Journal of Receptors and Signal Transduction

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Gene Expression Profiling of Porcine Alveolar Macrophages After Antibody-Mediated Cross-Linking of Sialoadhesin (Sn, Siglec-1)

SEM GENINI*a; ROBERTO MALINVERNI*a; PETER L. DELPUTTE b; SILVIA FIORENTINI a; ALESSANDRA STELLA a; SARA BOTTI a; HANS J. NAUWYNCK b ; ELISABETTA GIUFFRA a a Parco Tecnologico Padano-CERSA, Lodi, Italy b Laboratory of Virology, Department of Virology, Parasitology, and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Online Publication Date: 01 May 2008 To cite this Article: GENINI*, SEM, MALINVERNI*, ROBERTO, DELPUTTE, PETER L., FIORENTINI, SILVIA, STELLA, ALESSANDRA, BOTTI, SARA, NAUWYNCK, HANS J. and GIUFFRA, ELISABETTA (2008) 'Gene Expression Profiling of Porcine Alveolar Macrophages After Antibody-Mediated Cross-Linking of Sialoadhesin (Sn, Siglec-1)', Journal of Receptors and Signal Transduction, 28:3, 185 — 243 To link to this article: DOI: 10.1080/10799890802084226 URL: http://dx.doi.org/10.1080/10799890802084226

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Journal of Receptors and Signal Transduction, 28:185–243, 2008 C Informa Healthcare USA, Inc. Copyright ISSN: 1079-9893 print / 1532-4281 online DOI: 10.1080/10799890802084226

Gene Expression Profiling of Porcine Alveolar Macrophages After Antibody-Mediated Cross-Linking of Sialoadhesin (Sn, Siglec-1) Sem Genini,1* Roberto Malinverni,1* Peter L. Delputte,2 Silvia Fiorentini,1 Alessandra Stella,1 Sara Botti,1 Hans J. Nauwynck,2 and Elisabetta Giuffra1 1

Parco Tecnologico Padano-CERSA, Lodi, Italy Laboratory of Virology, Department of Virology, Parasitology, and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

2

Sialoadhesin (Sn) is the prototypic member of the Siglecs, a family of receptors mainly involved in cell-cell interactions. For several Siglecs, but not for Sn, intracellular signaling functions have been described. Because antibody-mediated cross-linking of surface transmembrane proteins is a powerful technique to investigate cell-molecular events, Sn expressed on porcine alveolar macrophages (PAM) was cross-linked with the antibody 41D3, and the expression profiles were compared with mock-treated macrophages by microarray analysis. Gene ontology analysis of 479 differentially expressed transcripts identified gene categories related to membrane localization, signal transduction, receptor and communication activities. Analyses of the human KEGG pathway database identified MAP kinase signaling, regulation of actin cytoskeleton, adipocytokine signaling, and wnt signaling as significantly altered pathways, supporting a role for Sn as intracellular signaling molecule. Real-time PCR of a subset of modulated genes confirmed these results and highlighted the reliability of a short-term cross-linking treatment for transcriptomic analysis of receptor functions. Key Words: Receptor-antibody cross-linking; Signal transduction; Gene pathways.

∗

Equal contribution. Address correspondence to Sem Genini, Livestock Genomics I, Parco Tecnologico Padano-CERSA, Via A. Einstein, 26900 Lodi, Italy. Fax: +39.0371.4662349; E-mail: [email protected]

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INTRODUCTION Sialoadhesin (Sn, Siglec-1, CD169) is the prototypic receptor of the Siglec family of sialic-acid-binding immunoglobulin-like lectins. It is a transmembrane glycoprotein of about 200 kDa and 1709 amino acids, originally described and cloned in mice (1,2) with structural features (a very large extracellular region with 17 Ig-like domains, an N-terminal V-set domain containing the sialic acid-binding site, and an unusually large number of C2-set domains) thought to be essential for mediating adhesive functions in cell-cell interactions (2,3). The short cytoplasmic tail of Sn (47 amino acids) is poorly conserved between human, mouse, and pig (4,5), suggesting that its primary function is in cell-cell or cellmatrix interactions. Moreover, Sn shows specific expression on macrophages of the hematopoietic and lymphoid tissues and on inflammatory macrophages, suggesting a specific role of this receptor in the pro-inflammatory functions of macrophages (4,6). Sn binds sialylated ligands specifically recognizing sialic acid in the α2,3glycosidic linkage of the Neu5Ac type (7). Moreover, Sn binding to several membrane receptors via a sialic acid-independent mechanism has been described (8,9). Sn is involved in the attachment and internalization of sialylated pathogens, such as Neisseria meningitidis (10) and Trypanosoma cruzi (11). In swine, Sn mediates the internalization of the porcine respiratory and reproductive syndrome virus (PRRSV) into the primary target cell, the alveolar macrophage, and it has been shown that the interactions between sialic acids on the porcine arterivirus and Sn are essential for PRRSV infection (12,13). In addition, the porcine Sn-specific monoclonal antibody 41D3 was shown to be internalized in Sn-expressing macrophages (5). Although Sn was previously described as a non-endocytic receptor, these data suggest its involvement in an endocytic process. Studies revealed that several Siglecs undergo endocytosis and possibly mediate intracellular signaling events in the hematopoietic, nervous, and immune systems [reviewed by Crocker et al. (6)]. As lectin-like receptors, they are involved in signal transduction by possibly mimicking the function of a growth factor receptor, providing an alternative or additional signaling potential to the cell (14). Among the 15 functional Siglec receptors already described in great apes, Siglec-14 was recently shown to be the first human Siglec to have an activating signaling potential (15). On the contrary, Siglec-2 (16), Siglec-3 (17), Siglec-7 and Siglec-9 (18), and Siglec-10 (19) were found to have inhibitory functions due to the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains (i.e., missing in Sn). In particular, antibody-mediated cross-linking experiments showed that Siglecs elicit signaling in the cells expressing these molecules (20). Cross-linking of Siglec-3 and Siglec-7 led to negative regulation of cellular activities (21,22), death of eosinophils (23), and neutrophils (24) was observed after cross-linking incubation with anti-Siglec-8


