Abstract
The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.
Highlights
The molecular mechanisms by which LPS and other ligands stimulate Toll-like receptors (TLRs) leading to a specific pattern of gene expression are well understood [7], detailed information about its negative regulation is lacking
We have shown that ATP promotes non-directional migration and acts as an extracellular regulator of TLR signaling in peripheral human monocytes
In chemotaxis assays we found that fMLP and ATP promoted migration, checkerboard analyses revealed that monocytes could not sense ATP concentration gradients
Summary
The molecular mechanisms by which LPS and other ligands stimulate TLRs leading to a specific pattern of gene expression are well understood [7], detailed information about its negative regulation is lacking. Following stimulation of TLRs in monocytes or macrophages, expression of IRAK-M increases, which has been shown to regulate negatively TLR signaling, as has up-regulation of single immunoglobulin IL-1 receptor-related molecule (SIGIRR) [10] and suppressor of cytokine signaling-1 (SOCS1) [11, 12] The latter, though, probably only dampens secondary (interferon--dependent) pathways [13, 14]. ATP has been shown to inhibit LPS-induced secretion of the proinflammatory cytokines TNF␣ and IL-12 [15,16,17], which raises the possibility that ATP may act, among other roles, as a “host tissue damage” signal for innate immune cells and inhibit TLR signaling in a negative feedback fashion To test this possibility and to understand better the effect of extracellular ATP on innate immunity, we have investigated the effects of nucleotide receptor activation on the migration, second messenger signaling, and cytokine secretion of human monocytes in the absence and presence of various TLR ligands
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