Is interleukin 6 the physiological regulator of thrombopoiesis?

Interleukin 6 (IL-6) is a multifunctional cytokine that also influences megakaryocyte (MK) development. To delineate the relationship between IL-6 and thrombopoietin (TPO), the putative physiological regulator of MK maturation, serum IL-6 levels and platelet counts were correlated in various clinical disorders. IL-6 was measured by a [3H] thymidine incorporation assay using the IL-6-dependent B9 cell line; 1 U is approximately equal to 1 pg/ml of a recombinant (r)IL-6 standard. Specificity of the assay was confirmed by neutralizing rIL-6 and selected sera containing IL-6 activity with anti-IL-6 antibody. Samples (n = 120) were obtained from normal individuals and patients with leukemia, myeloproliferative and rheumatologic disorders, solid tumors, and after bone marrow transplantation and chemotherapy. Patients were also grouped as to whether they had an ongoing inflammatory process, that is, an active infection, solid tumor malignancy, or rheumatological disorder. Serum IL-6 levels were 4.6 +/- 1.4 U/ml for normal individuals and ranged up to 14.8 x baseline; moderate increases (greater than 2 x normal) were found in 21.5% of all patients. Whereas only 39% of thrombocytopenic sera (less than 150,000 platelets) had elevated IL-6 levels, 91% of these sera were from patients with an ongoing inflammatory process. Only 29% of the thrombocytotic sera (greater than 400,000) had elevated IL-6 levels, but 86% of these sera were from patients suffering from concurrent inflammation. Overall, 80% of all patients with elevated serum IL-6 had definitive ongoing inflammatory processes. There was no inverse relationship between platelet numbers and IL-6 levels. Thus, the idea that IL-6 is TPO appears doubtful. However, production of IL-6 during inflammation may result in increased platelet numbers and account for the secondary thrombocytosis observed in some patients.

Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes.

We tested the hypothesis that anti-placental folate receptor (PFR) antiserum-mediated effects on hematopoietic progenitor cells in vitro of increased cell proliferation and megaloblastic morphology were independent responses. We determined that (a) purified IgG from anti-PFR antiserum reacted with purified apo- and holo-PFR and specifically immunoprecipitated a single (44-kD) iodinated moiety on cell surfaces of low density mononuclear cells (LDMNC); (b) when retained in culture during in vitro hematopoiesis, anti-PFR IgG (in contrast to PFR-neutralized anti-PFR IgG and nonimmune IgG) consistently led to increased cloning efficiency of colony forming unit-erythroid (CFU-E), burst forming unit-E (BFU-E), CFU-granulocyte macrophage (CFU-GM), and CFU-GEM megakaryocyte (CFU-GEMM), and objectively defined megaloblastic changes in orthochromatic normoblasts from CFU-E- and BFU-E-derived colonies; (c) when anti-PFR antiserum was removed after initial (less than 1 h) incubation with LDMNC, a cell proliferation response was induced, but megaloblastic changes were not evident. (d) Conversely, delay at 4 degrees C for 24 h before long-term plating with antiserum resulted in megaloblastosis without increased cell proliferation; (e) however, 500-fold molar excess extracellular folate concentrations completely abrogated the expected anti-PFR antiserum-induced megaloblastic changes, without altering cell proliferative responses. Thus, although cell proliferative and megaloblastic changes are induced after short-term and prolonged interaction of antibody with folate receptors on hematopoietic progenitors, respectively, they are independent effects.

Effects of thrombocytopoiesis-stimulating factor on terminal cytoplasmic maturation of human megakaryocytes.

Aplastic anemia serum (AAS) contains humoral factors that alter both proliferation and maturation of human megakaryocytes (MK). The ability of AAS to augment MK colony formation (colony-forming unit, CFU-MK) was neutralized by an antiserum against MK colony-stimulating factor (MK-CSF), a glycoprotein isolated from AAS. The adsorbed AAS still retained the ability to accelerate cytoplasmic maturation of recognizable MK. Similar experiments were done with thrombocytopoiesis-stimulating factor (TSF) and an anti-TSF antiserum to further define the activity in AAS responsible for accelerating cytoplasmic maturation. Bone marrow fractions enriched for recognizable human MK, but devoid of CFU-MK, were obtained by centrifugal elutriation and placed in short-term liquid cultures. MK progressed through identifiable maturation stages (1-4) more quickly in the presence of either TSF or AAS. TSF slightly enhanced the cloning efficiencies of CFU-MK, but did not alter the number of MK in individual colonies derived from non-adherent, low-density, T-cell-depleted bone marrow. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and crude AAS substantially augmented both MK colony formation and cells per colony. TSF also doubled the percent 35S incorporation into platelets of immunothrombocythemic mice, but stimulation was completely abolished by anti-TSF. Anti-TSF antiserum was then used to analyze the promotion of MK colony formation by cytokines. Cloning efficiencies of CFU-MK were reduced to baseline values when TSF was pretreated with anti-TSF; however, the MK colony-stimulating activity (MK-CSA) of GM-CSF, IL-3, or AAS was not altered by adsorption with anti-TSF. In contrast, the cytoplasmic maturation of recognizable MK was slower, and fewer mature stage-4 cells were present at days 1-3 in AAS adsorbed with anti-TSF than MK cultured in AAS treated with normal rabbit serum or untreated AAS. Therefore, TSF appears to be a major factor in AAS that accelerates terminal maturation of human MK. TSF primarily affects megakaryocytopoiesis by promoting MK maturation rather than enhancing CFU-MK proliferation.

