The immunosuppressive effects of dimethylnitrosamine (DMN) and 2-acetylaminofluorene (AAF) were investigated using various immunological parameters. DMN did not cause any suppression of in vitro antibody responses and mitogen-induced proliferation of splenocytes without metabolic activation or co-cultured with liver enzyme fractions (S-9). When the splenocytes were co-cultured with mouse hepatocytes along with DMN for 4hr, DMN markedly suppressed the response to SRBC, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS. The proliferative responses of splenocytes to B cell mitogen lipopolysaccharide (LPS) and T cell mitogen concanavalin A (ConA) were also inhibited by DMN after co-culture with hepatocytes. The suppression by DMN was related to the rocking speed during the 4hr co-culture period and was optimally produced when the cultures were not rocked. Addition of serum into the medium (10% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response. However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the response by DMN, indicating that DNA represents the primary macromolecular target for the reactive intermediate of DMN.
To elucidate the dependence of intact hepatocytes in vitro activation of DMN to immunosuppressive forms, S-9 fractions from both phenobarbital (PB)-and isopropanol (iso)-pretreated mice were used. These S-9 displaced significantly greater demethylase activity than uninduced S-9 fractions. However, when incubated with spleen cells, neither S-9 preparation was capable of activating DMN to a immunosuppressive forms. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. These results indicate that the in vitro activation of DMN to an immunosuppressive form is dependent on intact cells and on cell-cell contact or close proximity of activating and target cells.
In order to confirm that suppression of in vitro antibody responses was mediated via reactive metabolites released from hepatocytes, C14-DMN and demethylase inhibitors were introduced to this study. A significant increase in the binding of DMN metabolites to TCA precipitable material in splenocytes was induced when 94 uM C14-DMN was added to the co-culture medium. The amount of 14C-DMN bound to TCA ppt in splenocytes increased in a time-dependent manner up to 4hr incubation. Aminoacetonitrile (AAN) reversed the suppression by low concentrations of DMN (0.1 mM to 5 mM) and inhibited the binding of C14-DMN to both hepatocytes and splenocytes.
Unlike DMN, AAF did not produce the suppression of in vitro antibody response to SRBC up to 50 mg/kg while proliferative response of spleen cells to ConA was marginally inhibited at highest dose after 14 day treatment. Direct addition of AAF into spleen cell suspensions resulted in the suppression of the polyclonal response to LPS and T-dependent response to SRBC. AAF also produced the inhibition of lymphoproliferative response to LPS and ConA. Time course studies indicated that maximum suppression of the proliferative response to mitogen stimulation was obtained when AAF was added at the initiation of cultures and suppression was not produced when AAF was added 24 hr before harvesting the 72 hr cultures. In order to determine the role for metabolic activation on AAF-induced suppression of antibody response to LPS, splenocytes were co-cultured with hepatocytes along with AAF for 4 hr. AAF did not produce the suppression of the response in 4 hr co-culture system up to 100 uM AAF. The suppression was even reversed when AAF was not removed after 4 hr culture period. On the contrary, unscheduled DNA synthesis by AAF was induced in a dose dependent manner in the same co-culture system. These results suggest that the in vitro immunosuppression by AAF may not be mediated by the metabolites implicated in its mutagenicity.
본 연구에서는 일차배양 간세포와 비장세포의 공동배양을 개발하고 이 공동배양 system을 이용하여 dimethylnitrosamine (DMN) 과 2-acetylaminofluorene (AAF) 의 면역억제 작용과 그 작용기전에 관하여 연구하였다. DMN 그 자체는 면역억제 작용을 보이지 않았으며, 간세포 존재하에서만 대사활성이 되어 DNP-Ficoll 에 대한 T-independent 항체반응과 SRBC 에 대한 T-dependent 항체반응을 억제하였으며 또한 mitogen에 의한 비장세포의 증식반응도 억제하였다. DMN의 체외 면역 반응 억제효과는 공동배양시 rocking 속도에 따라 달랐으며, fasting rocking (24 rocks/min) 조건에서는 항체반응을 억제하지 않았고 slow rocking (8 rocks/min)과 rocking을 하지 않았을 경우에만 억제작용을 나타내었다. Rocking 하지 않은 조건에서 면역억제 작용이 최대로 나타났다. S9 fraction을 물질 대사계로 이용하였을 때는 DMN의 대사는 일어나나 면역억제 작용은 보이지 않았고, intact 간세포 존재하에서만 DMN이 면역 억제 효과를 보여주었다. 또한, 비장세포와 간세포를 agar층으로 분리시켜 배양할 때는 DMN에 의한 면역 억제 효과가 관측되지 않은 점으로 보아 비장세포와 간세포가 직접 접촉하거나 아주 근접한 거리에 있어야만 DMN의 면역 억제 작용이 나타나는 것으로 사료된다. DMN에 의한 면역억제 작용의 기전을 규명하기 위하여 C-DMN과 DMN demethylase inhibitor를 이용하여 실험 하였다. 비장세포를 간세포와 공동배양시 DMN에 의한 비장세포내 물질의 alkylation이 증대되었으며, 이 alkylation은 배양시간에 비례하여 증가됨을 관찰하였다. 또한, DMN demethylase inhibitor인 AAN은 DMN에 의한 antibody response 억제작용을 방지하였고 alkylation도 저하시켰다.
AAF는 생체내 투여시는 면역 억제 작용을 보이지 않았으며 DMN 과는 다르게 체외면역 반응에서는 대사물이 아닌 AAF 자체가 면역 억제 효과를 나타내었다. AAF처리 시간에 따라 그 억제효과가 변하는데 초기세포 activation 과정에 가장 영향을 주는 것으로 관찰되었다. AAF의 대사가 면역 억제 작용에 주는 영향을 알아보기 위하여 비장세포와 간세포의 공동 배양system을 이용하였다. AAF를 4시간 처리하여도 아무런 억제효과를 나타내지 않았으며 AAF 대사물이 억제효과를 보이지 않은 것으로 관찰되었다. 반면에 비장세포를 간세포와 공동배양시 AAF는 UDS를 증가시키는 것으로 관찰되었다.
위 결과를 종합하여 보면 DMN은 간세포에서 대사가 되고 이 대사물질이 비장세포로 이행되어 면역 억제 작용을 나타내고 반면에 AAF는 그 자체가 면역억제 효과를 나타내며 유전 독성을 나타내는 대사물질은 면역 억제 효과가 없는 것으로 판명되었다.
