ヒト末梢リンパ球の幼若化反応におよぼす性ステロイドの影響に関する研究

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To examine the immunobiological implication of sex steroids during pregnancy, the effect of sex steroids on the incorporation of 3 H-thymidine into lymphocytes was investigated in both phytohemaggltinin (PHA) -induced blastic transformation of lymphocytes and the two-way mixed lymphocyte reaction (MLR). Heparinized venous blood was taken from informed normal healthy men and nonpregnant women who were not taking oral contraceptives. Lymphocyte suspensions were prepared on a ficoll-metrizoate gradient centrifugation, and quadruplicate cultures were performed in flat-bottomed microtest plates. A culture mixture (0.2ml) containing 105 responder lymphocytes, 3μg of PHA and various concentrations of steroid or 7.5 × 104 responder lymphocytes from two individuals and various concentrations of steroid was placed in each well. Cultures were conducted in culture medium (RPMI-1640) supplemented with 20% of heat-inactivated autoserum and incubated in a humidified atmosphere of 5% CO2-95% air in an incubator at 37°C, for 3 days for PHA or 6 days for MLR. Before termination of culture, 1.25μCi or 0.5μCi of 3 H-thymidine was added to each well and incubation was continued for 24hr for PHA or 18hr for MLR. Incorporation of 3 H-thymidine into the DNA of lymphocytes was determined by a liquid scintillation counter. Results were expressed as average counts per minute of quadruplicate cultures. Sex steroid hormones tested were estrone, estradiol-17β, estriol, testosterone, androstendione, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone and 20α-dihydroprogesterone, in the concentration range between 10-1ng/ml and 104ng/ml. Each steroid was dissolved in ethanol and diluted with medium. A preliminary experiment to test the effect of ethanol confirmed that the ethanol concentrations present in cultures had no appreciable effect on the incorporation of 3 H-thymidine. To test the cytotoxicity of sex steroids toward lymphocytes, cell viability was assessed by trypan blue dye exclusion under conditions identical to those used for MLR. Although the suppressive effect of progesterone on MLR was less apparent than that on the lymphocyte blastogenic response to PHA, it was statistically significant at concentrations above 2 × 103 ng/ml. The suppressive effect increased with increase of concentration and reached a maximum at 104 ng/ml. The reported concentration of progesterone in the placental tissue is between 2 × 103 and 6 × 103 ng/g wet tissue throughout pregnancy, which agrees well with the effective concentration of progesterone described above. Estradiol-17β had an inhibitory effect on MLR at 104 ng/ml but no apparent effect on the lymphocyte blastogenic response to PHA at this concentration. The reason estradiol-17βsuppressed MLR but not the lymphocyte blastogenic response stimulated by PHA is unknown. On the other hand, 20α-dihydroprogesterone had a remarkable suppressive effect on the lymphocyte blastogenic response to PHA but no effect on MLR. The reason for this discrepancy is also unknown. The other 6 hormones tested had neither a suppressive nor enhansive effect on the lymphocyte blastogenic response to PHA or allogeneic cell stimulation. These results, together with the fact that the highest concentration of progesterone in the human placenta occurs in the earliest period of pregnancy, suggest that the nonspecific immunosuppressive property of progesterone could be an important factor in the initial development of the biological relationship between mother and fetus.

著者

小林八郎
京都大学医学部婦人科学産科学教室

ヒト末梢リンパ球の幼若化反応におよぼす性ステロイドの影響に関する研究

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