Chemokine receptor expression on human eosinophils from peripheral blood and bronchoalveolar lavage fluid after segmental antigen challenge

BACKGROUND: The recruitment of circulating eosinophils to the lung is a characteristic feature of allergic airway inflammation. Chemokine receptors likely play a role in this complex process. However, reports of chemokine receptor expression on human eosinophils are conflicting. OBJECTIVE: The aim of this study was to determine whether the chemokine receptor profile of human eosinophils change when these cells are recruited to the airway after an antigen challenge and development of an allergic inflammatory response. METHODS: Blood and bronchoalveolar lavage (BAL) cells were obtained from 13 allergic subjects 48 hours after segmental bronchoprovocation with antigen. The CC chemokine receptor (CCR) 1 to 7, 9, and CXC chemokine receptor (CXCR) 1 to 4 were determined by flow cytometric analysis of whole blood and unseparated BAL cells. RESULTS: Compared with their circulating counterparts, airway eosinophils had decreased CCR3 and increased CCR4, CCR9, and CXCR3 expression on their cell surface. Furthermore, expression of CCR3, CCR4, and CXCR3 was significantly correlated with the percentage of eosinophils in BAL fluid at 48 hours. Eosinophils also expressed CXCR4, but this receptor did not change after antigen-induced recruitment to the airway. In contrast, the expression of CCR1, CCR2, CCR5, CCR6, CCR7, CXCR1, and CXCR2 remained undetectable on either blood or BAL eosinophils. CONCLUSIONS: Our data suggest that recruitment of eosinophils to the airway is associated with a modulation of their chemokine receptor profiles. These changes in chemokine receptors could be involved in determining eosinophil function and antigen-induced airway inflammation.     

Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.     

The role of prostaglandins in the endothelium-mediated vasodilatory response to hypoxia

The effect of intraluminal hypoxia on vascular tone and the release of prostaglandins (PG) I₂ and E₂ were investigated in intact isolated segments of canine femoral and coronary arteries as well as in the rat tail artery. Perfusion with hypoxic Tyrode's solution (pO₂ 20–40 mm Hg) evoked a marked vasodilation of the segments, precontracted with norepinephrine or serotonin. Simultaneously, a 2–3-fold increase in the release of 6-keto-PGF₁ (the stable hydrolysis product of PGI₂) could be observed. In parallel to 6-keto-PGF₁, smaller quantities of PGE₂ were released. Removal of the endothelium as well as pretreatment with indomethacin abolished both, the dilatory response and the PG-release. After administration of verapamil as well as 3,4,5-trimethoxybenzoic acid 8-diethyl-aminooctylester (TMB-8) (which binds intracellular calcium) the PG-increase was abolished and hypoxic dilatation could no longer be elicited, although the vessel had still a capacity to dilate. Exogenous administration of PGI₂ and PGE₂ showed that in canine femoral and coronary arteries PGI₂ was the most effective vasodilating prostaglandin, while in the rat tail artery PGE₂ had a 10-fold higher dilating potency compared to PGI₂. At very high concentrations both PGI₂ and PGE₂ caused vasoconstriction. Our experiments suggest that the hypoxic endothelium-dependent dilatation may be mediated by an increased PG-release. Hypoxia-induced transmembrane calcium influx into the endothelial cells seems to be the trigger reaction.Supported by the Deutsche Forschungsgemeinschaft (Bu 436/2-1)     

Efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency

BACKGROUND: Common variable immunodeficiency (CVID) is a primary immune disorder characterized by antibody deficiency and a decrease in serum IgG and IgA, IgM, or both levels at least 2 SDs below the mean for age and not attributed to other known immunologic disorders. These patients often present with frequent and severe episodes of pneumonia before diagnosis. The standard treatment, intravenous immunoglobulin (IVIG), has been available for the past 20 years. No large-scale study has compared the incidence of pneumonia in these patients before and after IVIG treatment. OBJECTIVE: The aim of this study was to document the effectiveness of intravenous immunoglobulin treatment on the incidence of pneumonia in patients with CVID. METHODS: We performed chart reviews and interviews of patients with laboratory-confirmed CVID seen at our clinical center. The number of episodes of pneumonia was documented before and after treatment with immunoglobulin replacement therapy. RESULTS: The histories of 50 patients were reviewed (mean current age, 42 +/- 16.3 years; age range, 10-78 years; 20 male and 30 female patients). Forty-two (84%) of the 50 patients with CVID had pneumonia at least once before receiving immunoglobulin treatment, and 11 of 42 of these patients had multiple episodes. After treatment with gamma globulin over a mean period of 6.6 +/- 5.2 years (range, <1-20 years), the number of patients experiencing pneumonia significantly decreased to 11 (22%) of 50. In most cases these patients had pneumonia in the first year of immunoglobulin treatment. CONCLUSION: The treatment of CVID with IVIG significantly reduces the incidence of pneumonia.     

Effect of influenza A virus on leukocyte histamine release

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.     

Application of a comprehensive subtelomere array in clinical diagnosis of mental retardation

In 2-8% of patients with mental retardation, small copy number changes in the subtelomeric region are thought to be the underlying cause. As detection of these genomic rearrangements is labour intensive using FISH, we constructed and validated a high-density BAC/PAC array covering the first 5 Mb of all subtelomeric regions and applied it in our routine screening of patients with idiopathic mental retardation for submicroscopic telomeric rearrangements. The present study shows the efficiency of this comprehensive subtelomere array in detecting terminal deletions and duplications but also small interstitial subtelomeric rearrangements, starting from small amounts of DNA. With our array, the size of the affected segments, at least those smaller than 5 Mb, can be determined simultaneously in the same experiment. In the first 100 patient samples analysed in our diagnostic practice by the use of this comprehensive telomere array, we found three patients with deletions in 3p, 10q and 15q, respectively, four patients with duplications in 9p, 12p, 21q and Xp, respectively, and one patient with a del 6q/dup 16q. The patients with del 3p and 10q and dup 12p had interstitial rearrangements that would have been missed with techniques using one probe per subtelomeric region chosen close to the telomere.     

The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells

In the present study we determined whether the endothelium-derived hyperpolarizing factor (EDHF), in addition to its acute effects on vascular tone, activates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of porcine coronary arteries under conditions of combined nitric oxide (NO) synthase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate was applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mitogen-activated protein (MAP) kinases, extracellular signal regulated kinases 1 and 2 (Erk1/2) and p38, as well as protein kinase B/Akt. The constituent responsible for Erk1/2 phosphorylation was identified as a cytochrome P450 (CYP) metabolite, as Erk1/2 activation was attenuated by pretreating the EDHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C antisense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial cells treated with the CYP inducer beta-naphthoflavone. The CYP 2C product 11,12-epoxyeicosatrienoic acid (11,12-EET) also activated tyrosine kinases, Erk1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine coronary artery endothelial cells resulted in an increase in endothelial 11,12-EET production and Erk1/2 phosphorylation compared to that detected in untreated cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but was significantly enhanced in cells overexpressing CYP 2C8. These observations indicate that EDHF/11,12-EET is not simply a vasodilator and that its continuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation.