The metabolism and subsequent immunosuppressive effects of 2-acetylaminofluorene (AAF), cyclophosphamide (CP) were investigated in mouse splenocytes, in mixed cultures of mouse splenocytes and rat hepatocytes (coculture). This immunosuppression was identified by the quantitative analysis of x mRNA using immunoglobulin x constant region specific human genomic DNA probes in hybridization.
Also, to determine whether new protein synthesis is required for immunosuppression, we treated mouse splenocytes with LPS and AAF (or CP) in the presence of cycloheximide (CHX, 10㎍/ml).
Direct addition of AAF into culture medium resulted in the decrease of x mRNA, but the metabolism of AAF was not required in suppression of x mRNA and CP, vice versa. That is, CP was activated by hepatocytes to an immunosuppressive form that caused the suppression of the in vitro antibody response to the B cell-dependent antigen, lipopolysaccharide (LPS). But the dose-response of CP showed that only 1mM, not 0.01mM and 0.1mM CP, decreased x mRNA. It seems that immunosuppression by CP is caused by the cytotoxicity rather than the inhibition of transcription of the immunoglobulin gene. Also, we found that a simultaneous treatment of AAF and LPS largely decreased x mRNA level. Therefore, it seems that AAF may block the early activation step in antibody gene expression activation mechanism by LPS.
We identified that new protein synthesis is not required for immunosuppression by CP, AAF and unstimulated spleen cells also contain relatively high steady-state ievel of immunoglobulin mRNA when cultured for 48 hr, but they do not secrete antibody at a high rate until stimulated by LPS. In conclusion, the decreased level of immunoglobulin x mRNA can be interpreted as a failure of cell activation and splicing of primary transcripts.
본 연구에서는 mouse (Balb/c) 비장세포 배양에서 면역억제제로 알려진 Cyclophosphamide와 2-acetylaminofluorene에 의한 면역억제 작용 기작을 알아 보았다.
실험 결과, CP 그 자체는 immunoglobulin light chain 유전자의 k mRNA 수준의 감소 현상을 보이지 않았으며, 간세포 존재하에서만 대사활성이 되어 k mRNA수준을 감소시켰다. 그러나 CP의 dose-response실험 결과, 1 mM이상 처리해야 k mRNA수준의 감소가 나타났다. 아마도 CP에 의한 면역억제작용은 transcription 저해에 의한 것보다는 cytotoxicity에 의한 세포의 죽음때문인것 같다 (CP 1mM처리시, 얻을 수 있는 전체 RNA양이 세포의 죽음때문에 다른 group에 비해서 1/10 수준이다).
반대로 AAF는 그 자체에 의해서 k mRNA수준을 감소시켰으며, 간세포에 의해서 대사 활성이 되면 억제 작용이 사라졌다. 또한 AAF는 immunoglobulin 유전자 발현 과정의 초기 활성화 단계를 저해하는 것으로 생각된다.
그리고, AAF와 CP에 의한 k mRNA감소는 새로운 단백질의 합성이 필요없음을 알아내었다. 한편 mitigen이 존재하지 않는 상태하에서도 48시간이상 배양하면 일정수준의 k mRNA가 발현이 되지만, plaque assay를 통한 지금까지의 실험 결과를 보면 항원이 존재하기 전까지는 완전한 형태의 항체 형성은 없는 것 같다.
