Effect of hypoxia on the hematopoietic and immune modulator preprotachykinin-I.

OBJECTIVES To determine the effect of hypoxia on bone marrow mononuclear cells (BMMCs) and their ability to proliferate into granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid burst-forming units (BFU-Es) and to determine the role of the neuroimmune and hematopoietic mediator, substance P. DESIGN Controlled in vitro study. SETTING University research laboratory. MATERIALS Bone marrow aspirates were obtained from the posterior iliac crests of healthy volunteers after obtaining informed consent. INTERVENTIONS The BMMCs were divided into the following groups: (1) normoxia, (2) two hours of hypoxia, and (3) six hours of hypoxia. Additional BMMCs were purified before the period of hypoxia, while others were incubated with neurokinin (NK) receptor antagonists. In other experiments, bone marrow stroma was grown to confluence and randomized to the following groups: (1) normoxia, (2) hypoxia, (3) normoxia and interleukin (IL) 1, and (4) hypoxia and IL-1. All groups were cultured for 2, 6, 12, or 24 hours. MAIN OUTCOME MEASURES The formation of CFU-GMs and BFU-Es was measured after 10 to 14 days of incubation of the BMMCs. The messenger RNA of the preprotachykinin-I (PPT-I) gene and the NK-1 and NK-2 receptors was detected by using semiquantitative reverse transcriptase-polymerase chain reaction or Northern blot analysis on bone marrow stroma. The immunoreactivity of substance P in bone marrow stroma was measured by competitive enzyme-linked immunosorbent assay. RESULTS Hypoxia resulted in a 110% increase in the number of CFU-GMs and a 78% increase in the number of BFU-E colonies at 6 hours (both P<.05). Elimination of the stromal elements by purification abrogated the increase in colony formation to nonhypoxic levels. Hypoxia induced PPT-I gene expression at 24 hours; however, no PPT-I expression was found in the hypoxic group incubated with IL-1. The receptor, NK-1, was found to be equal in both hypoxic groups; NK-2 was found to have a 4-fold increase in the hypoxia and IL-1 group over the hypoxia alone group and normoxia and IL-1 group. The levels of substance P immunoreactivity were found to be similar in all groups. Incubation of BMMCs with NK receptor antagonists to NK-1 alone or NK-1 and NK-2 decreased the number of CFU-GM and BFU-E colonies similar to the level in controls. CONCLUSIONS These results indicate that hypoxia has a role in the proliferation and control of CFU-GMs and BFU-Es. This control seems to be mediated through the bone marrow stroma and modulated by NK receptors and induction of PPT-I. The neuropeptide, substance P, probably has a role but is clearly not the only mediator involved.