Characterization of the human burst-forming unit-megakaryocyte.

Two classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit-megakaryocyte (BFU-MK)-derived colonies appeared after 21 days. CFU-MK-derived colonies were primarily unifocal and composed of 11.6 +/- 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 +/- 0.4 foci and 108.6 +/- 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA-1, CD34, clone My10) and a major histocompatibility class II locus, HLA-DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogenous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony-stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1 alpha, IL-4, and purified thrombocytopoiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.

Serum from patients with various thrombopoietic disorders alters terminal cytoplasmic maturation of human megakaryocytes in vitro.

Human bone marrow was depleted of progenitors (CFU-MK), but enriched for recognizable megakaryocytes (MK), and placed in cultures with serum from either normal donors (NABS) or patients with primary (PTS) or secondary (STS) thrombocytosis, autoimmune thrombocytopenia (ATS) or aplastic anemia (AAS). Mean MK diameters shifted during the 3-4 days of incubation. Endomitotic figure were visible and mean ploidy increased slightly during cytoplasmic maturation, where decreases in immature cells (stages 1 and 2) were accompanied by increases in the mature MK (stages 3 and 4). Cytoplasmic maturation was faster in AAS, ATS and STS than PTS or NABS; mean size and ploidy were similar in all cultures. Recognizable MK were not forced to undergo additional endoreduplication in response to stimulation. Only AAS augmented MK colony formation, which indicated that at least two humoral factors can regulate megakaryocytopoiesis at separate levels, the progenitors and morphologically recognizable MK.

Analysis of phorbol ester stimulated human megakaryocyte development.

Megakaryocytes are relatively rare components of human bone marrow, making the study of their maturation difficult. Phorbol esters can act as differentiating agents in a number of cell systems including murine megakaryocytes. We report the effects of phorbol esters on the previously described long-term human megakaryocytic leukemia cell culture, EST-IU. While two nontransforming phorbols fail to affect these cells, the transforming phorbol 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a phenotype with characteristics of more mature megakaryocytes in a dose-related manner. This phenotype includes an increased adherence to untreated plastic or glass, polyploidization, an increase in cell size, and increased expression of both platelet glycoproteins and factor VIII-related antigen. Two-color flow cytometric analysis allowed simultaneous determinations of DNA content and the expression of surface membrane antigens or alpha-granule constituents, providing evidence that nuclear, membrane, and cytoplasmic maturation occur in parallel in this cellular system. TPA-induced maturation of EST-IU cells provides an important new cellular model for the further study of human megakaryocyte development.

Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor.

Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with 125I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with 125I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor with an approximate Ka of 10(8)-10(9) M-1. CFU-GM expressed an average of 3783 +/- 4 receptors per cell; normal bone marrow cells, 1518 +/- 242 receptors per cell; and peritoneal exudate cells, 2025 +/- 216 receptors per cell. Affinity crosslinking studies demonstrated that 125I-labeled GM-CSF bound specifically to two species of Mr 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The Mr 70,000 species is thought to be a proteolytic fragment of the intact Mr 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

Interactions between purified murine colony-stimulating factors (natural CSF-1, recombinant GM-CSF, and recombinant IL-3) on the in vitro proliferation of purified murine granulocyte-macrophage progenitor cells.

Purified preparations of natural CSF-1 (nCSF-1), recombinant GM-CSF (rGM-CSF), and recombinant IL-3 (rIL-3), alone and in combination, were investigated for their proliferative effects on highly enriched murine granulocyte-macrophage progenitor cells (CFU-GM). These CFU-GM had cloning efficiencies of 62%-95% in the presence of 10% (vol/vol) pokeweed mitogen-stimulated spleen cell-conditioned medium, and contained few, if any (less than or equal to 3%), contaminating morphologically recognizable monocytes or lymphocytes. The combination of low concentrations of nCSF-1 plus rIL-3, or nCSF-1 plus rGM-CSF, increased colony number greater than additively compared to the sum of colony formation with each factor alone, whereas total aggregate (colony plus cluster) number increased additively. At plateau concentrations, the previous CSF combinations increased colony number additively. Colony size was increased when nCSF-1 plus either rGM-CSF or rIL-3 were added simultaneously at either low or plateau concentrations, when compared to the size of colonies with any of the CSFs alone. Addition of rGM-CSF plus rIL-3 demonstrated no cooperative proliferative effect on either colony number or size. It is likely that these effects are mediated at the progenitor cell level and do not require accessory cell participation.

