Bone marrow stromal dysfunction in mice administered cytosine arabinoside.

OBJECTIVE: The aim of the study was to investigate ex-vivo the bone marrow (BM) stroma of mice under conditions of low- and high-dose cytosine arabinoside (Ara-C), a cycle-specific drug (S-phase) and to assess possible stromal damage, apart from the killing of hematopoietic cells. Stroma consists of mesenchymal elements generally not in the cell cycle; therefore it could not be a target for the killing effect of Ara- C. MATERIALS AND METHODS: The stromal function was studied by the following: the incidence of stromal stem cells, i.e. CFU-F; formation of stromal layers under growth conditions of long-term culture (LTC) followed by irradiation and overlayering of test cells in contact and non-contact co-cultures; subsequent culture of the test cells in a semi-solid medium to assay the incidence of hyperproliferative potential cells (HPPC); production of GM-CSF, IL-3, IL-4, IL-6 and IFNgamma in the conditioned medium (CM) of confluent stromal layers. All tests and assays were carried out on BM specimens, 1-4 d after Ara-C administration and on controls. RESULTS: Low-dose Ara-C induces a marked decrease of CFU-F, compensated by cycle induction of pre-CFU-F, young-type stromal stem cells. High-dose Ara-C causes a CFU-F decrease to almost zero level. The time length to layer confluency is normal after low-dose Ara-C ( approximately 10 d) and prolonged after a high dose ( approximately 30 d). The confluent layers from mice receiving low- or high-dose Ara-C support hematopoiesis adequately. Among the growth factors and cytokines assayed, only IL-6 is detected in CM layers. IL-6 decreases after a low dose of Ara-C and increases after a high dose. The cause of IL-6 fluctuations is yet to be investigated. It is, however, evident that IL-6 is not an essential factor in support of hematopoiesis. CONCLUSIONS: Taken together, the current study in mice indicates that Ara-C administration, in particular a high dose, induces bone marrow stromal damage and/or disfunction. The long period of time to reach layer confluency after a high Ara-C dose might reflect the in-vivo situation of slow stromal regeneration.

High-efficiency retroviral gene transfer into murine high-proliferative-potential cells cycle-activated by cytosine arabinoside.

We investigated cytosine arabinoside (Ara-C) as a potential agent for in vivo cycle activation of hematopoietic progenitors for the purpose of retroviral-mediated gene transfer. C57Bl mice were treated intraperitoneally with one of three regimens of Ara-C: a single 1,750 mg/kg dose (regimen 1), a 1,750 mg/kg dose on day 0, and a 1,500 mg/kg dose on day 2 (LD50) (regimen 2), or a 1,750 mg/kg dose on day 0 and a 1,500 mg/kg dose on day 3 (regimen 3). The high-proliferative-potential cells (HPPC)/10(5) cells were 47.0 +/- 7.5 pretreatment. The post-treatment HPPC cloning efficiencies were 40.6 +/- 3.4, 83.6 +/- 6.1, and 20.4 +/- 3.2 HPPC/10(5) cells on days 1, 2, and 4, respectively, with regimen 1; 60.0 +/- 7.9, 194.0 +/- 9.6, and 103.0 +/- 11.0 HPPC/10(5) cells 1, 2, and 4 days after the second Ara-C dose, respectively, with regimen 2; and 266 +/- 13.4, 132 +/- 23.9, and 118.0 +/- 5.7/10(5) cells 1, 2, and 4 days after the second Ara-C dose, respectively, with regimen 3. The transduction efficiency of HPPC from untreated animals with N2 viral supernatant was 4.9 +/- 5.8%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine and growth factor gene expression by bone marrow stroma of mice with damaged hematopoiesis and during regeneration.

A study of gene expression of GM-CSF, IL-3, c-kit ligand, and IL-1 by bone marrow stromal cells of mice who were treated with an LD50 cytosine arabinoside (Ara-C) dose was carried out. It was shown previously that this dose causes extensive hematopoietic damage which is followed by regeneration in surviving mice. Using PCR, we demonstrated GM-CSF and IL-1 expression by adherent cells taken from marrow fibroblast layers following Ara-C-induced hematopoietic damage and during marrow regeneration, while expression in control layers was not detected. The IL-3 gene was not expressed either by layers of Ara-C-treated mice or by controls, probably due to the absence of T-lymphocytes in 2-4 week old cultures. The c-kit ligand gene was expressed by layers during marrow regeneration and by control layers, but was absent during the stage of hematopoietic damage. In parallel, in vitro cytokine production was evaluated. While IL-3 and GM-CSF were not present in the conditioned medium of marrow fibroblast layers either from Ara-C-treated mice or controls, IL-1 was found in low concentrations in cultures from Ara-C-treated animals. We conclude that GM-CSF, c-kit ligand and IL-1 have important roles in sustaining marrow regeneration following extensive damage. The role of IL-3 in marrow regeneration cannot be assessed in fibroblast layers of 2-4 week incubation where T-lymphocytes are generally absent.

