AN ANTISENSE OLIGODEOXYNUCLEOTIDE COMPLEMENTARY TO CORTICOTROPIN-RELEASING HORMONE MESSENGER-RNA INHIBITS RAT SPLENOCYTE PROLIFERATION IN-VITRO

Expression of neuropeptides in immune tissues has been implicated in t he paracrine control of immune functions. The effects of the endogenou s splenic neuropeptide corticotropin-releasine hormone (CRH) on immune cell proliferation were investigated by incubating splenocytes from a dult male Wistar rats in vitro with a specific antisense oligodeoxynuc leotide probe complementary to CRH mRNA. Incubation of cells with 1 mu g/ml phosphodiester antisense probe for 24 h prior to stimulation wit h concanavalin A (Con A) resulted in a 30-65% decrease in H-3-thymidin e uptake compared to controls. In spleen cells incubated with a random base sequence (nonsense) probe the uptake of H-3-thymidine was not di fferent to that in control cells. Incubation of cells with either anti sense or nonsense phosphorothioate-protected probes resulted in variab le uptake of H-3-thymidine, demonstrating that these probes, unlike th e phosphodiester probes, have non-specific effects on cells. Addition of synthetic CRH to the cells incubated with the antisense phosphodies ter probe partially restored the proliferative response of splenocytes to Con A. Immunoreactive (ir) CRH measured by radioimmunoassay in spl enocytes incubated with the antisense probe was significantly less tha n ir-CRH in splenocytes incubated with the nonsense probe or without p robe, indicating that the expression of splenic CRH mRNA was specifica lly impaired. This attenuation of the cell proliferative response foll owing reduced expression of splenic ir-CRH provides functional evidenc e for the involvement of endogenously synthesised immune ir-CRH in spl enocyte activation.