Gonzalo de la Rosa

DC-SIGN (CD209) Expression Is IL-4 Dependent and Is Negatively Regulated by IFN, TGF-β, 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-α, IFN-γ, and TGF-β 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-β 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.

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.

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.

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 β2 and β1 integrins to transmigrate, whereas CD11c+ DCs may use integrin‐independent mechanisms to migrate across activated endothelium.

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.

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.

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

HMGN1 is a novel alarmin that signals through TLR4 and is required for LPS-induced immune responses in vivo.

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.

β-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 β-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.

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).