Physiopathological studies on granulocyte-macrophage colony stimulating factor and multi colony stimulating factor producing leukemia, L8313, induced by irradiation of C3H mice

Radiation-induced L8313 leukemia bearing mice (L8313 mice) had marked granulo-cytosis with splenomegaly. Hemopoietic stem cells and progenitors increased in the spleen but not in the bone marrow. Spleen conditioned-medium and serum from L8313 mice induced the formation of granulocyte-macrophage colonies (CFU-GM), erythroid bursts (BFU-E) and mixed colonies (CFU-Mix). Bone marrow conditioned medium did not show such activity. A cell line (STIL-3) was established from the spleen cells of L8313 mice. Surface marker analysis showed that the established cells were suppressor T cell. The cells produced IL-3 and GM-CSF in vitro, and induce essentially the same “leukemic” response in recipient mice. Inoculation of STIL-3 in diffusion chamber also induced leukemoid reaction, i.e. a marked granulocytosis with splenomegaly. Therefore, L8313 leukemia may be linked to an abnormality of growth and production of hemopoietic factors in hemopoetic regulatory cells.     

Establishment of a hemopoietic stimulating factor producing murine leukemia cell lines: pathogenesis of granulocytosis in L8313 bearing mice

Thy 1.2+ cells of L8313 leukemia bearing mice were previously shown to produce granulocyte macrophage colony stimulating activity (GM-CSA) and interleukin-3 (IL-3). Cell lines with the Thy 1.2+ phenotype were established from spleen cells of the leukemic mice, and were designated STIL-3 C5 and STIL-3 D10. They produced and released GM-CSA and IL-3 activities in culture supernatant. C5 cells were phenotypically Thy 1.2+ and Lyt 2.1+ with rearrangement of the T-cell receptor beta chain gene also being identified. D10 was Thy 1.2+ and L3T4+ with T-cell receptor beta chain gene rearrangement not detectable. Since the same sized rearranged bands were observed between C5 and L8313 leukemic mouse spleen cells, it is indicated that C5 cells are derived from the leukemic cells. Inoculation of these cells into mice induced granulocytosis. These results indicated that L8313, which was thought to be a "granulocytic leukemia", is actually a T-cell leukemia, whose granulocytosis is a leukemoid reaction induced by normal hemopoietic cells responding to the hemopoietic stimulating factors, GM-CSA and IL-3, produced by the leukemic T cells. Thus, L8313 should be known as a T-cell leukemia associated with granulocytosis.     

Acceleration of a murine T-cell leukemia associated with loss of interleukin-3 producing activity

L-8313 is a murine T-cell leukemia cell line the cells of which constitutively produce interleukin-3 (IL-3). S2-8313 is a unique subline of L-8313 which has lost IL-3 producing activity. Although the growth of S2-8313 cells in the bone marrow is comparable to that of L-8313 cells, the survival time of C3H mice grafted with S2-8313 cells is significantly shorter than that of C3H mice grafted with L-8313 cells. The accelerated death observed in C3H mice bearing S2-8313 cells is attributable to early development of granulocytopenia and thrombocytopenia, which resulted from the loss of the IL-3 producing activity.     

Distribution of a hematopoietic-specific differentiation antigen of K562 cells in the human myeloid and lymphoid cell lineages

BALB/c mice were immunized with uninduced K562 erythroleukemia cells and hybridomas were isolated after fusion of immune spleen cells to P3/NS1 murine myeloma cells. One selected hybrid, designated 10L-30, secreted an antibody of subclass immunoglobulin G2a which was specific for hematopoietic cells. Analysis of 10L-30 binding by complement-mediated cytotoxicity, indirect immunofluorescence, solid-phase radioimmunoassay, and mixed hemadsorption assay indicated that the 10L-30 antigen was expressed on the myeloid cell lines K562, KG-1A, KG-1, some B- and T-lymphoid cell lines, and all normal human peripheral blood T-lymphocyte samples tested, but was absent on the more differentiated myeloid cell lines HL-60, ML-2, ML-3, and normal blood granulocytes. Induction of erythroid differentiation in hemin-treated K562 cells caused a 10-fold reduction in 10L-30 binding. Human erythroid and granulocytic progenitor cells, platelets, erythrocytes, and reticulocytes were nonreactive, as were a variety of nonhematopoietic human tumor cell lines. Freshly isolated leukemic bone marrow samples from patients with M5 (2 of 5), M6 (2 of 2), acute lymphoid leukemia (9 of 14), and chronic myeloid leukemia in lymphoid blast crisis (1 of 1) were 10L-30 positive. The combined evidence indicates that the 10L-30 antigen is a normal, hematopoietic-specific differentiation antigen which is strongly expressed on both immature cells of the myeloid lineage and more generally in lymphoid ontogeny. The 10L-30 antigen may be a useful marker of both normal and leukemic hematopoietic differentiation.     

