Research Paper Mediators of Inflammation, 7, 31–33 (1998)

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.


Introduction
Human bone marrow stromal cells regulate haematopoiesis by interacting directly with marrow haematopoietic progenitors and/or by releasing cytokines. 1,2 Lipox ygenase metabolites of arachidonic acid (AA) such as leukotriene B 4 (LTB 4 ), LTC 4 , lipoxin A 4 (LXA 4 ), LXB 4 , 12-hydroxyeicosatetraenoic acid , and 15-HETE are produced by human marrow mononuclear cells. 3,4 Several of these AA metabolites act on the grow th of human myeloid and erythroid progenitors in semi-solid culture medium. [5][6][7] At this time no study has reported the role of AA metabolites on the grow th of human marrow stromal cells. These results could be of interest since the lipidic compound platelet-activating factor (PAF) stimulates [ 3 H]-thymidine incorporation in marrow stromal cell cultures. 8 In this study we have assessed the effect of LTB 4 , LTC 4 , LXA 4 , LXB 4 , 12-HETE, 15-HETE, and of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the growth of human marrow stromal cell cultures and fresh human mononuclear marrow cells.

Cell cultures
These experiments were performed according to the Helsinki recommendations. Bone marrow sternal cells were harvested from untreated patients referred for diagnosis. Cells collected by aspiration into heparinized tubes were isolated by separation on a Ficoll gradient (400 3 g , 20 min), and washed tw ice with Hanks's balanced salts solution. Mononuclear marrow cells were used when the cell morphology was normal.
Cultures of human bone marrow stromal cells (mostly fibroblast-like cells) were established from marrow mononuclear cells seeded in 75 cm 2 culture flasks in RPMI 1640 w ith 20% fetal calf serum (FCS) (Gibco, Cergy Pontoise, France), penicillin (100 U/ml) and streptomycin (100 m g/ml) (culture medium) at 37°C in 5% CO 2 in air as previously described. 9 After one week, non-adherent cells were removed from culture flasks. Adherent cells were grown to confluence for 4-5 weeks with weekly changes of medium and were subcultured after trypsin treatment (0.05% trypsin for 5 min). The cells used in these experiments were at the first passage. In these experimental conditions more than 99.8% of cells were CD2and CD22indicating the absence of Tand B-cells on the layers and 4% of cells were CD14 + and CD33 + indicating a monocytic/macrophagic lineage. 10,11 Cell proliferation In a separate set of experiments, human marrow stromal cells (in triplicate samples) were harvested after trypsin treatment (0.05% trypsin for 5 min at 37°C) and counted by using a haemocytometer. Results (in cell number) were compared by Mann-Whitney U-test.
As reported in Table 3, only LTB 4 had a significant effect on the [ 3 H]-thymidine incorporation of freshly isolated human marrow mononuclear cells. NDGA (1 m M) had no effect (P > 0.05, four experiments) on their thymidine incorporation (90326 ± 9262 dpm vs. 81712 ± 8532 dpm for NDGA-treated cells and control cells, respectively).

Discussion
Studies have reported the positive or negative effects of lipoxygenase metabolites of AA on cell proliferation. Thus, 12-HETE and 15-HETE stimulate [ 3 H]-thymidine incorporation in mammary tumour cells, 12 and endothelial cells, 13 but inhibit it in neuroblastoma cell cultures. 14 LTB 4 and LTC 4 stimulate DNA synthesis in cultured arterial smooth muscle cells, 15 while 12-HETE decreases the grow th of aortic smooth muscle cells. 16 These latter results are of interest since bone marrow stromal cells share numerous phenotypic similarities w ith vascular smooth muscle cells. 17 The growth of fresh marrow mononuclear cells is not affected by lipoxygenase metabolites of AA.
V. Despla t et al.
Although statistically significant, the small decrease of incorporation of thymidine with LTB 4 brings some doubts on its physiological meaning. Micromolar concentrations of LTB 4 , LXA 4 , LXB 4 and 15-HETE decrease [ 3 H]-thymidine incorporation in human bone marrow stromal cell cultures. However the fact that no dose -response curve was found and that no effect was documented on cell number cast some doubts on the physiological meaning of the observed effects on thymidine incorporation. In contrast to the other AA metabolites, 12-HETE inhibits in a dosedependent manner thymidine incorporation in marrow stromal cell cultures. However no effect was found on cell counts. An explanation might be that [ 3 H]-thymidine incorporation is not only an index of cell proliferation but may also reflect intracellular events other than cell division such as diffusion of DNA precursors. 18 Another explanation for these results might be that only a small percentage of cells were proliferating and that cell counts were not sensitive enough to detect changes. Taken together our results suggest no important role of exogenous lipoxygenase metabolites of AA in the grow th of human bone marrow stromal cells and mononuclear marrow cells in vitro . Moreover results with NDGA suggest that endogenous lipox ygenase metabolites had no role on FCS-induced cell grow th. These results markedly differ from data showing that PAF stimulates [ 3 H]-thymidine incorporation in freshly isolated human mononuclear marrow cells, 19 and marrow stromal cell cultures. 8