New technology for the depletion of T cells from soybean lectin agglutinated, HLA-matched bone marrow grafts for leukemia: initial laboratory and clinical results.
Explore the source record for details and available documents.
Adolescent↗
Biomedical subjects
H Castro-Malaspina
Publications and source records attributed to H Castro-Malaspina.
Immunosuppression in aplastic anemia--postponing the inevitable?
Explore the source record for details and available documents.
Anemia, Aplastic↗
Total body irradiation for bone marrow transplantation: the Memorial Sloan-Kettering Cancer Center experience.
In May 1979, Memorial Sloan-Kettering embarked on a programme of hyperfractionated TBI (HFTBI), 1320 cGy in 11 fractions over 4 days with partial lung shielding (1 HVL), followed by cyclophosphamide (60 mg/kg/d x 2d) for cytoreduction prior to allogeneic bone marrow transplantation (BMT). Anterior and posterior chest wall electron "boosts" were given to the areas blocked (600 cGy in 2 fractions) on the last two days of treatment. Since then, we have treated over 600 patients with HFTBI, the majority for allogeneic BMT. Several modifications have occurred over the years. We have added a "boost" electron dose of 400 cGy to the testes in all male leukemic patients; this reduced testicular relapses from a rate of 14% (4/28) to 0%. In an attempt to increase engraftment of T-depleted BMTs, we added one additional fraction; since our present dose/fraction was also increased to 125 cGy, we now deliver a total dose of 1500 cGy in 12 fractions over 4 days for allogeneic transplants. Tolerance to HFTBI has been excellent relative to the single dose (SD) regimen utilised prior to May, 1979. The incidence of fatal interstitial pneumonitis (IP) decreased from 50% in the SD regimen to 18% after the introduction of HFTBI. In children, the incidence of IP was only 4% with HFTBI. With the introduction of T-depleted marrows, fatal IP in adults has decreased also, e.g. to less than 10% in CML patients. With conventional BMT after HFTBI, relapse at 5 years has been exceedingly low (e.g. in children, 13% for ALL, 2nd remission and 0% for AML, 1st remission) and engraftment has been 100%. With matched T-depleted BMT, rejections have occurred in 15% overall; the incidence of graft failure has not been reduced by the higher dose of HFTBI. Relapses in this setting are equivalent to relapses with conventional BMT for AML, but appear to be increased for ALL. Radiobiological findings related to HFTBI will also be discussed.
Bone Marrow Transplantation↗
Graft failure after T-cell-depleted human leukocyte antigen identical marrow transplants for leukemia: I. Analysis of risk factors and results of secondary transplants.
Risk factors for graft failure were analyzed in 122 recipients of an allogeneic T-cell-depleted human leukocyte antigen (HLA)-identical sibling marrow transplant as treatment for leukemia. In each case pretransplant immunosuppression included 1,375 to 1,500 cGy hyperfractionated total body irradiation and cyclophosphamide (60 mg/kg/d x 2). No patient received immunosuppression prosttransplant for graft-versus-host disease (GVHD) prophylaxis. Nineteen patients in this group experienced graft failure. The major factors associated with graft failure were transplants from male donors and the age of the patient (or donor). Among male recipients of male donor-derived grafts a low dose per kilogram of nucleated cells, progenitor cells (colony forming unit-GM) and T cells was also associated with graft failure. Additional irradiation to 1,500 cGy, high dose corticosteroids posttransplant, and additional peripheral blood donor T cells did not decrease the incidence of graft failure. In addition, type of leukemia, time from diagnosis to transplant, an intact spleen, or the presence of antidonor leukocyte antibodies did not correlate with graft failure. To ensure engraftment of secondary transplants, further immunosuppression was necessary but was poorly tolerated. However, engraftment and survival could be achieved with an immunosuppressive regimen in which antithymocyte globulin and high dose methylprednisolone were administered both before and after infusions of secondary partially T-cell-depleted marrow grafts.
Age Factors↗
Immunosuppression prior to marrow transplantation for sensitized aplastic anemia patients: comparison of TLI with TBI.