Differences in resistance of metastatic tumor cells and cells from local tumor growth to cytotoxicity of natural killer cells.

Cells from local tumor growth (L-3LL) were compared to metastatic tumor cells (M-3LL) for their susceptibility to the cytotoxicity of natural killer (NK) cells. M-3LL cells were more resistant in vitro to NK cells from normal spleens than were L-3LL cells. A similar phenomenon of relative resistance of metastatic cells to NK activity was found when L-3LL and M-3LL cells were admixed with normal spleen cells and then inoculated into syngeneic mice. Because hybrid resistance was shown to be based on mechanisms that in principle are similar to mechanisms involved in NK activity, we tested the growth of M-3LL and L-3LL cells in semiallogeneic F1 mice. The in vitro effect of NK cells from semiallogeneic mice on M-3LL and L-3LL cells was tested in parallel. In vitro tests showed that irrespective of the haplotype of the spleen cell donors, L-3LL cells were more susceptible to NK activity than were M-3LL cells. In vivo experiments indicated that whereas M-3LL and L-3LL cells grew similarly in syngeneic recipients, M-3LL cells grew far more in F1 mice than did L-3LL cells. Thus metastatic cells are more resistant to NK activity than are cells of the local tumor growth. This relative resistance may determine, among other factors, the metastatic spread and progression of tumor cells.     

Hemopoietic features of splenectomized mice bearing IL-3 producing T cell leukemia

Hemopoietic responses were studied in splenectomized mice after transplantation of the hemopoietic colony stimulating activity producing leukemia, L8313. In non-splenectomized mice, the number of peripheral leukocytes was elevated to 4 × 10⁵/mm³ after transplantation. In contrast, it was below 1.7 × 10⁴/mm³ in splenectomized mice. Total cell, hemopoietic stem cell and progenitor cell numbers in the bone marrow decreased in both groups. Serum from splenectomized mice contained multi-colony stimulating activity although less effective than that from non-splenectomized mice. Extramedullary hemopoiesis was observed in splenectomized mice. These results suggest that invasion of leukemic cells to bone marrow results in a damage of hemopoietic microenvironment.     

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).     

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long‐term hematopoiesis

Adult long‐term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self‐renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase‐activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane‐targeted C3G (C3G‐F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long‐term hematopoiesis of all cell lineages. C3G‐F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G‐F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G‐F expression caused a markedly prolonged activation of c‐Kit receptor and downstream signaling through SCF ligation. A minor population of C3G‐F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long‐term adult hematopoiesis.     

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.     

Protective and restorative role of AS101 in combination with chemotherapy

The immunomodulator AS101 has been found previously by us to stimulate the secretion of high levels of interleukin 1 and colony stimulating factor (CSF) in vitro, as well as the production of CSF in vivo in mice models. These cytokines are known to induce proliferation and differentiation of hematopoietic progenitor cells from the spleen and bone marrow (BM) and to protect mice from DNA-damaging agents. The present studies were designed to evaluate the effects of prolonged treatment with AS101 on myelopoiesis, BM cellularity, and CSF secretion in mice treated with a sublethal dose of cyclophosphamide (CYP) and on the survival of mice undergoing treatment with lethal doses of this compound. In this model, the hematopoietic progenitors were suppressed during the overbound phase of myelopoiesis resulting from the cytotoxic effects of CYP. This allowed the detection of a significant proliferative effect of AS101 in vivo on BM colony-forming units granulocyte-macrophage progenitor cells, BM cellularity, and the secretion of CSF. Moreover, AS101 protected these animals from the lethal effects of high doses of CYP. These protective effects were demonstrable only when AS101 was administered to mice prior to CYP treatment. The only exception was CSF secretion by spleen cells that had been reconstituted when AS101 was administered both prior to and following CYP treatment. AS101 was found to have a synergistic effect with CYP in the treatment of tumor-bearing mice, suggesting that the combination of these two modalities provides a more effective treatment of their tumors. These results strongly suggest an immunoregulatory role for AS101 in counteracting the chemotherapy-induced hematopoietic suppression as well as usefulness as adjunct treatment of cancer when used in combination with CYP.     

The influence in vivo of natural murine interleukin-3 on the proliferation of myeloid progenitor cells in mice recovering from sublethal dosages of cyclophosphamide

Purified natural murine interleukin-3 (IL-3) was assessed for its effects in vivo in mice pretreated 7 days earlier with a sublethal dosage of cyclophosphamide. The multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells in these mice were in a slowly- or non-cycling state. Three hours after the i.v. administration of 200 units IL-3 into these mice, the hematopoietic progenitor cells in the marrow and spleen were placed into rapid cell-cycle. At this time, no effects were noted on marrow or spleen nucleated cellularity, numbers of progenitor cells per organ, or peripheral blood counts. No endotoxin was detected in the IL-3 preparation, by Limulus lysate assay. Treatment of IL-3 in vitro at 100 degrees C for 20 min partially decreased its colony stimulating activity in vitro and completely inactivated its proliferation stimulating effects in vivo. These findings suggest that the effects of IL-3 in vivo were not due to contaminating endotoxin or to a non-specific protein effect. These studies do not allow us to conclude whether the effects of IL-3 in vivo are directly on the progenitors and/or are indirect effects mediated by accessory cells.     

