Increased IL-6 Receptor Proteolytic Cleavage in Mononuclear Cells from Patients with Essential Thrombocythemia.

Recently we described increased IL-6 soluble receptor (IL-6sR) levels in plasma of essential thrombocythemia (ET) patients as well as elevated IL-6sR release by cultured mononuclear cells. IL-6sR can be generated by two different mechanisms: alternative splicing and proteolytic cleavage. Here we evaluated the participation of metalloproteases responsible for the proteolytic cleavage in the increase of IL-6sR levels produced by cultured cells. We studied expression changes in anchored IL-6 receptor (IL-6R) in CD3 lymphocytes as well as monocytes before and after cell culture. We also assessed the IL-6sR levels in the supernatant of cell culture in the presence and absence of a metalloprotease inhibitor, TAPI, that prevents most of the IL-6sR shedding from the anchored receptor. Seventeen patients with ET were studied, 14 without treatment and 3 under anagrelide treatment. Blood samples were obtained under sterility conditions and mononuclear cells were separated by Ficoll-Hypaque gradient. Afterwards, cells were cultured during 24 hs in IMDM supplemented with 10% FCS in the presence and absence of 150 mMol/L TAPI and IL-6sR released to culture media was measured by ELISA technique. Anchored IL-6R was evaluated before and after cell culture by flow cytometry using anti-CD126-PE. Monocyte and lymphocyte populations were selected using anti-CD14 and anti-CD3 respectively. IL-6sR levels in supernatant of mononuclear cells from 10 untreated ET patients were found increased, 122.5pg/ml (74–248 pg/ml) (median and range), compared to 9 normal controls, 83 pg/ml (22–130 pg/ml), p=0.011. In the presence of TAPI, patients and normal controls showed similar results, 54.95 pg/ml (18–137 pg/ml) and 48 pg/ml (18–96 pg/ml), respectively, p=NS. The difference between the amount of IL-6sR released in the absence and the presence of TAPI (D) would correspond to IL-6sR generated by proteolytic cleavage. This D was statistically higher in ET patients (61 pg/ml, 18–111 pg/ml) than in normal controls (28 pg/ml, 2–87 pg/ml), p=0.034. Expression of IL-6R on monocyte and lymphocytes from 5 untreated and 3 anagrelide treated ET patients before and after cell culture was similar to that of normal controls as shown by flow cytometry analysis. In conclusion, we found that proteolytic cleavage of IL-6R is increased in cultured mononuclear cells from patients with ET although the steady-state of IL-6R on monocytes and lymphocytes membrane is not altered.