LILRA5 is expressed by synovial tissue ... - Wiley Online Library

Eur. J. Immunol. 2008. 38: 3459–3473

DOI 10.1002/eji.200838415

Immunomodulation

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis, selectively induces proinflammatory cytokines and IL-10 and is regulated by TNF-a, IL-10 and IFN-c Ainslie Mitchell1, Carles Rentero2, Yasumi Endoh1, Kenneth Hsu1, Katharina Gaus2, Carolyn Geczy1, H. Patrick McNeil1, Luis Borges3 and Nicodemus Tedla1 1

Centre for Infection and Inflammation Research, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia 2 Centre for Vascular Research, University of New South Wales, Sydney, NSW, Australia 3 Amgen Inc. Seattle, WA, USA Leukocyte immunoglobulin-like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane-bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross-linking of LILRA5 on monocytes induced production of pro-inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common c chain of the FcR and LILRA5 cross-linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro-inflammatory cytokines as well as IL-10. LILRA5 mRNA and protein expression was tightly regulated by TNF-a, IL-10 and IFN-c. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.

Key words: Cytokines . Leukocyte immunoglobulin-like receptors . Macrophages . Rheumatoid arthritis

Introduction The leukocyte immunoglobulin-like receptors (LILR or LIR), also named Ig-like transcripts and CD85 antigens [1], are a family of

Correspondence: Nicodemus Tedla e-mail: n.tedla@unsw.edu.au

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

immunoglobulin-like receptors primarily co-expressed on the surface of leukocytes [1, 2]. They mediate inhibitory or activating functions through cytoplasmic domains containing two to four immunoreceptor tyrosine-based inhibitory motifs, or through association with proteins that contain immunoreceptor tyrosinebased activating motifs (ITAM) in their cytoplasmic domain [1–3]. Peripheral blood monocytes express mRNAs and/or protein for most LILR, whereas T cells have a highly restricted

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expression pattern [2], suggesting that these receptors might be important regulators in primary (innate) host defense. The activating leukocytes immunoglobullin-like receptor A5(LILRA5) is expressed as membrane-bound and soluble forms [4]; four different forms of LILRA5 have been identified, two membrane-bound, with expected sizes of the unprocessed protein of 32.7 and 31.5 kDa, respectively, and two soluble forms with expected sizes of 29 and 27.8 kDa respectively [4]. The transmembrane forms of LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region that is likely to mediate its association with a yet-to-be identified ITAM-containing adaptor protein [4]. LILRA5 is mostly expressed by monocytes and neutrophils [4]. Although the natural ligand for LILRA5 is unknown, cross-linking of this receptor on monocytes using a specific mAb induced calcium flux and production of TNF-a, IL-1b and IL-6, suggesting a role in inflammation [4]. However, the downstream signaling cascade triggered by LILRA5 is not well established. Furthermore, expression of LILRA5 in diseased tissue with an inflammatory component and its regulation in this context are unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis, with destruction of juxta-articular cartilage and bone, likely mediated by lipid mediators, cytokines, and proteases released from inflammatory leukocytes [5, 6]. The mechanisms regulating leukocyte activation in rheumatoid synovium are not fully elucidated. We previously demonstrated increased expression of activating LILRA2 and inhibitory LILRB2 on monocytes and neutrophils in synovial tissue from patients with active RA [7]. Furthermore, levels of LILR expression were significantly reduced in synovial tissue from patients who responded to treatment with disease-modifying anti-rheumatic drugs [8]. Monocytes treated with the commonly used disease-modifying anti-rheumatic drug, corticosteroid, in vitro significantly repressed LILRA2-mediated TNF-a production [8]. Here we demonstrate extensive LILRA5 expression on CD681 macrophages in rheumatoid synovial tissue, whereas there were relatively few LILRA51 CD68 cells in osteoarthritis (OA), which is characterized by minimal inflammation. We further establish that LILRA5 was constitutively expressed by peripheral blood monocytes from patients with RA and control subjects and is associated with the ITAM of the Fc receptor common g chain. Cross-linking of LILRA5 on the surface of monocytes increased phosphorylation of tyrosine kinases, leading to early and selective production of pro-inflammatory cytokines (TNF-a, IL-1b, IL-6) followed by delayed induction of IL-10. We investigated the regulation of LILRA5 expression on monocytes by various cytokines and upon differentiation to macrophages. LILRA5 mRNA and protein expression were strongly regulated upon differentiation to macrophages and by treatment of monocytes with TNF-a, IL-10 or IFN-g in vitro, indicating that expression of this receptor is tightly regulated by cytokines produced upon its activation. Key events of RA involve activation of macrophages and release of inflammatory mediators such as TNF-a [9] that are induced by LILRA5 cross-linking. Taken together our results suggest that LILRA5 may contribute to the pathogenesis of RA and could potentially be a therapeutic target.


