• Tuberculosis and Respiratory Diseases
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• v.45 no.3
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• pp.553-564
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• 1998

Background: The immediate hoot response to LPS is the production of proinflammatory cytokines that act as intercellular mediators in inflammatory reactions, including acute lung injury. These "early response" cytokines transmit signals from recognition cells to target or effector cells. This host response is further amplified by the expression of leukocyte chemoattractants, growth factors, and adhesion molecules, resulting in an array of proinflammatory events. This experiment was performed to define the lung origin of proinflammatory cytokines, such as TNF-$\alpha$, IL 6 in early periods of endotoxin induced acute lung injury (ALI). Method: The healthy male Sprague-Dawley, weighted 150 - 250g, were divided into saline control (NC) and endotoxemia-induced ALI (ETX-), and leukopenic endotoxemia-induced ALI (CPA-ETX-Group) which was induced by cyclophosphamide, 70 mg/kg i.p. injection. Acute lung injury was evoked by LPS, 5 mg/kg, intravenously administered. Bronchoalveolar lavage was performed at 0, 3, 6 h after LPS-treated to estimate the influx of phagocytes and concentration of total protein, and cytokines as TNF-$\alpha$ and IL 6 by a bioassy using MIT method. We also examined the localization of TNF-$\alpha$ and IL 6 protein in endotoxemia-challenged lung tissue by immunohistochemical stain (IH). Results: The total cell, macrophage and PMN count in BALF were elavated in ETX group compared to NC(p<0.05). In CPA-ETX group, total cell and macrophage count in BALF were not changed compared to NC. but PMN count was markedly reduced and it took part in less than 0.1 % of total BAL cells (p<0.01). The protein concentration in BALF were significantly increased in ETX and CPA-ETX group Compared to NC (p<0.05), but there was significant difference between ETX- and CPA-ETX group only at 6 h (p<0.05). This observation suggested that even if PMNs are involved in the pathogenesis of acute lung injury, their role cannot be viewed as essential The concentration of TNF-$\alpha$ and IL 6 in BALF was significantly increased in the ETX- and CPA-ETX group compared to NC. There was no difference between ETX- and CPA-ETX group. In IH, anti-TNF-$\alpha$- and anti-IL 6 antibody was strongly localized at interstitial monocytes and alveolar macrophages in endotoxemia-challenged lung tissue. From above point of view, activated alveolar macrophage/monocyte considered as a prominent source of proinflammatory cytokines in endotoxemia-challenged lung injury. Conclusion: The prominent source of proinflammatory cytokines in early periods of endotoxemia-induced lung injury will be the activated resident macrophages like an alveolar macrophage and interstitial monocytes. The pulmonary macrophage/monocyte will impact the initiation and continuance of lung injury without PMNs's certain inflammatory role, particularly in endotoxemia-induced acute lung injury.

• Tuberculosis and Respiratory Diseases
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• v.53 no.5
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• pp.497-509
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• 2002

