Cytokine regulation of the human burst-forming unit-megakaryocyte

The human burst-forming unit-megakaryocyte (BFU-MK) is a primitive megakaryocytic progenitor cell. A marrow cell population enriched for BFU-MK (CD34+ DR-) was obtained by monoclonal antibody labeling and fluorescence-activated cell sorting. CD34+DR- cells were assayed in a serum-depleted, fibrin clot culture system. Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant interleukin-3 (rIL-3), and megakaryocyte colony-stimulating factor (MK-CSF), partially purified from human plasma, were each individually capable of promoting BFU-MK-derived colony formation. Recombinant erythropoietin, rG-CSF, rIL-4, rIL-6, and thrombocytopiesis stimulating factor, partially purified from human embryonic kidney cell conditioned media, had no stimulatory effect on BFU-MK-derived colony formation when added alone or in various combinations with either GM-CSF, IL-3, or MK-CSF, GM-CSF and IL-3, GM-CSF and MK-CSF, but not IL-3 and MK-CSF had additive actions in promoting BFU-MK-derived colony formation, rIL-1 alpha had no influence alone on BFU-MK cloning efficiency, but had a dose-dependent, synergistic effect with IL-3, but not with GM-CSF or MK-CSF. The synergistic relationship between IL-1 alpha and IL-3 was abrogated by addition of an IL-1 alpha neutralizing antibody but not by a GM-CSF neutralizing antiserum, suggesting that IL-1 alpha acts directly on the BFU-MK and not by stimulating marrow auxiliary cells to secondarily release additional cytokines. Information presented here indicates that the regulatory influence, acting on the different stages of megakaryocyte development, are stage-specific and accomplished by multiple cytokines.     

Large granular lymphocytes have a promoting activity on human peripheral blood erythroid burst-forming units

Peripheral blood mononuclear cells were fractionated according to the expression of a variety of surface markers, and the fractions obtained were tested for erythroid burst-forming unit (BFU-E) colony formation. BFU-Es were detected in the HLA-DR+ non-T cell fraction, but gave rise to optimum colony numbers only in the presence of a nonadherent, relatively radioresistant cell. This accessory cell was found among the HLA-DR- non-T, non-B cells, a fraction that was particularly enriched in large granular lymphocytes (LGLs). Experiments carried out to assess directly the surface markers of the accessory cell revealed an FcR+, OKM1+, Leu 7+, Leu 11+, OKT4-, OKT8- surface phenotype, which is consistent with that of the majority of LGLs. Peripheral blood LGLs, purified by Percoll density gradient, proved very efficient in promoting optimal BFU-E colony formation. All of these results indicate that LGLs have a potent erythroid burst-promoting activity. Such activity is probably mediated through the release of soluble factors, as shown by the observation that LGL culture supernatants were as effective as LGLs in sustaining colony formation.     

Effect of estrogens and progesterone on human peripheral erythroid burst-forming unit (BFU-E) growth

Variations in the number of peripheral burst-forming unit--erythroid (BFU-E) of healthy women were observed during a prolonged period of observation. These differences were related to different phases of the menstrual cycle. A peak in the number of BFU-E occurred on day 14 of the cycle corresponding to the serum 17-beta-estradiol peak. The effect of estrogens and progesterone on the in vitro growth of peripheral BFU-E of healthy women was assayed. Estrogens demonstrated a stimulatory and progesterone an inhibitory effect in total lymphomonocyte cultures, whereas neither hormone had an effect in monocyte-depleted cultures. Prostaglandin E1 (PGE1) which is known to be secreted by monocytes, stimulated the in vitro growth of peripheral BFU-E. These data suggest that estrogens and progesterone could have a role in the in vitro growth of peripheral BFU-E, probably mediated by monocytes.     

In vitro culture of colony forming unit-megakaryocyte (CFU-MK) in fetal alloimmune thrombocytopenia

