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ABSTRACT: Urethane, which is used as an anesthetic for animal experiments, causes inflammation and cancer in the lung. BALB/c mice received 1 mg/g of urethane once a week for four consecutive weeks via intraperitoneal injections developed interstitial infiltration of inflammatory cells and tumors in the lung. However, the intracellular signaling events which urethane causes inflammation and cancer are largely unknown. Here we show that urethane caused overproduction of reactive oxygen species (ROS) in RAW 264.7 macrophages and A549 lung epithelial cells. Pretreatment of these cells with the antioxidant N-acetylcysteine attenuated the urethane-induced ROS production. Urethane increased heme oxygenase-1 expression to protect these cells from cytotoxicity caused by overproduced ROS. In addition, urethane activated extracellular signal-regulated kinase (ERK) in both cell types. Overall, our data imply that urethane stimulates ROS production and ERK activation in macrophages and lung epithelial cells, and the overproduced ROS and activated ERK may promote tumor formation in the lung.
Archives of Pharmacal Research 04/2013; · 1.59 Impact Factor
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Kyoung Soo Kim,
Hyun-Mi Choi,
Hye-In Ji, Chaekyun Kim,
Jung Yeon Kim,
Ran Song,
So-Mi Kim,
Yeon-Ah Lee,
Sang-Hoon Lee,
Hyung-In Yang,
Myung Chul Yoo,
Seung-Jae Hong
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ABSTRACT: We investigated whether taurine chloramine (TauCl), which is -endogenously produced by immune cells such as macrophages that infiltrate adipose tissue, affects the differentiation of preadipocytes into adipocytes or modulates the expression of adipokines in adipocytes. To study the physiological effects of TauCl on human adipocyte differentiation and adipokine expression, preadipocytes were cultured under differentiation conditions for 14 days in the presence or the absence of TauCl. Differentiated adipocytes were also treated with TauCl in the presence or the absence of IL-1β (1 ng/ml) for 7 days. The culture supernatants were analyzed for adipokines such as adiponectin, leptin, IL-6, and IL-8. At concentrations of 400-600 μM, TauCl significantly inhibited the differentiation of human preadipocytes into adipocytes in a dose-dependent manner. It did not induce the dedifferentiation of adipocytes or inhibit fat accumulation in adipocytes. Expression of major transcription factors of adipogenesis and adipocyte marker genes was decreased after treatment with TauCl, in agreement with its inhibition of -differentiation. These results suggest that TauCl may inhibit the differentiation of -preadipocytes into adipocytes. Thus, TauCl or more stable derivatives of TauCl could potentially be a safe drug therapy for obesity-related diseases.
Advances in experimental medicine and biology 01/2013; 775:247-57. · 1.09 Impact Factor
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ABSTRACT: Taurine chloramine (TauCl) is produced by activated neutrophils in the inflammatory joint cavities of rheumatoid arthritis and is known to have anti-inflammatory and anti-arthritic effects. However, the mechanisms underlying the anti-arthritic effect of TauCl have not been elucidated. Here, we investigated that mechanism using a collagen-induced arthritis (CIA) mouse model. DBA/1J mice were immunized with bovine type II collagen to induce CIA and were given daily subcutaneous injections of TauCl from the day of first collagen immunization. Severity of arthritis was scored by paw swelling and the arthritis score system. At the 8th week, mice were sacrificed for histological examination using hematoxylin and eosin, safranin-O and tartrate-resistant acid phosphatase (TRAP) staining. Effects of TauCl on osteoclastogenesis from bone marrow-derived preosteoclasts and proliferation of the lymphocytes obtained from spleens of CIA mice were determined. TauCl significantly attenuated the severity of paw swelling and reduced arthritis score in CIA mice. TauCl treated CIA mice showed significant reductions of synovial inflammation, cartilage damage and bone erosion. The number of TRAP-positive cells in the joints of TauCl treated CIA mice was reduced. TauCl inhibited osteoclastogenesis from the RANKL treated bone marrow-derived preosteoclasts in a dose-dependent manner. TauCl also inhibited the proliferation of splenic lymphocytes obtained from CIA mice. In conclusion, TauCl attenuated the severity of CIA by inhibiting lymphocyte proliferation and osteoclastogenesis. Combined our results suggest that TauCl produced endogenously in inflamed joints may suppress the development of rheumatoid arthritis and that TauCl may be used for therapeutic treatment of rheumatoid arthritis.
