Signaling Through Purinergic Receptor P2Y2 Enhances Macrophage IL-1ß Production.

The release of nucleotides during necrosis or apoptosis has been described to have both proinflammatory and anti-inflammatory effect on the surrounding cells. Here we describe how low concentrations of UTP and ATP applied during macrophage priming enhance IL-1ß production when subsequently the NLRP3 inflammasome is activated in murine resident peritoneal macrophages. Deficiency or pharmacological inhibition of the purinergic receptor P2Y2 reverted the increase of IL-1ß release induced by nucleotides. IL-1ß increase was found dependent on the expression of Il1b gene and probably involving JNK activity. On the contrary, nucleotides decreased the production of a different proinflammatory cytokines such as TNF-α. These results suggest that nucleotides could shape the response of macrophages to obtain a unique proinflammatory signature that might be relevant in unrevealing specific inflammatory conditions.

Essential role of Elmo1 in Dock2-dependent lymphocyte migration.

Elmo1 and Elmo2 are highly homologous cytoplasmic adaptor proteins that interact with Dock family guanine nucleotide exchange factors to promote activation of the small GTPase Rac. In T lymphocytes, Dock2 is essential for CCR7- and CXCR4-dependent Rac activation and chemotaxis, but the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. In this article, we show that endogenous Elmo1, but not Elmo2, interacts constitutively with Dock2 in mouse and human primary T cells. CD4(+) T cells from Elmo1(-/-) mice were profoundly impaired in polarization, Rac activation, and chemotaxis in response to CCR7 and CXCR4 stimulation. Transfection of full-length Elmo1, but not Elmo2 or a Dock2-binding mutant of Elmo1, rescued defective migration of Elmo1(-/-) T cells. Interestingly, Dock2 protein levels were reduced by 4-fold in Elmo1(-/-) lymphocytes despite normal levels of Dock2 mRNA. Dock2 polyubiquitination was increased in Elmo1(-/-) T cells, and treatment with proteasome inhibitors partially restored Dock2 levels in Elmo1(-/-) T cells. Finally, we show that Dock2 is directly ubiquitinated in CD4(+) T cells and that Elmo1 expression in heterologous cells inhibits ubiquitination of Dock2. Taken together, these findings reveal a previously unknown, nonredundant role for Elmo1 in controlling Dock2 levels and Dock2-dependent T cell migration in primary lymphocytes. Inhibition of Dock2 has therapeutic potential as a means to control recruitment of pathogenic lymphocytes in diseased tissues. This work provides valuable insights into the molecular regulation of Dock2 by Elmo1 that can be used to design improved inhibitors that target the Elmo-Dock-Rac signaling complex.

ß-Defensin 2 and 3 promote the uptake of self or CpG DNA, enhance IFN-α production by human plasmacytoid dendritic cells, and promote inflammation.

Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. In this article, we present a novel role for two potent alarmins, human ß-defensin 2 and 3 (HBD2 and 3), in promoting IFN-α production by human plasmacytoid dendritic cells. We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA-induced IFN-α production in a TLR9-dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal titration calorimetry studies to elucidate the nature of HBD3/CpG complexes demonstrate involvement of enthalpy-driven interactions, in addition to hydrophobic interactions, with the formation of complexes at a molar ratio of 2:1 defensin/CpG. The i.v. administration of HBD3/CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α, and IL-10 in serum, associated with an increased recruitment of APCs in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses to OVA, compared with OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus, the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells, resulting in an enhanced immune response.

High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses.

Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses. HMGN1 induced DC maturation via TLR4 (Toll-like receptor 4), recruitment of APCs at sites of injection, and activation of NF-κB and multiple mitogen-activated protein kinases in DCs. HMGN1 promoted antigen-specific immune response upon co-administration with antigens, and Hmgn1(-/-) mice developed greatly reduced antigen-specific antibody and T cell responses when immunized with antigens in the presence of lipopolysaccharide (LPS). The impaired ability of Hmgn1(-/-) mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from nonleukocytes was critical for the induction of antigen-specific antibody and T cell responses. Thus, extracellular HMGN1 acts as a novel alarmin critical for LPS-induced development of innate and adaptive immune responses.

Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin.

Granulysin (GNLY), an antimicrobial protein present in the granules of human cytotoxic T lymphocytes and natural killer (NK) cells, is produced as an intact 15-kDa form that is cleaved to yield a 9-kDa form. Alarmins are endogenous mediators that can induce recruitment and activation of antigen-presenting cells (APCs) and consequently promote the generation of immune response. We hypothesized that GNLY might function as an alarmin. Here, we report that both 9- and 15-kDa forms of recombinant GNLY-induced in vitro chemotaxis and activation of both human and mouse dendritic cells (DCs), recruited inflammatory leucocytes, including APCs in mice, and promoted antigen-specific immune responses upon coadministration with an antigen. GNLY-induced APC recruitment and activation required the presence of Toll-like receptor 4. The observed activity of recombinant GNLY was not due to endotoxin contamination. The capability of the supernatant of GNLY-expressing HuT78 cells to activate DC was blocked by anti-GNLY antibodies. Finally we present evidence that supernatants of degranulated human NK92 or primary NK cells also activated DCs in a GNLY- and Toll-like receptor 4-dependent manner, indicating the physiologic relevance of our findings. Thus, GNLY is the first identified lymphocyte-derived alarmin capable of promoting APC recruitment, activation, and antigen-specific immune response.

The alarmin functions of high-mobility group proteins.

High-mobility group (HMG) proteins are non-histone nuclear proteins that bind nucleosomes and regulate chromosome architecture and gene transcription. Over the past decade, numerous studies have established that some HMG proteins can be released extracellularly and demonstrate distinct extracellular biological activities. Here, we will give a brief overview of HMG proteins and highlight their participation in innate/inflammatory and adaptive immune responses. They have the activities of alarmins, which are endogenous mediators that are rapidly released in response to danger signals initiated by infection and/or tissue damage and are capable of activating innate and adaptive immunity by promoting the recruitment and activation of antigen-presenting cells (APCs).

Alarmins link neutrophils and dendritic cells.

Neutrophils are the first major population of leukocyte to infiltrate infected or injured tissues and are crucial for initiating host innate defense and adaptive immunity. Although the contribution of neutrophils to innate immune defense is mediated predominantly by phagocytosis and killing of microorganisms, neutrophils also participate in the induction of adaptive immune responses. At sites of infection and/or injury, neutrophils release numerous mediators upon degranulation or death, among these are alarmins which have a characteristic dual capacity to mobilize and activate antigen-presenting cells. We describe here how alarmins released by neutrophil degranulation and/or death can link neutrophils to dendritic cells by promoting their recruitment and activation, resulting in the augmentation of innate and adaptive immune responses.

Lactoferrin acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses.

Lactoferrin is an 80-kDa iron-binding protein present at high concentrations in milk and in the granules of neutrophils. It possesses multiple activities, including antibacterial, antiviral, antifungal, and even antitumor effects. Most of its antimicrobial effects are due to direct interaction with pathogens, but a few reports show that it has direct interactions with cells of the immune system. In this study, we show the ability of recombinant human lactoferrin (talactoferrin alfa (TLF)) to chemoattract monocytes. What is more, addition of TLF to human peripheral blood or monocyte-derived dendritic cell cultures resulted in cell maturation, as evidenced by up-regulated expression of CD80, CD83, and CD86, production of proinflammatory cytokines, and increased capacity to stimulate the proliferation of allogeneic lymphocytes. When injected into the mouse peritoneal cavity, lactoferrin also caused a marked recruitment of neutrophils and macrophages. Immunization of mice with OVA in the presence of TLF promoted Th1-polarized Ag-specific immune responses. These results suggest that lactoferrin contributes to the activation of both the innate and adaptive immune responses by promoting the recruitment of leukocytes and activation of dendritic cells.

Rapamycin inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells.

