Anti-Apoptotic Effect of Soluble IL-6 Receptor on Megakaryocytic Lineage in Physiologic Conditions and Essential Thrombocythemia.

Abstract Interleukin 6 (IL-6) plays a stimulatory role in megakaryocytic development acting through a specific receptor (IL-6Ra) and a signal transducing unit, gp130. A soluble IL-6R (IL-6sR) has also been described which retains the ability to bind IL-6 and associate to gp130 acting agonistically on cells that do not express IL-6R. We have previously described increased IL-6sR in essential thrombocythemia (ET), an illness characterized by megakaryocytic hyperplasia and high platelet count. The aim of this study was to evaluate the participation of IL-6sR in CD34+ cell survival and in platelet signaling in physiologic conditions and in ET. Therefore we studied: The ability of IL-6/IL-6sR to protect CD34+ cells from death by evaluating propidium iodine (PI) uptake, anexin-V binding and caspase 3 activation. We worked with normal CD34+ cells obtained from umbilical blood cord and with peripheral blood CD34+ cells from ET patients. Cells were purified by immunomagnetic means and cultured for 16 hours in the presence of culture media (IMDM), 100 ng/ml IL-6, 100 ng/ml thrombopoietin (Tpo) or 100 ng/ml IL-6 + 200 ng/ml IL-6sR (IL-6/IL-6sR). After that, PI uptake, anexin-V binding and activated caspase 3 were evaluated by flow cytometry using standard techniques. Besides, we have measured IL-6Ra on CD34+ cell surface by flow cytometry. JAK-2 and STAT-3 phosphorylation in normal and ET platelets. After stimulation with 100 ng/ml IL-6 plus 200 ng/ml IL-6sR or 100 ng/ml IL-6 alone, washed platelets were evaluated by western-blot with antibodies against phosphorylated and unphosphorylated proteins. Normal cells treated with Tpo and IL-6/IL-6sR displayed lower PI uptake than those cultured in IMDM or IL-6, while 3 patients did not show this pattern. Mean values are shown in table 1. Anexin-V binding did not differ among these four conditions. Next, we evaluated activated caspase 3 in CD34+ cells treated with either IL-6 or IL-6/IL-6sR. Mean flourescence intensity (Gm) from cells cultured in the presence of IL-6sR/IL-6 was lower than that of cells cultured in IL-6, in controls (p=0.002, Wilcoxon signed runk sum test) as well as ET patients (p=0.002). Median values are shown in table 2. A trend to increased apoptosis was found in controls compared to ET samples (p=0.051 for IL-6/IL-6sR; p=0.074 for IL-6). Normal CD34+ cells from five blood cord samples showed 0.12% (0–2.5) of positivity for IL-6Ra, while CD34+ cells from ET patients had 4.0% (0.73–24) (n=8, p=0.023, Wilcoxon rank sum test). We found an increased STAT-3 phosphorylation status in ET platelets (n=3) compared to normal controls (n=2) when platelets were stimulated with IL-6/IL-6sR, while JAK2 was found equally phosphorylated in these conditions. On the contrary, the same samples challenged with 100 ng/ml Tpo displayed similar intensity in STAT-3 phosphorylation. We conclude that IL-6/IL-6sR can protect normal and ET CD34+ cells from apoptosis more efficiently than IL-6. Besides, CD34+ cells from ET seem to be more resistant to apoptosis than their normal counterpart when cultured in the presence either of IL-6 or IL-6/IL-6sR. This effect could be due to an increased IL-6Ra expression on cell surface. Table 1 % of PI uptake IMDM IL-6 Tpo IL-6/IL-6sR (1) differed significantly from (2) p<0.05, one way AOV. Patients n=3 58.1 53.9 55.4 46.6 Controls n=6 57.1 (2) 50.5 (2) 43.1 (1) 41.2 (1) Table 2 Activated caspase 3 (Gm) IL-6/IL-6sR IL-6 Patients n=5 50.0 70.7 Controls n=7 117.7 123.9