CLEC-2 is a phagocytic activation receptor expressed on murine peripheral blood neutrophils

Abstract

CLEC-2 is a member of the "dectin-1 cluster" of C-type lectin-like receptors and was originally thought to be restricted to platelets. In this study, we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of Ab-coated beads and the production of proinflammatory cytokines, including TNF-alpha, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like dectin-1, CLEC-2 can recruit the signaling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.

FIGURE 1

mCLEC-2 is expressed on the surface of peripheral blood neutrophils. A, Anti-HA staining of EL4 cells, as determined by flow cytometry, showing expression of HA-tagged mCLEC-2 (filled histogram) at the cell surface. The vector-only control cells are indicated by the unfilled histogram. B, The monoclonal and affinity-purified polyclonal antibody raised against CLEC-2 specifically recognise EL4 cells transduced with HA-tagged mCLEC-2 (filled histogram), but not vector-only control cells (unfilled histogram) as determined by flow cytometry. C, mCLEC-2 is expressed on the surface of CD61^highSSC ^low platelets (filled histogram). D, Expression of mCLEC-2 (filled histogram) on CD11b⁺Gr-1⁺ peripheral blood neutrophils from BALB/c, C57BL/6 and 129/Sv mice, as indicated. E, mCLEC-2 is only weakly expressed on CD11b⁺Gr-1⁺ bone marrow cells and F, elicited inflammatory neutrophils. In C to F, the polyclonal antibody was used for staining and isotype control staining is indicated by the unfilled histogram. The data shown are representative of at least three independent experiments. G, Regulation of mCLEC-2 expression on neutrophils and monocytes following stimulation with various TLR agonists, as detected with monoclonal anti-CLEC-2 by flow cytometry. The data show results of PBLs pooled from 18 mice.

FIGURE 2

mCLEC-2 mediates phagocytosis. A, Quantitation of zymosan binding by transduced NIH3T3 fibroblasts. B, FACS-based analysis of phagocytosis, showing the extent of zymosan internalisation (grey histograms) by NIH3T3 cells expressing the constructs, as indicated. Cytochalsin D (unfilled histograms) was used to inhibit actin polymerisation and served as a control in this assay. The histograms shown are representative of at least three independent experiments, and the bars indicate the percentage of cells with internalised particles. C, Confocal image demonstrating FITC-zymosan uptake by RAW264.7 cells expressing the chimera, but not by cells expressing the Y7F chimera. D, FACS-based analysis demonstrating uptake of anti-CLEC-2 coated Dynabeads (grey histogram) by NIH3T3 cells expressing the chimeric receptor. Cytochalsin D treated cells (black histogram) were included as a control. E, Confocal image showing an NIH3T3 cell expressing mCLEC-2 internalising a FITC-labelled anti-CLEC-2 coated Dynabead. Inset shows the presence of an actin-based (red) phagoyctic cup around the particle. F, In comparison to isotype-coated beads, granulocytes specifically recognise anti-CLEC-2 and anti-Dectin-1 coated Dynbabeads. G, Cells binding the anti-CLEC-2 coated Dynabeads internalise these particles in an actin dependent fashion. *, p <0.05 versus control (Student’s t test).

FIGURE 3

mCLEC-2 can induce pro-inflammatory cytokine production. A, Addition of rhodocytin or LPS to purified neutrophils induces the production of TNFα. B, Quantitation of zymosan (zy) binding and C, zymosan induced TNFα production, by transduced RAW264.7 macrophages, in the presence or absence of soluble β-glucan (βG), as indicated. The data shown are the mean ± SD and are representative of at least three independent experiments. *, p <0.05 versus control (Student’s t test).

FIGURE 4

mCLEC-2 can recruit and signal via Syk kinase. A, Western blotting of immunoprecipitates from RAW macrophages using phosphorylated (YP) and unphosphorylated peptides (Y), corresponding to the cytoplasmic tail of mCLEC-2 or Dectin-1. B, Western blotting of anti-HA immunoprecipitates from A20 cells expressing HA-tagged mCLEC-2. Cells were either unstimulated (US) or stimulated with pervanadate (S). Blots were probed with anti-phosphotyrosine (αPY) and anti-Syk as indicated. C, IL-2 production following zymosan stimulation of Syk-deficient (Syk-) and Syk-sufficient. (Syk+) B-cells, transduced with the chimeric receptor. The data shown are the mean ±SD and are representative of at least three independent experiments. *, p <0.05 versus C35 Syk^- cells (Student’s t test). D, Confocal image demonstrating the recruitment of phospho-Syk (red) by RAW264.7 macrophages expressing the chimeric receptor following stimulation with FITC-zymosan. The nucleus is stained with Hoechst dye (blue).

FIGURE 5

mCLEC-2 does not induce the respiratory burst. A, The respiratory burst in various RAW264.7 transfectants, measured by assessing the conversion of dihydrorhodamine 123 to rhodamine by flow cytometry, following stimulation with zymosan. The data are expressed as mean fluorescent intensity (MFI). The data shown are the mean ± SD and are representative of at least three independent experiments. *, p <0.05 versus control (Student’s t test). B, The respiratory burst in peripheral blood neutrophils, determined as in (A), following stimulation with rhodocytin or PMA. Number indicates mean fluorescent intensity.