Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

Absence of mitochondrial superoxide dismutase results in a murine hemolytic anemia responsive to therapy with a catalytic antioxidant.

Manganese superoxide dismutase 2 (SOD2) is a critical component of the mitochondrial pathway for detoxification of O2(-), and targeted disruption of this locus leads to embryonic or neonatal lethality in mice. To follow the effects of SOD2 deficiency in cells over a longer time course, we created hematopoietic chimeras in which all blood cells are derived from fetal liver stem cells of Sod2 knockout, heterozygous, or wild-type littermates. Stem cells of each genotype efficiently rescued hematopoiesis and allowed long-term survival of lethally irradiated host animals. Peripheral blood analysis of leukocyte populations revealed no differences in reconstitution kinetics of T cells, B cells, or myeloid cells when comparing Sod2(+/+), Sod2(-/-), and Sod2(+/-) fetal liver recipients. However, animals receiving Sod2(-/-) cells were persistently anemic, with findings suggestive of a hemolytic process. Loss of SOD2 in erythroid progenitor cells results in enhanced protein oxidative damage, altered membrane deformation, and reduced survival of red cells. Treatment of anemic animals with Euk-8, a catalytic antioxidant with both SOD and catalase activities, significantly corrected this oxidative stress-induced condition. Such therapy may prove useful in treatment of human disorders such as sideroblastic anemia, which SOD2 deficiency most closely resembles.

Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells.

Adenovirus vectors have been used to transfer genes into both hematopoietic progenitor cells and tumor cells, including carcinoma cells that have metastasized to bone marrow (BM). However, the relative susceptibility of different subsets of hematopoietic cells is unknown. In permissive cells adenoviral-mediated gene transfer is mediated by the coxsackievirus and adenovirus receptor (CAR) protein and alpha(v) integrins expressed on the cell surface of the target cells. This prompted us to investigate the expression of CAR on subpopulations of hematopoietic cells, determine whether this protein played a role in adenovirus-mediated gene transfer of hematopoietic cells and whether we could modulate CAR to enhance gene transfer efficiency. In this report we show that CAR is expressed on approximately 40% of all human BM cells, including erythroid and myeloid cells, but not lymphoid cells. Of the CD34(+) cells, 10%-15% expressed CAR, but this did not include most colony-forming progenitor cells, nor the most primitive CD38(-) subpopulation. The presence of CAR correlated well with gene transfer efficiency, but we were unable to induce CAR expression on immature, noncommitted progenitor cells. In conclusion, our results show that primitive hematopoietic progenitor cells lack CAR expression, but that expression is acquired during erythroid and myeloid differentiation.

Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP.

Mice with monoallelic inactivation of the CBP gene develop highly penetrant, multilineage defects in hematopoietic differentiation and, with advancing age, an increased incidence of hematologic malignancies. The latter are characterized, at least in some cases, by loss of heterozygosity (LOH) at the CBP locus. No such pathology was observed in wild-type or p300 heterozygous null mice of the same age and genetic background. Thus, a full complement of CBP, but not p300, is required for normal hematopoietic differentiation. These results also provide the first experimental evidence for the hypothesis that CBP has tumor-suppressing activity.

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity.

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.

Essential role for the p55 tumor necrosis factor receptor in regulating hematopoiesis at a stem cell level.

Hematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-alpha as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55(-/-) mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75(-/-) or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55(-/-) mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

One-day ex vivo culture allows effective gene transfer into human nonobese diabetic/severe combined immune-deficient repopulating cells using high-titer vesicular stomatitis virus G protein pseudotyped retrovirus.

Retrovirus-mediated gene transfer into long-lived human pluripotent hematopoietic stem cells (HSCs) is a widely sought but elusive goal. A major problem is the quiescent nature of most HSCs, with the perceived requirement for ex vivo prestimulation in cytokines to induce stem cell cycling and allow stable gene integration. However, ex vivo culture may impair stem cell function, and could explain the disappointing clinical results in many current gene transfer trials. To address this possibility, we examined the ex vivo survival of nonobese diabetic/severe combined immune-deficient (NOD/SCID) repopulating cells (SRCs) over 3 days. After 1 day of culture, the SRC number and proliferation declined twofold, and was further reduced by day 3; self-renewal was only detectable in noncultured cells. To determine if the period of ex vivo culture could be shortened, we used a vesicular stomatitis virus G protein (VSV-G) pseudotyped retrovirus vector that was concentrated to high titer. The results showed that gene transfer rates were similar without or with 48 hours prestimulation. Thus, the use of high-titer VSV-G pseudotyped retrovirus may minimize the loss of HSCs during culture, because efficient gene transfer can be obtained without the need for extended ex vivo culture.

