• Journal of Periodontal and Implant Science
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• v.36 no.3
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• pp.579-590
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• 2006

1. 연구배경 교원질 분해작용을 하는 호중구의 세포질 효소인 기질금속단백분해효소-8은 치주질환, 류마티스 관절염, 그리고 궤양결장염과 같은 염증성 질환에서 농도가 증가한다고 알려져 있다. 최근에는 A. actinomycetemcomitans의 leukotoxin이 사람호중구에서 기질금속단백분해효소-8의 분비를 유도하는 것이 보고되었다. 이 연구의 목적은 선천면역 체계에서 세포표면 항원무리14, Toll-like 수용기, 그리고 $NF-{\kappa}$ B경로를 통하여 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비 여부와 세포기전을 알아보고자 하였다. 2. 연구재료 및 방법 건강한 개인 제공자(남자 13명, 여자 3명)로부터 얻은 개개인의 20ml 말초혈액을 제조사의 지침에 따라 호중구를 추출한 후 항세포표면 항원무리14와 함께 $4^{\circ}C$에서 30분간 전배양 한 후, $37^{\circ}C$에서 9시간 동안 배양시켰다. 추출한 호중구에 Toll-like 수용기 억제제 또는 $NF-{\kappa}$ B억제제인 TPCK를 첨가한 후 $37^{\circ}C$에서 1시간 동안 전배양하고 $37^{\circ}C$에서 9시간 동안 배양시켰다. 호중구에 세포뼈대 억제제인 cholchicine, nocodazole, demecolcine, 그리고 cytochalasin B를 A. actinomycetemcomitans의 지질다당질과 함께 $37^{\circ}C$에서 9시간 동안 배양시켰다. 기질금속단백분해효소-8 분비량은 효소면역측정법을 통해 결정하였다. 통계처리는 일원배치 분산분석법을 이용하였다(p<0.05). 3. 결과 A. actinomycetemcomitans 지질다당질은 기질금속단백분해효소-8의 분비를 증가시켰다. 기질금속단백분해효소-8의 분비는 항세포표면 항원무리14에 의해서 억제되었지만, 항 Toll-like 수용기2, 항 Toll-like 수용기4 항체는 억제시키지 못했다. $NF-{\kappa}$ B 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 $NF-{\kappa}$ B 결합 활성도와 기질금속단백분해효소-8 분비를 억제하였다. 미세섬유 중합반응 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비를 억제시켰으나, 미세관 중합반응억제제는 억제시키지 못했다. 4. 결론 위의 연구결과를 종합하여 볼 때, 기질금속단백분해효소-8은 A. actinomycetemcomitans의 지질다당질로 유도되며, 세포표면 항원무리-$NF-{\kappa}$ B 경로를 통하여 분비되고, 이 분비 과정은 미세섬유 계통이 관여하는 것으로 보인다.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.35-40
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• 2002

Background: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail. Methods: In this study CD152 expression in both $CD4^+$ and $CD8^+$ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex. Results: Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were $CD152^+$ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in $CD8^+$ cells and about 60% of $CD8^+$ cells were $CD152^+$ at this stage. High molecular weight (>350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non-reducing condition. Conclusion: It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.235-241
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• 2003

Background: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-$A^*0201$ restricted CD8+ T cells ($ThyA_{30-38}$, $RpoB_{127-135}$, $85B_{15-23}$, $PstA1_{75-83}$). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. Methods: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD+healthy people using IFN-${\gamma}$elispot assay, intracellular staining and HLA-A2 dimer staining. Results: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in $1.7{\times}10^5$ PBMC based on ex vivo IFN-${\gamma}$ elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD+ people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-${\gamma}$ upon antigenic stimulation in PPD+ donors. Lastly, HLA-$A^*0201$ dimer assays indicated that $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors produced IFN-${\gamma}$ upon peptide stimulation. Conclusion: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD + people; however, the CD8+ T cell population is functionally heterogeneous.

