The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region. (9/1018)

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2-expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5' end of the IS first exon direct expression of beta-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5' end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5' to the IS exon.  (+info)

Schistosome-infected IL-4 receptor knockout (KO) mice, in contrast to IL-4 KO mice, fail to develop granulomatous pathology while maintaining the same lymphokine expression profile. (10/1018)

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Ralpha-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Ralpha KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Ralpha-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-gamma secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Ralpha KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Ralpha-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Ralpha+ non-T cell effector population.  (+info)

ANG II- and TxA(2)-induced mesenteric vasoconstriction in rats is mediated by separate cell signaling pathways. (11/1018)

Studies in vitro have demonstrated that vasoconstrictor agents increase intracellular Ca(2+) and activate protein kinase C (PKC) to elevate vascular tone. The aim of the present study was to determine the importance of these signaling pathways for angiotensin II (ANG II) and thromboxane A(2) (TxA(2)) in regulating mesenteric blood flow (MBF) in vivo. In anesthetized rats increasing doses of ANG II or the TxA(2) agonist U-46619 were administered into the superior mesenteric artery to reduce MBF. Intra-arterial infusion of inhibitors served to examine the contribution of different pathways: 8-(diethylamino)octyl 3,4,5-trimethoxybenoate hydrochloride (TMB-8) to inhibit intracellular Ca(2+) release, nifedipine to block transmembrane Ca(2+) influx through the L-type Ca(2+) channel, and staurosporine to inhibit PKC. Each of the inhibitors attenuated ANG II-induced reductions in MBF, and all dose-response curves were shifted to the right to an approximately threefold higher ANG II dose. Combinations of the inhibitors revealed that their effects were additive; together they abolished the vasoconstrictor action of ANG II completely. In contrast, the dose-response curve for U-46619 was not affected by any of the inhibitors infused either separately or together. The results demonstrate that a rise in intracellular Ca(2+) and activation of PKC are major mediators of the vasoconstrictor effect of ANG II in mesenteric circulation, but they play a subordinate role, if any, for the effects of TxA(2). Because TxA(2) plays a major role only under pathological conditions, the uncontrolled vasoconstriction appears to be associated with the recruitment of novel signal transduction pathways.  (+info)

Concentration-dependent effects of bradykinin on leukocyte recruitment and venular hemodynamics in rat mesentery. (12/1018)

The results of several recent studies indicate that bradykinin protects tissues against the deleterious effects of ischemia-reperfusion (I/R). However, other studies indicate that bradykinin can act as a proinflammatory agent, inducing P-selectin expression, the formation of chemotactic stimuli, and endothelial barrier disruption. In the present study, we used intravital microscopic techniques to examine the dose-dependent effects of bradykinin on leukocyte-endothelial cell interactions, the formation of platelet-leukocyte aggregates, and venular hemodynamics in rat mesentery in an attempt to explain these divergent findings. Superfusion of the mesentery with low concentrations of bradykinin (/=10(-6) M) decreased V(RBC), increased the number of platelet-leukocyte aggregates, and induced leukocyte adhesion in single postcapillary venules. The formation of platelet-leukocyte aggregates and increased leukocyte adhesion induced by high-dose bradykinin were attenuated by administration of a B(2)-receptor (HOE-140) or a platelet-activating factor (PAF, WEB-2086) antagonist. Thus these adhesive interactions induced by high-dose bradykinin appear to be mediated by a mechanism that is dependent on B(2)-receptor activation and the formation of PAF or PAF-like lipids. The effects of bradykinin on venular V(RBC) and blood flow were also concentration dependent, with low doses producing nitric oxide-mediated vasodilation, whereas high doses decreased V(RBC) by a mechanism that is PAF independent.  (+info)

Flow dynamics of QW7437, a new dodecafluoropentane ultrasound contrast agent, in the microcirculation: microvascular mechanisms for persistent tissue echo enhancement. (13/1018)

OBJECTIVES: The purpose of this study was to test the hypothesis that a subgroup of QW7437 microbubbles, dodecafluoropentane-based ultrasound contrast microspheres, resides for prolonged periods in the microvasculature. BACKGROUND: QW7437 produces echo enhancement in myocardium which may persist relatively longer than opacification in the left ventricular cavity. The mechanism for this persistent enhancement remains unknown. METHODS: The transit of fluorescently labeled erythrocytes was examined by fluorescence intravital microscopy in the microvessels in five rat mesenteries. Ten rats were used to observe the behavior of fluorescently labeled QW7437 microbubbles in the mesenteric microcirculation. RESULTS: There was no significant change in erythrocyte velocity in the arterioles and venules after the administration of QW7437 microbubbles (0.05 ml/kg) preactivated by negative hydrodynamic pressure. Of 552 microbubbles observed in four arterioles and five capillaries, 549 (99.5%) passed without stoppage (> or = 0.1 s stoppage); only one stopped transiently in arteriole and two in capillaries, each for <0.5 s. Sixty-five of 478 microbubbles (13.6%) observed in six postcapillary venules 11 to 30 microm in diameter and 24 of 408 microbubbles (5.9%) in four venules 31 to 50 microm in diameter stopped transiently (0.1 to 180 s) with an attachment to venular endothelium; the remaining microbubbles passed through the venules without stoppage. CONCLUSIONS: Prolonged survival as microbubbles in the circulation and transient stoppage of a subgroup of microbubbles in the microvasculature, particularly in venules, are potential mechanisms for the persistent tissue echo enhancement by QW7437 microbubbles during contrast echocardiography.  (+info)

