Synergistic production of TNF\u3b1 and IFN\u3b1 by human pDCs incubated with IFN\u3bb3 and IL-3

In this study, we investigated whether IFN\u3bb3 and IL-3 reciprocally influence their capacity to activate various functions of human plasmacytoid dendritic cells (pDCs). In fact, we preliminarily observed that IFN\u3bb3 upregulates the expression of the IL-3R\u3b1 (CD123), while IL-3 augments the expression of IFN\u3bbR1 in pDCs. As a result, we found that combination of IFN\u3bb3 and IL-3 induces a strong potentiation in the production of TNF\u3b1, IFN\u3b1, as well as in the expression of Interferon-Stimulated Gene (ISG) mRNAs by pDCs, as compared to either IFN\u3bb3 or IL-3 alone. In such regard, we found that endogenous IFN\u3b1 autocrinally promotes the expression of ISG mRNAs in IL-3-, but not in IFN\u3bb3 plus IL-3-, treated pDCs. Moreover, we uncovered that the production of IFN\u3b1 by IFN\u3bb3 plus IL-3-treated pDCs is mostly dependent on endogenously produced TNF\u3b1. Altogether, our data demonstrate that IFN\u3bb3 and IL-3 collaborate to promote, at maximal levels, discrete functional responses of human pDCs

Generation of Biologically Active Angiostatin Kringle 1–3 by Activated Human Neutrophils

AbstractThe contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1–3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1–3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule

G-CSF–stimulated Neutrophils Are a Prominent Source of Functional BLyS

B lymphocyte stimulator (BLyS) is a novel member of the TNF ligand superfamily that is important in B cell maturation and survival. We demonstrate that human neutrophils, after incubation with G-CSF or, less efficiently, IFNγ, express high levels of BLyS mRNA and release elevated amounts of biologically active BLyS. In contrast, surface expression of the membrane-bound BLyS was not detected in activated neutrophils. Indeed, in neutrophils, uniquely among other myeloid cells, soluble BLyS is processed intracellularly by a furin-type convertase. Worthy of note, the absolute capacity of G-CSF–stimulated neutrophils to release BLyS was similar to that of activated monocytes or dendritic cells, suggesting that neutrophils might represent an important source of BLyS. In this regard, we show that BLyS serum levels as well as neutrophil-associated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. In addition, serum obtained from two of these patients induced a remarkable accumulation of neutrophil-associated BLyS in vitro. This effect was neutralized by anti–G-CSF antibodies, indicating that G-CSF, present in the serum, stimulated neutrophils to produce BLyS. Collectively, our findings suggest that neutrophils, through the production of BLyS, might play an unsuspected role in the regulation of B cell homeostasis

