Megakaryocyte Colony-stimulating Activity Research Articles

Murine megakaryocyte colony stimulating factor: Its relationship to interleukin 3

A protocol for the partial purification of murine megakaryocyte colony stimulating factor from WEHI-3 cell conditioned medium is described. The procedure involving separation by anion exchange chromatography and molecular sieving on Sephadex G100, gave a 260-fold purification over starting material, as determined by in-vitro megakaryocyte colony formation. Fifty percent of maximum colony numbers were obtained at a dose of 1 μg protein/ml. Electrophoretic analysis of the partially purified preparation followed by silver nitrate staining revealed four major protein bands with apparent molecular weights of 67 Kd, 43 Kd, 38 Kd and 28 Kd. All megakaryocyte colony stimulating activity and interleukin 3 activity was found in the 22–25 Kd fraction from a preparative SDS-polyacrylamide gel using non-reducing conditions, a molecular weight range that coincided with the 28-Kd molecule obtained when reducing conditions were employed.

Role of phorbol diesters in in vitro murine megakaryocyte colony formation.

In vitro megakaryocyte differentiation is regulated by two activities: a megakaryocyte colony-stimulating activity (Mk-CSA), which is required for proliferation, and an auxiliary factor, megakaryocyte potentiating activity, which plays a role in later differentiation events. Tumor-promoting phorbol esters alter many cellular differentiation-related events. Thus, it was hypothesized that phorbol esters may bring about megakaryocyte differentiation in vitro. 4 beta-Phorbol 12-myristate 13-acetate (PMA), when co-cultured with a source of Mk-CSA, stimulated a threefold increase in colony numbers. Co-culture of PMA and megakaryocyte potentiator activity did not stimulate colony formation, thus eliminating any action of PMA as an Mk-CSA. The direct effect of PMA on the formation of megakaryocyte colonies was established by (a) the function of PMA as a megakaryocyte potentiator in serum-free experiments, (b) the ability of PMA to stimulate megakaryocyte colony formation using bone marrow cells depleted of populations known to produce potentiating activity, (c) the inability of bone marrow adherent cells previously treated with phorbol, 12,13-dibutyrate (PDBu) to augment megakaryocyte colony formation, and (d) the ability of PMA to induce the growth of immature megakaryocytes into large single megakaryocytes. Structure:activity experiments resulted in equivalent activities for PMA and PDBu, whereas the nontumor promoter phorbol 12,13-diacetate and phorbol itself lacked activity. The observations in this study indicate that phorbol esters can bring about megakaryocyte differentiation, and during colony formation, can induce effects identical to those brought about by biological sources of megakaryocyte potentiator activity.

Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

Hematopoietic Regulatory Factors Produced in Long-Term Murine Bone Marrow Cultures and the Effect of In Vitro Irradiation

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M-CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M-CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to "fat" cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

In vitro studies of megakaryocytopoiesis in thrombocytotic disorders of man

Increased numbers of bone marrow megakaryocytes and thrombocytosis are frequently observed in patients with myeloproliferative disorders (MPD). Increased marrow megakaryocytes and thrombocytosis are also noted in a variety of inflammatory and neoplastic disease leading to the phenomenon of reactive thrombocytosis (RT). The pathogenesis of this finding remains incompletely understood. Using methodology developed in our laboratory, we investigated the causative role of megakaryocyte colony-stimulating activity (Meg-CSA) in generating this phenomenon. We also examined the cloning efficiency of colony-forming units-megakaryocyte (CFU-M) and their responsiveness to an exogenous source of Meg-CSA in patients with these diseases. The results of our investigations suggest that: (1) increased production of Meg-CSA is not responsible for the megakaryocyte hyperplasia and thrombocytosis noted in these patients; (2) the intrinsic stem cell defect described in MPD appears to affect the CFU-M of these patients as well, resulting in an effective expansion of the CFU-M pool with consequent megakaryocyte hyperplasia and thrombocytosis; (3) the CFU-M of patients with MPD remain responsive to an exogenous source of Meg-CSA, suggesting that this megakaryocyte hyperplasia may not be entirely autonomous of its effects; and (4) the CFU-M pool in RT is normal both in size and responsiveness to Meg-CSA, suggesting that in these disorders, the stimulus leading to megakaryocyte hyperplasia and thrombocytosis is active at the post-CFU-M level of megakaryocyte differentiation.

