Chemotactic activity of extracellular nucleotideson human immune cells.

Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation.     

HMGB1 and cord blood: its role as immuno-adjuvant factor in innate immunity

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4(+) T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.     

Innate immunity: structure and function of TLRs

The innate immune system provides the first line of defence against infection. Through a limited number of germline-encoded receptors called pattern recognition receptors (PRRs), innate cells recognize and are activated by highly conserved structures expressed by large group of microorganisms called pathogen-associated molecular patterns (PAMPs). PRRs are involved either in recognition (scavenger receptors, C-type lectins) or in cell activation (Toll-like receptors or TLR, helicases and NOD molecules). TLRs play a pivotal role in cell activation in response to PAMPs. TLR are type I transmembrane proteins characterized by an intracellular Toll/IL 1 receptor homology domain that are expressed by innate immune cells (dendritic cells, macrophages, NK cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). In all the cell types analyzed, TLR agonists, alone or in combination with costimulatory molecules, induce cell activation. The crucial role played by TLR in immune cell activation has been detailed in dendritic cells. A TLR-dependent activation of dendritic cells is required to induce their maturation and migration to regional lymph nodes and to activate na?ve T cells. The ability of different cell types to respond to TLR agonists is related to the pattern of expression of the TLRs and its regulation as well as their intracellular localization. Recent studies suggest that the nature of the endocytic and signaling receptors engaged by PAMPs may determine the nature of the immune response generated against the microbial molecules, highlighting the role of TLRs as molecular interfaces between innate and adaptive immunity. In this review are summarized the main biological properties of the TLR molecules.     

3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.     

Cathelicidin antimicrobial peptides block dendritic cell TLR4 activation and allergic contact sensitization

Cathelicidins are antimicrobial peptides of the innate immune system that establish an antimicrobial barrier at epithelial interfaces and have been proposed to have a proinflammatory function. We studied the role of cathelicidin in allergic contact dermatitis, a model requiring dendritic cells of the innate immune response and T cells of the adaptive immune response. Deletion of the murine cathelicidin gene Cnlp enhanced an allergic contact response, whereas local administration of cathelicidin before sensitization inhibited the allergic response. Cathelicidins inhibited TLR4 but not TLR2 mediated induction of dendritic cell maturation and cytokine release, and this inhibition was associated with an alteration of cell membrane function and structure. Further analysis in vivo connected these observations because inhibition of sensitization by exogenous cathelicidin was dependent on the presence of functional TLR4. These observations provide evidence that cathelicidin antimicrobial peptides mediate an anti-inflammatory response in part by their activity at the membrane.     

High mobility group B1 protein suppresses the human plasmacytoid dendritic cell response to TLR9 agonists

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-alpha, IL-6, TNF-alpha, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-gamma-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.     

CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation

The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.     

Adhesion molecules: Key players in Mesenchymal stem cell-mediated immunosuppression

Adhesion molecules are known to -be important components of an active T-cell mediated immune response. Signals generated at a site of inflammation cause circulating T cells to respond by rolling, arrest and then transmigration through the endothelium, all of which are mediated by adhesion molecules. Consequently, strategies have been developed to treat immune disorders with specific antibodies that block the interaction of adhesion molecules. However, the therapeutic effects of such remedies are not always achieved. Our recent investigations have revealed that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) work together with chemokines to induce immunosuppression mediated by Mesenchymal stem cells (MSCs), thus demonstrating the dual role of adhesion molecules in immune responses. Since MSCs represent an important component of the stromal cells in an inflammatory microenvironment, our findings provide novel information for understanding the regulation of immune responses and for designing new strategies to treat immune disorders.Adhesion molecules are cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM). There are four families of adhesion molecules: immunoglobulin-like adhesion molecules, integrins, cadherins and selectins. Most of them are typical transmembrane proteins that have cytoplasmic, transmembrane and extracellular domains. In the immune system, cell adhesion plays a critical role in initiating and sustaining an effective immune response against foreign pathogens.¹ Based on our recent data, we discuss herein the role of immunoglobulin superfamily cell adhesion molecules, ICAM1 and VCAM-1, in the immunosuppression mediated by Mesenchymal stem cells.     

A tumor-associated glycoprotein that blocks MHC class II-dependent antigen presentation by dendritic cells

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.     

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrowderived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCsandTh2lymphocytes, (iii) proliferation ofTh2cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.     

Neutrophils, dendritic cells and Toxoplasma

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.     

Liposomal vaccines--targeting the delivery of antigen

Vaccines that can prime the adaptive immune system for a quick and effective response against a pathogen or tumor cells, require the generation of antigen (Ag)-specific memory T and B cells. The unique ability of dendritic cells (DCs) to activate na?ve T cells, implies a key role for DCs in this process. The generation of tumor-specific CD8(+) cytotoxic T cells (CTLs) is dependent on both T cell stimulation with Ag (peptide-MHC-complexes) and costimulation. Interestingly, tumor cells that lack expression of T cell costimulatory molecules become highly immunogenic when transfected to express such molecules on their surface. Adoptive immunotherapy with Ag-pulsed DCs also is a strategy showing promise as a treatment for cancer. The use of such cell-based vaccines, however, is cumbersome and expensive to use clinically, and/or may carry risks due to genetic manipulations. Liposomes are particulate vesicular lipid structures that can incorporate Ag, immunomodulatory factors and targeting molecules, and hence can serve as potent vaccines. Similarly, Ag-containing plasma membrane vesicles (PMV) derived from tumor cells can be modified to incorporate a T cell costimulatory molecule to provide both TCR stimulation, and costimulation. PMVs also can be modified to contain IFN-gamma and molecules for targeting DCs, permitting delivery of both Ag and a DC maturation signal for initiating an effective immune response. Our results show that use of such agents as vaccines can induce potent anti-tumor immune responses and immunotherapeutic effects in tumor models, and provide a strategy for the development of effective vaccines and immunotherapies for cancer and infectious diseases.     

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.     

Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine.

The absence of surface costimulatory molecules explains in part the lack of an effective anti-tumor immune response in tumor-bearing animals, even though unique tumor antigens may be presented by class I MHC. We determined that the immunogenicity of a murine neuroblastoma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electrically induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized mice were significantly protected from challenge with Neuro-2a. We propose that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means to produce patient-specific tumor vaccines.     

LIMLE,a New Molecule Over-Expressed following Activation,Is Involved in the Stimulatory Properties of Dendritic Cells

Dendritic cells are sentinels of the immune system distributed throughout the body, that following danger signals will migrate to secondary lymphoid organs to induce effector T cell responses. We have identified, in a rodent model of graft rejection, a new molecule expressed by dendritic cells that we have named LIMLE (RGD1310371). To characterize this new molecule, we analyzed its regulation of expression and its function. We observed that LIMLE mRNAs were rapidly and strongly up regulated in dendritic cells following inflammatory stimulation. We demonstrated that LIMLE inhibition does not alter dendritic cell maturation or cytokine production following Toll-like-receptor stimulation. However, it reduces their ability to stimulate effector T cells in a mixed leukocyte reaction or T cell receptor transgenic system. Interestingly, we observed that LIMLE protein localized with actin at some areas under the plasma membrane. Moreover, LIMLE is highly expressed in testis, trachea, lung and ciliated cells and it has been shown that cilia formation bears similarities to formation of the immunological synapse which is required for the T cell activation by dendritic cells. Taken together, these data suggest a role for LIMLE in specialized structures of the cytoskeleton that are important for dynamic cellular events such as immune synapse formation. In the future, LIMLE may represent a new target to reduce the capacity of dendritic cells to stimulate T cells and to regulate an immune response.     

RUFY4: Immunity piggybacking on autophagy?

Although autophagy is a highly conserved mechanism among species and cell types, few are the molecules involved with the autophagic process that display cell- or tissue- specific expression. We have unraveled the positive regulatory role on autophagy of RUFY4 (RUN and FYVE domain containing 4), which is expressed in subsets of immune cells, including dendritic cells (DCs). DCs orchestrate the eradication of pathogens by coordinating the action of the different cell types involved in microbe recognition and destruction during the immune response. To fulfill this function, DC display particular regulation of their endocytic and autophagy pathways in response to the immune environment. Autophagy flux is downmodulated in DCs upon microbe sensing, but is remarkably augmented, when cells are differentiated in the presence of the pleiotropic cytokine IL4 (interleukin 4). From gene expression studies aimed at comparing the impact of IL4 on DC differentiation, we identified RUFY4, as a novel regulator that augments autophagy flux and, when overexpressed, induces drastic membrane redistribution and strongly tethers lysosomes. RUFY4 is therefore one of the few known positive regulators of autophagy that is expressed in a cell-specific manner or under specific immunological conditions associated with IL4 expression such as allergic asthma.     

Understanding the cell biology of antigen presentation: the dendritic cell contribution

The study of the cell biology of antigen processing and presentation has greatly contributed to our understanding of the immune response. The work of many immunologically inclined cell biologists has also permitted us to gain new insights on cellular mechanisms shared by many cell types. Dendritic cells are master regulators of the immune system and consequently have received a lot of attention in recent years. With the aim of controlling antigen processing and presentation, the solutions used by dendritic cells to respond to environmental changes are numerous and surprising. In the presence of pathogens, dendritic cells regulate strongly their endocytic pathway by interfering with uptake, proteolysis, membrane dynamics and transport in and out of the lysosome to become the most potent antigen-presenting cells known.     

Reactive oxygen species activate human peripheral blood dendritic cells

This study investigates the effects of hydrogen peroxide, a potent oxygen free radical donor, on the phenotype and function of dendritic cells differentiated from peripheral blood precursors. We report that hydrogen peroxide induces an up-regulation of several dendritic cell surface markers involved in interaction with T cells, including MHC Class II molecules (DQ and DR) and the co-stimulatory molecules CD40 and CD86. Moreover we have observed that H₂O₂-treated dendritic cells are more efficient in promoting T cell proliferation than normal dendritic cells and that this enhancement can be blocked using the free radical scavenger agent N-acetylcysteine. Oxygen free radicals are a common by-product of inflammation, and our results suggest they may play an important role in activation of sentinel dendritic cells, linking tissue damage to the initiation of an adaptive immune response.     

Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune responses efficiently, have developed a unique membrane transport pathway linking the lumen of endocytic compartments and the cytosol. Endosome-to-cytosol transport is restricted to dendritic cells, specific to internalized antigens and selective for the size of the transported molecules. Thus, in dendritic cells, internalized antigens gain access to the cytosolic antigen-processing machinery and to the conventional MHC class I antigen-presentation pathway.     

Mechanisms of cellular communication through intercellular protein transfer

In a multicellular system, cellular communication is a must for orchestration and coordination of cellular events. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the intercellular communication. An intercellular exchange of proteins or intact membrane patches is a ubiquitous phenomenon, and has been the subject of renewed interest, particularly in the context of immune cells. Recent evidence implicates that intercellular protein transfers, including trogocytosis is an important mechanism of the immune system to modulate immune responses and transferred proteins can also contribute to pathology. It has been demonstrated that intercellular protein transfer can be through the internalization/pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we will discuss the important findings concerning intercellular protein transfers, possible mechanisms and highlight their physiological relevance to the immune system, with special reference to T cells such as the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules.