주인 HT-29에 이질아메바 영양형을 실험적으로 노출시킨 뒤 발현되는 IL-8 mRNA를 역전사 중합효소법(reverse transcriptional polymerase chain reaction, RT-PCR)으로 검사함과 동시에 발현된 IL-8 mRNA를 인공적으로 합성시킨 표준 RNA와 RT-PCR법을 이용하여 정량하였다. 실험 결과 이질아메바 영양형에 노출된 30분 후 부터 IL-8 mRNA가 발현되기 시작하였다. 그리고 그 발현 분자수는 노출 시간의 증가에 따라 계속 증가하여 3시간 대에는 3.1×10(7) molecules/㎍ total RNA를 나타내었다. 동시에 IL-8 mRNA의 발현은 노출시킨 이질아메바 영양형의 수에 비례하였다. 즉, HT-29/아메바 영양형의 비율이 10:1인 경우 IL-8 mRNA의 발현 분자수는 1.2×10(7) molecules/㎍ total RNA로 나타났다. 이와같은 IL-8 mRNA의 발현은 IL-8 단백질 분비로 이어짐을 ELISA 검사로 확인할 수 있었다. 한편 이질아메바 파쇄액(lysate)도 대장상피세포주인 Caco-2에서 IL-8 mRNA발현을 유도하였다. 결론적으로 본 실험은 이질아메바 감염 초기에 대장상피세포로 부터 IL-8이 발현되며 이에 의하여 염증반응이 촉발될 가능성이 있음을 시사해 준다. The protozoan parasite, Entamoeba histolytica, is one of major causative agents of intestinal disease all over the world. In acute experimental infection, the early host response to E. histolytica is characterized by an infiltration of neutrophils. However, the chemotactic signal for this response is not well known. Based on the finding that human epithelial cells produce the potent neutrophil chemoattractant and activator, interlukin-8 (IL-8), IL-8 gene expression was examined thoroughly in human colon epithelial cells exposed to E. histolytica trophozoites. Cellular RNAs were extracted from HT-29 or Caco-2 human colon epithelial cells exposed to E. histolytica trophozoities for 30 minutes. 1 and 3 hours. IL-8 mRNA transcripts were measured by reverse transcriptional polymerase chain reaction (RT-PCR) using synthetic standard RNA. The number of IL-8 mRNA molecules increased from 30 minutes to 3 hours of exposure period, reaching 3.1×10(7) molecules/㎍ of total RNA. Expression pattern of IL-8 mRNA transcripts was parallel to the amounts of IL-8 protein measured by enzyme-linked immunosorbent assay (ELISA). Lysates of E. histolytica also induced expression of mRNA for IL-8 in colon epithelial cells. These results suggest that acute inflammatory reaction by E. hisstolytica may be initially triggered by proinflammatory cytokines such as IL-8 secreted from epithelial cells of the colon.

IL-15에 의한 류마티스관절염 환자 활막 섬유모세포에서의 SDF-1 유도

박영은 ( Young Eun Park ),김성일 ( Sung Il Kim ),박성후 ( Seong Hu Park ),백승훈 ( Seung Hoon Baek ),오혜좌 ( Hye Jwa Oh ),허양미 ( Yang Mi Heo ),조미라 ( Mi La Cho ) 대한류마티스학회 2010 대한류마티스학회지 Vol.17 No.3

Objective: Interleukin-15 (IL-15) recruits and activates synovial T cells, and IL-15 plays an important role in amplifying and perpetuating inflammation in the pathogenesis of rheumatoid arthritis (RA). Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for memory T cells in the inflamed RA synovium. This study investigated the effect of IL-15 on SDF-1 production in RA fibroblast-like synoviocytes (FLS). Methods: The expressions of IL-15 and SDF-1 were determined from the synovium of patients with RA and osteoarthritis (OA) by performing immunohistochemistry. The expressions of SDF-1 was measured from the RA FLS that were cultured with IL-15 and IL-17 by real-time RT-PCR and ELISA. The SDF-1 expression was also measured, via ELISA, from the RA FLS stimulated by IL-15 together with the inhibitors of such intracellular signal molecules as phosphatidylinositol 3-kinase (PI 3-kinase, LY294002), STAT3 (AG490), MAP Kinase (PD98059), NF-κB (parthenolide) and activator protein 1 (AP-1, curcumin). Results: IL-15 and SDF-1 were mainly expressed in the RA synovium compared to that of the OA synovium. IL-15 increased the SDF-1 expressions and it, and had an additive effect with IL-17 on the SDF-1 expressions in the cultured RA FLS. The IL-15 induced increase of the SDF-1 expression in the cultured RA FLS was blocked by the inhibitors of PI 3-kinase, NF-κB and AP-1. Conclusion: The SDF-1 expression was increased in the RA synovium and it was up-regulated by IL-15 in the RA FLS through the PI 3-kinase, NF-κB, and AP-1 pathways. These results imply that the IL-15 induced increase of the SDF-1 expressions may be involved in the immunopathogenesis of RA.