Eur. J. Immunol. 2008. 38: 3459–3473

Results Expression of LILRA5 by human macrophages in vivo We first examined the expression of LILRA5 in chronic inflammatory disease (RA), degenerative joint disease with minimal inflammation (OA) and healthy tissue (Table 1). Immunohistochemistry of synovial tissue obtained from patients with RA showed extensive LILRA5 staining in CD681 cells in the synovium (Fig. 1A–D) (Table 2). In contrast, there was limited LILRA5 and CD68 staining in OA synovium (Fig. 1E and F). No LILRA5 reactivity was observed in normal synovium (Fig. 1G, inset). Staining of RA tissue with isotype-matched control mAb showed no non-specific reactivity (Fig. 1G) Double immunofluorescence staining using Alexa 488-conjugated anti-CD68 and Alexa 568-conjugated anti-LILRA5 mAb confirmed that the majority of macrophages in RA tissue were LILRA51 (Fig. 1H). Quantitative analysis confirmed significantly more CD681 cells in RA synovium (median count of 39.776.6 cells/high power field (HPF); n 5 14) compared with 19.171.6 cells/HPF in OA (n 5 10) and 5.870.6 cells/HPF in normal tissue (n 5 6) (Fig. 2A). Similarly, there were significantly more LILRA51 cells in RA synovium, with a median count of 30.576.8 compared with 3.270.5 in OA and none in normal synovium (Fig. 2A). Although not all macrophages in the synovial tissue from patients were LILRA51, a higher proportion of LILRA51 macrophages were evident in RA (68.5%77.0%) compared with OA synovium (12.6%76.3%). LILRA5 expression strongly correlated with macrophage numbers in synovial tissue from patients with RA (R2 5 0.95) but not in patients with OA (R2 5 0.23) (Fig. 2B). Moreover, there was a correlation between disease activity scores (DAS) and numbers of LILRA51 cells in synovial tissue from patients with RA (R2 5 0.80) (Fig. 2C). Western blotting using anti-LILRA5 mAb detected an immunoreactive component at 25 kDa, consistent with the mass of soluble LILRA5, in cell-free synovial fluid from patients with RA but not OA (Fig. 2D).

Surface expression of LILRA5 on PBMC To explore whether LILRA5 is differentially expressed on PBMC from patients with RA compared with healthy controls, we recruited six patients with active RA (DAS42.6) and 10 age- and sex-matched controls. Flow cytometric analysis of PBMC from healthy subjects and patients with active RA showed high intensity surface expression of LILRA5 by 495% of monocytes (Fig. 3A) but not by T cells, B cells or NK cells (data not shown). No significant differences in the levels of LILRA5 expression were found on monocytes from healthy donors (MFI of 64.075.2) compared with those from patients with active RA (MFI 66.473.9) (Fig. 3B and C). Moreover, a surface LILRA5 protein of 40 kDa was immunoprecipitated from biotin-labeled primary monocytes from healthy subjects and from patients with RA using anti-LILRA5 mAb (Fig. 3D). Western blot analysis using a polyclonal Ab against the common g chain of Fc receptors

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Eur. J. Immunol. 2008. 38: 3459–3473

Immunomodulation

Table 1. Demography of synovial tissue donors

Mean age Mean duration of disease (years) M:F ratio a)

RA (n 5 14)

OA (n 5 10)

Normal (n 5 6)

60.3715.7 9.576.6 1:3.6

68.7710.8 8.275.7 1:1.5

38.779.8 3–45a) 6:0

Days.

detected an 15 kDa immunoreactive band in protein precipitated with anti-LILRA5 from normal and patients but not IgG1 control (Fig. 3D), suggesting that LILRA5 uses the ITAMcontaining Fc g receptor to transduce activation signals.

