Mechanisms Of Antigen Uptake Research Articles

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

BackgroundThe innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays.Methodology/Principal FindingsPooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes.Conclusions/SignificanceZebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.

Dendritic Cell immuno‐activating receptor 1 – Characterization of a novel member of the C‐type Lectin family

C‐type lectins (CLEC) comprise a superfamily of cell surface proteins, whose multifunctional capacity has been shown to address both, innate and adaptive immune response. Here, we describe mDCAR1 a novel CLEC belonging to the recently defined murine dendritic cell (DC) immuno‐receptor (mDCIR)/DC immuno‐activating receptor (mDCAR) family.mDCAR1 expression was found to be restricted to mononuclear phagocytes in bone marrow. Extensive phenotypical characterization defined them as CCR2−CD62L−CX3CR1+Ly6C− cells, thereby belonging to the recently described ‘resident’ monocytes, which correspond to CD14+ CD16+ human monocytes. In contrast, the CCR2+CD62L+CX3CR1lowLy6C+ ‘inflammatory’ monocytes were mDCAR1−. Noteworthy, in thymus, mDCAR1 expression was found on CD11chighCD8alpha+CD205+CD11b−B220−BP‐1+ lymphoid‐related DC, which play an important role in central tolerance induction.With respect to the classical function of CLEC as antigen uptake receptors for pathogens and self‐proteins, mimicking the ligation of mDCAR1 by labeling with specific mAb shows the internalization of the mDCAR1/ligand complex. Crosslinking of mDCAR1 further initiates signal transduction resulting in tyrosine‐phosphorylation of proteins.The expression pattern of mDCAR1 and the evidence of a classical antigen‐uptake mechanism are first steps to uncover the role of this representative of the CLEC receptor family.

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.

Abstracts

The dendritic cell (DC) specific pathogen-recognition receptor DCSIGN binds and internalizes antigens for degradation. The organization of DC-SIGN in microdomains is crucial for the binding and the internalization of virus particles, suggesting that these multimolecular assemblies act as docking site for pathogens like HIV-1 to invade the host. We have recently shown that DC-SIGN potentially associates with lipid rafts [1] and clathrin coated pits [2]. Nevertheless, the nano-scale organization of DC-SIGN with respect to these lipid domains remains largely unresolved. To map the nanolandscape distribution of DC-SIGN on DC cell membranes we are exploiting state-of-the-art microscopic imaging techniques. Single fluorescent molecule detection together with multicolor labeling offers the possibility to elucidate organization and co-localization at <100 nm spatial resolution. Currently we are investigating the potential association of DC-SIGN with lipid rafts using a near-field optical microscope working under physiological conditions. Additionally we are planning a three color experiment to resolve the nano-landscape of DC-SIGN, lipid rafts and clathrin coated pits before and during endocytosis. The possible change in organization and association of DC-SIGN is essential for the understanding of the antigen uptake mechanism(s) from the membrane of DCs. [1] A. Cambi, et al., Journal of Cell Biology, 164 , 145 (2004). [2] A. Cambi, et al., Nanoletters, In Press.

