Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor

Abstract

Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of the immune system. Here, we report a mouse orthologue of the human activating paired immunoglobulin-like type 2 receptor (PILR) beta, which was cloned from a cDNA library of natural killer (NK) cells based on its ability to associate with the DAP12 signaling adaptor protein. The activating PILRbeta was expressed not only on NK cells but also on dendritic cells and macrophages. Furthermore, we have identified a novel CD99-like molecule as a ligand for the activating PILRbeta and inhibitory PILRalpha receptors. Transcripts of PILR ligand are present in many tissues, including some T cell lines. Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta. Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

Figure 1.

Amino acid comparison between mouse PILRα and PILRβ. Amino acid alignment of mouse PILRα and PILRβ is shown. Signal sequence and transmembrane region are indicated by double and single underline, respectively. A charged residue (K, bold and italicized) in transmembrane domain of PILRβ and the ITIM sequence in the cytoplasmic domain of PILRα are indicated (bold letters). Identical amino acids between PILRβ and PILRα are indicated by asterisks. GenBank/EMBL/DDBJ accession nos. of mouse PILRα and PILRβ are NM_153510 and AB122024/NM_133209, respectively.

Figure 2.

(A) Role of DAP12 in the cell surface expression and signal transduction of PILRβ, FLAG-PILRβ (solid line), and FLAG-PILRα (dashed line) was transfected into the MA5.8 cell line. Parental MA5.8 (dotted line) and the transfectants were stained with anti-FLAG mAb. Mean fluorescence intensities of anti-FLAG mAb staining for parental MA5.8, FLAG-PILRα, and FLAG-PILRβ–transfected cells were 2.93, 27.0, and 369.1, respectively. (B) DAP12, CD3ζ, or FcRγ were transfected into MA5.8-expressing FLAG-PILRβ using the pMx-IRES-GFP retrovirus vector. Transfectants were stained with anti-FLAG mAb and expression of FLAG on GFP-expressing cells is shown (solid line). FLAG expression on a mock transfectant was overlaid in this figure (dotted line). Mean fluorescence intensities of anti-FLAG mAb staining for FLAG-PILRβ–expressing cells transfected with DAP12, CD3ζ, and FcRγ were 1137.7, 56.4, and 33.9, respectively. (C) IL-2 production by MA5.8 cells and MA5.8 transfected with FLAG-PILRβ and DAP12, CD3ζ, or FcRγ and cultured for 1 d in the presence of immobilized anti-FLAG mAb (shaded bar) or control mAb (unshaded bar). IL-2 in the culture supernatants was measured by ELISA.

Figure 3.

Transcription of PILRα and PILRβ in various tissues and purified populations. Transcription of PILRα and PILRβ in various tissues (A) and purified populations (B) was analyzed by real-time quantitative RT-PCR. Transcription of β-actin was used as a standard, and relative expression levels of PILRα and PILRβ compared with amounts in the spleen are shown. BM-DC, bone marrow–derived dendritic cells. S. intestine, small intestine.

Figure 4.

Expression of PILR-L. The cell lines indicated were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. Fluorescence intensities of PE-stained cells are shown.

Figure 5.

Molecular cloning of mouse PILR-L. (A) Nucleic and predicted amino acid sequences of mouse PILR-L are shown. The signal sequence and transmembrane domain are indicated by double and single underline, respectively. Sequence data of PILR-L are available from GenBank/EMBL/DDBJ under accession no. AB122023. (B) Ba/F3 and 293T cells were transfected with mouse PILR-L. Parental cells and the PILR-L transfectants were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. (C) SDS-PAGE analysis of PILR-L. PILR-L transfected (+) and parental (−) Ba/F3 cells were surface biotinylated, and cell lysates were precipitated with PILRα-Ig, PILRβ-Ig, or control Ig fusion protein as indicated. Precipitates were electrophoresed by SDS-PAGE and biotinylated proteins were detected by using horseradish peroxidase–conjugated streptavidin and the enhanced chemiluminescence detection reagent.

Figure 6.

Transcription of PILR-L in various tissues. Transcription of PILRα and PILRβ in various tissues was analyzed by real-time quantitative RT-PCR. Transcription of β-actin was used as a standard and relative expression levels of PILR-L to the amounts present in spleen are shown. S. intestine, small intestine.

Figure 7.

Cytotoxicity of NK cells against PILR-L–expressing cells. (A) Cytotoxicity of mouse PILRβ-transfected human NKL (left) or mock-transfected NKL cells (right) against mouse PILR-L–transfected 293T cells (closed circles) or parental 293T cells (open circles) is shown. (B) Cytotoxicity of IL-2 expanded NK cells from wild-type mice (left) or DAP12-deficient mice (right) against PILR-L–transfected Ba/F3 cells (closed circles) or parental Ba/F3 cells (open circles) is shown.

Figure 8.

Activation of DCs by PILR-L–expressing cells. 5 × 10⁵ BM-derived DCs were cocultured with PILR-L–transfected 293T cells (closed circles) or parental 293T cells (open circles) at the indicated cell number for 1 d. BM-derived DCs were also stimulated with 1 μg/ml LPS as a positive control. Nitric oxide and mouse TNF-α produced in the culture supernatants were measured. Amounts of nitric oxide and TNF-α produced by DCs cultured in medium only were 1.5 ± 0.1 μM and 48.5 ± 7.7 pg/ml, respectively, and those produced by DCs upon LPS stimulation were 59.1 ± 2.8 μM and 2048 ± 127.9 pg/ml, respectively.