The enhanced IL-l8 production by UVB irradiation requires ROI and AP-1 signaling in human keratinocyte cell line (HaCaT)

Cho, Daeho,Kang, Jae Seung,Park, Jong Hoon,Kim, Young-In,Hahm, Eunsil,Lee, Junechul,Yang, Yoolhee,Jeon, Junho,Song, HyunKeun,Park, Hyunjeong,Kim, Taesung,Pang, Saic,Kim, Chul-Woo,Hwang, Young Il,Lee, 전남대학교 약품개발연구소 2002 약품개발연구지 Vol.11 No.-

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N-acetyl-ι-cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.

담배 니코틴에 의한 사람 태아 성상세포에서 종양괴사인자(TNF-α)의 발현 억제작용

손일홍,이성익,양현덕,한선정,석승한,이재규,김재현,박주영,문형인,이성수,Son, Il-Hong,Lee, Sung-Ik,Yang, Hyun-Duk,Han, Sun-Jung,Suk, Seung-Han,Lee, Jai-Kyoo,Kim, Jae-Hyun,Park, Joo-Young,Moon, Hyung-In,Lee, Sung-Soo 대한화학회 2007 대한화학회지 Vol.51 No.3

니코틴은 사람 대식세포에서 interleukin 2 (IL-2)와 종양괴사인자 (tumor necrosis factor-alpha; TNF-α) 가 생성되는 것을 억제하는데, 이러한 억제작용은 cytokine 유전자 발현 중 전사단계에서 전사인자의 활성을 억제함으로써 일어난다. 이러한 니코틴의 면역반응 억제작용은 아프타성궤양 및 궤양성대장염, 알레르기성폐 포염, 건초열 등에서도 보고되고 있다. 만일 중추신경계에서도 위와 같은 니코틴의 면역억제 작용이 일어난 다면 다발성경화증과 같은 면역반응 매개질환의 치료에 새로운 전기가 마련될 수 있을 것이다. 본 연구에서 는 중추신경계의 여러 면역반응 매개질환의 병태생리에 대한 이해를 넓히고자, 이미 알려진 니코틴의 cytokine 생성억제가 사람 중추신경계의 성상세포에서도 일어남을 확인하고 그 억제기전을 밝히고자 하였다. 이를 위 하여 사람 태아 성상세포에 다양한 농도의 니코틴과 IL-1β를 처리한 다음 TNF-α mRNA의 발현 정도와 NF- κB의 활성을 비교, 분석하여 다음과 같은 결과를 얻었다. 1. 사람 태아 성상세포를 0.1-20 μg/ml의 니코틴으로 처리해 본 결과 10 μg/ml 이상의 농도에서 세포독성능이 나타나기 시작하였다. 2. 사람 태아 성상세포에 IL- 1β를 처리하면 2시간만에 TNF-α mRNA가 최대로 발현되었으며 그 이후로는 점진적으로 감소하였다. 3. 사 람 태아 성상세포를 1 및 0.1 μg/ml의 니코틴으로 전처리한 후 IL-1β로 자극한 군에서는 IL-1β 단독 처리군에 비해 TNF-α mRNA의 발현이 감소하는 양상을 보였다. 1 μg/ml의 니코틴을 처리한 경우에는 8시간 이후부터 TNF-α mRNA의 발현이 현저하게 감소하여 12시간에 최대로 감소하였다. 또한 0.1 μg/ml의 니코틴을 처리한 군에서는 24시간에 가장 현저하게 감소하였다. 4. 성상세포에 IL-1β로 처리한 군에서는 강력한 NF-κB의 활성 을 확인할 수 있었으며, 니코틴을 전처리하고 IL-1β 자극한 군에서는 NF-B의 활성이 감소하였다. 결론적으로 일정농도 이상의 니코틴은 세포독성효과를 나타내나 적정한 농도와 시간 경과후 니코틴은 사람 태아 성상세포에서 IL-1β에 의해 유도되는 TNF-α의 발현 감소를 유도하며, 이는 NF-κB의 활성을 감소시킴으로써 나타난다고 생각된다. The Tumor necrosis factor-α, (TNF-α), is involved in the pathogenesis of multiple sclerosis and contributes to the degeneration of oligodendrocytes as well as neurons. Nicotine has been found to have immunosuppressive and inflammation-suppressing effects. Astrocytes, the major glial cells in the CNS, are capable of producing TNF-α at both the mRNA and protein levels in response to interleukin-1 (IL-1) or TNF-α. Nicotine has been shown to influence glial cell functions. To order to explore the role of astrocytes in the production of TNF-α, astrocytes were pretreated with nicotine and are stimulated with IL-1β to determine their effects on TNF-α production. The results are as follows. Cytotoxic effects of nicotine on human fetal astrocytes were noted above 10 μg/ml of nicotine. The effect of IL-1β on TNF-α mRNA expression in primary cultured human fetal astrocytes was maximal at 2 h after IL- 1β(100 pg/ml) treatment. Human fetal astrocytes were pretreated with 0.1, 1, and 10 μg/ml of nicotine and then stimulated with IL-1β (100 pg/ml) for 2 h. The inhibitory effect of nicotine on expressions of TNF-α mRNA in human fetal astrocytes with pretreated 0.1 μg/ml of nicotine is first noted at 8 hr, and the inhibitory effect is maximal at 12 h. The inhibitory effect at 1 μg/ml of nicotine is inhibited maximal at 24 h. Nicotine at 0.1, 1 and 10 μg/ml concentrations significantly inhibits IL-1β-induced NF-κB activation. Collectively, this study indicates that nicotine might inhibit the expression of TNF-α in activated human fetal astrocytes.

