Ivan N. Rich

The Na(+)/H(+) exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pH(i)). It is a ubiquitous, amiloride-sensitive, growth factor-activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pH(i) than normal hematopoietic tissue. We then show that a direct correlation exists between pH(i) and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na(+)/H(+) exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pH(i) and induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pH(i) decreases, apoptosis-measured by annexin-V and TUNEL methodologies-rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents. (Blood. 2000;95:1427-1434)

A physiologic role for lactoferrin (Lf) has been implicated by (1) its antibacterial effect and (2) its involvement as a negative-feedback regulator for colony stimulating factor (CSF) and, therefore, granulocyte production. The isolation and purification of endotoxin-free, species-specific mouse and human Lf have enabled a study of the role of Lf both in vitro and in vivo. Injection of Salmonella typhimurium or LPS into mice resulted in a dose-dependent increase in plasma Lf. Treating normal and neutropenic mice with LPS showed that the plasma Lf level was directly related to the number of granulocytes found in the peripheral blood. The effect of neutropenia did not inhibit release of Lf. By incubating mouse bone marrow and adherent peritoneal cells with 0.1 microM mouse or human Lf in the absence or presence of the prostaglandin synthesis inhibitor, indomethacin (1.0 microM), no evidence could be obtained in support of a negative-feedback regulation of CSF. In fact, rather than an inhibition of CSF, the production of the latter was found to be stimulated from both cell types. Injection of endotoxin-free, mouse Lf (2 mg/animal) into mice at concentrations in the same order of magnitude as that found during bacterial infection, resulted in an increase in CSF production by 12 hours and prior to the increase in bone marrow granulocyte-macrophage progenitor cells (GM-CFC) at 48 hours. The results do not support a negative-feedback regulation of CSF by macrophages. Instead, they can be incorporated into a "demand signal" model for CSF production by macrophages.

The effect of reduced oxygen tension and the role of cellular components known to protect the cell against oxygen toxicity has been studied with respect to erythropoietic colony formation in vitro. Alphathioglycerol can be partially replaced by vitamin E and completely replaced by reduced glutathione (GSH) at physiological concentrations. Incubation of bone marrow and fetal liver early (BFU-E) and late (CFU-E) erythropoietic progenitor cells, in the presence of GSH, in an atmosphere containing 5% oxygen, 5% carbon dioxide and 90% nitrogen, as opposed to air supplemented with 5% carbon dioxide, resulted in an increase in colony numbers and response to erythropoietin (Epo). The number of colonies derived from bone marrow and fetal liver CFU-E increased by 1.2--2.8-fold with a relative Epo sensitivity increase of 3.5--4-fold. Bursts obtained from bone marrow and fetal liver BFU-E increased from 2.6- to 3.8-fold with an increased response to Epo of 2--3-fold. The effects of GSH and low oxygen tension are interpreted as causing a reduction in oxygen toxicity of the cells, thereby increasing the life span in vitro and so increasing the number of cells capable of forming colonies. The heightened response of BFU-E to Epo, analogous to the effect seen for CFU-E, implies that BFU-E may be responsive to physiological Epo concentrations at physiological oxygen tensions.

Macrophages derived from unstimulated and unseparated mouse bone marrow cells cultivated on hydrophobic foils can release hemopoietic regulator molecules into the surrounding medium. To prove that one of these regulators exists in macrophages in vitro, in situ hybridization using a 1.2-kb erythropoietin (Epo) gene probe was employed. The probe was biotinylated and the signal developed using a streptavidin-gold reagent. Observation was performed using reflection-contrast microscopy. The results indicate that from a 98% pure population of macrophages, 34% F4/80 (mouse, macrophage-specific antigenic determinant)-positive macrophages exhibited Epo gene expression. The technique was also applied to normal, steady-state mouse bone marrow in which approximately 10% of the cells are F4/80-positive and of which about 3% demonstrated simultaneous Epo gene expression. As positive control, kidneys from anemic mice were hybridized with the biotin-labeled Epo DNA. A second positive control utilized biotin-labeled actin DNA hybridized to cultured macrophages and normal bone marrow cells. The accumulating information, demonstrating that the unstimulated kidney does not express the Epo gene, indicates that Epo is produced in other areas of the body under normal, steady-state conditions. The present results show that 1) macrophages can express the Epo gene, 2) this function is carried out by a subpopulation of macrophages, and 3) bone marrow macrophages in vivo may be responsible for the Epo production-target cell mechanism evoked by short-range and/or cell-to-cell interactions under normal, steady-state conditions.