• Applied Microscopy
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• v.30 no.2
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• pp.173-183
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• 2000

This study was designed to observe the ultrastructural localization of synoviocytes, which are concerned with the function of phagocytic synovial cells (type A synoviocytes, macrophage-like synoviocytes), in the knee joint of the human for CD14 and CD105 by cryo-immune-electron microscopic technique. The synovium were dissected and fixed for two hours (in 4% paraformaldehyde and 0.1% glutaraldehyde mixture), and were immerged in 2.3 M sucrose and 20% PVP solution. Finally, they were cut with the cryoultramicrotome and labelled with primary antibodies (monoclonal mouse anti-human CD14, monoclonal mouse anti-human CD105 (endoglin) and secondary (donkey anti-mouse IgG) tagged with 6 nm colloidal gold particles. The tissues were observed under transmission electron microscope. This study was resulted as follows. 1. In the synovium of the human knee joint, CD14+ cells were identified. These cells showed phagocytic synovial cell's features. In the phagocytic synoviocyte, the distributions of CD14 were marked in the cytoplasm, around vacuoles, and in cytoplasmic process, but not detected inside of vacuoles. 2. In the synovium of the human knee joint, CD105+ cells were identified. These cells were recognized endothelial cells and phagocytic synovial cells. In the phagocytic synovial cells, the distributions of CD105 (endoglin) were marked in cytoplasic process, around vacuoles, and in cell membrane, but not detected inside of vacuoles. On the basis of above findings, it is obvious that phagocytic synovial cells were marked at CD 14 and CD 105, and might be play the role of activated macrophages or phagocytes in the synovial membrane.

• YAKHAK HOEJI
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• v.51 no.6
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• pp.482-489
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• 2007

Kaempferitrin, isolated from Kenaf (Hibiscus cannabinus), was examined to evaluate its modulatory effects on antigen-presenting cell functions of macrophages/monocytes such as phagocytosis of foreign materials, up-regulation of costimulatory molecules (CD40, CD80 and CD86), adhesion molecule activation, and antigen processing and presentation. Kaempferitrin strongly blocked up-regulation of CD40, CD80 and CD86, but not pattern recognition receptor (PRR) (e.g., TLR2). It also suppressed functional activation of CD29 (${\beta}1$-integrins), as assessed by cell-cell adhesion assay, required for T cell-antigen-presenting cell (APC) interaction. Furthermore, this compound did not block a simple activation of CD29, as assessed by cell-fibronectin adhesion assay. However, the compound did not diminish phagocytic uptake, an initial step for antigen processing, and ROS generation in RAW264.7 cells. In particular, to understand molecular mechanism of kaempferitrin-mediated inhibition, the regulatory role of LPS-induced signaling events was examined using immunoblotting analysis. Interestingly, this compound dose dependently suppressed the phosphorylation of $I{\kappa}B{\alpha}$, Src, Akt and Syk, demonstrating that it can negatively modulate the activation of these signaling enzymes. Therefore, our data suggested that kaempferitrin may be involved in regulating APC function-relevant immune responses of macrophages and monocytes by regulating intracellular signaling.

• IMMUNE NETWORK
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• v.3 no.4
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• pp.287-294
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• 2003