Gene Expression Profiling of Macrophages After Sialoadhesin Stimulation

and anti-Siglec-9 antibodies, respectively. Cross-linking with epitope-specific anti-Siglec-5 monoclonal antibodies caused an augmented neutrophil oxidative burst activity (25). Furthermore, Tateno et al. (26) showed that Siglec-2 and Siglec-F have clathrin-dependent and clathrin-independent mechanisms of endocytosis, respectively. The aim of this work was to unravel a potential signaling role also for Sn, using genome-wide expression analysis of gene pathways that are modulated in macrophages after antibody-induced cross-linking of the Sn receptor. Transcriptional profiles were investigated by using the Affymetrix platform, recently described as an appropriate tool for swine expression studies (27), by comparing anti-Sn mAb 41D3-mediated cross-linking vs. a control isotypematched mAb treatment. The obtained results provide new information on the (patho-)physiological role of Sn in the macrophage and a reference basis for the study of gene expression changes induced by Sn-dependent entry of the porcine arterivirus.

MATERIALS AND METHODS Samples and Treatments Porcine alveolar macrophages (PAM) were collected, as previously described (28), from six hybrid piglets originating from a Pietrain boar and two not parentally related sows (three piglets for each sow), belonging to the rattlerow seghers genetic line (cross-breeding between English Landrace, Belgian Landrace, Large White, and a synthetic company Landrace). PAM cells were grown for 24 hr before treatment as previously shown (12). To cross-link Sn, PAM were incubated at 37◦ C with either 50 µg/mL purified anti-Sn monoclonal antibody 41D3 (group A), or 50 µg/mL of the isotypematched (immunoglobulin G1) control/mock monoclonal antibody 13D12, directed against pseudorabies virus glycoprotein gD (group B) (29). Two hours R after treatment the cells were collected in TriZol , and the RNA was extracted and stored under standard procedures (Invitrogen Life Technologies, Inc., Paisley, UK). A cleanup step using QIAGEN RNeasy columns was performed before proceeding to sample quantification and quality control with an Agilent 2001 Bioanalyzer (Agilent Technologies GmbH, Waldbronn, Germany) using RNA 6000 Nanochips. Reverse transcription (RT) of the total RNA and synthesis of biotin-labeled cRNA with one round of amplification was carried out following the standard Affymetrix one-cycle protocol.

cRNA Hybridization on Affymetrix Gene Chips The transcriptional profiles of group A and group B (six biological repli R cates each) were compared on 12 Affymetrix GeneChip Porcine Arrays,

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containing 23,937 probe sets representing pig transcripts and control sequences (Affymetrix Inc., Santa Clara, CA, USA). Affymetrix chip hybridization and scanning of the samples were performed in accordance with standard Affymetrix protocols published previously (30) using an Affymetrix GeneChip Scanner 3000 7G.

Microarray Data Analysis The calculations of the emission intensities used the GeneChip Operating Software (GCOS v.1.4) algorithm. Raw cell files were then imported into the statistical programming environment R (31) for further analysis with tools available from the open-source Bioconductor Project (32). Expression data of the 12 slides were normalized and converted to log2 as a set using the robust multi-chip average (RMA) method (33) implemented in the Bioconductor package RMA. Linear modeling to calculate gene-wise P-values was carried out using the package linear modeling for microarray analysis (LIMMA) (34). Differentially expressed genes were ranked based on moderated t-statistics. The value of significance P was set to ≤0.02 for the initial analysis of differential expression and to