Potentiation of the oxidative burst of human neutrophils. A signaling role for L-selectin.
Production of reactive oxygen intermediates (ROI) by the NADPH oxidase of neutrophils is a major mechanism of bacterial killing and, in pathologic circumstances, tissue damage. Integrins and selectins participate in neutrophil adhesion but may also play a role in intracellular signaling. The role of L-selectin in ROI production and Ca2+ signaling in suspended neutrophils was examined using the DREG series of anti-L-selectin antibodies. NADPH oxidase activation was assessed in three ways: H2O2 production using either scopoletin or dihydrorhodamine and O2- production using cytochrome c. Alterations in [Ca2+]i were measured using Fura 2-AM and fluorescence spectrophotometry. Cross-linking of L-selectin with DREG and 2 degrees antibody did not trigger production of H2O2 by itself but significantly enhanced the subsequent response to two soluble activating agents; the formyl peptide formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor (TNF). Potentiation of the oxidative burst was observed using F(ab')2 fragments but not with irrelevant antibodies and was observed whether 2 degrees antibody was added before or after fMLP. Cross-linking of L-selectin also triggered a rise in [Ca2+]i, due, in part, to release from intracellular stores. The intracellular Ca2+ chelator BAPTA blocked both the rise in [Ca2+]i and the potentiation of the oxidative burst in response to fMLP or TNF. We conclude that cross-linking of L-selectin induces intracellular signals, including release of Ca2+, which may contribute to potentiation of the oxidative burst.
Waddell
T K
TK
Department of Medicine, University of Toronto, Ontario, Canada.
Fialkow
L
L
Chan
C K
CK
Kishimoto
T K
TK
Downey
G P
GP
D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000097:blood; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000097:blood / Q000276:immunology / Q000502:physiology*; D003432:Cross-Linking Reagents; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D019041:L-Selectin; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012235:Rhodamines / Q000494:pharmacology; D012603:Scopoletin / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000906:Antibodies; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D012235:Rhodamines; D014409:Tumor Necrosis Factor-alpha; C049925:fura-2-am; C058319:dihydrorhodamine 123; D013481:Superoxides; D019041:L-Selectin; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D012603:Scopoletin; D002118:Calcium; D016257:Fura-2