Fusion of a Signal Sequence to the Interleukin-1β 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 proform intracellularly (ic). Several cell types have the capacity to produce IL-1, but release has been shown to be restricted predominantly to monocytes/macrophages and associated with apoptosis of the producer cell. 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-1β release during long-lasting immune responses, we have established cells transfected with IL-1β cDNA constructs. To construct a hybrid gene for IL-1β release, the signal sequence from the related IL-1 receptor antagonist was fused to the gene encoding the 17-kDa mature form of IL-1β. A murine fibroblast cell line was transduced with retroviral technique and analyzed for the expression of human IL-1β, with or without a signal sequence (ssIL-1β and IL-1β, respectively). The fibroblasts transduced with either IL-1β or ssIL-1β expressed similar levels of human IL-1β mRNA. High levels of IL-1 bioactivity were recorded in freeze–thaw extracts from cells expressing the IL-1β protein ic, and in supernatants of ssIL-1β-transduced cells, which indicates that the initial formation of a proform of IL-1β is not required for correct folding of the protein. Treatment of ssIL-1β-transduced cells with Brefeldin A (BFA), an inhibitor of protein transport in the endoplasmatic reticulum, induced accumulation of the protein ic. BFA treatment did not affect IL-1β-transduced cells, while lipopolysaccharide-activated human monocytes increased the secretion of IL-1β. Cytoplasmic staining of single cells demonstrated that expression of the ssIL-1β gene directed the protein to a perinuclear Golgi-like compartment, whereas cells transduced with IL-1β cDNA showed a diffuse cytoplasmic distribution pattern. Secretion of IL-1β from human monocytes was under certain conditions accompanied by cell death. In contrast, in the fibroblast cell line transduced to secrete IL-1β, no accompanying cell death could be detected. Gene targeting of IL-1 to the secretory or cytoplasmic pathway may be useful for elucidating the role of IL-1 in T cell–APC interactions, avoiding cell death of the producer cells.