Interference of a human leukocyte interferon preparation with stimulatory activities in leukocyte-conditioned medium for human hematopoietic stem cells

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of multilineage hemopoietic progenitors derived from human bone marrow. However, PHA-LCM prepared in the presence of a human leukocyte interferon preparation does not support mixed colony formation. Crude PHA-LCM preparations were characterized by gel filtration, affinity chromatography, and gel electrophoresis. The elution profile on Sephacryl S-300 of PHA-LCM prepared without interferon showed a distinct peak that stimulated the growth of pluripotent stem cells (CFU-gemm) and committed precursors (CFU-c, BFU-e). Gel filtration of PHA-LCM, prepared with 1000 U/ml of interferon, revealed a change in the elution profile. The eluted material demonstrated no growth-promoting activities. We conclude that the abolished stimulatory activity of PHA-LCM, prepared with human leukocyte interferon, might be due to a reduced production of stimulatory molecules, suggesting that interferon interferes with the molecular events required for colony formation of committed and noncommitted hemopoietic progenitors.     

Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte-macrophage-megakaryocyte (GMM) differentiation

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GEMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.     

Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU-S) and committed progenitors (CFU-C1, CFU-C2, BFU-E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti-GM-1.2 and anti-PGP-1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light-activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU-S) and progenitors. Anti-GM-1.2 stained only mature myeloid cells, not CFU-S or the in vitro colony-forming cells. Anti-PGP-1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU-S were intermediate, CFU-C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two-step sorting procedure. First, the 6% most WGA-binding cells with high FLS and low PLS were sorted out. A 10-15-fold enrichment of progenitors and CFU-S was obtained. Next, these cells were restained with anti-GM-1.2 or anti-PGP-1 and again fractionated on the FACS. The GM-1.2-negative cells were then another four- to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony-forming unit. With anti-PGP-1, CFU-C2 could be partly separated from more early cells such as CFU-S and BFU-E.     

Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction?

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.     

Human thymic epithelial cells produce granulocyte and macrophage colony-stimulating factors

The development of culture conditions for growing normal human thymic epithelial (TE) cells free from contamination with other stromal cells has allowed us to identify and characterize TE cell-derived cytokines. In this study, we report that cultured human TE cells produced CSF that supported the growth of clonal hematopoietic progenitor cells in the light density fraction of human bone marrow cells. Thymic epithelial supernatants (TES) induced growth of granulocyte/macrophage colonies (CFU-GM), mixed granulocyte/erythrocyte/monocyte/megakaryocyte colonies (CFU-GEMM), and early burst-forming unit erythroid colonies (BFU-E). In addition, TES induced differentiation of the promyelocyte leukemic cell line HL-60 and stimulated growth of both granulocyte (CFU-G) and monocyte (CFU-M) colonies from murine bone marrow cells. Using anion exchange column chromatography, pluripotent CSF activities in TES were separated and shown to be distinct from an IL-1-like cytokine that has been shown as a TE cell-derived cytokine (TE-IL-1). Colony-stimulating activity supporting the growth of bone marrow CFU-GEMM, BFU-E, and CFU-GM co-eluted at 150 to 180 mM NaCl. A separate peak of CFU-GM-stimulating activity eluted early in the gradient at 20 mM NaCl. In Northern blot analysis of enriched RNA, synthetic oligonucleotide probes complementary to human G-CSF and M-CSF coding sequence each hybridized with a single RNA species of 1.7 and 4.4 kb, respectively. These data suggest that normal human TE cells synthesize G-CSF and M-CSF that promote differentiation of non-lymphoid hematopoietic cell precursors.     

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

Recombinant human tumor necrosis factors alpha and beta stimulate fibroblasts to produce hemopoietic growth factors in vitro

The influences of TNF alpha and TNF beta were evaluated for their stimulatory and inhibitory effects on in vitro colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. Both TNF alpha and TNF beta induced fibroblasts to produce stimulators of CFU-GM, BFU-E, and CFU-GEMM in a dose-dependent fashion. Similar results were seen when equivalent concentrations of TNF alpha and TNF beta were used. Prior incubation of the TNF alpha and TNF beta with their respective antibodies inactivated the ability of the TNF preparations to induce the release of granulocyte-macrophage, erythroid, and multipotential colony-stimulating activity from fibroblasts. In addition, incubation of the TNF-induced fibroblast supernatant with antibody before colony assay resulted in enhanced colony formation, suggesting that the TNF carried over into the colony assay suppressed colony formation. Additional proof of this suppression by TNF was evident when TNF was added directly to the CFU-GM, BFU-E, and CFU-GEMM colony assays. IL-1 does not appear to function as an intermediary in growth factor production by fibroblasts stimulated with TNF because antibody to IL-1 displayed no effect. Furthermore, assay of TNF-induced fibroblast supernatant was negative for IL-1. These results suggest that TNF alpha and TNF beta exert both a positive and negative influence on in vitro hemopoietic colony formation.     