Summary . Perinatal alloimmune thrombocytopenia (PAITP) causes intracranial haemorrhage in the fetus and neonate. However, the severity of the thromobocytopenia correlates poorly with maternal anti-platelet antibody titres. To test the hypothesis that reduced platelet production contributes to fetal thrombocytopenia in PAITP, maternal sera from three HPA-la-negative mothers whose pregnancies were complicated by anti-HPA-la (two severe cases, one mild case) were added to colony forming unit-megakaryocyte (CFU-MK) cultures from HPA-la positive and negative individuals. Sera from the two severely affected pregnancies containing anti-HPA-la caused 66–100% inhibition of HPA-la-positive fetal and neonatal CFU-MK, whereas CFU-MK from two HPA-la-negative mothers were not inhibited by the anti-HPA-la-containing sera. Maternal serum from the case of mild PAITP caused only mild inhibition of HPA-la-positive cord and adult CFU-MK and did not inhibit HPA-la-positive fetal CFU-MK. Taken together, these findings suggest that reduced megakaryocyte production contributes to fetal thrombocytopenia due to maternal anti-HPA-la antibodies and also that the degree of CFU-MK inhibition correlates with severity of fetal thrombocytopenia.     

Vanadate mimics the effect of stem cell factor on highly purified human erythroid burst-forming units in vitro, but not the effect of erythropoietin.

When orthovanadate, an inhibitor of protein tyrosine phosphatase activity, was added to highly purified human blood erythroid burst-forming units (BFU-E) a marked increase in the number and size of erythroid bursts was evident at an optimum concentration of 4 microM. Because BFU-E are stimulated by stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (EP), this effect could occur through an enhancement of any one of these pathways. However, no effect was observed on human erythroid colony-forming units (CFU-E), indicating that vanadate was not potentiating the effect of EP. The time course of decline in BFU-E, when vanadate was added in vitro on successive days, followed the time course produced by delayed addition of SCF but not IL-3. In addition, vanadate markedly enhanced the effect of an optimal concentration of IL-3, but it could only enhance the effect of SCF when SCF concentrations were less than optimum. These experiments demonstrate that vanadate markedly stimulates the number and size of human BFU-E in vitro and that it mimics the effect of SCF. Vanadate may be acting as a phosphatase inhibitor that potentiates the kinase activity induced by SCF, but elucidation of its specific biochemical effects on these cells awaits further investigation.     

Deficiency of alpha-spectrin synthesis in burst-forming units-erythroid in lethal hereditary spherocytosis

A child diagnosed in utero with hydrops fetalis and a hematocrit of 6.4% was studied to determine the etiology of the anemia. Fetal red blood cells (RBCs) obtained during in utero transfusion had extremely abnormal osmotic fragility. A maternal history of mild autosomal dominant hereditary spherocytosis was present, and the father, who was hematologically normal, had a slightly abnormal osmotic fragility test. The patient was transfusion dependent after birth, with circulating nucleated RBCs but less than 1% reticulocytes. The patient's anemia failed to respond to splenectomy. Because mature RBCs of the patient were not available for study, progenitor-derived erythroblasts grown in culture were investigated. Immunodot assays of the patient's progenitor-derived cells showed a total cell spectrin content 26% of normal. Immunoprecipitation of whole burst-forming units-erythroid-derived cells and solubilized membranes from cells pulse-labeled with 35S-methionine showed a severe deficiency in alpha-spectrin synthesis and a markedly reduced amount of alpha- and beta-spectrin on cell membranes. No alpha-spectrin degradation products were found within the cells or were produced during membrane preparation. Ankyrin content and band 3 synthesis were not different from control. Inheritance of two genetic defects causing severely reduced alpha-spectrin synthesis is proposed as the cause of the lethal anemia, resulting in cell fragmentation during precursor enucleation or during egress from bone marrow.     

Kit ligand in synergy with interleukin-3 amplifies the erythropoietin- independent, globin-synthesizing progeny of normal human burst-forming units-erythroid in suspension cultures: physiologic implications

Although the proliferative effects of hematopoietic cytokines on erythroid progenitors are well known, parameters that influence the initiation of expression of specialized or lineage-restricted genes are not clear. We have studied the acquisition of erythroid-differentiative features from enriched populations of human early erythroid progenitors (burst-forming unit-erythroid [BFUe]) in suspension culture and the influence of several cytokines on this process. In suspension cultures containing no erythropoietin (Epo), we have found that kit ligand (KL) in synergy with interleukin-3 not only increases the proliferation of cells and of progenitors but also consistently amplifies a population of cells that contain globin within 1 week. Our experiments suggest that neither extraneously provided nor endogenously produced Epo is critical for the generation of globin-synthesizing cells. Globin- producing cells generated mostly from late BFUe or pre-CFUe with a CD34- /EP-1+ phenotype in this system do not all express a well-coordinated erythroid program accompanied by heme or glycophorin A expression and most die maintaining an immature state. Therefore, conditions that are responsible for initiation of globin expression in these cells are not sufficient to carry them to terminal maturation. The data point to an expanded target cell population for KL, as they suggest an influence of KL on survival and/or amplification of late erythroid cells previously thought to be influenced only by Epo. Our results in aggregate are of relevance to the physiology of normal erythropoiesis and the role of Epo and KL in the initial stages of lineage-restricted gene expression. In addition, they provide insight into the understanding of anemia in W and Steel mutants in which expansion of the late erythroid progenitor pool, normally dependent on the synergistic action of KL and Epo, is curtailed.     

