Antibody binding to porcine sialoadhesin reduces phagocytic capacity without affecting other macrophage effector functions

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Abstract

Sialoadhesin (Sn) is a macrophage-restricted endocytic receptor involved in cell–cell, cell–matrix and cell–pathogen interactions. Recently, porcine Sn (pSn) was shown to be involved in signaling and lately Sn is gaining interest as a potential target for immunotherapy. However, little is known about the effect of ligand binding to Sn on macrophage effector functions. In this study, we tested the effect of antibody binding to pSn on macrophage viability, phagocytosis of microspheres, uptake and processing of soluble antigens, reactive oxygen/nitrogen species production, MHC I and MHC II cell surface expression and cytokine production. This was done by treatment of porcine primary alveolar macrophages with the pSn-specific mAb 41D3, or an isotype-matched control mAb. No significant effect on most effector functions under study was observed, except for a significant reduction of phagocytosis. Thus, antibody binding to pSn can downregulate phagocytosis, which could have implications on homeostasis, infectious and immune diseases, and immunotherapy.

Highlights

Sn is a macrophage-restricted endocytic receptor and a target for immunotherapy. ► The effect of mAb binding to pSn on macrophage viability and functions was tested. ► Phagocytosis, OVA-DQ processing, ROI and cytokine production, MHC I/II expression. ► mAb binding to Sn causes a decrease in phagocytosis, not affecting other functions. ► Possible implications on homeostasis, infectious/immune diseases, immunotherapy.

Introduction

Macrophages perform essential roles in the immune system of humans and animals in steady state conditions and inflammation [1], [2]. In the innate immune system they play pivotal roles in the cellular host defence against infection and in tissue remodeling and repair. The overproduction of chemokines and inflammatory cytokines by macrophages during inflammation is a crucial event for triggering the progressive recruitment and activation of immune cells [3], where they are at the crossroads with adaptive immunity. Additionally, they are specialized in chemotaxis, killing of invading microorganisms, uptake of microbes, infected or apoptotic cells and molecules, repairing damaged tissues, controlling the inflammatory response and preventing excessive tissue damage by their anti-inflammatory properties [4], [5], [6], [7], [8], [9]. Another important macrophage property at the interface between the innate and the adaptive immune system is the processing and presentation of endogenous or internalized antigens [10], [11], [12] or the presentation of captured antigens [13], [14]. These various functions can be exerted by different populations of functionally heterogeneous macrophages, which is reflected by their phenotypical diversity [1]. Depending on their tissue site and activation status, a range of macrophages can be found, from resting resident to fully activated inflammatory macrophages [9], each expressing various receptors enabling them to exercise their respective functions [15].

Sialoadhesin (Sn, Siglec-1, CD169) is a macrophage-restricted receptor that was first described as a sialic acid-dependent sheep erythrocyte receptor (SER) involved in lymphocyte interactions [16], [17], [18]. Sn is a member of the family of sialic acid-binding immunoglobulin (Ig)-like lectins (siglecs) [19], which are expressed in a cell type specific manner on cells of the hematopoietic and immune system [20], with the exception of MAG (Siglec-4), which is expressed in the nervous system [21], and Siglec-6, expressed in human placenta [22]. The expression of Sn is highly regulated and under normal physiological conditions Sn can only be found at high levels on distinct subsets of tissue-resident macrophages, but not on its precursor cells, the monocytes [17], [23]. Upon exposure to inflammatory stimuli, however, Sn can rapidly be upregulated in macrophages and monocytes, suggesting a role for Sn in macrophage-mediated inflammatory responses [20]. Abundant Sn expression is seen in chronic inflammatory diseases such as rheumatoid arthritis and atherosclerosis [24], [25], and on tumor-infiltrating macrophages in breast cancer tumors [26].

