Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines

Abstract

Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn.

Activation of CD8+ T Cell Responses after Melanoma Antigen Targeting to CD169+ Antigen Presenting Cells in Mice and Humans

“…Our data show that there is uptake and internalization of both receptors upon Ab binding, although DC-SIGN is internalized faster and to a greater extent. Internalization rates and routing differences could potentially depend on the Abs used or the region where they bind to [66,67]. Alternatively, lower internalization rates of human CD169 compared to DC-SIGN might suggest an Ag retention function for human CD169 similar to that observed for mouse CD169 [32,68].…”

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

“…To inhibit antibody-induced internalization, two strategies were carried out as previously reported (57). First, inhibitors were used in different concentrations before and during the internalization assay.…”

Targeting of sialoadhesin-expressing macrophages through antibody-conjugated (polyethylene glycol) poly(lactic-co-glycolic acid) nanoparticles

“…RPMI cell medium enriched with 10% (v/v) iFBS, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, 1% (v/v) glutamine, and 10% (v/v) L929 supernatant was added after 1 to 6 h and refreshed after another 24 h of incubation time. On day 6, macrophages were stimulated with mouse IFN-alpha (100 IU/mL) to induce Sn expression (De Schryver et al 2017a , b ). Macrophages were cultured for 7 days in total before endocytosis experiments were performed.…”