Impaired steel factor responsiveness differentially affects the detection and long-term maintenance of fetal liver hematopoietic stem cells in vivo.

The results of previous studies have shown that the development of hematopoiesis during fetal life can occur in the absence of Steel factor (SF) signaling. On the other hand, impairment of this mechanism can severely compromise the ability of cells from adult bone marrow to regenerate hematopoiesis on their transplantation into myeloablated recipients. This apparent paradox could result from changes during ontogeny in the responsiveness of hematopoietic stem cells to regulators that may substitute for SF as well as from differences in the availability of such factors during embryogenesis and in the myeloablated adult. To investigate these possibilities, we studied the effect of W41 and W42 mutations on the numbers, phenotype, and posttransplant self-renewal behavior of primitive hematopoietic cells present in the fetal liver (FL) of 14.5-day-old mouse embryos. In W41/ W41 FL, day-12 spleen colony-forming units and long-term culture-initiating cells appeared both quantitatively and qualitatively similar to their counterparts in the FL of +/+ embryos. W41/W41 FL also contained near normal numbers (approximately 50% of controls) of transplantable lymphomyeloid stem cells with competitive reconstituting ability in myeloablated adult +/+ recipients (as assessed for up to at least 16 weeks posttransplant). Moreover, both the original phenotype of these W41/W41 competitive repopulating units (CRUs) and their clonal posttransplant output of mature progeny were normal. Similarly, when myeloablated adult +/+ mice were cotransplanted with 5 x 10(4) +/+ FL cells and a sevenfold to 70-fold excess of W41/W41 FL CRUs, the contribution of the +/+ FL CRUs to the circulating white blood cell count present 5 weeks later was markedly reduced as compared with that of mice that received only +/+ FL cells. However, over the next 3 months, the proportion of mature white blood cells that were derived from +/+ precursors increased significantly (P < .002) in all groups (to > or = 30%), indicating that the ability to sustain hematopoiesis beyond 5 weeks is more SF-dependent than the ability to initially reconstitute both lymphoid and myeloid compartments. Cells from individual FL of W42/+ matings also showed an initial ability (at 7 to 8 weeks posttransplant) to competitively repopulate both lymphoid and myeloid compartments of myeloablated +/+ adult recipients. However, in contrast to recipients of normal or W41/W41 FL cells, the repopulation obtained with the W42 mutant stem cells was transient. Secondary transplants confirmed the inability of the W42 mutant cells to regenerate or even maintain a population of transplantable stem cells. Taken together with previous results from studies of CRUs in adult W mice, these findings support the concept of changes in the way hematopoietic stem cells at different stages of development respond to the stimulatory conditions evoked in the myeloablated recipient. In addition, they provide the first definitive evidence that SF is a limiting physiological regulator of sustained hematopoietic stem cell self-renewal in vivo.

The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts.

Varying, limiting numbers of unseparated or purified cells (Ly-5.1), either from 14.5-day-old fetal liver (FL) or from adult bone marrow (BM) were coinjected with 10(5) unseparated BM cells (Ly-5.2) into lethally irradiated adult C57B1/6 recipients (Ly-5.2). The kinetics of donor cell repopulation of the lymphoid and myeloid compartments by Ly-5.1+ donor hematopoietic stem cells (ie, competitive repopulation units [CRU]) were monitored at various time points after the transplantation by Ly-5 analysis of the peripheral white blood cells (WBC). Recipients that had received on average less than 2 adult BM or FL CRU did not show a significant difference in the level of donor-reconstitution when analyzed 4 weeks after the transplantation, However, at 8 and 16 weeks, the FL recipients showed a significantly higher percentage of donor-derived nucleated peripheral blood cells than did the recipients of adult BM cells. Analysis of individual mice showed that approximately 80% of the recipients of FL CRU showed an increase in mature WBC output between 4 and 8 weeks after transplantation, whereas this occurred in less than 40% in the recipients of adult BM cells. In addition to this effect on mature cell output, the cellularity of the reconstituted BM was significantly higher in recipients of FL CRU than in recipients of adult BM CRU, even at 7 to 9 months after transplantation, which is consistent with an increased clonal expansion of FL CRU. When marrow cells from primary recipients of FL CRU were injected into secondary recipients, a significantly higher percentage of these mice showed donor-reconstitution of their lymphoid and myeloid compartments (P < .01) and to a greater extent (P < .008) as compared with mice that had received marrow cells from primary recipients of similar numbers of adult BM CRU. Taken together, these results show that individual FL CRU exhibit a greater proliferative activity in vivo than similar cells from adult BM that is accompanied by a greater production of daughter CRU.