Reciprocal differentiation of immunosuppressive CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs) and proinflammatory IL-17-producing cells (Th17) from naïve CD4 cells is contingent upon the cytokine environment. Using MACS-purified CD4 cells, we found that rapamycin and cyclosporine A (CsA) potently inhibited the TGFbeta and IL-6-induced generation of IL-17-producing cells. Intriguingly, rapamycin promoted, while CsA markedly inhibited, TGFbeta-mediated generation of Tregs. The aforementioned effects of rapamycin and CsA were also observed for Flow-sorted CD4(+)CD25(-) T cells, indicating that the effect of these two immunosuppressive agents was based on their action on de novo generation of Tregs and Th17 cells from naïve CD4 cells. Our observation suggests a distinct mode of immunosuppressive action and tolerance induction by rapamycin and CsA. The capacity of rapamycin to generate immunosuppressive Tregs and to suppress differentiation of pathogenic Th17 cells furthers our understanding of the basis for the therapeutic immunosuppressive effects of rapamycin in patients with autoimmune diseases and allo-transplantation reactions.

Defensin participation in innate and adaptive immunity.

Defensins are endogenous, small, cysteine-rich antimicrobial peptides that are produced by leukocytes and epithelial cells. Substantial evidence accumulated in recent years indicates that mammalian defensins are multifunctional and, by interacting with host cell receptor(s), participate in both the innate and adaptive antimicrobial immunity of the host. A better understanding of the function of defensins in immunity has implications for the development of potential clinical therapeutics for the treatment of infection or cancer. Here we will briefly outline the classification, genes, expression, and structure of mammalian defensins and focus on their roles in innate and adaptive immune response of the host.

Alarmins initiate host defense.

In response to infection and/or tissue injury, cells of the host innate immune system rapidly produce a variety of structurally distinct mediators (we elect to call alarmins) that not only function as potent effectors of innate defense but also act to alarm the immune system by promoting the recruitment and activation of host leukocytes through interaction with distinct receptors. Alarmins are capable of activating antigen-presenting cells (APCs) and enhancing the development of antigen-specific immune responses. Here, we discuss the characteristics of several alarmins, a variety of potential alarmin candidates and potential implications of alarmins.

Regulated recruitment of DC-SIGN to cell-cell contact regions during zymosan-induced human dendritic cell aggregation.

Zymosan is a beta-glucan, mannan-rich yeast particle widely used to activate the inflammatory response of immune cells. We studied the zymosan-binding potential of human dendritic cells (hDCs) by using specific carbohydrate inhibitors and blocking monoclonal antibodies. We show that DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) is a major nonopsonic recognition receptor for zymosan on hDCs. Indeed, blocking of DC-SIGN inhibited the inflammatory response of DCs to zymosan. We compared the zymosan-binding capacity of hDC-SIGN to that of Dectin-1 and complement receptor 3 (CR3), which are receptors involved in the nonopsonic recognition of these yeast-derived particles. Dectin-1- and DC-SIGN-K562 cells bound to zymosan particles, whereas CR3-K562 cells did not. DC-SIGN and Dectin-1 were also expressed in COS cells to compare their ability to trigger particle internalization in a nonphagocytic cell line. DC-SIGN transfectants were unable to internalize bound particles, indicating that DC-SIGN is primarily involved in recognition but not in particle internalization. Zymosan induced a rapid DC aggregation that was accompanied by a dramatic change of DC-SIGN distribution in the plasma membrane. Under resting conditions, DC-SIGN was diffusely distributed through the cell surface, displaying clusters at the free leading edge. Upon zymosan treatment, DC-SIGN was markedly redistributed to cell-cell contacts, supporting an adhesion role in DC-DC interactions. The mechanism(s) supporting DC-SIGN-mediated intercellular adhesion were further investigated by using DC-SIGN-K562 aggregation. DC-SIGN was highly concentrated at points of cell-cell contact, suggesting a role for enhanced avidity during DC-SIGN-mediated intercellular adhesion.

Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti-MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)-stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-alpha (TNF-alpha)-activated endothelium is also inhibited by anti-MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1-stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on alpha4beta1 and alpha5beta1 integrins. Binding of monocytes to TNF-alpha-activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation.

Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells.

