Leukocyte-conditioned Medium Research Articles

Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose Tissue Cells: Paracrine Consequences on Vascular Endothelium.

Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin-sensitizer, anti-inflammatory and anti-atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200, or 295 mg/dl glucose, with or without macrophage-conditioned medium (MCM). Expression of adiponectin, GLUT-4 and the insulin receptor was analyzed by real-time PCR. The paracrine effect of stromal cells was determined in co-cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte-conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U-shaped dose-response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis-induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell-conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis-induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression.

Scientific Scholarship and Impact Factors

Scientific Scholarship and Impact Factors

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Long‐lasting partial remission by Interferon‐alpha treatment in a child with essential thrombocythemia

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder characterized by sustained thrombocytosis, isolated hyperplasia of megakaryocytic lineage, and association with thrombotic or bleeding episodes. It is extremely rare in childhood and frequently presents without evident clinical signs. We describe a 3-year-old girl with severe headache and dizziness suffering from ET, who was treated with Interferon-alpha-2a (IFN) based on the potent effect of this agent to inhibit myeloid colonies induced by phytohemagglutinin A stimulated leukocyte conditioned medium (PHA-LCM). Bone-marrow-derived mononuclear cells of this patient did not exhibit spontaneous colony formation but responded to recombinant human (rh) erythropoietin (EPO), rh granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and stem-cell factor in addition to PHA-LCM. After 65 months of in vivo IFN treatment, the patient experienced a sustained partial remission with platelet counts varying between 400 and 600 x 10(3)/microl.

Clonogenic growth patterns correlate with chemotherapy response in acute myeloid leukaemia

Cytosine arabinoside and anthracycline-containing regimens induce remission in upwards of 60% of previously untreated patients with adult acute myeloid leukaemia (AML). Despite this, in addition to primary drug resistance, the majority of these patients relapse. Reliable methods for uniformly recognising these two subgroups at presentation do not exist and therefore a further attempt has been made to relate in vitro toxicity, using a clonogenic assay, to clinical outcome. In 10 normal controls and 12 chemotherapy naïve cases, mononuclear cells harvested by density gradient separation were resuspended at a concentration of 2 × 105/ml and quadruplicates of 250 μl per well cultured in methycellulose containing foetal calf serum and phytohaemagglutinin stimulated leucocyte conditioned medium. Cell kill was determined for cytosine arabinoside, daunorubicin and etoposide either singly or in combination using both a pulsed and continuous exposure. Aggregates were scored after seven days and three distinct patterns recognised. The patients all received the same drugs in a standard protocol and achievement of complete remission correlated with growth pattern. The survival of normal marrow colony-forming cells or GM-CFUc and the leukemic equivalent designated L-CFUc were assessed and a sensitivity index (SI) determined as a ratio of these two values in which more reproducible results were found when the drug was continuously present. It is concluded that the microculture technique is feasible and clearly demonstrates chemotherapy effect but no correlation was demonstrated with clinical outcome. This is a negative pilot study and, as a means of recognising drug sensitivity or resistance, should be discarded in favour of currently available molecular techniques.

Effect of myelopoietic and pleiotropic cytokines on colony formation by blast cells of children with acute lymphoblastic leukemia.

The aim of this study was to see whether pleiotropic or myeloid hematopoietic growth factors, which do not stimulate normal lymphoid cells, can induce proliferation of blast cells of the acute lymphoid leukemia (ALL) of childhood. Bone marrow cells of 13 children with untreated ALL (nine common ALL, two myeloid antigen positive ALL and two early T-cell ALL) formed colonies of leukemic blast cells in primary methylcellulose cultures. Spontaneous growth was observed in three of 13 cases, whereas phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM), a conventional source of various natural human cytokines, induced colony formation in ten of 13 cases. A similar rate of responsiveness was seen with recombinant human granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF); a combination of these three cytokines induced colony formation in all cases studied. The effect of these growth factors on colony formation seemed to be dose-dependent in some cases. Of the stimuli studied, GM-CSF induced the smallest number of colonies, whereas the effects of G-CSF, SCF and PHA-LCM were similar in this respect. Combination of cytokines proved to be even more efficient in inducing clonal proliferation of leukemic lymphoblasts. In double combinations, G-CSF and GM-CSF as well as G-CSF and SCF were able to potentiate each other's effects. Triple combination of these cytokines mediated the most potent growth stimulus. Our results demonstrate that myeloid and pleiotropic cytokines are able to stimulate clonal proliferation of pediatric leukemic lymphoblasts. This may present a potential hazard to children with ALL while on adjuvant therapy with hematopoietic growth factors. In vitro colony assays performed prior to or in parallel with the administration of hematopoietic growth factors to ALL patients may help to forecast their possible effects on leukemic cells in vivo.

