FUSION OF A SIGNAL SEQUENCE TO THE INTERLEUKIN-1-BETA GENE DIRECTS THE PROTEIN FROM CYTOPLASMIC ACCUMULATION TO EXTRACELLULAR RELEASE

Interleukin (IL)-1 differs from most other cytokines by the lack of a signal sequence, which results in the retention of the immature prefor m intracellularly (ic). Several cell types have the capacity to produc e IL-1, but release has been shown to be restricted predominantly to m onocytes/macrophages and associated with apoptosis of the producer cel l. These features have limited the studies on IL-1 in early T cell-APC interactions. To develop a model for studying the biological effects of IL-1 beta release during long-lasting immune responses, we have est ablished cells transfected with IL-1 beta cDNA constructs. To construc t a hybrid gene for IL-1 beta release, the signal sequence from the re lated IL-1 receptor antagonist was fused to the gene encoding the 17-k Da mature form of IL-1 beta. A murine fibroblast cell line was transdu ced with retroviral technique and analyzed for the expression of human IL-1 beta, with or without a signal sequence (ssIL-1 beta and IL-1 be ta, respectively). The fibroblasts transduced with either IL-1 beta or ssIL-1 beta expressed similar levels of human IL-1 beta mRNA. High le vels of IL-1 bioactivity were recorded in freeze-thaw extracts from ce lls expressing the IL-1 beta protein ic, and in supernatants of ssIL-1 beta-transduced cells, which indicates that the initial formation of a preform of IL-1 beta is not required for correct folding of the prot ein. Treatment of ssIL-1 beta-transduced cells with Brefeldin A (BFA), an inhibitor of protein transport in the endoplasmatic reticulum, ind uced accumulation of the protein ic. BFA treatment did not affect IL-1 beta-transduced cells, while lipopolysaccharide-activated human monoc ytes increased the secretion of IL-1 beta. Cytoplasmic staining of sin gle cells demonstrated that expression of the ssIL-1 beta gene directe d the protein to a perinuclear Golgi-like compartment, whereas cells t ransduced with IL-1 beta cDNA showed a diffuse cytoplasmic distributio n pattern. Secretion of IL-1 beta from human monocytes was under certa in conditions accompanied by cell death. In contrast, in the fibroblas t cell line transduced to secrete IL-1 beta, no accompanying cell deat h could be detected. Gene targeting of IL-1 to the secretory or cytopl asmic pathway may be useful for elucidating the role of IL-1 in T cell -APC interactions, avoiding cell death of the producer cells. (C) 1996 Academic Press, Inc.