• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.1-6
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• 2004

The human chemokine, the short version of leukotactin-1(shLkn-1;molecular weight=7.2 kD and 66 amino acids), was expressed and secreted into a culture medium using the me-thylotrophic yeast, Pichia pastoris. The recombinant shLkn-1 was purified from the culture supernatant using a simple two-step procedure consisting of cation exchange and reverse phase chromatography(RPC), in which shLkn-1 was highly purified (99.5%) with a high recovery yield of 82.7%. The C-terminal truncated derivative of shLkn-1 was found in the supernatant and was separated by RPC. The physicochemical properties of the purified shLkn-1 were verified to be the same as expected. The biological activity of the purified recombinant shLkn-1 was also quantified using a chemotaxis assay. It was observed that the recombinant shLkn-1 had the maximum migration activity at a concentration of 10nM, as potent as MIP-1${\alpha}$.

• IMMUNE NETWORK
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• 제8권3호
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• pp.90-97
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• 2008

Background: Hypersensitivity pneumonitis (HP) comprises a group of lung diseases resulting from repeated inhalation of various antigens such as Saccharopolyspora rectivirgula (SR). HP is categorized as a Th1 disease. Therefore, it has been suggested that IL-4, Th2 type cytokine, plays a protective role in the development of HP. However, the functional role of IL-4 in HP has not been extensively investigated in vivo. Therefore, we investigated the functional role of IL-4 in HP using IL-4 knockout (KO) mice. Methods: HP was induced by repeated exposure to SR in C57BL/6 (B6) and IL-4 KO (C57BL/6 background) mice. Results: IL-4 KO mice aggravated HP in terms of histological alteration, SR-specific immune responses, and inflammatory cell infiltration in the lungs compared with B6 mice. IL-4 KO mice produced high levels of IFN-${\gamma}$, TGF-${\beta}$ and TNF-${\alpha}$ in the lungs, whereas B6 mice showed the enhanced production of IL-4. Moreover, chemokines such as MIP-1${\alpha}$, MCP-1, and RANTES were highly expressed in IL-4 KO mice. IFN-${\gamma}$-secreting CD4, CD8 T cells, and neutrophils were enhanced in the bronchoalveolar lavage fluid (BALF) of IL-4 KO mice than those of B6 mice. The administration of recombinant(r) IL-4 restored these immunologic parameters in IL-4 KO mice. Conclusion: These results indicate that IL-4 plays a suppressive role in SR-induced HP by attenuating Th1-dominant immune responses.

• 대한한방부인과학회지
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• 제26권4호
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• pp.66-81
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• 2013

Purpose: This study was carried out to investigate the anti-inflammatory effects of Ligustri Lucidi Fructus water extract (LF) in the lipopolysaccharide (LPS)-induced mouse macrophages RAW 264.7 cell. Methods: Ligustri Lucidi Fructus was extracted with distilled water (2,000 ml) for 2 hours. In order to evaluate cytotoxicity of LF, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. To investigate anti-inflammatory effects of LF, the concentration of nitric oxide (NO) was measured with NO assay, cytokine was measured by Bio-Plex cytokine assay, and intracellular calcium (Ca) was measured with Fluo-4 Ca assay in RAW 264.7 cell. And when p-value is below 0.05, it is judged to have the significant difference statistically (P<0.05). Results: 1. LF showed no cytotoxicity. 2. LF inhibited significantly the production of NO at the concentration of 25, 50 and $100{\mu}g/ml$. 3. LF inhibited significantly the production of interleukin (IL)-4, macrophage inflammatory protein (MIP)-$1{\alpha}$, granulocyte colony stimulating factor (G-CSF) at the concentration of 25, 50, 100 and $200{\mu}g/ml$. 4. LF inhibited significantly the production of granulocyte macrophage-colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) at the concentration of 50, 100 and $200{\mu}g/ml$, the interferon (IFN)-${\gamma}$ at 25, 50 and $100{\mu}g/ml$ respectively. 5. LF inhibited significantly the production of IL-$1{\beta}$ at the concentration of 50 and $200{\mu}g/ml$, the IL-5 at 25 and $100{\mu}g/ml$, the IL-12p70, MIP-$1{\beta}$ at 50 and $100{\mu}g/ml$, the regulated on activation, normal T cell expressed and secrete d (RANTES) at 100 and $200{\mu}g/ml$ respectively. 6. LF inhibited significantly the production of IL-10, interferon gamma-induced protein (IP)-10 at the concentration of $200{\mu}g/ml$. 7. LF inhibited significantly the production of intracellular Ca at the concentration of 25, 50, 100 and $200{\mu}g/ml$. Conclusions: These results suggest that LF has anti-inflammatory effect and immuno-modulating activity.

• IMMUNE NETWORK
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• 제1권3호
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• pp.230-235
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• 2001

Background: Finding of the regulation of various gene expression by cytokine including $IFN-{\gamma}$ in hematopoietic stem cell will light up the understanding of pathogenesis of aplastic anemia in various aspects. To study on aplastic anemia, however, we have to circumvent the difficulty of directly obtaining bone marrow stem cells from the patient. Therefore, we tried to find out a cell can replace the bone marrow stem cells for study on cell signaling pathway and regulation of gene expression by $IFN-{\gamma}$. Materials and Methods: HL-60 cells, of 20 ng/mL of $IFN-{\gamma}$. Total RNA was isolated from the cells and RT-PCR of the indoleamine 2,3-dioxygenase (IDO), $IFN-{\gamma}$, TNF-${\alpha}$, $MIP-1{\alpha}$, and $TGF-{\beta}2$ was carried out for the estimation of the gene expression. Results: $IFN-{\gamma}$ induced IDO gene expression of mononuclear cells from umbilical cord blood showed similar pattern as compared to that of bone marrow. Whether $INF-{\gamma}$ was treated or not, $TNF-{\alpha}$ was expressed in both mononuclear cells from umbilical cord blood and bone marrow. However, HL-60 cells showed different expression patterns. HL-60 cells would express neither IDO nor $TNF-{\alpha}$ even under the culture with 20ng/mL of $IFN-{\gamma}$. Conclusion: Our results showed bone marrow can be replaced with mononuclear cells from umbilical cord blood in the study on the relation between aplastic anemia and $IFN-{\gamma}$ including $IFN-{\gamma}$ cell signaling pathway.

• 대한의생명과학회지
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• 제16권3호
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• pp.179-185
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• 2010

Benzene is known as a ubiquitous air pollutant and has a carcinogenic influence on the human body. Benzene is also metabolized to other volatile organic compounds (VOCs) in the body such as phenol and hydroquinone (HQ). The metabolites are accumulated and further oxidized by myeloperoxidase in bone marrow. They act as toxic agents and cause a variety of diseases, including cancer, atopic dermatitis and asthma. In this study, we examined the effects of benzene and its metabolites on proliferation, differentiation and chemotaxis of EoL-1 cells, the human eosinophilic leukemia cell line. These chemicals had no effect on the proliferation of EoL-1 cells. Benzene decreased the differentiation of EoL-1 cells induced by butyric acid. HQ was induced the cell death during butyric acid-induced EoL-1 cell differentiation. In a chemotaxis experiment, benzene, phenol and HQ enhanced the cell migration induced by Lkn-1 but not by MCP-1, eotaxin, MIP-$1{\alpha}$ and RANTES. These findings provide the effect of VOCs on the regulation of eosinophil-involved immune response.

• 대한의생명과학회지
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• 제15권1호
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• pp.93-96
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• 2009

House dust mites (HDMs) play an important role in the occurrence of allergic diseases such as asthma and atopic dermatitis. Dermatophagoides pteronyssinus (Der p) is one of the most prevalent HDMs. It mediates the activation of T cells and monocytes, and induces the elevation of immunoglobulin E levels in allergic diseases. However, the effects of Der p on human monocytes have not been fully understood. In the present study, we investigated whether or not Der p has a great effect on the chemotactic activity of the human monocytic cell line, THP-1 cells, as induced by CC chemokines. We also show that the Der p extract (DpE) increased the chemotactic activity of THP-1 cells in response to MCP-1, RANTES, MIP-1${\alpha}$, and TARC, but had no effect on the expressions of CC chemokine receptors (CCRs) binding to CC chemokines in THP-1 cells. Protease inhibitors, such as aprotinin and E64, blocked the increased chemotaxis, while cytoplasmic $Ca^{2+}$ influx mediated by these chemokines was inhibited by DpE. These results indicate that DpE increases the chemotactic activity of THP-1 cells in response to CC chemokines by regulating the cells' protease-dependent mechanism. This finding may be useful in identifying the pathogenesis of allergic diseases induced by Der p.

• 대한의생명과학회지
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• 제12권3호
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• pp.145-151
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• 2006

Leukotactin-l (Lkn-l )/CCL15 has been known as a potent chemoattractant of leukocytes. However, the precise function of Lkn-l in human neutrophils has not been explained well. In the present study, we investigated the contribution of Lkn-1 in chemotactic activity of human neutrophils. Both CCR1 and CCR3 mRNA expressions are strongly expressed in human neutrophils but CCR2 protein expression was uniquely detected on the cell surface. Lkn-l binding to CCR1 and CCR3 induced chemotactic activity of neutrophils. Chemotactic index of Lkn-l was comparable to that of IL-8. $MIP-1{\alpha}/CCL3$ binding to CCR1 and CCR5 has no effect on neutrophil migration. Cell migration, in response to Lkn-l, was blocked by pertussis toxin (Ptx), a $G_o/G_i$ protein inhibitor, and U73122, a phospholipase C(PLC) inhibitor but not by protein kinase C inhibitor such as rottlerin, and Ro-31-8425. Taken together, our results demonstrate that Lkn-l transduces the chemotaxis signal through $G_o/G_i$ protein and PLC. This finding provides the molecular mechanism by which Lkn-l may contribute to neutrophil movement into the site of inflammation.

