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Hong, Soon-Sun,Choi, Jung Ho,Lee, Sung Yoon,Park, Yeon-Hwa,Park, Kyung-Yeon,Lee, Joo Young,Kim, Juyoung,Gajulapati, Veeraswamy,Goo, Ja-Il,Singh, Sarbjit,Lee, Kyeong,Kim, Young-Kook,Im, So Hee,Ahn, Sun The American Association of Immunologists, Inc. 2015 JOURNAL OF IMMUNOLOGY Vol.195 No.1
<P>IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified-LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6-dependent TF-1 cell proliferation. LMT-28 antagonized IL-6-induced TNF-alpha production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6R alpha, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6R alpha complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.</P>
목적: Interleukin 9(IL-9)는 Th2 싸이토카인의 일종으로서 알레르기 염증반응의 병태생리에 관여하는 것으로 알려져 있다. 본 연구에서는 IL-9이 주요 알레르기 염증세포의 하나인 인간 비만세포에 어떠한 영향을 주는지를 알아보기 위하여 시행되었다. 방법: 본 연구에서는 인간 제대혈에서 CD34(+) 세포를 분리한 후 stem sell factor(SCF), IL-3, IL-6를 투여함으로써 비만세포를 선택적으로 배양하였다. 8주간 배양이 끝난 후 10 ×10^4개의 세포를 분주하여 배양조건을 SCF(100 ng/mL)만 투여하는 군, IL-9(50 ng/mL)만 투여하는 군, SCF(100 ng/mL)와 IL-9(50 ng/mL)을 병합투여하는 군의 3개 군으로 분리하였다. 4주간 배양을 하여 세포수를 측정하였고, PI 염색으로 사멸을 관찰하였으며, 인간 IgE를 투여한 후 분비되는 히스타민의 양을 측정하였다. 결과: 세포 수는 SCF 단독군에 비해 SCF+IL-9 병합군에서 의미있게 증가하였으며,(P<0.05) 이는 사멸의 억제와 관련되었다. 히스타민의 분비량은 배양조건에 따른 차이를 보이지 않았다. 결론: 본 연구의 결과는 IL-9이 알레르기 질환이 있는 표적기관에서 비만세포의 사멸을 억제하여 세포 수를 증가시킴으로써 알레르기 염증반응에 관여함을 시사한다. Purpose : Interleukin-9(IL-9), one of Th2-type cytokines, might be important in the pathophysiology of allergic diseases. We investigated the effect of IL-9 on human mast cells by assessing sell proliferation and histamine release. Methods : Human umbilical cord blood cells were cultured in the presence of stem cell factor(SCF, 100 ng/mL) and IL-6(50 ng/mL) in liquid medium for 8 weeks. Then these cells were divided into 3 aliquots. Each aliquot was cultured for 4 more weeks in different conditions : SCF alone(100 ng/mL), IL-9 alone(50 ng/mL) and SCF+IL-9. Cell numbers were counted using hemocytometer. For evaluation of apoptosis, DNA fragmentation was determined by propidium iodide(PI) staining and flow cytometric analysis. Histamine concentration was measured by ELISA after stimulation with human IgE and anti-human IgE. Results : Cell numbers increased significantly when they were cultured in the presence of SCF and IL-9 compared with SCF alone(P<0.05). Proliferation of mast cells was mediated by decreased apoptosis. Histamine release in activated mast cells was not different regardless of incubation with IL-9. Conclusion : IL-9 might be involved in allergic inflammation via proliferation of mast cells in target tissue.
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This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.
Artemisiae Asiaticae Herba (AAH) has been used to remedy of symptoms such as bleeding, dysmenorrhea, eczema and itchy skin in Oriental Medicine. In this study, we investigated the protective effect of AAH on allergic response. The effect of AAH was analyzed by ELISA and RT-PCR in RBL-2H3 cells. We investigated cell viability, β -hexosaminidase and histamine as markers of degranulation, production of IL-4 and TNF-α, and gene expression of HDC2, cytokines and FcεRI αβγ subunit. We found that AAH suppressed β-hexosaminidase and histamine release, the production of IL-4 and TNF-α in RBL-2H3 by the anti-DNP IgE plus DNP-HSA stimulation. AAH also significantly decreased cytokine mRNA expressions, such as IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-13, TNF-α, and GM-CSF, and increased cytokine mRNA expressions of IL-10 in RBL-2H3. In addition, AAH suppressed mRNA expression of FcεRI αβγ subunit on cell surface. Our results indicate that AAH protects against allergic response and exerts an anti-inflammatory effect through the inhibition of degranulation and production of cytokines and expression of FcεRI αβγ subunit.
Objective: Unlike other soluble receptors, the soluble interleukin-6 receptor (sIL-6R) cooperates with IL-6 to activate gp130 of effector cell. As the IL-6 and sIL-6R are important in the rheumatoid disease, this study was designed to measure concentration of IL-6 and sIL-6R in synovium and synovial fluid of the degenerative arthritis. Methods: The synovium and synovial fluid were obtained during total knee replacement arthroplasty. The synovium was taken from eleven patients, and synovial fluid taken from sixteen patients. Same patients between two groups were seven. Tissue cultures of the synovial tissues were done with 10% FBS for 72 hours. After irrigation, thery were incubated for 48 hours without FBS, and the culture media and the synovial fluid were collected after centrifuged at 2500rpm for 10 minutes. The level of IL-6 and sIL-6R were measured by quantitative sandwich enzyme immunoassay technique. Results: In the synovium, the IL-6 level was 5.1±0.12ng/ml, and the sIL-6R level was 0.41±0.25ng/ml. In the synovial fluid, the IL-6 level was 0.09± 0.15ng/ml, and the sIL-6R level was 10.37±3.28ng/ml. These results show that IL-6 concentration was measured highly in two groups, especially in synovium (sixty times), and the sIL-6R concentration was measured significantly high in synovial fluid (twenty-five times). Conclusion: The IL-6 and sIL-6R were elevated in degenerative arthrits. We confirmed the source of IL-6 was synovium (very high in synovial tissue culture media), but we need further study for the source of sIL-6R as it was remarkably elevated as IL-6 and its level was lower than serum.
We performed this study to investigate the feature of rejection in porcine-to-rat corneal orthotopic transplantation and to evaluate the effect of cyclosporine and mycophenolate on the xeno-rejection. Orthotopic corneal transplantation was done at 91 Sprague-Dawley rats, and they were divided into 10 groups based on the combination of immunosuppressants including dexamethasone, cyclosporine, and mycophenolate mofetil. Graft survival was analyzed and grafted eyes were examined with Hematoxylin & Eosin and CD4 or CD8 staining. Enzyme-linked immunosorbent assays were done for interleukin-2 (IL-2), IL-4, IL-5, IL-10, and interferon (IFN)-γin cornea, lacrimal gland, and cervical lymph nodes. The longest median survival of the immune suppressant group was 11.00±1.96 days, which showed no statistical differences compared with that of control (8.00±1.52 days). The neutrophils were prominent in the early phase but soon gave way to the monocytes. The number of CD8+ cells was higher than that of CD4+ cells. IL-2 and IFN-γmarkedly increased at 10 to13 days in cornea, lacrimal glands, and cervical lymph nodes, which showed a decrease with immunosuppressants except in the cornea. In conclusion, cyclosporine and mycophenolate could not prevent the rejection in porcine to rat orthotopic corneal xenograft associated with infiltraton of CD8+ and innate immune cells.
Purpose: Podocytes are important architectures that maintain the crucial roles of glomerular filtration barrier functions. Despite this structural importance, however, the mechanisms of the changes in podocytes that can be an important pathogenesis of minimal change nephrotic syndrome (MCNS) are not clear yet. The aim of this study was to investigate whether apoptosis is induced by interleukin (IL)-13 in cultured human podocytes. Methods: Human podocytes were treated with different IL-13 doses and apoptotic cells were analyzed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) and fluorescence-activated cell sorting (FACS). Results: The IL-13 increased the number of TUNEL-positive cells in a dose-dependent manner at 6 and 18 hours (P<0.05 and P<0.05, respectively). The apoptosis rate was appeared to be increased slightly in the IL-13-stimulated podocytes (8.63%, 13.02%, and 14.46%; 3, 10 and 30 ng/mL, respectively) than in the control cells (7.66%) at 12 hours by FACS assay. Conclusion: Our study revealed that IL-13 expression may increase podocyte apoptosis. Blocking the IL-13 signal pathway can potentially play an important role in regulating the apoptosis of podocytes.
