Kazuki Murakami

THROMBIN STIMULATES PRODUCTION OF INTERLEUKIN-8 IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

Interleukin‐8 (IL‐8) is regarded as an important mediator of inflammation because of its potent and specific chemotactic activity on neutrophils. In the present investigation, human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce IL‐8, in a dose‐ and time‐dependent manner. After stimulation with 10 U/ml thrombin for 24 hr, the level of IL‐8 in the conditioned medium was 14 ng/ml, or enough to elicit PMN chemotaxis in vitro. Northern blot analysis revealed that thrombin as well as IL‐1β elevates the level of IL‐8 mRNA preceding the formation of IL‐8 protein. A synthetic peptide SFLLRN [human thrombin receptor‐activating peptide (TRAP)] was found to mimic the action of thrombin. Preincubation with anti‐thrombin compounds such as hirudin and antithrombin‐III–heparin almost completely suppressed the action of thrombin without affecting the actions of other stimuli including IL‐1β, phorbol 12‐myristate 13‐acetate (PMA) and TRAP. Diisopropylfluorophosphate‐treated thrombin did not stimulate IL‐8 production. Calphostin‐C, a protein kinase C (PKC) inhibitor, attenuated the production of IL‐8 by thrombin, TRAP and PMA, but left the action of IL‐1β unchanged. These results strongly suggest that catalytic activation of thrombin receptor by thrombin results in PKC‐dependent IL‐8 production accompanied by an increase in IL‐8 mRNA level.

Proliferative Effect of Dextran Sulfate Sodium (DSS)‐Pulsed Macrophages on T Cells from Mice with DSS‐Induced Colitis and Inhibition of Effect by IgG

The authors have previously reported that homologous immunoglobulin (Ig)G reduces the occurrence of dextran sulfate sodium (DSS)‐induced colitis, mainly by suppressing recruitment of immunocompetent cells into colitis lesions. However, the mechanisms of cell recruitment and of its suppression by IgG remain unclear. In addressing these questions, this study focused on the activation of T cells in the presence of macrophages. The authors found that [3H]‐thymidine uptake of T cells from DSS‐induced colitis mice, but not from normal mice, was significantly enhanced when cultured with DSS‐pulsed macrophages. From the profile of cytokine production, it was suggested that T helper 1 (Th1)‐type cells become predominant during stimulation. Addition of homologous IgG significantly suppressed T cell proliferation in a dose‐dependent manner, while no suppressive effect was observed with heterologous IgG. Mouse IgG F(ab′)2, but not Fc, fragments partially mimicked the suppressive effect of whole IgG. These findings provide evidence that Th1‐type cells may play an important role in the development of DSS‐induced colitis and that homologous IgG exerts its protective action at least in part through the F(ab′)2 portion.

Thrombin induces GRO α MGSA production in human umbilical vein endothelial cells

Abstract Thrombin, besides being a potent coagulation factor, exerts influence on endothelial and leukocyte functions and may thus be involved in the regulation of inflammatory reactions. The present study investigated whether thrombin stimulates the production of growth-related cytokine/melanoma growth-stimulatory activity ( GRO α MGSA ) in endothelial cells. Human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce GRO α MGSA in a dose- and time-dependent manner. This action of thrombin was completely suppressed by preincubation with either hirudin or antithrombin-III (AT-III)-heparin. Interestingly, the thrombin receptor-activating peptide SFLLRN mimicked the action of thrombin. In addition, staurosporine, a protein kinase C (PKC) inhibitor, attenuated the production of GRO α MGSA by thrombin, SFLLRN and phorbol 12-myristate 13-acetate (PMA), but left the action of interleukin-1β (IL-1β) unchanged. These results suggest that catalytic activation of thrombin receptor by thrombin results in GRO α MGSA production, at least in part, via a pathway involving PKC in HUVEC.

Metalloproteinase inhibitor prevents hepatic injury in endotoxemic mice

This study was conducted to examine of [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)-succinyl]-L- phenylalanine-N-methylamide (GI 129471), a matrix metalloproteinase inhibitor, for its effects on increase of serum pro-inflammatory cytokine levels as well as hepatic injury in D-galactosamine plus lipopolysaccharide-injected mice. In vitro experiments showed that GI 129471 was able to inhibit the elevation of tumor necrosis factor-alpha (TNF-alpha) in LPS-stimulated human and mouse whole blood with IC50 values of 370 nM and 260 nM, respectively. When administrated i.p. at 40 mg/kg, GI 129471 significantly reduced serum TNF-alpha level but not other pro-inflammatory cytokines in D-galactosamine plus lipopolysaccharide-injected mice. Treatment of mice with GI 129471 also reduced biochemical indices of hepatic injury to the normal level. Histopathological findings indicated that GI 129471 treatment can prevent severe centrilobular necrosis in liver. These results suggest that release of TNF-alpha from lipopolysaccharide-stimulated cells is the critical step leading to hepatic injury in endotoxemia and that a matrix metalloproteinase inhibitor with an inhibitory action on this step may be a promising drug for the clinical treatment of endotoxemia accompanied by hepatic injury.

Intravenous immunoglobulin preparation prevents the production of pro-inflammatory cytokines by modulating NFκB and MAPKs pathways in the human monocytic THP-1 cells stimulated with procalcitonin.