Macrophage polarization in health and disease

Macrophages are terminally differentiated cells of the mononuclear phagocyte system that also encompasses dendritic cells, circulating blood monocytes, and committed myeloid progenitor cells in the bone marrow. Both macrophages and their monocytic precursors can change their functional state in response to microenvironmental cues exhibiting a marked heterogeneity. However, there are still uncertainties regarding distinct expression patterns of surface markers that clearly define macrophage subsets, particularly in the case of human macrophages. In addition to their tissue distribution, macrophages can be functionally polarized into M1 (proinflammatory) and M2 (alternatively activated) as well as regulatory cells in response to both exogenous infections and solid tumors as well as by systems biology approaches. KEYWORDS: Macrophage, polarization, M1/M2, HIV, tumors, TAMs (tumor-associated macrophages), regulatory macrophages Correspondence should be addressed to Guido Poli, [email protected] Copyright © 2011 Luca Cassetta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Published by TheScientificWorldJOURNAL; http://www.tswj.com/ TheScientificWorldJOURNAL (2011) 11, 2391-2402 MACROPHAGE POLARIZATION: DOGMA OR REALITY? Proinflammatory, "classical Activation" of macrophages, which was delineated in early studies from the 1960s Indeed, "classically" and "alternatively" activated macrophages have been designated as "M1" and "M2" macrophages, respectively, by analogy to the Th1/Th2 division of labor of CD4 helper T cells A more flexible classification has been suggested recently by mouse studies in which macrophages are considered as part of a continuum having a range of overlapping functions and in which classically activated, wound-healing, and regulatory macrophages occupy different points of the spectrum MACROPHAGE PLASTICITY: AN OBSTACLE TO STUDY MACROPHAGE POLARIZATION? Unlike lymphocytes where phenotypic changes are largely "fixed" by chromatin modifications after exposure to polarizing cytokines, macrophages have a plastic gene expression profile that is influenced by the type, concentration, and longevity of exposure to the stimulating agents, as documented extensively There is a high number of factors contributing to diversity of macrophage function, including the synergistic or antagonistic effects of different cytokines and related signals on their differential expression, chemokines, hormones (including adrenergic and cholinergic agonists), TLR ligands, and other endogenous ligands (e.g., histamine, integrin ligands, peroxisome proliferator-activated receptor ligands, apoptotic cells); this plethora of signals underlines the fact that macrophages can display a large number of distinct, functional patterns that have not yet been completely defined. Furthermore, identical macrophages placed in different microenvironments display different functions in response to a common stimulus. Stimulation of macrophages with functionally opposite cytokines, such as IFN-γ and IL-4, initiates signal cascades that results in differential modulation (enhancement or inhibition) of different genes at the transcriptional or posttranscriptional level (e.g., stabilization or destabilization of mRNA). Unless the signal cascade triggered an apoptotic cascade, macrophages will eventually revert to their original, functional status after the cytokine signaling ceases. In vivo or in vitro treatment of macrophages with cytokines alters their functional response pattern to LPS. However, if the cytokines are washed away after incubation and macrophages are then maintained in the absence of cytokines for 1-2 days before LPS stimulation, the functional response pattern is usually identical to that of macrophages that had not been prestimulated with the cytokine. A similar reversion to basal macrophage phenotype is observed when IL-4 and granulocyte macrophage-colony-stimulating factor (GM-CSF) are removed from human monocyte-derived, immature dendritic cells (iDCs) and the cells are resuspended in a neutral environment Therefore, most Th1 and Th2 cytokines do not seem to induce a stable differentiation of macrophages into distinct subsets, but they rather promote a transient functional pattern of responses that return to basal levels in a few (3-7) days. MARKERS OF MACROPHAGE POLARIZATION: STILL AN OPEN CHASE One of the most debated issues in the context of human macrophage polarization is the identification of unique or restricted markers to be used for research and clinical purposes. Innovative approaches, including intravital imaging and other in vivo techniques, will be of great help in the identification of "real" subsets of macrophages in addition to more static antigens expressed on their cellular surface following cell polarization. An example of this broader approach is summarized by the identification of at least 6 different subsets of mouse tumor-associated macrophages (TAMs) based on their distinct functional features CONTRIBUTION OF MACROPHAGE POLARIZATION TO INNATE IMMUNE RESPONSES REGULATORY MACROPHAGES (RMs) As mentioned, diversity is a key feature of macrophage activation. In addition to M1 and M2 macrophages, RMs have recently emerged as an important population of cells that play a pivotal role in limiting inflammation during innate and adaptive immune responses Interestingly, a recent study has identified a subpopulation of Foxp3 + macrophages in the mouse 2394 TheScientificWorldJOURNAL (2011TheScientificWorldJOURNAL ( ) 11, 2391TheScientificWorldJOURNAL ( -2402 Manrique et al. reported that F4/80 + Foxp3 − cells could be converted into FoxP3 + cells by stimulation with TGF-β, Vascular Endothelial Growth Factor (VEGF), or TLR ligands Although cell activation is critical for the induction of an effective immune response to pathogens or tumors, inappropriate and sustained activation/polarization of macrophages leads to tissue damage, immune dysfunction, and disease. As with exacerbated M1 and M2 responses, dysfunctional regulatory responses contribute to tumor progression and growth (as discussed below) and can predispose the host to infection. Several pathogens including Staphylococcus aureus MACROPHAGE DIVERSITY IN SYSTEMS BIOLOGY Systems biology approaches have provided important insights into the heterogeneity of mononuclear phagocyte populations, the plasticity of macrophage activation, and the molecular pathways associated with polarization. Transcriptome profiling has been commonly used to examine networks of molecules and transcription factors linked to activation. Using this approach, Martinez et al. obtained a comprehensive global view of human macrophage polarization Epigenetic studies have begun to unravel how polarized macrophages acquire and maintain their activation phenotype. M2 genes in mice, including Chi3l3, Retnla, and Arg-1, were shown to be epigenetically regulated as a result of signal transducer and activator of transcription 6-(STAT6-) dependent induction of the H3K27 demethylase Jmjd3 Thus, systems biology will keep providing a constantly updated global view of the networks regulating or involved in macrophage polarization, allowing us to evaluate key issues related to macrophage heterogeneity and plasticity. 2395 TheScientificWorldJOURNAL (2011TheScientificWorldJOURNAL ( ) 11, 2391TheScientificWorldJOURNAL ( -2402 MACROPHAGE POLARIZATION IN CANCER BIOLOGY: A MATTER OF GOOD OR BAD EDUCATION All solid tumors recruit monocytes and local macrophages into their microenvironment making them TAM; it is being increasingly clear that TAMs play several, sometimes opposite, roles during tumor development. Originally it was believed that these cells were attempting to reject the immunologically non-self entity made of transformed cells (that frequently lose or modify their MHC profile). Indeed, macrophages can effectively kill tumor cells in vitro In conclusion, tumors can affect macrophages playing with their impressive plastic nature in order to modify the microenvironment and consequently alter the function and the strength of the cellular and innate immune response. These studies together provide useful models to investigate how cancer cells (and viruses, as discussed later on) crosstalk with macrophages; the potential application of this information will be the identification of soluble factors or inhibitors that will "reeducate" macrophages against pathogens and neoplastic lesions

mRNA expression and release of interleukin-8 induced by serum amyloid A in neutrophils and monocytes.

The acute phase response is a systemic reaction to inflammatory processes characterized by multiple physiological adaptations, including the hepatic synthesis of acute-phase proteins. In humans, serum amyloid A (SAA) is one of the most prominent of these proteins. Despite the huge increase of serum levels of SAA in inflammation, its biological role remains to be elucidated, even though SAA is undoubtedly active in neutrophils. In a previous study, we reported that SAA induces the release of tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-8 from human blood neutrophils. Here, we extend our earlier study, focusing on the effect of SAA on neutrophil IL-8 transcription and on the signaling pathways involved. We demonstrate herein that SAA, in relatively low concentrations (0.4-100 microg/ml) compared with those found in plasma in inflammatory conditions, induces a dose-dependent release of IL-8 from neutrophils. The p38 mitogen-activated protein kinase inhibitor SB 203580 inhibits the IL-8 mRNA expression and the release of protein from neutrophils. The release of IL-8 from SAA-stimulated neutrophils is strongly suppressed by the addition of N-acetyl-l-cysteine, alpha-mercaptoethanol, glutathione, and dexamethasone. SAA also induces IL-8 expression and release from monocytes. In conclusion, SAA appears to be an important mediator of the inflammatory process, possibly contributing to the pool of IL-8 produced in chronic diseases, which may play a role in degenerative diseases

RelB activation in anti-inflammatory decidual endothelial cells: a master plan to avoid pregnancy failure?

It is known that excessive inflammation at fetal-maternal interface is a key contributor in a compromised pregnancy. Female genital tract is constantly in contact with microorganisms and several strategies must be adopted to avoid pregnancy failure. Decidual endothelial cells (DECs) lining decidual microvascular vessels are the first cells that interact with pro-inflammatory stimuli released into the environment by microorganisms derived from gestational tissues or systemic circulation. Here, we show that DECs are hypo-responsive to LPS stimulation in terms of IL-6, CXCL8 and CCL2 production. Our results demonstrate that DECs express low levels of TLR4 and are characterized by a strong constitutive activation of the non-canonical NF-\u3baB pathway and a low responsiveness of the canonical pathway to LPS. In conclusion, DECs show a unique hypo-responsive phenotype to the pro-inflammatory stimulus LPS in order to control the inflammatory response at feto-maternal interface

Myeloid cells, BAFF, and IFN-γ establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient mice

Autoimmunity is traditionally attributed to altered lymphoid cell selection and/or tolerance, whereas the contribution of innate immune cells is less well understood. Autoimmunity is also associated with increased levels of B cell–activating factor of the TNF family (BAFF; also known as B lymphocyte stimulator), a cytokine that promotes survival of self-reactive B cell clones. We describe an important role for myeloid cells in autoimmune disease progression. Using Lyn-deficient mice, we show that overproduction of BAFF by hyperactive myeloid cells contributes to inflammation and autoimmunity in part by acting directly on T cells to induce the release of IFN-γ. Genetic deletion of IFN-γ or reduction of BAFF activity, achieved by either reducing myeloid cell hyperproduction or by treating with an anti-BAFF monoclonal antibody, reduced disease development in lyn−/− mice. The increased production of IFN-γ in lyn−/− mice feeds back on the myeloid cells to further stimulate BAFF release. Expression of BAFF receptor on T cells was required for their full activation and IFN-γ release. Overall, our data suggest that the reciprocal production of BAFF and IFN-γ establishes an inflammatory loop between myeloid cells and T cells that exacerbates autoimmunity in this model. Our findings uncover an important pathological role of BAFF in autoimmune disorders

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBP beta, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kappa B (NF-kappa B) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation

Helicobacter pylori-derived neutrophil-activating protein increases the lifespan of monocytes and neutrophils

P>An invariable feature of Helicobacter pylori-infected gastric mucosa is the persistent infiltration of inflammatory cells. The neutrophil-activating protein (HP-NAP) has a pivotal role in triggering and orchestrating the phlogistic process associated with H. pylori infection. Aim of this study was to address whether HP-NAP might further contribute to the inflammation by increasing the lifespan of inflammatory cells. We report that HP-NAP is able to prolong the lifespan of monocytes, in parallel with the induction of the anti-apoptotic proteins A1, Mcl-1, Bcl-2 and Bcl-X(L). This effect does not result from a direct action on the apoptotic machinery, but rather it requires the release of endogenous pro-survival factors, such as interleukin-1 beta, which probably acts in synergy with other unidentified mediators. We also report that HP-NAP promotes the survival of Ficoll-purified neutrophils in a monocyte-dependent fashion: indeed, mononuclear cell depletion of Ficoll-purified neutrophils completely abolished the pro-survival effect by HP-NAP. In conclusion, our data reinforce the notion that HP-NAP has a pivotal role in sustaining a prolonged activation of myeloid cells