Activation of monocytes by LILRA5 cross-linking in vitro To determine the functional significance of LILRA5 expression of on monocytes and macrophages, and its association with the ITAMcontaining Fc common g chain, we examined the responses of monocytes upon engagement of LILRA5. Cross-linking of LILRA5 on the surface of monocytes showed selective and dose-dependent induction of three pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) but only one (IL-10) of the five immunomodulatory cytokines (IL-10, IL-12, IL-13, IL-17 and IFN-g) was significantly up-regulated (Table 3). There was substantial down-regulation of constitutively produced IL-12 and IL-13 (Table 3). No changes in the levels of IL-17 or IFN-g were observed. Time-course studies using an optimal dose of anti-LILRA5 mAb (1 mg/mL) showed significant up-regulation of TNF-a, peaking at 12 h, followed by a delayed increase in IL-10 over 24–48 h (Fig. 4A and B). In contrast, cross-linking with an anti-MHCclass I or control IgG1 mAb did not affect cytokine induction. LILRA5 cross-linking on monocytes from patients with active RA (DAS44.6) generated slightly higher levels of TNF-a than monocytes from patients in remission (DASo2.6) or normal controls, but levels did not reach statistical significance (Fig. 4C).

TNF-a production in response to LILRA5 cross-linking is mediated by protein tyrosine kinase (PTK) and MAPK pathways We next examined whether monocyte activation in response to LILRA-5 cross-linking was via activation of specific PTK signaling cascades. Western blot analysis using anti-phospho-Src family Ab showed strong phosphorylation of 57–60 kDa proteins in monocytes that were briefly cross-linked with anti-LILRA5 mAb that were minimal in those cross-linked with anti-MHC class I mAb, control IgG1 or secondary Ab alone (Fig. 5A). Cross-linking with LILRA5 also caused significant dose-dependent phosphorylation of Syk, a key signaling molecule in tyrosine kinasemediated activation cascades (Fig. 5B). One hour pre-incubation of monocytes with the Src inhibitor (PP2) or Syk inhibitor II significantly reduced TNF-a production by 62 and 56%,


respectively, upon subsequent LILRA5 cross-linking for 12 h (Fig. 5C). Moreover, treatment with PD98059 (ERK inhibitor), SB202180 or SB202190 (p38 inhibitors) for 12 h also significantly inhibited TNF-a production by LILRA5 cross-linked monocytes (Fig. 5D), by 44, 69 and 70%, respectively. Treatment of cells with PP3 (a control for PP2) or vehicle control (0.05% DMSO) had no effect. These results suggest that LILRA5 crosslinking activates ITAM-mediated signaling, leading to cytokine production via the PTK and MAPK pathways.

Expression of LILRA5 in cultured monocytes and in vitro differentiated macrophages To compare LILRA5 expression on human monocytes and macrophages, fresh PBMC were stained for LILRA5 or cultured for 24 h or differentiated to macrophages for 5 days using GMCSF. Short-term (24 h) culture and differentiation to macrophages caused significant down-regulation of LILRA5 surface expression (Fig. 6A and B). Consistent with the protein data, mRNA encoding membrane-bound LILRA5 was also significantly down-regulated in macrophages (20–40 fold) and cultured monocytes (10–15 fold) compared with fresh monocytes (Fig. 6C and D). A similar trend was observed when RNA was analysed by qRT-PCR using gene-specific primers for soluble LILRA5, which indicated 10–15 fold down-regulation in macrophages compared with monocytes (data not shown). These findings suggest that LILRA5 expression depends on a specific microenvironment in vivo such as the constitutive presence of ligand(s), interaction with other cells, matrix proteins or soluble mediators.

Regulation of LILRA5 expression in monocytes by cytokines TNF-a is an important cytokine in the pathogenesis of RA [1, 6, 10] and IL-10 play critical roles in immune regulation [11, 12]. Production of both cytokines was strongly up-regulated by monocytes upon LILRA5 cross-linking. IFN-g is a key modulator of monocyte/macrophage functions [12]. Thus, we evaluated the ability of these cytokines to regulate LILRA5 mRNA and protein expression in monocytes. Monocytes purified from PBMC by negative selection using MACS beads were treated with TNF-a, IFN-g or IL-10 for 6–24 h and LILRA5 mRNA analysed by gene-specific primers for membrane and soluble LILRA5 using

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Eur. J. Immunol. 2008. 38: 3459–3473