A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse.

Previous studies have shown that stem cells able to competitively reconstitute the hematopoietic system of lethally irradiated mice (competitive repopulating units [CRU]) can be obtained in highly purified form from adult mouse bone marrow (BM) by the isolation of cells with a Sca-1+Lin-WGA+ phenotype. We now report on the phenotypic characteristics of CRU from day-14.5 murine fetal liver (FL). Our results confirm previous reports of similarities between the two CRU populations but also reveal a few striking differences. Both were found to express the Sca-1 antigen (SCA-1+ and surface molecules that bind wheat germ agglutinin (WGA+), and both show an absence or low expression of a number of markers characteristic of mature hematopoietic cells: B220, Gr-1,ly-1 and Ter119 (together termed Lin*-). Limiting dilution analysis of recipients transplanted with purified Sca-1+Lin*- FL cells with intermediate forward- and side-scatter properties showed that the frequency of CRU in this FL subpopulation was one in 39 cells. This represents an enrichment of approximately 450-fold over the labeled but unseparated FL starting population (one in 17,300 total FL cells). These FL CRU also resembled their counterparts in adult BM in that they expressed high levels of MHC class I and CD43 and intermediate levels of heat-stable antigen (HSA) and c-kit and did not express, or expressed at a low level, Thy-1.2, CD71, and the antigen recognized by the Fall-3 monoclonal antibody (mAb). In contrast, a high percentage of the Sca-1+Lin*- cells isolated from 14.5-day-old FL stained with the AA4.1, anti-Mac-1, and the anti-CD45RB mAbs and retained Rhodamine 123 (Rh123(bright)), whereas the Sca-1+Lin-WGA+ CRU-containing fraction of adult BM cells was found to be AA4.1-, Mac-1-, CD45RB-, and Rh123(dull). These differences in phenotype between CRU in FL and adult BM indicate changes that occur during ontogeny in cells that are similar with respect to their totipotentiality and long-term repopulating potential and complement parallel observations of functional differences between these two populations of CRU.

Culture of purified stem cells from fetal liver results in loss of in vivo repopulating potential.

The development of in vitro conditions that promote a numerical expansion of hematopoietic stem cells (HSCs) with long-term reconstituting ability has been a long-standing goal in experimental hematology. In previous studies, we showed that input numbers of such cells, i.e., competitive repopulating units (CRU), could be maintained for 2 weeks when purified Sca-1 + Lin-WGA + adult bone marrow (BM) cells were cultured in serum-free and stromal cell-free cultures containing Steel factor (SF), interleukin 6 (IL-6), and erythropoietin (Epo). In separate studies, we showed that limiting numbers of purified fetal liver (FL) cells that are highly enriched for CRU display a higher proliferative and self-renewal potential in vivo compared with similar cells purified from adult BM. These findings prompted us to explore the possibility of achieving a numerical expansion of purified FL cells in culture. Although we observed an extensive increase in the number of nucleated FL cells in all culture conditions tested, none of the cultures, including cultures in serum-containing medium and cocultures on a preestablished feeder layer of BM stromal cells of S17 cells, sustained the expected expansion or even supported the maintenance of input numbers of FL CRU. Single cell cultures showed that the production of nucleated cells by purified Sca-1 ++ Lin.-AA4.1 + FL cells stayed behind that of purified Sca-1 + Lin-WGA + adult BM cells. Taken together, our results show that a variety of culture conditions tested, including conditions that support maintenance and limited expansion of adult BM CRU do not support the production of repopulating stem cells from FL. Because such expansion can be observed in vivo, this system appears useful to search for novel culture conditions and-perhaps yet unidentified-cytokines or other microenvironment-related factors that may be required for FL CRU to prevent loss of repopulation potential in vitro and allow these cells to exhibit their expected self-renewal potential.

Studies of W mutant mice provide evidence for alternate mechanisms capable of activating hematopoietic stem cells.

