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Retagging Identifies Dendritic Cell-specific Intercellular Adhesion Molecule-3 (ICAM3)-grabbing Non-integrin (DC-SIGN) Protein as a Novel Receptor for a Major Allergen from House Dust Mite*

  • Mohamed Emara
    Footnotes
    Affiliations
    School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Pierre-Joseph Royer
    Footnotes
    Affiliations
    School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Jafar Mahdavi
    Affiliations
    School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Farouk Shakib
    Affiliations
    School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, United Kingdom

    Respiratory Biomedical Research Unit, The University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Amir M. Ghaemmaghami
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-0-115-823-0730; Fax: 44-0-115-823-0759
    Affiliations
    School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, United Kingdom

    Respiratory Biomedical Research Unit, The University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Author Footnotes
    * This study was supported in part by the University of Nottingham School of Molecular Medical Sciences.
    1 Present address: Faculty of Pharmacy, Helwan University, Helwan, Egypt.
    2 Recipient of a Wellcome Trust Value In People (VIP) award.
Open AccessPublished:December 28, 2011DOI:https://doi.org/10.1074/jbc.M111.312520
      Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.

      Introduction

      Dendritic cells (DCs)
      The abbreviations used are: DC
      dendritic cell
      CLR
      C-type lectin receptor
      CT
      control
      DC-SIGN
      dendritic cell-specific ICAM-3-grabbing non-integrin
      ICAM
      intercellular adhesion molecule
      MR
      mannose receptor
      qRT-PCR
      quantitative real time PCR
      retagging
      receptor activity-directed affinity tagging
      sDC-SIGN
      soluble DC-SIGN.
      act as sentinels of the immune system and serve as a bridge between innate and adaptive immunity. Internalization of antigens by DCs is an important step in the sequence of events that leads to the induction of the adaptive immune response (
      • Banchereau J.
      • Briere F.
      • Caux C.
      • Davoust J.
      • Lebecque S.
      • Liu Y.J.
      • Pulendran B.
      • Palucka K.
      Immunobiology of dendritic cells.
      ). DCs can efficiently sample their microenvironment using a plethora of receptors such as C-type lectin receptors (CLRs), Toll-like receptors, or scavenger receptors (
      • Figdor C.G.
      • van Kooyk Y.
      • Adema G.J.
      C-type lectin receptors on dendritic cells and Langerhans cells.
      ). Immature DCs take up antigens in peripheral tissues, process them into peptides, and then migrate to lymph nodes where they acquire a fully mature status capable of stimulating naïve T cells (
      • Lambrecht B.N.
      Allergen uptake and presentation by dendritic cells.
      ,
      • Lanzavecchia A.
      Mechanisms of antigen uptake for presentation.
      ). Immature DCs are characterized by their superior capacity for antigen uptake which can be attributed to the numerous CLRs that are highly expressed on these cells. These CLRs include mannose receptor (MR, CD206), dendritic cell-specific intracellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN, CD209), and dendritic and epithelial cells, 205 kDa (DEC-205, CD205) (
      • Geijtenbeek T.B.
      • van Vliet S.J.
      • Engering A.
      • 't Hart B.A.
      • van Kooyk Y.
      Self- and nonself-recognition by C-type lectins on dendritic cells.
      ,
      • van Kooyk Y.
      C-type lectins on dendritic cells: key modulators for the induction of immune responses.
      ,
      • van Vliet S.J.
      • García-Vallejo J.J.
      • van Kooyk Y.
      Dendritic cells and C-type lectin receptors: coupling innate to adaptive immune responses.
      ). C-type lectins are calcium-dependent carbohydrate-binding glycoproteins with a wide range of biological functions characterized by the presence of at least one carbohydrate recognition domain that interacts with and recognizes carbohydrates via either mannose or galactose side chains (
      • Cambi A.
      • Koopman M.
      • Figdor C.G.
      How C-type lectins detect pathogens.
      ,
      • Drickamer K.
      C-type lectin-like domains.
      ,
      • Drickamer K.
