Pluripotent haemopoietic stem cell entry into DNA synthesis in vitro after stimulation by Ara-C treatment in vivo

Non-cycling pluripotent bone marrow stem cells (CFU-S), taken 3 hrs after injection of 20 mg of Ara-C, have been shown to enter DNA synthesis at 1 to 3 hr after being cultured in alpha-medium. This phenomenon was observed when bone marrow was incubated as a plug, but not when incubated as a cell suspension in the present experimental conditions. These results suggest that a medullary structure is necessary in order to observe this effect and/or that accessory cells are destroyed during the process of single cell preparation.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

In vivo localization and growth of the 70Z/3 pre-B-cell line

The 70Z/3 pre-B-cell line has long been used as a model for understanding the nature and mode of action of differentiation-inducing stimuli as well as mechanisms which control immunoglobulin light chain gene expression. This study is a first appraisal of the localization, growth, and differentiation of the cell line in vivo. At 24 hr after intravenous injection, radiolabeled 70Z/3 cells localized efficiently to the bone marrow and analysis by flow cytometry revealed that fluorescein isothiocyanate-labeled cells localized to bone marrow and spleen in a ratio of 2:1. Growth of the cell line paralleled the localization pattern. When the cells were given intravenously, bone marrow contained 100% of tumor cells at a time when a majority of spleen cells were still normal. Tumor cells were found in the blood only at end-stage disease in a minority of animals. Because 70Z/3 cells differentiate in vitro in response to a variety of factors, it is possible that exposure to the in vivo environment would have a similar effect. When blast cells from heavily infiltrated bone marrow and spleen were analyzed for the expression of a panel of B-lymphocyte lineage surface antigens, however, there was no evidence for surface kappa induction. Inductive stimuli may be present in limiting quantities in vivo or overridden by by negative feed-back control mechanisms. This information provides a basis for in vivo experimentation with the inducible 70Z/3 cell line and raises issues concerning normal mechanisms which control B-lineage cell differentiation.     

Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation. The estimated minimal frequency of NK cell progenitors was reduced from 1/11,800 in control to 1/41,900 in IL-3-exposed mice. IL-3 may take part in the homeostasis of NK cells by the down-regulation of their progenitors.     

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.     

The mast cell-committed progenitor. In vitro generation of committed progenitors from bone marrow

Mast cell-committed progenitors are detected in the unique microenvironment of the mesenteric lymph node (MLN) of Nippostrongylus brasiliensis-infected mice but not in naive bone marrow. We have determined that MLN cells, after infection, produce high levels of IL-3, IL-4, and IgE, presumably in the form of immune complexes with antigens produced by the infecting helminth. After N. brasiliensis infection, peak production of these factors occurs several days before the peak appearance of mast cell-committed progenitors in the MLN. To determine if these factors play a role in mast cell commitment, we recreated these conditions, in vitro. Naive bone marrow cells were cultured with combinations of IL-3, IL-4, and IgE immune complexes, or on IgE-coated plates, and then assayed for acquisition of the ability to form mast cell colonies when supplemented with fibroblast-conditioned medium alone. IL-3 and IgE immune complexes, and, unexpectedly, IgE immune complexes alone were found to be capable of producing mast cell-committed progenitors, i.e., cells responsive to fibroblast-conditioned medium alone, from bone marrow, whereas IL-4 did not enhance production of mast cell-committed progenitors from bone marrow. Production of IFN-gamma peaked at the same time point as committed progenitor activity and may be responsible for down regulating the response.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells

Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autologue bone graft. Activity of the Alkaline Phosphatase enzyme were measurement on 0.5, 14, 21, 30 day and increased activity were detected day 14 on animals treated with bone marrow MSCs compared with day 30 on iliac crest treated animals. That results indicates strong osteoinduction activity of the experimental bone marrow MSCs models exposed to the rhBMP-7 factor Comparing ALP activity, that model showed superiorly results than control group. That result initiates us in opinion that MSCs alone should be alternative for the autolologue bone transplantation and in vitro study we isolated singles MSCs from the bone marrow of rat's tibia and femora and cultivated according to the method of Maniatopoulos et all. The small initial colonies of fibroblast like cells were photo-documented after 2 days of primary culture. Such isolated and cultivated MSCs in future studies will be exposed to the growth factors to differentiate in osteoblast and indicate their clinically potential as alternative for conventional medicine and autologue bone transplantation. That new horizons have potential to minimize surgery and patient donor morbidity, with more success treatment in bone regenerative and metabolism diseases.     

