Nitric oxide production in human macrophagic cells phagocytizing opsonized zymosan: direct characterization by measurement of the luminol dependent chemiluminescence.
When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.
Gross
A
A
INSERM U431, Université de Montpellier II, France.
Dugas
N
N
Spiesser
S
S
Vouldoukis
I
I
Damais
C
C
Kolb
J P
JP
Dugas
B
B
Dornand
J
J
D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002474:Cell-Free System; D016166:Free Radical Scavengers; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007948:Leukemia, Monocytic, Acute / Q000473:pathology; D008163:Luminescent Measurements; D008165:Luminol; D016403:Lymphoma, Large B-Cell, Diffuse / Q000473:pathology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000187:drug effects; D009566:Nitrates / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism; D015054:Zymosan / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology
D016166:Free Radical Scavengers; D006146:Guanidines; D007074:Immunoglobulin G; D009566:Nitrates; D009895:Opsonin Proteins; D013481:Superoxides; D019820:Xanthine; C039642:peroxynitric acid; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007073:Immunoglobulin E; D014212:Tretinoin; D008165:Luminol; D015054:Zymosan; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D002117:Calcitriol; C004479:pimagedine