From May 1980 through July 1986, 26 patients with severe aplastic anemia, sensitized with multiple transfusions of blood products, were treated on either of two immunosuppressive regimens in preparation for bone marrow transplantation from a matched donor. There were 10 patients treated with total body irradiation (TBI), 200 cGy/fraction X 4 daily fractions (800 cGy total dose), followed by cyclophosphamide, 60 mg/kg/d X 2 d. An additional 16 patients were treated with total lymphoid irradiation (TLI) [or, if they were infants, a modified TLI or thoracoabdominal irradiation (TAI)], 100 cGy/fraction, 3 fractions/d X 2 d (600 cGy total dose), followed by cyclophosphamide, 40 mg/kg/d X 4 d. The extent of immunosuppression was similar in both groups as measured by peripheral blood lymphocyte depression at the completion of the course of irradiation (5% of initial concentration for TBI and 24% for TLI), neutrophil engraftment (10/10 for TBI and 15/16 for TLI), and time to neutrophil engraftment (median of 22 d for TBI and 17 d for TLI). Marrow and peripheral blood cytogenetic analysis for assessment of percent donor cells was also compared in those patients in whom it was available. 2/2 patients studied with TBI had 100% donor cells, whereas 6/11 with TLI had 100% donor cells. Of the five who did not, three were stable mixed chimeras with greater than or equal to 70% donor cells, one became a mixed chimera with about 50% donor cells, but became aplastic again after Cyclosporine A cessation 5 mo post-transplant, and the fifth reverted to all host cells by d. 18 post-transplant. Overall actuarial survival at 2 years was 56% in the TLI group compared with 30% in the TBI group although this was not statistically significant. No survival decrement has been seen after 2 years in either group. There was less long-term morbidity in the TLI group compared with TBI although the numbers of surviving patients are small. With no difference in engraftment or survival, it is suggested that, for sensitized severe aplastic anemia patients, who are to receive a non-T cell-depleted marrow from a matched donor, prudent cytoreduction should include a fractionated, moderate dose irradiation regimen with maximum organ sparing, that is either TLI or TAI.
Adolescent↗
In vitro interferon-gamma production by cultured T-cells in severe aplastic anaemia: correlation with granulomonopoietic inhibition in patients who respond to anti-thymocyte globulin.
T-cell-mediated inhibition of granulomonopoietic progenitors (CFU-GM) was studied in vitro in 27 patients with severe aplastic anaemia (AA). In nine out of 13 responders to anti-thymocyte globulin (ATG), cultured T-cells obtained prior to therapy, as well as their conditioned medium strongly suppressed both normal allogeneic and autologous CFU-GM (the latter obtained after marrow recovery). Addition of anti-interferon-gamma to the cultures abolished the suppressive effect on CFU-GM. After ATG therapy, no similar inhibitory effect was detected. Employing the panning method with monoclonal antibodies (CD4+ for inducer/helper and CD8+ for cytotoxic/suppressor T-cells) we were able to show that the cells responsible for in vitro CFU-GM inhibition were included in the cytotoxic/suppressor T-cell subpopulation. Cultured T-cells and their conditioned medium obtained from 14 non-responders to ATG did not show CFU-GM suppression. The mean interferon (IFN) levels in the T-cell conditioned media of ATG-responders was 625 +/- 125 mu/ml while in non-responders the level was 45 +/- 15 mu/ml (normal control levels 43 +/- 24 mu/ml). Freshly isolated peripheral blood lymphocytes from either group did not show any in vitro inhibitory effect. The response rate to ATG was statistically significant when the generation in culture of high versus low IFN production was compared (P = 0.0001). Experiments with T-cells obtained from heavily transfused thalassaemia major, and myelodysplastic syndrome patients, as well as normal volunteers, also did not demonstrate any suppression of CFU-GM. Our results indicate that the response rate to ATG is significantly higher in patients with AA who have an abnormal regulation of interferon-gamma (g-IFN) production.
Adult↗
Autologous bone marrow transplantation for patients with poor-prognosis lymphoma.
Review of prognostic factors at Memorial Hospital in New York City has shown that adult patients with large-cell lymphoma (diffuse histiocytic lymphoma by Rappaport classification) who have high lactic dehydrogenase (LDH) and/or bulky mediastinal or abdominal disease are destined to do poorly with conventional combination chemotherapy, with a 2-year disease-free survival of about 20%. Patients who relapse after conventional combination chemotherapy have a similar poor prognosis. Thirty-one such patients with lymphoma were studied to evaluate the efficacy of intensive radiotherapy (hyperfractionated total body irradiation [TBI] [1,320 rad]), and cyclophosphamide (60 mg/kg/d for two days) followed by autologous bone marrow transplantation (ABMT). Our results show a disease-free survival advantage (P = .002) for 14 patients who underwent ABMT immediately after induction of remission with 79% surviving at a median follow-up 49.2+ months, compared with a median survival of 5.2 months for 17 patients administered ABMT while in relapse and/or after failing conventional treatment. Our results support the use of aggressive therapy as early treatment for patients with poor prognostic features.
Adolescent↗
Down regulation of myelopoiesis by mediators inhibiting the production of macrophage-derived granulomonopoietic enhancing activity (GM-EA).
Monocyte-derived lipid-containing macrophages (MDLMs) constitutively synthesize a granulomonopoietic enhancing activity (GM-EA) that potentiates the function of granulocyte-macrophage colony-stimulating activity (GM-CSA). In the study reported, we show that GM-EA is distinct from interleukin-1 (IL-1) in biochemical and functional properties and that its production is negatively regulated by several mediators. Thus, MDLM cultures pretreated with interferon-gamma (IFN-gamma, 3 to 900 U/mL), prostaglandin E2 (PGE2, 10(-13) to 10(-8) mol/L), or lactoferrin (LF, 10(-13) to 10(-8) mol/L) invariably produced less GM-EA than untreated controls. The relative potency of inhibition was in the order of IFN-gamma greater than or equal to PGE2 greater than LF. The extent of the inhibitory effects was proportional to dosage and the duration of treatment and could be observed following only a brief exposure (two hours) of the MDLMS to physiologic doses of the mediators. Under optimal conditions, IFN-gamma (300 U/mL for 24 to 48 hours) and PGE2 (10(-9) mol/L for 24 to 48 hours) could totally abrogate the ability of the MDLMs to produce GM-EA. However, the drug-inhibited MDLMs could be reactivated to produce GM-EA by treatment with zymosan (60 micrograms/mL). These results demonstrate that a mechanism for the control of myelopoiesis by mediators such as IFN-gamma, PGE2, and LF may involve the inhibition of GM-EA production. Furthermore, this negative feedback control is reversible and can be overridden when a proper stimulatory signal is given.
Cells, Cultured↗
Graft failures after T cell depleted marrow transplants for leukemia: clinical and in vitro characteristics.
Explore the source record for details and available documents.
Antigens, Differentiation↗
Host origin of the human hematopoietic microenvironment following allogeneic bone marrow transplantation.
The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA-identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.
Adolescent↗
Effects of low dose rate irradiation on human marrow hematopoietic and microenvironmental cells: sparing effect upon survival of stromal and leukemic cells.
The effects of different dose rates of in vitro irradiation on the proliferative capacity of marrow stromal, hematopoietic and leukemic colony-forming cells (CFC) are described. Marrow cell suspensions, HL-60 cells and trypsin-dispersed fibroblasts were irradiated at 5 or 45 cGy/min and then assayed for CFC. Irradiation at low (5 cGy/min) compared to high (45 cGy/min) dose rate showed a significant difference in survival of stromal and of HL-60 cells, but not of hematopoietic progenitors: the respective D0 values were 170 and 120 (p = 0.003) for marrow fibroblastic progenitors (CFU-F); 145 and 110 (p = 0.005) for passaged marrow fibroblasts (CFU-F); 170 and 140 (p = 0.045) for HL-60 cells; 85 and 85 for multipotential CFC (CFU-mix); 125 and 120 for erythroid progenitors (BFU-E); and 115 and 120 for granulomonopoietic progenitors (CFU-GM) (p = 0.5 for hematopoietic clonogenic cells). Marrow suspensions did not establish confluent stromal layers in long-term marrow cultures following irradiation with 600 cGy at 45 cGy/min, whereas after 840 cGy at 5 cGy/min confluent stromal layers were obtained. This indicates that low dose rate-sparing effect applies to all stromal cell progenitors. Confluent stromal layers derived from progenitors surviving irradiation sustained hematopoiesis as well as controls when co-cultured with fresh hematopoietic cells. Adherent layers in long-term marrow cultures irradiated after establishment with doses less than or equal to 1500 cGy at 5 or 45 cGy/min also showed normal hematopoietic supportive function when co-cultured with freshly isolated hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone Marrow↗
Myeloablation with diaziquone: in vitro assessment.