Reticulum cell neoplasms induced in C57BL/6 mice by cultured virus grown in stromal hematopoietic cell lines.

Thirty-one adherent cell lines have been established from the spleens, lymph nodes, and bone marrow of C57BL/6 mice carrying radiation leukemia virus (Duplan isolate)-induced reticulum cell neoplasms (RCN). The cell lines had a stable epithelial or fibroblastoid morphology, Supernatant virus from these lines induced splenic and lymph node RCN in 100% of inoculated C57BL/6 mice within 30 days. The disease was generalized and involved many organs. The monolayer cells themselves were not tumor cells and induced RCN through infection of the host with RCN virus. Simultaneous inoculation of in vitro-grown RCN-inducing virus any thymic lymphosarcoma virus induced each disease independently with unaltered incidence, latency period, and organ involvement; no mutual enhancement or inhibition was found, thus two separate mechanisms of action were indicated. Reextraction of the viruses from spleen, lymph nodes, and thymus gland indicated the specific organotropism of each agent. All the adherent cell lines that were derived from hematopoietic tissues produced ample, potent RCN-inducing virus. This high success rate suggests that in the hematopoietic organs the stromal, fibroblastoid cells are a natural habitat for the RCN-inducing virus. The RCN-inducing virus species may well be synthesized in these hematopoietic stromal cells. RCN-inducing virus from culture supernatants contained high-titer infectious ecotropic and xenotropic virus that was titrated. The cultures are being used to clone the RCN-inducing virus and to establish the virologic and molecular properties that endow it with specific RCN-inducing capacity.     

Isolation from the spleen of myeloproliferative sarcoma virus-infected mice of a myelomonocytic tumor cell line secreting hematopoietic colony stimulating factors

Clonogenic tumor cells were detected in the spleen of DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). These cells could be isolated because of their ability to proliferate on the greater omentum of sublethally irradiated isogenic recipient mice. We have established a permanent suspension myelomonocytic cell line in vitro (TE8), from a tumor obtained after subcutaneous transplantation of the omental tumor masses. The TE8 cells are clonogenic in semi-solid medium containing agarose. The colonies thus obtained gave rise to myelomonocytic cell lines growing in suspension in liquid medium. One of those cloned cell lines, C1(1011) was studied in details. It is exclusively composed of initial donor cells, it is tumorigenic in vivo, clonogenic in vitro and produces MPSV. It also secretes a colony stimulating activity (CSA), and an activity termed mixed colony promoting activity (MPA), which enables pluripotent hematopoietic stem cells to proliferate and differentiate. It differentiates through the granulomacrophage cell line independently of exogenous factors other than those which may be present in the serum.     

The spleen in Friend leukemia. II. Nonleukemic nature of spleen stroma.

Pieces of Friend leukemic spleens implanted sc into mice regenerated into normal-appearing spleens if animals were protected against the leukemia. In unimmunized animals, the implant regenerated normally, but was subsequently infiltrated by leukemia cells. This leukemia cell infiltration required at least 30% stromal regeneration of the implant. The hematopoietic regeneration was primarily a repopulation with cells from the host animal, not with cells indigenous to the donor spleen. The development of the implant, whether normal or leukemic, was retarded by the presence of the host spleen, whether leukemic or normal. These studies strongly suggested that the stromal cells of the spleen are not directly involved in the virus-induced leukemic process but acted as supporting structure for the malignant cells.     

Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease.

Evidence is continuing to accumulate that the FMS-like tyrosine kinase 3 (FLT3) receptor plays an important role in acute leukemias. Acute myeloid leukemia patients often express constitutive active mutant forms of the receptor in their leukemic cells. A t(12;13)(p13;q12) translocation between Tel and the FLT3 receptor was recently described in a patient with myeloproliferative disease (MPD). Here a Tel-FLT3 construct mimicking this fusion protein was used to generate transgenic mice. The fusion protein was previously found to constitutively activate FLT3 signaling and transform Ba/F3 cells. Expression of the fusion protein in the transgenic mice was found in all tissues assayed including spleen, bone marrow (BM), thymus and liver. These mice developed splenomegaly and had a high incidence of MPD with extramedullary hematopoiesis in the liver and lymph nodes. Spleens also had increased dendritic and natural killer cell populations. In vitro analysis of the hematopoietic progenitor cells derived from Tel-FLT3 transgenic mice showed a significant increase in the number of CFU-GM in the BM, and CFU-GM, BFU-E and CFU-GEMM in the spleen. BM also showed significant increases of in vivo CFU-S colonies. Thus, transgenic mice expressing constitutively activated Tel-FLT3 develop MPD with a long latency and also result in the expansion of the hematopoietic stem/progenitor cells.     