Effects of megakaryocyte colony-stimulating factor on terminal cytoplasmic maturation of human megakaryocytes.

Suspensions of enriched human megakaryocytes (MK) devoid of MK progenitors (CFU-MK) undergo complete cytoplasmic maturation in vitro. MK were cultured in the presence of normal human AB serum (NABS) to mimic "normal" development. The rate of maturation was not statistically altered by higher concentrations (10%-20%-30%) of NABS, or by the addition of bovine serum albumin (1.5%-3.0%), but was accelerated in the presence of aplastic anemia serum (AAS). Sera from eight different patients with severe aplastic anemia were effective in accelerating terminal differentiation. MK-CSF, a glycoprotein isolated from AAS, specifically augments MK colony formation by two- to sixfold. Similar doses of MK-CSF were ineffective in altering terminal cytoplasmic maturation. Anti-MK-CSF, a polyclonal antibody prepared against purified MK-CSF, neutralizes the ability of both purified MK-CSF and AAS to promote MK colony formation. However, AAS adsorbed with anti-MK-CSF still retained its ability to accelerate terminal differentiation. Apparently, AAS contains at least two separate humoral factors, which can regulate in vitro human megakaryocytopoiesis: MK-CSF, which stimulates proliferation of the progenitors (CFU-MK), and a maturation factor, which accelerates cytoplasmic maturation of morphologically recognizable megakaryocytes.

Purification of murine bone-marrow-derived granulocyte-macrophage colony-forming cells.

Previous attempts to purify progenitor cells that form colonies and clusters of granulocytes and/or macrophages (CFU-GM) from adult murine bone marrow have had limited success because of the paucity of these cells. In the present paper we report studies with a rapid, reproducible method involving pretreatment of mice, three days prior to sacrifice, with 200 mg/kg of Cytoxan (cyclophosphamide), density separation on Ficoll-Hypaque, and counterflow centrifugal elutriation, that yielded highly enriched populations of CFU-GM. The peak CFU-GM-containing fraction (FR-28) eluted at a flow rate of 28 ml/min and contained very little contamination by other in vitro colony-forming cells (BFU-E, CFU-GEMM, CFU-MK). FR-28 contained 0.54% +/- 0.30% (16 experiments) of the unfractionated post-Cytoxan bone marrow nucleated cells and lacked significant contamination by lymphocytes and monocytes. The mean CFU-GM cloning efficiency of FR-28 was 44% +/- 9% in agar (11 experiments) and 75% +/- 10% in agarose (nine experiments). CFU-GM from FR-28 demonstrated linear plating characteristics even at very low cell density (25 cells), and formed colonies and clusters of granulocytes, macrophages, or both in the same proportions as did unfractionated post-Cytoxan or untreated bone marrow. Approximately 10% (assuming a seeding efficiency of 10%) of FR-28 cells were in vivo spleen colony-forming cells (CFU-S) measured at day 12. These results represent the highest degree of purity (up to 94%) of CFU-GM thus far reported and should prove useful in studies of this cell population.

Terminal cytoplasmic maturation of human megakaryocytes in vitro.

Several studies suggest that serum factors (thrombopoietins) regulate thrombopoiesis by altering the number, size, ploidy, and maturation rate of megakaryocytes (MK). Various in vivo systems have been used to quantitate these events. In this study, an in vitro system was developed to monitor terminal cytoplasmic maturation of isolated human MK. MK enriched by elutriation, which eliminated the MK progenitors, were suspended in culture with serum from either normal donors (NABS) or patients with aplastic anemia (AAS). In cultures composed of small platelet glycoprotein-positive mononuclear cells and morphologically immature MK, development was characterized by sequential shifts in MK through morphologically recognizable maturation stages I, II, III, and IV over eight days of incubation (I and II only; then I, II, III; II, III, IV; III and IV; then IV only). Platelet formation coincided with the appearances of stage IV cells. Cultures composed of a mixture of all stages followed a similar maturation sequence, only at an accelerated rate. AAS resulted in the more rapid appearances of the mature cells in either system. This study indicates that human MK can undergo terminal cytoplasmic maturation in vitro, and that altering culture conditions (AAS for NABS) can accelerate the rate of maturation. Three major events occur during megakaryocytopoiesis: proliferation of the progenitor cells, polyploidization, and cytoplasmic maturation. Now it is possible to study the terminal steps of differentiation independent of proliferative events.

Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X 10(-8) M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethane-sulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

Isolation of human megakaryocytes by density centrifugation and counterflow centrigual elutriation.

Density centrifugation and counterflow centrifugal elutriation were utilized to prepare enriched fractions of megakaryocytes from human bone marrow aspirates. This separation method enriched megakaryocytes in initial marrow aspirates by 116- to 463-fold. Approximately 63% of megakaryocytes were recovered from the initial samples, composing 18.7% of the nucleated cells in the final preparations. Mean megakaryocyte diameters of 51.6 micron and 33.8 micron were obtained from fixed and unfixed cellular specimens, respectively. Smaller platelet glycoprotein-positive mononuclear cells with a mean diameter of 20.5 micron were found in the highest concentrations in this final fraction. These cells presumably represent immature megakaryocytic forms. Counterflow centrifugal elutriation provides a means of isolating enriched populations of marrow megakaryocytes. This accessibility to viable populations of human megakaryocytes will allow additional investigation of the terminal events of megakaryocyte development.

Eimeria strangfordensis sp. n., Eimeria barleyi sp. n., and eimeria antonellii sp. n. from the eastern woodrat (Neotoma floridana) from Pennsylvania.

Three new eimerian species are described from the eastern woodrat, Neotoma floridana, in Pennsylvania. Sporulated oocysts of Eimeria strangfordensis sp. n. are broadly ellipsoidal, 25.0 to 31.7 (29.1) x 20.5 to 26.2 (24.1) with subspherical to ovoid sporocysts, 10.6 to 17.2 (13.4) x 7.4 to 16.4 (10.9). Oocyst wall is thick, rough, pitted, and two layered with no micropyle. Oocyst and sporocyst residuum and Stieda body are present; polar granule is absent. Sporulated oocysts of Eimeria barleyi sp. n. are broadly ellipsoidal, 18.0 to 24.8 (21.7) x 16.2 to 21.2 (18.4) with ovoid sporocysts, 9.8 to 11.9 (10.7) x 7.4 to 8.7 (8.0). Oocyst wall is rough, pitted, and two layered with micropyle. Polar granule, Stieda and substiedal bodies, and sporocyst residuum are present; oocyst residuum is absent. Sporulated oocysts of Eimeria antonellii sp. n. are spherical to ellipsoidal, 14.8 to 23.8 (18.2) x 11.9 to 20.5 (14.8) with ovoid sporocysts, 7.4 to 9.8 (8.5) x 4.5 to 6.6 (5.6). Oocyst wall is smooth, and single layered with no micropyle. Polar granule, oocyst and sporocyst residuum, and Stieda body are present.

Eimeria clethrionomysis sp. n., Eimeria gallatii sp. n., Eimeria pileata sp. n. and Eimeria marconii sp. n. from the red-backed vole Clethrionomys gapperi Vigors, from Pennsylvania.

Four new eimerian species are described from red-backed voles, Clethrionomys gapperi in Pennsylvania. Sporulated oocysts of Eimeria clethrionomyis sp. n. are ellipsoidal, 18.8 (16.5-21.5) x 14.9 (14.0-16.5) with elongate, ovoid sporocysts, 10.6 (9.5-12.0) x6.1 (5.5-7.0). The oocyst wall is smooth, with 2 layers, and thins, with terminal cap at one or both ends. Polar granules, dark Stieda body and sporocyst residuum are present. The oocyst residuum is absent. Sporulated oocysts of Eimeria gallatii sp. n. are ellipsoidal, 27.7 (21-32) x 19.3 (17-24) with ovoid sporocysts, 13.5 (12-15) x 8.8 (8-10). The oocyst wall is smooth, 2-layered, with a micropyle and thin wall at the end opposite the micropyle. Polar granules, Stieda body and sporocyst residuum are present. The oocyst residuum is atypical, of cobwebby material. Sporulated oocysts of Eimeria pileata sp. n. are subspherical to spherical, 25.2 (20.5-29.5) x 22.5 (19.5-25.5) with ellipsoidal sporocysts, 13.4(10.5-15.0) x 8.4 (7.5-9.5). The oocyst wall is rough, pitted, striated, 2-layered, with no micropyle. Polar granules, oocyst and sporocyst residuum, Stieda body and stiedal cap are present. Sporulated oocysts of Eimeria marconii sp. n. are ellipsoidal, 13.0 (10.5-15-0) x 10.6 (9.5-12.0) with elongate, ovoid sporocysts, 7.7 (7.0-8.5) x 4.2 (3.0-4.5). The oocyst wall is smooth, single-layered, with no micropyle. Polar granules, dark Stiedal body and sporocyst residuum are present. There is no oocyst residuum.