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.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

Increased intracellular growth of Mycobacterium avium in HIV-1 exposed monocyte-derived dendritic cells

Dendritic cells (DC) are the most potent antigen-presenting cells, and form a link between the innate and adaptive immune system. They sample the periphery of the body for antigens and present them to T cells to elicit a proper immune response. It has been shown that dendritic cells phagocytose mycobacteria, but there have been conflicting reports as to whether the bacteria are capable of intracellular replication in DCs. Mycobacterium avium is a facultative intracellular bacterium, part of the Mycobacterium avium complex (MAC) of mycobacteria and are commonly seen as opportunistic pathogens in patients infected by Human immunodeficiency virus type 1 (HIV-1). To clarify the issue of whether DCs are capable of controlling the intracellular growth of M. avium and whether this control is lost upon HIV-1 exposure, we investigated the intracellular replication of M. avium in monocyte-derived dendritic cells and compared it to bacterial growth in dendritic cultures exposed to HIV-1 for 24 h. Our results show that exposure of DCs to HIV-1 promotes or facilitates the intracellular growth of M. avium.     

Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.     

Dendritic cells and their role in immune reactions of atherosclerosis

Dendritic cells were discovered and recognized as antigen-presenting cells in 1973. Since then, a large volume of information has been accumulated showing the role of dendritic cells as a key element connecting the innate and adaptive immunity. Today, dendritic cells are considered to be dedicated sensors of the immune system that are capable of recognizing both antigen amounts and antigen persistence via complex mechanisms that involve decoding and integration of various signals received in a receptor-dependant manner. The tissue microenvironment plays an important role in the modulation of effector functions of dendritic cells, inducing either activation or suppression of immune reactions. Dendritic cells maintain homeostasis and are involved in a number of diseases, including infectious diseases and cancer. The presence of dendritic cells in arteries was reported in 1995, and, since then, the involvement of dendritic cells in atherogenesis has been evaluated. This review briefly describes the current knowledge of dendritic cells and their role in atherosclerosis.     

Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.     

Natural adjuvants: endogenous activators of dendritic cells.

Dendritic cells, the most potent antigen-presenting cells, need to be activated before they can function to initiate an immune response. We report here that, in the absence of any foreign substances, dendritic cells can be activated by endogenous signals received from cells that are stressed, virally infected or killed necrotically, but not by healthy cells or those dying apoptotically. Injected in vivo with an antigen, the endogenous activating substances can function as natural adjuvants to stimulate a primary immune response, and they may represent the natural initiators of transplant rejection, spontaneous tumor rejection, and some forms of autoimmunity.     

The Role of Exosomes in Pancreatic Cancer Microenvironment

Exosomes are nanovesicles shed by cells as a means of communication with other cells. Exosomes contain mRNAs, microRNAs (miRs) and functional proteins. In the present paper, we develop a mathematical model of tumor–immune interaction by means of exosomes shed by pancreatic cancer cells and dendritic cells. Cancer cells’ exosomes contain miRs that promote their proliferation and that inhibit immune response by dendritic cells, and by CD4+ and CD8+ T cells. Dendritic cells release exosomes with proteins that induce apoptosis of cancer cells and that block regulatory T cells. Simulations of the model show how the size of the pancreatic cancer can be determined by measurement of specific miRs (miR-21 and miR-203 in the case of pancreatic cancer), suggesting these miRs as biomarkers for cancer.     

Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.     

Dendritic cell immunotherapy induces antitumour response in parathyroid carcinoma and neuroendocrine pancreas carcinoma.

Parathyroid carcinomas and neuroendocrine carcinomas of the pancreas are rare malignancies in humans. Because of their low radio- and chemosensibility, they fail to respond to conventional therapy. We therefore tested a dendritic cell immunotherapy in an attempt to control the tumour growth in two patients. Studies on mice and humans have demonstrated the potent capacity of dendritic cells to induce specific antitumour immunity. Mature dendritic cells were generated from peripheral blood monocytes in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4 and tumour necrosis factor alpha. Dendritic cells were either loaded with parathyroid hormone (PTH) or with (pancreas) tumour-derived lysate (TL), respectively, and were delivered by subcutaneous injections. All immunizations were well tolerated with no side effects, and were administered on an outpatient basis. After repeated vaccinations, specific in vivo immune response was demonstrated by positive delayed-type hypersensitivity (DTH) toward PTH or TL, demonstrating the efficient generation of antigen-specific memory T-cells. DTH reactivity was accompanied by a significant decrease of tumour markers in both patients. This approach might be generally applicable to other advanced, radio- and chemotherapy-resistant endocrine malignancies.     

