DICHOTOMY OF RESPONSE OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TO NOCARDIA WATER SOLUBLE MITOGEN WHEN CULTURED WITH AUTOLOGOUS SERUM VERSUS FETAL CALF SERUM: CORRELATION OF INHIBITION OF TRITIUM-THYMIDINE UPTAKE WITH DNASE ACTIVITY OF THE SERUM AND PRESENCE

摘要

Culture of human peripheral blood lymphocytes (HPBL) with Nocardia water soluble mitogen (NWSM) in autologous serum (AS) supplemented cultures resulted in increasing ('3)H-thymidine (('3)H-TdR) incorporation with increasing concentration of mitogen, plateauing at a mitogen construction of 31 to 833 ug/ml. When the cells were cultured with fetal calf serum (FCS), the backgrounds were higher and the ('3)H-TdR uptake did not significantly exceed the background at any dose of the mitogen but fell precipitiously at the high doses of the mitogen. After optimizing the response to NWSM in AS supplemented cultures, the focus of the research concerned the explanation of the differences in response using FCS versus AS for supplementation of the culture media. The differences were not due to an alteration of the proliferation kinetics or to a protective or positive interaction of human sera with NWSM. The degree of inhibition of ('3)H-TdR at high doses of NWSM correlated with the concentration of FCS. However, the NWSM-induced ('3)H-TdR uptake per cell did not correlate with the number of blast cells in FCS supplemented cultures.;A rudimentary analysis of the constituents of NWSM releaved that the mitogen could have large amounts of DNA (up to 55% of the dry weight). Concurrently it was found that serum had a DNA degrading activity. This activity was significantly greater in FCS than in human sera. The major products of serum degradation of DNA were the nucleosides, including thymidine which could competitively inhibit the uptake of labeled thymidine by pokeweed mitogen (PWM) stimulated HPBL. DNA competitively inhibits ('3)H-TdR uptake by PWM stimulated cells and this is dependent on the DNase activity of the serum supplement of the culture.;NWSM free of DNA did not demonstrate the high dose inhibition of ('3)H-TdR uptake when cultured with FCS. The re-addition of DNA restored the high dose inhibition.;In conclusion, soluble DNA can cause an inhibition artifact in ('3)H-TdR uptake assays which correlates in degree with the DNase activity of the serum present and may be misinterpreted as immunosuppression.