Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60 . The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and polymorphonuclear leukocytes (PMN) over 9 d of culture in 1 .3% dimethylsulfoxide (DM SO). As the HL-60 cells mature, the rate of 02 production increases 18-fold, with a progressive shortening of the lag time required for activation . Hexosemonophosphate shunt activity rises concomitantly . Ingestion of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities . Degranulation, as measured by release of R-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective . However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of 02 generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100% that of normal PMN . DMSO-induced differentiation of HL-60 cells is a promising model for myeloid development .KEY WORDS phagocytosis " superoxide granulocyte -lysosome -lactoferrin A human promyelocytic leukemia cell line, HL-60, was recently established from the peripheral blood of a patient with acute promyelocytic leukemia (9) . It has maintained continuous growth in J . CELL. BIOLOGY © The Rockefeller University Press -0021-9525/79/08/0315/08 $1 .00 Volume 82 August 1979 315-322 suspension culture in the absence of added conditioned medium or colony-stimulating factor for over 18 mo and is tumorigenic in athymic nude mice (10). The majority of HL-60 cells are promyelocytic in morphology and histochemistry, but 4-15% of them show morphologic characteristics of more mature myeloid cells: myelocytes, meta-315 on

A B S T R A C T Stimulation of guinea pig granulocytes by digitonin results in superoxide (O°) generation. A continuous assay shows that there is a lag between the addition of digitonin and the onset of O2 production. The rate of activation of the O2 generating system is dependent upon the concentration of digitonin and the temperature. The final linear rate of O2 production is affected by the concentration of digitonin, temperature, pH, and the presence of exogenous reduced pyridine nucleotides. Thus, factors which alter either the activation process or the activity of the°2 generating system can affect°2 production by stimulated granulocytes.

No abstract

Phagocytic cells generate superoxide (02) as a part of the respiratory burst of phagocytosis. We studied human granulocyte #{176}2 production in vitro in response to phorbol myristate acetate, a soluble fatty acid ester that stimulates 02 generation. The properties of this compound permit the adaptation of a continuous assay of 0 production to the study of the activity and activation (i.e., the change from the resting to the active state) of the 02-generating system. The two processes, measured respectively as the rate of 02 production and the lag time for its attainment, respond differently to manipulations involving temperature. pH, phorbol myristate acetate concentration, N-ethyl maleimide inhibition. and energy metabolism. Changes in pH profoundly influence the rate without changing the lag time. Increases in temperature above 3TC depress the rate but continue to shorten the lag time. Thus. the activation and activity of the 02-generating system appear to have different pH and temperature dependency. In addition. they have different dose-response curves for phorbol myristate acetate stimulation: at high concentrations the rate reaches a plateau but the lag time continues to shorten. Activity of the human 02-generating system is sensitive to metabolic inhibitors. such as N-ethyl maleimide. 2-deoxyglucose, and the combination of 2-deoxyglucose with dinitrophenol or cyanide. The rate of activation (lag time) is unaffected by these compounds. These findings show that the activity of the human 02-generating system and its activation are separable processes. Furthermore. comparison of the above characteristics of human peripheral blood and guinea pig peritoneal exudate granulocytes reveals several important differences. Hospital Medical ('enter and Sidne8' Farber ('ancer institute.

We studied three children with chronic gastrointestinal disease who had been on intravenous hyperalimentation for periods of time ranging from 4 to 23 months. Each child was found to have low plasma and red blood cell glutathione peroxidase activity. This was associated, in the two children tested, with a marked deficiency of serum selenium. Their plasma glutathione peroxidase levels ranged between 4 and 24% of normal and their red blood cell levels ranged between 4 and 14% of normal. The intravenous alimentation was then supplemented with sodium selenite (240 micrograms Se/d). Within 4-5 weeks, the plasma glutathione peroxidase activity returned to normal. Red cell glutathione peroxidase activity remained essentially unchanged for 4-6 weeks, after which it increased over the following 3-4 months. Red cells were separated by density on a continuous Percoll-diatrizoate gradient. In normal individuals, the specific activity of glutathione peroxidase did not differ across the gradient despite a 2.5-fold difference in the specific activity of pyruvate kinase. When studied initially, glutathione peroxidase activity from the deficient patients did not change across the gradient. As the red cell enzyme activity increased with selenium repletion, the highest specific activity was initially found at the top of the gradient (youngest cells). After 3-4 months of supplementation, the specific activity became equal across the gradient. Thus, with selenium repletion, there is a rapid increase in plasma glutathione peroxidase activity, a 4-6 week lag prior to an increase in red cell enzyme activity, and the increase in red cell activity is due to newly synthesized red cells made in the presence of selenium.