Association of Increased Pulmonary Interleukin-6 with the Priming Effect of Intra-Amniotic Lipopolysaccharide on Hyperoxic Lung Injury in a Rat Model of Bronchopulmonary Dysplasia

Kim, Do-Hyun,Choi, Chang Won,Kim, Ee-Kyung,Kim, Han-Suk,Kim, Beyong Il,Choi, Jung-Hwan,Lee, Myong Jin,Yang, Eun Gyeong S. Karger AG 2010 NEONATOLOGY Vol.98 No.1

<P><I>Background:</I> The authors previously demonstrated the priming effect of intra-amniotic lipopolysaccharide (LPS) on hyperoxic lung injury in a rat model of bronchopulmonary dysplasia (BPD). <I>Objectives:</I> To investigate the mechanism underlying this priming effect by determining biochemical profiles in a rat model of BPD. <I>Methods:</I> The rat model involved intra-amniotic LPS administration and postnatal hyperoxia (85%). The mRNA expressions of <I>interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), basic fibroblast growth factor (bFGF),</I> and <I>transforming growth factor </I>β<SUB><I>1</I></SUB><I> (TGF-</I>β<SUB><I>1</I></SUB><I>),</I> as well as the protein levels of IL-6, VEGF, and protein carbonyl in lung tissue were compared between the LPS plus hyperoxia, the LPS only, the hyperoxia only, and the control groups. <I>Results:</I> Morphometric analysis of lung tissues demonstrated that alveolarization was significantly inhibited only in the LPS plus hyperoxia group. IL-6 protein levels and its mRNA expression in the lungs were significantly increased only in the LPS plus hyperoxia group. Neither LPS nor hyperoxia increased IL-6 in the lungs independently. <I>bFGF</I> mRNA expression was significantly decreased in the LPS-treated groups. VEGF protein levels were significantly reduced by hyperoxia, whereas protein carbonyl levels were increased by intra-amniotic LPS or hyperoxia. No additional significant change to VEGF or protein carbonyl levels was produced by intra-amniotic LPS or hyperoxia. There were no significant differences in the mRNA expressions of <I>VEGF, VEGFR-2,</I> and <I>TGF-</I>β<SUB><I>1</I></SUB>. <I>Conclusions:</I> The priming effect of intra-amniotic LPS on hyperoxic lung injury may be associated with IL-6 elevation in the lungs.</P><P>Copyright © 2009 S. Karger AG, Basel</P>

