Anna Morelli

Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses

Sphingosine 1‐phosphate (S1P) is a potent extracellular lysolipid phosphoric acid mediator that is released after IgE‐stimulation of mast cells. Here we investigated the biological activity and intracellular signaling of S1P on human dendritic cells (DC), which are specialized antigen presenting cells with the ability to migrate into peripheral tissues and lymph nodes, as well as control the activation of naive T cells. We show that immature and mature DC express the mRNA for different S1P receptors, such as endothelial differentiation gene (EDG)‐1, EDG‐3, EDG‐5, and EDG‐6. In immature DC, S1P stimulated pertussis toxin‐sensitive Ca2+ increase actin‐polymerization and chemotaxis. These responses were lost by DC matured with lipopolysaccharide. In maturing DC, however, S1P inhibited the secretion of tumor necrosis factor α and interleukin (IL)‐12, whereas it enhanced secretion of IL‐10. As a consequence, mature DC exposed to S1P showed a reduced and increased capacity to generate allogeneic Th1 and Th2 responses, respectively. In summary, our study implicates that S1P might regulate the trafficking of DC and ultimately favor Th2 lymphocyte‐dominated immunity.

The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release

We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca2+, membrane depolarization, and secretion of inflammatory cytokines. The ATP‐activated Ca2+ change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X7, as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X7 receptor ligand 2′,3′‐(4‐benzoylbenzoyl)‐ATP was a better agonist then ATP for Ca2+increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X7 antagonist KN‐62 inhibited both permeabilization and Ca2+ changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y1, P2Y2, P2Y5, P2Y11 and P2X1, P2X4, P2X7. Finally, stimulation of LPS‐matured cells with ATP triggered release of IL‐1 β and TNF‐a. Purinoceptors may provide a new avenue to modulation of dendritic cells function.—Ferrari, D., La Sala, A., Chiozzi, P., Morelli, A., Falzoni, S., Girolomoni, G., Idzko, M., Dichmann, S., Norgauer, J., Di Virgilio, F. The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release. FASEB J. 14, 2466–2476 (2000)

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells

The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. The A3 subtype was found by means of RT‐PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]‐MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 1.9±0.2 nM and Bmax of 1.3±0.1 pmol mg−1 of protein. The pharmacological profile of [3H]‐MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that [3H]‐MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy‐ and enthalpy‐driven, according with that found in cells expressing the recombinant human A3 subtype. In functional assays the high affinity A3 agonists Cl‐IB‐MECA and IB‐MECA were able to inhibit cyclic AMP accumulation and stimulate Ca2+ release from intracellular Ca2+ pools followed by Ca2+ influx. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]‐DPCPX binding with a KD of 0.9±0.1 nM and Bmax of 42±3 fmol mg−1 of protein. A2A receptors were studied with [3H]‐SCH 58261 binding and revealed a KD of 2.5±0.3 nM and a Bmax of 1.4±0.2 pmol mg−1 of protein. In conclusion, by means of the first antagonist radioligand [3H]‐MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

High glucose modulates P2X7 receptor-mediated function in human primary fibroblasts

Aims/hypothesis. Purinergic receptors are a family of newly characterized plasma membrane molecules involved in several and as yet only partially known cellular functions such as vascular reactivity, apoptosis and cytokine secretion. Little is known about the effect extracellular microenvironment has on their function. Fibroblasts share several features with smooth muscle cells and are an important constituent of the atherosclerotic plaque. Our aim was to evaluate the effect of high glucose concentration on ATP-mediated responses in human fibroblasts.¶Methods. Fibroblasts were obtained by skin biopsies and grown at two different glucose concentrations. We evaluated receptor expression by RT-PCR and immunoblotting and receptor localization by immunofluorescence. Plasma membrane potential and calcium changes were measured by fluorescent indicators. Apoptosis was determined by ethidium bromide staining and caspase-3 activation.¶Results. We show that cells grown in a medium with high glucose concentration underwent great ATP-mediated morphological changes, enhanced apoptosis, caspase 3 activation and interleukin-6 release. We identified P2X7 as the main purinergic receptor involved in these responses. Furthermore, high glucose concentration triggered the assembly of P2X7 into ring-like structures located at the periphery of the cells.¶Conclusion/interpretation. Given that ATP is frequently released into the extracellular milieu upon cell and tissue damage, secretory exocytosis or activation of plasma membrane transporters, we hypothesize that ATP receptors participate in the pathogenesis of vascular complications of diabetes. [Diabetologia (2000) 43: 1248–1256]

Enhanced P2X7 Activity in Human Fibroblasts From Diabetic Patients A Possible Pathogenetic Mechanism for Vascular Damage in Diabetes

Objective—We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes. Methods and Results—Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3× as much ATP into the supernatants compared with fibroblasts from healthy subjects. Conclusions—We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Defective P2Y purinergic receptor function: a possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT‐PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2‐deoxy‐D‐[1‐3H]glucose (2‐DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X‐mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co‐incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two‐fold larger amount of ATP compared to controls. Pre‐treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y‐dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes. J. Cell. Physiol. 197: 435–444, 2003© 2003 Wiley‐Liss, Inc.