Effects of endothelin on spontaneous contractions in lymph vessels. (14/1018)

A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10(-10) and 10(-9) M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ET(A)-receptor antagonist BQ-123 (5 x 10(-7) M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10(-8)-10(-7) M), a selective ET(B)-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME) (10(-5) M) but unaffected by aspirin (10(-5) M). Additional treatment with L-arginine (10(-4) M) completely reversed the L-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ET(A) receptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ET(B) receptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.  (+info)

Evidence that the ATP-induced increase in vasomotion of guinea-pig mesenteric lymphatics involves an endothelium-dependent release of thromboxane A2. (15/1018)

1. Experiments were made to investigate mechanisms by which adenosine 5'-trisphosphate (ATP) enhanced vasomotion in mesenteric lymphatic vessels isolated from young guinea-pigs. 2. ATP (10-8 - 10-3 M) caused a concentration-dependent increase of perfusion-induced vasomotion with the endothelium mediating a fundamental role at low ATP concentrations (10-8 - 10-6 M). 3. The response to 10-6 M ATP showed tachyphylaxis when applied at intervals of 10 min but not at intervals of 20 or 30 min. 4. Suramin (10-4 M) or reactive blue 2 (3x10-5 M) but not PPADS (3x10-5 M) abolished the excitatory response to 10-6 M ATP confirming an involvement of P2 purinoceptors. 5. The excitatory response to 10-6 M ATP was abolished by treatment with either pertussis toxin (100 ng ml-1), antiflammin-1 (10-9 M), indomethacin (3x10-6 M) or SQ29548 (3x10-7 M), inhibitors of specific G proteins, phospholipase A2, cyclo-oxygenase and thromboxane A2 receptors respectively. 6. ATP simultaneously induced a suramin-sensitive inhibitory response, which was normally masked by the excitatory response. ATP-induced inhibition was mediated by endothelium-derived nitric oxide (EDNO) as the response was abolished by NG-nitro-L-arginine (L-NOARG; 10-4 M), an inhibitor of nitric oxide synthase. 7. We conclude that ATP modulates lymphatic vasomotion by endothelium-dependent and endothelium-independent mechanisms. One of these is a dominant excitation caused through endothelial P2 purinoceptors which because of an involvement of a pertussis toxin sensitive G-protein may be of the P2Y receptor subtype. Their stimulation increases synthesis of phospholipase A2 and production of thromboxane A2, an arachidonic acid metabolite which acts as an endothelium-derived excitatory factor.  (+info)

PR-39, a proline/arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. (16/1018)

We and others have previously demonstrated that intestinal ischemia-reperfusion (I/R) is associated with a large increase in oxidant production that contributes to microvascular barrier disruption in the small bowel. It has been suggested that the bulk of tissue damage during reperfusion can be attributed to adherent, activated neutrophils. From these observations, we hypothesized that pretreatment with PR-39, an endogenous neutrophil antibacterial peptide that is also a potent inhibitor of the neutrophil NADPH oxidase, would prevent postischemic oxidant production and the development of oxidant-dependent sequelae to I/R such as increased venular protein leakage. To test this postulate, oxidant production, venular protein leakage, leukocyte adhesion, and leukocyte emigration were monitored during reperfusion in control (no ischemia) rat mesenteric venules and in mesenteric venules subjected to I/R alone or PR-39 + I/R. Treatment with a single intravenous bolus injection of PR-39 (administered at a dose to achieve an initial blood concentration of 5 microM) abolished I/R-induced leukocyte adhesion and emigration in vivo. In vitro studies indicated that PR-39 prevents platelet-activating factor-induced neutrophil chemotaxis as well as phorbol myristate acetate (PMA)-stimulated intercellular adhesion molecule-1 expression by cultured endothelial cells. PR-39 pretreatment of rat neutrophils also blocked PMA-stimulated neutrophil adhesion to activated endothelial monolayers. In vivo, I/R was associated with a marked and progressive increase in oxidant production and venular protein leakage during reperfusion, effects that were abolished by PR-39 treatment. The results of this study indicate that PR-39 completely abolishes postischemic leukocyte adhesion and emigration. The time course for inhibition of oxidant production by PR-39 suggests that its antiadhesive properties account for this effect of the peptide. PR-39 may thus be therapeutically useful for prevention of neutrophil adhesion and activation during the postischemic inflammatory response.  (+info)