The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP

Jeong

The Journal of Immunology

et al. 2011

100

Chemokines play critical roles in inflammation by recruiting inflammatory cells to injury sites. In this study, we found that UDP induced expression of chemokines CCL2 (MCP-1) and CCL3 (MIP-1α) in microglia, astrocytes, and slice cultures by activation of P2Y6. Interestingly, CCL2 was more highly expressed than CCL3. However, CCL2 synthesis kinetics in response to UDP differed in microglia and astrocytes; microglia rapidly produced small amounts of CCL2, whereas astrocytes continuously synthesized large amounts of CCL2, resulting in a high ultimate level of the chemokine. UDP-induced chemokine expression was reduced in the presence of a specific antagonist of P2Y6 (MRS2578) or small interfering RNA directed against the P2Y6 gene. Inhibition of phospholipase C and calcium increase, downstream signaling pathways of Gq-coupled P2Y6, reduced UDP-induced chemokine expression. UDP activated two calcium-activated transcription factors, NFATc1 and c2. Furthermore, inhibitors of calcineurin (a phosphatase activating NFAT) and NFAT reduced UDP-induced chemokine synthesis. We also found, using a transmigration assay, that UDP-treated astrocytes recruited monocytes. These results suggest that UDP induces chemokine expression in microglia and astrocytes of the injured brain by activation of P2Y6 receptors.

Section: Discussionmentioning

confidence: 99%

Uridine 5′-Diphosphate Induces Chemokine Expression in Microglia and Astrocytes through Activation of the P2Y6 Receptor

Jeong

The Journal of Immunology

et al. 2011

100

“…This nucleotide receptor is also required for IL-8 secretion due to TLR4 responding to LPS of Gram-negative bacteria in both THP-1 and human primary monocytes [14,15,30]. The P2Y 6 receptor has also been shown to activate the release of other cytokines and chemokines, such as TNF-a, MCP-1 and IP-10 from U937 cells, and CCL20 (MIP-3a) from human monocytes and monocytederived dendritic cells [30,31]. It is noteworthy that in the latter cells, CCL20 release could also be induced by ATP-gS, suggesting that again two or more P2 receptors can be involved [31].…”

Section: Discussionmentioning

confidence: 99%

Concomitant activation of P2Y₂ and P2Y₆ receptors on monocytes is required for TLR1/2‐induced neutrophil migration by regulating IL‐8 secretion

et al. 2009

Extracellular nucleotides regulate a variety of cellular responses involved in inflammation via the activation of P2 receptors. Here, we show that nucleotides regulate TLR2-induced neutrophil migration both in vivo and in vitro. The nucleotide scavenger apyrase inhibited neutrophil recruitment in murine air pouches injected with the TLR2 agonist Pam 3 CSK 4 . In agreement, the supernatants of either human primary monocytes or monocytic cells (THP-1 and U937) treated with Pam 3 CSK 4 recruited significantly fewer neutrophils when the former cells were treated in the presence of apyrase. As demonstrated with inhibitory Ab, these supernatants induced neutrophil migration due to IL-8 secretion. In addition, IL-8 secretion was markedly diminished by the non-selective P2 receptor antagonists reactive blue 2 and suramin, and by a selective P2Y 6 antagonist, MRS2578. Selective antagonists of P2Y 1 (MRS2500) and P2Y 11 (NF157) did not affect IL-8 release. The knockdown of either P2Y 2 or P2Y 6 with specific shRNA diminished IL-8 secretion from Pam 3 CSK 4 -treated THP-1 cells. Altogether, these results show that extracellular nucleotides, via P2Y 2 and P2Y 6 receptors, regulate neutrophil migration by controlling TLR2-induced IL-8 release from human monocytes. In line with our previous work on TLR4, this study further supports the importance of nucleotides in bacterial-induced neutrophil migration.Key words: Cell trafficking . Human monocytes . Inflammation . Neutrophils . Pam 3 CSK 4 IntroductionMonocytes play an important role in immune defences against invading bacteria and viruses via TLR activation [1]. TLR recognize highly conserved structural motifs expressed by microbes called PAMP. Monocytes express various TLR including TLR4 (that is activated by LPS, a cell wall component of Gramnegative bacteria), TLR2 (activated by peptidoglycan and lipopeptide, components of Gram-positive bacteria), TLR5 (activated by the bacterial flagellin) and TLR7/8 (activated by single-stranded viral RNA) [2][3][4][5]. In response to TLR activation by PAMP, monocytes release various cytokines and chemokines that orchestrate the innate immune responses [6][7][8]. For example, these cells secrete large amounts of the chemokine IL-8 that plays a major role in neutrophil recruitment at sites of infection [9].In addition to TLR, monocytes abundantly express P2 receptors that are activated by extracellular nucleotides. P2 receptors are divided into two subfamilies: the G-protein-coupled receptors (P2Y 1,2,4,6,[11][12][13][14] ) and the ligand-gated ion channels (P2X 1-7 ). The P2Y subtypes differ in their selectivity toward adenine (ATP, ADP) and uracil nucleotides (UTP, UDP) while all P2X receptors are activated by ATP [10,11]. Human primary monocytes and monocytic cell lines (THP-1 and U937) express the mRNA of various P2 receptors of which P2X 1,7 and P2Y 2,6,11 are common to all these cells [12,13]. There is growing evidence indicating that Results Extracellular nucleotides are involved in Pam 3 CSK 4 -induced neutrophil migration in vi...

“…2+ assays were carried out as previously described with modifications [26] . Freshly isolated human neutrophils were resuspended at 1 × 10 6 cells/mL in cell culture medium (RPMI 1640 with 2 mmol/L GlutaMAX and 5% fetal bovine serum) and incubated at 37℃, 50 mL/L CO2, for 1 h. Cells were spun and resuspended at 1 × 10 6 cells/mL in a 1:1 mixture of cell culture medium and Calcium-4 no wash dye (Molecular Devices, Sunnyvale, CA, USA) containing a final concentration of 5 mmol/L probenecid (Sigma).…”

Section: Calcium Flux Assay Intracellular Camentioning

confidence: 99%

Short-chain fatty acids act as antiinﬂammatory mediatorsby regulating prostaglandin E2 and cytokines

Cox¹,

Jackson²,

Stanton³

et al. 2009

WJG

Self Cite

308

248

AIM:To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E2 (PGE2), cytokines and chemokines in human monocytes. METHODS:Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative realtime polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE2, cytokines and chemokines in the supernatant.The effect of SCFAs in vivo was examined by intraplantar injection into rat paws. RESULTS:Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE2 and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE2 production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE2, MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-α and interferon-γ in human PBMC. Finally, we show that SCFAs and LPS can induce PGE2 production in vivo by intraplantar injection into rat paws (P < 0.01). CONCLUSION:SCFAs can have distinct antiinflammatory activities due to their regulation of PGE2, cytokine and chemokine release from human immune cells.