Background : The mechanisms through which cellular activation results in intracellular mycobacterial killing is only partially understood. However, in vitro studies of human immunity to Mycobacterium tuberculosis have been largely modeled on the work reported by Crowle, which is complicated by several factors. The whole blood culture is simple and allows the simultaneous analysis of the relationship between bacterial killing and the effect of effector cells and humoral factors. In this study, we attempted to determine the extent to which M. tuberculosis is killed in a human whole blood culture and to explore the role of the host and microbial factor in this process. Methods : The PPD positive subject were compared to the umbilical cord blood and patients with tuberculosis, diabetes and lung cancer. The culture is performed using heparinized whole blood diluted with a culture medium and infected with a low number of M. avium or M. tuberculosis $H_{37}Ra$ for 4 days by rotating the culture in a $37^{\circ}C$, 5% $CO_2$ incubator. In some experiments, methlprednisolone- or pentoxifyline were used to inhibit the immune response. To assess the role of the T-cell subsets, CD4+, CD8+ T-cells or both were removed from the blood using magnetic beads. The ${\Delta}$ log killing ratio was defined using a CFU assay as the difference in the log number of viable organisms in the completed culture compared to the inoculum. Results : 1. A trend was noted toward the improved killing of mycobacteria in PPD+ subjects comparing to the umbilical cord blood but there was no specific difference in the patients with tuberculosis, diabetes and lung cancer. 2. Methylprednisolone and pentoxifyline adversely affected the killing in the PPD+ subjects umbilical cord blood and patients with tuberculosis. 3. The deletion of CD4+ or CD8+ T-lymphocytes adversely affected the killing of M. avium and M. tuberculosis $H_{37}Ra$ by PPD+ subjects. Deletion of both cell types had an additive effect, particularly in M. tuberculosis $H_{37}Ra$. 4. A significantly improved mycobacterial killing was noted after chemotherapy in patients with tuberculosis and the ${\Delta}$ logKR continuously decreased in a 3 and 4 days of whole blood culture. Conclusion : The in vitro bactericidal assay by human whole blood culture model was settled using a CFU assay. However, the host immunity to M. tuberculosis was not apparent in the human whole blood culture bactericidal assay, and patients with tuberculosis showed markedly improved bacterial killing after anti-tuberculous chemotherapy compared to before. The simplicity of a whole blood culture facilitates its inclusion in a clinical trial and it may have a potential role as a surrogate marker in a TB vaccine trial.

• Tuberculosis and Respiratory Diseases
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• v.41 no.2
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• pp.87-96
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• 1994

Background : Since tumor necrosis factor was discovered in 1975, TNF has been well known about its cytotoxic effect on tumor cells in vivo and in vitro. According to the recent improvement of molecular biological techinques, it is possible that exogenous TNF gene is transferred to tumor cells and is expressed in theirs. By virtue of TNF gene transfer, we have expected that TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells in vivo without systemic side effect. The expected mechanisms in which antitumor effects of TNF expressed in TNF-gene-transferred tumor cells are working would be as followings. In the first mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells around(like homicide). In the second mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill themselves(like suicide). In the third mechanism, TNF expressed in TNF-gene-transferred tumor cells would recruit immune effector cells and kill tumor cells indirectly. In the last mechanism, TNF expressed in TNF-gene-transferred tumor cells would augment cytokine such as interferon-$\gamma$ to kill tumor cells. Among these four mechanisms of antitumor effect, only the second mechanism has not been established yet. Therefore, to elucidate the second mechanism, We performed this study. Method : We transferred TNF-$\alpha$ gene to NCI-H2058, a human mesothelioma cell line and WEHI164, a murine fibrosarcoma cell line by using retroviral vector(pLT12SNTNF). And, We determined by using MTT assay whether TNF expressed in TNF-gene-transferred tumor cell lines would kill themselves like suicide or not. Then, if TNF-gene-transferred tumor cell lines would not suicide themselves, I would know more about the TNF sensitivity of TNF-gene-transferred tumor cell lines to exogenous TNF also by MTT assay. Result : NCI-H2058 and WEHI164 which were sensitive to TNF, became far less sensitive to endogenous and exogenous TNF after being transferred TNF-$\alpha$ gene to. Conclusion : TNF-gene-transfer to NCI-H2058 and WEHI164 gave them resistance to TNF.

