Effect of 1,25-dihydroxyvitamin D3, lipopolysaccharide, or lipoteichoic acid on the expression of NADPH oxidase components in cultured human monocytes.
Human blood monocytes in culture gradually lose their capability to produce superoxide when stimulated. Addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3], 2.5 ng/ml LPS, or 25 ng/ml lipotechoic acid (LTA) prevented this decrease in monocyte respiratory burst when added at initiation of culture. Monocytes cultured for 4 days in the presence of 1,25 (OH)2D3, LPS, or LTA retained the ability to produce superoxide and 1,25 (OH)2D3 actually increased the oxidative capacity compared with fresh monocytes. Furthermore, addition of 1,25(OH)2D3, LPS, or LTA to monocytes at day 4 of culture restored the oxidase activity by day 7 to the levels seen with fresh monocytes. Cytosol or membrane fractions from monocytes cultured with or without the agents indicated above were tested in a cell-free assay of superoxide production by mixing with fresh monocyte membrane or cytosol fractions, respectively. Membrane oxidase activity from these cultured monocytes showed only minimal changes regardless of the agent present or absent from the culture. However, the activity of cytosol fractions from these same cultured monocytes showed substantial differences depending upon the culture conditions and these changes correlated closely with changes in activity of the intact monocytes from which the cytosols were derived. Immunoblot analysis of monocyte membranes and cytosol was used to assess the amount of the 91-kDa and 22-kDa subunits of membrane oxidase cytochrome 558 (gp91 and p22), and the 47-kDa and 67-kDa cytosol oxidase factors (p47 and p67) in the different culture conditions. The gp91 and p22 decreased during culture of monocytes, although the decrease was only apparent by 7 days in culture. When 1,25 (OH)2D3, LPS, or LTA were present at the initiation of culture, then an increase in gp91 and p22 was seen as compared with fresh monocytes at day 4. When these agents were added at day 4 of culture, the levels of gp91 and p22 at day 7 were similar to fresh monocytes. The p67 cytosol oxidase component showed little change in amount by immunoblot regardless of the time in culture or whether 1,25(OH)2D3, LPS, or LTA were present. In contrast, the p47 cytosol oxidase factor decreased by day 4 of culture and was markedly depressed by day 7. Monocytes cultured in the presence of either 1,25(OH)2D3, LPS, or LTA preserved the expression of p47, whereas addition of these agents to monocytes at day 4 of culture restored the expression of p47 by day 7 to the level of fresh monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Levy
R
R
Infectious Diseases Laboratory, Soroka Medical Center, Beer-Sheva, Israel.
Malech
H L
HL
D002117:Calcitriol / Q000494:pharmacology*; D002474:Cell-Free System; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009000:Monocytes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D013682:Teichoic Acids / Q000494:pharmacology*
D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D013682:Teichoic Acids; D013481:Superoxides; C009900:lipoteichoic acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D002117:Calcitriol