European journal of pharmacology 07/2011; 668(1-2):325-30. · 2.59 Impact Factor
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ABSTRACT: Taurine chloramine is the major chloramine generated in activated neutrophils via the reaction between the overproduced hypochlorous acid and the stored taurine. Taurine chloramine has anti-inflammatory and cytoprotective effects in inflamed tissues by inhibiting the production of inflammatory mediators. Taurine chloramine increases heme oxygenase activity and also protects against hydrogen peroxide (H(2)O(2))-derived necrosis in macrophages. In this study, we examined further whether taurine chloramine could protect RAW 264.7 macrophages from apoptosis caused by H(2)O(2). Macrophages treated with 0.4 mM H(2)O(2) underwent apoptosis without showing immediate signs of necrosis, and the cells pretreated with taurine chloramine were protected from the H(2)O(2)-derived apoptosis. Taurine chloramine increased heme oxygenase-1 expression and heme oxygenase activity. The taurine chloramine-derived upregulation of heme oxygenase-1 expression was blocked by inhibition of ERK phosphorylation. Taurine chloramine decreased cellular glutathione (GSH) levels initially, but the GSH level increased above the control level by 10 h. Taurine chloramine also increased catalase expression and protected macrophages from the apoptotic effect of H(2)O(2). Combined, these results indicate that the taurine chloramine, produced and released endogenously by the activated neutrophils, can protect the macrophages in inflamed tissues from the H(2)O(2)-derived apoptosis not only by increasing the expression of cytoprotective enzymes like heme oxygenase-1 and catalase, but also by increasing the intracellular antioxidant GSH level.
Journal of Clinical Biochemistry and Nutrition 07/2011; 49(1):50-6. · 1.98 Impact Factor
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ABSTRACT: Taurine chloramine (TauCl) is produced abundantly in activated neutrophils by a reaction between the stored taurine and the newly produced HOCl by the myeloperoxidase system, and is much less oxidizing or toxic than HOCl. TauCl has been shown to provide cytoprotection against inflammatory tissue injury by inhibiting the overproduction of inflammatory mediators. The result of this study shows that TauCl upregulated the expression of heme oxygenase (HO)-1 and increased HO activity in RAW 264.7 macrophages, while taurine had no effect. TauCl by itself generated reactive oxygen species (ROS) in macrophages and diminished total glutathione (GSH) level initially. TauCl increased the nuclear translocation of NF-E2-related factor 2 (Nrf2) and enhanced its binding to the anti-oxidant response element (ARE). This, in turn, was responsible for the upregulation of HO-1 expression. In summary, TauCl generated ROS in RAW 264.7 macrophages and decreased cellular GSH level initially. This was responsible for the nuclear translocation of Nrf2 and its binding to ARE promoted the expression of HO-1 and increased HO activity. Thus, TauCl-derived elevation of HO activity may play an essential role in the adaptive cytoprotection of inflammatory tissues.
International immunopharmacology 04/2010; 10(4):440-6. · 2.21 Impact Factor
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ABSTRACT: Adiponectin greatly stimulated the expression of matrix metalloproteinases (MMPs) in fibroblast-like synoviocytes (FLSs) as did IL-1beta. We wondered whether taurine chloramine (TauCl) inhibits the production of MMPs stimulated by adiponectin in the same pattern as by IL-1beta stimulation in vitro
Synovial cells from rheumatoid arthritis (RA) patients were treated with adiponectin or interleukin (IL)-1beta for 24 hr in the presence or absence of TauCl. The culture supernatant was collected and the levels of MMPs were measured by enzyme-linked immunosorbent assay (ELISA). The IkappaB signaling pathways stimulated by adiponectin were studied and the levels of NF-kappaB in the nuclei of the cells were analyzed by ELISA.
TauCl (600 microM) inhibited MMP-13, but not MMP-1, expression in IL-1beta-stimulated RA FLSs. However, TauCl at the same concentration significantly inhibited the production of both adiponectin-stimulated MMP-1 and MMP-13 expression. TauCl inhibited the degradation of IkappaB-alpha stimulated by adiponectin, but not by IL-1beta. Similarly, the level of NF-kappaB in the nucleus was increased by adiponectin stimulation and was inhibited by 600 microM TauCl. However, the levels of NF-kappaB increased by IL-1beta stimulation were not inhibited by 600 microM TauCl.