Minimal residual disease in vitro of a pre-B-lymphoma.

We have studied the survival of clonogenic neoplastic cells of a murine pre-B-lymphoma (BCl-1) of the spleen in culture. We have found quantitative deficiencies such as reduced surface adherence of stromal cells and impaired CFU-F (colony forming units-fibroblast) and pre-CFU-F colony and layer formation in stromal cultures of lymphoma bearing spleen, as compared to cultures from normal spleen. There are two populations of clonogenic BCl-1 lymphoma cells surviving in culture: one population is surface adherent, and the other is non-adherent. Both populations transmit the lymphoma to healthy indicator mice. We hope that this model will be helpful in studying minimal residual leukemic disease [1] in culture.

Improved prognosis in mice with advanced myeloid leukemia following administration of GM-CSF and cytosine arabinoside.

Acute myeloid leukemia (AML) was induced in C57Bl mice through the i.v. innoculation of C-1498 cell line. One week later, i.e. at mid-term disease, the leukemic mice received an i.p. injection of 200 ng rmGM-CSF and 24 h later, two consecutive i.p. cytosine arabinoside (ara-C) injections at 6 h intervals (2 x 200 mg/kg). The leukemic mice received 3-4 weekly courses of combined therapy and survived 4-5 weeks following leukemia induction. Control mice received ara-C only and survived 2-3 weeks. Moreover, leukemic mice administered both GM-CSF and ara-C had a lower marrow leukemic load than mice treated with ara-C only. From these findings, we conclude that therapy of murine AML with combined rmGM-CSF and ara-C is more effective than ara-C only. Leukemic mice treated with GM-CSF and ara-C had a longer life expectancy and a smaller leukemic load than mice administered ara-C only.

Bone marrow stromal elements in murine leukemia: decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages.

A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF alpha in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicitly, reduced CSF production.

Cumulative bone marrow stromal damage caused by X-irradiation and cytosine-arabinoside in leukemic mice.

A study of treated murine acute myeloid leukemia (AML) with an emphasis on the bone marrow stromal function is reported. Leukemia was induced in C57Bl mice through intraperitoneal (i.p.) inoculation of C-1498 myelogenous leukemic cells. The leukemic mice were administered: (1) total body lethal X-irradiation (t.b.i.); (2) two i.p. cytosine-arabinoside (Ara-C) injections followed by X-irradiation. Control mice received similar regimens. Bone marrow of experimental and control mice was processed for stromal cell cultures (SCC) and in vitro engraftment of hematopoietic cells onto the cultures. The results of this study indicate that the bone marrow stromal deficiency which occurs in leukemia is aggravated by Ara-C and irradiation treatments. Moreover, SCC of treated leukemic mice sustain in vitro hematopoiesis only to a limited degree. Stromal deficiency, as possible cause for graft failure in bone marrow transplanted leukemic patients, is discussed.

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture.

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.

Haemophagocytosis and other haematological aspects of Mycobacterium lepraemurium disease of mice.

CBA mice inoculated i.v. with 2 x 10(8) viable MLM were observed at intervals for 5 months. In the bone marrow, haematopoietic cells were progressively displaced by MLM-laden phagocytes; depletion of erythroid cells began earlier and was more pronounced than that of myeloid cells. Transiently, mild anaemia and profound leucopenia were noted. The spleen was enlarged and the site of increasing histiocytosis and extramedullary haematopoiesis which was accompanied by displacement of splenic parenchyma. The liver was enlarged and its parenchyma contained scattered islands of haematopoietic elements. Lymph node cells had been largely replaced by MLM-laden macrophages by the end of the process. Thus, MLM infection exerts important effects on haematopoiesis of susceptible mice and is accompanied by active extramedullary haematopoiesis. In addition, the haemophagocytosis, observed most commonly in immuno-compromised patients infected with certain viral or mycobacterial pathogens, was observed late in the course of MLM disease of mice.

Enhanced capacity of cytosine-arabinoside-treated murine bone marrow to maintain hematopoiesis in long-term culture.

A study of bone marrow of C57B1 mice administered cytosine-arabinoside (Ara-C) was carried out in long-term bone marrow culture (LTBMC). Two days after administration of two consecutive i.p. Ara-C injections (200 mg/kg each) at 6-h intervals, the bone marrow becomes hypocellular, yet in the process of regeneration, with an enriched and/or concentrated content of progenitors and stem cells. Ara-C-treated marrow was observed to sustain hematopoiesis in vitro better than physiological marrow; it produced a higher cell yield, a higher proportion of young-type myeloid cells, and higher levels of granulocyte-macrophage colony-forming cells and colony-forming units in diffusion chamber than control marrow. In addition, stromal cell cultures (SCC), devoid of hematopoiesis and engrafted with hematopoietic cells from LTBMC of Ara-C-treated marrow, were observed to produce hematopoietic cells for longer periods of time than SCC engrafted with control cells. In view of its increased capacity for regeneration, it is suggested that regenerative marrow should be used in autologous bone marrow transplantation in humans.