A

B

C

D

E

F

G

H

* * d *

10 mm Figure 1. Immunohistochemical staining showing extensive expression of LILRA5 in synovial tissue from a patient with active RA (DAS 5 6.2) (A) and moderate expression in a patient with low DAS (2.7) (C). Staining of adjacent sections with anti-CD68 mAb showed that the majority of LILRA51 cells were CD681 macrophages (B and D). Synovial tissue from a patient with OA stained for LILRA5 and CD68 (E and F), respectively. LILRA5 was not expressed despite the presence of CD681 cells along the synovial lining. Rheumatoid synovial tissue stained with isotype-matched negative control (G) and normal synovial tissue stained with anti-LILRA5 (G, inset) showed no specific immunoreactive cells. Red is positive staining with blue hematoxylin counterstain (400 magnification). Double immunofluorescence staining of RA synovial tissue (H) using Alexa 488-conjugated anti-CD68 (green arrows), Alexa 568-conjugated anti-LILRA5 (red asterisk) and nuclear staining with DAPI (blue) showing the majority of macrophages were LILRA51 (yellow, d).


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Eur. J. Immunol. 2008. 38: 3459–3473

Immunomodulation

Table 2. Clinical profile of patients with RA Patients

Age (years)

Gender

Duration (years)

Treatment at the time of biopsy

Disease activity score

Median LILRA51 cells/mm2

RA1 RA2 RA3 RA4 RA5 RA6 RA7 RA8 RA9 RA10 RA11 RA12 RA13 RA14

60 55 80 52 58 64 73 83 43 48 78 75 36 39

Female Female Female Female Male Male Female Male Female Female Female Female Female Female

2 14 2 23 12 13 18 2 6 15 10 4 7 4

MTXa), IMb) Gold SSZc), NSAIDSd) IM Gold, Prede) IM Gold SSZ MTX, IM Gold Nil MTX, IM Gold MTX, Pred MTX, IM Gold, Pred CyAf), Pred MTX, Pred Nil MTX, IM Gold

0.8 1.2 1.4 2.1 2.5 2.8 4.6 4.8 5.3 5.3 5.4 5.6 5.9 6.0

9 2 26 10 19 32 48 26 29 23 58 50 60 38

a)

Methotrexate. Intramuscular. c) Sulfasalazine. d) Non-steroidal anti-inflammatory drugs. e) Prednisolone. f) Cyclosphorine A. b)

C

50 40

CD68

7

LILRA5

6

Disease activity score

Mean cell count (+SEM)/HPF

A

30 *

20 10

#

**

0 OA (n=10)

Normal (n=6)

B Median macrophage count per HPF

5 4 3 2 1 0

RA (n=14)

R = 0.80

0

10

20

30 40 50 Median LILRA5 count

60

70

D 80

MW

70

R = 0.95

OA1

OA2

RA1

RA2

RA3

RA4

25kDa

60

20kDa

50 40 30

R = 0.23

20 10

5

0 0

10

20

30 40 50 Median LILRA5 count

60

70

Figure 2. Quantitative analysis of macrophages in synovial tissue showed significantly more macrophages in patients with RA compared with patients with OA (po0.05) or control subjects (po0.01). Similarly, the number of LILRA51 cells was significantly higher in patients with RA compared with patients with OA and control subjects (]po0.01) (A). LILRA5 expression in synovial tissue from patients with RA (B) but not with OA (B, inset) significantly correlated with median macrophage counts (Spearman r 5 0.95, po0.0001). The numbers of LILRA51 cells and DAS in patients with RA were also statistically significant (Spearman r 5 0.80, po0.0005) (C). Western blotting with anti- LILRA5 mAb showed a strong 25 kDa immunoreactive protein consistent with the mass of the soluble/cleaved form of LILRA5 in synovial fluid from patients with RA but not from OA (D).