      • Fadden A.J.
      Genomic analysis of C-type lectins.
      ,
      • McGreal E.P.
      • Miller J.L.
      • Gordon S.
      Ligand recognition by antigen-presenting cell C-type lectin receptors.
      ).
      DCs have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive (
      • Broide D.H.
      • Finkelman F.
      • Bochner B.S.
      • Rothenberg M.E.
      Advances in mechanisms of asthma, allergy, and immunology in 2010.
      ,
      • Eisenbarth S.C.
      • Piggott D.A.
      • Bottomly K.
      The master regulators of allergic inflammation: dendritic cells in Th2 sensitization.
      ). Given the importance of antigen recognition and uptake by DCs on downstream events leading to T cell differentiation, there is considerable interest in identifying potential receptors for allergens on DCs. Within this context, we and others have shown that MR is partially involved in the uptake of Der p 1, the major allergen from house dust mite (
      • Deslée G.
      • Charbonnier A.S.
      • Hammad H.
      • Angyalosi G.
      • Tillie-Leblond I.
      • Mantovani A.
      • Tonnel A.B.
      • Pestel J.
      Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.
      ,
      • Royer P.J.
      • Emara M.
      • Yang C.
      • Al-Ghouleh A.
      • Tighe P.
      • Jones N.
      • Sewell H.F.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.
      ). Blocking MR by mannan (
      • Deslée G.
      • Charbonnier A.S.
      • Hammad H.
      • Angyalosi G.
      • Tillie-Leblond I.
      • Mantovani A.
      • Tonnel A.B.
      • Pestel J.
      Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.
      ) or its down-regulation using siRNA (
      • Royer P.J.
      • Emara M.
      • Yang C.
      • Al-Ghouleh A.
      • Tighe P.
      • Jones N.
      • Sewell H.F.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.
      ) leads to approximately 60–70% reduction in Der p 1 uptake by human monocyte-derived DCs. The residual uptake after blocking MR does not seem to be due to macropinocytosis by DCs, and as such it is reasonable to suggest the presence of other putative allergen receptors on DCs. In this study, using a number of different approaches including confocal microscopy, receptor activity-directed affinity tagging (retagging) (
      • Ilver D.
      • Arnqvist A.
      • Ogren J.
      • Frick I.M.
      • Kersulyte D.
      • Incecik E.T.
      • Berg D.E.
      • Covacci A.
      • Engstrand L.
      • Borén T.
      Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.
      ,
      • Mahdavi J.
      • Sondén B.
      • Hurtig M.
      • Olfat F.O.
      • Forsberg L.
      • Roche N.
      • Angstrom J.
      • Larsson T.
      • Teneberg S.
      • Karlsson K.A.
      • Altraja S.
      • Wadström T.
      • Kersulyte D.
      • Berg D.E.
      • Dubois A.
      • Petersson C.
      • Magnusson K.E.
      • Norberg T.
      • Lindh F.
      • Lundskog B.B.
      • Arnqvist A.
      • Hammarström L.
      • Borén T.
      Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation.
      ) and gene silencing, we show that Der p 1 and Can f 1 uptake by human DCs is also mediated by DC-SIGN. Although down-regulation of MR inhibits Th2 differentiation (
      • Royer P.J.
      • Emara M.
      • Yang C.
      • Al-Ghouleh A.
      • Tighe P.
      • Jones N.
      • Sewell H.F.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.
      ,
      • Emara M.
      • Royer P.J.
      • Abbas Z.
      • Sewell H.F.
      • Mohamed G.G.
      • Singh S.
      • Peel S.
      • Fox J.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      Recognition of the major cat allergen Fel d 1 through the cysteine-rich domain of the mannose receptor determines its allergenicity.
      ), intriguingly we have shown that knocking down DC-SIGN expression on human DCs leads to a bias toward Th2 cell differentiation in autologous DC-T cell co-cultures, suggesting an antagonistic relationship between the two main allergen receptors expressed on DC surface.
      Early events at the interface of allergens and DCs play a key role in downstream events leading to allergic sensitization. Therefore, identifying receptors that are involved in the initial recognition and uptake of allergens by DCs would not only lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization but also pave the way for the rational design of novel intervention strategies.