Characterization of human iodothyronine sulfotransferases

Sulfation is an important pathway of thyroid hormone metabolism that facilitates the degradation of the hormone by the type I iodothyronine deiodinase, but little is known about which human sulfotransferase isoenzymes are involved. We have investigated the sulfation of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2) by human liver and kidney cytosol as well as by recombinant human SULT1A1 and SULT1A3, previously known as phenol-preferring and monoamine-preferring phenol sulfotransferase, respectively. In all cases, the substrate preference was 3,3'-T2 > rT3 > T3 > T4. The apparent Km values of 3,3'-T2 and T3 [at 50 micromol/L 3'-phosphoadenosine-5'-phosphosulfate (PAPS)] were 1.02 and 54.9 micromol/L for liver cytosol, 0.64 and 27.8 micromol/L for kidney cytosol, 0.14 and 29.1 micromol/L for SULT1A1, and 33 and 112 micromol/L for SULT1A3, respectively. The apparent Km of PAPS (at 0.1 micromol/L 3,3'-T2) was 6.0 micromol/L for liver cytosol, 9.0 micromol/L for kidney cytosol, 0.65 micromol/L for SULT1A1, and 2.7 micromol/L for SULT1A3. The sulfation of 3,3'-T2 was inhibited by the other iodothyronines in a concentration-dependent manner. The inhibition profiles of the 3,3'-T2 sulfotransferase activities of liver and kidney cytosol obtained by addition of 10 micromol/L of the various analogs were better correlated with the inhibition profile of SULT1A1 than with that of SULT1A3. These results indicate similar substrate specificities for iodothyronine sulfation by native human liver and kidney sulfotransferases and recombinant SULT1A1 and SULT1A3. Of the latter, SULT1A1 clearly shows the highest affinity for both iodothyronines and PAPS, but it remains to be established whether it is the prominent isoenzyme for sulfation of thyroid hormone in human liver and kidney.     

Characterization of the prolactin receptor in human prostate

The uptake of 125I-labelled human prolactin by subcellular fractions obtained from the human hyperplastic prostate was investigated and the specific binding sites were characterized. The binding was both time- and temperature-dependent with maximum specific uptake achieved after incubation for 20 h at 4 degrees C (dissociation constant = 2.4 x 10(-10) mol/l). The present studies also indicate that the bulk of the specific prolactin binding was confined to the 105 000 and 15 000 g membrane fractions whilst the cytosol and nuclear pellet exhibited a lower capacity for the peptide hormone. Attempts to optimize the binding revealed that pretreatment of the subcellular fractions with 4 mol MgCl2/l quadrupled the binding sites; a similar effect was produced after pretreatment with dextran-coated charcoal, but the changes were less pronounced. Furthermore, our studies suggest that the binding was specific for the human prolactin, with other hormones such as ovine prolactin, human LH, human FSH and human GH showing little or no competition. The addition of magnesium and copper ions to the incubation medium also markedly increased the specific binding, whereas calcium, manganese and EDTA inhibited the prolactin uptake. Freezing and storage of tissue at -70 degrees C did not greatly affect the binding sites. The results suggest that we are clearly dealing with a specific prolactin-binding protein.     

Characterization of the IgG-Fc receptor on human platelets

To determine quantitatively the number and avidity of receptors for the Fc portion of IgG on human platelets, we have measured the binding to platelets of human monomeric monoclonal IgG, and of small covalently crosslinked polymers of IgG1 labeled with 125I. The binding of labeled IgG1 monomers to platelets is too weak to permit quantitation. The binding of dimers or larger polymers of IgG1 is much more avid (greater at 4 degrees C than 37 degrees C), is readily reversible, and is saturable. The number of receptor sites ranges from 400 to 2000 per platelet and the mean equilibrium association constant (Ka) for the binding of dimers at 4 degrees C is 2.2 x 10(7) M-1 +/- 0.9 x 10(7) M- 1. The binding is specific for the Fc portion of IgG, and IgG1 and IgG3 bind to the receptors much more avidly than IgG2 or IgG4. Unlabeled IgG1 dimers are about 7--8-fold more potent in inhibiting binding than are IgG1 monomers, and larger polymers are even more potent than dimers. Thus, the Fc receptors on platelets bind human IgG1 with the same specificity and similar avidity as Fc receptors on polymorphonuclear leukocytes (PMNs), but PMNs have about 300-fold more receptors per unit of surface area than platelets.     

