Externalization of the Leaderless Cytokine IL-1F6 Occurs in Response to Lipopolysaccharide/ATP Activation of Transduced Bone Marrow Macrophages

Abstract

An interesting trait shared by many members of the IL-1 cytokine family is the absence of a signal sequence that can direct the newly synthesized polypeptides to the endoplasmic reticulum. As a result, these cytokines accumulate intracellularly. Recent studies investigating IL-1beta export established that its release is facilitated via activation of an intracellular multiprotein complex termed the inflammasome. The purpose of the current study was to explore the mechanism by which murine IL-1F6 is released from bone marrow-derived macrophages (BMDMs) and to compare this mechanism to that used by IL-1beta. BMDMs were engineered to overexpress IL-1F6 by retroviral transduction; cells overexpressing GFP also were generated to provide a noncytokine comparator. The transduced cells constitutively expressed IL-1F6 and GFP, but they did not constitutively release these polypeptides to the medium. Enhanced release of IL-1F6 was achieved by treating with LPS followed by ATP-induced activation of the P2X(7) receptor; GFP also was released under these conditions. No obvious proteolytic cleavage of IL-1F6 was noted following P2X(7) receptor-induced release. Stimulus-induced release of IL-1F6 and GFP demonstrated comparable susceptibility to pharmacological modulation. Therefore, transduced IL-1F6 is released in parallel with endogenous mature IL-1beta from LPS/ATP-treated BMDMs, but this externalization process is not selective for cytokines as a noncytokine (GFP) shows similar behavior. These findings suggest that IL-1F6 can be externalized via a stimulus-coupled mechanism comparable to that used by IL-1beta, and they provide additional insight into the complex cellular processes controlling posttranslational processing of the IL-1 cytokine family.