Prostaglandin E2 is generally required for human dendritic cell migration and exerts its effect via EP2 and EP4 receptors

The control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. In this study, we demonstrate that the migration of human monocyte-derived (Mo)DCs as well as of ex vivo peripheral blood DCs toward CCL21, CXCL12, and C5a is stringently dependent on the presence of the proinflammatory mediator PGE2, although DCs expressed CXCR4 and C5aR on their surface and DC maturation was accompanied by CCR7 up-regulation independently of PGE2. The necessity of exogenous PGE2 for DC migration is not due to the suppression of PGE2 synthesis by IL-4, which is used for MoDC differentiation, because maturation-induced endogenous production of PGE2 cannot promote DC migration. Surprisingly, PGE2 was absolutely required at early time points of maturation to enable MoDC chemotaxis, whereas PGE2 addition during terminal maturation events was ineffective. In contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination. Our results provide clear evidence that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for peripheral blood myeloid DCs.     

Extracellular adenine nucleotides inhibit the release of major monocyte recruiters by human monocyte-derived dendritic cells

Extracellular ATP is known to affect the maturation of monocyte-derived dendritic cells mainly by regulation of cytokines and costimulatory molecules. The present study describes the inhibition of MCP-1 (CCL2) and MIP-1alpha (CCL3) release by human monocyte-derived dendritic cells in response to adenine nucleotides. Our pharmacological data support the involvement of P2Y11 and P2Y1 purinergic receptors in the downregulation of these major monocyte recruiters. Migration assays have demonstrated that supernatants of dendritic cells treated with adenine nucleotides or anti-MCP-1/MIP-1alpha blocking antibodies display a strongly reduced capacity to attract monocytes and immature dendritic cells.     

The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.     

Insulin regulation of MCP-1 in human adipose tissue of obese and lean women

CCL2 (MCP-1, monocyte chemoattractant protein 1) and CCL3 (MIP-1alpha, macrophage inflammatory protein 1alpha) are required for macrophage infiltration in adipose tissue. Insulin increases CCL2 expression in adipose tissue and in serum more in insulin-resistant obese than in insulin-sensitive lean mice, but whether this is true in humans is unknown. We compared basal expression and insulin regulation of CCL2 and CCL3 in adipose tissue and MCP-1 and MIP-1alpha in serum between insulin-resistant and insulin-sensitive human subjects. Subcutaneous adipose tissue biopsies and blood samples were obtained before and at the end of 6 h of in vivo euglycemic hyperinsulinemia (maintained by the insulin clamp technique) in 11 lean insulin-sensitive and 10 obese insulin-resistant women, and before and after a 6-h saline infusion in 8 women. Adipose tissue mRNA concentrations of monocyte/macrophage markers CD68, EMR1, ITGAM, ADAM8, chemokines CCL2 and CCL3, and housekeeping gene ribosomal protein large P0 (RPLP0) were measured by means of real-time PCR at baseline. In addition, mRNA concentrations of CCL2, CCL3, and RPLP0 were measured after insulin infusion. Levels of MCP-1 and MIP-1alpha were determined in serum, and protein concentration of MCP-1 was determined in adipose tissue at baseline and after insulin infusion. Basally, expression of the macrophage markers CD68 and EMR1 were increased in adipose tissue of insulin-resistant subjects. Insulin increased MCP-1 gene and protein expression significantly more in the insulin-resistant than in the insulin-sensitive subjects. Basally expression of CCL2 and CCL3 and expression of macrophage markers CD68 and ITGAM were significantly correlated. In serum, MCP-1 decreased significantly in insulin-sensitive but not insulin-resistant subjects. MIP-1alpha was undetectable in serum. Insulin regulation of CCL2 differs between insulin-sensitive and -resistant subjects in a direction that could exacerbate adipose tissue inflammation.     

