CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

Abstract

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.

Figure 1

Triggering of CD300a results in the suppression of lipopolysaccharide (LPS) -induced matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) expression. (a) Cells were stained with anti-CD300a monoclonal antibody (mAb; empty area) for flow cytometry. Background fluorescence levels (black area) were obtained by staining the cells with isotype-matching mouse IgG. (b) Cells were pre-treated with 0·1, 0·3 or 1 μg/ml anti-CD300a mAb or 1 μg/ml mouse IgG (M) for 30 min and then stimulated with 1 μg/ml of LPS. Culture supernatants were collected 24 hr after activation for the analysis of MMP-9/MMP-2 activity using gelatin zymography and measurement of IL-8 concentrations using double sandwich ELISA. Data points are represented as a percentage of positive control (LPS-treated samples) (n = 3, ***P < 0·001 when compared with positive control samples).

Figure 2

Treatment with anti-CD300a monoclonal antibody (mAb) blocks the expression of interleukin-8 (IL-8) and matrix metalloproteinase 9 (MMP-9) in cells stimulated with ligands for Toll-like receptor 9 (TLR9) but not TLR3. THP-1 cells were pre-treated with 1 μg/ml anti-CD300a, anti-CD300f mAb or mouse IgG (M) for 30 min and then stimulated with 1 μg/ml CpG ODN or Poly I:C. Culture supernatants were collected 24 hr after activation for the measurement of MMP-9 activity and IL-8 concentrations (n = 3, ***P < 0·001 when compared with corresponding positive control samples).

Figure 3

Treatment with anti-CD300a monoclonal antibody (mAb) blocks the expression of interleukin-8 (IL-8) in U937 cells stimulated with lipopolysaccharide (LPS) and CpG PDN, but not PolyI:C. (a) U937 cells were analysed for their expression levels of CD300a using flow cytometry. Background fluorescence levels (black area) were obtained by staining the cells with isotype-matching mouse IgG. (b) Cells were pre-treated with 1 μg/ml of anti-CD300a mAb or mouse IgG for 30 min and then stimulated with 1 μg/ml of LPS, CpG ODN or Poly I:C. Culture supernatants were collected 24 hr after activation for the measurement of IL-8 concentrations (n = 3, *P < 0·05 and ***P < 0·001 when compared with corresponding positive control samples).

Figure 4

CD300a blocks Toll-like receptor (TLR) -mediated nuclear factor-κB (NF-κB) activation through inhibition of a MyD88-mediated but not a toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF) -mediated pathway. (a) 293T cells were transfected with empty vector (control) or CD300a expression vector. The expression levels of CD300a were tested after 48 hr using flow cytometry. Background fluorescence levels (black area) were obtained by staining the cells with isotype-matching mouse IgG. (b) 293T cells were transfected with a luciferase reporter plasmid containing NF-κB binding sites (4 ×) and an expression plasmid containing Renilla-luciferase as an internal control. For the induction of NF-κB activity, cells were transfected with the constitutively active forms of TLR4, MyD88, TRIF or tumour necrosis factor receptor-associated factor 6 (TRAF6); 0·2 μg/well). For inhibition, CD300a expression construct (0·2 μg/well) was co-transfected. Relative luciferase activity (RLA) was determined at 24 hr after transfection after normalization with Renilla-luciferase activity (n = 3, **P < 0·01 and ***P < 0·001 when compared with corresponding positive control).

Figure 5

CD300a and CD300f differentially affects Toll-like receptor (TLR) -mediated activation of toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF). 293T cells were transfected with a luciferase reporter plasmid containing nuclear factor-κB (NF-κB) binding sites (4 ×) and an expression plasmid containing Renilla-luciferase as an internal control. For the induction of NF-κB activity, cells were co-transfected with the constitutively active forms of TLR4, MyD88, TRIF or tumour necrosis factor receptor-associated factor 6 (TRAF6) (0·2 μg/well). For inhibition, cells were co-transfected with 0·1, 0·2, 0·3 and 0·4 μg/well of expression plasmids for CD300a, full-length CD300f, or 0·4 μg/well of cytoplasmic deletion mutant of CD300f (Δ). Relative luciferase activity (RLA) was determined at 24 hr after transfection (n = 3, *P < 0·05 when compared with corresponding positive control, **P < 0·01; ***P < 0·001).

Figure 6

Synthetic peptides containing immunoreceptor tyrosine-based inhibitory motif-like sequences of CD300a mimics the inhibitory activities of CD300a. (a) THP-1 cells were pre-treated with 1 μg/ml anti-CD300a mAb or 5 μm synthetic peptides as indicated for 30 min. Cells were then stimulated with 1 μg/ml lipopolysaccharide (LPS), CpG ODN or PolyI:C for 24 hr. Culture supernatants were then collected for the measurement of IL-8 concentrations using ELISA. (b) 293T cells were transfected with a luciferase reporter plasmid containing nuclear factor-κB (NF-κB) binding sites (4 ×) and an expression plasmid containing Renilla-luciferase as an internal control. For the induction of NF-κB activity, cells were co-transfected with the constitutively active forms of Toll-like receptor 4 (TLR4), MyD88 or toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF). Three hours after transfection, cells were treated with 5 μm synthetic peptides for additional 20 hr before measurement of relative luciferase activity (RLA; n = 3, ***P<0·001 when compared with corresponding positive control samples).

Figure 7

Inhibitory function of CD300a and CD300f is differentially regulated by specific inhibitors for SHP-1 and SHP-2. (a) THP-1 cells were sequentially pre-treated with 100 nm of PP2 or 0·2% DMSO (vehicle control, VC) for 30 min, followed by 1 μg/ml of monoclonal antibodies (mAbs) against CD300a or CD300f. Finally, cells were stimulated with 1 μg/ml lipopolysaccharide (LPS) for 24 hr. Culture supernatants were collected in 24 hr for the measurement of interleukin-8 (IL-8) concentrations. (b) THP-1 cells were sequentially pre-treated with 1 mm of PTP inhibitor III (PTP-III), 1 mm of PTP inhibitor IV (PTP-IV) or 0·2% DMSO for 30 min, followed by 1 μg/ml of mAb against CD300a or CD300f. Finally, cells were stimulated with 1 μg/ml of LPS for 24 hr. Culture supernatants were collected for the measurement of IL-8 concentrations (n = 3, ***P<0·001 when compared with corresponding control samples treated with LPS and anti-CD300a or anti-CD300f mAb).

Figure 8

Triggering CD300f stimulates both SHP-1 and SHP-2 while CD300a stimulates only SHP-1. THP-1 cells were treated with 1 μg/ml monoclonal antibodies (mAbs) against CD300f or CD300a for the indicated times. Cell lysates were then collected for Western blot analysis for SHP-1/SHP-2 and their phosphorylated forms. The experiment was performed twice with essentially the same results.

Figure 9

Inhibitors of SHP-1 and SHP-2 differentially block the inhibitory activities of CD300f under various Toll-like receptor (TLR) ligands. THP-1 cells were sequentially pre-treated with 1 mm of PTP inhibitor III (PIII), 1 mm of PTP inhibitor IV (PIV) or 0·2% DMSO (vehicle control, VC) for 30 min, followed by 1 μg/ml mIgG or monoclonal antibody (mAb) against CD300f for another 30 min. Finally, cells were stimulated with 1 μg/ml lipopolysaccharide (LPS), CpG ODN or PolyI:C for 24 hr. Culture supernatants were collected for the measurement of IL-8 concentrations (n = 3, **P < 0·01 and ***P<0·001 when compared with control samples treated with corresponding TLR ligand and anti-CD300f mAb).