The Effects of HIV Infection on Oral Mucosal Immunity

Oral mucosal infections, especially candidiasis, are a feature of HIV disease, suggesting that compromised mucosal immunity within the oral cavity is a consequence of the viral infection. However, how this mucosal immunity is compromised and at what stage of HIV infection this occurs are unclear. Better understanding of the protection of the oral cavity against infection has allowed us to gain some insight into the local consequences of HIV infection. From a humoral perpective, IgA2 subclasses are reduced in HIV infection in saliva, and total secretory IgA levels are reduced in later disease. Similarly, mucosal antibody responses appear near normal in early HIV infection but reduced in AIDS. There is now convincing evidence that salivary IgA can be neutralizing to HIV 1 and HIV 2, as well as block epithelial transmigration. Oral cellular immunity is also affected by HIV infection. Transmission of HIV from one oral cell type to another appears to be confirmed by work showing that HIV can bind to or infect epithelial cells, Langerhans cells, and other mucosal cells. CXCR4 tropic (via GalCer and CXCR4) and dual tropic HIV strains have been shown to be able to infect normal human oral keratinocytes (NHOKs), and infectious HIV virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection. There is evidence of up-regulation of various receptors, including HIV receptors, on the surface of oral epithelium, and the epithelium may become more permeable. HIV may exploit this antigen uptake mechanism to cross epithelial barriers during co-infection with damage-inducing pathogens such as Candida. Immune responsiveness to many of the co-pathogens associated with HIV has been demonstrated to depend on a family of innate recognition molecules, known as Toll-like receptors (TLR), and recognition of a single pathogen can involve activation of multiple TLRs. Consequently, TLR-pathogen interactions could play an indirect but major role in regulating HIV-associated disease in the oral cavity. Thus, HIV infection appears to have both direct and indirect effects on oral mucosal immunity, affecting both cellular and humoral immunity as well as both specific and innate immunity.

Dynamic Properties of Antigen Uptake and Communication Between Dendritic Cells

We have examined mechanisms of antigen uptake by dendritic cells (DC). While multiple experimental approaches have been used, live cell imaging has been especially informative, and is reviewed here. DC were shown to bind to and internalize bacteria, but not inert particles, demonstrating an unexpected selectivity that included sensing of secreted bacterial products and subsequent cell activation. This led to the observation that induced calcium fluxes in these cells could be propagated to other cells via thin membrane connections termed tunneling nanotubules that were shown to form an extensive network between cells, and are also present in other cell types. These data are discussed in terms of DC function, and also demonstrate the power of live cell imaging in elucidating complex biological phenomena that are not readily revealed using static imaging approaches or by biochemical analyses.

Cytopathic and non-cytopathic bovine viral diarrhoea virus biotypes affect fluid phase uptake and mannose receptor-mediated endocytosis in bovine monocytes.

We have used non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea viruses (BVDV) to determine how the two biotypes affect mannose receptor (MR)-mediated endocytosis and fluid phase uptake in bovine monocytes. We have demonstrated that endocytosis in uninfected monocytes after 1 h of culture was mediated by the MR and fluid phase uptake, and after 24 h of culture it was mediated via fluid phase uptake only. Both cp and ncp BVDV affected the mechanisms of antigen uptake in monocytes. Endocytosis in BVDV infected monocytes, unlike in uninfected cells, was MR-independent and mediated by fluid phase uptake after 1 h of infection. The 24-h-BVDV infection changed the antigen uptake mechanisms to become MR- and fluid phase uptake-dependent. We conclude that antigen uptake, an important antigen presenting cell (APC) function, is affected in the early stage of BVDV infection during the first 24 h, with both BVDV biotypes, cp and ncp, having similar effects on monocyte antigen uptake in cattle. By influencing the early antigen uptake function of APC, BVDV might disrupt the function of monocytes as professional APC and contribute to the specific immunotolerance to BVDV.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8 + T cells and induce cytolytic effector T cells in vitro.

Intestinal IgE secretion in food allergy: Potential role for CD23 isoforms on intestinal epithelial cells