Demonstration of a protector effect of interleukin-1 against hematologic toxicity of azidothymidine (AZT)]

3'-azido-3'-deoxythymidine (Azidothymidine or AZT) has attained wide critical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, treatment with AZT is associated with the development of severe hematopoietic toxicity. The AZT sensitivity of marrow progenitors was different with an IC 50 of 10(-8) M and 10(-6) M for respectively BFU-E and CFU-GM/GEMM. Data reported here show that recombinant human interleukin-1 alpha (IL-1 alpha), a pleiotropic cytokine, was demonstrated to be efficient to protect normal human as well as murine hematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E) from the toxic effect of AZT. The maximal effect was observed with 30 U/ml (Human cells) or 100 U/ml (murine cells) IL-1 alpha for BFU-E and CFU-GM/GEMM, with a marked effect at 1 U/ml. The results demonstrate that marrow progenitors respond differently to AZT and point out the potential efficacy of IL-1 alpha to enhance the proliferation of hematopoietic stem cells treated with growth factors (IL-3, erythropoietin) and to minimize the hematopoietic toxicity associated with AZT treatment.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Cryopreservation of human megakaryocytic progenitor cells (CFU-MK): influence of culture conditions

The survival of human megakaryocytic progenitor cells (CFU-MK) after freezing using a two-step cooling technique was studied in a methylcellulose culture system, using different stimulating activities and their combinations. The best growth stimulating activity for CFU-MK was found in plasma from aplastic patients (PAP). PAP was roughly three times more potent than optimal doses of human recombinant interleukin 3 (IL3), itself two times more active than our batch of phytohemagglutinin stimulated leukocyte conditioned medium (PHA-LCM). The addition of erythropoietin (EPO) to PHA-LCM doubled the number of megakaryocytic colonies, but had no effect on the number of CFU-MK stimulated by IL3. Of the two batches of PAP used, one was optimal and the other was significantly improved by the addition of PHA-LCM. The percentage recovery of CFU-MK after freezing reached 70 to 80% in cultures stimulated by PHA-LCM and IL3 +/- EPO, and 58% in PAP-supplemented cultures. However, this last value was not significantly different from the previous ones. The percentage recovery of CFU-MK in each condition tested was similar to that obtained with the other myeloid progenitors, CFU-GM and BFU-E. However, the high CFU-MK stimulating activity of PAP is hindered by its high variability and shortage in supply. Thus it seems more appropriate to recommend the use of recombinant human IL3 because of its easily standardizable and consistent CFU-MK stimulating activity.     

Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies

Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10 DR+) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-alpha, recombinant human interferon-gamma, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-alpha and interferon-gamma and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of 1,25-dihydroxyvitamin D3 on hematopoiesis in long-term human bone marrow cultures

The modulatory effect of 1,25-dihydroxyvitamin D3 (vit D) on the growth of myeloid progenitors and on the composition of the stromal layer in human bone marrow long-term cultures was studied. Vit D (2 X 10(-8) M) caused an enhancement in myeloid progenitor cell (CFU-C) growth in the nonadherent and adherent layers during the entire 5-week incubation period. The vitamin did not alter the differentiation pattern of CFU-C (monocyte-macrophage progenitors CFU-M, granulocytic progenitors CFU-G, or monocyte-granulocyte progenitors CFU-GM). Vit D caused a marked increase in the percentage of lipid-containing cells in the adherent layer and an increase in the number of cells that specifically bound My4 monoclonal antibody (McAb), that reacted positively to fluoride-sensitive alpha-naphthyl acetate esterase, and that phagocytosed Candida albicans (CA). Concentrated supernatants harvested from control cultures showed significant levels of myeloid colony stimulating factor (CSF) activity. The addition of vit D to cultures for 5 weeks did not alter CSF levels. These results suggest that vit D may play a role in hematopoiesis by acting directly on the progenitor cells or via the stromal cell production of stimulatory factor(s).     

A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture

We recently identified a murine hemopoietic stem cell colony which consists of undifferentiated (blast) cells and appears to be more primitive than CFU-GEMM in the stem cell hierarchy. The progenitors for the colony which we termed “stem cell colony” possess an extensive self-renewal capacity and the ability to generate many secondary multipotential hemopoietic colonies in culture. We replated a total of 68 stem cell colonies from cultures of murine spleen cells and analyzed the number of stem cell–and granulocyte(neutrophil)-erythrocyte-macrophage-megakaryocyte (GEMM) colony-forming cells in individual stem cell colonies. Of the 68 stem cell colonies, 35 contained progenitors (abbreviated as “S”-cells) for stem cell colonies. The distributions of S-cells and CFU-GEMM in individual stem cell colonies were extremely heterogeneous. Neither the frequency distributions of S-cells nor CFU-GEMM in stem cell colonies could be fitted well by Poisson distribution. Rather, the frequency distribution of the s-cells could be approximated by a geometric distribution and that of CFU-GEMM by an exponential distribution, both of which are variates of the gamma distribution. Our observations are in agreement with those on the distributions of CFU-S in individual spleen colonies and provided support for a stochastic model for stem cell self-renewal and commitment in culture. Application of the theory of the branching process to the distribution of S-cells revealed a distributional parameter “p” of 0.589 which is also in agreement with the earlier report on the p value for reproduction of CFU-S.     

