Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.     

Characteristics of proliferation and differentiation of spleen colony-forming cells from bone marrow

By using c-band staining or sex chromosome identification techniques, we have demonstrated that some of the spleen colony-forming cells from normal bone marrow have the potential to form both myeloid and lymphoid elements.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

The sensitivity of G0-state haemopoietic spleen colony-forming cells to a stimulus for proliferation

Haemopoietic spleen colony-forming units (CFU-s) close to the axis (axial CFU-s) of the long bones have a high probability of self-renewal. They are pluripotent cells and are largely in a G0-State. By contrast, CFU-s close to the bone surface (marginal CFU-s) have a lower probability of self-renewal and are probably more mature, though still pluripotent. Most CFU-s proliferation arises in this zone. As a consequence, marginal CFU-s tend to have shorter G0 histories than do axial CFU-s. Femoral marrow was, therefore, divided into axial and marginal populations and the sensitivity of the CFU-s to an endogenous CFU-s-specific proliferation-stimulating factor was assessed and compared by the tritiated thymidine suicide technique. It was found that axial CFU-s are considerably more resistant to stimulation than are marginal CFU-s in that larger doses for longer periods of exposure are required to increase the proliferative activity of the cells. This behaviour is consistent with the suggestion that cells with a low division probability exist in deeper levels of the quiescent G0-state. Although this hypothesis was developed from the behaviour of cells maintained in culture under sub-optimal physiological conditions, this phenomenon appears, in vivo, to be a characteristic of the stem cell population of haemopoietic tissue; their high resistance to stimulation maintaining the axial CFU-s in a quiescent state.     

The production of factors regulating the proliferation of haemopoietic spleen colony-forming cells by bone marrow macrophages

Media conditioned by normal murine bone marrow cells contain an inhibitor of haemopoietic spleen colony-forming cell proliferation that is concentrated in a nominal 50-100K fraction. Media conditioned by regenerating marrow cells contain a proliferation-stimulatory activity that is concentrated in a nominal 30-50K fraction. Cell separation experiments demonstrated that the activities are produced by adherent, phagocytic, radioresistant, Thy 1.2- Fc+, F4/80+ cells. Cultured macrophages, obtained from long-term marrow cultures or derived from progenitor cells in methyl cellulose cultures are also capable of producing inhibitory and stimulatory activities. The results are consistent with macrophages being an important source of stem cell proliferation regulators in the bone marrow.     

Recovery of proliferative capacity of agar colony-forming cells and spleen colony-forming cells following ionizing radiation or vinblastine

The effects of sublethal radiation and the mitotic inhibitor, vinblastine sulphate, on the number of cells in mouse bone marrow capable upon transplantation of forming macroscopic colonies on the surface of the spleens of irradiated recipient mice (CFU) and on the number of cells capable of forming colonies in soft agar after cell culture (ACFU) were studied as a function of time after injury. The results show that ACFU are radiosensitive and vinblastine-sensitive cells, comparable in sensitivity to erythropoietin-sensitive cells. The temporal pattern of recovery following radiation of ACFU, different from that for CFU, is compatible with the concept that these are two distinct but closely related stem cell populations. The relevance of these findings to models of hematopoiesis and to studies on the precursors of macrophages and monocytes in inflammatory exudates is discussed.     

Haemopoietic stem cells: spleen colony-forming cells are normally actively proliferating

Abstract. The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.     

Haemopoietic stem cells: spleen colony-forming cells are normally actively proliferating

The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Effect of the dose of injected hematopoietic cells on the number of daughter splenic colony-forming cells in spleen colonies

The number of daughter CFU-S per spleen colony was critically dependent on the injected cell dose and the time of registration. The influence of these factors on the measurement of self-maintenance CFU-S ability is discussed.     

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.     

Effect of bone marrow cells on colony-forming stromal cells in guinea pigs and on the proliferation of their cultured descendents

In the presence of irradiated bone marrow cells the efficiency of stromal colony formation increases from 0.8 +/- 0.2 to 3.6 +/- 0.4 per 10(4) explanted bone marrow cells. The growth-stimulating activity of bone marrow cells on passaged bone marrow fibroblasts depends on growth conditions of passages to which irradiated bone marrow cells are added. The response of proliferating bone marrow fibroblasts to stimulating activity of bone marrow cells is low, while addition of bone marrow cells to fibroblast cultures stimulates their proliferation.     

The distribution of colony-forming cells in the kidney

Abstract. A method is described for producing outgrowths of small nephron segments (average 24 cells) in culture. The method was used to estimate an overall colony-forming efficiency of 4.6% for cells constituting the segments. Efficiency was found to be lower for thick segments (1%) than for thin segments (6%) from Henle's loop. The latter higher level indicates that precursor cells are concentrated near the middle of the nephron. For comparison, a two-dose irradiation technique was used to calculate a mean number of 5 ± 2 (SE) clonogens per segment producing outgrowths. This tended to be higher than the value of about 1 calculated from the 65% of segments producing outgrowths, as expected if the remaining segments contained no clonogens.