ObjectiveIn the previous investigations, we showed that intravenous immunoglobulin (IVIG) prevented cytokine release in procalcitonin (PCT)-stimulated monocytic cells. The aim of the present study was to investigate the underlying mechanisms of inhibition of IVIG on cytokine production in PCT-stimulated THP-1 cells.MethodsTHP-1 cells treated with phorbol myristate acetate were stimulated with PCT. The protein levels of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and high-mobility group box 1 (HMGB1)] in the culture supernatants were determined using enzyme-linked immunosorbent assay kits. The mRNA level of TNF-α was determined by reverse transcription-polymerase chain reaction. The phosphorylations of nuclear factor kappa B (NFκB) and the mitogen-activated protein kinases (MAPKs) were determined by Western blotting.ResultsIVIG reduced mRNA expression and protein production of TNF-α in PCT-stimulated THP-1 cells. Not only IVIG but also both the Fc fragment and the F(ab’)2 fragment inhibited PCT-induced TNF-α, IL-6, and HMGB1 production. Furthermore, IVIG and its fragments suppressed PCT-induced phosphorylations of NFκB, p38 MAPK, and c-Jun N-terminal kinase.ConclusionsOur results indicate that IVIG prevents PCT-induced cytokine production mediated by not only the Fab region but also the Fc region. The activity of IVIG and its fragments might be regulated by inhibiting NFκB and MAPKs pathways in THP-1 cells.

Intravenous immunoglobulin prevents release of proinflammatory cytokines in human monocytic cells stimulated with procalcitonin

ObjectiveThe aim of this study was to investigate whether the stimulation of monocytic cells with procalcitonin (PCT) results in the release of proinflammatory cytokines. The effects of intravenous immunoglobulin (IVIG) on the production of cytokines from the cells stimulated with PCT were also studied.Materials and methodsCultured monocytic cells [THP-1 cells treated with phorbol myristate acetate or peripheral blood mononuclear cells (PBMCs)] were stimulated with PCT. The protein levels of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and high mobility group box-1] in the culture supernatants were determined by ELISA kits. The concentration of PCT-specific IgG antibody in IVIG was measured using a specific ELISA.ResultsPCT induced the release of cytokines from THP-1 cells in a time- and dose-dependent manner. IVIG inhibited the release of cytokines from the cells stimulated with PCT. It was confirmed that IVIG also inhibited TNF-α release in the same dose range for PBMCs stimulated with PCT. The presence of PCT-specific IgG antibody was detected in the tested IVIG, which might be one of the mechanisms.ConclusionsPCT induced the release of proinflammatory cytokines from THP-1 cells and PBMCs. The function of PCT was prevented by the presence of IVIG.

Thrombin induces GROαMGSA production in human umbilical vein endothelial cells

Abstract Thrombin, besides being a potent coagulation factor, exerts influence on endothelial and leukocyte functions and may thus be involved in the regulation of inflammatory reactions. The present study investigated whether thrombin stimulates the production of growth-related cytokine/melanoma growth-stimulatory activity ( GRO α MGSA ) in endothelial cells. Human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce GRO α MGSA in a dose- and time-dependent manner. This action of thrombin was completely suppressed by preincubation with either hirudin or antithrombin-III (AT-III)-heparin. Interestingly, the thrombin receptor-activating peptide SFLLRN mimicked the action of thrombin. In addition, staurosporine, a protein kinase C (PKC) inhibitor, attenuated the production of GRO α MGSA by thrombin, SFLLRN and phorbol 12-myristate 13-acetate (PMA), but left the action of interleukin-1β (IL-1β) unchanged. These results suggest that catalytic activation of thrombin receptor by thrombin results in GRO α MGSA production, at least in part, via a pathway involving PKC in HUVEC.

POTENT INHIBITION OF THE PROINFLAMMATORY RESPONSES OF HUMAN POLYMORPHONUCLEAR CELLS BY PROSTAGLANDIN E1 BUT NOT BY PGI2 OR CARBACYCLIN

The effects of prostaglandin E1 (PGE1) on adhesion of activated human polymorphonuclear cells (HPMNs) to human umbilical vein endothelial cells (HUVECs) and on chemotaxis of HPMNs were compared with those of prostaglandin I2 (PGI2) or its chemically stable analog carbacyclin. Although HUVECs treated with PGI2 inhibited only fMLP-activated HPMN adhesion, both HUVECs and HPMNs treated with PGE1 equally inhibited HPMN adhesion, thus indicating the appropriate reactions to endothelium-leukocyte interaction by PGE1. In the presence of a nonspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), PGE1 inhibited HPMN chemotaxis induced by either fMLP or LTB4, whereas both PGI2 and carbacyclin had no effect. To examine whether the response of PMNs to PGs is species-specific, intracellular cAMP accumulation in response to PGs was determined in human, guinea-pig, and rat isolated PMNs. Although PGE1 increased cAMP levels in all PMNs tested, both PGI2 and carbacyclin increased cAMP only in rat PMNs. These results demonstrate that HPMN cAMP formation can be affected by PGE1 but not by PGI2. The findings help our understanding of the clinical use of PGs in patients with peripheral arterial occlusive diseases.

Thrombin induces production in human umbilical vein endothelial cells

Abstract Thrombin, besides being a potent coagulation factor, exerts influence on endothelial and leukocyte functions and may thus be involved in the regulation of inflammatory reactions. The present study investigated whether thrombin stimulates the production of growth-related cytokine/melanoma growth-stimulatory activity ( GRO α MGSA ) in endothelial cells. Human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce GRO α MGSA in a dose- and time-dependent manner. This action of thrombin was completely suppressed by preincubation with either hirudin or antithrombin-III (AT-III)-heparin. Interestingly, the thrombin receptor-activating peptide SFLLRN mimicked the action of thrombin. In addition, staurosporine, a protein kinase C (PKC) inhibitor, attenuated the production of GRO α MGSA by thrombin, SFLLRN and phorbol 12-myristate 13-acetate (PMA), but left the action of interleukin-1β (IL-1β) unchanged. These results suggest that catalytic activation of thrombin receptor by thrombin results in GRO α MGSA production, at least in part, via a pathway involving PKC in HUVEC.