• Journal of Life Science
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• v.21 no.3
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• pp.393-399
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• 2011

The mast cell is one of the major effector cells in inflammatory reactions and can be found in most tissues throughout the body. Activated mast cells can produce histamine, as well as a wide variety of other inflammatory mediators such as eicosanoids, proteoglycans, proteases, and several pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-${\alpha}$, and interleukins (IL-6), IL-8, IL-4, IL-13. In the present study, we isolated two bacterial strains (J80 and G147) from fermented soybean and Jeotgal, and investigated the inhibitory effects of their extracts which were prepared by several pretreatment methods (sonication for 20 min, heating at $100^{\circ}C$ for 30 min, autoclaving at $121^{\circ}C$ for 15 min) on the mast cell-mediated inflammatory response. The pretreated bacterial extracts had no cytotoxicity against Human Mast Cell (HMC-1). Among various pretreatments, the extracts treated at $100^{\circ}C$ showed highest inhibition of histamine release (J80, 28.46%; G147, 41.14%). The J80 and G147 extracts treated at $100^{\circ}C$ resulted in the inhibition of IL-6 secretion by 38.46% and 56.45%, respectively. The J80 extract treated at $100^{\circ}C$ resulted in the inhibition of TNF-${\alpha}$ secretion by 66.67%, but G147 extract showed the highest inhibition effect by 41.1% when treated with sonication. These results suggest that bacterial extracts treated at $100^{\circ}C$ have a higher level of anti-inflammatory effects than other treatments such as sonication or autoclaving.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.379-390
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• 1997

Background : It has long been suggested that neutrophils and their products are implicated as the central mediators of the acute lung injuries. Contrary to the dominant role of neutrophils in ARDS, many cases of ARDS has occurred in the setting of severe neutropenia without pulmonary neutrophil infiltration. Therefore it is certain that effector cell(s) other than neutrophil play an important role in the pathogenesis of ARDS. This experiment was performed to define the mechanism of ARDS in the setting of neutropenia, 1) by comparing the severity of endotoxin-induced lung injury, 2) by measurement of hydrogen peroxide production and cytokine concentration in the bronchoalveolar lavage cells and fluids obtained from different rats with and without cyclophosphamide-pretreatment. Method : The male Sprague-Dawleys were divided into the normal control (NC)-, endotoxin (ETX)-, and cyclophosphamide (CPA)-group in which neutropenia was induced by injecting cyclophosphamide intraperitoneally. Acute lung injury was evoked by injecting lipopolysaccharide (LPS) into a tail vein. The bronchoalveolar lavage (BAL) was performed at 3 and 6 hour after administration of LPS to measure the change of cell counts and concentrations of protein and cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Hydrogen peroxide (HPO) production from BAL cells was measured at 6 hour after LPS administration by phenol red microassay with and without zymosan stimulation. Results : The results were as follows. A change of leukocyte counts in the peripheral blood after treatment with CPA : More than 95% of total leukocytes and neutrophils were reduced after CPA administration, resulting in severe neutropenia. A change of BAL cells : In the ETX-group, the number of total cells (p < 0.01) and of macrophage and neutrophil (p < 0.05) were increased at 3 and 6 hour after LPS administration compared to those of NC-group. In the CPA-group, the number of total leukocyte and macrophage were not changed after LPS administration, but neutrophil counts were significantly reduced and it took part in less than 0.1% of total BAL cells (p < 0.01 vs NC-group). BAL cells in this group were almost all macrophages (99.7%). A change of protein concentration in the BALF : In the ETX-group, protein concentration was increased at 3 hour and was more increased at 6 hour after LPS administration (p < 0.05 and < 0.01 vs NC-group, respectively). In the CPA-group, it was also significantly elevated at 3 hour after LPS administration (p < 0.05 vs NC-group), but the value was statistically not different from that of ETX-group. The value measured at 6 hour after LPS administration in the CPA-group became lower than that of ETX-group (p < 0.05), but showed still a higher value compared to that of NC-group (p < 0.05). A change of cytokine concentration in the BALF : TNF -alpha and IL-6 were elevated in the ETX - and CPA-group compared to those of NC-group at both time intervals. There was no statistical difference in the values of both cytokines between the ETX- and CPA-groups. Measurement of hydrogen peroxide production from BAL cells : There was no intergroup difference of HPO production from resting cells. HPO production after incubation with opsonized zymosan was significantly elevated in all groups. The percent increment of HPO production was highest in the ETX-group (89.0%, p < 0.0008 vs NC-group), and was 42.85 in the CPA-group (p = 0.003 vs NC-group ). Conclusion : Acute lung injury in the setting of neutropenia might be caused by functional activation of resident alveolar macrophages.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.360-378
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• 1997

