Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells.
We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults. Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane. We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C. The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN). However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05). We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN. However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph. Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner. Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.
Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
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Qing
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Howlett
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Bortolussi
D000209:Acute-Phase Proteins; D000328:Adult; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005312:Fetal Blood; D006801:Humans; D007231:Infant, Newborn; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism; D010949:Plasma; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D001386:Azides; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein; C047258:2-(4-azidosalicylamido)-1,3'-dithiopropionate-lipopolysaccharide