Eighth Leucocyte Differentiation Antigen Workshop DC section summary

Dendritic cells (DC) are specialist antigen presenting cells that play a role in the initiation of innate and adaptive immune response. At the seventh Human Leucocyte Differentiation Antigen workshop, these intriguing cell populations were included as a separate lineage of leucocytes. This paper reports the studies performed in the eighth Human Leucocyte Differentiation Antigen workshop as part of the DC section. Many investigators currently focus on DC that are derived from a number of different leucocyte populations, including those that are differentiated in vitro and cells that are purified ex vivo. The DC section assessed the surface expression of different leucocyte surface molecules on a range of different DC populations. The results summarise the expression of each molecule on dendritic cell populations and differences between different DC preparations. Eleven new CDs were allocated on the basis of monoclonal antibodies and molecular information that identify known cell surface molecules expressed by dendritic cells. This paper gives a brief review of the work that was performed during the HLDA8 and a summary of the CDs represented by submitted mAb.     

Rapid downregulation of antigen processing enzymes in ex vivo generated human monocyte derived dendritic cells occur endogenously in extended cultures

Dendritic cells, the most potent antigen presenting cells, have been shown in murine models to induce immune responses against many antigens. Their role in the initiation of antitumour immunity has received enormous attention. Their ability to process and present antigen is dependent on their state of maturation. This study examines the activity of human monocyte-derived dendritic cells at two different time points and the corresponding changes in the proteolytic enzyme activity. Dendritic cells were produced from peripheral blood mononuclear cells of normal volunteers. Plastic adherent cells were cultured for 5 or 7 days with recombinant human (rh)GM-CSF and rhIL-4. Flow cytometry showed that day 5 dendritic cells (DC) were less mature than day 7 DC as indicated by the expression of CD1a, CD11c, CD14, CD80, CD83, CD86 and MHC-II. Proteolytic activity of the enzymes cathepsin C and cathepsin G and phagocytosis of particulate antigens also showed significant differences between d5 dendritic cells and d7 dendritic cells. Allogeneic costimulatory activity of d7 dendritic cells was also significantly increased. Induction of immunity requires active presentation of antigens by antigen processing cells on their MHC-I and/or MHC-II molecules. Study of peptide carriers and peptide precursor molecules showed a significant decrease in CLIP levels in the day 7 DC, suggesting their decreased ability to process antigens but no difference in their ability to load MHC-II molecules. These findings indicate that the length of time in culture, in the absence of exogenous maturation - inducing stimuli affects dendritic cell maturation. Intracellular enzymatic activities of dendritic cells also changed rapidly with small changes in phenotype.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

Toll-like receptor 4 is required for optimal development of Th2 immune responses: role of dendritic cells

LPS potently induces dendritic cell maturation and the production of proinflammatory cytokines, such as IL-12, by activation of Toll-like receptor 4 (TLR4). Since IL-12 is important for the generation and maintenance of Th1 responses and may also inhibit Th2 cell generation from naive CD4 T cell precursors, it has been inferred that TLR4 signaling would have similar effects via the induction of IL-12 secretion. Surprisingly, we found that TLR4-defective mice subjected to sensitization and pulmonary challenge with a protein allergen had reductions in airway inflammation with eosinophils, allergen-specific IgE levels, and Th2 cytokine production, compared with wild-type mice. These reduced responses were attributable, at least in part, to decreased dendritic cell function: Dendritic cells from TLR4-defective mice expressed lower levels of CD86, a costimulatory molecule important for Th2 responses. They also induced less Th2 cytokine production by antigenically naive CD4 T cells in vitro and mediated diminished CD4 T cell Ag-specific pulmonary inflammation in vivo. These results indicate that TLR4 is required for optimal Th2 responses to Ags from nonpathogenic sources and suggest a role for TLR4 ligands, such as LPS derived from commensal bacteria or endogenously derived ligands, in maturation of the innate immune system before pathogen exposure.     

