Andrea la Sala

Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions

IL-10 is a critical cytokine that blocks the maturation of dendritic cells (DCs), but the relevance of autocrine IL-10 on DC functions has not been investigated. In this study, we found that immature monocyte-derived DCs released low but sizeable amounts of IL-10. After stimulation with bacteria, LPS, lipoteichoic acid, or soluble CD40 ligand, DCs secreted high levels of IL-10. Addition of an anti-IL-10-neutralizing Ab to immature DCs as well as to soluble CD40 ligand- or LPS-maturing DCs led to enhanced expression of surface CD83, CD80, CD86, and MHC molecules and markedly augmented release of TNF-α and IL-12, but diminished IL-10 mRNA expression. Moreover, DCs treated with anti-IL-10 Ab showed an increased capacity to activate allogeneic T cells and primed naive T cells to a more prominent Th1 polarization. DC maturation and IL-10 neutralization were associated with enhanced accumulation of the IL-10 receptor binding chain (IL-10R1) mRNA and intracellular IL-10R1 protein. In contrast, surface IL-10R1 and IL-10 binding activity diminished in mature DCs. These results indicate that autocrine IL-10 prevents spontaneous maturation of DCs in vitro, limits LPS- and CD40-mediated maturation, and increases IL-10 production by DCs. Moreover, IL-10R expression appears to be regulated by both transcriptional and posttranscriptional mechanisms. Endogenous IL-10 and IL-10R can be relevant targets for the manipulation of DC functions.

Atrial fibrillation after isolated coronary surgery affects late survival.

Background— Atrial fibrillation (AF) after coronary artery bypass graft surgery is a difficult problem and a continuing source of morbidity and mortality. However, the prognostic implications of postoperative AF are still in dispute. Our aim was to ascertain the impact of AF after coronary artery bypass graft on postoperative survival and to assess its prognostic role in cause-specific mortality. Methods and Results— We conducted a prospective observational study of 1832 patients undergoing isolated coronary artery bypass graft between January 2000 and December 2005 at 2 cardiac surgery centers in northern Italy. Patients affected by postoperative AF were identified and followed up until death or study end (April 30, 2007). A total of 570 patients (31%) developed AF after coronary surgery. Patients affected by postoperative AF experienced a longer hospital stay (7 days [25th to 75th percentile, 7 to 10 days] versus 7 days [25th to 75th percentile, 6 to 8 days]; P<0.001). Hospital mortality also was higher in AF patients (3.3% versus 0.5%; P<0.001). On discharge, 1806 patients were alive; 143 were lost to follow-up. The remaining 1663 were followed up for a median of 51 months (25th to 75th percentile, 41 to 63 months); 126 of them died after a median of 14 months (25th to 75th percentile, 5 to 32 months). Long-term mortality rates were significantly higher for patients with postoperative AF (2.99 per 100 person-years; 95% confidence interval, 2.33 to 3.84; 61 deaths) compared with those without the arrhythmia (1.34 per 100 person-years; 95% confidence interval, 1.05 to 1.71; 65 deaths), with an adjusted hazard ratio of 2.13 (P<0.001) and 2.56 (P=0.001) when also accounting for the prescription of warfarin at discharge. With Cox regression, patients with AF were shown to be at higher risk of dying from embolism (adjusted hazard ratio, 4.33; 95% confidence interval, 1.78 to 10.52) but not from other causes. Conclusions— Postoperative AF affects early and late mortality after isolated coronary artery bypass graft surgery. Patients affected by AF are at higher risk of fatal embolic events. Careful postoperative surveillance with a specific antiarrhythmic and antithrombotic prophylaxis, aimed at reducing AF and its complications, is recommended.

Extracellular ATP Induces a Distorted Maturation of Dendritic Cells and Inhibits Their Capacity to Initiate Th1 Responses

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1α, IL-1β, TNF-α, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA+ T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-γ and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-α and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.

