Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y2 receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing

Microparticle formation is independent of intracellular calcium levels.

<p><i>Panels A–C</i>: HEK293-NK1R cells were loaded with fluo-4 and fura red in (A) HBSS containing Ca²⁺/Mg²⁺, (B) Ca²⁺/Mg²⁺ free HBSS with EDTA, or (C) Ca²⁺/Mg²⁺ free HBSS with EDTA and BAPTA-AM. Cells were then stimulated with SP and intracellular calcium levels were measurement by flow cytometry. Cells incubated with EDTA and EDTA and BAPTA showed very low levels of intracellular calcium, indicating that calcium was effectively chelated and they responded to SP treatment with very small intracellular calcium increase. <i>Panel D)</i> Microparticles were counted by flow cytometry 10 minutes after addition of SP. The number of microparticles was relatively unaffected by EDTA and BAPTA, indicating that this response is independent of intracellular calcium levels.</p

Extracellular Nucleotide UTP Regulate Lipopolisaccharide-Induced Cytokine Production in Human Neutrophils.

Abstract Extracellular nucleotides can function as paracrine and autocrine mediators that signal through P2Y nucleotide receptors. Human polymorphonuclear (PMN) cells express P2Y receptors on the cell surface but the role of these G protein-coupled receptors in human neutrophils is not well established. Interleukin-8 (IL-8), which is a CXC-chemokine that induces adherence of neutrophils to vascular endothelium and extravasation into tissue, is produced by macrophages and endothelium. Additionally, neutrophils can synthesize and release interleukin-8 upon stimulation with lipopolisacharides (LPS) but the intracellular signaling pathways involved in this process are not clearly understood. LPS is a potent activator of macrophages and is responsible for the initiation the septic shock, increasing the secretion of various cytokines. Here we present evidence that LPS (1 ng/ml to 100 ng/ml) causes IL-8 production in a dose- and time- dependent manner and demonstrate that LPS itself is responsible for mitogen-activated protein kinase (MAPK) ERK1/2 phosphorylation. We used an enzyme immunosorbent-linked assay (ELISA) to determinate the amount of IL-8 released by isolated human neutrophils and present evidence that extracellular nucleotide uridine triphosphate (UTP) (1 μM/ml to 100 μM/ml) can inhibit IL-8 production in human neutrophils in a dose- and time- dependent manner. The addition of ATP had a similar inhibitory effect suggesting that nucleotides act on neutrophils via the P2Y2 receptor, which is equally activated by UTP and ATP. A cytokine array was used to detect the release of 42 different cytokines in the extracellular medium. IL-8 and IL-6 ere the major cytokines detected in the medium, though other cytokines were detected in lower concentrations. In the current study, we demonstrated that extracellular nucleotides may have an important role in regulating IL-8 production in neutrophils and potentially modulating the inflammatory response.</jats:p

Substance P–mediated chemokine production promotes monocyte migration

Abstract The neuropeptide SP has physiologic and pathophysiologic roles in CNS and peripheral tissues and is involved in crosstalk between nervous and immune systems in various conditions, including HIV and SIV infection. Increased SP levels were demonstrated in plasma of HIV+ individuals as well as in the CNS of SIV-infected, nonhuman primates. SP increases HIV infection in macrophages through interaction with its receptor, NK1R. The SP effect on immune system is both pro- and anti-inflammatory and includes up-regulation of a number of cytokines and cell receptors. The main goal of this study was to determine whether there is interplay between monocyte exposure to SP and recruitment into sites of inflammation. We now demonstrate that exposure of either human macrophages or PBMCs to SP leads to increased production of chemokines, including MCP-1, for which expression is limited to cells of the myeloid lineage. This effect is inhibited by the NK1R antagonist, aprepitant. Exposure to conditioned medium derived from SP-treated PBMCs resulted in increased monocyte migration through semipermeable membranes and an in vitro human BBB model. Monocyte migration was blocked by anti–MCP-1 antibodies. Our results suggest that increased SP levels associated with HIV and other inflammatory conditions may contribute to increased monocyte migration into the CNS and other tissues through a MCP-1–dependent mechanism.</jats:p