• Korean Journal of Acupuncture
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• 제27권3호
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• pp.109-118
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• 2010

Objectives : The purpose of this study is to investigate the effect of Bacillus-fermented Scutellariae Radix acupuncture solution (SB) on chemokine and growth factor production in RAW 264.7 mouse macrophages stimulated by lipopolysaccharide (LPS). Methods : Productions of chemokine and growth factor were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on xMAP$^{(R)}$ technology. Firstly, cell culture supernatant was obtained after treatment with LPS (1 ${\mu}g$/mL) and SB for 24 hours. Then, it was incubated with the antibody-conjugated beads for 30 minutes. Detection antibody was then added and incubated for 30 minutes. After incubated for 30 minutes, strepavidin-conjugated phycoerythrin (SAPE) was added. After another 30 minutes incubation, the level of SAPE fluorescence was analyzed in Bio-plex Suspension Array System. Results : The results of the experiment are as follows. 1. SB significantly inhibited the LPS-induced production of vascular endothelial growth factor (VEGF), interferon-inducible protein (IP)-10, and granulocyte-colony stimulating factor (G-CSF) at the concentration of 25, 50, 100, and 200 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). 2. SB significantly inhibited the LPS-induced production of Eotaxin at the concentration of 25, 100, and 200 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). 3. SB significantly inhibited the LPS-induced production of MIP-$1\alpha$ at the concentration of 25 and 100 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). Conclusions : These results suggest that SB has immuno-modulatory property related with its inhibition of VEGF, IP-10, G-CSF, and Eotaxin production in macrophages.

• IMMUNE NETWORK
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• 제7권1호
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• pp.18-25
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• 2007

Background: Although glucocorticoids (GCs) are effective in controlling asthma in the majority of patients, a subset of asthmatics fails to demonstrate a satisfactory response, even to systemic GC therapy. This population is referred to as being "steroid-resistant". The actual mechanism underlying steroid resistance in asthma remains to be elucidated. Methods: We have investigated how dexamethasone (DEX) regulates asthmatic phenotypes in a murine model of asthma, in which mice received i.p. immunization twice, followed by two bronchoprovocations with aerosolized OVA with a one-week interval, which we have recently described. Results: Pretreatment with DEX resulted in an inhibition of NF-${\kappa}B$ activation in asthmatic lungs, and also inhibited bronchoalveolar lavage (BAL) levels of NF-${\kappa}B$-dependent cytokines such as TNF-${\alpha}$ and CC chemokines [eotaxin and monocyte chemotactic protein (MCP)-1]. DEX was effective in suppressing airway hyperresponsiveness (AHR) at 10 h, Th2-dependent asthmatic phenotypes such as airway eosinophilia, BAL levels of Th2 cytokines (IL-5 and IL-13), and mucin production. However, DEX failed to suppress BAL levels of CXC chemokines [macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC)] and airway neutrophilia. Conclusion: Airway neutrophilia is among the phenomena observed in patients with severe GC-resistant asthma. This study will provide insight into the molecular basis for airway neutrophila seen in steroid-resistant asthma. Further studies are required to delineate the underlying mechanism of CXC chemokine expression in asthma.

• Pediatric Infection and Vaccine
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• 제26권1호
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• pp.51-59
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• 2019

목적: 소아 마이코플라즈마 폐렴의 임상 중증도와 cytokine, chemokine의 상관 관계를 살펴보았다. 방법: 대상 환아의 임상소견과 검사소견을 후향적으로 조사하였고, interleukin (IL)-6, IL-8, IL-10, IL-18, inducible protein (IP)-10, macrophage inflammatory protein $(MIP)-1{\beta}$와 tumor necrosis factor $(TNF)-{\alpha}$를 비교 분석하였다. 결과: 총 72명이 포함되었고, 흉부 사진에서 대엽성 병변을 보이는 경우(29명)에서 기관지-미만성 병변을 보이는 경우(43명)보다 erythrocyte sedimentation rate (ESR), C-reactive protein (CRP)와 IL-18 수치가 의미 있게 높았다. 하지만, 스테로이드 사용 여부에 따른 차이는 보이지 않았다. CRP, ESR, lactate dehydrogenase (LDH), IL-18 그리고 IP-10 수치는 입원 전 발열 기간과 양의 상관관계를 보였다. 또한 ESR과 CRP 수치는 IL-18과, LDH는 IP-10과 양의 상관관계를 보였다. 결론: CRP, ESR, IL-18 그리고 IP-10 수치는 대엽성 폐렴이나 긴 발열 기간과 같은 질병의 중증도와 연관성을 가진다.