Rationale Anaphylactic reactions can occur extremely rapidly after ingestion of food allergens. A CD23/IgE-dependent antigen uptake mechanism is required for rapid hypersensitivity reactions in rodent models of food allergy. The mechanism leading to lumenal IgE remains unknown. Methods Naïve and sensitized (OVA and casein) C3H/HeJ mice were used. Specific IgE was measured in intestinal lavage from mice, and in stool samples from food allergic patients. Intestinal secretion of IgE and IgG were compared by injection of mice with 125I-IgE or -IgG and measurement of 125I in intestinal lavage and stool. CD23 expression was determined by immunohistochemistry and RT-PCR. IgE uptake by intestinal epithelial cells (IEC) was determined by confocal microscopy. Results Allergen-specific IgE was detected in stool samples from food-allergic patients, and in intestinal lavage fluid from sensitized mice. Sensitized mice secreted greater levels of 125I-IgE, and to a lesser extent 125I-IgG, into the intestinal lumen compared to naïve mice. Sensitization was associated with upregulated CD23 expression on mouse IEC. RT-PCR analysis of CD23 isoform expression in IEC obtained from patients with food allergic disorders or normal controls demonstrated that the CD23a isoform was constitutively expressed, while the CD23b isoform was present only in IEC from food allergic patients. Polarized caco-2 cells transfected with the CD23a isoform rapidly internalized FITC-IgE from the apical, but not the basolateral domain. Conclusions Allergen-specific IgE is secreted into the intestinal lumen in sensitized individuals, and may facilitate uptake of antigen by interaction with CD23a. Upregulation of the CD23b isoform may be responsible for the secretion of IgE into the intestinal lumen.

Increased efficacy of immersion vaccination in fish with hyperosmotic pretreatment

Immersion vaccination is common practice in aquaculture, because of its convenience for mass vaccination with sufficient protection. However, the mechanisms of antigen uptake and presentation, resulting in a protective immune response and the role of the innate immune system therein are largely unknown. The impact of immersion vaccination on fish physiology and on the ensuing innate and specific immune response was characterized with fluorescently labeled particulate and soluble model antigens. Vaccination of common carp by direct immersion (DI) or hyperosmotic immersion (HI; direct immersion, preceded by a brief immersion in a hypertonic solution) greatly enhanced the uptake of soluble, but not particulate antigen through temporary disruption of the integrity of the epithelia of gills and skin. Damage induced is mild and does not impose additional stress over the handling associated with immersion vaccination. Especially HI briefly but strongly activates the innate immune system. We conclude that HI more effectively increased the uptake of vaccine and enhanced the efficacy by which vaccine components are processed and presented by the innate immune system, dually enhancing the mucosal immune response. Understanding the mechanisms involved in uptake and processing of vaccine in the early phase of the immune response will greatly benefit the design of immersion vaccination.

Fusion of interleukin-2 to subunit antigens increase their antigenicity in vitro due to an interleukin-2 receptor beta-mediated antigen uptake mechanism.

Subunit vaccines, based on one or more epitopes, offer advantages over whole vaccines in terms of safety but are less antigenic. We investigated whether fusion of the cytokine interleukin-2 (IL-2) to influenza-derived subunit antigens could increase their antigenicity. The fusion of IL-2 to the subunit antigens increased their antigenicity in vitro. Encapsulation of the subunit antigen in liposomes also increased its antigenicity in vitro, yet encapsulation of the subunit IL-2 fusion did not. The use of anti-IL-2 receptor beta (IL-2Rbeta) antibody to block the receptor subunit on macrophages suggested that the adjuvancy exerted by IL-2 in our in vitro system is due to, at least in part, a previously unreported IL-2Rbeta-mediated antigen uptake mechanism.

Intestinal barrier function.

Intestinal barrier function regulates transport and host defense mechanisms at the mucosal interface with the outside world. Transcellular and paracellular fluxes are tightly controlled by membrane pumps, ion channels and tight junctions, adapting permeability to physiological needs. Food and microbial antigens are under constant surveillance of the mucosal immune system. Tolerance against commensals and immunity against pathogens require intact antigen uptake, recognition, processing and response mechanisms. Disturbance at any level, but particularly bacterial translocation due to increased permeability and breakdown of oral tolerance due to compromised epithelial and T cell interaction, can result in inflammation and tissue damage. New therapeutic approaches including probiotics and peptides to restore disrupted barrier function are evolving.

Dendritic cells derived from multiple myeloma patients efficiently internalize different classes of myeloma protein.