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A

Eur. J. Immunol. 2008. 38: 3459–3473

B

Control

RA 95.6%

200

94.2%

α-LILRA5

Counts

CD14-PE 100

101

102

103 104 100

101

102

103

IgG1

104

α-LILRA5-Alexa488 100

3.4%

101

102

103

104

Log Fluorescence intensity-Alexa488

IgG2b-PE

1.1%

100

101

102

103 104 100

101

102

103

104

IgG1-Alexa488

D

C 75

Normal PBMC LILRA5 IgG1

RA PBMC IgG1 LILRA5

50

70 MFI + SD

3464

IP with α-A5

37

65 60

20

55 50

WB with α-Fcγ

10 Control (n=10)

RA (n=6)

Figure 3. A. Representative Flow cytometric analysis of LILRA5 expression on the surface of peripheral blood monocytes from a patient with RA and an age- and sex-matched control subject. More than 95% of CD141 cells were strongly LILRA51 (upper panel). Lower panels show cells double stained with isotype and fluorochrome-matched negative control antibodies. (B) Histograms on the left-hand side show cells from a patient and control subject stained with isotype-matched control mAb and histograms on the right-hand side show cells from a patient with RA (bold) and a control subject (non-bold) stained with anti-LILRA5 mAb. There was no significant difference in proportions (A) and intensity (B and C) of LILRA51 cells between patients and control subjects. (D) Immunoprecipitation with anti-LILRA5 but not isotype-matched control mAb pulled down a single 40 kDa LILRA5 membrane protein from biotin-labeled monocytes in patients and control subjects. Western blotting of the immunoprecipitated protein showed co-precipitation of LILRA5 with the ITAM-containing Fc common g chain (lower panel).

Table 3. Dose-dependent production of cytokines by PBMC 24 h after LILRA5 cross-linking as determined by multiplex assay (n 5 3)

Anti-LILRA5 antibody (mg/mL)a)

Cytokine (pg/mL)

TNF-a IL-1b IL-6b) IL-10 IL-12 IL-13 IL-17 IFN-g IL-2 a) b)

0

0.01

0.1

1.0

10

42.1713.8 52.1711.9 0.4270.1 13.972.9 112.2711.9 9.572.9 46.4710.1 302.2798.7 46.876.2

58.1720.7 10.575.0 0.6370.1 14.671.9 28.372.0 7.570.3 32.270.2 242.273.1 2.570.2

93.2725.7 73.7710.6 2.7370.8 20.276.4 30.470.3 4.472.7 31.5711.5 226.9765.3 14.6710.2

819.67168.8 290.3727.8 20.671.4 90.770.2 2.771.8 1.470.4 32.371.7 139.3712.3 0.470.3

850.6722.0 395.6784.2 20.671.2 102.279.5 3.671.8 0.170.1 41.170.4 220.3711.6 0.470.1

Cells treated with corresponding doses of IgG1 control did not cause non-specific cytokine production. ng/mL.


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Eur. J. Immunol. 2008. 38: 3459–3473

TNFα (pg/ml)

A

Immunomodulation

4000 3000

IgG1 α-MHC class I α-LILRA5

** **

2000

*

* *

1000 0 6

B

12

24

48

IL-10 (pg/ml)

72

350 **

300 *

250

*

200 150

IgG1 α-MHC class I α-LILRA5

*

100 50 0

6

12

24 Time (hr)

48

TNFα (pg/ml)

C 4000

72

α-MHC class I α-LILRA5

3000 2000 1000 0 Normal

RA in remission

TNF-a in a manner similar to membrane bound LILRA5 (data not shown). Treatment of monocytes for 24 h with IL-10 or IFN-g, but not TNF-a, substantially up-regulated the expression of membrane-bound LILRA5 protein (Fig. 7C). Analysis of supernatants from cultured cells detected an 25 kDa protein consistent with cleaved/soluble LILRA5 product (Fig. 7D). Analysis of immunoreactive bands by densitometry showed that IL-10, IFN-g or TNF-a increased levels 8.070.2, 5.570.6 and 4.170.1 fold, respectively (Fig. 7D).

Active RA

Figure 4. Cross-linking of LILRA5 on peripheral blood monocytes from healthy subjects with 1 mg/mL anti-LILRA5 mAb caused significant induction of TNF-a at 6–12 h (A) and significant increases in IL-10 at later time points (B) (n 5 5). (C) Monocytes from patients with RA in remission (mean DAS 2.470.23) produced similar quantities of TNF-a as normal controls, whereas those with active disease (mean DAS 5.171.06) generated more TNF-a but was not statistically significant (n 5 4 per group). Treatment of cells with 1 mg/mL irrelevant IgG1 or antiMHC class I mAb (1 mg/mL) did not substantially induce cytokine production confirming specificity. Results are presented as mean7SEM of 4–5 independent experiments. po0.05; po0.01 compared with corresponding IgG1 control.

qRT-PCR. We observed consistent dose- and time-dependent up-regulation of constitutively expressed membrane LILRA5 mRNA in monocytes treated with IFN-g and IL-10, whereas TNF-a marginally decreased constitutive expression (Fig. 7A). However, TNF-a significantly abrogated IL-10-mediated up-regulation of LILRA5 mRNA (Fig. 7B). The IL-10-mediated up-regulation was significantly higher (15 fold increase) and peaked earlier (6 h) than IFN-g-mediated induction that peaked at 12 h with a maximum increase of 7 fold compared with untreated control cells (Fig. 7B). The mRNA for soluble LILRA5 was also constitutively expressed by untreated monocytes, albeit at substantially lower levels compared with mRNA encoding membrane LILRA5 and this was regulated by IL-10, IFN-g and


Discussion RA manifests as chronic synovial inflammation characterized by synovial pannus formation and destruction of articular surfaces [5, 6]. Activated synovial macrophages are the primary source of TNF-a and IL-1b that are central in the pathogenesis of the disease [5, 6, 10]. Importantly, these cells produce tissuedegrading enzymes responsible for joint destruction [5, 6]. However, despite the key role of activated macrophages in the pathogenesis of RA, the mechanism(s) and regulation of their activation are not fully elucidated and the underlying etiology of RA is unknown. Inflammatory responses by macrophages in RA are likely to be regulated by a network of inhibitory and activating signals. The chronic relapsing and remitting nature of the disease suggests that a balance between activation and inhibitory signals may determine the extent of macrophage activation at a given time. Since LILR can modulate the activity of inflammatory cells in vitro, we predicted that they may regulate macrophage activation in RA and contribute to disease pathogenesis. Here, for the first time we demonstrate extensive expression of LILRA5 on macrophages in rheumatoid synovium. We previously reported the expression of two inhibitory (LILRB2 and B3) and an activating LILRA2 in synovial tissue of patients with active RA [7]. However, unlike LILRA5, which is primarily expressed by macrophages, these receptors were expressed by diverse leukocytes types and by non-leukocytic cells such as endothelial cells and synoviocytes [7], suggesting that LILRA5 may be mainly involved in macrophage activation. LILRA5 expression strongly correlated with numbers of CD681 macrophages and disease severity (Fig. 1 and 2; Table 2). In contrast, very limited expression of LILRA5 was seen in synovial tissue obtained from patients with OA, despite the presence of substantial numbers of CD681 cells (Fig. 1 and 2). Western blotting using anti-LILRA5 detected an 25 kDa LILRA5-reactive component in cell-free synovial fluid from patients with RA, but not from patients with OA (Fig. 2D). Since the size of unprocessed, soluble LILRA5 is 28–29 kDa, and the post-translationally modified protein is expected to be even larger, the smaller-than-expected protein found in synovial fluid from patients with RA is likely to be a product shed from the surface of activated macrophages or a proteolytically processed, truncated soluble form. Although soluble/cleaved LILRA5 may act as antagonist to the membrane-bound protein by competing for the same ligand(s),

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A

B α-MHC

9000

IgG1 α-LILRA5

60

WB with anti-pSrc Ab

42

WB with anti-β actin Ab

Fluorescence value (Mean +SEM)

None

**

Phosphorylated Syk

8000 7000 6000 5000 4000

*

3000 2000 1000 0 IgG1 α−LILRA5 α−LILRA5 (1µg/ml) (10µg/ml) (10µg/ml)

C

α−MHC (10µg/ml)

D 6000 1 No treatment 4000

2 PP3 3 PP2 * 4 DMSO 5 Syk inhibitor II 1µM

**

2000

TNFα (pg/ml)

6000

TNFα (pg/ml)

3466

1 No treatment 4000

2 DMSO 3 PD 98059

* 2000

**

4 SB 202190 ** 5 SB 202180

4

5

6 Syk inhibitor II 10µM 0

IgG1 1

2

3

4

5

6

α-LILRA5

0 IgG1

1

2

3

α-LILRA5

Figure 5. Cross-linking of LILRA5 activated ITAM-mediated signaling on freshly isolated monocytes of healthy subjects via strong phosphorylation of Src family kinases determined by Western blotting (A) and Syk determined by Bioplex assay (B) (n 5 4). Cells treated with anti-MHC class I mAb, isotype-matched IgG control or cells alone had minimally phosphorylated Src or Syk kinases. po0.05; po0.01 compared with corresponding IgG1 control. Pre-incubation of monocytes with Src inhibitor PP2 (50 mM) or Syk inhibitor II (10 mM) significantly down-regulated LILRA5 mediated TNF-a production compared with corresponding PP3 and DMSO controls (C) (n 5 3). Inhibitors of ERK (PD98059, 10 mM) and p38 (SB202190 or SB202180, 1 mM) significantly reduced TNF-a levels compared with DMSO control (D) (n 5 3). Results are presented as mean7SEM of 3–4 independent experiments.po0.05; po0.01.