In Vitro Studies of Megakaryocytopoiesis in Thrombocytotic Disorders of Man

Increased numbers of bone marrow megakaryocytes and thrombocytosis are frequently observed in patients with myeloproliferative disorders (MPD). Increased marrow megakaryocytes and thrombocytosis are also noted in a variety of inflammatory and neoplastic disease leading to the phenomenon of reactive thrombocytosis (RT). The pathogenesis of this finding remains incompletely understood. Using methodology developed in our laboratory, we investigated the causative role of megakaryocyte colony-stimulating activity (Meg-CSA) in generating this phenomenon. We also examined the cloning efficiency of colony-forming units-megakaryocyte (CFU-M) and their responsiveness to an exogenous source of Meg-CSA in patients with these diseases. The results of our investigations suggest that: (1) increased production of Meg-CSA is not responsible for the megakaryocyte hyperplasia and thrombocytosis noted in these patients; (2) the intrinsic stem cell defect described in MPD appears to affect the CFU-M of these patients as well, resulting in an effective expansion of the CFU-M pool with consequent megakaryocyte hyperplasia and thrombocytosis; (3) the CFU-M of patients with MPD remain responsive to an exogenous source of Meg-CSA, suggesting that this megakaryocyte hyperplasia may not be entirely autonomous of its effects; and (4) the CFU-M pool in RT is normal both in size and responsiveness to Meg-CSA, suggesting that in these disorders, the stimulus leading to megakaryocyte hyperplasia and thrombocytosis is active at the post-CFU-M level of megakaryocyte differentiation.

Acquired Amegakaryocytic Thrombocytopenic Purpura: A Syndrome of Diverse Etiologies

AbstractThe possible pathogenetic mechanisms responsible for the production of acquired amegakaryocytic thrombocytopenic purpura (AATP) were investigated in a group of patients with this disorder. Absence of megakaryocytes and small platelet glycoprotein-bearing mononuclear cells, as determined by immunochemical staining of patient marrows with an antisera to platelet glycoproteins, suggested that the defect in AATP occurs in an early progenitor cell of the megakaryocytic lineage. Using an in vitro clonal assay system for megakaryocytic progenitor cells or megakaryocyte colony-forming units (CFU-M), the proliferative capacity of AATP marrow cells was then assessed. Bone marrow cells from three of four patients formed virtually no megakaryocyte colonies, suggesting that in these individuals the AATP was due to an intrinsic defect in the CFU-M. Bone marrow cells from an additional patient, however, formed 12% of the normal numbers of colonies, providing evidence for at least partial integrity of the CFU-M compartment in this patient. Serum specimens from all six patients were screened for their capacity to alter in vitro megakaryocyte colony formation. Five of six sera enhanced colony formation in a stepwise fashion, demonstrating appropriately elevated levels of megakaryocyte colony-stimulating activity. The serum of the patient with partial integrity of the CFU-M compartment, however, stimulated colony formation only at low concentrations. At higher concentrations, this patient’s serum actually inhibited the number of colonies cloned, suggesting the presence of a humoral inhibitor to CFU-M. Serum samples from all patients were further screened for such humoral inhibitors of megakaryocyte colony formation using a cytotoxicity assay. The patient whose serum was inhibitory to CFU-M at high concentrations was indeed found to have a complement-dependent serum IgG inhibitor that was cytotoxic to allogeneic and autologous marrow CFU-M but did not alter erythroid colony formation. These studies suggest that AATP can be due to at least two mechanisms: either an intrinsic defect at the level of the CFU-M or a circulating cytotoxic autoantibody directed against the CFU-M.

Assay of an activity in the serum of patients with disorders of thrombopoiesis that stimulates formation of megakaryocytic colonies.

We have recently described an in vitro clonal assay system for human megakaryocyte-progenitor cells or megakaryocytic colony-forming units (CFU-M). Serum specimens from patients with quantitative platelet disorders were screened for the capacity to alter in vitro megakaryocyte-colony formation. Serum from 11 patients with hypomegakaryocytic thrombocytopenia significantly enhanced the formation of CFU-M-derived colonies (200 to 1840 per cent). Neither serum from eight patients with thrombocytopenia and normal or increased numbers of marrow megakaryocytes nor serum from 11 patients with thrombocytosis altered colony formation. This stimulatory activity has been termed megakaryocytic-colony-stimulating activity (Meg-CSA). The number of megakaryocytic colonies formed was directly proportional to the quantity of stimulatory serum added. Meg-CSA levels appeared to be inversely related to marrow megakaryocyte numbers. The variations in Meg-CSA levels that were detected in different disease states suggest that alterations in the production of this stem-cell regulator have physiologic importance.