The ability of Steel Factor (SF) to stimulate colony formation and progenitor cell generation by hemopoietic stem cells (HSCs) in vitro in the absence of interleukin 3 (IL-3) was investigated. IL-3 was required for HSC proliferation, and no or restricted proliferation occurred in the presence of SF, IL-6, IL-11, or IL-12 as single factors or in combination. Neutralizing concentrations of anti-transforming growth factor (TGF)-beta 1 antibodies enhanced progenitor cell generation 2-3-fold in the presence of IL-3, but 75 to over 300-fold when cultures contained at least SF in the absence of IL-3. Exogenous TGF-beta 1 fully abrogated the antibody effects. In the presence of antibodies to TGF-beta 1, SF alone stimulated the delayed formation of small blast cell colonies and SF synergized with IL-6, IL-11, or IL-12 to greatly hasten colony formation, enhance colony number and size, and increase colony forming unit-culture (CFU-C) output from suspension cultures of enriched HSC populations. Secondary CFU-C colonies were significantly larger when IL-3 was absent during the suspension culture phase. Single cell and limiting dilution analysis using a homogenous colony forming unit-spleen (CFU-S) day-12 population and an 800-fold enriched long-term repopulating HSC fraction, respectively, indicated that TGF-beta 1 was an autocrine product of these HSC subsets. Addition of nucleosides, insulin, extra glucose, or serum could not replace the effects of the anti-TGF-beta 1 antibody. While these data offer one possible explanation for reports on the inability of SF to stimulate HSC proliferation, they present the basis for a novel model of the regulation of HSC activation wherein: 1) close-range interactions of HSCs with mesenchymal stromal cells do not exclusively determine maintenance of HSC quiescence; 2) competence acquisition by dormant HSCs may involve the down-regulation or inactivation of autocrine TGF-beta 1; and 3) SF may act as a primary growth factor rather than exclusively as a synergistic cytokine.

No abstract

The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2 and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5-1%, whereas glutaraldehyde should be used at concentrations between 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.

SUMMARYThe immunoregulatory cytokine, interleukin-10 (IL-10), has been shown to inhibit the maturation of human myeloid dendritic cells (DC). In the present study, we demonstrate that IL-10 has paradoxical effects on the maturation of murine myeloid bone marrow-derived DC. On the one hand, IL-10 inhibits the maturation of murine myeloid DC. The addition of IL-10 to granulocyte±macrophage colony-stimulating factor (GM-CSF)-supported murine BM-derived DC cultures reduced the frequency of major histocompatibility complex (MHC) class II bright cells. These IL-10-pretreated DC have a reduced capacity to stimulate T cells in an allogeneic mixed leucocyte reaction. On the other hand, however, and in contrast to the effects of IL-10 on human DC, we found that the addition of IL-10 from the initiation of the culture onwards induced an up-regulation of the expression of the costimulatory molecules CD40, CD80 and CD86 on murine myeloid DC, as compared to DC generated with GM-CSF only. Moreover, a subpopulation of IL-10-pretreated MHC class II dim DC lacked the capacity to take up dextran-¯uorescein isothiocyanate (FITC), a feature of DC maturation. Taken together, our data demonstrate that the generation of murine myeloid DC in the presence of IL-10 results in a population of incompletely matured MHC class II dim CD80 CD86 DC. These DC lack T-cell stimulatory capacity, suggesting a role for IL-10 in conferring tolerogenic properties on murine myeloid DC.