IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion.

Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFα and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. Science. 310:1492–1495). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFα, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFα and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFα delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/ golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of fulllength GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for ␣2,6-sialyltransferase, ␤-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the Nterminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex. Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton. Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens.

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Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either "White" or "Black". We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype-phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively.

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