Effects of T cells and monocytes on globin chain synthesis in erythroid bursts cultured from human peripheral blood burst-forming units (BFU-e)

Globin chain synthesis was studied in mature erythroid bursts cultured from the peripheral blood null cells of 21 normal individuals. Although coculture of autologous T-lymphocytes with null cells resulted in a fivefold increase in the yield of erythroid bursts, the proportion of gamma chains synthesized (gamma/gamma + beta) was not significantly different from that seen without T cells. Coculture with autologous monocytes also resulted in increased burst-forming unit (BFU-e) proliferation but the ratio gamma/gamma + beta (0.25 +/- 0.03) was significantly higher than that seen with null cells alone (0.08 +/- 0.006) or with T cells (0.10 +/- 0.008). The relative increase in gamma-chain synthesis correlated with the severity of megaloblastic changes in erythroid progeny of BFU-e (P less than 0.01) but showed no significant relationship to the extent of colony maturation assessed by erythroblast maturity.     

Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit

Thrombopoietin (Tpo), the ligand for the c-Mpl receptor, is a major regulator of megakaryopoiesis. Treatment of mice with Tpo raises the platelet count fourfold within a few days. Conversely, c-mpl knock-out mice have platelet counts that are 15% that of normal. The subunit structure of the c-Mpl receptor is not fully understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind to receptors that use gp130 as a signal transduction subunit. For these reasons, we determined whether gp130 function was required for Tpo-induced signal transduction. Murine marrow cells were cultured in semi-solid media in the presence of Tpo or IL-3, with or without a neutralizing anti-gp130 monoclonal antibody (RX187) or a soluble form of c-Mpl receptor (soluble Mpl) that blocks Tpo bioactivity, and the numbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted on day 5. Murine marrow cells were also cultured in suspension under serum-free conditions for 5 days, and megakaryocyte DNA content was measured by flow cytometry, as an index of nuclear maturation. The addition of RX187 did not block Tpo-induced CFU-Meg colony growth nor CFU-Meg nuclear maturation in suspension culture. However, IL-3-induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth, and partially blocked IL- 3-stimulated CFU-Meg growth. Thus the effects of Tpo on megakaryopoiesis in vitro do not depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp 130 serves as a beta chain for the c-Mpl receptor, as Tpo signalling is unimpaired in the presence of RX187. In contrast, the effects of IL-3 on CFU-Meg growth are mediated in part through Tpo and through gp130-signalling cytokines.     

Characterization of the endocannabinoid system in early human pregnancy

In recent years, it has been demonstrated that high circulating levels of the endogenous cannabinoid anandamide, resulting from low expression of its metabolizing enzyme fatty acid amide hydrolase (FAAH), may contribute to spontaneous miscarriage and poor outcome in women undergoing in vitro fertilization. The site of action of this compound, however, has not been determined. In this study, we examined the distribution of the cannabinoid receptors, CB1 and CB2, and the endocannabinoid-metabolizing enzyme FAAH in first trimester human placenta. Here, we show that FAAH is expressed throughout the human first trimester placenta, in extravillous trophoblast columns, villous cytotrophoblasts, syncytiotrophoblasts, and macrophages. Furthermore, FAAH mRNA levels appear to be regulated during gestation, with levels peaking at 11 wk before declining again. The immune system-associated cannabinoid CB2 receptors were localized only to placental macrophages. Interestingly, the cannabinoid receptor CB1 was not identified in first trimester placenta despite having previously been shown to be present in placental tissues at term. These findings suggest that the placenta may form a barrier preventing maternal-fetal transfer of anandamide and/or modulate local levels of anandamide by regulation of FAAH expression with gestation.     

