A ceramide‐1‐phosphate analogue, PCERA‐1, simultaneously suppresses tumour necrosis factor‐α and induces interleukin‐10 production in activated macrophages

Abstract

SummaryTight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-a blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-a in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-a suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-jB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the antiinflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-a suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.

Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1)

“…Importantly, the correct stereochemistry is essential for the activity of PCERA-1, as the [1S,2R] stereoisomer is two orders of magnitude more potent than the [1S,2S] stereoisomer (Matsui et al, 2002d), indicating that this phospholipid-like molecule binds a protein target, rather than affects membrane structure in a nonspecific manner. The identical dose-response curves obtained for the independent modulation of TNF- and IL-10 production in LPS-stimulated macrophages by PCERA-1, suggests that a single protein target mediates both activities (Goldsmith et al, 2008).…”

“…Moreover, since de-phosphorylation of PCERA-1 would give rise to a cellpermeable CERA-1, one can predict that if PCERA-1 crosses the cell membrane by the mechanism suggested above for C1P, then exogenous CERA-1 should be at least as active as A c c e p t e d M a n u s c r i p t 7 exogenous PCERA-1. However, CERA-1 was found to be essentially inactive, both in-vivo Matsui et al, 2002c;Matsui et al, 2002d), and in-vitro (Goldsmith et al, 2008), suggesting that PCERA-1 acts in an extra-cellular manner. It should be noted however that exogenous phospholipids may also enter the cell by alternative mechanisms such as endocytosis (Boudker and Futerman, 1993) or via the scavenger receptor (Adachi and Tsujimoto, 2006).…”

“…However, it was found that only macrophages co-incubated with LPS and PCERA-1 show modulated TNF- production (relative to LPS-stimulated macrophages), and that preincubation is not advantageous for PCERA-1 activity (Goldsmith et al, 2008). In that sense PCERA-1 resembled PGE 2 , which suppresses TNF- production in an extra-cellular manner via membrane receptors, and differed from the cell-permeable dexamethasone, which suppresses TNF- production via a nuclear receptor, and was therefore resistant to washing (Goldsmith et al, 2008).…”

“…The anti-inflammatory activity of PCERA-1 has been demonstrated in the mouse RAW264.7 macrophage cell line (Goldsmith et al, 2008) as well as in primary mouse macrophages ). Of note, isolated blood monocytes were inert to PCERA-1 but acquired sensitivity to PCERA-1 upon M-CSF-induced differentiation into macrophages .…”

“…3) PCERA-1 rapidly elevates intra-cellular cAMP level in macrophages (Goldsmith et al, 2008), suggesting it activates a GPCR upstream to adenylyl cyclase (AC).…”