TauCl more effectively inhibited MMPs expression induced by adiponectin than that by IL-1beta in RA FLS, suggesting that TauCl plays an important role in down-regulating the expression of MMPs in arthritic joints.
Journal of Biomedical Science 01/2010; 17 Suppl 1:S27. · 2.01 Impact Factor
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ABSTRACT: Hypochlorous acid (HOCl) is toxic and causes cell death. However, this effect is inhibited by reaction with taurine, which generates taurine chloramine (TauCl), thereby protecting the cells from HOCl-generated toxicity. TauCl has been shown to inhibit the production of inflammatory mediators like O(2) (*-), H(2)O(2) and NO. In this study, RAW 264.7 macrophages treated with TauCl were protected from death caused by H(2)O(2). TauCl increased the expression of peroxiredoxin-1, thioredoxin-1 and heme oxygenase (HO)-1, the anti-oxidant enzymes normally induced by activation of NF-E2-related factor-2 (Nrf2). TauCl increased nuclear translocation of Nrf2 and binding to the anti-oxidant response element. These data suggest that TauCl produced abundantly in the activated neutrophils and released to surrounding cells in the inflamed tissues may induce the expression of cytoprotective anti-oxidant enzymes. Elevation of HO activity via induction of HO-1 expression within neighboring cells may provide protection from cytotoxicity caused by inflammatory oxidants like H(2)O(2).
Journal of Clinical Biochemistry and Nutrition 08/2009; 45(1):37-43. · 1.98 Impact Factor
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ABSTRACT: Taurine is abundantly present in phagocytic cells and provides protection against cytotoxicity caused by reactive oxygen species (ROS). The reaction between taurine and HOCl, a toxic product of the myeloperoxidase (MPO) system, generates a more stable and less toxic product, taurine chloramine (TauCl). TauCl has also been shown to inhibit the production of superoxide anion (O2-) and nitric oxide (NO). In this review, we compare the effect of taurine and TauCl on the production of these reactive species in phagocytes. First, TauCl inhibit PMA-derived O2- production and this is associated with inhibition of p47phox phosphorylation and of p47phox and p67phox translocation. Second, TauCl inhibits LPS-induced iNOS expression and NO production. This occurs by direct inhibition of Ras activation, ERK1/2 phosphorylation and NF-kappaB activation. Third, TauCl by itself increases the expression of heme oxygenase-1 (HO-1) and enhances HO activity. Carbon monoxide (CO), a product of HO activity, is able to inhibit both O2- and NO production. Combined, these effects of TauCl appear to provide cytoprotection against the inadvertent cytotoxicity caused by overproduction of O2- and NO.
Advances in experimental medicine and biology 02/2009; 643:463-72. · 1.09 Impact Factor
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ABSTRACT: Inflammatory cells use glucose as a primary source of metabolic energy, and thus increased uptake of glucose and high rates of glycolysis are characteristics of inflamed cells. Taurine chloramine (TauCl) is the product of a reaction between cellular taurine and hypochlorous acid (HOCl/OCl-), the latter produced by the halide-dependent myeloperoxidase (MPO) system in inflammatory cells. Taurine, a major metabolite of cysteine, protects cells from inflammatory injury by removing toxic hypochlorous acid formed by the MPO system, and also by inhibiting the production of inflammatory mediators. In the present study, we examined the effect of TauCl on glucose uptake and the expression of the glucose transporter 1 (GLUT1) in RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS). Glucose uptake was measured by employing labeled glucose analogue [18F]-2-fluoro-2-deoxy-D-glucose (FDG). Stimulation RAW 264.7 cells with LPS increased glucose uptake and led to an upregulation in GLUT1 expression, effects that were abrogated in macrophages treated with TauCl. These data suggest that TauCl can inhibit LPS-mediated enhancement of glucose uptake through inhibition of the upregulation of glucose transporter expression in activated macrophages. This represents one of the mechanisms by which TauCl modulates inflammatory cell function.