Background: In kidney transplantation, donor specific transfusion may induce tolerance as a result of some immune regulatory cells against the graft. In organ transplantation, the immune state arises from a relationship between the immunocompromised graft and the immunocompetent host. However, a reverse immunological situation exists between the graft and the host in hematopoietic stem cell transplantation (HSCT). In addition, early IL-2 injections after an allogeneic murine HSCT have been shown to prevent lethal graft versus host disease (GVHD) due to CD4+ cells. We investigated the induction of the regulatory CD4+CD25+ cells after a transfusion of irradiated recipient cells with IL-2 into a donor. Methods: The splenocytes (SP) were obtained from 6 week-old BALB/c mice ($H-2^d$) and irradiated as a single cell suspension. The donor mice (C3H/He, $H-2^k$) received $5{\times}10^6$ irradiated SP, and 5,000 IU IL-2 injected intraperitoneally on the day prior to HSCT. The CD4+CD25+ cell populations in SP treated C3H/He were analyzed. In order to determine the in vivo effect of CD4+CD25+ cells, the lethally irradiated BALB/c were transplanted with $1{\times}10^7$ donor BM and $5{\times}10^6$ CD4+CD25+ cells. The other recipient mice received either $1{\times}10^7$ donor BM with $5{\times}10^6$ CD4+ CD25- cells or the untreated SP. The survival and GVHD was assessed daily by a clinical scoring system. Results: In the MLR assay, BALB/c SP was used as a stimulator with C3H/He SP, as a responder, with or without treatment. The inhibition of proliferation was $30.0{\pm}13%$ compared to the control. In addition, the MLR with either the CD4+CD25+ or CD4+CD25- cells, which were isolated by MidiMacs, from the C3H/He SP treated with the recipient SP and IL-2 was evaluated. The donor SP treated with the recipient cells and IL-2 contained more CD4+CD25+ cells ($5.4{\pm}1.5%$) than the untreated mice SP ($1.4{\pm}0.3%$)(P<0.01). There was a profound inhibition in the CD4+CD25+ cells ($61.1{\pm}6.1%$), but a marked proliferation in the CD4+CD25- cells ($129.8{\pm}65.2%$). Mice in the CD4+CD25+ group showed low GVHD scores and a slow progression from the post-HSCT day 4 to day 9, but those in the control and CD4+CD25- groups had a high score and rapid progression (P<0.001). The probability of survival was 83.3% in the CD4+CD25+ group until post-HSC day 35 and all mice in the control and CD4+CD25- groups died on post-HSCT day 8 or 9 (P=0.0105). Conclusion: Donor graft engineering with irradiated recipient SP and IL-2 (recipient specific transfusion) can induce abundant regulatory CD4+CD25+ cells to prevent GVHD.

• Journal of Chest Surgery
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• v.43 no.5
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• pp.499-505
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• 2010

Background: There have been several reports using animal experiments that CD1-restricted T-cells have a key role in tumor immunity. To address this issue, we studied the expression of markers for CD1c+ myeloid dendritic cells (DCs) isolated from peripheral blood in the clinical setting. Material and Method: A total of 24 patients with radiologically suspected or histologically confirmed lung cancer who underwent pulmonary resection were enrolled in this study. The patients were divided according to histology findings into three groups: primary adenocarcinoma of lung (PACL), primary squamous cell carcinoma of lung (PSqCL) and benign lung disease (BLD). We obtained 20 mL of peripheral venous blood from patients using heparin-coated syringes. Using flow-cytometry after labeling with monoclonal antibodies, data acquisition and analysis were done. Result: The ratio of CD1c+CD19- dendritic cells to CD1c+ dendritic cells were not significantly different between the three groups. CD40 (p=0.171), CD86 (p=0.037) and HLA-DR (p=0.036) were less expressed in the PACL than the BLD group. Expression of CD40 (p=0.319), CD86 (p=0.036) and HLA-DR (p=0.085) were less expressed in the PACL than the PSqCL group, but the differences were only significant for CD86. Expression of co-stimulatory markers was not different between the PSqCL and BLD groups. Expression of markers for activated DCs were dramatically lower in the PACL group than in groups with other histology (CD40 (p=0.005), CD86 (p=0.013) HLA-DR (p=0.004). Conclusion: These results suggest the possibility that CD1c+ myeloid DCs participate in control of the tumor immunity system and that low expression of markers results in lack of an immune response triggered by dendritic cells in adenocarcinoma of the lung.

• Journal of Veterinary Clinics
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• v.23 no.1
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• pp.9-13
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• 2006

Anaplasma phagocytophilum major surface protein-2 (Msp2 or p44) is the immunodominant outer membrane protein of the bacterium. Recently, we disclosed that Msp2 was an A. phagocytophilum adhesin for binding to host neutrophils and HL-60 cells, probably mediated by attachment to platelet selectin glycoprotein ligand-1 (PSGL-1). In this study, we further elucidated that Msp2 bound to PSGL-1/FucT IV-transfected BJAB but not nontransfected BJAB cells. Binding of recombinant Msp2 or cell (lee bacteria to the surface of PSGL-1/FucT IV-transfected BJAB cells was significantly higher than to nontransfected BJAB cells (p<0.01 and p<0.01). Also, Msp2 monoclonal antibody and soluble recombinant Msp2 as antagonist led to concentration-dependent reductions in A. phagocytophilum adhesln (p<0.05 and p<0.01) to transfected BJAB cells. Thus, we conclude that Msp2 of. A. phagocytophilum acts as an adhesin by which the bacterium binds to PSGL-1 on host neutrophils and myeloid cells.

