Phagocytosis of malarial pigment hemozoin inhibits NADPH-oxidase activity in human monocyte-derived macrophages.
Upon stimulation, inactive subunits of monocyte NADPH oxidase (NOX) are assembled in the membrane to generate the active enzyme responsible for oxidative burst. Phosphorylation of the 47 kDa NOX cytoplasmic subunit (47 kDa band) by protein kinase C (PKC) is important for NOX assembly and activation. Alternatively, NOX is activated in vitro by sodium dodecyl sulfate (SDS) or amphiphiles via a phosphorylation-independent mechanism. Previous data indicate that phagocytosis of malarial pigment hemozoin inhibits oxidative burst and PKC activity (Schwarzer, E., Turrini, F., Giribaldi, G., Cappadoro, M. and Arese, P. (1993) Biochim. Biophys. Acta, 1181, 51-54). We show here that SDS-stimulated NOX activity and phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst dropped by 54% and 46% of control values 2 h after hemozoin phagocytosis, respectively. SDS-stimulated NOX activity remained roughly constant until 12 h, whereas oxidative burst dropped further by approx. 60% and 75% of control values 6 h and 12 h after hemozoin phagocytosis. Reconstitution experiments indicate that damage was localized to cytosolic NOX subunit(s). Membrane assembly of active NOX was defective in PMA-(PKC-dependent stimulation) and FMLP-(PKC-dependent and independent stimulation) stimulated hemozoin-fed monocytes. Labeling experiments with [32P]orthophosphate or [gamma-32P]ATP showed that endogenous PKC-dependent phosphorylation of the 47 kDa band was unaffected 12 h and impaired only 24 h after hemozoin phagocytosis. Thus, only long-term inhibition of NOX may additionally depend on superimposed PKC inhibition.
Schwarzer
E
E
Dipartimento di Genetica, Biologia, Chimica Medica, Università di Torino, Italy.
Arese
P
P
D000328:Adult; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D005260:Female; D006420:Hemeproteins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019255:NADPH Oxidases; D009281:Naphthalenes / Q000494:pharmacology; D010587:Phagocytosis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D016897:Respiratory Burst / Q000187:drug effects; D013997:Time Factors
D006420:Hemeproteins; D009281:Naphthalenes; C061465:calphostin complex; C020753:hemozoin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C