The Effects of Ketorolac Tromethamine and Baicalein on the Levels of Inflammatory Factors in Human Synoviocytes

Yang, Jae-Heon,Yun, Mi-Young,Lee, Nam-Hee,Kim, Dae-Keun,Kim, Young-Il,Noh, Young-Hee,Kim, Tae-Youl,Yoon, Se-Won,Shin, Sang-Chul 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.11

This study examined the effects of ketorolac tromethamine (KT) and baicalein (BE) on the levels of inflammatory factors in human synoviocytes. The fibroblast-like synoviocytes (FLS) cells were used to determine the possible regulatory effects of KT and BE (KTBE) on the levels of inflammatory factors in FLS cells. In addition, the levels of TNF-$\alpha$, IL-6, and IL-$1{\beta}$ mRNA expression in FLS cells induced by a TNF-$\alpha$ and IL-$1{\beta}$ co-treatment were largely inhibited by a KTBE treatment. The level of FLS cells proliferation was increased by IL-$1{\beta}$ and TNF-$\alpha$, and strongly inhibited by KTBE treatment. The production of oxygen species (ROS) was inhibited by KTBE in FLS cells. KTBE appears to regulate the levels of mRNA that are important for regulating RA progression.

Interaction between CD40L and CD40 Mediates Hepatic Exosomal Delivery to Kupffer Cells in Alcoholic Liver Disease

( Kyurae Kim ),( Jun-hee Lee ),( Young-ri Shim ),( Hee-hoon Kim ),( Ye Eun Kim ),( Keungmo Yang ),( Tom Ryu ),( Won-il Jeong ) 대한간학회 2020 춘·추계 학술대회 (KASL) Vol.2020 No.1

Aims: Cluster of differentiation 40 (CD40) is a costimulatory molecule on antigen presenting cells including macrophages. An interesting study reported that extracellular vesicles (EVs) containing CD40L promote macrophage activation through CD40, thereby accelerating alcoholic liver diseases (ALD) in mice and patients. However, other effects of CD40L-expressing EV on Kupffer cells (KCs) have not been investigated clearly. Here, we explored CD40-mediated delivery of hepatic exosomes and its effects on KCs in acute alcoholic liver injury. Methods: To induce acute liver injury, binge ethanol drinking (4 g/kg, 40% ethanol) was performed by oral gavage into wildtype (WT) and CD40 knockout (KO) mice. Interleukin-17A (IL- 17A) positive cells were analyzed by flow cytometry. Isolated hepatocytes, KCs and DiI-stained exosomes, neutralizing antibody and dynasore were used for in vitro experiments. Results: Although the number of exosomes and mRNA expression of CD40L in hepatocytes were significantly increased by ethanol exposure, protein levels of CD40L in ethanol-induced exosomes were similar with controls, reflecting proportional increase of CD40L to the numbers of exosomes. However, freshly isolated KCs from ethanol-fed WT mice exhibited increased expression of CD40 (protein receptor of CD40L). In vitro, ethanol-induced exosomes increased CD40 expression in KCs by a TLR3-dependent manner. Moreover, DiI-stained exosomes were successfully delivered to WT KCs, but not in CD40-deficient KCs. In addition, treatments with neutralizing antibody of CD40 and dynamin inhibitor (dynasore) decreased internalization of hepatic exosomes into KCs, thereby reducing IL-1β production in KCs. Furthermore, binge ethanol drinking increased IL-17A production of γδ T cells in WT mice but not in CD40 KO mice. Conclusions: Alcohol-induced hepatic exosomes could be delivered to KCs through a CD40L/CD40-dependent endocytic uptake and they stimulate IL-1β expression in KCs, subsequently leading to IL-17A production in γδ T cells in ALD. Thus, CD40L/ CD40 axis could be a potential target to reduce IL-17A production in ALD.

