CD40L stimulates the production of TPO by stromal cells.

(A) ELISA on 48-hour-old conditioned medium in ECs, BMEC line LTBMCs, bone marrow-derived myofibroblasts cultured in the presence of 10% supernatant of nontransfected COS cell (left histogram), or with 10% supernatant of CD40L-CD8α–transfected COS cells (right histogram). No TPO was detected in untransfected or transfected COS cell supernatants. Results are expressed as the mean ± SD for 3 experiments. (B) Western blotting showing TPO protein in HUVEC- and LTBMC-culture medium and increased production by CD40L. 1, recombinant TPO protein, 2 and 4, LTBMCs and HUVECs, respectively, cultured in the presence of 10% supernatant of nontransfected COS cells; 3 and 5, LTBMCs and HUVECs respectively, cultured with 10% supernatant of CD40L-CD8α– transfected COS cells. TPO was found migrating at an Mr of 58 to 68 kd. An additional species migrating at an Mr of approximately 90 kd (visible in track 1) can be seen on overloading and/or overexposure. We do not know the nature of this high-molecular weight species. Dotted line indicates the level of the lowest TPO standard (31.2 pg/mL).