Dendritic cells as sensors of environmental perturbations

Dendritic cells were discovered 25 years ago as professional antigen presenting cells bridging together innate and adaptive immunity. Recently additional functions of dendritic cells have been uncovered indicating a relevant role of dendritic cells in immune system regulation. Indeed, they are the professional sensors of the immune system that can detect perturbations caused by non-self infectious as well as self non-infectious signals in most tissues. Dendritic cells discriminate both antigen amounts and antigen persistence through their receptor repertoire via the integration of different signaling pathways. The environment plays an essential role in conditioning the effector functions of dendritic cells leading either to the activation or suppression of adaptive immunity.     

Functional identification of dendritic cells in the teleost model, rainbow trout (Oncorhynchus mykiss)

Dendritic cells are specialized antigen presenting cells that bridge innate and adaptive immunity in mammals. This link between the ancient innate immune system and the more evolutionarily recent adaptive immune system is of particular interest in fish, the oldest vertebrates to have both innate and adaptive immunity. It is unknown whether dendritic cells co-evolved with the adaptive response, or if the connection between innate and adaptive immunity relied on a fundamentally different cell type early in evolution. We approached this question using the teleost model organism, rainbow trout (Oncorhynchus mykiss), with the aim of identifying dendritic cells based on their ability to stimulate naïve T cells. Adapting mammalian protocols for the generation of dendritic cells, we established a method of culturing highly motile, non-adherent cells from trout hematopoietic tissue that had irregular membrane processes and expressed surface MHCII. When side-by-side mixed leukocyte reactions were performed, these cells stimulated greater proliferation than B cells or macrophages, demonstrating their specialized ability to present antigen and therefore their functional homology to mammalian dendritic cells. Trout dendritic cells were then further analyzed to determine if they exhibited other features of mammalian dendritic cells. Trout dendritic cells were found to have many of the hallmarks of mammalian DCs including tree-like morphology, the expression of dendritic cell markers, the ability to phagocytose small particles, activation by toll-like receptor-ligands, and the ability to migrate in vivo. As in mammals, trout dendritic cells could be isolated directly from the spleen, or larger numbers could be derived from hematopoietic tissue and peripheral blood mononuclear cells in vitro.     

ILT3+/ILT4+ tolerogenic dendritic cells and their influence on allograft survival

Dendritic cells, the most powerful antigen-presenting cells, are important for triggering of the immune responses to allo-antigens. However, they also play a fundamental role in the peripheral tolerance maintenance. Tolerance is enhanced by the presence on the dendritic cell surface of the inhibitor receptors ILT3 and ILT4. They recruit protein tyrosine-phosphatases to their ITIM domains and inhibit antigen-presenting cell activation, leading T cell hypo-responsivensess. Moreover, these receptors favor a bidirectional interaction with T-suppressor and T-regulator cells, generating an antigen-specific immunoregulator cascade, in which the dendritic cell behaves as a tolerogenic cell. In the current review, analysis is centered on the biology and behavior of the tolerogenic dendritic cells that express high levels of ILT3 and ILT4. Some molecular and genetics aspects of these receptors are discussed as well as their importance in the modulation of the allo-specific antigen immune response to transplants.     

Nitric oxide inhibits IFN-alpha production of human plasmacytoid dendritic cells partly via a guanosine 3',5'-cyclic monophosphate-dependent pathway

NO, a free radical gas, is known to be critically involved not only in vascular relaxation but also in host defense. Besides direct bactericidal effects, NO has been shown to inhibit Th1 responses and modulate immune responses in vivo, although the precise mechanism is unclear. In this study, we examined the effect of NO on human plasmacytoid dendritic cells (pDCs) to explore the possibility that NO might affect innate as well as adaptive immunity through pDCs. We found that NO suppressed IFN-alpha production of pDCs partly via a cGMP-dependent mechanism, which was accompanied by down-regulation of IFN regulatory factor 7 expression. Furthermore, treatment of pDCs with NO decreased production of IL-6 and TNF-alpha and up-regulated OX40 ligand expression. In accordance with these changes, pDCs treated with NO plus CpG-oligodeoxynucleotide AAC-30 promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Moreover, pDCs did not express inducible NO synthase even after treatment with AAC-30, LPS, and several cytokines. These results suggest that exogenous NO and its second messenger, cGMP, alter innate as well as adaptive immune response through modulating the functions of pDCs and may be involved in the pathogenesis of certain Th2-dominant allergic diseases.     

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.