Host response to malaria during pregnancy: placental monocyte recruitment is associated with elevated beta chemokine expression

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

Monocyte chemoattractant protein-1 selectively inhibits the acquisition of CD40 ligand-dependent IL-12-producing capacity of monocyte-derived dendritic cells and modulates Th1 immune response

Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, also displays immunoregulatory functions and may be involved in Th subset differentiation. In this study, we examined the effects of MCP-1 on the cytokine-driven differentiation of monocytes into dendritic cells (DCs), the most potent APCs for naive T cells. We found that DCs generated in the presence of MCP-1 displayed a markedly reduced production of IL-12 in response to CD40 ligand but not in response to Staphylococcus aureus stimulation in the presence or absence of IFN-gamma. The production of IL-10, a potent endogenous IL-12 inhibitor, was not affected by MCP-1. Whereas the inhibitory activity of MCP-1 on IL-12 production by monocytes was sensitive to pertussis toxin, its effects on DC differentiation were pertussis toxin resistant. MCP-1 did not affect the surface phenotype and T cell-stimulating activity of DCs, but most interestingly, naive T cells stimulated with MCP-1-primed DCs produced much less IFN-gamma but the same levels of IL-13. Taken together, our results indicated that MCP-1 modulates the differentiation of monocytes into DCs and may thereby inhibit Th1 cell development.     

Alterations in the secretory pattern of dermal dendritic cells following melanin uptake

Under a variety of circumstances, melanin occurs in the dermal compartment of the skin, being mostly observed in cells that have been termed melanophages, some of which have been identified as dermal dendritic cells. We analysed changes in the expression and secretion pattern of cytokines by dendritic cells after the uptake of melanin from various sources. Dendritic cells were derived from human primary blood monocytes or from the human monocytic cell line THP-1. Melanin uptake increased the secretion of the chemokines MIP-1β (CCL4) and MCP-1 (CCL2). The higher MIP-1β secretion was accompanied by higher MIP-1β gene expression. Elevation of MIP-1β secretion was dependent on the uptake of melanin but could not be induced by the phagocytosis of latex beads, indicating that the phagocytic process itself was not sufficient to increase the secretion of this cytokine. The data thus show that the uptake of melanin changes the cytokine expression and secretion pattern of dendritic-like cells.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

Extracellular nucleotides regulate CCL20 release from human primary airway epithelial cells, monocytes and monocyte-derived dendritic cells

Extracellular nucleotides regulate ion transport and mucociliary clearance in human airway epithelial cells (HAECs) via the activation of P2 receptors, especially P2Y(2). Therefore, P2Y(2) receptor agonists represent potential pharmacotherapeutic agents to treat cystic fibrosis (CF). Nucleotides also modulate inflammatory properties of immune cells like dendritic cells (DCs), which play an important role in mucosal immunity. Using DNA-microarray experiments, quantitative RT-PCR and cytokine measurements, we show here that UTP up-regulated approximately 2- to 3-fold the antimicrobial chemokine CCL20 expression and release in primary HAECs cultured on permeable supports at an air-liquid interface (ALI). Both P2Y(2) (ATPgammaS, UTP, INS365) and P2Y(6) (UDP, INS48823) agonists increased CCL20 release. UTP-induced CCL20 release was insensitive to NF-kappaB pathway inhibitors but sensitive to inhibitors of ERK1/2 and p38/MAPK pathways. Furthermore, UTP had no effect on interleukin-(IL)-8 release and reduced the release of both CCL20 and IL-8 induced by TNF-alpha and LPS. Accordingly, UTP reduced the capacity of basolateral supernatants of HAECs treated with TNF-alpha or LPS to induce the chemoattraction of both CD4(+) T lymphocytes and neutrophils. In addition, we show that, in monocyte-derived DCs, ATPgammaS, and UDP but not UTP/INS365-stimulated CCL20 release. Likewise, UDP but not ATPgammaS was also able to increase CCL20 release from monocytes. Pharmacological experiments suggested an involvement of P2Y(11) or P2Y(6) receptors through NF-kappaB, ERK1/2, and p38/MAPK pathways. Altogether, our data demonstrate that nucleotides may modulate chemokine release and leukocyte recruitment in inflamed airways by acting on both epithelial and immune cells. Our results could be relevant for further clinical investigations in CF.     

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.     