Background/Aims: Interleukin-8 (IL-8) has been reported to play a critical role in Helicobacter pylori (H. pylori)-associated gastric mucosal damage. H. pylori exist in both bacillary and coccoid forms in the stomach. In contrast to bacillary forms, it is not clear whether coccoid forms stimulate gastric epithelial cells to produce IL-8. This study was conducted to investigate the effects of coccoid forms on IL-8 production. Methods: H. pylori strains of ATCC 43504, ATCC 43526 and three clinical isolates were used in the present study. Coccoid forms were induced by culturing bacillary forms of H. pylori for more than 84 hr. After co-culture of two human gastric adenocarcinoma cel lines (KATO III and AGS) with five strains of H. pylori, the levels of IL-8 were determined in th supernatants by enzyme linked immunosorbent assay. Results: The levels of IL-8 in KATO III and AGS cells were markedly elevated up to 6-9 hr after co-culture with the bacillary forms. The IL-8 levels produced in both cell lines by the coccoid forms were significantly lower than those by the bacillary forms in all strains. Conclusions: These results suggest that coccoid forms are much less implicated in IL-8- mediated gastric mucosal damage than bacillary forms.
<P>We performed this study to investigate the feature of rejection in porcine-to-rat corneal orthotopic transplantation and to evaluate the effect of cyclosporine and mycophenolate on the xeno-rejection. Orthotopic corneal transplantation was done at 91 Sprague-Dawley rats, and they were divided into 10 groups based on the combination of immunosuppressants including dexamethasone, cyclosporine, and mycophenolate mofetil. Graft survival was analyzed and grafted eyes were examined with Hematoxylin & Eosin and CD4 or CD8 staining. Enzyme-linked immunosorbent assays were done for interleukin-2 (IL-2), IL-4, IL-5, IL-10, and interferon (IFN)-γ in cornea, lacrimal gland, and cervical lymph nodes. The longest median survival of the immune suppressant group was 11.00±1.96 days, which showed no statistical differences compared with that of control (8.00±1.52 days). The neutrophils were prominent in the early phase but soon gave way to the monocytes. The number of CD8+ cells was higher than that of CD4+ cells. IL-2 and IFN-γ markedly increased at 10 to13 days in cornea, lacrimal glands, and cervical lymph nodes, which showed a decrease with immunosuppressants except in the cornea. In conclusion, cyclosporine and mycophenolate could not prevent the rejection in porcine to rat orthotopic corneal xenograft associated with infiltraton of CD8+ and innate immune cells.</P>
This study is to investigate the effects of Chinemys reevesii (CR) on allergic inflammation mechanism related chronic dermatitis. To investigate the effects of CR, we study inhibitory effect of CR on the levels of pro-inflammatory cytokines released from RAW 264.7 cell stimulated with lipopolysaccaride (LPS), and EoL-1, THP-1, Jutkat cell stimulated with Dermatophagoides pteronyssinus (DP), and LPS induced acute inflammatory BALB/c mouse model. CR reduced the levels of IL-1β released from RAW 264.7 cell stimulated with LPS at 20 ug/ml, 10 ug/ml concentration. CR significantly reduced the levels of MCP-1 released from EoL-1 cell, IL-6 from THP-1 cell, and IL-4, IL-5, TNF-α from Jutkat cell stimulated with DP at all the concentration. CR significantly reduced the levels of TNF-α, IL-6, IL-1β, in LPS induced inflammatory BALB/c mouse model, in a dose-dependent manner. These results suggested that CR has suppressive effects on pro-inflammatory cytokines in various inflammation related cell lines through the regulation of immune system. CR could be a therapeutic agent for treatment of chronic inflammatory dermatitis in the future.