Immunology Letters 131 (2010) 89–96
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Human dendritic cells contain cell surface sialyltransferase activity M. Guadalupe Cabral a, A. Rita Piteira a, Zélia Silva a, Dário Ligeiro b, Reinhard Brossmer c, Paula A. Videira a,∗ a
CEDOC, Departamento de Imunologia, Faculdade de Ciências Médicas, FCM, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal Centro de Histocompatibilidade do Sul, Lisboa, Portugal c Biochemistry Centre, University of Heidelberg, Heidelberg, Germany b
a r t i c l e
i n f o
Article history: Received 11 November 2009 Received in revised form 22 February 2010 Accepted 24 February 2010 Available online 3 March 2010 Keywords: Ecto-sialyltransferase Sialic acid Dendritic cell Endocytosis
a b s t r a c t Human monocyte-derived dendritic cells (mo-DCs) express highly sialylated structures, with recognized but poorly understood function in maturation, immunogenicity and endocytosis capacity. We have previously shown that mo-DCs surface sialylation is changeable upon different stimuli, which led us to hypothesise the existence of cell surface (non-intracellular) sialyltransferases, rapidly restoring or altering mo-DC surface sialylation, thus modulating specific functions. Here, we demonstrate that, in the presence of exogenous CMP-Neu5Ac, mo-DCs incorporate considerable amounts of sialic acids into cell surface, predominantly when mo-DCs were previously desialylated or matured. This is a genuine sialyltransferase activity, confirmed by specific inhibition assays, which is not influenced by secreted enzymes. Functionally, the ecto-sialyltransferase activity causes a significant down-regulation of mo-DCs endocytic capacity, without affecting the maturation state. These findings suggest that ecto-sialyltransferases participate in a dynamic control of mo-DC sialylation, with functional repercussions. This activity is possibly related with specific physiological and pathological conditions, as inflammation and infection, contributing to protection and homeostasis regulation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs) are special antigen-presenting cells that uptake, process and present antigens to lymphocytes and regulate cellular and humoral immune responses [1]. Their role in initiating specific immunity against many agents, including tumor antigens, has encouraged its inclusion in immunotherapy. At present, different DC-subsets, including monocyte-derived DCs (mo-DCs) are used in immune adoptive vaccine protocols to treat cancer patients [2]. However, DC-based therapy still faces several problems that hinder its efficiency. In fact, these cells often show defective maturation and antigen presentation, which results in the induction and maintenance of tolerance to tumor antigens [3], a situation counterproductive to the therapeutic value of DC therapy. Sialic acids or N-acetylneuraminic acids are a family of sugars, typically found on the outermost ends of cell surface glycoconjugates. Due to this position, they are key structural determinants for a number of cell surface receptors, involved in the immune response, such as the siglecs and selectins (reviewed in [4,5]). Sialic acid moieties also prevent the deposition of complement on the cell
∗ Corresponding author. Tel.: +351 218 803 045; fax: +351 218 853 480. E-mail address:
[email protected] (P.A. Videira). 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.02.009
surface and are also ligands for a diverse array of exogenous lectins from other host cells and invasive pathogens [6]. We and other groups have confirmed evidence that sialic acids influences DC immune response [7–10]. Immature mo-DC surface has a high ␣-2,3- and ␣-2,6-sialylation content, which is changeable upon different maturation stimuli [8,9,11]. Removing the sialic acid from mo-DCs, by bacterial sialidase treatment rapidly diminishes the mo-DC capacity for endocytosis [9]. This phenomenon seems to be in part associated with the DC-maturation triggering, as evidenced by the increased MHC and costimulatory molecules’ expression [10]. Sialidase-treated mo-DCs also show increased gene transcription of pro-inflammatory and Th1promoting cytokines and induce higher proliferative response of T lymphocytes. Experiments with mouse models suggested that the sialylated glycans mediated by ST3Gal.I and ST6Gal.I sialyltransferases are at the basis of the observed maturation induction mechanism [10]. Thus, modifications of the mo-DC surface sialylation result in the modulation of different immunological aspects, such as differentiation, endocytosis capacity and maturation and may provide an additional layer to regulate its immunobiology. Sialyltransferases are usually integral membrane proteins of the Golgi apparatus and generate sialic acid content and diversity during the biosynthesis of glycoconjugates and restore it during the recycling of surface receptors [12]. Nevertheless, other forms of
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sialyltransferases have been described. For instance, secretion of sialyltransferases into the serum is considered an integral part of the systemic inflammatory responses [13,14]. These soluble enzymes are originated from membrane-bound forms after proteolytic cleavage of the NH2 -terminal signal anchor [15]. There is also growing evidence that sialyltransferases may be present on the cell surface of some leukocytes, such as neutrophils and B lymphocytes, promoting a rapid modulation of surface sialylation without de novo synthesis of glycoconjugates [16–18]. In neutrophils, these ecto-sialyltransferases seem to modulate the ability of these cells to adhere and migrate across endothelium [16]. Nevertheless, the data about the distribution, role and nature of these surface sialyltransferases is still very scarce. Because mo-DCs express a highly sialylated cell surface, whose sialic acid content is modulated and influences different functionalities, we hypothesize, in this work, that mo-DCs also express ecto-sialyltransferases which rapidly modulate or restores the surface content of this sugar. Through the use of a very sensitive fluorescence-based sialyltransferase assay, we were able to determine cell surface sialylation by flow cytometry and microscopy, and thus prove that mo-DC exhibit a surface enzymatic activity that rapidly restores its sialic acid content and is inhibited by the sialyltransferase inhibitor, CTP [19,20]. In addition, given the important DC functions which are modulated by sialylation [9], we attempted to address different functional aspects by analysing whether surface sialyltransferase activity is influenced by maturation and if this activity affects mo-DC capacity for endocytosis.
2. Materials and methods 2.1. Media and reagents RPMI 1640 supplemented with 10% foetal calf serum (FCS) from Sigma (St. Louis, MO, USA), 2 mM l-glutamine, 1% nonessential amino acids, 1% pyruvate, 100 g/ml penicillin/streptomycin and 50 M 2-mercaptoethanol, all from Gibco–Invitrogen (Painsley, UK) was used throughout this work, for cell culture. Recombinant human IL-4 (rhIL-4) and GM-CSF (rhGM-CSF) were purchased from R&D Systems (Minneapolis, MN, USA). Cytidine-5 -monophospho5-fluoresceinylthioureido-N-aminoacetylneuraminic acid (CMP-5FITC-Neu) (CMP contamination in each preparation
