Fibrinogen cooperates with cytokines to induce interleukin-6 receptor mRNA expression in human hematopoietic CD34+ progenitors.

We have previously demonstrated that purified human fibrinogen (Fg), a major plasma component removed during serum preparation, shows mitogenic properties towards lymphoma cells and normal human hematopoietic progenitors. Indeed, adding Fg with IL-3 to a serum-containing medium stimulates growth of human CD34+ progenitors. In this report, we show in serum-free medium, that this stimulating effect only occurs in the presence of IL-6. To clarify the cooperative effect between Fg and IL-6, the kinetics of IL-6 receptor (IL-6R) mRNA expression in CD34+ cells have been analyzed by semi-quantitative in situ hybridization. In the presence of both IL-3 and Fg, more cells express IL-6R mRNA, and this expression per cell is significantly greater than with each factor added separately. These results suggest that Fg does not promote the growth of normal cells by itself, but sensitizes the cells to cytokines. Fg behaves not as a "progression" factor but as a typical "competence" factor, which induces a faster and greater IL-6R expression in early human hematopoietic progenitors by cooperating with other cytokines.

Transforming growth factor-beta: pleiotropic role in the regulation of hematopoiesis.

Hematopoiesis is a remarkable cell-renewal process that leads to the continuous generation of large numbers of multiple mature cell types, starting from a relatively small stem cell compartment. A highly complex but efficient regulatory network is necessary to tightly control this production and to maintain the hematopoietic tissue in homeostasis. During the last 3 decades, constantly growing numbers of molecules involved in this regulation have been identified. They include soluble cytokines and growth factors, cell-cell interaction molecules, and extracellular matrix components, which provide a multifunctional scaffolding specific for each tissue. The cloning of numerous growth factors and their mass production have led to their possible use for both fundamental research and clinical application.

TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation.

Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-(beta)1 (TGF-(beta)1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-beta signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-(beta)1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-(beta)1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells. Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-(beta)1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-(beta)1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-(beta)1 may be independent. These data provide additional evidence that TGF-(beta)1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM-CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Purified fibrinogen at concentrations of 3-30 nM has been found to stimulate continuous growth of human lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphoma-derived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 10(5) cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, approximately equal to 50 granulocyte-macrophage colonies per 10(5) cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.