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia.

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.

Immune impairments and antibodies to HTLVIII/LAV in asymptomatic male homosexuals in Israel: relevance to the risk of acquired immune deficiency syndrome (AIDS).

We have studied 288 Israeli asymptomatic male homosexuals (MHS) to determine the prevalence of antibodies to HTLVI and HTLVIII and their correlation with impairments of the immune system and serum interferon (IFN). Seropositivity for HTLVI, HTLVIII, or both was found in 1.4, 8.3, and 0%, respectively. Significant decreases in the total peripheral T cells, TH cells, and TH/TS ratio as well as elevated alpha IFN serum levels were found in the MHS group in comparison with normal controls. Although no difference in the prevalence of either immune derangements or elevated serum IFN was observed between HTLVIII/LAV-seropositive and HTLVIII/LAV-seronegative MHS, the decreases in total T cells, TH cells, and TH/TS ratios were significantly greater in the seropositive MHS. These results indicate that (a) immune impairments and IFN system activation occur commonly in homosexuals, precede their exposure to HTLVIII/LAV, and probably reflect this group's increased risk for AIDS and (b) HTLVIII/LAV infection of MHS aggravates further their preexisting immune impairments.

PIP: In 1983, researchers studied 288 asymptomatic Israeli male homosexuals (mean age, 30.8 years) to determine the prevalence of antibodies to HTLVI and HTLVIII and their correlation with impairments of the immune system and serum interferon (IFN). These men reported an average of 11.8 years of homosexual experience and an average of 42 partners/year. 26% of the participants had mild inguinal lymphoadenopathy in the beginning of the study and 8 of the 26% later developed generalized lymphoadenopathy. Further, HTLVIII infection progressed to AIDS in 3 of the 288 men during follow-up observations. Laboratory personnel tested each participant's blood for HTLVI and HTLVIII antibodies by using the ELISA technique and positive results were confirmed by immunoblotting, titration, and specific competition with either HTLVI or HTLVIII. The seropositivity prevalence for HTLVI, HTLVIII, or both was 1.4, 8.3, and 0% respectively. No antibodies to either virus were present in the control subjects. Researchers noted significant increases in the level of serum IFN (2SD) in 50% of both seronegative and seropositive homosexuals, when compared with the control group. Significantly more lymphoid population impairment existed in the HTLVIII seropositive homosexuals than in the HTLVIII seronegative homosexuals, but more lymphoid population impairment was found among the HTLVIII seronegative homosexuals than the controls. Therefore, this study provides evidence that such abnormalities in seronegative individuals are present prior to any exposure to the HTLVIII virus. Thus, infection with the virus probably aggravates the preexisting immune impairments, leading eventually to the development of AIDS.

CFU-F circulating in cord blood.

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors.

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.

Effects of chemotherapy on bone marrow stroma in mice with acute myelogenous leukemia. Correlation with CFU-C and CFU-D.

This study describes changes in bone marrow stroma in murine acute myelogenous leukemia (AML). The AML was induced in C57B1 mice by intravenous (i.v.) transfusion of C4198 myelogenous leukemic cells. In untreated leukemic mice, the colony-forming unit fibroblasts (CFU-F) were severely inhibited. In leukemic mice treated by three chemotherapy protocols of cytosine-arabinoside (Ara-C) and adriamycin there was a 200% increase in the life span as compared to untreated leukemic animals and marked reduction of marrow leukemic load. In these mice the stromal inhibition was temporarily relieved, expressed by peaks of CFU-F2-3 days following each protocol. In between the peaks, CFU-F decreased to subnormal levels, remaining low to the end of the disease. In normal mice administered a similar chemotherapy regimen, there were peaks of CFU-F activation after each protocol and normal levels in between the peaks. Granulocyte/macrophage progenitors (CFU-C) of leukemic-treated and normal-treated mice showed increased levels following each chemotherapy protocol. Whereas CFU-C decreased below normal levels in leukemic mice towards the end of the disease, the level of these progenitors remained high in normal mice receiving Ara-C and adriamycin. Colony-forming units in diffusion chamber (CFU-D) showed mild fluctuations in both leukemic and normal mice receiving three protocols of Ara-C and adriamycin. It is possible that despite treatment, the bone marrow stroma in leukemia becomes irreversibly deficient towards the end of the disease and cannot support the residual normal hematopoiesis.