All trans retinoic acid abrogates spontaneous monocytic growth in juvenile chronic myelomonocytic leukaemia.

All trans retinoic acid, the active metabolite of vitamin A, exerts profound effects on cell differentiation. On normal myeloid progenitors, retinoids switch the differentiation program of granulo-macrophagic progenitors towards the granulocytic lineage and consequently reduce CFU-M colony formation. Bone marrow and peripheral blood mononuclear cells from children with Juvenile Chronic Myelomonocytic Leukaemia show typical spontaneous monocytic growth. We questioned whether in this disease, retinoids could switch myelomonocytic growth and inhibit the abnormal CFU-M colony proliferation. Ten JCML samples were studied in the presence of ATRA in methyl cellulose colony assay, before (CFU-C) or after (pre-CFU) liquid suspension culture. In vitro characteristics of JCML such as spontaneous monocytic growth in the absence of growth factor was noted in all patients. In the presence of leucocyte-conditioned medium, nine samples showed only CFU-M growth and one sample CFU-GM growth. Incubation with ATRA inhibited CFU-M colony formation in nine cases. Enhancement of granulocytic differentiation (CFU-G) was noted in nine cases. ATRA also inhibited CD34+ JCML monocytic growth and GM-CSF hypersensitivity. These data suggest that, in JCML progenitors, retinoid pathways are functional and inhibition of immature monocytic progenitors cells may be achieved with retinoids, without impeding granulocytic cell growth.

Lymphocytes stimulate dehydroepiandrosterone production through direct cellular contact with adrenal zona reticularis cells: a novel mechanism of immune-endocrine interaction.

Adrenal androgen production was reduced by 80% in patients receiving T lymphocyte-suppressive medications compared to that in age-matched controls. In vitro, however, neither tacrolimus nor cyclosporin A reduced dehydroepiandrosterone (DHEA) release by adrenocortical cells. Therefore, we examined the potential role of lymphocytes in adrenal androgen production, using cocultures of human T lymphocytes and adrenocortical primary or transformed cells. Co-cultures led to a 4-fold elevation of DHEA levels (490.4 +/- 94.8% over basal), which was greater than the increase observed after the addition of maximal concentrations of ACTH (117.4 +/- 14.8%). Separation of cells by semipermeable membranes abolished this effect, and transfer of leukocyte-conditioned medium had little androgen-stimulating effect. These data suggested that the observed stimulation of androgen secretion required cell contact rather than soluble paracrine factor(s). Furthermore, we examined human adrenal glands for the presence of T lymphocytes and contact between these cells and steroid-secreting cells of the zona reticularis. Indeed, T lymphocytes expressing CD4 and CD8 antigens were present within human adrenal zona reticularis by immunohistochemical subtyping. Electron microscopic analyses demonstrated direct cell-cell contact between T lymphocytes and adrenocortical cells in situ. This study provides evidence for a novel mechanism of immune-endocrine interactions of direct T lymphocyte-adrenocortical cell contact-mediated stimulation of adrenal androgen secretion.

In vitro effect of ketone bodies, glucocorticosteroids and bovine pregnancy-associated glycoprotein on cultures of bone marrow progenitor cells of cows and calves

Changes in the number, maturity and function of neutrophils, concomitant changes in plasma concentrations of hormones and metabolites, and the increased susceptibility of cows to infectious diseases around parturition, led us to investigate the effect of β-hydroxybutyric acid (BHBA), acetoacetic acid (AcAc), hydrocortisone-21-acetate (HCAc) and bovine pregnancy-associated glycoprotein (bPAG) on the proliferation of bovine bone marrow progenitor cells in methylcellulose in vitro cultures. Myeloid progenitors were stimulated with concanavalin A-stimulated leukocyte conditioned medium (LCM) and erythroid progenitors with erythropoetin in the presence of hemin. Erythroid and myeloid colonies were scored after five and seven days, respectively. BHBA and AcAc induced inhibitory effects on the proliferation of bovine bone marrow cells at concentrations of 1.0, 2.5, and 5.0 mM. HCAc significantly inhibited growth of progenitors at concentrations of 10, 20, 50, and 100 ng/ml, and bPAG at concentrations of 2400 and 3000 ng/ml. The results of this study suggest that in the cow high concentrations of BHBA, AcAc, HCAc and bPAG, which can be reached in the circulation around calving, could alter the number of circulating neutrophils after parturition. This phenomenon might contribute to the increased susceptibility of dairy cows to environmental mastitis.

Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers.

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.

Culture requirements and cytochemical characteristics of colony forming units-granulocyte/monocyte in cynomolgus monkeys

For the preclinical safety assessment of recombinant human haematopoietic growth factors, the in vitro bone marrow progenitor cell assays could provide valuable information that can be used to select appropriate biologically responsive animal species and to predict the potential toxicity and dosage selection for in-life toxicology studies. The cynomolgus monkey is a commonly used non-human primate model for the assessment of efficacy and toxicity of such compounds; however, the information regarding their bone marrow progenitor cell assays is scarce. This study evaluated the culture requirements and the morphological and cytochemical characteristics of colony forming units-granulocyte/monocyte (CFU-GM) for this species. The optimal CFU-GM growth was achieved in methylcellulose cultures of minimum essential medium-alpha (MEM-alpha) that contained 5% leucocyte-conditioned medium (LCM) and 30% heat inactivated horse serum (HIHS), after 14 days of incubation in a humidified atmosphere. A triple cytochemical staining method using naphthol-ASD chloroacetate esterase (CAE), alphanaphthol acetate esterase (ANAE) and luxol fast blue (LFB) was used to characterise the monocyte/macrophage, neutrophil and eosinophil colonies, respectively. This cytochemical method showed the presence of approximately 15% pure neutrophil, 61% pure monocyte/macrophage, 4% pure eosinophil, 15% mixed granulocyte/monocyte (positive for at least two stains) and 3.5% unstained colonies in 14-day-old cultures. The monocyte, neutrophil and mixed colonies were smaller in diameter, compact and contained a larger number of cells than the eosinophil colonies. Since eosinophil colonies exhibit lower cell density, they may be easily missed in total CFU counts performed at low magnification. These in vitro assays could be used to predict the potency of and lineage-specific response to haematopoietic growth factors in cynomolgus monkeys.

Differential effects of recombinant human interleukin-13 on the in vitro growth of human haemopoietic progenitor cells.

Effects of recombinant human interleukin (IL)-13 on in vitro haemopoiesis from non-adherent mononuclear cells (NAMC) or highly enriched CD34+ cells of human cord blood (CB) were studied. IL-13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34+ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemagglutinin-stimulated human peripheral blood leucocyte-conditioned medium (PHA-LCM) in a dose-dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA-LCM, demonstrated an increased MK colony number in the presence of IL-13, especially in combination with IL-3. However, IL-13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte-macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL-3, GM-CSF, Epo, G-CSF or in combination was significantly inhibited by the addition of IL-13. On the other hand, high concentrations (100 ng/ml) of IL-13 were needed to cause a slight inhibition on the growth of BFU-E-derived colonies under the same methylcellulose culture. These results indicate that IL-13, alone and synergistically with the effect of IL-3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.

In vitro autonomous proliferation in ANLL: Clinical and biological significance

In acute non-lymphoblastic leukemia (ANLL) progenitor cells frequently display a certain degree of autonomous growth. The aim of the present work was to analyze the autonomous proliterative capacity of leukemic progenitors in both de novo and secondary to myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS), acute myeloid leukemias and to correlate with clinical and biological characteristics of the disease. Clonogenic assays with and without leukocyte conditioned medium with PHA (LCM-PHA) were performed and the autonomous proliferation index (API) calculated in a series of 50 patients (34 de novo ANLL, eight secondary to MPD and eight secondary to MDS). Patients were divided into two groups according to their API, low (⩽0.4) or high (>0.4). Autonomous growth was observed in 84% of cases studied (82% in de novo ANLL, 75% secondary to MDS and 100% secondary to MPD). The group with the highest API (29 patients) had increased levels of hemoglobin ( P = 0.006) and platelets ( P = 0.01). A high API was also associated with an immature phenotype of blast cells ( P = 0.02). Upon analyzing the de novo ANLL separately we observed that a high API correlated with high Hb values ( P = 0.02), a lower rate of complete remission (42% vs 61%) and a lower survival rate (medium of 3 vs 10 months). These findings suggest that the capacity for autonomous proliferation can condition the clinical and biological profile of the disease.