Myeloma protein is a unique tumor antigen that can be used to devise tumor-specific vaccination strategies. As dendritic cells (DCs) are extremely potent at inducing T-cell responses, clinical protocols have been designed using myeloma protein-pulsed DCs to elicit anti-tumor cell responses in vivo. To optimize antigen pulsing of DCs, we investigated mechanisms of antigen uptake and evaluated various laboratory parameters including class of myeloma protein, antigen exposure time, and DC maturational stage.DCs were generated by culturing peripheral blood stem cells from myeloma patients in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Myeloma proteins were labeled with fluorescein isothiocyanate (FITC) and internalization of protein by DCs was measured by flow cytometry.IgG, IgA, and free-kappa light chain myeloma proteins were all rapidly internalized by DCs in a time-dependent fashion. Maturation of DCs with tumor necrosis factor-alpha (TNF-alpha) resulted in diminished uptake. Endocytosis of myeloma protein by DCs was primarily mediated by fluid-phase macropinocytosis based on morphology, nonsaturable uptake kinetics, and sensitivity to drugs that inhibit membrane ruffling. Pulse-chase experiments revealed that the majority of internalized myeloma protein disappeared within 4 hours but was retained in the presence of chloroquine, indicating antigen processing had occurred. Cultured DCs from myeloma patients are functional and can efficiently endocytose different classes of myeloma protein by the mechanism of macropinocytosis. This demonstrates the feasibility of using all classes of myeloma protein for producing DC vaccines, and defines culture conditions for optimizing antigen loading of DCs for induction of anti-myeloma responses.

Mechanisms of allergen-specific immunotherapy.

Mechanisms of allergen-specific immunotherapy.

Atopic dermatitis: from the genes to skin lesions.

Atopic dermatitis: from the genes to skin lesions.

Dendritic cell maturation and antigen presentation in the absence of invariant chain.

In immature dendritic cells (DCs), major histocompatibility complex class II molecules accumulate in peptide-loading compartments and, during DC maturation, are exported to the cell surface in response to inflammatory stimuli. Moreover, it has recently been proposed that DCs have specific mechanisms of antigen uptake and delivery into major histocompatibility complex class II-loading compartments. B cells bearing a genetically disrupted invariant chain gene (Ii -/-) show alterations in the transport and function of class II molecules. We herein report that DCs derived from Ii -/- H2(k) but not Ii -/- H2(b) mice undergo normal maturation in response to tumor necrosis factor alpha and show a high degree of class II surface expression. Class II molecules are accumulated in cathepsin D- and H2-M-positive compartments in immature Ii -/- DC and, during DC maturation, are exported to the cell membrane as compact dimers. Ii -/- DCs present putative Ii-dependent hen egg lysozyme-derived epitopes to T cells. These data support the existence of Ii-independent molecular requirements for class II transport and peptide loading in DCs.

Distinction of mechanism of antigen uptake and stimulation of T cells in cultured bovine dendritic cells

Distinction of mechanism of antigen uptake and stimulation of T cells in cultured bovine dendritic cells

Distinction of mechanism of antigen uptake and stimulation of T cells in cultured bovine dendritic cells

Distinction of mechanism of antigen uptake and stimulation of T cells in cultured bovine dendritic cells

Origin, maturation and antigen presenting function of dendritic cells.

Dendritic cells are cells specialized for antigen capture, migration and T cell stimulation. Recent advances have been made in understanding their origin, their heterogeneity, the mechanism of antigen uptake, and the signals that induce their migration and maturation into immunostimulatory antigen-presenting cells. Dendritic cells represent the natural adjuvants for T cell responses and their therapeutic exploitation in the near future is foreseen.

Mechanisms of antigen uptake for presentation

There is growing evidence that different antigen-presenting cells use specialized mechanisms for antigen uptake. Macropinocytosis and the activity of the mannose receptor have been identified as efficient mechanisms of antigen capture in dendritic cells. The mechanism of uptake determines the intracellular compartment to which antigen is delivered and may determine the type of T-cell epitopes generated. New pathways for presentation of exogenous antigens on MHC class I and II molecules have been identified. These findings provide new insights into antigen presentation in vivo and will be instrumental in designing better methods of vaccination.