Bone marrow stromal deficiency in acute myeloid leukemia in mice.

A study of bone marrow stroma in acute myeloid leukemia (AML) in mice was carried out. AML was induced in C57B1 mice by i.v. inoculation of the C1498 cell line. There was a direct correlation between the marrow leukemic load and the degree of marrow stromal deficiency. This was expressed by reduced in vitro fibroblastoid colony formation. In co-cultures of normal mouse bone marrow and leukemic cells, and also in cultures of normal marrow with added conditioned medium (CM) of leukemic cells, marked inhibition of fibroblast-colony-forming units (CFU-F) from normal marrow was observed. In additional experiments, leukemic mice were treated with two consecutive injections of cytosine arabinoside (Ara-C) (2 X 200mg/kg) and sacrificed 48 h later. Bone marrow samples of treated animals formed in vitro an increased number of fibroblastoid colonies despite relatively high levels of marrow leukemia. It is concluded that: (a) there is a direct correlation between the incidence of marrow leukemic cells and the degree of stromal deficiency; (b) leukemic cells produce in vitro--and probably in vivo CFU-F inhibitory factors and (c) administration of restricted doses of cytosine arabinoside to leukemic mice reduces the marrow leukemic cell content to some extent and increases the capacity of CFU-F to form fibroblastoid colonies in vitro.

Incidence and characteristics of colony-forming units fibroblasts (CFU-F) in the bone marrow of weanling mice treated with cytosine arabinoside and phenylhydrazine: correlation with CFU-S, progenitors of diffusion-chamber colonies (CFU-D), and CFU-C.

A study of murine adherent marrow cells (AMC) under conditions of high and low concentrations of hematopoietic stem cells and progenitors was carried out. In one group of weanling mice, decreased marrow cellularity and increased concentrations of CFU-S, CFU-D, and CFU-C were observed two days after administration of two consecutive intraperitoneal (i.p.) cytosine arabinoside (Ara-C) injections (2 x 200 ng/kg, at 6 h interval). Fibroblast colony-forming units (CFU-F) of this marrow were studied. In a second group of mice, which were given three i.p. phenylhydrazine (PHZ) injections (3 x 60 mg/kg on days 0, 1, and 3), CFU-S and CFU-C levels were unchanged or lowered 6 days after the start of PHZ administration. In these animals, however, the number of CFU-D was four times higher than in controls. The study of CFU-F in experiments of groups 1 and 2 indicated a high concentration of these progenitors in group 1 and lower concentration in group 2. Furthermore, fibroblastoid colonies produced in vitro by CFU-F of Ara-C-treated marrow were significantly larger than colonies from control marrow, and they markedly inhibited G/M colonies in split-phase agar cultures. By contrast, fibroblastoid colonies produced by PHZ-treated marrow were of regular size and did not inhibit G/M colonies from test bone marrow.

Differential characterization of colony-forming units in diffusion chambers from pluripotent stem cells and progenitors of granulocytes/macrophages.

Colony-forming units in diffusion chambers (CFU-D), pluripotent stem cells (CFU-S) and colony-forming units in agar cultures (CFU-C) were studied in the bone marrow of weanling mice injected twice with hydroxyurea (HU). CFU-D, CFU-S and CFU-C were assayed 2 h, 2 days and 4 days after HU administration. After 2 h the total number of CFU-D/femur marrow cells did not change, while CFU-S and CFU-C decreased to 16.6 and 13.5% of control levels, respectively. On Day 2, CFU-D and CFU-S increased to 25 and 16.6%, respectively, above control levels and CFU-C was only 52% of the normal level. Four days after HU administration, CFU-S decreased steeply to 7.6%, CFU-D 80% and CFU-C to 32% of their pretreatment levels. Bone marrow of HU-treated weanling mice was also incubated in liquid medium where CFU-S decreased more rapidly than did CFU-D. It is concluded that CFU-D in weanling mice represent a noncycling and more primitive stem cell pool than CFU-S and probably constitute a reserve pool, the mobilization of which is secondary to that of CFU-S.

A comparison of spontaneous and CSF added CFU-MG colony formation in healthy, sick and hypotrophic pre-term infants.

Myelopoietic progenitor cells (CFU-MG) have been studied from peripheral blood of healthy, sick, and hypotrophic pre-term infants. Methylcellulose cultures were prepared simultaneously with and without exogenous colony stimulating factor. It was found that large numbers of circulating CFU-M are present at birth in healthy infants, smaller numbers in sick infants, and very few in hypotrophic infants. Exogenous CSF increases the number of colonies in cultures of healthy infants at birth. A limiting factor in spontaneous colony formation is the production of CSF by the cells in culture. This is particularly evident in sick infants. During the postnatal course similar levels of circulating CFUc, higher than in adult blood, are found in all three groups of pre-term infants.