      DISCUSSION

      We have previously shown that the uptake of a number of glycoallergens such as Der p 1 is mediated through MR; however, MR seems to be responsible for only approximately 60% of this uptake (
      • Royer P.J.
      • Emara M.
      • Yang C.
      • Al-Ghouleh A.
      • Tighe P.
      • Jones N.
      • Sewell H.F.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.
      ). Previous work (
      • Deslée G.
      • Charbonnier A.S.
      • Hammad H.
      • Angyalosi G.
      • Tillie-Leblond I.
      • Mantovani A.
      • Tonnel A.B.
      • Pestel J.
      Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.
      ) suggested that the residual Der p 1 uptake does not seem to be mediated through macropinocytosis, which is in line with our own observations (data not shown). Almost all endocytic pathways are sensitive to cholesterol perturbation (
      • Mayor S.
      • Pagano R.E.
      Pathways of clathrin-independent endocytosis.
      ), and clathrin-dependent (
      • Subtil A.
      • Gaidarov I.
      • Kobylarz K.
      • Lampson M.A.
      • Keen J.H.
      • McGraw T.E.
      Acute cholesterol depletion inhibits clathrin-coated pit budding.
      ) and -independent pathways (
      • Naslavsky N.
      • Weigert R.
      • Donaldson J.G.
      Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements.
      ) are both inhibited by the removal of cholesterol. In this study, using cyclodextran (
      • Gold S.
      • Monaghan P.
      • Mertens P.
      • Jackson T.
      A clathrin-independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of BHK cells.
      ), we depleted the DC membrane from cholesterol thereby disrupting all receptor-mediated endocytosis (
      • Mayor S.
      • Pagano R.E.
      Pathways of clathrin-independent endocytosis.
      ). This led to almost complete abrogation of Der p 1 uptake by DCs compared with control cultures which were not treated by cyclodextran. These data clearly suggest the presence of other putative receptors on DCs that are capable of allergen uptake.
      To identify such receptors we used retagging, which is mainly based on labeling the protein of interest with a multifunctional cross-linker composed of a biotin tag disulfide bonded to a photoreactive group. Thus, allowing the labeled allergen to interact with the cells and then subjecting the cells to UV irradiation will enable the photoreactive group to form a covalent bond with structures in the immediate vicinity. The bound receptor could then be extracted and identified by mass spectroscopy (
      • Ilver D.
      • Arnqvist A.
      • Ogren J.
      • Frick I.M.
      • Kersulyte D.
      • Incecik E.T.
      • Berg D.E.
      • Covacci A.
      • Engstrand L.
      • Borén T.
      Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.
      ,
      • Mahdavi J.
      • Sondén B.
      • Hurtig M.
      • Olfat F.O.
      • Forsberg L.
      • Roche N.
      • Angstrom J.
      • Larsson T.
      • Teneberg S.
      • Karlsson K.A.
      • Altraja S.
      • Wadström T.
      • Kersulyte D.
      • Berg D.E.
      • Dubois A.
      • Petersson C.
      • Magnusson K.E.
      • Norberg T.
      • Lindh F.
      • Lundskog B.B.
      • Arnqvist A.
      • Hammarström L.
      • Borén T.
      Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation.
      ). Using this technique, we identified DC-SIGN as a novel receptor for the major house dust mite and dog allergens, Der p 1 and Can f 1, respectively.
      Having identified DC-SIGN as a potential receptor for Der p 1 on DCs, we set out to investigate its role in Der p 1 uptake by DCs. The functional importance and contribution of DC-SIGN to Der p 1 uptake by human DCs were assessed using different, but complementary approaches. First, we investigated the binding of DC-SIGN to different allergens using ELISA. Der p 1, Can f 1, and Ara h 1 were all found to bind to DC-SIGN in contrast to the cat allergen Fel d 1 which showed no binding. Another, perhaps more physiologically relevant, approach is to study the binding of Der p 1 to DC-SIGN using 3T3/DC-SIGN cells (
      • Wu L.
      • Martin T.D.
      • Vazeux R.
      • Unutmaz D.
      • KewalRamani V.N.
      Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGN interactions with ICAM-3 do not promote human immunodeficiency virus type 1 transmission.
      ,
      • Furmonaviciene R.
      • Ghaemmaghami A.M.
      • Boyd S.E.
      • Jones N.S.
      • Bailey K.
      • Willis A.C.
      • Sewell H.F.
      • Mitchell D.A.
      • Shakib F.
      The protease allergen Der p 1 cleaves cell surface DC-SIGN and DC-SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses.
      ). This showed that Cy5 Der p 1 binds to 3T3 fibroblasts expressing DC-SIGN, but not to mock cells as detected by flow cytometry. Incubation with EDTA completely abrogated the binding, indicating that binding is Ca2+-dependent.
      The above binding data prompted us to investigate whether DC-SIGN is involved in allergen uptake by human DCs. Based on the carbohydrate specificity of Lewis-x for DC-SIGN (
      • Appelmelk B.J.
      • van Die I.
      • van Vliet S.J.
      • Vandenbroucke-Grauls C.M.
      • Geijtenbeek T.B.
      • van Kooyk Y.
      Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing non-integrin on dendritic cells.
      ,
      • Feinberg H.
      • Mitchell D.A.
      • Drickamer K.
