Modified hemoglobins produce venular interendothelial gaps and albumin leakage in the rat mesentery.
(25/1340)
Cross-linked hemoglobin (alphaalpha-Hb) and polyethylene glycol (PEG)-conjugated Hb have both been considered as possible "blood substitutes." Previously, we showed that PEG-Hb extravasates rapidly in the intestinal mucosa and causes transient epithelial sloughing, resulting in temporary opening of the intestinal epithelial barrier. In the present study, the rat mesenteric preparation was used to quantify the effects of the two Hbs on microvascular leakage to albumin and to investigate possible changes in the integrity of the interendothelial cell junctions and the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with HEPES-buffered saline (HBS) containing 0.5 mg/ml BSA and 2 mg/ml alphaalpha-Hb (n = 16) or PEG-Hb (n = 5) for 2 or 10 min. Controls (n = 4) just received HBS-BSA. In some experiments (n = 9 for alphaalpha-Hb; n = 5 for PEG-Hb), the perfusate was then replaced by FITC-albumin in HBS-BSA for the next 3 min. The vasculature was then perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In the remaining experiments, the mesenteric microvasculature was stained with silver nitrate to determine the number of endothelial junctional gaps per length of venules. Both Hbs increased the number and area of leaks per micrometer of venular length compared with control, but alphaalpha-Hb increased to a greater extent than PEG-Hb. Formation of leaks was accompanied by changes in the endothelial actin cytoskeleton and by an increased number of endothelial gaps. Mast cell degranulation was significantly greater (P < 0.05) in Hb-treated preparations compared with controls, but there was no direct correlation between sites of degranulation and albumin leakage. These Hbs appear to induce venular leakage in the mesentery by mechanisms similar to those previously observed after treatment with histamine or nitric oxide synthase inhibitors. (+info)
CD45 is essential for Fc epsilon RI signaling by ZAP70, but not Syk, in Syk-negative mast cells.
(26/1340)
The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore Fc epsilon RI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FcepsilonRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated Fc epsilon RI gamma-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling. (+info)
Respiratory syncytial virus stimulates neutrophil degranulation and chemokine release.
(27/1340)
Neutrophil infiltration of the airways is a common finding in respiratory syncytial virus (RSV) bronchiolitis. Neutrophil-derived chemokines and neutrophil granule contents can cause further inflammation, hyperresponsiveness, and damage of the airways. In this study, peripheral blood neutrophils incubated with RSV (multiplicity of infection (MOI) = 10) induced IL-8, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and myeloperoxidase (MPO) release. In contrast, LPS induced only chemokine but not MPO release. RSV-induced chemokine and MPO release was noncytotoxic as assessed by trypan blue exclusion. The mechanism of RSV-induced chemokine release was shown to be transcription dependent since cytokine mRNA synthesis was increased with RSV stimulation and the process was inhibited by actinomycin-D. In addition, the effect of dexamethasone (dex) on mediator release was also studied. Dex significantly inhibited chemokine release but did not inhibit MPO release. The mechanism of inhibition of the release of these chemokines is probably posttranscriptional since the mRNA synthesis was not inhibited by dex. We conclude that the release of chemokines (IL-8, MIP-1alpha, MIP-1beta) and granule enzymes (MPO) by RSV-stimulated neutrophils may contribute to the pulmonary pathology in RSV bronchiolitis. These in vitro findings showing that dex failed to consistently inhibit all the RSV-induced release of neutrophil inflammatory mediators may explain the variable efficacy of corticosteroids in the treatment of RSV bronchiolitis. (+info)
Von Willebrand factor receptor GPIb alpha is expressed by human factor XIIIa-positive dermal dendrocytes and is upregulated by mast cell degranulation.
(28/1340)
GPIb alpha, a glycoprotein component of the GPIb-IX-V complex, serves as a platelet membrane receptor that mediates adhesion to von Willebrand factor normally present in the vascular subendothelium. Recent data have demonstrated that GPIb alpha is not restricted to platelets, but is also expressed by endothelium in vitro. In this study, we describe the expression and distribution of GPIb alpha in normal adult and neonatal human skin. GPIb alpha is present, as detected by immunohistochemistry, on endothelial cells and on highly dendritic cells localized within the perivascular space, dermal-epidermal junction, and reticular dermis. By dual-labeling immunofluorescence and confocal microscopy, GPIb alpha-positive cells within the dermal interstitium are demonstrated to represent factor XIIIa-positive dermal dendrocytes. In organ cultures of neonatal human foreskin, mast cell degranulation induced by either substance P or compound 48/80 resulted in transiently increased GPIb alpha expression by dermal dendrocytes. Because the GPIb-IX-V complex plays a part in regulating hemostasis and may be important for cellular interactions with extracellular matrix molecules, these data provide additional insight into the potential function of FXIIIa-positive dermal dendrocytes in skin remodeling and repair. (+info)
Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.
