Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Abstract

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.

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

“…1C). By flow-cytometric analysis, the surface-bound antibodies were measured before and after induction of internalization to allow a more quantitative measurement of internalization (33,34). For HEp-2 cells and A549 cells, the mean fluorescence intensity (MFI) of surface RSV proteins after the addition of antibodies was reduced to 44% and 40%, respectively, showing that internalization occurs but that not all RSV proteins are internalized (Fig.…”

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

“…Earlier studies showed that pCD163 cooperated with another putative receptor, porcine sialoadhesin (Sn/CD169) (23), to facilitate the PRRSV infection, where the former contributed to the uncoating of PRRSV while the latter promoted the attachment and internalization (invasion) of the virus (40)(41)(42)(43)(44)(45)(46)(47)(48). However, one study showed that Sn gene knockout pigs were infected by PRRSV as usual (49), and a recent study using geneedited pigs demonstrates the indispensability of pCD163 (28).…”

Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27

“…It is believed to primarily function in mediating macrophage-pathogen adhesion and phagocytosis [30] as well as in promoting antigen presentation [31]. Siglec1 does not contain any known cytosolic motifs to mediate inhibitory signaling itself, and the interaction between Siglec1 and intracellular regulators is poorly understood.…”