      • Weis W.I.
      Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR.
      ), we therefore sought to target DC-SIGN on DCs using a natural ligand (Lewis-x antigen) to investigate further the impact of DC-SIGN engagement on Der p 1 uptake by DCs. Results obtained demonstrated dose-dependent and significant inhibition of Der p 1 uptake by Lewis-x with nearly 20% inhibition with highest concentration (20 mg/ml) of Lewis-x.
      DC-SIGN has also been shown to mediate the uptake of various allergens such as the major peanut allergen (Ara h 1) (
      • Shreffler W.G.
      • Castro R.R.
      • Kucuk Z.Y.
      • Charlop-Powers Z.
      • Grishina G.
      • Yoo S.
      • Burks A.W.
      • Sampson H.A.
      The major glycoprotein allergen from Arachis hypogaea, Ara h 1, is a ligand of dendritic cell-specific ICAM-grabbing nonintegrin and acts as a Th2 adjuvant in vitro.
      ), as well as the Bermuda grass pollen allergen (BG-60) and the major group 2 allergen from house dust mite (Der p 2) (
      • Hsu S.C.
      • Chen C.H.
      • Tsai S.H.
      • Kawasaki H.
      • Hung C.H.
      • Chu Y.T.
      • Chang H.W.
      • Zhou Y.
      • Fu J.
      • Plunkett B.
      • Su S.N.
      • Vieths S.
      • Lee R.T.
      • Lee Y.C.
      • Huang S.K.
      Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells.
      ). As such, this and other reports clearly underscore the potential of CLRs (in particular MR and DC-SIGN), which are highly expressed on the surface of antigen-presenting cells, to serve as common receptors for recognition of a wide range of glycosylated allergens. Intriguingly, we have also shown for the first time that down-regulation of DC-SIGN expression through siRNA leads to a bias toward Th2 polarization in autologous DC-T cell co-cultures. This is in contrast with our earlier work showing that down-regulation of MR, a major Der p 1 receptor expressed on DCs, leads to an opposite effect, i.e. bias toward Th1 polarization (
      • Royer P.J.
      • Emara M.
      • Yang C.
      • Al-Ghouleh A.
      • Tighe P.
      • Jones N.
      • Sewell H.F.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.
      ,
      • Emara M.
      • Royer P.J.
      • Abbas Z.
      • Sewell H.F.
      • Mohamed G.G.
      • Singh S.
      • Peel S.
      • Fox J.
      • Shakib F.
      • Martinez-Pomares L.
      • Ghaemmaghami A.M.
      Recognition of the major cat allergen Fel d 1 through the cysteine-rich domain of the mannose receptor determines its allergenicity.
      ). This could be partly explained by data showing that differentiation of naive T cells toward Th2 occurs upon co-stimulation through ICAM-1 and ICAM-2 instead of ICAM-3, which is thought to be the main DC-SIGN counterstructure on T cells (
      • Bleijs D.A.
      • de Waal-Malefyt R.
      • Figdor C.G.
      • van Kooyk Y.
      Co-stimulation of T cells results in distinct IL-10 and TNF-α cytokine profiles dependent on binding to ICAM-1, ICAM-2, or ICAM-3.
      ). Within this context, it is interesting to note that we have shown previously (
      • Furmonaviciene R.
      • Ghaemmaghami A.M.
      • Boyd S.E.
      • Jones N.S.
      • Bailey K.
      • Willis A.C.
      • Sewell H.F.
      • Mitchell D.A.
      • Shakib F.
      The protease allergen Der p 1 cleaves cell surface DC-SIGN and DC-SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses.
      ) that Der p 1, a cysteine protease, in its enzymatically active form, can cleave DC-SIGN (its “Th1-promoting” receptor) but not MR (its “Th2-promoting” receptor), and this will further amplify its alleregenicity. Although the exact mechanism of polarized Th2 cell differentiation in the absence of DC-SIGN is yet to be determined, these data clearly indicate that glycoallergen uptake by DCs and events leading to downstream Th2 polarization are complex and involve at least two receptors, MR and DC-SIGN, whose engagement by allergen leads to distinct and possibly antagonistic signaling events. Therefore, it is reasonable to assume that the overall fate of T cells in response to allergen exposure in different individuals could at least be partly determined by differential level of MR and DC-SIGN expression on DC subsets. In this connection, it is interesting to note that MR expression has been shown to be higher in atopic individuals (
      • Deslée G.
      • Charbonnier A.S.
      • Hammad H.
      • Angyalosi G.
      • Tillie-Leblond I.
      • Mantovani A.
      • Tonnel A.B.
      • Pestel J.
      Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.
      ).
      Early events at the interface of DCs and allergens are clearly of great importance in determining T cell polarization. In this study, we have identified a novel receptor for two major allergens from house dust mite and dog, Der p 1 and Can f 1, respectively. This new insight could lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization, and this should hopefully pave the way for the rational design of novel intervention strategies.

      Acknowledgments

      We thank Kevin Bailey and Matt Carlile (School of Biomedical Sciences, Post-Genomic Technologies Facility, University of Nottingham) for technical assistance with regard to mass spectrometry experiments. We also thank the Proteomics Core Facility at Sahlgrenska Academy, Gothenburg University.

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