Effect of high-mobility group box 1 on keratinocytes and fibroblasts

( Chan-yang Lee ),( In-hye Kang ),( Seung-min Oh ),( Jin-woo Lee ),( Young Il Kim ),( Ki-heon Jeong ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Background: High-mobility group protein B1 (HMGB1) is a physiological activator of immune responses. In patients with chronic inflammatory skin disorders including lupus, atopic dermatitis, and psoriasis, HMGB1 is increased in the cutaneous lesions. Objectives: This study was designed to investigate the effect of HMGB1 on keratinocytes and fibroblasts. Methods: In this study, keratinocytes and fibroblasts were exposed to narrow band ultraviolet B (NBUVB). Thereafter, release of HMGB1 were measured. Then keratinocytes and fibroblasts were treated with recombinant HMGB1 (rHMGB1) and production of inflammatory factors such as interleukin (IL)-1β, IL-6, IL-8, IL-18, chemokine (C-X-C motif) ligand (CXCL)1, CXCL2 and tumor necrosis factor (TNF)-α were examined. Results: 24h after NBUVB irradiation, the level of HMGB1 mRNA was increased in the fibroblasts but it was decreased in the keratinocytes. Extracellular level of HMGB1 was significantly increased in both keratinocytes and fibroblasts, 72h after NBUVB irradiation. After rHMGB1 treatment, proinflammatory molecule such as CXCL-1, IL-6, IL-8 and IL-18 were significantly increased in the keratinocytes and CXCL-1, IL-6 and IL-8 were significantly increased in the fibroblasts. Conclusion: HMGB1 can be released from keratinocytes and fibroblasts by external stress such as NBUVB irradiation and leads to activation of inflammatory signaling pathways in the keratinocytes and fibroblasts.

Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10

Seung-Ha Yang,Min-Jung Park,Il-Hee Yoon,Su-Young Kim,So-Hee Hong,Jin-Young Shin,Hye-Young Nam,김용희,Bongi Kim,박정규 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.5

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naïve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naïve and pre-activated T cells in which IL-10 and IDO play important roles.

복막 중피 세포에서 IL-1β 자극에 의한 MCP-1과 RANTES의 생성

송인숙,이상구,박정식,양원석,김순배,윤견일 대한신장학회 2000 Kidney Research and Clinical Practice Vol.19 No.5

Human peritoneal mesothelial cells may have a great potential to secrete chemokines, growth factors, adhesion molecules, and various cytokines stimulated with proinflammatory cytokines during peritoneal infection. In the course of peritonitis, rapid neutrophil cell influx and subsequent monocytic cell influx can be observed. It has been demonstrated that human peritoneal mesothelial cells secrete a C-X-C chemokine, IL-8, which contributes to the recruitment of neutrophil influx during peritoneal infection. However, the production and role of C-C chemokines have not been fully defined in human peritoneal mesothelial cells. This study was performed to evaluate the production of MCP-1 and RANTES and their influence on the chemotaxis of monocytes when human peritoneal mesothelial cells were stimulated with IL-1β. Mesothelial cells obtained by enzymatic digestion of pieces of human omentum and stimulated with a various doses and times of IL-1β. The expression of MCP-1 and RANTES mRNA was measured by Northern bloassay and the expression of their proteins was analyzed by ELISA. To evaluate their function, monocytes chemotaxis assay was performed using a 48-well chemotactic chamber. Cultured human peritoneal mesothelial cells appeared to be polygonal at confluence using phase contrast microscope. Indirect immunofluorescent staining demonstrated that the mesothelial cells reacted positively with anti-cytokeratin antibody and anti-vimentin antibody. The expression of MCP-1 and RANTES mRNA increased in response to IL-1β in time and dose dependent manner. The protein levels of MCP-1 and RANTES with stimulation of 1.0ng/mL of IL-1β for 24 hours were higher than those without(30.0±2.22 vs 3.55±0.74ng/105cells and 1.53±0.41 vs 0.11±0.02ng/105cells respectively, p$lt;0.05, n=6). Chemotaxis assay showed that the supernatants from human peritoneal mesothelial cells with stimulation of IL-1β for 24 hours had significantly higher chemotaxis of monocytes than those without(71±3.4% vs 50±2.9%, p$lt;0.05, n=6). Coincubation of sup with stimulation and antibodies to MCP-1 or RANTES(20μL/mL, lOμL/mL, respectively) resulted in a significant inhibition of chemotaxis of monocytes by 33% and 12%(47±3.1% and 62±3.0% respectively, p$lt;0.05, n=6). Human peritoneal mesothelial cells are capable of the expression of MCP-1 and RANTES mRNA and the production of their proteins in response to IL-1β. Functionally, mesothelial cells derived Mand RANTES may contribute to the recruitment of monocytes and amplify the inflammatory process. Thus, human peritoneal mesothelial cells play an important role during peritoneal infection.