Kayoko Maehara

Inactivation of interleukin-8 by aminopeptidase N (CD13).

Aminopeptidase (APN) was found to degrade interleukin‐8 (IL‐8) and inactivate its chemotactic activity. The chemotactic activity of IL‐8 was decreased by APN or neutrophil plasma membranes dose‐ and time‐dependently. The chemotactic activity was not inactivated in the presence of bestatin or WM15 monoclonal antibody. The expression of IL‐8 was measured by flow cytometry. On lipopolysaccharide (LPS) stimulation, IL‐8 expression increased for 60 min and then decreased markedly. In contrast, on treatment with LPS and bestatin, the expression of IL‐8 increased continuously for at least 120 min. These results suggest that the expression and release of IL‐8 from phagocytic cells are regulated by the proteolytic effect of APN on IL‐8. J. Leukoc. Biol. 57: 129–134; 1995.

Interleukin-8 induces cervical ripening in rabbits

OBJECTIVE The purpose of this study was to determine whether cervical ripening can be induced in rabbits by interleukin-8. STUDY DESIGN Nonpregnant and pregnant rabbits were treated for 5 days with vaginal suppositories containing 100 ng of interleukin-8. Collagen and glycosaminoglycan concentration in the cervices were assessed histologically by picrosirius red and alcian blue, and the mean optical density was calculated. The mean neutrophil count in five random fields was calculated from each biopsy specimen. RESULTS Interleukin-8 induced softening and dilatation of the rabbit cervices. Water content was significantly increased (p < 0.0001 and p < 0.001, respectively). Cervical collagen concentration was found to be significantly decreased (p < 0.0004 and p < 0.0001, respectively). Glycosaminoglycan concentration was significantly increased in nonpregnant and pregnant cervices (p < 0.0009 and p < 0.1, respectively). The mean number of neutrophils was significantly increased (p < 0.0005 and p < 0.006, respectively). CONCLUSION Interleukin-8 can induce cervical ripening in nonpregnant and pregnant rabbits.

Why (−)deprenyl prolongs survivals of experimental animals: Increase of anti-oxidant enzymes in brain and other body tissues as well as mobilization of various humoral factors may lead to systemic anti-aging effects

(--)Deprenyl, a monoamine oxidase B (MAO B) inhibitor is known to upregulate activities of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in brain dopaminergic regions. The drug is also the sole chemical which has been repeatedly shown to increase life spans of several animal species including rats, mice, hamsters and dogs. Further, the drug was recently found to enhance anti-oxidant enzyme activities not only in brain dopaminergic regions but also in extra-brain tissues such as the heart, kidneys, adrenal glands and the spleen. We and others have also observed mobilization of many humoral factors (interferone (INF)-gamma, tumor necrosis factor (TNF)-alpha, interleukine (IL)-1beta,2,6, trophic factors, etc.) and enhancement of natural killer (NK) cell functions by (-)deprenyl administration. An apparent extension of life spans of experimental animals reported in the past may be better explained by these new observations that (-)deprenyl upregulate SOD and CAT activities not only in the brain but also in extra-brain vital organs and involve anti-tumorigenic as well as immunomodulatory effect as well. These combined drug effects may lead to the protection of the homeostatic regulations of the neuro-immuno-endocrine axis of an organism against aging.

Stretching of fetal membranes increases the concentration of interleukin-8 and collagenase activity.

OBJECTIVE The aim of this study was to determine whether stretching of fetal membranes can increase interleukin-8 concentration and collagenase activity. STUDY DESIGN Strips of whole fetal membranes, amnion, or muscles of the lower uterine segment were stretched for 2 or 4 hours. Interleukin-8 and collagenase activity were measured in homogenized control and stretched samples. Immunohistochemical staining for interleukin-8 was carried out. RESULTS The interleukin-8 concentration increased significantly after the whole fetal membranes were stretched for 2 and 4 hours (p <0.0007 and 0.001, respectively). Also, stretching of amnion and muscles of the lower uterine segment for 2 and 4 hours increased the concentration of interleukin-8 significantly (p <0.0007 after 2 and 4 hours, respectively). Collagenase activity was significantly increased after stretching of amnion, amniochorion, and muscles of the lower uterine segment for 4 hours (p <0.0007, 0.006, and 0.0007, respectively). After stretching, samples were darkly stained for interleukin-8 compared with control nonstretched samples. CONCLUSION Stretching of amnion, amniochorion, and muscles of the lower uterine segment increased interleukin-8 production and collagenase activity.

Cold-induced stress stimulates the sympathetic nervous system, causing hypertension and proteinuria in rats

Objective To determine whether cold-stress stimulation of the soles of the paws would produce a preeclampsialike syndrome in rats. Methods Pregnant or nonpregnant rats were kept in 0°C floor and 23°C room temperature cages (the cold-stressed group) or in 23°C floor and 23°C room temperature cages (the control group) for 2 weeks. Their blood pressure, proteinuria, and plasma catecholamines were measured, and histologic studies were performed on all groups. Results There were no significant differences in systolic blood pressure between the two groups during the first week of the experimental period; however, during the last week of gestation the blood pressure of the cold-stressed group did not fall and was significantly higher than that of the control group. A significant increase in urinary protein excretion was observed in the cold-stimulated pregnant rats, in contrast to the control rats. The concentrations of norepinephrine and epinephrine in the cold-stressed pregnant rats were markedly higher than those in the control rats. A decrease in trophoblast invasion, congestion, and fibrinoid deposits of the labyrinth were observed in the cold-stressed rats. A marked increase in subendothelial fibrinoid deposits in the glomerular capillary was found only in the cold- stressed pregnant rats. The blood pressure, biochemical parameters, and histologic findings in the nonpregnant rats were almost the same as those in the pregnant rats. Conclusion Chronic local cold stimulation of the soles of the paws induces preeclampsia-like phenomena in pregnant and nonpregnant rats, and this model suggests that the cause of preeclampsia is involved in chronic stimulation of the sympathetic nerve.

