Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia.

Hematopoietic progenitor cells in 2 myeloproliferative disorders, juvenile chronic myelomonocytic leukemia and polycythemia vera, are known to be hypersensitive to cytokines that control normal progenitor cell proliferation, differentiation, and survival in their respective granulocyte/macrophage and erythroid lineages. Because thrombopoietin controls these functions in the normal megakaryocytic lineage, we asked the question: Are megakaryocytic progenitor cells in the myeloproliferative disorder essential thrombocythemia (ET) hypersensitive to thrombopoietin? Peripheral blood mononuclear cells from patients with ET, or secondary (reactive) thrombocytosis (2 degrees T), or healthy volunteers were grown in strictly serum-free agarose culture containing interleukin 3 (IL-3) and all-trans-retinoic acid, with various concentrations of PEG-rHu megakaryocyte growth and development factor (MGDF). The concentration of cytokine at half-maximum colony number served as a measure of progenitor cell sensitivity. Hypersensitivity to PEG-rHu MGDF was found in circulating progenitors from 18 of 20 (90%) informative patients with presumptive diagnosis ET, 1 of 8 (12.5%) 2 degrees T patients, and none of the 22 healthy volunteers. Median MGDF sensitivity ratio in ET patients was approximately 53 times greater than in the controls. This hypersensitivity, which was also directed to rHu thrombopoietin, was highly specific with respect to cytokine, disease, and cell lineage. We propose that, despite their single pluripotential cell origin, the different clinicopathologic phenotypes in different chronic myeloproliferative disorders are determined by lineage-restricted hypersensitivities of hematopoietic progenitor cells to endogenous cytokines. This work emphasizes the importance of stringent serum-free conditions for revealing true sensitivities to cytokines. The findings also offer a basis for evolving a positive test for ET, a diagnosis now made essentially by exclusion.

Increased basal and induced tyrosine phosphorylation of the insulin-like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera.

We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum-free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

Circulating erythroid progenitors in polycythemia vera are hypersensitive to insulin-like growth factor-1 in vitro: studies in an improved serum-free medium.

We have investigated the question of erythropoietin (Epo) hypersensitivity versus Epo independence as the basis for the endogenous erythroid bursts (EEBs) that develop in cultures without added Epo from hematopoietic cells of polycythemia vera (PV) patients. Using an improved serum-free (SF) medium containing interleukin (IL)-3, but no insulin-like growth factor-1 (IGF-1), and devoid of contaminants that influence erythropoiesis, we compared circulating normal and PV early erythroid progenitors (BFU-E) with respect to their responses in vitro to recombinant human (rHu) Epo. Cultures were seeded with Ficoll-Hypaque density-separated peripheral blood (PB) mononuclear cells (MNCs), and erythroid bursts, together with their component colonies of > or = 50 cells, were scored in situ at 13 to 16 days of culture. The Epo dose-response curve of BFU-E from PV patients was found to be statistically indistinguishable from that of normal subjects. This observation provides compelling evidence against the Epo-hypersensitivity hypothesis. In the complete SF medium minus Epo, the sensitivity of BFU-E to IGF-1 was much greater in PV than in normals, the dose-response curve being shifted to the left by at least 2 orders of magnitude. These data show that the erythroid progenitor cell response in PV is hypersensitive to IGF-1, and independent of Epo. The data also emphasize the importance of truly SF medium conditions for assessment of progenitor cell sensitivities to recombinant growth factors. Depletion of adherent cells totally prevented erythroid burst formation by normal circulating progenitors, but did not prevent the hypersensitive response to IGF-1 of such cells from PV patients. Hence, again unlike its normal counterpart, the progenitor cell response in PV appears to be independent of adherent cell control.

Retinyl acetate and all-trans-retinoic acid enhance erythroid colony formation in vitro by circulating human progenitors in an improved serum-free medium.

Retinyl acetate (RA) dramatically increased the production of early (d16) erythroid colonies in vitro by circulating human progenitor cells growing in an improved serum-free (SF) medium. In the absence of either erythropoietin (Epo) or insulin-like growth factor I (IGF-I), RA alone was able to induce the hemoglobinization of cells in these erythroid colonies. RA synergized with Epo or with IGF-I to yield increased numbers of well-hemoglobinized early colonies. In the presence of defined burst promoting activity (BPA) provided by recombinant human interleukin 3 (rHuIL-3) and hemin, RA and all-trans-retinoic acid (ATRA) were identical with respect to their differentiation-inducing function for early erythroid colonies. ATRA increased the number of these colonies in a concentration-dependent manner, with maximal stimulation (3.5-fold) occurring at 30 nM in the presence of 5.5 ng/ml IL-3, 0.1 mM hemin, 3.0 U/ml Epo and 30 nM IGF-I. This appears to be the first demonstration of erythropoietic activity of two metabolic derivatives of vitamin A in SF medium.

Production of erythropoietic bursts by progenitor cells from adult human peripheral blood in an improved serum-free medium: role of insulinlike growth factor 1.

Several culture media for the growth of human circulating erythroid burst-forming units (BFU-E) that have been claimed to be "serum-free" ("SF") have actually included albumin preparations known to be contaminated with an undefined burst-promoting activity (BPA); a BPA has also been found in the preparations of other "SF" medium components. This has precluded reliable investigation of the growth factor (GF) requirements of these progenitors. Using a defatted, BPA-free bovine serum albumin (BSA) and the recombinant human growth factors (GFs) erythropoietin (rHu Epo), insulinlike growth factor 1 (rHu IGF-1), and interleukin-3 (rHu IL-3), we have developed an improved serum-free (SF) medium for the production of erythroid bursts from normal adult human peripheral blood mononuclear cells (PBMNC), which requires both hemin and retinyl acetate for its optimal performance. In the presence of BSA without IL-3 or Epo, no burst or colony formation was observed. With IL-3 and Epo alone, only a small number of day 14 erythroid colonies was obtained (12 +/- 1/10(5) PBMNC). Addition of hemin (0.1 mmol/L) allowed the direct scoring of day 14 hemoglobinized colonies and increased their number sevenfold (86 +/- 5). Inclusion of retinyl acetate at physiologic concentrations further augmented the number of colonies threefold to fourfold. Under these apparently optimal conditions, we found that IGF-I could entirely replace Epo. However, IGF-I required a 100-fold higher molar concentration than that of Epo to reach maximal stimulation. The combined effect of Epo and IGF-I was found to be less than the sum of their individual effects, suggesting an overlap in the sensitivities of erythroid progenitors to these GFs. The colony-forming efficiencies of erythroid progenitors in the improved SF medium was very high: 700 single, day 14 erythroid colonies/10(5) PB MNC (at 0.25 mmol/L hemin) distributed as 126 clusters (bursts), with a mean of 5.6 component colonies per burst. These findings show that IGF-I has an Epo-like activity that targets circulating early erythroid progenitors or their progeny, providing strong evidence for the existence of an Epo-independent pathway for normal human adult erythropoiesis, possibly operative when Epo levels are low.

Specific binding of 125I-rErythropoietin to Friend polycythemia virus-transformed erythroleukemia cells purified by centrifugal elutriation.

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. These belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.