Blood erythroid progenitors (CFU-E and BFU-E) in acute lymphoblastic leukemias

Circulating erythroid progenitors from 14 patients with acute lymphoblastic leukemia (ALL) and from 8 healthy subjects were studied in culture to determine the frequency and size of CFU-E- and BFU-E-derived colonies. Cells were cultured in a plasma clot system, and hemoglobinized colonies identified by diaminobenzidine reaction. The numbers of CFU-E and BFU-E per milliliter of peripheral blood were greatly increased in 10 patients when compared to controls. In 13 patients, the size distribution of BFU-E-derived colonies, analyzed by counting the number of subunits in each colony, was also found to differ significantly from controls, with a large excess of small colonies and a low percentage or a total lack of large colonies. This abnormal BFU-E size distribution was partially corrected, in the 5 patients tested, by the addition to the culture medium of 10% phytohemagglutinin-leukocyte-conditioned medium (PHA-LCM). Bone marrow crowding out of the normal progenitors, as well as disturbances in the cellular interactions involved in their normal development, most likely explain these results and these factors could be implicated in the frequent pancytopenia of ALL.     

The cellular basis of self renewal in culture by human acute myeloblastic leukemia blast cell progenitors

Blast cells from patients with Acute Myeloblastic Leukemia (AML) were separated according to cell size using velocity sedimentation under unit gravity. Fractions obtained in this way were plated in methyl cellulose with a growth stimulator present in media conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM). Colonies of blast cells form under these conditions. Pooled cell suspensions from such colonies were plated in microwells; the plating efficiency of such suspensions is a measure of blast progenitor self-renewal occurring in the original blast colonies. Self-renewal assays on each fraction indicated that self renewal among blast progenitors is heterogeneously distributed with subpopulations differing in renewal capacities. The results are consistent with the view that blast cell subpopulations in AML undergo a series of transitions associated with decreasing self renewal capacity, analogous to that observed in normal hemopoiesis, where proliferative capacity decreases with increasing differentiation.     

Myeloproliferative sarcoma virus (MPSV), a new tool for the study of hematopoiesis

MPSV induces a myeloproliferative syndrome in susceptible mice associated with an invasion of hematopoietic and nonhematopoietic organs with tumor nodules. The effect of the virus on the various hematopoietic precursors (CFU-S, CFU-C, CFU-E, BFU-E) was studied in vivo in the spleen, blood, and bone marrow, and in vitro, using colony assays in semisolid medium. After in vivo and in vitro infection MPSV induces the appearance of CFU-C, independent of added colony-stimulating activity and of pure and mixed BFU-E, independent of burst-promoting activity. MPSV also induces in vivo an amplification of the size and concentration of the hematopoietic system, including hematopoietic stem cells. MPSV infection may also alter the hemapoietic microenvironment. Modification of the disease by total body irradiation followed by bone marrow stem cell reconstitution or by splenectomy is compatible with mediation of the virus effect at the level of hematopoietic microenvironment. MPSV may constitute a new tool to study the regulation of murine hematopoiesis and viral genetic information, which can specifically induce characteristic disturbances of this system.     

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.     

Differentiation of CD34+ cells from human cord blood and murine bone marrow is suppressed by C6 beta-chemokines

Several recently identified chemokines, Lkn-1, CKbeta8-1, MRP-2, and Mu C10 (MRP-1), are classified as C6 beta-chemokines. All of these chemokines have been found to suppress colony formation by bone marrow (BM) myeloid progenitors. Since cord blood (CB), like BM, contains CD34-positive cells, we examined the effects of these chemokines on CD34+ cells isolated from human CB. Lkn-1 and CKbeta8-1 suppressed colony formation by multi-potential granulocyte erythroid mega-karyocyte macrophages (CFU-GEMM), granulocyte-macrophages (CFU-GM), and erythroid (BFU-E) cells among the CD34+ cells from CB. CC chemokine receptor 1 (CCR1) that is known to be a receptor for Lkn-1 and CKbeta8-1 in neutrophils, monocytes, and lymphocytes, was also present on the surface of CD34+ cells from CB. Taken together these results suggest that Lkn-1 and CKbeta8-1 are active in inhibiting myeloid progenitor cells from both BM and CB. Macrophage inflammatory protein related protein-2 (mMRP-2) and Mu C10 (mMRP-1), which are murine C6 beta-chemokines, also inhibited colony formation by CB CD34+ cells. The inhibitory activity of these chemokines suggests that they may protect hematopoietic progenitors from the cytotoxic effects of the antiblastic drugs used in cancer therapy.