Sn is a type I transmembrane protein with a very large extracellular region, consisting of 1 N-terminal variable (V) Ig-like domain followed by 16 constant Ig-like domains of type C2 [27]. The extended structure of Sn is thought to promote interactions of macrophages with host cells and with the extracellular matrix, which relate to homeostasis and immunity [23], [28]. Via its distal N-terminal domain Sn can bind promiscuously to many sialylated glycoconjugates, with a preference for O-linked oligosaccharides terminating in Neu5Acα2,3Galβ1,3GalNAc [29], [30]. Besides this, binding of Sn to membrane receptors via a sialic acid-independent mechanism has also been described [31], [32]. In addition to its role in cell–cell interactions, Sn has also been shown to facilitate interactions with a variety of pathogens including viruses (HIV-1, human rhinoviruses and PRRSV; [33], [34], [35]), bacteria (Campylobacter jejuni and Neisseria meningitides; [36], [37]) and a parasite (Trypanosoma cruzi; [38]). Porcine Sn (pSn) was shown to be a clathrin-dependent endocytic receptor and to mediate uptake of sialylated bacterial and viral pathogens [36], [39]. Furthermore, it was established that porcine primary alveolar macrophages (PAM) expressing pSn internalized pSn upon binding of a pSn-specific monoclonal antibody (mAb) [39], [40], [41]. Additionally, it was assessed that a pSn-specific immunotoxin and an immunoconjugate of the model antigen human serum albumin linked to a pSn-specific mAb were also internalized into porcine PAM together with pSn [41]. Overall, these data demonstrate the involvement of Sn in internalization processes. Given its restricted expression pattern and its potential to internalize, Sn is gaining interest as a potential target in immunotherapy [40], [41], [42], [43], [44], [45]. The receptor is well suited for a ‘Trojan horse’ strategy, whereby therapeutic agents conjugated to an antibody or a multimeric glycan ligand are carried into the cell [41], [42], [45], [46]. Sn could be used to specifically target antigens, toxins, drugs or other molecules to macrophages, either to specifically eliminate, activate or immunomodulate these cells or to prevent ligand binding to Sn.

Apart from its large extracellular domain, Sn also contains a transmembrane domain and a short cytoplasmic domain, which is poorly conserved between mammalian species [28]. In contrast to most other siglecs, these domains are devoid of tyrosine-based motifs that are implicated in signal transduction and endocytosis, and do not associate with the DAP-12 (12 kDa DNAX-activating protein) adaptor implicated in both positive and negative immunoregulation and endocytosis [28], [46]. Recently however, antibody binding to pSn on PAM was shown to be associated with subtle alterations of MAPK (mitogen-activated protein kinase), adipocytokine and Wnt signaling and regulation of the actin cytoskeleton [47]. This indicates that antibody binding to pSn activates signaling in PAM, which could affect macrophage effector functions through interference with signaling pathways. So far, little is known about the role of Sn in the regulation of macrophage effector functions. Seen the gaining interest of using Sn as a targeting molecule, the growing number of cells and pathogens engaging Sn and the induction of signaling upon antibody binding to pSn, the present study aimed to characterize the effect of antibody binding to pSn on macrophage viability, sialoadhesin cell surface expression, phagocytosis of particulate antigens, uptake and processing of soluble antigens, reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, MHC I and MHC II cell surface expression and cytokine production.

Section snippets

Cells and antibodies

Porcine primary alveolar macrophages (PAM) were obtained and cultivated as described earlier [48]. The IgG1 mouse monoclonal anti-porcine Sn antibody (mAb) 41D3 was used as a ligand to bind pSn and for visualization of pSn cell surface expression [39], [49]. MAb 13D12 was used as an isotype-matched irrelevant control mAb [50]. MHC I antigen was visualized using the IgG2a mouse mAb PT85A (VMRD, Pullman, WA, USA), directed against the heavy chain of MHC I. MHC II antigen was detected using the IgG

Antibody binding to pSn has no effect on macrophage viability

To assess the effect of antibody binding to pSn on macrophage viability, flow cytometry was performed upon live-dead staining of macrophages treated with the Sn-specific mAb 41D3. No differences in macrophage viability were detected for mAb 41D3 treated macrophages versus isotype-matched control mAb treated groups in response to all studied concentrations (Fig. 1A). In addition, treatment of macrophages with the highest mAb dose did not have any cytotoxic effect up to 72 h after starting the

Discussion

Macrophages are considered important target cells for selective elimination, activation or immunomodulation due to their importance in homeostasis and host defence, their migratory capacity, their implications in infectious diseases, cancer and inflammatory disorders and since immunity to a specific antigen might be modulated upon targeting of this antigen to antigen-presenting cells [58], [59], [60], [61], [62]. Since Sn is an endocytic receptor and given its restricted expression pattern,

Role of the funding source

M. De Baere was financially supported by the Flemish Institute for the Promotion of Innovation by Science and technology (I.W.T.-Flanders; SB 71434). H. Van Gorp was financially supported by a post-doctoral grant from the Special Research Fund of Ghent University. This research was further supported by the Industrial Research Fund (IOF) of Ghent University (www.techtransfer.ugent.be) and the Flemish Institute for the Promotion of Innovation by Science and Technology (IWT – SBO 80046) (www.iwt.be

Disclosure statement

Three authors (Miet De Baere, Peter L. Delputte and Hans J. Nauwynck) are listed as inventors on a patent application related to the work described in this study, which has been submitted through Ghent University.

Acknowledgments

We would like to thank C. Vanmaercke, C. Boone, D. Helderweirt, T. Van Gaever, L. Sys and N. Dennequin for excellent technical assistance, Professor H. Hammad for assistance with the confocal microscopical analysis, and Professor H.W. Favoreel and Dr. I. Hoebeke for their useful comments on the manuscript.

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