(29/1340)
Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) >> N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s). (+info)
Augmentation of eosinophil degranulation and LTC(4) secretion by integrin-mediated endothelial cell adhesion.
(30/1340)
We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C(4) and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 +/- 15.3 (BSA control) to 778 +/- 225 ng/10(6) cells for eosinophils exposed to interleukin-1alpha-treated HUVECs (P < 0.05) and also caused a twofold increase in stimulated LTC(4) secretion (P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC(4) from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to alpha(4)- or beta(2)-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC(4) and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion. (+info)
Evidence that mast cell degranulation, histamine and tumour necrosis factor alpha release occur in LPS-induced plasma leakage in rat skin.
(31/1340)
1. In the present study we investigated the role of mast cells during inflammation in rat skin. As the release of several pro-inflammatory mediators, such as histamine and tumour necrosis factor alpha (TNFalpha), occurs following mast cell activation we studied whether mast cell degranulation and the release of both histamine (H) and TNFalpha occurred in a model of lipopolysaccharide (LPS)-induced plasma leakage in rat skin. 2. Plasma leakage in the rat skin was measured over a period of 2 h as the local accumulation of intravenous injection of 125I-human serum albumin (125I-HSA) in response to intradermal injection of LPS. LPS (10 microg site-1) produced an increase of plasma leakage (50.1+/-2.3 microl site-1) as compared to saline (9.0+/-3.2 microl site-1). Histological analysis of rat tissue showed that LPS induced a remarkable mast cell degranulation (59.8+/-2.1%) as compared to saline (13.5+/-2.2%). 3. Ketotifen (10-9 - 10-7 mol site-1), a well-known mast cell-membrane stabilizer, produced a dose-related inhibition of LPS-induced plasma leakage by 36+/-3.5%, 47+/-4.0%, 60+/-3.3% respectively. In addition, ketotifen (10-7 mol site-1) inhibited mast cell degranulation by 59. 2+/-2.7%. 4. Chlorpheniramine maleate (CPM) (10-9 - 10-7 mol site-1), an H1 histamine receptor antagonist only partially inhibited LPS-induced plasma leakage in rat skin (38+/-1.1% at the highest dose). Furthermore, CPM (10-7 mol site-1) did not prevent mast cell degranulation. 5. A polyclonal antibody against TNFalpha (1:500, 1:100, 1:50 v v-1 dilution), locally injected, decreased LPS-induced plasma leakage in the skin by 15+/-2.0%, 24+/-2.1% and 50+/-3.0% respectively. 6. Taken together these results suggest that LPS-induced plasma leakage in rat skin is mediated, at least in part, by mast cell degranulation and by the release of histamine and TNFalpha from these cells. (+info)
A(3) adenosine receptor activation attenuates neutrophil function and neutrophil-mediated reperfusion injury.
(32/1340)
This study tested the hypothesis that A(3) adenosine receptors inhibit neutrophil (PMN) function and PMN-mediated reperfusion injury. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), an A(3) agonist, did not attenuate superoxide production or myeloperoxidase release from stimulated PMNs. However, Cl-IB-MECA reduced platelet-activating factor-stimulated PMN adherence to coronary endothelium at low concentrations: 52 +/- 27, 45 +/- 10, and 87 +/- 23 PMNs/mm(2) at 0.1, 1.0, and 10 nM vs. 422 +/- 64 PMNs/mm(2) with platelet-activating factor alone. This inhibition was not blocked by A(1) (5 microM KW-3902) or A(2a) (5 microM KF-21326) antagonists: 44 +/- 3 and 43 +/- 2 PMNs/mm(2), respectively. Endothelial pretreatment with 1 nM Cl-IB-MECA reduced PMN adherence, which was reversed by the A(3) antagonist MRS-1220 (100 nM). PMN-mediated reperfusion injury was initiated in isolated rabbit hearts by infusion of 28 x 10(6) PMNs/min for 10 min early in reperfusion. PMNs caused a significant decrease in recovery of left ventricular developed pressure and positive and negative time derivatives of pressure (23 +/- 3, 25 +/- 3, and 23 +/- 3% of baseline, respectively) vs. buffer-perfused hearts (43 +/- 7, 44 +/- 7, and 45 +/- 6%, respectively). Cl-IB-MECA (10 nM) given at reperfusion attenuated the PMN-mediated loss of contractile recovery (40 +/- 3, 46 +/- 5, and 42 +/- 4% of baseline). Cl-IB-MECA reduced myeloperoxidase release activity (5.3 +/- 0.6 absorbance units/min) and CD18-positive cells (54 +/- 9 cells/slide) compared with the untreated PMN group (17.9 +/- 1.7 absorbance units/min and 183 +/- 68 cells/slide). We conclude that Cl-IB-MECA attenuates reperfusion injury by decreasing PMN-endothelial cell interactions. These results suggest that the A(3) adenosine receptor may be a novel therapeutic target for treatment of myocardial ischemia and reperfusion. (+info)