In Vitro Model of Metastasis to Bone Marrow Mediates Prostate Cancer Castration Resistant Growth through Paracrine and Extracellular Matrix Factors

The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

In vitro hemopoiesis in human micro long-term bone marrow cultures recharged with either allogeneic, T-cell-depleted allogeneic, or syngeneic bone marrow cells

The production of granulocyte-macrophage colony-forming cells (GM-CFC) and the proliferation period in human long-term bone marrow cultures are inferior to murine cultures. There is also evidence that recharge of the cultures after establishing confluent stromal layers will not greatly improve myelopoiesis. Data in the literature indicate that PHA-responsive T lymphocytes persist for up to 5 weeks in human but not in murine long-term marrow cultures. We therefore analyzed the effects of recharging micro long-term bone marrow cultures with bone marrow cell samples depleted by T lymphocytes. Depletion was performed in a complement-mediated cytotoxicity assay by applying the monoclonal antibody CAMPATH-1. Our data show that regardless of whether T cells were removed only at recharge, at both initiation and recharge, or only at initiation, obvious enhancement could neither be achieved in the GM-CFC production nor in the proliferation period. Furthermore, no advantage was seen when using syngeneic marrow cells. We conclude that in allogeneic long-term marrow cultures hemopoiesis is not limited by immunological incompatibilities.     

Regulation of natural killer progenitors. Studies with a novel immunomodulator with distinct effects at the precursor level

NK cells originate from progenitors in the bone marrow and maturate independently of other lymphoid organs. NK cell maturation represents an important site for regulation of the level of NK activity and constitutes a potentially interesting target for therapeutic intervention. The effect of the immunomodulator Linomide (carboxamide-3-quinoline) on the regeneration of NK cells was studied in vivo after depletion of mature NK cells. Linomide significantly, although to various extent, accelerated the maturation of NK cells after specific depletion with antibodies to asialomonoganglioside, treatment with cyclophosphamide or lethal irradiation and syngeneic bone marrow grafting. Examination of the target cell spectrum lysed by spleen effector cells during Linomide treatment as well as studies of phenotype, clearly indicated that the effector cells studied were NK cells. Treatment of mice for 4 days with Linomide increased the frequency of bone marrow NK cell progenitors from 1/11,900 to 1/6,000 as judged by limiting dilution analysis. Direct addition of Linomide in vitro had no effect on cultures of mature NK cells from spleen, but had an additive effect to IL-2 on the generation of NK cells when added to bone marrow cultures. Our study indicates that different mechanisms exist for the regulation of progenitor and mature NK cells, and that the immunomodulator Linomide represent a potentially important tool for investigating the mechanisms governing NK cell maturation.     

Growth Fraction of Myelocytes In Normal Human Granulopoiesis

Abstract. The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [³H]-thymidine ([³H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G₁ phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. the evolution of the LI and of the mean number of grains per cell was monitored in [³H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. the generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

Biological time and the effects of hydroxyurea on DNA synthetic activity of bone marrow and tumor cells in mice bearing the Ehrlich ascites carcinoma

The objective of this experiment was to attempt to induce, with hydroxyurea (HU), significant quantitative differences in the level of DNA-synthetic activity (DNA-SA) between a neoplastic cell population (the Ehrlich ascites carcinoma or EAC) and bone marrow in the same animal. Mice bearing a 5-day-old EAC were standardized to and kept on an LD 12:12 cycle (light 0600-1800 hr). They were treated with 500 mg/kg HU at 0500 hr (23 hr after lights on, or HALO) or at 1700 hr (11 HALO). DNA-SA was determined by liquid scintillation counting of 3H-thymidine incorporation into chemically isolated DNA. DNA-SA in bone marrow and EAC cells was monitored over the next 60 hr with subgroups of ten mice each killed every 3 hr beginning 3 hr after treatment with HU. The circadian system of the host influenced the response of the bone marrow to HU; i.e., the response to HU administered at 0500 hr was different both qualitatively and quantitatively from that for HU given at 1700 hr. Comparisons of DNA-SA in bone marrow and EAC from the same animal revealed time points after treatment with HU when DNA-SA in the EAC was high, but DNA-SA in bone marrow was low. These differences in the level of DNA-SA between a tumor cell population and bone marrow should be of therapeutic value; i.e., executor doses of anti-DNA-SA drugs such as cytosine arabinoside could be given at that point in time after treatment with HU when DNA-SA in the tumor was high, but DNA-SA in the bone marrow was low.     

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.     

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

The effect of natural killer cells on the development of syngeneic hematopoietic progenitors

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.