• Biomedical Science Letters
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• v.1 no.1
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• pp.19-25
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• 1995

Characterization of immune cell subpopulations in the bovine was performed using a direct immunofluorescence technique adaptable for routine and repeated monitoring. This whole blood procedure is faster and requires less volume than conventional density gradient isolation methods. Low inter-and intra-animal variations were seen in hematology parameters and in CD4, CD8 and CD2 lymphocyte subtypes. CD4 values were 30% of lymphocytes in male and 32% in females. Thriteen percent were CD8 in males and 13% in females. CD4: CD8 ratios were approximately two in both sexes. Fiffty three percent were CD2 in males and 54% in females. The mean RBC counts of peripheral blood were 7.20$\times10^6/{mm}^3$ for male cattle and 6.36$\times10^6/{mm}^3$ for females. The mean WBC counts were 8.09$\times10^3/{mm}^3$ for males and 7.09$\times10^3/{mm}^3$ for females. The percent of lymphocytes(63-65%) was higher than the percent of neutrophils(17-18%), the percent of eosinophils(11-15%), te percent of monocytes(4-5%), and percent of basophiles(1%).

• Tuberculosis and Respiratory Diseases
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• v.45 no.4
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• pp.846-860
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• 1998

Background: Endotoxin (LPS : lipopolysaccharide), a potent activator of immune system, can induce acute and chronic inflammation through the production of cytokines by a variety of cells, such as monocytes, endothelial cells, lymphocytes, eosinophils, neutrophils and fibroblasts. LPS stimulate the mononucelar cells by two different pathway, the CD14 dependent and independent way, of which the former has been well documented, but not the latter. LPS binds to the LPS-binding protein (LBP), in serum, to make the LPS-LBP complex which interacts with CD14 molecules on the mononuclear cell surface in peripheral blood or is transported to the tissues. In case of high concentration of LPS, LPS can stimulate directly the macrophages without LBP. We investigated to detect the generation of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-$\alpha$ and fibrogenic cytokine, TGF-$\beta$, by peripheral blood mononuclear cells (PBMC) after LPS stimulation under serum-free conditions, which lacks LBPs. Methods : PBMC were obtained by centrifugation on Ficoll Hypaque solution of peripheral venous bloods from healthy normal subjects, then stimulated in the presence of LPS (0.1 ${\mu}g/mL$ to 100 ${\mu}g/mL$ ). The activities of IL-1, IL-6, TNF, and TGF-$\beta$ were measured by bioassaies using cytokines - dependent proliferating or inhibiting cell lines. The cellular sources producing the cytokines was investigated by immunohistochemical stains and in situ hybridization. Results : PBMC started to produce IL-6, TNF-$\alpha$ and TGF-$\beta$ in 1 hr, 4 hrs and 8hrs, respectively, after LPS stimulation. The production of IL-6, TNF-$\alpha$ and TGF-$\beta$ continuously increased 96 hrs after stimulation of LPS. The amount of production was 19.8 ng/ml of IL-6 by $10^5$ PBMC, 4.1 ng/mL of TNF by $10^6$ PBMC and 34.4 pg/mL of TGF-$\beta$ by $2{\times}10^6$ PBMC. The immunoreactivity to IL-6, TNF-$\alpha$ and TGF-$\beta$ were detected on monocytes in LPS-stimulated PBMC. Some of lymphocytes showed positive immunoreactivity to TGF-$\beta$. Double immunohistochemical stain showed that IL-1$\beta$, IL-6, TNF-$\alpha$ expression was not associated with CD14 postivity on monocytes. IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$mRNA expression were same as observed in immunoreactivity for each cytokines. Conclusion: When monocytes are stimulated with LPS under serum-free conditions, IL-6 and TNF-$\alpha$ are secreted in early stage of inflammation. In contrast, the secretion of TGF-$\beta$ arise in the late stages and that is maintained after 96 hrs. The main cells releasing IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$ are monocytes, but also lymphocytes can secret TGF-$\beta$.