Advances in experimental medicine and biology 02/2009; 643:473-80. · 1.09 Impact Factor
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ABSTRACT: Fluoxetine is a selective serotonin reuptake inhibitor that is widely used in the treatment of major depression including after stroke. In this study, we tested whether fluoxetine protects neuronal death in a rat cerebral ischemia model of middle cerebral artery occlusion (MCAO). The administration of fluoxetine intravenously (10 mg/kg) at 30 min, 3 hr, or 6 hr after MCAO reduced infarct volumes to 21.2+/-6.7%, 14.5+/-3.0%, and 22.8+/-2.9%, respectively, of that of the untreated control. Moreover, the neuroprotective effect of fluoxetine was evident when it was administered as late as 9 hr after MCAO/reperfusion. These neuroprotective effects were accompanied by improvement of motor impairment and neurological deficits. The fluoxetine-treated brain was found to show marked repressions of microglia activation, neutrophil infiltration, and proinflammatory marker expressions. Moreover, fluoxetine suppressed NF-kappaB activity dose-dependently in the postischemic brain and also in lipopolysaccharide-treated primary microglia and neutrophil cultures, suggesting that NF-kappaB activity inhibition explains in part its anti-inflammatory effect. These results demonstrate that curative treatment of fluoxetine affords strong protection against delayed cerebral ischemic injury, and that these neuroprotective effects might be associated with its anti-inflammatory effects.
Journal of Neuroscience Research 11/2008; 87(4):1037-45. · 2.74 Impact Factor
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Yu Li,
Jingliang Yan,
Pradip De,
Hua-Chen Chang,
Akira Yamauchi,
Kent W Christopherson,
Nivanka C Paranavitana,
Xiaodong Peng, Chaekyun Kim,
Veerendra Munugalavadla,
Veerendra Munugulavadla,
Reuben Kapur,
Hanying Chen,
Weinian Shou,
James C Stone,
Mark H Kaplan,
Mary C Dinauer,
Donald L Durden,
Lawrence A Quilliam
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ABSTRACT: The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a-/- embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a-/- mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a-/- mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.
The Journal of Immunology 01/2008; 179(12):8322-31. · 5.79 Impact Factor
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Kyoung Soo Kim,
Eun Kyung Park,
Seung Min Ju,
Hye-Sook Jung,
Jun Soo Bang, Chaekyun Kim,
Yeon-Ah Lee,
Seung-Jae Hong,
Sang-Hoon Lee,
Hyung-In Yang,
Myung Chul Yoo
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ABSTRACT: It has been suggested that taurine chloramine (TauCl) plays an important role in the downregulation of proinflammatory mediators. However, little is known about its effect on the expression of matrix metalloproteinases (MMPs). In this study, we investigated the effects of TauCl on synovial expression of MMPs. The effects of TauCl on MMP expression in IL-1beta stimulated fibroblast-like synoviocytes (FLSs) were studied using the following techniques. Real-time PCR and semi-quantitative PCR were employed to analyze the mRNA expression of MMPs. ELISA was used to determine protein levels of MMPs. Western blot analyses were performed to analyze the mitogen-activated protein kinase and inhibitor of nuclear factor-kappaB (IkappaB) kinase signalling pathways. Finally, electrophoretic mobility shift assay and immunohistochemistry were used to assess localization of transcription factors. IL-1beta increased the transcriptional and translational levels of MMP-1 and MMP-13 in rheumatoid arthritis FLSs, whereas the levels of MMP-2 and MMP-9 were unaffected. TauCl at a concentration of 400 to 600 micromol/l greatly inhibited the transcriptional and translational expression of MMP-13, but the expression of MMP-1 was significantly inhibited at 800 micromol/l. At a concentration of 600 micromol/l, TauCl did not significantly inhibit phosphorylation of mitogen-activated protein kinase or IkappaB degradation in IL-1beta stimulated rheumatoid arthritis FLSs. The degradation of IkappaB was significantly inhibited at a TauCl concentration of 800 micromol/l. The inhibitory effect of TauCl on IkappaB degradation was confirmed by electrophoretic mobility shift assay and immunochemical staining for localization of nuclear factor-kappaB. TauCl differentially inhibits the expression of MMP-1 and MMP-13, and inhibits expression of MMP-1 primarily through the inhibition of IkappaB degradation, whereas it inhibits expression of MMP-13 through signalling pathways other than the IkappaB pathway.