Acquisition of CCR7 expression is an important phenotype change during dendritic cell (DC) maturation that endows these cells with the capability to migrate to lymph nodes. We have analyzed the possible role of CCR7 on the regulation of the survival of DCs. Stimulation with CCR7 ligands CCL19 and CCL21 inhibits apoptotic hallmarks of serum-deprived DCs, including membrane phosphatidylserine exposure, loss of mitochondria membrane potential, increased membrane blebs, and nuclear changes. Both chemokines induced a rapid activation of phosphatidylinositol 3'-kinase/Akt1 (PI3K/Akt1), with a prolonged and persistent activation of Akt1. Interference with PI3K, Gi, or G protein betagamma subunits abrogated the effects of the chemokines on Akt1 activation and on survival. In contrast, inhibition of extracellular signal-related kinase 1/2 (Erk1/2), p38, or c-Jun N-terminal kinase (JNK) was ineffective. Nuclear factor-kappaB (NFkappaB) was involved in the antiapoptotic effects of chemokines because inhibition of NFkappaB blunted the effects of CCL19 and CCL21 on survival. Furthermore, chemokines induced down-regulation of the NFkappaB inhibitor IkappaB, an increase of NFkappaB DNA-binding capability, and translocation of the NFkappaB subunit p65 to the nucleus. In summary, in addition to its well-established role in chemotaxis, we show that CCR7 also induces antiapoptotic signaling in mature DCs.

The neuronal protein Kidins220 localizes in a raft compartment at the leading edge of motile immature dendritic cells.

Kidins220, a protein predominantly expressed in neural tissues, is the first physiological substrate for protein kinase D (PKD). We show that Kidins220 is expressed in monocyte-derived and in peripheral blood immature dendritic cells (im DC). Immature DC (im DC) migrate onto extracellular matrices changing cyclically from a highly polarized morphology (monopolar (MP) stage) to a morphologically symmetrical shape (bipolar (BP) stage). Kidins220 was localized on membrane protrusions at the leading edge or on both poles in MP and BP cells, respectively. CD43, CD44, ICAM-3 and DC-SIGN, and signaling molecules PKD, Arp2/3 were found at the leading edge in MP or on both edges in BP cells, showing an intriguing parallelism between morphology and localization of molecular components on the poles of the motile DC. F-actin co-localized and it was necessary for Kidins220 localization on the membrane in MP and BP cells. Kidins220 was also found in a raft compartment. Disruption of rafts with methyl-beta-cyclodextrin induced rounding of the cells, inhibition of motility and lost of Kidins220 polarization. Our results describe for the first time the molecular components of the poles of motile im DC and indicate that a novel neuronal protein may be an important component among these molecules.

Migration of human blood dendritic cells across endothelial cell monolayers: adhesion molecules and chemokines involved in subset-specific transmigration.

Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium.

DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents.

Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a monocyte-derived dendritic cell (MDDC)-specific lectin which participates in dendritic cell (DC) migration and DC-T lymphocyte interactions at the initiation of immune responses and enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to generate a high level of DC-SIGN mRNA and cell surface expression on immature MDDC. IL-4 was capable of inducing DC-SIGN expression on monocytes without affecting the expression of other MDDC differentiation markers. By contrast, IFN-alpha, IFN-gamma, and TGF-beta were identified as negative regulators of DC-SIGN expression, as they prevented the IL-4-dependent induction of DC-SIGN mRNA on monocytes, and a similar inhibitory effect was exerted by dexamethasone, an inhibitor of the monocyte-MDDC differentiation pathway. The relevance of the inhibitory action of dexamethasone, IFN, and TGF-beta on DC-SIGN expression was emphasized by their ability to inhibit the DC-SIGN-dependent HIV-1 binding to differentiating MDDC. These results demonstrate that DC-SIGN, considered as a MDDC differentiation marker, is a molecule specifically expressed on IL-4-treated monocytes, and whose expression is subjected to a tight regulation by numerous cytokines and growth factors. This feature might help in the development of strategies to modulate the DC-SIGN-dependent cell surface attachment of HIV for therapeutic purposes.