Granulocyte-monocyte colony forming unit content of autologous bone marrow transplants in patients with haematological malignancy.

Cell viability and number of granulocyte-monocyte colony forming units (CFU-GM) were systematically assessed in 57 patients who had undergone transplantation of the autologous bone marrow for treatment of haematologic malignancies. Bone marrow cell cultivation in agarose with feeder layers appeared inferior to that performed in agarose with recombinant human granulocyte-monocyte colony stimulating factor and methylcellulose with phytohaemaglutinin leukocyte-conditioned medium. Since the transplant cells were frozen in liquid nitrogen between harvesting and reinfusion, the following samples were tested: buffy coat cells, buffy coat cells immediately after addition of dimethylsulphoxide, cell sample that had been frozen for 24 hours, and frozen transplant cells at the time of thawing and transplantation. Each procedural step decreased both cell viability and the number of CFU-GM, but since the lymphohaematologic recovery in all patients followed the pattern reported in the literature for high-quality transplants, we concluded that our transplants retained the necessary number of progenitor cells. It appears that the best strategy for dynamic assessment of the transplant quality would be to perform tests after every step of the transplant processing. Cell viability and number of progenitors per body weight in transplants were also found to be associated with probability of neutrophil reconstitution after bone marrow reinfusion.

Growth factors controlling interleukin-4 action on hematopoietic progenitors

We investigated the effect of interleukin-4 (IL-4) on human hematopoietic progenitors using low-density bone marrow cells from 29 hematologically normal donors. We found that IL-4 could either inhibit or stimulate cell growth, depending upon the other constituents of the culture medium. At concentrations ranging from 0.1 to 10.0 micrograms/ml, it significantly inhibited colony-forming units granulocyte-macrophage (CFU-GM) in the presence of either fetal calf serum alone, erythropoietin, leukocyte-conditioned medium prepared with phytohemagglutinin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (SCF), in a dose-dependent fashion. In contrast, IL-4 stimulated CFU-GM colony multiplication in the presence of granulocyte colony-stimulating factor (G-CSF). Similar but less significant inhibitory effects were exerted by IL-4 on burst-forming units-erythroid (BFU-E). The growth-suppressive effect of IL-4 was partially reversed by IL-1 beta, and to a lesser extent by IL-6. When tested by enzyme-linked immunosorbent assay (ELISA), IL-4 suppressed cellular IL-1 beta production, and, similar to IL-4, anti-IL-1 beta-neutralizing antibodies inhibited CFU-GM colony growth, suggesting that the inhibition of endogenous IL-1 beta is a factor in regulating the IL-4 effect. Furthermore, in the absence of exogenous growth factors, IL-4 inhibited CFU-GM colony growth when anti-G-CSF neutralizing antibodies were also present. Therefore, we tested the effect of IL-4 on G-CSF receptors and found that 6- or 24-h incubation of low-density marrow cells with 1.0 microgram/ml IL-4 resulted in up-regulation of G-CSF receptors. Taken together, these results suggest that IL-4 possesses a dual modulatory role in the hematopoietic system via interaction with various cytokines.

Costimulatory signals are required for optimal proliferation of human natural killer cells.

