Co-expression of several human syntaxin genes in neutrophils and differentiating HL-60 cells: variant isoforms and detection of syntaxin 1. (57/19639)

Syntaxins are major components of vesicle trafficking and their pattern of expression depends on the cell type. Using reverse transcriptase-polymerase chain reaction (RT-PCR), cloning, and sequencing techniques, we have found that human neutrophils and neutrophil-differentiated HL-60 cells co-express syntaxins 1A, 3, 4, 5, 6, 7, 9, 11, and 16. These genes are also expressed in human peripheral blood lymphocytes and SH-SY5Y neuroblastoma cells, which, unlike neutrophils, also expressed syntaxin 10. We have identified two isoforms of syntaxin 3. Syntaxin 3A, similar to the previously reported syntaxin 3, and the novel isoform syntaxin 3B, which is identical to syntaxin 3A but lacks 37 amino acid residues at the carboxy-terminal region. Syntaxin 1 was mainly located to neutrophil granule membranes by confocal microscopy and by immunoblotting of subcellular fractions. These data indicate that syntaxin 1 cannot be considered specific to neural tissues. The level of expression of syntaxins 3, 4, 6, and 11 was increased during neutrophil differentiation of HL-60 cells, whereas that of syntaxins 1A, 5, 9, and 16 was unchanged. Syntaxin 7 was not expressed in undifferentiated HL-60 cells, but its expression was induced on neutrophil differentiation. The expression of several syntaxin genes in human neutrophils could be related to the high secretory capacity of these cells as well as to the presence of different cytoplasmic granules with distinct exocytic capabilities.  (+info)

Activation of the neutrophil respiratory burst oxidase. (58/19639)

The neutrophil respiratory burst oxidase is a multicomponent activatable enzyme comprising one of the major phagocyte antimicrobial systems. In the genetic disorder chronic granulomatous disease, absent oxidase function is associated with recurrent, severe, and often life-threatening infections. The components of the oxidase system include both membrane-bound and soluble cytosolic proteins. A primary feature of stimulus-dependent activation is the translocation of a complex of cytosolic factors to the membrane, where they associate with a flavocytochrome enzyme. Interactions among the various oxidase components occur through a number of specific regions, including SH3 domains and proline-rich motifs. The fully assembled complex functions as an electron transport system, moving electrons from cytosolic NADPH to molecular oxygen to form superoxide, which, along with subsequent reactive products, exerts microbicidal and cytotoxic activities.  (+info)

Progress in gene therapy for chronic granulomatous disease. (59/19639)

Progress in development of gene therapy for chronic granulomatous disease (CGD), an inherited defect in leukocyte oxidase deficiency, is reviewed. The use of retrovirus vectors to transfer oxidase enzyme subunit cDNA sequence into hematopoietic progenitors results in correction of oxidase activity in neutrophils differentiating from transduced progenitors. In CGD mouse knockouts (X-linked gp91phox-deficient CGD and autosomal recessive p47phox-deficient CGD), gene therapy correction of the CGD defect resulted in appearance of oxidase-normal neutrophils in the peripheral blood and increased host resistance to challenge with fungi or bacteria. In a phase I clinical trial of ex vivo gene therapy of p47phox-deficient CGD, prolonged production (2-6 months) of a low number (1:5000) of oxidase-normal neutrophils was achieved. This therapy might prove beneficial in a setting of prolonged infection in CGD patients, in which even transient production of autologous gene-corrected neutrophils might augment host defense.  (+info)

Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor: comparisons and potential for use in the treatment of infections in nonneutropenic patients. (60/19639)

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhance the antimicrobial functions of mature neutrophils. G-CSF differs from GM-CSF in its specificity of action on developing and mature neutrophils, its effects on neutrophil kinetics, and its toxicity profile. The toxicity profile of recombinant (r) GM-CSF is consistent with priming of macrophages for increased formation and release of inflammatory cytokines, whereas rG-CSF induces production of antiinflammatory factors, such as interleukin-1 receptor antagonist and soluble tumor necrosis factor receptors, and is protective against endotoxin- and sepsis-induced organ injury. The low toxicity of rG-CSF, results of animal models of infection, and extensive experience with neutropenic subjects have promoted clinical studies in nonneutropenic subjects, which indicate that rG-CSF may be beneficial as adjunctive therapy for treatment of serious bacterial and opportunistic fungal infections in nonneutropenic patients, including those with alterations in neutrophil function.  (+info)

Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance. (61/19639)

Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-kappaB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO-induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin-specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti-E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-kappaB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil-endothelial adhesion response.  (+info)

Defensins impair phagocytic killing by neutrophils in biomaterial-related infection. (62/19639)

The implantation of foreign material carries a risk of infection which frequently is resistant to all treatment short of removing the implant. We have previously shown that these materials activate neutrophils by contact, leading to production of oxygen free radicals accompanied by release of granule products. Such activation further results in depletion of local host defenses, including the capacity of biomaterial-activated neutrophils to kill bacteria. Among the granule products released from neutrophils are small cationic antibacterial peptides (human neutrophil peptides [HNP]) known as defensins. Here we tested the hypothesis that defensins, released from activated neutrophils onto the surface of biomaterials, might play a role in the deactivation of subsequent neutrophil populations. Incubation of neutrophils with purified HNP resulted in a dose-related impairment of stimulus-induced oxygen radical production and of phagocytic killing. Furthermore, fresh neutrophils added to biomaterial-associated neutrophils exhibited impaired phagocytic killing. This impairment could be abrogated by antibody to HNP but not by an irrelevant antibody. Taken together, these observations support the idea that neutrophils activated at a material surface can create, by means of HNP release, an environment hostile to their microbicidal function and that of their infiltrating brethren.  (+info)

Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. (63/19639)

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.  (+info)

Alkaline conditions accelerate polymorphonuclear leukocyte apoptosis in vitro. (64/19639)

Apoptosis was monitored in polymorphonuclear leukocytes (PMNs) cultured under mildly acidic, neutral, and alkaline conditions. Within 3 h, 9.0% of the PMNs underwent apoptosis at pH 6.7, as did 12% at pH 7.2, 38% at pH 7.7, and 60% at pH 8.2. Inhibitors of serine proteases, caspase-1, or caspase-3 significantly inhibited PMN apoptosis at pH 8.2, suggesting an involvement by these enzymes.  (+info)