Localization and Post-Golgi Trafficking of Tumor Necrosis Factor-alpha in Macrophages

Abstract

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.

Endocytosis of Uncleaved Tumor Necrosis Factor-α in Macrophages

“…Additionally, in monocytes it has been demonstrated that pro-TNF-␣ normally has a relatively short half-life of approximately 15 minutes, but in the presence of inhibitor its half-life in cell extracts is extended for up to one hour (Solomon et al, 1997). However, our pulse-chase and immunofluorescence studies in RAW macrophages show that pro-TNF-␣ has a half-life of at least 30 minutes under physiologic conditions (Shurety et al, 2000). Furthermore, in inhibitor-treated macrophages a significant pool of pro-TNF-␣ is retained for much longer times on the surface and in endosomes.…”

“…Endocytosis and recycling of pro-TNF-␣ is a possible mechanism for modulating levels and functions of surface exposed TNF-␣ and of TNF-␣ available for cleavage. In support of this concept, we have previously shown that macrophages activated by the distinct cascades associated with either LPS and interferon-␥ or LPS alone have different trafficking of TNF-␣ (Shurety et al, 2000). Interferon-␥ induces the trafficking of some TNF-␣ to endosomal structures that may be used to sequester or reroute surface TNF-␣ to produce a different biological action.…”

“…In unstimulated cells and in cells exposed only to the inhibitor BB-3103, faint diffuse immunoreactivity was detected. This may represent low constitutive levels of TNF-␣ in or on the cells (Shurety et al, 2000). After exposure to 100 ng/ml of LPS for 30 minutes to 2 hours, intense immunoreactivity of TNF-␣ appeared in the Golgi region.…”

“…The murine macrophage cell line RAW264 is widely used in studies of the regulation of TNF-␣ production (Darville et al, 1994;Kol et al, 1998). Exposure of RAW264 cells to bacterial lipopolysaccharide (LPS) leads to the rapid appearance of TNF-␣ mRNA (Chang et al, 1999) and a rapid accumulation of TNF-␣ protein in the Golgi complex (Shurety et al, 2000). The swift induction of relatively large quantities of this protein in mouse macrophages makes this an ideal system to dissect and characterize the intracellular trafficking of TNF-␣.…”

“…The efficiency of TNF-␣ processing is normally high, up to 90% of pro-TNF-␣ is converted to the 17 kd form for acute release by activated monocytes (Solomon et al, 1997). The cleavage of pro-TNF-␣ is thought to occur at the cell surface (Black et al, 1997;Decoster et al, 1995;Glaser et al, 1999), although there is also evidence for earlier proteolytic processing at the level of the Golgi complex (Jue et al, 1990;Shurety et al, 2000;Solomon et al, 1997;Watanabe et al, 1998). Secreted 17 kd TNF-␣ can act locally in autocrine and paracrine modes and at long distances in an endocrine mode by binding to the two known TNF-␣ receptors (55 kd and 75 kd) to initiate the acute systemic effects of TNF-␣ (Vassalli, 1996).…”