UNLABELLED: The promising antineoplastic agent diaziquone is associated with prolonged aplasia and rare instances of bone marrow necrosis, but only mild extramedullary toxicity. To explore the drug's potential as a myeloablative agent prior to bone marrow transplantation, we compared its effects on hematopoietic versus marrow stromal cells. After short-term (one to six hours) or prolonged (three to seven days) exposure to the drug, marrow was assayed for hematopoietic (CFU-Mix, BFU-E, CFU-GM) and stromal (CFU-F) colony-forming cells and studied in long-term marrow culture (LTMC). One- and three-hour treatments produced little cytotoxicity, even at 5000 ng/mL. After six-hour treatments with this dose, marrow was depleted of CFU-Mix, BFU-E, and CFU-GM, but produced CFU-GM in LTMCs, indicating an ongoing input of CFU-GM from a surviving pre-CFU-Mix population. In contrast, elimination of the latter may be inferred from the absence of CFU-GM in LTMCs exposed for three to seven days to diaziquone at only 150 ng/mL. Under these conditions, CFU-F recovery was 40% and adherent stromal layers in LTMCs were similar to untreated controls regarding rate of development and cellular composition. Our in vitro pre-CFU-Mix-ablative regimen correlates with clinical data that show prolonged but reversible myelosuppression at steady-state diaziquone plasma levels of 101 +/- 10 ng/mL (mean +/- standard error of mean) during 7-day constant infusions. IN CONCLUSION: hematopoietic cells are more sensitive than marrow stromal cells to the dose- and highly time-dependent cytotoxicity of diaziquone, a direct drug-induced noxious effect on the marrow microenvironment is an unlikely cause of the isolated episodes of marrow necrosis after the use of diaziquone in vivo, and prolonged infusion of diaziquone represents an attractive means for achieving myeloablation in selected clinical situations.
Aziridines↗
Preclinical assessment of purging with VP-16-213: key role for long-term marrow cultures.
An evaluation of the effects of VP-16 on normal human marrow cells and representative lymphoma-leukemia cell lines was performed to assess this agent's applicability to ex vivo marrow purging. Tumoricidal dose curves were defined using malignant lymphoid (SK-DHL2 and Reh) and myeloid (HL-60) cells admixed with a 20-fold excess of irradiated marrow cells to simulate a borderline remission marrow. One-hour treatments yielded ID50 of less than 5 mumol/L of VP-16 for clonogenic units from each cell line; rare-to-zero clonogenic units survived exposure to 50 to 100 mumol/L. CFU-Mix, BFU-E, and CFU-GM were equal in their sensitivity to VP-16 (ID50s25 to 30 mumol/L). Marrows treated with 75 mumol/L were completely depleted of these colony-forming cells but produced CFU-GM in one-stage long-term marrow cultures (LTMCs). This dose had little adverse effect on the proliferative capacity of marrow stromal progenitors, as measured by CFU-F (ID50 271 mumol/L) and by the unperturbed development of adherent layers in LTMCs. Furthermore, these stromal layers were able to support hematopoiesis as well as controls in co-culture experiments with autologous marrow cells (two-stage LTMCs). In conclusion, doses of VP-16 that cleanse marrow of lymphoma-leukemia cells spare hematopoietic and stromal progenitors as demonstrated by LTMCs. These data favor the use of VP-16 in the clinical autotransplant setting.
Bone Marrow↗
Radiobiological properties of the human hematopoietic microenvironment: contrasting sensitivities of proliferative capacity and hematopoietic function to in vitro irradiation.
We describe the effects of in vitro irradiation on the proliferative capacity and hematopoietic supportive function of human marrow stromal cells. To assess the effects on the proliferative capacity of stromal progenitors and differentiated fibroblasts, marrow cell suspensions and trypsin-dispersed marrow fibroblasts were treated with a single dose of gamma radiation at 100 rad/min. Fibroblastic progenitors (CFU-F) showed an exponential decrease in colony formation with increasing doses of irradiation, with a Do slightly higher than that of granulomonopoietic progenitors (CFU-GM); Do values for CFU-F and CFU-GM were 130 and 115, respectively. However, although the CFU-F survival curve exhibited a shoulder (n = 1.3), the CFU-GM curve did not (n = 1.0), indicating that only fibroblastic progenitors have the potential to repair irradiation-induced damage. Passaged marrow fibroblast colony-forming cells also showed a shouldered exponential survival curve with a Do of 110 and n value of 1.4. Marrow stromal progenitors giving rise to adherent layers in long-term marrow cultures also demonstrated a highly radiosensitive proliferative capacity. Stromal layers derived from irradiated marrow suspensions failed to establish adherent layers after relatively low doses of irradiation (over 240 rad) in a dose-response manner. To assess any functional damage in stromal progenitors surviving irradiation, stromal layers derived from marrow suspensions irradiated up to 240 rad were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged CFU-GM production. Confluent stromal layers derived from irradiated marrow suspensions sustained CFU-GM production as well as controls. To study the effects of irradiation on the hematopoietic supportive capacity of established marrow-derived stromal layers, 4 to 6-week-old adherent layers were irradiated as described and cocultured with autologous marrow cells enriched for colony-forming cells. Stromal layers irradiated up to 1,320 rad sustained prolonged CFU-GM production, indicating that the hematopoietic supportive function remained intact at this dose of irradiation. In conclusion, we demonstrated that the proliferative capacity of human marrow stromal progenitors, as well as that of their differentiated descendants, is quite sensitive to in vitro radiation, while the hematopoietic supportive function of differentiated stromal cells is relatively resistant to the effects of radiation.