Background : Severe acute lung injury(ALI), also known as the adult respiratory distress syndrome(ARDS), is a heterogenous nature of dynamic and explosive clinical synrome that exacts a mortality of approximately 50%. Endotoxin(ETX) is an abundant component of the outer membrane of gram-negative bacteria capable of inducing severe lung injury in gram-negative sepsis and gram-negative bacterial pneumonia, which are among the most common predisposing causes of ARDS. The influx of PMNs into airway tissue is a pathological hallmark of LPS-induced lung injury. And there is a substantial evidence suggesting that cytokines are important mediators of lung injury in gram-negative sepsis. However, the kinetics of phagocytes and cytokines by an exact time sequence and their respective pathogenic importance remain to be elucidated. This study was performed to investigate the role of phagocytes and proinflammatory cytokines in ETX-induced ALI through a time course of changes in the concentration of protein, $TNF{\alpha}$ and IL-6, and counts of total and its differential cells in BALF. The consecutive histologic findings were also evaluated. Method : The experimental animals, healthy male Sprague-Dawley, weighted $200{\pm}50g$, were divided into control- and ALI- group. ALI was induced by an intravenous administration of ETX, 5mg/kg. Above mentioned all parameters were examined at 0(control), 3, 6, 24, 72 h after administration of ETX. $TNF{\alpha}$ and IL-6 cone. in BALF were measured by a bioassay. Results : The protein concentration and total leukocyte count(TC) in BALF was significantly increased at 3h compared to controls(p < 0.05). The protein conc. was significantly elavated during observation period, but TC was significantly decreased at 72h(p < 0.05 vs. 24h). There was a close relationship between TC and protein cone. in BALF(r = 0.65, p < 0.001). The PMN and monocyte count was well correlated with TC in BALF, and the correlation of PMN(r = 0.97, p < 0.001) appeared to be more meaningful than that of monocyte(r = 0.61, p < 0.001). There was also a significant correlation between protein cone. and PMN or monocyte count in BALF(PMN vs. monocyte : r = 0.55, p < 0.005 vs. r = 0.64, p < 0.001). The count of monocyte was significantly elavated during observation period though a meaningful reduction of PMN count in BALF at 72h, this observation suggested that monocyte may, at least, partipate in the process of lung injury steadly. In this study, there was no relationship between IL-6 and $TNF{\alpha}$ cone., and $TNF{\alpha}$ but not IL-6 was correlated with TC(r = 0.61, p < 0.05) and monocyte(r = 0.67, p < 0.05) in BALF only at 3, 6h after ETX introduced. In particular, the IL-6 cone. increased earlier and rapidly peaked than $TNF{\alpha}$ cone. in BALF. In histologic findings, the cell counts of lung slices were increased from 3 to 72h(p < 0.001 vs. NC). Alveolar wall-thickness was increased from 6 to 24h(p < 0.001 vs. NC). There was a significant correlation between the cell counts of lung slices and alveolar wall-thickness(r= 0.61, p < 0.001). This result suggested that the cellular infiltrations might be followed by the alterations of interstitium, and the edematous change of alveolar wall might be most rapidly recovered to its normal condition in the process of repair. Conclusion : We concluded that although the role of PMN is partly certain in ETX-induced ALI, it is somewhat inadequate to its known major impact on ALL Alveolar macrophage and/or non-immune cells such as pulmonary endothelial or epithelial cells, may be more importantly contributed to the initiation and perpetual progression of ETX-induced ALI. The IL-6 in ETX-induced ALI was independent to $TNF{\alpha}$, measured by a bioassay in BALF. The early rise in IL-6 in BALF implies multiple origins of the IL-6.