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
Known: LAIR-1（leukocyte-associated Ig-like receptor-1）is an ITIMcontaining 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.
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.
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
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
3. Results
3.1. LAIR-2 is expressed by immune cells
Cell lines
Total RNA
LAIR-2 specific primers
RT-PCR
3.2. LAIR-2 is a secreted protein
His-tagged LAIR-2a
Wild type LAIR-2b
239T cells
anti-LAIR-2
mAbs
Flow
cytometry
supernatant
FMU-LAIR2.1 anti-LAIR-2 mAbs
peroxidase-conjugated rabbit antimouse mAb as secondary Ab
Western blot
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
Collagen Ⅰand collagen Ⅲ
surface plasmon resonance
3.4. LAIR-2 prevents binding of hLAIR-1 to its ligand
HT29 cells
Incubate with
unlabeled LAIR-2-IgG
Incubate with biotinylated
hLAIR-1-IgG
Can express endogenous
hLAIR-1 ligands
Incubate with
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.
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
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
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.
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
3.6. LAIR-2 is produced by primary cells and in vivo
PBMC
stimulated
CD3+CD4+ T cells
with PMA and
+
+
CD3 CD8 T cells
ionomycin
+
CD3 CD19 B cells
day 4
CD3-CD16+CD56+ NK cells
CD3-CD14+ monocytes
Sandwich ELISA
To detecte LAIR-2
expression
10 healthy individuals and
6 pregnant women
14 patients with RA and
16patients with OA
Plasma, serum and urine
SF
Sandwich ELISA
Sandwich ELISA