CD300c is an activating receptor expressed on human monocytes

Abstract

Human CD300 molecules comprise a family of receptors that regulate many immune cell processes. They are mostly expressed on myeloid cells, although expression of two members, CD300a and CD300c, has also been described on lymphocytes. However, due to the lack of specific antibodies that distinguish between these two receptors, it has been difficult to determine the expression pattern and function of CD300a and CD300c in primary cells. Here, we have identified a specific monoclonal antibody, clone TX45, that recognizes only CD300c and show that within freshly isolated blood leukocytes, monocytes are the only cells that express CD300c on the cell surface. In vitro differentiation experiments revealed that CD300c is differentially expressed on different monocyte-derived cells, including macrophages and dendritic cells. Furthermore, TLR ligands LPS and flagellin dynamically regulate the expression of CD300c. Cross-linking of this receptor with clone TX45 monoclonal antibody induced calcium mobilization, upregulation of the costimulatory molecule CD86 and the production of inflammatory cytokines. Importantly, LPS-mediated production of inflammatory cytokines by monocytes was further enhanced if CD300c was simultaneously engaged by the agonist antibody. Altogether, our results show that human CD300c is an activating receptor expressed on monocytes and that it has a potential role in inflammatory responses.

Fig. 1

Monoclonal antibody clone TX45 binds specifically to CD300c. a 293T cells were transiently transfected with empty vector (upper panels), vectors expressing full-length CD300c (middle panels) or CD300a (lower panels). After 16 h of transfection, cells were stained with the following monoclonal antibodies: anti-CD300c, clone TX45 (left lane); anti-CD300a/c, clone E59.126 (middle lane), and anti-CD300a/c, clone TX49 (right lane). Shaded histograms represent unstained cells and the open histograms represent the staining with the specific antibodies. These data are representative of 3 independent experiments. b YTS cells stably transfected with empty vector (upper panel), vector expressing CD300c (middle panel) and CD300a (lower panel) were stained with anti-CD300c, clone TX45 (left lane) and anti-CD300a/c, clone E59.126 (middle lane). The shaded histograms represent the isotype-matched controls and the open histograms the staining with the specific monoclonal antibodies. Results are representative of 3 independent experiments.

Fig. 2

CD300c is expressed on human monocytes and monocyte-derived cells. a Cells were isolated from normal healthy donors and stained with specific antibodies to distinguish different cell subsets. In addition, cells were stained with anti-CD300a/c (clone E59.126) and anti-CD300c (clone TX45) monoclonal antibodies. Shaded histograms represent the staining with isotype-matched controls and empty histograms the staining with anti-CD300a/c (clone E59.126; upper panel) and anti-CD300c (clone TX45; lower panel). Data from a representative out of 6 are shown. b Enriched monocytes were differentiated into mDC and macrophages according to the protocol described in the Materials and Methods. The expression of CD300c was determined using anti-CD300c (clone TX45) monoclonal antibody. The shaded histograms represent the staining with isotype-matched control and the open histograms correspond to the specific staining with anti-CD300c (clone TX45). Results from a representative out of 4 are shown.

Fig. 3

Expression of CD300c is regulated on monocytes by LPS. a Enriched monocytes from healthy donors were either left untreated (Alone) or treated with LPS for 2 and 24 h. RNA was extracted and the levels of CD300c mRNA were determined by real-time PCR. Graph bars represent the average ± SEM. Data are from 5 independent experiments. b Enriched monocytes were either left untreated or treated with LPS for 2 and 24 h. The cells were harvested and checked for CD300c cell surface expression. The shaded histograms represent the staining with the isotype control (MOPC-21) and the open histograms represent the staining with anti-CD300c (clone TX45) monoclonal antibody. The numbers indicate the values of median fluorescence intensity. Results are representative of 5 healthy donors.

Fig. 4

Cross-linking of CD300c induces calcium mobilization and upregulation of CD86. a Enriched monocytes from human healthy donors were loaded with Fluo-4 and Fura-Red. To establish a baseline, monocytes were first acquired using a FACSCalibur for 30 s at which point the primary antibodies, anti-CD300c (clone TX45) or isotype-matched control (clone MOPC-21), were added. Then, at 60 s, the primary antibodies were cross-linked with goat anti-mouse IgG F(ab′)₂ and fluorescence was measured. Ca²⁺ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. Results are representative of 3 independent experiments. b Enriched monocytes from human healthy donors were left either nonstimulated (None) or stimulated with plate-bound isotype control antibody, MOPC-21 (Isotype), and anti-CD300c monoclonal antibody, clone TX45 (TX45) for 24 h. Then, cells were harvested, stained and analyzed by flow cytometry. Shaded histograms correspond to unstained cells and open histograms to the specific staining with anti-CD86 monoclonal antibody. Numbers indicate the values of the median fluorescence intensity. These results are representative of 3 independent experiments from 3 different healthy donors.

Fig. 5

Cross-linking of CD300c in monocytes induces the secretion of inflammatory cytokines. Freshly isolated monocytes from healthy donors were either stimulated with plate-bound isotype-matched control antibody, MOPC-21 (empty bars), or with anti-CD300c antibody, TX45 (black bars), in the absence (Untreated) or presence of LPS for 24 h. Culture supernatants were harvested and tested for the secretion of human inflammatory cytokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of cytokines: TNF-α, IL-1β and IL-10 (pg/ml), and IL-8 and IL-6 (ng/ml). Graph bars represent the average ± SEM. Data are from 5 independent experiments.