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The Journal of Immunology, 2008

The Journal of Immunology, 2008. The Soluble Leukocyte-Associated Ig-Like Receptor (LAIR)-2 Antagonizes the Collagen/LAIR-1 Inhibitory Immune. Xu Zhenjie. 2011,09,06. 1. Background.

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The Journal of Immunology, 2008

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  1. The Journal of Immunology, 2008 The Soluble Leukocyte-Associated Ig-Like Receptor (LAIR)-2 Antagonizes the Collagen/LAIR-1 Inhibitory Immune Xu Zhenjie 2011,09,06

  2. 1. Background • Known: LAIR-1（leukocyte-associated Ig-like receptor-1）is an ITIM-containing inhibitory immune receptor. Collagens are high-affinity ligands for the LAIR-1 molecules. • Unknown: Whether LAIR-2 is produced in vivo is unknown. Whether LAIR-2 is a secreted protein. Whether it may function as a soluble competitor for the interaction between collagens and hLAIR-1. • Findings：a novel mechanism of immune regulation： sLAIR-2 regulated the inhibitory potential of the membrane-bound hLAIR-1 via competition for the same ligands.

  3. 2. Materials and methods Cells lines Human monocytic U937 cells; HEK293T human embryonic kidney cells; Human colon carcinomas lines HT29, DLD-1, LS174, SW480, and HCT116; SKBR3 human breast cancer line; YT.2C2 human NK-like cells; 721.221 lymphoblastoid cells; THP-1 human monocyte-like cells; Jurkat human T cells; CEM human leukemia T cells; human erythroleukemia K562 cells. K562 cells and 2B4 NFAT-GFP T cell reporter cells were stably transfected with hLAIR-1 or hLAIR-1a-CD3 respectively.

  4. 2. Materials and methods Subjects: 10 healthy individuals (5 males and 5 females) and 6 healthy pregnant women (16–37 wk of gestation) volunteers. 14 patients suffering from rheumatoid arthritis (RA) and 16 patients suffering from osteoarthritis (OA). Method: RT-PCR Western blot analysis Surface plasmon resonance (表面等离子共振)? Flow cytometry Reporter cell assay ? Sandwich ELISA

  5. contents 1. LAIR-2 is expressed by immune cells. 2. LAIR-2 is a secreted protein. 3. LAIR-2 is a high-affinity collagen receptor. 4. LAIR-2 prevents binding of hLAIR-1 to its ligand. 5. LAIR-2 prevents hLAIR-1 cross-linking by collagens. 6. LAIR-2 is produced by primary cells and in vivo. LAIR-2 is a secreted protein; and it functions as a soluble competitor for the interaction between collagens and hLAIR-1

  6. 3. Results 3.1. LAIR-2 is expressed by immune cells Cell lines Total RNA LAIR-2 specific primers RT-PCR

  7. 3.2. LAIR-2 is a secreted protein His-tagged LAIR-2a Wild type LAIR-2b 239T cells supernatant anti-LAIR-2 mAbs FMU-LAIR2.1 anti-LAIR-2 mAbs peroxidase-conjugated rabbit anti-mouse mAb as secondary Ab Flow cytometry Western blot

  8. 3.3. LAIR-2 is a high-affinity collagen receptor To measured binding and affinity of LAIR-2-IgG fusion proteins to collagen: LAIR-2-IgG fusion proteins surface plasmon resonance Collagen Ⅰand collagen Ⅲ

  9. 3.4. LAIR-2 prevents binding of hLAIR-1 to its ligand Can express endogenous hLAIR-1 ligands HT29 cells Incubate with unlabeled LAIR-2-IgG Incubate with hLAIR-1-IgG Incubate with biotinylated hLAIR-1-IgG Incubate with biotinylated LAIR-2-IgG Both human LAIR-1 and LAIR-2 fusion proteins bind the same sites on collagen molecules.

  10. Oregon Green 488-labeled collagen IV Incubate with LAIR-2-IgG Incubate with control Ig fusion proteins (isotype) Incubate with K562 cells or LAIR-1-transfected K562 cells

  11. As documented before, hLAIR-1-transfected K562 cells bind firmly to plate-bound collagens I and III. plate-bound collagens I and III Incubate with LAIR-2-IgG Incubate with irrelevant fusion protein Incubate with hLAIR-1-transfected K562 cells

  12. To assess whether wild-type LAIR-2 protein is capable of interfering with the hLAIR-1/collagen interaction: plate-bound collagens I and III concentrated supernatant from LAIR-2-transfected 293T cells control supernatant Incubate with hLAIR-1-transfected K562 cells LAIR-2 binds the same sites on various collagen species as hLAIR-1, suggesting that LAIR-2 may function as a soluble competitor to hLAIR-1/collagen binding.

  13. 3.5. LAIR-2 prevents hLAIR-1 cross-linking by collagens By using NFAT-GFP reporter cells expressing a chimeric protein containing the extracellular domain of hLAIR-1 and the transmembrane and intracellular domain of CD3ζ, we have shown that collagen I and III are capable to functionally cross-link hLAIR-1. plate-bound collagens I and III

  14. 3.6. LAIR-2 is produced by primary cells and in vivo stimulated with PMA and ionomycin CD3+CD4+ T cells anti-CD4/ CD8/CD3/CD19 Abs CD3+CD8+ T cells Sandwich ELISA CD3-CD19+ B cells day 4 PBMC CD3-CD16+CD56+ NK cells anti-CD16/ CD14/CD3/CD56 Abs To detecteLAIR-2 expression CD3-CD14+ monocytes

  15. 14 patients with RA and 16patients with OA 10 healthy individuals and 6 pregnant women SF Plasma, serum and urine Sandwich ELISA Sandwich ELISA

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