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.