Arthritis research & therapy 02/2007; 9(4):R80. · 4.27 Impact Factor
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ABSTRACT: Neutrophils produce microbicidal oxidants to destroy the invading pathogens using nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a membrane-associated enzyme complex that generates superoxide anion (O(2)(-)). Upon stimulation, the cytosolic components of NADPH oxidase, p47(phox) and p67(phox) and the small GTPase Rac move to phagosomal and plasma membranes where they become associated with the membrane components of NADPH oxidase, gp91(phox) and p22(phox) and express enzyme activity. We previously showed that taurine chloramine (Tau-Cl) inhibits O(2)(-) production in mouse peritoneal neutrophils (Kim, 1996). In the present study, we investigated the mechanisms underlying Tau-Cl-derived inhibition on O(2)(-) production using a human myeloid leukemia cell line, PLB-985 cell, which has been differentiated into neutrophil-like cell. Tau-Cl inhibited the phorbol myristate acetate (PMA)-elicited O(2)(-) production as previously observed in murine peritoneal neutrophils. Translocation of p47(phox), p67(phox) and Rac was increased in response to PMA, and Tau-Cl inhibited the PMA-stimulated translocation of p47(phox) and p67(phox) to plasma membrane without affecting the translocation of Rac. In addition, Tau-Cl inhibited the PMA-derived phosphorylation of p47(phox), a requirement for the translocation of cytosolic NADPH oxidase component to the plasma membrane. These results suggest that Tau-Cl inhibits PMA-elicited O(2)(-) production in PLB-985 granulocytes by inhibiting phosphorylation of p47(phox) and translocation of p47(phox) and p67(phox), eventually blocking the assembly of NADPH oxidase complex.
International Immunopharmacology 10/2006; 6(9):1431-40. · 2.38 Impact Factor
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Advances in experimental medicine and biology 02/2006; 583:493-8. · 1.09 Impact Factor
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Chaekyun Kim
Advances in experimental medicine and biology 02/2006; 583:213-7. · 1.09 Impact Factor
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ABSTRACT: Human newborn infants display a variety of immunodeficiencies of immaturity, including diminished neutrophil adhesion, chemotaxis, and migration. Rac2, a guanosine triphosphate-binding protein, is an essential regulator of human neutrophil migration and chemotaxis. Since human subjects and mice deficient in Rac2 display deficiencies in neutrophil functions similar to newborn infants, we postulated that newborn neutrophils may be deficient in Rac2.
The aim of the study was to measure Rac1 and Rac2 concentrations in neutrophils from umbilical cord blood.
Neutrophils from cord and adult blood were isolated, total cell lysates extracted, and Rac protein concentrations determined using Western blot analysis.
Rac2 concentrations were significantly lower in the neutrophil protein lysates isolated from cord blood compared to adult blood despite similar levels of Rac1.
Diminished Rac2 expression in cord blood neutrophils may contribute to the defects observed in cord blood neutrophil function.
Biology of the Neonate 02/2006; 90(3):156-9. · 1.90 Impact Factor
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ABSTRACT: Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.
Biochemical Pharmacology 02/2006; 71(3):307-18. · 4.70 Impact Factor
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ABSTRACT: Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.
Journal of Leukocyte Biology 02/2006; 79(1):223-34. · 4.99 Impact Factor
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ABSTRACT: Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O2(-). However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620nm. The absorbance of dissolved NBT increased in proportion to cell number (r = 0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O2(-). The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.
Journal of Immunoassay and Immunochemistry 02/2006; 27(1):31-44. · 0.69 Impact Factor
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ABSTRACT: Taurine is an abundant free amino acid in inflammatory cells that protects cells from inflammatory damages. Although the protection mechanism remains unclear, taurine chloramine (Tau-Cl) produced by the reaction between taurine and hypochlorous acid in neutrophils plays an important role. In this study, we investigated the mechanism(s) by which Tau-Cl inhibits LPS-induced NO production in macrophages. Tau-Cl inhibited LPS-induced iNOS expression and NO production in RAW 264.7 cells. LPS treatment elevated the level of active Ras-GTP, and Tau-Cl inhibited LPS-induced Ras activation. Tau-Cl also inhibited ERK1/2 activation in a dose-dependent manner in both RAW 264.7 cells and murine peritoneal macrophages, whereas it did not exert any effect on p38 MAPK activation. Furthermore, Tau-Cl inhibited NF-kappaB activation without affecting AP-1 activity. These results suggest that Tau-Cl suppresses LPS-induced NO production by inhibiting specific signaling pathways. Thus, Tau-Cl protects cells from inflammatory injury resulting from overproduction of NO in a signaling pathway-specific manner.
Biochemical Pharmacology 12/2005; 70(9):1352-60. · 4.70 Impact Factor