Subpopulations of human dendritic cells display a distinct phenotype and bind differentially to proteins of the extracellular matrix

CD1a(pos) dendritic cells (DCs) and Langerhans cells (LCs) are highly specialized antigen-presenting cells mainly localized in the skin. Various cells have been identified as precursors of cutaneous DCs, but the definitive precursor subpopulations remain to be defined and characterized in detail. In this study, DCs were generated in vitro from monocytes (monocyte-derived DCs, MoDCs) and from CD34(pos) stem cells (CD34(pos) cell-derived DCs, CD34DCs). By virtue of their CD14 and CD1a expression, four CD34DC subpopulations were characterized while MoDCs contain three different subpopulations. Of these, CD14-expressing cells are considered to be precursors of fully differentiated DCs, which themselves are CD14(neg)CD1a(pos). Both, MoDCs and CD34DCs expressed the alpha integrins LFA-1, Mac-1, CR4, VLA-4, VLA-5 and the beta2 integrin CD18. CD34DCs and MoDCs were negative for VLA-3, whereas MoDCs, but not CD34DCs expressed VLA-6. Phenotypic and functional characterization of the cells generated herein at earlier time points revealed that DCs at day 3 of culture may reflect the in vivo situation more closely than at day 7. Adhesion of DC precursors to endothelial cells and to components of the extracellular matrix is a prerequisite for their migration towards the epidermis. To this end, we investigated adhesion of CD34DCs and MoDCs to components of the cutaneous extracellular matrix. Distinct DC subsets showed a differential binding pattern to proteins of the extracellular matrix. MoDCs and CD34DCs bound preferentially to laminin 332 via CD49f and to fibronectin via CD49e, but only weakly to laminin 111 or to collagens. While CD14(pos) cells preferentially bound to laminin 332, CD1a(pos) cells adhered to fibronectin. In summary, subpopulations of CD34DCs and MoDCs are phenotypically related to each other, but not identical and display differential binding to components of the extracellular matrix.     

The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells

Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiating inflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized by an increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immune response in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on human DCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in the absence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellin induced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptor CCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-alpha/CXCL1, GRO-beta/CXCL2, GRO-gamma/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), and immature DCs (macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4), but not chemokines active on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-gamma/CXCL9, IFN-inducible T cell alpha chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine induced by IFN-gamma, and IFN-inducible T cell alpha chemoattractant expression, whereas stimulation with IFN-beta or flagellin alone failed to induce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with a concomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not express TLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human but not murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immune response.     

Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells

BACKGROUND: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. METHODS: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). RESULTS: mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. CONCLUSIONS: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.     

Alterations in Gene Array Patterns in Dendritic Cells from Aged Humans

Dendritic cells (DCs) are major antigen-presenting cells that play a key role in initiating and regulating innate and adaptive immune responses. DCs are critical mediators of tolerance and immunity. The functional properties of DCs decline with age. The purpose of this study was to define the age-associated molecular changes in DCs by gene array analysis using Affymatrix GeneChips. The expression levels of a total of 260 genes (1.8%) were significantly different (144 down-regulated and 116 upregulated) in monocyte-derived DCs (MoDCs) from aged compared to young human donors. Of the 260 differentially expressed genes, 24% were down-regulated by more than 3-fold, suggesting that a large reduction in expression occurred for a notable number of genes in the aged. Our results suggest that the genes involved in immune response to pathogens, cell migration and T cell priming display significant age-related changes. Furthermore, downregulated genes involved in cell cycle arrest and DNA replication may play a critical role in aging-associated genetic instability. These changes in gene expression provide molecular based evidence for age-associated functional abnormalities in human DCs that may be responsible for the defects in adaptive immunity observed in the elderly.     

Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro

The clinical picture of severe acute respiratory syndrome (SARS) is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS coronavirus (SARS-CoV)-host cell interaction, we investigated the induction of chemokines, adhesion molecules, and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) by SARS-CoV. Immunohistochemistry revealed neutrophil, macrophage, and CD8 T-cell infiltration in the lung autopsy of a SARS patient who died during the acute phase of illness. Additionally, pneumocytes and macrophages in the patient's lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that the A549 and THP-1 cell lines were susceptible to SARS-CoV. A549 cells produced CCL2/monocyte chemoattractant protein 1 (MCP-1) and CXCL8/interleukin-8 (IL-8) after interaction with SARS-CoV and expressed P-selectin and VCAM-1. Moreover, SARS-CoV induced THP-1 cells to express CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1alpha, CXCL10/IP-10, CCL4/MIP-1beta, and CCL5/RANTES, which attracted neutrophils, monocytes, and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Our in vitro experiments modeling infection in humans together with the study of a lung biopsy of a patient who died during the early phase of infection demonstrated that SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive for immune cell migration and accumulation that eventually leads to lung injury.     

Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2

Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.