Exploring the mechanisms of membrane blebbing and their relationship to interleukin-1beta release in murine macrophages

The proinflammatory cytokine interleukin-1beta (IL-1beta) plays an important role in the innate immune system, inducing fever and activation of the vascular endothelium. However, the mechanism by which IL-1beta is secreted remains a mystery given that it is expressed in the cytosol and therefore not targeted through the endoplasmic reticulum/Golgi secretory pathways. Furthermore, IL-1beta exists as an inactive, 33 kDa procytokine which must be cleaved to its secreted, biologically active 17 kDa form by caspase-1, itself a complexly regulated zymogen. Given that ATP activation of the P2X7R on LPS-primed macrophages can facilitate secretion of IL-1beta, while simultaneously mediating membrane bleb formation, we hypothesized that membrane blebbing plays an important role in IL-1beta secretion. To test this hypothesis, we used time-lapse videomicroscopy to assess the response of macrophages to various, bleb-inducing stimuli coupled with biochemical analyses of IL-1beta release. We found that P2X7R stimulation induces different forms of membrane blebbing, depending on cell type and extracellular ionic milieu. In HEK-P2X7 cells in BSS, this blebbing was characteristically zeiotic, while in macrophages in BSS, this blebbing was morphologically distinct, prompting us to name it "tethered." We further demonstrated that P2X7R stimulation led to activation of the small GTPase Rho, which in turn signaled the formation ROCK-dependent blebs. Finally, we showed inhibition of tethered membrane blebbing had no effect on ATP-induced IL-1beta release, nor did blockade of IL-1beta release by caspase-inhibition impact tethered membrane blebbing. In a separate study, we demonstrated that removal of halide anions from the extracellular BSS media led to ATP-induced formation of ROCK-independent dilated blebs and biphasic release of IL-1beta whereby one phase was blocked by the tyrphostin tyrosine kinase inhibitor AG126 while the second phase was lytic and blocked by glycine. Finally, we demonstrated that maitotoxin, which recruits the same pore-forming moiety as the P2X7R, can also facilitate massive dilated membrane blebbing and IL-1beta release. This release can be blocked by AG126, but is primarily lytic in nature, since it can be blocked by glycine. In summary, we have shown that membrane blebbing and IL-1beta secretion are regulated by many common signaling pathways but can be pharmacologically dissociated.