Previous studies have suggested that Steel factor (SF) can influence the behavior of many types of hematopoietic progenitor cells both in vivo and in vitro, although whether these may include the most primitive populations of totipotent repopulating cells remains controversial. To approach this question, we measured the number of Sca1+Lin-WGA+ cells, the number of cells with demonstrable myeloid (long-term culture-initiating cell [LTC-IC]) or both myeloid and lymphoid (LTC-IC(ML)) potential in 4- to 5-week-old long-term cultures containing irradiated primary marrow feeder layers, and the number of multilineage long-term in vivo repopulating cells (competitive repopulating unit [CRU]) present in the marrow of W42/+ or W41/W41 mice compared to +/+ controls. There was no significant effect of either of these W mutations on the number of Sca1+Lin-WGA+ cells and, in W41/W41 mice, neither LTC-IC nor LTC-IC(ML) populations appeared to be affected. On the other hand, although W41/W41 and W42/+ cells could both be detected in the in vivo CRU assay, their numbers were markedly reduced (17- and seven-fold, respectively) in spite of the fact that both of these W mutant genotypes contained near normal numbers of day-9 and -12 colony-forming units-spleen (CFU-S). In vitro quantitation of erythroid (burst-forming units-erythroid [BFU-E]), granulopoietic (CFU-granulocyte/macrophage [CFU-GM]), multilineage (CFU-granulocyte/erythrocyte/monocyte/macrophage [CFU-GEMM]), and pre-B clonogenic progenitors (CFU-pre-B) also revealed no differences in the numbers (or proliferative potential) of any of these cells when W41/W41 or W42/+ and normal mice were compared, although day 3 BFU-E from both types of W mutant mice showed no response to the typical enhancing effect exerted by SF on their +/+ counterparts. Taken together, these findings are consistent with the view that SF activation of c-kit receptor-induced signaling events is not a rate-limiting mechanism controlling red blood cell production during normal development until hematopoietic cells differentiate beyond the day-3 BFU-E stage. Nevertheless, normal hematopoietic stem cells do appear to be responsive to SF, since their W mutant counterparts display a disadvantage in the in vivo setting which is exaggerated under conditions of hematopoietic regeneration. On the other hand, alternative mechanisms also appear to contribute to the regulation of hematopoietic stem cell numbers in vivo and to their detection as LTC-IC in vitro.

Nonlinear effects of radiation dose on donor-cell reconstitution by limited numbers of purified stem cells.

Despite the increasing use of bone marrow transplantation (BMT) as a treatment for a wide variety of diseases, the numbers and types of cells required for both rapid and sustained recovery of hematopoiesis are not well defined. To investigate further the potential of transplants consisting of highly enriched populations of long-term repopulating cells, we transplanted a series of Ly-5.2 mice given various doses (750, 800, 850, 900, or 950 cGy) of total-body irradiation (TBI) with 30 or 90 Sca-1+Lin-WGA+ marrow cells isolated from congenic Ly-5.1 donors. As expected, mature progeny derived from these cells, belonging to both myeloid and lymphoid compartments, could be detected with increasing case in recipients given radiation doses from 750 to 900 cGy TBI. Surprisingly, expression of this potential was significantly reduced in mice that had received 950 cGy TBI. This contrasts with the capacity of the same number of purified Sca-1+Lin-WGA+ cells to generate readily detectable progeny in 950 cGy treated mice given a simultaneous transplant of 10(5) normal marrow cells or 2 x 10(5) serially passaged marrow cells. We suggest that this variable behavior of purified stem cells in differently treated recipients may reflect radiation dose-dependent differences in the types or levels of expression of factors that regulate transplanted stem cell proliferation and differentiation in vivo and that above a certain threshold radiation dose, this may result in an irreversible loss of long-term reconstituting potential. Regardless of the nature of the underlying mechanism, this study shows that the extent of donor repopulation after BMT can be a function not only of the number of stem cells transplanted but also of the conditioning of the recipient and whether other cell types are also injected.

Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow "switch" cultures.

In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for > or = 7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in > 70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.

Amplification of Sca-1+ Lin- WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential.

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

Monocytic differentiation induction of HL-60 cells by MC 903, a novel vitamin D analogue.