Alerting and tuning the immune response by extracellular nucleotides

The interplay between pro‐ and anti‐inflammatory mechanisms during inflammatory and immune responses is critical for avoiding excessive tissue damage. Extracellular nucleotides (e.g., adenosine 5′‐triphosphate) may represent constitutive signals that can alert the immune system of abnormal cell death. Relatively high doses of nucleotides induce rapid release of proinflammatory mediators and favor pathogen killing. However, recent findings on antigen presenting cells, particularly dendritic cells, revealed a more complex role for these molecules. Chronic exposure to low‐dose nucleotides can redirect cellular responses to prototypic activation stimuli, leading to suppressed inflammation and immune deviation.

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)

G(i)-protein-dependent inhibition of IL-12 production is mediated by activation of the phosphatidylinositol 3-kinase-protein 3 kinase B/Akt pathway and JNK.

Ligands for certain Gi-protein-coupled receptors (GiPCRs) potently inhibit the production of IL-12 by human monocytes. We addressed the intracellular signaling mechanisms by which this occurs using primary human cells. Stimulation with the GiPCR ligands C5a and 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purine-9-y1]-N-methyl-β-d-ribofuranuronamide (IB-MECA) blocked the production of IL-12 p70 by human monocytes stimulated with LPS and IFN-γ. In addition, C5a reduced the expression of mRNA for IL-12 p35, p40, IL-23 p19, and IL-27 p28. This effect was due neither to a down-regulation of TLR4 or IFN-γ receptor on the cell surface nor to interference with IFN-γ signaling, because IFN-γ-induced up-regulation of HLA-DR and CD40 were unaffected. C5a or IB-MECA activated the PI3K/Akt signaling pathway and induced the phosphorylation of the MAPK p38, ERK, and JNK. Inhibition of the PI3K/Akt signaling pathway with wortmannin or an inhibitor of Akt activity, and inhibition of JNK but not ERK prevented IL-12 and IL-23 suppression by C5a. These data extend observations on IL-12 suppression by C5a to IL-23 and IL-27, and are the first to demonstrate the intracellular signaling events leading to IL-12 and IL-23 inhibition after GiPCR activation.

Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis

Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high‐affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up‐regulates the expression of the anti‐apoptotic Bcl‐2 family members, Bcl‐2, Bcl‐XL, and Bfl‐1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen‐presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.

Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8.

Antibodies to Complement Receptor 3 Treat Established Inflammation in Murine Models of Colitis and a Novel Model of Psoriasiform Dermatitis

Prior studies indicated the ability of Abs to complement receptor 3 (CR3, CD11b/CD18) to suppress the production of IL-12 from immune cells. Therefore, we tested the ability of an anti-CR3 Ab (clone M1/70) to treat established IL-12-dependent Th1-mediated inflammation in murine models. Systemic administration of anti-CR3 significantly ameliorated established intestinal inflammation following the intrarectal administration of trinitrobenzene sulfonic acid (TNBS-colitis), as well as colitis and skin inflammation in C57BL/10 RAG-2−/− mice reconstituted with CD4+CD45RBhigh T cells. The hyperproliferative skin inflammation in this novel murine model demonstrated many characteristics of human psoriasis, and was prevented by the adoptive transfer of CD45RBlow T cells. In vitro and in vivo studies suggest that anti-CR3 treatment may act, at least in part, by directly inhibiting IL-12 production by APCs. Administration of anti-CR3 may be a useful therapeutic approach to consider for the treatment of inflammatory bowel disease and psoriasis in humans.

Activation of human eosinophils via P2 receptors: novel findings and future perspectives.

A growing body of information indicates that release of intracellular nucleotides represents an important way to modulate several cell pathways in physiological or pathological conditions. Nucleotides released as a consequence of cell damage, cell stress, bacterial infection, or other noxious stimuli signal at a class of plasma membrane receptors—P2 receptors—activating diverse intracellular pathways in many tissues and organs. For example, nucleotides secreted in the airway system control chloride/liquid secretion, goblet cell degranulation, and ciliary beat frequency. Several studies indicate that nucleotides play a role in airway diseases through their action on multiple cell types, including mast cells, dendritic cells, neurons, and eosinophils. Recent work by us and other groups led to the identification and characterization of P2 receptors expressed by human eosinophils. In this review, we will summarize recent developments in this field and put forward a hypothesis about the role of P2 receptors in pathophysiological conditions where eosinophils are major players.