Characterization of the insulin receptor kinase from human erythrocytes

The insulin receptor from human erythrocytes was studied for receptor-associated tyrosine kinase activity. Receptor phosphorylation was performed by incubation of the receptor with [gamma-32P]ATP and Mn2+ in the presence or absence of insulin, and the phosphorylated receptor was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In Triton X-100-solubilized, and partially purified, receptor preparations, insulin stimulated the phosphorylation of a 95,000 dalton protein in a dose-dependent fashion. Immunoprecipitation with antiinsulin receptor antibodies indicates that this 95,000 dalton protein corresponds to the beta-subunit of the insulin receptor. Phosphoaminoacid analysis revealed that 32P incorporation occurred predominantly on tyrosine residues of the beta-subunit. In addition, the insulin receptor kinase catalyzed the phosphorylation of Histone H2b. Dose response curves for insulin-stimulated phosphorylation of the beta-subunit and Histone H2b were sigmoidal. Both half-maximal effects were observed at 3 X 10(-9) M insulin with maximal effects at 10(-6) M. When insulin binding was examined under the same conditions as phosphorylation, the dose-response curves for receptor occupancy and the kinase activation were nearly superimposable, indicating few or no spare receptors for this response to insulin. Insulin-stimulated receptor phosphorylation was diminished by treatment with N-ethylmaleimide, indicating that the receptor possesses sulfhydryl groups, which are important for its enzyme activity. This study demonstrates that insulin receptor kinase from human erythrocytes shares characteristics which are similar to those found in other classical insulin target cells and tissues.     

Characterization of the human gut microbiome during travelers' diarrhea

Alterations in the gut microbiota are correlated with ailments such as obesity, inflammatory bowel disease, and diarrhea. Up to 60% of individuals traveling from industrialized to developing countries acquire a form of secretory diarrhea known as travelers' diarrhea (TD), and enterotoxigenic Escherichia coli (ETEC) and norovirus (NoV) are the leading causative pathogens. Presumably, TD alters the gut microbiome, however the effect of TD on gut communities has not been studied. We report the first analysis of bacterial gut populations associated with TD. We examined and compared the gut microbiomes of individuals who developed TD associated with ETEC, NoV, or mixed pathogens, and TD with no pathogen identified, to healthy travelers. We observed a signature dysbiotic gut microbiome profile of high Firmicutes:Bacteroidetes ratios in the travelers who developed diarrhea, regardless of etiologic agent or presence of a pathogen. There was no significant difference in α-diversity among travelers. The bacterial composition of the microbiota of the healthy travelers was similar to the diarrheal groups, however the β-diversity of the healthy travelers was significantly different than any pathogen-associated TD group. Further comparison of the healthy traveler microbiota to those from healthy subjects who were part of the Human Microbiome Project also revealed a significantly higher Firmicutes:Bacteriodetes ratio in the healthy travelers and significantly different β-diversity. Thus, the composition of the gut microbiome in healthy, diarrhea-free travelers has characteristics of a dysbiotic gut, suggesting that these alterations could be associated with factors such as travel.     

Characterization of the human gut microbiome during travelers' diarrhea

Alterations in the gut microbiota are correlated with ailments such as obesity, inflammatory bowel disease, and diarrhea. Up to 60% of individuals traveling from industrialized to developing countries acquire a form of secretory diarrhea known as travelers'' diarrhea (TD), and enterotoxigenic Escherichia coli (ETEC) and norovirus (NoV) are the leading causative pathogens. Presumably, TD alters the gut microbiome, however the effect of TD on gut communities has not been studied. We report the first analysis of bacterial gut populations associated with TD. We examined and compared the gut microbiomes of individuals who developed TD associated with ETEC, NoV, or mixed pathogens, and TD with no pathogen identified, to healthy travelers. We observed a signature dysbiotic gut microbiome profile of high Firmicutes:Bacteroidetes ratios in the travelers who developed diarrhea, regardless of etiologic agent or presence of a pathogen. There was no significant difference in α-diversity among travelers. The bacterial composition of the microbiota of the healthy travelers was similar to the diarrheal groups, however the β-diversity of the healthy travelers was significantly different than any pathogen-associated TD group. Further comparison of the healthy traveler microbiota to those from healthy subjects who were part of the Human Microbiome Project also revealed a significantly higher Firmicutes:Bacteriodetes ratio in the healthy travelers and significantly different β-diversity. Thus, the composition of the gut microbiome in healthy, diarrhea-free travelers has characteristics of a dysbiotic gut, suggesting that these alterations could be associated with factors such as travel.