The mannose receptor C-type (MRC1) is an endocytic pattern recognition receptor expressed on the surface of human macrophages, important to innate and adaptive immune responses. More recently the MRC1 has been identified in a shed form following cleavage by metalloproteases. Serological levels of mammalian soluble MRC1 can be detected by ELISA and have been shown to increase in accordance with inflammation, which can be replicated in vitro with stimulation by IL-4 and GM-CSF. This study aims to elucidate the potential solubility of the chicken MRC1L-C; a paralogue from the expanded MRC1L repertoire in birds, which displays a distinctly short cytoplasmic domain and uniquely, minimal expression in tissues. Initial attempts to identify a soluble MRC1L-C shed in vitro were unsuccessful when assayed in this study by a novel competitive ELISA. Conversely immunofluorescent staining supported the hypothesis, revealing no surface expression of MRC1L-C compared to other paralogues, despite evident protein synthesis. To mimic inflammation, cells were conditioned with IL-4 and GM-CSF before analysis by SDS-PAGE. Coomassie stain revealed several bands, likely reflecting remnants of culture medium. However, a plausible band at 100-150kDa was uniquely observed for cells stimulated with an increased concentration of IL-4 (125ng/mL). Whilst aligning with human and murine reports, the banding was faint and not specifically detected. Despite using positive controls to trial initial western blot analysis, the proteins were poorly or not detected. When ran under semi-reducing conditions the MRC1L-C control was detected with a weak signal, emphasising the need to optimise loading concentration. Whilst this study provides initial evidence for the soluble nature of MRC1L-C, further research is needed to improve the stimulation of in vitro MRC1L cell models and optimise appropriate assays for the specific detection of shed paralogue.
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