A NF-?B p65 subunit is indispensable for activating manganese superoxide: Dismutase gene transcription mediated by tumor necrosis factor-?

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), and lipopolysaccharide (LPS). Recently, a TNF‐responsive element (TNFRE) was identified within the second intron of the murine Mn‐SOD gene. The 5′ CCAAT/enhancer binding protein (C/EBP)‐related region within the TNFRE was responsive to TNF, whereas the 3′ NF‐κB‐related region alone was not. This report describes the minimal promoter region of the Mn‐SOD gene and investigates the cis‐acting elements and trans‐acting factors responsible for TNF‐α‐induced Mn‐SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF‐α stimulation, p50 and p65 NF‐κB subunits bound specifically to the newly identified NF‐κB transcription factor‐binding site, distinct from the previously described NF‐κB site, within the intronic enhancer region. In addition, site‐directed mutagenesis and cotransfection studies demonstrated that the NF‐κB p65 subunit enhanced the transcriptional activity of the Mn‐SOD gene through the newly identified NF‐κB site. These results show that a NF‐κB p65 subunit is mainly involved in the molecular mechanisms controlling TNF‐α‐mediated Mn‐SOD gene transcription. J. Cell. Biochem. 77:474–486, 2000.

Cooperative interaction of NF-κB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-γ

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by pro‐inflammatory cytokines. We investigated the cis‐acting elements within a tumor necrosis factor‐responsive element (TNFRE) which was identified in the second intron of the murine Mn‐SOD gene. Site‐directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF‐κB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis‐acting elements may be required for induction of Mn‐SOD gene expression mediated by lipopolysaccharide and interferon‐γ.

Effects of histone acetylation on transcriptional regulation of manganese superoxide dismutase gene.

To better understand the link between chromatin modification and manganese superoxide dismutase (Mn-SOD) gene expression, we have investigated the level of histone acetylation at Mn-SOD proximal promoter. TSA induced the expression of Mn-SOD mRNA and its transcriptional activity in C2C12 cells. Sp1 binding sites in the proximal promoter region of Mn-SOD were transcriptionally responsive to TSA by transfection studies. We have detected a localized acetylation of histones H3 and H4, in vivo occupation by Sp1, early growth responsive-1 (Egr-1), and histone deacetylase-1 (HDAC1) in the proximal promoter region of Mn-SOD gene using chromatin immunoprecipitation assays. Our findings indicate that Mn-SOD gene expression is repressed by Sp1-HDAC1 complex. This repression is released by a localized histone acetylation and at least in parts a displacement by Egr-1 in response to TSA.

Activated neutrophil by endothelin-1 caused tissue damage in human umbilical cord

Immunostaining of human neutrophils incubated with endothelin-1 (ET-1) showed intense and spreading pattern of anti human granulocyte elastase within the cytosol. That reflected neutrophil activation followed by the release of granule contents by ET-1. In contrast, PBS (phosphate buffered saline) treated neutrophils showed localized and faintly stained granules. Intracellular calcium in fura-2 loaded neutrophils was measured at 340/380 nm. A dose and time dependent increase in intracellular calcium by ET-1 occurred in human single neutrophil. Elastase activity assay was done with chromogenic substrate S2484. ET-1 induces dose and time dependent increase in elastase activity in neutrophil suspensions like ionophore A23187. A similar time dependent increase in elastase activity was retained even after repeated wash and ET-1 treatment. That confirmed the viability of most of the neutrophils after each treatment. In umbilical cord preparations, ET-1 treated neutrophils could migrate from the venous lumen into the tissue matrix of the umbilical cords. Hematoxylin and eosin staining revealed a massive tissue destruction in ET-1 activated neutrophil treated cords when compared to sham control and untreated neutrophil injected cords. Immunostaining with monoclonal anti human elastase revealed an intense staining in former sections when compared to the others. We suggest that ET-1 activated neutrophil might play a major role in endothelial injury and tissue damage in conditions with high blood level of endothelin.

Coagulation in vivo microcirculation and in vitro caused by endothelin-1

This study was designed to elucidate the participation of endothelin-1(ET-1) in vivo and in vitro coagulation. The microvascular hemodynamic changes in terms of intravascular thrombus formation in rat mesentery induced by the superfusion of ET-1 (0.5, 1 and 2 pmol) were visualized by an intravital microscope system assisted by television-video tape recorder system. In addition to vasoconstriction we observed the blockade of circulation by clumps resembling thrombus in a dose dependent fashion by ET-1. Thrombus formation could be attenuated by pretreatment with superfusion of 3.8% Na citrate solution but not by the prior superfusion of 1 to 3 ng of nitroglycerine. Thrombus formation was found after the administration of 10 microliters of CaCl2 (100 nM) solution in Na citrate (3.8%, 20 microliters) and ET-1 treated field. In vitro study, a dose dependent increase in TAT (thrombin-antithrombin complexes) and decrease in AT III (antithrombin III) (%) activity, the prolongation of PT (prothrombin time) and APTT (activated partial thromboplastin time) was found by administering ET-1 immediately in native (unanticoagulated) blood in silicon coated test tubes (p < 0.05; n = 6). However in citrated blood, TAT complexes, AT III (%) activity, PT and APTT were not significantly changed after administration of the same doses of ET-1 (p > 0.05; n = 6). Therefore, this study suggested that endothelin-1 caused intravascular thrombosis and enhanced intra test tube coagulation which could be attenuated by blocking ionic calcium.