No abstract

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/ MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining

Key Points• Neutrophilic granulocytes stimulated with opsonized particles produce microvesicles (MVs) that are able to impair bacterial growth.• Antibacterial effect correlates with number and size of aggregates between bacteria and MVs and depends on cytoskeletal reorganization of MVs.Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties. Only opsonized particles initiated the formation of microvesicles that were able to impair bacterial growth. The antibacterial effect of neutrophil-derived microvesicles was independent of production of toxic oxygen metabolites and opsonization or engulfment of the microbes, but depended on ␤ 2 integrin function, continuous actin remodeling, and on the glucose supply. Neutrophil-derived microvesicles were detected in the serum of healthy donors, and their number was significantly increased in the serum of bacteremic patients. We propose a new extracellular mechanism to restrict bacterial growth and dissemination. IntroductionCell-derived vesicles (such as exosomes, ectosomes, microvesicles, shedding microvesicles, and microparticles) represent a recently discovered mechanism for cell-cell communication. [1][2][3] Exosomes are small (50-100 nm) vesicles released from multivesicular bodies. 4 They are involved in antigen presentation [5][6][7] and cell-tocell transfer of receptors 8 or RNA, 9,10 thereby influencing or reprogramming neighboring cells and often promoting tumorigenesis. 8,11 Exosomes also play a role in host defense against microorganisms: tracheobronchial epithelial cells produce exosome-like vesicles with antiviral activity, 12 and macrophage-derived exosomes are able to transfer pathogen-associated molecular patterns of opportunistic intracellular pathogens to uninfected cells. 13 Larger vesicles, called microvesicles (MVs) or microparticles express tissue factor on their surface that is capable of initiating coagulation. 14 Both exosomes and MVs of different cellular origin were detected in various body fluids and selective enrichment was related to specific diseases. [15][16][17][18][19] Neutrophilic granulocytes (PMNs) play a critical role in innate immune mechanisms by engulfing, killing, and degrading various microorganisms. PMNs produce larger vesicles (named by the authors alternatively as ectosomes, microparticles, or MVs) after incubation with various stimuli. [19][20][21][22] Microparticles obtained from PMNs stimulated by chemotactic agents or phorbol esters activated cytokine (IL-6) secretion from endothelial cells 23 and platelets, 24 thereby contributing to the procoagulant effect of leukocytederived microparticles. 25 Chemotactic peptide-induced PMNectosomes increase the secretion of the anti-inflammatory cytokine transforming growth factor ␤ 26 and interfere with the maturatio...

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0.0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.

Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.

The cause of most cases of human obesity is unknown. Specific alterations in the activity of the autonomic nervous system may mediate and perhaps cause obesity in animal models. We therefore looked for alterations in autonomic activity in human obesity. Fifty-six healthy men with various percentages of body fat underwent autonomic testing while at rest. Significant correlations were found between the percentage of body fat and the variation in the R-R interval after beta-adrenergic blockade (r = -0.30, P less than 0.03), the heart rate (r = 0.30, P less than 0.03), the plasma norepinephrine concentration (r = -0.30, P less than 0.05), the plasma epinephrine concentration (r = -0.49, P less than 0.001), and the pupillary latency period (r = 0.39, P less than 0.01). Each of these variables reflects the activity of the sympathetic nervous system or parasympathetic nervous system or both. Depressions in sympathetic and parasympathetic activity were significantly but weakly associated with increasing percentages of body fat. These associations indicate that in obese persons, autonomic changes, though not necessarily causal, involve several organ systems. We suggest that autonomic alterations are important in human obesity, as they are in animal obesity. A disordered homeostatic mechanism may promote excessive storage of energy by decreasing sympathetic activity, while defending against weight gain by decreasing parasympathetic activity. The use of autonomic profiles holds promise for classifying human obesity and identifying obese patients at increased risk for various disorders.

Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Exposure to pro-inflammatory cytokines, chemokines, mitochondrial contents, and bacterial and viral products induces neutrophils to transition from a basal state into a primed one, which is currently defined as an enhanced response to activating stimuli. Although, typically associated with enhanced generation of reactive oxygen species (ROS) by the NADPH oxidase, primed neutrophils show enhanced responsiveness of exocytosis, NET formation, and chemotaxis. Phenotypic changes associated with priming also include activation of a subset of functions, including adhesion, transcription, metabolism, and rate of apoptosis. This review summarizes the breadth of phenotypic changes associated with priming and reviews current knowledge of the molecular mechanisms behind those changes. We conclude that the current definition of priming is too restrictive. Priming represents a combination of enhanced responsiveness and activated functions that regulate both adaptive and innate immune responses.