CD56dim NK cells, which comprise approximately 90% of human peripheral blood NK cells, respond to IL-2 with cytokine production, up-regulation of functionally relevant surface molecules, and augmented cytolytic activity. Nevertheless, CD56dim NK cells proliferate poorly in response to IL-2 alone. We found that other NK cell mitogens, including IL-4, IL-7, and IL-12, also induced little proliferation of CD56dim NK cells. Indeed, IL-2 stimulated at least 10-fold more NK cell proliferation than did IL-4, IL-7, or IL-12. In contrast, leukocyte-conditioned medium (LCM) induced two- to threefold greater proliferation of CD56dim NK cells than did optimal concentrations of IL-2. Although the calcium ionophore ionomycin did not stimulate proliferation by itself, it markedly augmented LCM-induced proliferation of CD56dim NK cells. Proliferation in response to either LCM alone or LCM together with ionomycin was almost completely abrogated by anti-IL-2R antibodies. Thus, IL-2 appears to be necessary but not sufficient for optimal proliferation of CD56dim NK cells. LCM-induced proliferation of ionomycin-activated CD56dim NK cells was inhibited 24% by anti-IL-1 heteroantisera and 57% by anti-TNF antisera; a combination of both antisera inhibited proliferation by 73%. Furthermore, although rIL-1 and TNF did not induce proliferation by themselves, both cytokines could augment IL-2-induced proliferation of resting or ionomycin-activated NK cells. Hence IL-1 and TNF do not appear to be primary NK cell mitogens, but rather accessory factors that can enhance IL-2-dependent NK cell proliferation. Stimulation through CD2 or CD16 Ag did not enhance LCM-induced NK cell proliferation. However, stimulation with NK-sensitive K562 cells strongly augmented CD56dim NK cell proliferation to LCM or to IL-2, IL-1, and TNF in combination. NK-resistant Daudi cells did not promote the proliferation of highly purified NK cells. Thus, NK cell proliferation may be enhanced by triggering through putative receptors for natural killing, and ionomycin may mimic such triggering. Although IL-2 by itself can induce NK cell proliferation, most NK cells resemble T and B lymphocytes in that they require multiple signals for optimal proliferation.

Burst-promoting activity in the serum of patients with chronic renal failure undergoing long-term hemodialysis.

Anemia in patients with chronic renal failure (CRF) is multifactorial, and while the majority will respond to the paternal administration of recombinant human erythropoietin (r-HuEPO), a role for coexistent plasma inhibitors and stimulators, such as burst-promoting activity (BPA), remains controversial. To evaluate the latter possibility, eight individuals with CRF on long-term hemodialysis, were studied before (mean hemoglobin 58.4 +/- 8.0 g/l and after 12 weeks of r-HuEPO therapy (mean hemoglobin 100.4 +/- 18.3 g/l). In vitro erythroid cultures using erythroid burst forming unit (BFU-E) and erythroid colony forming unit (CFU-E) assays were performed, plating 5 x 10(4) bone marrow mononuclear cells and comparing growth in heat-inactivated autologous serum with AB serum. Using Step III sheep erythropoietin (Connaught Laboratories, Willowdale, Ontario, Canada) (n = 4), mean BFU-E pre-therapy were 89.7 +/- 75.1, CFU-E were 418.5 +/- 150.6, whereas the corresponding figures in AB serum were 2.5 +/- 2.9 and 197 +/- 94.19, p = 0.1, p = 0.01, respectively. Similarly, with r-HuEPO (EPOCONN: Connaught Laboratories, Willowdale, Ontario, Canada) (n = 4), mean BFU-E were 145.25 +/- 103.3 in autologous serum and 31.0 +/- 56.75 in AB serum (p = 0.04). As controls, erythroid progenitors from two normal donors yielded 69 and 61 BFU-E colonies in autologous serum and 103 and 42 in AB serum; the corresponding CFU-E were 52 and 235 versus 136 and 137.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.

Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.

Inhibition of acute myelogenous leukemia blast proliferation by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors

Interleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (sIL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1 beta. A similar inhibitory effect was also noted with the use of anti-IL-1 beta neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, interleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1- inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 10(7) low-density AML marrow cells were tested for intrinsic IL-1 beta content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 10(7) cells from two normal marrow aspirates yielded 54.6 pg of IL-1 beta. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either sIL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates, and supernatants from nine (P less than .01) and six samples (P less than .037), respectively, and G-CSF concentration in lysates from six of nine samples (P less than .03), and in supernatants from five of six samples (P less than .06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.

Inhibition of Acute Myelogenous Leukemia Blast Proliferation by Interleukin-1 (IL-1) Receptor Antagonist and Soluble IL-1 Receptors

Abstractlnterleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (slL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (slL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1β. A similar inhibitory effect was also noted with the use of anti-IL-lβ neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, in-terleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1-inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 107 low-density AML marrow cells were tested for intrinsic IL-1β content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 107 cells from two normal marrow aspirates yielded 54.6 pg of IL-1β. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either slL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates and supernatants from nine (P <. 01) and six samples (P <. 037), respectively, and G-CSF concentration in lysates from six of nine samples (P <. 03), and in superna-tants from five of six samples (P <. 06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.