1.25 (OH)2D3 is a potent inducer of differentiation of leukaemic cells into a monocytic direction. However, therapeutic application is difficult because of the development of hypercalcaemia. We examined a novel vitamin D analogue, MC 903, which is at least 100 times less effective on calcium metabolism in rats than 1.25 (OH)2D3. Using the HL-60 cell line, differentiation was measured with a comprehensive panel of qualitative and quantitative parameters. Development of monocytic cells was shown morphologically, immunophenotypically and functionally by increased capability of reducing NBT (vs cultures without MC 903, p less than 0.0001) and by qualitatively and quantitatively increased non-specific esterase activity. Furthermore, a concomitant decreased activity of myeloperoxidase and lactate dehydrogenase was noticed. In conclusion, MC 903 is a potent inducer of monocytic differentiation, comparable with 1.25 (OH)2D3 and will therefore be an interesting and potential therapeutic agent for studies in human acute leukaemia.

Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 monoclonal antibodies. Analysis of requirements for human cytolytic T-lymphocyte differentiation.

Requirements for the induction of human cytolytic T-lymphocyte (CTL) activity were studied in a monocyte-free T-cell activation system that uses immobilized anti-CD3 monoclonal antibodies (mAb) as a stimulus. Alloreactive CTL with specificity for HLA-A and -B locus antigens could be demonstrated within 2 days after the initiation of activation. CTL induction in purified T cells initiated by an optimal concentration of immobilized anti-CD3 mAb was not enhanced by the addition of monocytes or exogeneous cytokines, whereas addition of anti-CD25 mAb largely blocked the response. Upon suboptimal anti-CD3 mAb stimulation, addition of recombinant interleukin (rIL)-2, rIL-1 and rIL-4, but not recombinant interferon-gamma (IFN-gamma) or rIL-6, potentiated the development of CTL activity. Finally it was shown that immobilized anti-CD3 mAb induced significant levels of CTL activity in both purified CD4+ and CD8+ cells. This study indicates that the requirement for cytokines in the differentiation of CTL precursors depends on the strength of the activation signal delivered through the T-cell receptor.

Immobilized anti-CD3 monoclonal antibodies induce accessory cell-independent lymphokine production, proliferation and helper activity in human T lymphocytes.

Monoclonal antibodies (mAb) directed against the human CD3 molecular complex are able, when immobilized on the plastic of microtitre wells, to induce accessory cell-independent T-cell proliferation. In this study, we show that the anti-CD3 mAb CLB-T3/3 induces strong T-cell stimulation that is proportional to the density of the immobilized antibody. T cells, optimally stimulated with plastic-immobilized CLB-T3/3, showed a five-fold higher proliferation compared to cells that were stimulated with soluble anti-CD3 in the presence of accessory cells. The difference in magnitude of proliferation was found to be correlated with the expression of the CD25 (TAC) antigen and the production of interleukin (IL)-2, but not with the number of high-avidity IL-2 receptors expressed on the surface of these differentially activated cells. In addition, immobilized CLB-T3/3 initiated the production of interferon-gamma (IFN-gamma), but not of IL-4, in purified T lymphocytes. Coated anti-CD3 mAb induced helper activity in T cells for IgM and IgG production by B lymphocytes. Whereas addition of IL-1 or IL-2 had only a moderate effect on T-cell proliferation induced by immobilized anti-CD3 mAb, helper activity was strongly enhanced in the presence of these factors. This T-cell activation system may prove useful for a standardized analysis of both activation requirements and immunoregulatory capacities of human T cells.

Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemic cells: reversal of the bleeding disorder by cell differentiation.

In the human promyelocytic cell line HL60, we observed both a strong procoagulant activity (PCA) on the cell membrane and proteolytic activity in the lysate of these cells. Because these cell-line cells are susceptible to differentiation to either a more mature granulocytic or monocytic form, we were able to study the hypothesis that the combination of PCA and proteolytic activity is confined to the promyelocyte. This may explain the severe coagulopathy seen in patients with acute promyelocytic leukemia. Cell differentiation in a myeloid direction induced by retinoic acid or DMSO led to a diminished PCA, while not affecting the fibrinolytic activity. On the other hand, monocytic differentiation obtained by culturing the cells in the presence of 1; 25 dihydroxy vitamin D3 led to the complete disappearance of the proteolytic activity of the cell lysate, although the procoagulant activity was still present. Furthermore, we found that the elastase activity almost disappeared after monocytic differentiation. We also studied the PCA, proteolytic activity, and elastase activity of blast cells of patients with acute myeloid leukemia. Only in patients with acute promyelocytic leukemia did we observe both a strong PCA and fibrinolytic activity. This supports our hypothesis that the combination of these activities is unique to the promyelocyte and may explain the observed bleeding complications in patients with acute promyelocytic leukemia.