Molecular mechanisms that set the stage for DC-T cell engagement

The unsurpassed capacity of dendritic cells (DC) to prime naive T cells is thought to depend on the formation of an immunological synapse. DC-SIGN, a C-type lectin exclusively expressed at the cell surface of DC, functions as an adhesion receptor facilitating T cell binding and priming through recognition of glycosylated ICAM-3 on naive T cells. Yet, DC-SIGN also mediates binding to pathogens such as HIV by recognizing glycosylated gp120. The scope of the present study was to investigate whether DC-SIGN upon recognition of its cellular ligand and pathogenic ligand affects DC synapse formation and activation/mobilization of other adhesion receptors such as LFA-1 to the cell contact site. Using a DC-SIGN deletion mutant, we show that DC-SIGN is a constitutively active receptor that mediates ligand binding independent of signaling through the cytoplasmic domain. Surprisingly, initial binding of gp120 to DC-SIGN did not result in increased adhesion levels of LFA-1 to its ligand ICAM-1 in both immature DC and Raji-DC-SIGN cells. However, ligand binding to DC-SIGN induced recruitment of LFA-1 to the adhesion site. Moreover, we could demonstrate that activation of LFA-1 results in DC-SIGN-LFA-1 co-clustering in the cell membrane. This triggers binding of ligands to LFA-1 that are shared with DC-SIGN, such as ICAM-3, but not of ligands that are not shared with DC-SIGN, such as ICAM-1. Thus, we propose that upon ligand binding DC-SIGN recruits LFA-1 to the contact site, resulting in the formation of DC-SIGN-LFA-1 co-clusters, in which the initial DC-SIGN-mediated interactions with ligand are transient and eventually shift to more stable LFA-1-dependent interactions.     

DC-SIGN specifically recognizes Streptococcus pneumoniae serotypes 3 and 14

The Gram-positive bacterium Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia. The ever-increasing frequency of antibiotic-resistant S. pneumoniae strains severely hampers effective treatments. Thus, a better understanding of the mechanisms involved in the pathogenesis of pneumococcal disease is needed; in particular, of the initial interactions that take place between the host and the bacterium. Recognition of pathogens by dendritic cells is one of the most crucial steps in the induction of an immune response. For efficient pathogen recognition, dendritic cells express various kinds of receptors, including the DC-specific C-type lectin DC-SIGN. Pathogens such as Mycobacterium tuberculosis and HIV target DC-SIGN to escape immunity. Here the in vitro binding of DC-SIGN with S. pneumoniae was investigated. DC-SIGN specifically interacts with S. pneumoniae serotype 3 and 14 in contrast to other serotypes such as 19F. While the data described here suggest that DC-SIGN interacts with S. pneumoniae serotype 14 through a ligand expressed by the capsular polysaccharide, the binding to S. pneumoniae serotype 3 appears to depend on an as yet unidentified ligand. Despite the binding capacity of the capsular polysaccharide of S. pneumoniae 14 to DC-SIGN, no immunomodulatory effects on the dendritic cells were observed. The immunological consequences of the serotype-specific capacity to interact with DC-SIGN should be further explored and might result in new insights in the development of new and more potent vaccines.     

E‐cadherin interactions are required for Langerhans cell differentiation

Human skin contains the following two distinct DC subsets: (i) Langerhans cells (LCs), expressing Langerin but not DC‐specific intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN), are predominantly localized in the epidermis; and (ii) dermal DCs, expressing DC‐SIGN but not Langerin, are observed mainly in the dermis. It is not known whether localization in the epidermis provides cues for LC differentiation. Here, we show that E‐cadherin expressed by epidermal keratinocytes (KCs) is crucial for differentiation of LCs. Monocytes differentiated into LC‐like cells in presence of IL‐4, GM‐CSF, and TGF‐β1. However, these LC‐like cells expressed not only Langerin but also DC‐SIGN. Notably, co‐culturing of these LC‐like cells with KCs expressing E‐cadherin or recombinant E‐cadherin strongly decreased expression of DC‐SIGN and further induced a phenotype similar to purified epidermal LCs. Moreover, pretreatment of LC‐like cells with anti‐E‐cadherin‐specific antibody completely abolished their Langerin expression, indicating the requirement of E‐cadherin–E‐cadherin interactions for the differentiation into Langerin⁺ cells. These findings suggest that E‐cadherin expressed by KCs provide environmental cues that induce differentiation of LCs in the epidermis.     

