Effect of nitric oxide production and oxygen tension on progenitor preservation in ex vivo culture

Abstract

Many cell types are capable of expressing inducible nitric oxide synthase (iNOS) in response to cytokines or endotoxin. We detected iNOS mRNA by reverse transcriptase polymerase chain reaction in CD34+ human bone marrow cells after 48-hour incubation with interferon-gamma (IFN-gamma)/tumor necrosis factor alpha (TNF-alpha) and IFN-gamma/lipopolysaccharide. Based on this finding, we examined the effect of nitric oxide (NO) on hematopoiesis and particularly on proliferation and survival of CD34+ marrow cells in in vitro culture. When CD34+ cells were cultured in the presence of an NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a dose-dependent inhibition of cell proliferation was observed. Addition of the selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysine hydrochloride (L-NIL) at a dose of 500 microM increased the cell counts by 23% (range 0-89%). The expansion of total CD34+ cell number was 4-fold with a hematopoietic cytokine cocktail compared to 5.2-fold with the addition of L-NIL to this combination. At days 7 and 14 of culture, SNAP induced apoptosis in CD34+ human bone marrow cells detected by an in situ terminal deoxynucleotidyl transferase assay. The apoptosis was partially inhibited with the addition of L-NIL. SNAP also inhibited cell cycling, as evidenced by a 56% decrease in the number of cells in S phase after 5 days of culture in the presence of SNAP. To investigate if NO production was dependent on oxygen tension, J774A mouse macrophage cells were induced with lipopolysaccharide/IFN-gamma, and nitrite production measured by the Griess reaction. Nitrite production was persistently less in 5% compared to 21% oxygen. CD34+ marrow cells from normal donors were also grown under variable oxygen tension, and the cell proliferation in 5% oxygen was significantly greater at both 7 and 14 days of culture. CFU-GM colony growth also was increased in the low oxygen setting. The concentration of various cytokines was measured in CD34+ progenitor culture supernatants, including interleukin (IL)-1 alpha, IL-1 beta and TNF-alpha. SNAP increased IL-1 alpha production by CD34+ cells as well as from light-density bone marrow cells, whereas the effect on IL-1 beta and TNF-alpha production was not significant. Manipulation of oxygen tension and the inhibition of production of reactive oxygen and nitrogen intermediates may have potential to optimize cell expansion and progenitor preservation in ex vivo culture systems.