TISSUE-SPECIFIC EXPRESSION AND CELLULAR LOCALIZATION OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED NAD:ARGININE ADP-RIBOSYLTRANSFERASE.

Mono ADP-ribosyltransferases (ADPRT) transfer the ADP-ribose moiety O fNAD to acceptor proteins. A glycosylphosphatidylinositol(GPI)- anchored ADPRT, originalyisolated from rabbit skeletal muscle, is conserved across species as evidenced on immunoblots where antibodies against rabbit skeletal muscle transferase reacted with partially purified ADPRT from canine,bovine and humanskeletal muscle. On Northern analysis, the skeletalmuscle ADPRTcDNA hybridized with a 1.6-kb band, strongly with mouse cardiac and skeletal muscle poly(A)+RNA and weakly with a 1.6-kb band from lung and lymphocyte mRNA. ADPRT activity was detected in human cells obtained by bronchoalveolarlavage (BAL).On immunoblots, a 40-kDa protein, reactive with anti-ADPRT antibodies, bound [32P]-NAD in an overlay assay. On immunohistochemical staining and confocal microscopic study of lung tisue, the epithelial layer was strongly reactive, with the immunoreactivity localized on the surface of intermediate bronchial epithelial cells and on the apical surface of ciliated cells, consistent with previous observations that GPI-linked proteins are targeted to the apical surface of polarized cells. Immunoreactivity was reduced by incubation of cells with phosphatidylinositol (PI)-specific phospholipase C, which releases the protein from the PI anchor. These data are consistent with the hypothesis that a GPI-anchored ADPRT, in addition to be found in muscle and lymphocytes, is a constituent of pulmonary epithelial cell membranes

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8

Pathobiology of neutrophil–epithelial interactions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134067/1/imr12446_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134067/2/imr12446.pd