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation

BACKGROUND: To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. METHODS: Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. RESULTS: Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). CONCLUSIONS: LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.     

Integrin mediated adhesion of mononuclear cells from patients with familial hypercholesterolemia

BACKGROUND: Low-density lipoproteins (LDL) can induce the adhesion of monocytes to endothelial cells. Monocytes of patients with familial hypercholesterolemia (FH) are exposed to high concentrations of LDL, and it has been reported that adhesiveness of these cells in hypercholesterolemic patients is enhanced. We investigated whether LFA-1 or VLA-4 mediated adhesion is altered in FH patients and whether HMG-CoA reductase inhibitors influence this adhesion. PATIENTS AND METHODS: LFA-1 and VLA-4 mediated adhesion to ICAM-1 and VCAM-1 coated beads was investigated using freshly isolated monocytes and T-lymphocytes from patients with homozygous FH, heterozygous FH (before and after cholesterol lowering treatment), and from controls. In addition, the expression of beta1- and beta2-integrins on these cells was determined. RESULTS: Both LFA-1 and VLA-4 mediated adhesion and integrin expression of monocytes and CD3+ cells from patients with homozygous FH and heterozygous FH was similar to that of monocytes from a control population. Treatment with HMG-CoA reductase inhibitors did not affect the adherence to ICAM-1 or VCAM-1, and did not influence the expression of integrins. CONCLUSIONS: In contrast to studies by others, we demonstrated in the present study that the actual LFA-1 and VLA-4 mediated adhesion of T-lymphocytes and monocytes is not altered in patients with FH.     

Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic alpha-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4(+) T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.     

Syndecan-3 is a dendritic cell-specific attachment receptor for HIV-1

Dendritic cells (DCs) efficiently capture HIV-1 and mediate transmission to T cells, but the underlying molecular mechanism is still being debated. The C-type lectin DC-SIGN is important in HIV-1 transmission by DCs. However, various studies strongly suggest that another HIV-1 receptor on DCs is involved in the capture of HIV-1. Here we have identified syndecan-3 as a major HIV-1 attachment receptor on DCs. Syndecan-3 is a DC-specific heparan sulfate (HS) proteoglycan that captures HIV-1 through interaction with the HIV-1 envelope glycoprotein gp120. Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transmission to T cells. Removal of the HSs from the cell surface by heparinase III or by silencing syndecan-3 by siRNA partially inhibited HIV-1 transmission by immature DCs, whereas neutralizing both syndecan-3 and DC-SIGN completely abrogated HIV-1 capture and subsequent transmission. Thus, HIV-1 exploits both syndecan-3 and DC-SIGN to mediate HIV-1 transmission, and an effective microbicide should target both syndecan-3 and DC-SIGN on DCs to prevent transmission.     

A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes

Several therapeutic self-proteins elicit immune responses when administered to patients. Such adverse immune responses reduce drug efficacy. To induce an immune response, a protein must interact with different immune cells, including antigen-presenting cells, T cells, and B cells. Each cell type recognizes distinct immunogenic patterns on antigens. Mannose-terminating glycans have been identified as pathogen-associated molecular patterns that are essential for internalization of microbes by antigen-presenting cells, leading to presentation. Here, we have investigated the importance of exposed mannosylation on an immunogenic therapeutic self-protein, procoagulant human factor VIII (FVIII). Administration of therapeutic FVIII to hemophilia A patients induces inhibitory anti-FVIII antibodies in up to 30% of the cases. We demonstrate that entry of FVIII into human dendritic cells (DC) leading to T cell activation, is mediated by mannose-terminating glycans on FVIII. Further, we identified macrophage mannose receptor (CD206) as a candidate endocytic receptor for FVIII on DC. Saturation of mannose receptors on DC with mannan, and enzymatic removal of mannosylated glycans from FVIII lead to reduced T cell activation. The interaction between FVIII and CD206 was blocked by VWF, suggesting that, under physiological conditions, the intrinsic mannose-dependent immunogenicity of FVIII is quenched by endogenous immunochaperones. These data provide a link between the mannosylation of therapeutic self-proteins and their iatrogenic immunogenicity. Such a link would be of special relevance in the context of replacement therapy where mechanisms of central and peripheral tolerance have not been established during ontogeny because of the absence of the antigen.     

