Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

<p>Abstract</p> <p>Background</p> <p>Neutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.</p> <p>Methods</p> <p>Human bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA's were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.</p> <p>Results</p> <p>Infection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.</p> <p>Conclusion</p> <p>Collectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.</p

Reduced Neutrophil Apoptosis in Diabetic Mice during Staphylococcal Infection Leads to Prolonged Tnfα Production and Reduced Neutrophil Clearance

Diabetes is a frequent underlying medical condition among individuals with Staphylococcus aureus infections, and diabetic patients often suffer from chronic inflammation and prolonged infections. Neutrophils are the most abundant inflammatory cells during the early stages of bacterial diseases, and previous studies have reported deficiencies in neutrophil function in diabetic hosts. We challenged age-matched hyperglycemic and normoglycemic NOD mice intraperitoneally with S. aureus and evaluated the fate of neutrophils recruited to the peritoneal cavity. Neutrophils were more abundant in the peritoneal fluids of infected diabetic mice by 48 h after bacterial inoculation, and they showed prolonged viability ex vivo compared to neutrophils from infected nondiabetic mice. These differences correlated with reduced apoptosis of neutrophils from diabetic mice and were dependent upon the presence of S. aureus and a functional neutrophil respiratory burst. Decreased apoptosis correlated with impaired clearance of neutrophils by macrophages both in vitro and in vivo and prolonged production of proinflammatory tumor necrosis factor alpha by neutrophils from diabetic mice. Our results suggest that defects in neutrophil apoptosis may contribute to the chronic inflammation and the inability to clear staphylococcal infections observed in diabetic patients

Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors

Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity.

Abstract The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.</jats:p

Interferon-gamma primes neutrophil-mediated gastric surface cell cytotoxicity

The T lymphocyte product interferon-gamma (IFN-gamma) upregulates or primes polymorphonuclear leukocyte (PMN) oxidative responses to the receptor-initiated stimulant N-formyl-methionyl-leucyl-phenylalanine (FMLP) but not to the transduction-mediated stimulant phorbol myristate acetate (PMA). We sought a functional correlation between IFN-gamma-induced oxidative priming of PMNs and PMN-mediated cytotoxicity, using an in vitro assay of 51Cr release from rabbit gastric surface cells. Compared with control PMNs, IFN-gamma-primed PMNs exhibited a significant increase in cytotoxicity when stimulated with FMLP, but not when stimulated with PMA. IFN-gamma-induced, FMLP-stimulated, PMN-mediated cytotoxicity was reduced by adding superoxide dismutase to scavenge superoxide anion or by adding catalase or glutathione peroxidase to scavenge hydrogen peroxide. Cytotoxicity was also reduced by inhibiting myeloperoxidase activity with azide or scavenging HOCl with alanine or methionine. Cytotoxicity was blocked by a monoclonal antibody against the CD11/CD18 integrin of PMNs. The results indicate that the immunoregulatory lymphokine IFN-gamma primes the FMLP-stimulated cytotoxic activity of PMNs via the increased generation of reactive oxygen metabolites and indicate that cytotoxicity may require effector-target cell adherence. Therefore, T lymphocyte-derived IFN-gamma may have a role in the pathogenesis of PMN-mediated injury to gastric and gastrointestinal tract mucosa

Anaphylaxis XVIII: Studies on Passive Sensitization of the Dog

Abstract The concept that a latent period for passive sensitization of the dog is essential has been widely accepted for many years. Lewis (1) states, “The earliest experiments in the transference of hypersusceptibility from one animal to another clearly showed that a definite interval of time must elapse between the injections of sensitizing antiserum and antigen.” He further states that the interval is usually 24 hours but may be reduced to 4 hours under optimum condition; that it is, “Generally agreed that the latent period cannot be entirely dispensed with.” Zinsser, Enders, and Fothergill (2) are of the same opinion since they state that in passive sensitization a definite period must elapse between the injection of sensitive blood and that of antigen. Apparently Dragstedt (3, 4) does not challenge these conclusions since he states that a latent period “… has been shown to be necessary for the guinea pig and the dog.”</jats:p