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

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED(50) of 15, 31, and 28 nM, respectively, with maximal response at 10(-7) M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC(50) for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [32P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr113. MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [32P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

Key Points Activation of endothelial cells by anti-β2GPI antibodies causes myosin RLC phosphorylation, leading to actin-myosin association. In response to anti-β2GPI antibodies, release of endothelial microparticles, but not E-selectin expression, requires actomyosin assembly.

As a global effort toward improving patient safety, a specific area of focus has been the early recognition and rapid intervention in deteriorating ward patients. This focus on "failure to rescue" has led to the construction of early warning/track-and-trigger systems. In this review article, we present a description of the data behind the creation and implementation of such systems, including multiple algorithms and strategies for deployment. Additionally, the strengths and weaknesses of the various systems and their evaluation in the literature are emphasized. Despite the limitations of the current literature, the potential benefit of these early warning/track-and-trigger systems to improve patient outcomes remains significant.