Nitric Oxide Production in Human Macrophagic Cells Phagocytizing Opsonized Zymosan: Direct Characterization by Measurement of the Luminol Dependent Chemilurninescence

Abstract

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1, 25-dihy-droxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide syn-thase (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, super-oxide 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 abroated 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 per-oxynitrite (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.