IL-18 associates to microvesicles shed from human macrophages by a LPS/TLR-4 independent mechanism in response to P2X receptor stimulation.

Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL-18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist 3'-benzoylbenzoyl adenosine 5'-triphosphate, while it was greatly reduced by P2X irreversible inhibitor-oxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003, and A-438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca(2+) ionophore A23187 mimicked the effect of 3'-benzoylbenzoyl adenosine 5'-triphosphate suggesting that an intracellular Ca(2+) increase was sufficient to evoke MV shedding. Caspase inhibitors Ac-YVAD-CMK or Z-YVAD-CMK did not block the cleavage of MV-associated pro-IL-18. Pro-IL-18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor with the TLR-4 intracellular inhibitor CLI-095. These data reveal a nucleotide-based mechanism responsible for the shedding of MV to which IL-18 is associated.

Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation.

It has been reported that ATP inhibits or stimulates lymphoid cell proliferation depending on the cellular subset analyzed. In this study, we show that ATP exerts strikingly opposite effects on anti-CD3/CD28-activated and regulatory CD4(+) T cells (T(regs)), based on nucleotide concentration. We demonstrate that physiological concentrations of extracellular ATP (1-50 nM) do not affect activated CD4(+) T cells and T(regs). Conversely, higher ATP concentrations have a bimodal effect on activated CD4(+) T cells. Whereas 250 nM ATP stimulates proliferation, cytokine release, expression of adhesion molecules, and adhesion, 1 mM ATP induces apoptosis and inhibits activated CD4(+) T cell functions. The expression analysis and pharmacological profile of purinergic P2 receptors for extracellular nucleotides suggest that activated CD4(+) T cells are induced to apoptosis via the upregulation and engagement of P2X7R and P2X4R. On the contrary, 1 mM ATP enhances proliferation, adhesion, migration, via P2Y2R activation, and immunosuppressive ability of T(regs). Similar results were obtained when activated CD4(+) T cells and T(regs) were exposed to ATP released by necrotized leukemic cells. Taken together, our results show that different concentrations of extracellular ATP modulate CD4(+) T cells according to their activated/regulatory status. Because extracellular ATP concentration highly increases in fast-growing tumors or hyperinflamed tissues, the manipulation of purinergic signaling might represent a new therapeutic target to shift the balance between activated CD4(+) T cells and T(regs).

Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice.

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.

Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines.

OBJECTIVE: Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions. MATERIALS AND METHODS: ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology. Clonogenic and migration assays in vitro, as well as xenotransplant experiments in vivo, were performed to evaluate the effects of ATP on hMSCs proliferation and BM homing. Enzyme-linked immunosorbent assays were used to assess hMSCs cytokines production, whereas T-cell cultures demonstrated the immunoregulatory activity of ATP-treated hMSCs. RESULTS: hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP-stimulated hMSCs underwent a downregulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly upregulated. The inhibitory activity of ATP on hMSCs proliferation was confirmed by assessing clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, the homing capacity of hMSCs to the BM of immunodeficient mice was significantly increased by pretreatment with ATP. Moreover, ATP increased the production of the proinflammatory cytokines interleukin-2, interferon-γ, and interleukin-12p70, while decreasing the anti-inflammatory cytokine interleukin-10, and this finding was associated with the reduced ability of MSCs to inhibit T-cell proliferation. CONCLUSIONS: Our data show that purinergic signaling modulates hMSCs functions and highlights a role for extracellular nucleotides in hMSCs biology.

ATP secreted by endothelial cells blocks CX3CL 1-elicited natural killer cell chemotaxis and cytotoxicity via P2Y11 receptor activation.

Endothelial cells (ECs) represent a major source of actively secreted adenosine triphosphate (ATP). Natural killer (NK) cells can mediate vascular injury in several pathologic conditions, including cytomegalovirus infection and vascular leak syndrome. We studied NK-cell expression of P2 receptors and the role of these nucleotide receptors in the regulation of endothelial-NK cell cross-talk. NK cells from healthy subjects expressed P2Y(1,2,4,6,11,12,13,14) and P2X(1,4,5,6,7) receptors. NK cells stimulated with ATP, but not uridine triphosphate, increased intracellular Ca²(+) and chemokinesis. Moreover, ATP, but not uridine triphosphate, inhibited NK chemotaxis in response to CX3CL1, whereas chemotaxis to CXCL12 was increased. CX3CL1 elicited killing of human umbilical vein ECs and human coronary artery ECs by NK cells. However, in the presence of ATP, CX3CL1 failed to stimulate killing of ECs. Such inhibitory effect was lost on exogenous addition of the ATP-hydrolyzing enzyme apyrase or by pharmacologic inhibition of the P2Y11R, and correlated with increased intracellular cyclic adenosine monophosphate concentrations induced by ATP or other P2Y11R agonists, including NAD(+). Extracellular ATP regulates NK-cell cytotoxicity via P2Y11R activation, protecting ECs from CX3CL1-elicited NK cell-mediated killing. These findings point out the P2Y11R as a potential target for pharmacologic intervention aimed at reducing NK-mediated vascular injury.

Extracellular ATP acting at the P2X7 receptor inhibits secretion of soluble HLA-G from human monocytes.

Bacterial LPS induces the release of ATP from immune cells. Accruing evidence suggests that extracellular ATP participates in the inflammatory response as a proinflammatory mediator by activating the inflammasome complex, inducing secretion of cytokines (IL-1, IL-18) and cell damaging agents such as oxygen radicals, cationic proteins, and metalloproteases. It is not known whether ATP can also act as a proinflammatory mediator by inhibiting production of molecules down-modulating the immune response. Here, we show that extracellular ATP impairs in an IL-10-dependent fashion the expression of the tolerogenic soluble and membrane-bound HLA-G Ag in human monocytes. The effect of ATP was mimicked by BzATP (3'-O-(4-benzoyl)benzoyl-ATP) and greatly reduced by pretreatment with oATP (periodate-oxidized ATP), KN-62 (1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine), and an anti-P2X(7) mAb, thus pointing to a specific role of the P2X(7) receptor. The effect of ATP was time- and dose-dependent and was not due to a decrease in expression of IL-10 receptor. Inhibition by ATP was reverted by supplementation of culture medium with exogenous IL-10. Due to the well-known immunosuppressive activity of IL-10 and soluble HLA-G, this novel effect of ATP might be relevant for the pathophysiology and therapy of inflammatory disorders.

The human cathelicidin LL-37 modulates the activities of the P2X7 receptor in a structure-dependent manner.

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X(7) receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X(7) blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-d analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X(7) was analyzed using P2X(7)-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca(2+) influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X(7). The activity of LL-37 had an absolute requirement for P2X(7) expression as it was blocked by the P2X(7) inhibitor KN-62, was absent in cells lacking P2X(7), and was restored by P2X(7) transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X(7) receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X(7) is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X(7).

Increased P2X7 receptor expression and function in thyroid papillary cancer: a new potential marker of the disease?

Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.

Stimulation of purinergic receptors modulates chemokine expression in human keratinocytes.

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-gamma-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5'-O-3-thiotriphosphate, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-gamma-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3'-phosphate 5'-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation.

The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration.

Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Galphai) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5'-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase-activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Galphai proteins and RhoGTPases.

Extracellular adenosine 5'-triphosphate modulates interleukin-6 production by human thyrocytes through functional purinergic P2 receptors.

We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X(3), P2X(5), P2X(6), P2X(7), and P2Y(1), P2Y(2), P2Y(4), and P2Y(11). Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca(2+) signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca(2+) ([Ca(2+)](i)) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca(2+)](i)) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.