Bone Marrow↗
Constitutive production of leukemia differentiation, colony-stimulating, erythroid burst-promoting, and pluripoietic factors by a human hepatoma cell line: characterization of the leukemia differentiation factor.
Conditioned medium (CM) obtained from a human hepatoma cell line, SK-HEP-1, contains colony-stimulating factors (CSFs) active on murine and human bone marrow-derived granulocyte and macrophage colony-forming units (CFU-GM) and a factor capable of inducing granulocyte-macrophage differentiation (GM-DF) of murine myelomonocytic leukemic cells WEHI-3B(D+) and human promyelocytic leukemic cells HL-60 when assayed in semisolid agar cultures. The human active granulocyte-macrophage colony-stimulating factor (GM-CSF) for day 7 CFU-GM and the GM-DF for WEHI-3B(D+) and for HL-60 are not separable by acrylamide agarose column chromatography, eluting at an apparent molecular weight between 20,000 and 35,000 daltons, or by isoelectric focusing (isoelectric point, pH 5.4). In addition, SK-HEP-1 CM contains erythroid burst-promoting activity (BPA) and a factor that promotes the growth of human mixed colonies. SK-HEP-1 cells, which grow as an adherent monolayer, appear not to be endothelial or monocytic in origin since by immunofluorescent staining they are negative for Ia (HLA-DR), monocyte antigen 1 and 2, lysozyme, and factor VIII-related antigen. Positive immunofluorescent staining for keratin and fibronectin suggests the possibility that SK-HEP-1 is an epithelial cell line. Constitutive production of GM-DF as well as other hematopoietic activities including GM-CSF, erythroid BPA, and an activity that promotes the growth of human mixed colony progenitors by a human epithelial tumor cell line, SK-HEP-1, suggests that this cell line is a valuable resource for both large-scale production of these factors and the cloning of the gene(s) that code for these regulators.
Animals↗
Biological characterization of a granulomonopoietic enhancing activity derived from cultured human lipid-containing macrophages.
We describe the biologic characteristics of an activity produced by human monocyte-derived lipid-containing cells (MDLCCs) that enhances the colony-forming capacity of granulocyte-macrophage progenitors (CFU-GM). Medium conditioned by well-developed MDLCCs (at day 21 to day 28 of cultivation) was added to bone marrow cultures containing GCT cell line-conditioned medium (GCT-CM) or other material as a source of granulocyte-macrophage colony-stimulating factors (GM-CSFs). MDLCC-conditioned medium (CM) had no detectable granulocyte-macrophage colony-stimulating activity (GM-CSA), but it contained an activity that enhanced the colony number in both day 7 and day 14 CFU-GM cultures. Dose-response curves for GCT-CM in the presence of MDLCC-CM demonstrated that this enhancing effect occurred at concentrations of GM-CSFs that stimulate maximal CFU-GM growth. This enhancing effect was seen with both granulocytic and monocytic progenitor cells. It was titratible and required the continuous presence of MDLCC-CM from initiation of culture. No enhancement was noted when MDLCC-CM was added 48 hours after plating. The enhancement still occurred when marrow cells were first incubated with MDLCC-CM and GCT-CM was added at later times. Neither the enhancing activity nor its production was dependent on horse serum contained in MDLCC culture medium. The enhancing effect was also seen when other sources of GM-CSA were used: medium conditioned by 5637 cell line, phytohemagglutinin-stimulated lymphocytes (PHAL), or placenta tissue. Furthermore, this enhancing activity appeared to be specific for CFU-GM. Addition of MDLCC-CM to mixed and erythroid cultures, stimulated by suboptimal and optimal concentrations of PHAL-CM did not modify the number of mixed colonies or erythroid bursts. This granulomonopoietic enhancing activity contained in MDLCC-CM was heat stable (56 degrees C and 75 degrees C for 30 minutes) and nondialyzable (3,500 and 14,000 molecular weight cut off tubing). Its production was increased by treating MDLCC with lipopolysaccharide (5 micrograms/mL) or zymosan (60 micrograms/mL) and inhibited by lactoferrin (10(-7) mol/L). The production of a granulomonopoietic enhancing activity by MDLCCs represents the demonstration of another positive feedback regulator of myelopoiesis involving the monocyte-macrophage system.
Cells, Cultured↗
Depletion of stromal cell elements in human marrow grafts separated by soybean agglutinin.
We report studies demonstrating the presence on human marrow stromal cells of binding sites for the soybean lectin (SBA). Marrow cells were separated by agglutination with SBA into an agglutinated cell (SBA+) fraction containing most mature hemic cells including T lymphocytes, and an unagglutinated cell (SBA-) fraction containing the hematopoietic stem cells. The vast majority of fibroblast colony-forming units (CFU-F) (97.2% +/- 1.1%) were in the SBA+ fraction. Mixing experiments using SBA+ and SBA- cells excluded the possibility that these results were caused by an unequal distribution of accessory cells having a regulatory effect on CFU-F growth. In the heterogeneous adherent layers of long-term marrow cultures derived from SBA+ and SBA- cells, endothelial cells, and adipocytes were found almost exclusively in cultures of SBA+ marrow cells. Immunofluorescence studies with fluorescein-labeled SBA revealed the staining of cultured marrow fibroblasts. This staining was inhibited by D-galactose, which is the sugar that specifically binds to SBA. Staining of marrow-derived heterogeneous adherent layers with fluorescein-labeled SBA demonstrated that cultured endothelial cells (identified by rhodamine-labeled antibodies against factor VIII-associated protein) and early adipocytes also had surface glycoproteins binding SBA, although its density on the latter cells was much less important. Thus, our data indicate that human marrow grafts processed with soybean lectin to remove T cells are also depleted of the cellular components of the marrow stroma.
Adipose Tissue↗
Long-term culture of human bone marrow macrophages: macrophage development is associated with the production of granulomonopoietic enhancing activity (GM-EA).
We describe a liquid culture system allowing the long-term maintenance and differentiation of human marrow monocytes into well-developed, lipid-containing macrophages, and we show that these cells produce a regulating activity that enhances the colony formation induced by granulocyte-macrophage colony-stimulating factors (GM-CSF). Adherent, low density (less than 1.074 g/cm3) human marrow cells were used as a source of monocytic cells. Of 12 different culture conditions tested, alpha-medium supplemented with 20% horse serum and incubation at 37 degrees C provided the best conditions for macrophage development, adipogenesis, and long-term culture. Neither insulin (1 to 10 micrograms/ml) nor hydrocortisone (10(-6) M) improved the lipid accumulation in cultures containing horse serum. Trypsin was employed to remove fibroblasts without detaching monocytic cells from the marrow-derived adherent cell layers. Marked structural and functional changes characterized the transformation of monocytes into lipid-containing macrophages. Cell enlargement up to seven or eight times by 21 to 28 days of culture was associated with an increase in small and medium-sized lipid granules, as well as in acid phosphatase and nonspecific esterase activities. Ultrastructurally, there was an increase in the number of mitochondria, Golgi apparatus, lysosomes, rough endoplasmic reticulum, and lipid inclusions, which remained of small size and did not coalesce to form larger inclusions. The absolute quantity of Fc receptors, Ia antigens, and antigens recognized by monoclonal antibodies 61D3 and 63D3 also increased as a function of cell size. As marrow monocyte-macrophage differentiation proceeded, a rapid decline in GM-CSF production was accompanied by an increase in an activity that by itself had no capacity to stimulate colony formation in CFU-GM cultures devoid of GM-CSF, but did enhance the colony formation induced by optimal concentrations of GM-CSF. Neither the enhancing activity nor its production was related to the horse serum present in the culture supernatants. The morphology of colonies enhanced by this activity was different from the morphologic spectrum in non-enhanced cultures. This granulomonopoietic enhancing activity (GM-EA) represents another positive feedback regulator of hematopoiesis derived from cells of the monocyte-macrophage system.
Adult↗