PMC

Production of infectious DV by primary human MO/Mφ at various differentiation stages. Peripheral blood MO were cultured for 1 day or 1, 3, or >6 weeks, washed, and then infected with DV at an MOI of 2 to 3 PFU per cell in the absence of serum. After 2.5 h of viral adsorption, the cells were washed, and the cultures were further incubated with fresh complete medium for 40 to 48 h. At that time, the culture supernatants were harvested and assayed for infectious-virus production. The results are expressed as the mean ± standard error of pooled data from the number of separate experiments shown in parentheses with cells obtained from up to 20 different donors.

Short-term (A) and long-term (B) production kinetics of various cytokines and chemokines in 7-day-old human MO/Mφ after DV infection. The cells were cultured and infected as described in the legend to Fig. . At the indicated times, the culture supernatants were harvested and assayed for release of infectious viruses, cytokines, and chemokines. The cryolysates of infected MO/Mφ were analyzed for intracellular infectious-virus titers. The results shown in panels A and B were derived separately from one of the representative experiments. Each time point represents the mean ± standard error of results from three independent wells. The levels of these cytokines and chemokines in the supernatants of mock-infected control cultures remained constant before and after infection over time: (A) average values were 462 to 600 pg/ml for IL-8, 336 to 617 pg/ml for RANTES, 82 to 196 pg/ml for MIP-1α, and below the detection limit for IFN-α (<5 pg/ml) and IL-12 (<2 pg/ml); (B) average values were 1.7 to 2.8 ng/ml for MIP-1α, 2.4 to 4.2 ng/ml for IL-8, and 0.32 to 0.35 ng/ml for RANTES.

Kinetics of virus replication and cytokine secretion in terminally differentiated human MO/Mφ after DV infection. Peripheral blood MO were cultured with α-MEM containing 10% heat-inactivated autologous human serum that was half-replaced with fresh complete α-MEM every 5 to 6 days to promote MO/Mφ differentiation. After 45 days of culture, the cells were washed and infected with DV at an MOI of 3 PFU per cell. Culture supernatants were harvested at the time points indicated and assayed for infectious-virus production and cytokine secretion. Cell cryolysates were analyzed for intracellular infectious-virus titers. For each time point, three separate wells were prepared and analyzed, and the results are expressed as the mean ± standard error. Some error bars are too small to be seen. The levels of these cytokines and chemokines in the supernatants of mock-infected control cultures remained constant after infection over time. The average values were 2.2 to 3.6 ng/ml for IL-8, 0.14 to 2.16 ng/ml for MIP-1α, 343 to 464 pg/ml for RANTES, and below the detection limit for IL-1β (<2 pg/ml), IL-12 (<2 pg/ml), TNF-α (<2 pg/ml), and IFN-α (<5 pg/ml). NA, not available.

Effects of LPS on DV replication and DV-induced IFN-α production in differentiated human MO/Mφ. Seven-day-old MO/Mφ were washed and then left uninfected or infected with DV at an MOI of 3 PFU per cell. After 2.5 h of viral adsorption, cells were washed and replenished with fresh complete medium. After 6 h of infection, the cultures were either left untreated (−/LPS) or treated with 5 μg of LPS per ml (+/LPS). Culture supernatants were harvested at different time points and analyzed for extracellular infectious-virus and IFN-α titers. Cell cryolysates in fresh α-MEM of the same volume were used for titration of intracellular infectious viruses. (A) LPS enhanced and sustained DV replication at later times postinfection. (B) Enhancing effect of LPS on DV-induced IFN-α secretion at early times postinfection. For each time point, three separate wells were prepared and analyzed, and the results are expressed as the mean ± standard error. Titers of IFN-α in the mock-infected control cultures were below the detection limit (<7 pg/ml). Representative data out of four independent experiments are shown. Some error bars are too small to be seen.

Morphological development and enzymatic and phagocytic activity of primary differentiated human MO/Mφ. Cell morphology of MO/Mφ cultured for 1 day (A), 2 to 3 days (B), 7 days (C), 10 days (D and F), 14 days (E), 45 days (G), and 50 days (H) is shown. MO/Mφ grew slowly for the first 4 to 5 days, and after 5 days of culture the cells enlarged rapidly and reached confluence at about day 7 to day 8. MNGC formed at ∼day 5, and huge, osteoclast-like MNGC were generated at ∼10 days of culture. After 6 weeks of culture, the cells decreased in size, and condensed cytoplasm was evident in every single cell as a feature of aging (G and H). Degeneration of MNGC was noticeable after 6 weeks of culture, in which the edge of the MNGC was vague (G) and disappeared with time (H). (F) Liu's stain of MNGC. (I) Nitroblue tetrazolium reduction by MNGC. (J) Nonspecific esterase staining of MNGC. Phagocytosis of yeasts by 9-day-old, differentiated mononuclear MO/Mφ (K) and MNGC (L) is shown. Magnifications: (A to C, G, and H) ×84; (D to F) ×126; (I to L) ×840.