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The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets
N Debili1, F Wendling, D Cosman
1INSERM U362, Institut Gustave Roussy, Villejuif, France.
Abstract:
The Mpl receptor (Mpl-R) is a cytokine receptor belonging to the hematopoietin receptor superfamily for which a ligand has been recently characterized. To study the lineage distribution of Mpl-R in normal hematopoietic cells, we developed a monoclonal antibody (designated M1 MoAb) by immunizing mice with a soluble form of the human Mpl-R protein. With few exceptions, Mpl-R was detected by indirect immunofluorescent analysis on all human leukemic hematopoietic cell lines with pluripotential and megakaryocytic phenotypes, but not on other cell lines. By immunoprecipitation and immunoblotting, M1 MoAb recognized a band at 82 to 84 kD corresponding to the expected size of the glycosylated receptor. Among normal hematopoietic cells, M1 MoAb strongly stained megakaryocytes (MK) and Mpl-R was detected on platelets by indirect immunofluorescence staining or immunoblotting. On purified CD34+ cells, less than 2% of the population was stained, but the labeling was weak and just above the threshold of detection. However, dual-labeling with the M1 and antiplatelet glycoprotein MoAbs showed that most Mpl-R+/CD34+ cells coexpressed CD41a, CD61, or CD42a, suggesting that cell surface appearance of Mpl-R and platelet glycoproteins could be coordinated. M1-positive and M1-negative subsets were sorted from purified CD34+ cell populations. Colony assays showed that the absolute number of hematopoietic progenitors was extremely low and no primitive progenitors were present in the CD34+/Mpl-R+ fraction. However, this cell fraction was significantly enriched in low proliferative colony-forming units-MK. When the CD34+/Mpl-R+ fraction was grown in liquid culture containing human aplastic serum and a combination of growth factors, mature MK were seen as early as day 4, whereas the predominant cell population was erythroblasts on day 8. Similar data were also obtained with the CD34+/Mpl-R- fraction with, however, a delay in the time of appearance of both MK and erythroblasts. In conclusion, Mpl-R is a cytokine receptor restricted to the MK cell lineage. Its expression is low on CD34+ cells and these cells mainly correspond to late MK progenitors and transitional cells. These data indicate that the action of the Mpl-R ligand might predominate during the late stages of human MK differentiation.
Insights
The Mpl receptor (Mpl-R) is primarily found on megakaryocytes and platelets, playing a key role in their development. Its expression on CD34+ cells indicates late-stage megakaryocyte progenitors.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- The Mpl receptor (Mpl-R) is a cytokine receptor within the hematopoietin receptor superfamily.
- A ligand for Mpl-R has been recently identified, prompting investigation into its role in hematopoiesis.
Purpose of the Study:
- To investigate the lineage distribution of Mpl-R in normal hematopoietic cells.
- To characterize the Mpl-R expression pattern on various hematopoietic cell types, including leukemic cell lines and normal progenitors.
Main Methods:
- Development of a monoclonal antibody (M1 MoAb) against human Mpl-R.
- Indirect immunofluorescence, immunoprecipitation, and immunoblotting to detect Mpl-R expression.
- Colony assays and liquid culture to assess the function of Mpl-R expressing cells.
Main Results:
- Mpl-R was detected on human leukemic cell lines with megakaryocytic phenotypes and on normal megakaryocytes and platelets.
- Mpl-R expression was low on CD34+ cells, with Mpl-R+/CD34+ cells coexpressing platelet glycoproteins.
- Mpl-R+/CD34+ cells were enriched in late megakaryocyte progenitors but lacked primitive progenitors.
Conclusions:
- Mpl-R is a cytokine receptor predominantly expressed in the megakaryocyte lineage.
- Mpl-R expression on CD34+ cells identifies late megakaryocyte progenitors and transitional cells.
- The Mpl-R ligand likely influences late-stage human megakaryocyte differentiation.