A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection

The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN-related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN-related molecule is L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1.     

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.Eukaryotic translation elongation factor 2 (eEF2) is a highly conserved protein and essential for protein biosynthesis. EEF2 enables peptide-chain elongation by translocating the peptide–tRNA complex from the A- to the P-site of the ribosome (1, 2). The diphthamide modification at His715 of human eEF2 (or at the corresponding position in other species) is conserved in all eukaryotes (3) and in archaeal counterparts. It is generated by proteins that are encoded by seven genes (4). Proteins encoded by dipthamide biosynthesis protein (DPH)1, DPH2, DPH3, and DPH4 (DNAJC24) attach a 3-amino-3-carboxypropyl (ACP) group to eEF2. This intermediate is converted by the methyltransferase DPH5 to diphthine, which is subsequently amidated to diphthamide by DPH6 and DPH7 (5).Diphthamide synthesis was previously described in yeast and other eukaryotes (4–6). However, the “complete picture” is (with the exception of the yeast pathway) to a large portion is composed of observations made in different cell types on single genes. Many reports related to diphthamide synthesis of mammalian cells describe “partial knockouts” and “partial phenotypes” (i.e., reduced levels but not complete loss of diphthamide modification or toxin sensitivities) (7–9). Because mammalian genomes are more complex than that of yeast, carrying extendend gene families, mammalian cells may compensate—at least to some degree—functional loss of genes that may be unique and essential in yeast. If and to what degree mammalian cells can compensate a partial or complete loss of DPH gene functionality (and with what consequences) is unknown to date.So far, the function of diphthamide on eEF2 also remained rather elusive. Reports indicate that it contributes to translation fidelity (10–13). On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are viable (5, 11, 14, 15). Animals with heterozygous DPH knockouts (DPHko) can be generated, but homozygous DPH1ko, DPH3ko, and DPH4ko are embryonic lethal (13, 16–18). Because these studies are based on inactivation of individual genes, it is difficult to discriminate between phenotypes caused by gene loss and phenotypes as a consequence of loss of diphthamide.Diphthamide-modified eEF2 is the target of ADP ribosylating toxins, including Pseudomonas exotoxin A (PE) and diphtheria toxin (DT) (19). These bacterial proteins enter cells and catalyze ADP ribosylation of diphthamide using nictotinamide adenine dinucleotide (NAD) as substrate (20, 21). This inactivates eEF2, arrests protein synthesis, and kills (14). Tumor-targeted PE and DT derivatives are applied in cancer therapies (22–28) and their efficacy depends on toxin sensitivity of target cells. Therefore, information about factors (and their relative contributions) that influences cellular sensitivities toward diphthamide-modifying toxins may predict therapy responses. For example, alterations in OVCA1 (human DPH1) were described for ovarian cancers (16, 29), yet it is not known if and to what degree such alterations would affect sensitivities of tumor cells toward PE-derived drugs.Here we describe MCF7 breast cancer cell line derivatives with heterozygous or complete DPH gene inactivations. These cells are applied to analyze the contributions of individual DPHs not only to diphthamide synthesis and toxin sensitivity, but also to address gene dose effects. Because the set of knockout cell lines is derived from the same parent cell and provides loss of diphthamide as common consequence of inactivation of different genes, these cells can also shed light on the biological relevance of the diphthamide modification.