GM-CSF secreted by murine adenocarcinoma cells modulates tumor progression and immune activity

During tumor development, growth factors may act in autocrine manner stimulating cell proliferation, or in paracrine manner affecting the microenvironment of the tumor and modulating the immune system. Murine mammary adenocarcinoma M3 tumor bearers develop lung metastases and leukocytosis during its evolution. Previously we described that M3 conditioned media enhanced metastasis incidence, when it was inoculated in tumor-operated mice. In the present study we determine that spleen cells from M3 tumor operated mice treated with M3 conditioned media, were able to transfer the capacity to enhance metastasis to other tumor operated mice. Spleen cells have immune suppressor activity that could be reversed by cyclophosfamide treatment. M3 tumor cells secrete GM-CSF, which is able to promote in vitro proliferation of M3 cells as well as spleen cells. This proliferation could be abrogated by the addition of anti-GM-CSF. We report that the GM-CSF secreted by M3 tumor cells had stimulatory activity on M3 tumor cell and lymphocyte proliferation.     

Negative and positive immunobiological responses in mice pretreated with Bacillus Calmette-Guérin cell wall

Spleen cells from C57BL/6 mice injected i.p. with Bacillus Calmette-Guérin cell walls (BCGcw) showed strongly depressed response to the T-cell mitogen phytohemagglutinin-P in vitro. Mitogen reactivity of normal spleen cells could be suppressed by the addition of spleen cells from BCGcw-treated mice. The suppressor cells mediating this effect appeared to belong to the plastic-adherent, radioresistant, and non-T-cell populations, maybe macrophages. Spleen cells from mice which had been passively transferred i.p. with the adherent cells from BCGcw-treated mice also showed the depressed mitogen response in vitro. Depressed T-cell reactivity of spleen cells obtained from animals immunized with BCGcw on a per-cell basis was also demonstrated in vivo: graft versus host reactivity of spleen cells obtained from animals immunized with BCGcw was depressed as compared to normal spleen cells. At times when strong suppressor cell activity could be detected in BCGcw-treated mice, activity of alloimmune cytotoxic lymphocytes generated in vivo by immunizing with X-irradiated allogeneic MH-134 tumor cells was weaker in BCGcw-pretreated mice than in untreated control groups (detected by means of 51Cr release assay). Furthermore, accelerated development of s.c. inoculated syngeneic B-16 melanoma cells was observed in BCGcw-pretreated mice. On the other hand, stronger resistance to i.v. inoculated B-16 tumor cells was observed in BCGcw-pretreated mice. BCGcw-treated mice responded normally to i.p. immunization with 2 X 10(8) sheep erythrocytes. Negative and positive immunobiological responses were observed in C57BL/6 mice pretreated with BCGcw.     

Characterization of lymphocyte subsets in spontaneous mouse mammary tumors and host lymphoid organs

The subsets of small lymphocytes (surface lgm-positive or B, Thy-I-positive or T and «double-negative» or null) appearing within spontaneous mammary tumors of C3H retired breeder female mice and in various lymphoid organs of the host at different stages of tumor development were characterized directly using a radioautographic method. Tumor-bearing mice showed early transient splenomegaly and progressive atrophy of the thymus. The proportion of T small lymphocytes increased with tumor age within the tumor and in the spleen, blood and thymus; then, except in the spleen, these proportions declined again by 8 weeks of tumor growth. The incidence of B small lymphocytes showed no change in the tumor or thymus; there was an increase in the blood after 3 weeks, and a small decrease in the spleen after 7 weeks. At all the sites examined, the proportion of null small lymphocytes declined from an initial high level observed in the elderly tumor-free control mice, and then recovered to control levels after 7 weeks. The absolute numbers of T and null cells in the spleen changed in parallel to their proportions, while splenic B-cell numbers increased at 1–3 weeks. In the thymus the absolute numbers of all subsets decreased, with a late recovery of null cell numbers. Age-matched control mice showed higher proportions of null cells in the spleen, blood and thymus, and lower proportions of T cells in the blood and spleen, than did young normal mice. This spontaneous tumor appears to be characterized by an increase in T cells, rather than in null cells, as observed for rapidly growing transplanted tumors. The null cell rise in the present case takes place before the clinical appearance of tumors. In both cases, however, tumor-infiltrating B cells exhibited low levels of surface Ig, possibly related to a low level of maturation. The functional significance of these findings remains to be determined.