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One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.

Myeloid leukemic progenitor cells proliferate in vitro in response to a variety of humoral factors, the most prominent among these being granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanism by which GM-CSF transduces its proliferative signal in acute myeloid leukemia has been extensively investigated over the past year. It is now known that the GM-CSF belongs to a new family of hematopoietic growth factor binding proteins which are characterized by a relatively short intracytoplasmic domain that lacks a tyrosine kinase sequence. Nevertheless, studies performed using GM-CSF-dependent leukemic cell lines demonstrate the appearance of several new phosphotyrosine species after GM-CSF exposure, suggesting that receptor activation is directly or indirectly linked to tyrosine kinase stimulation. Apart from the basic biology of GM-CSF-induced signal transduction, the ability of this factor to enhance the S-phase fraction of myeloid leukemia blasts may have important therapeutic implications. Clinical trials are currently being conducted in an attempt to determine whether GM-CSF is able to overcome kinetic resistance of leukemic myeloblasts to cell cycle-specific agents such as cytarabine in the treatment of patients with acute myeloid leukemia.

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors (Kd = 30-100 pM). By using radiolabeled GM-CSF with high specific activity, we have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations (95% +/- 1%, 34% +/- 5%, 48% +/- 8% receptor down-regulation, respectively). GM-CSF also down-regulated its own receptor on monocytes and myeloid progenitor cells. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, we also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

The expression of HLA-DR antigens by normal myeloid progenitor cells (CFU-GM) has been linked to inhibition of colony growth by prostaglandin E (PGE), while resistance to the inhibitory effects of PGE in chronic myeloid leukemia (CML) has been attributed to a lower fraction of HLA-DR+ CFU-GM in this disease. However, we have previously shown that virtually all CFU-GM in normal bone marrow (NBM) as well as CML peripheral blood express HLA-DR antigens, which raises the possibility that these surface molecules may not be the sole determinants of a progenitor cell's sensitivity to PGE. In order to evaluate the relationship between HLA-DR expression and prostaglandin inhibition, we partially purified NBM progenitor cells using fluorescence-activated cell sorting to prepare cell fractions with high and low HLA-DR antigen density. Normal progenitor cells with high DR density tended to form monocyte colonies in agar culture, whereas the low DR density fraction was enriched for granulocyte colony-forming cells. Inhibition by PGE was greatest in the high DR+ fraction and was largely restricted to monocyte progenitor cells. Inhibition of CFU-GM by PGE was less in CML than in NBM, but this decreased inhibition correlated with a significantly lower number of monocyte-CFU in CML. These data suggest that high HLA-DR antigen density may select for normal progenitor cells that are committed to monocyte differentiation and are, therefore, more likely to be inhibited by PGE. The relative deficit of monocyte progenitor cells in CML may partially explain the phenomenon of PGE resistance in this disease.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on bone-marrow and peripheral-blood progenitor cells was investigated in a three-phase study in 13 patients with sarcoma. In the first phase patients were given GM-CSF alone. In phase II, which started a week after completion of phase I, patients received a course of cytotoxic chemotherapy, then a course of GM-CSF. Phase III consisted only of cytotoxic chemotherapy. GM-CSF (phase I) alone produced an 18-fold increase in peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) and an 8-fold increase in erythroid burst-forming units (BFU-E) in the peripheral blood. GM-CSF had no effect on bone-marrow CFU-GM and BFU-E in the group as a whole. Three patients were investigated after phases II and III. GM-CSF increased the absolute number of peripheral blood CFU-GM by approximately 60-fold compared with the pretreatment baseline. These effects of GM-CSF may be of clinical importance with regard to facilitating the harvest of peripheral blood progenitor cells for autotransplantation.

In this study, we have examined the effects of autologous monocytes and T-lymphocytes on gamma-interferon (gamma-IFN)-induced inhibition of granulocyte-monocyte progenitor cells (CFU-GM) in vitro. Depletion of adherent cells from the mononuclear fraction of normal bone marrow (NBM) resulted in a significant reduction in the inhibitory effects of gamma-IFN on CFU-GM growth, whereas T-lymphocyte depletion had no effect. Adding back autologous monocytes to the underlayer fraction of agar culture resulted in a concentration-dependent enhancement of gamma-IFN-induced CFU-GM inhibition that did not require cell-cell contact. Adding back autologous T-lymphocytes had no effect and did not synergize with monocytes in enhancing gamma-IFN-induced inhibition. Based on the use of indomethacin and the pattern of CFU-GM subset growth, it was determined that prostaglandin E was unlikely to be the humoral inhibitory factor involved in this process. However, the effects of monocytes were completely reversed in the presence of a neutralizing monoclonal antibody to tumor necrosis factor (TNF), suggesting that monocyte-derived TNF was responsible for the enhancement of gamma-IFN-induced CFU-GM inhibition. This observation was further supported by the ability of gamma-IFN to induce an eightfold increase of baseline monocyte TNF secretion in agar culture. These data suggest that gamma-IFN may inhibit progenitor cell growth in vitro through indirect humoral mechanisms involving monocyte-derived TNF, as well as through direct inhibitory effects on CFU-GM proliferation. Because monocytes are a component of the bone marrow microenvironment, the ability of gamma-IFN to induce biologically relevant levels of monocyte-derived TNF may play an important role in the negative regulation of hematopoiesis.

The regulation of Ia (HLA-DR) antigen expression on myeloid progenitor cells may be closely related to the control of myelopoiesis in both normal individuals and chronic myeloid leukemia (CML) patients. In an effort to study directly the expression and behavior of Ia surface molecules on myeloid progenitor cells, we used an immunologic purification technique to enrich these cells approximately 100-fold from the peripheral blood of CML patients. The majority of cells in this blast population expressed HLA-DR antigens. Thirty percent to 40% of cells could form a granulocyte or monocyte colony in agar, and these cells tended to express the highest levels of HLA-DR. The number of HLA-DR molecules per cell increased about twofold as the cells tranversed the cell cycle from G0/G1 to G2/M. This was true for unstimulated cells or cells exposed to colony-stimulating factors. Some of this increase was related to a corresponding increase in cell size and is also seen with other cell surface antigens such as beta-2-microglobulin. Ia antigen expression was not modified by culture with colony-stimulating factors, fetal calf serum, or serum-free, prostaglandin-free medium for periods of up to 24 hours. These results demonstrate that Ia antigens are expressed on the myeloid progenitor cells of CML, are increased in the S and G2/M phases of the cell cycle, and are stable under most in vitro culture conditions for at least 24 hours of culture.