LILRA5-ligand(s) are not identified, precluding experiments aimed at determining its functional role in the pathogenesis of RA. The selective expression of LILRA5 in RA, but not in OA or normal tissue, suggests that this molecule is preferentially present in active inflammatory conditions characterized by increased leukocyte infiltration and that it plays a limited role in chronic degenerative conditions. This is supported by our data, showing that the levels of LILRA5 expression in RA synovial tissue correlated with disease severity and with the increased numbers of inflammatory cells seen in this condition (Fig. 2). Interestingly, no significant differences in the surface expression of LILRA5 on peripheral blood monocytes from patients with active RA or age and sex-matched healthy control subjects were found (Fig. 3). We and others previously showed similar expression patterns of LILRA2 in tissue versus blood, in patients with RA [8] and leprosy [13]. These results suggest that increased expression of LILRA5 in rheumatoid tissue might be due to the level of activation and/or differentiation of monocytes/macro-


phages within the tissue and/or due to increased recruitment of LILRA5-expressing monocytes from peripheral blood as these cells are the principal reservoirs for synovial tissue macrophages in RA [14, 15]. Although little is known regarding transcriptional regulation of LILR family receptors, there is sufficient evidence showing their differential expression upon cellular activation/ differentiation [16–22]. Here we show that a short (12–24 h) in vitro culture of monocytes, or differentiation of these cells to macrophages using GM-CSF, significantly down-regulated LILRA5 mRNA and protein (Fig. 6). The incongruity between the increased tissue expression of LILRA5 in macrophages in RA and its significant down-regulation upon activation/differentiation in vitro indicates that local factors such as LILRA5 ligand(s) and certain soluble mediators may be key determinants in its sustained expression in vivo. We investigated the effects of a key immuno-regulatory cytokine (IL-10) and two cytokines important in the pathogenesis of RA (TNF-a and IFN-g). For the first time, we demonstrated that

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Eur. J. Immunol. 2008. 38: 3459–3473

Immunomodulation

A

B 80 0 hr IgG1

24 hr

5 day 60

α-LILRA5

100 101 102

103

MFI +SD

Counts

200

104100 101

102

103

104100

101

102

103

104

40

* *

20

Log Fluorescence intensity-FITC 0 0 hr

5 days

D 0.2

Fold decrease in mRNA

LILRA5 to β-actin ratio

C

24 hr

0.15 0.1 0.05 0 0 hr

12 hr

24 hr

5 days

40 35 30 25 20 15 10 5 0

**

**

0 hr

24 hr

5 days

Figure 6. Analysis of LILRA5 expression on freshly isolated monocytes showed significant down-regulation of surface LILRA5 protein and mRNA after short-term culture in media containing autologous serum (24 h) or long-term culture with GM-CSF (5 days) determined by flow cytometry (5 104 cells/tube) (A and B) or qPCR (2 105 cells/time point) (C and D). (A) and (C) (qPCR in triplicate7SD) are representative data from monocytes of a single donor; (B) shows the average MFI (MFI7SD) of LILRA5 on the surface of monocytes from five independent donors and (D) is the average fold change in mRNA expression relative to the expression by fresh monocytes from four independent experiments (7SEM). po0.05; po0.01 compared with fresh monocytes (0 h).

the treatment of primary monocytes with IL-10 and IFN-g in vitro significantly up-regulated LILRA5 mRNA that encodes both membrane (Fig. 7) and soluble forms (data not shown). Moreover, IL-10 and IFN-g induced high levels of soluble and surface-expressed LILRA5 protein compared with controls (Fig. 7). Levels of LILRA5 on the surface of IL-10- or IFN-gtreated cells were similar to amounts expressed on freshly isolated monocytes, whereas mRNA levels of after IL-10 and IFN-g treatment were significantly higher than in fresh monocytes (Fig. 6 and 7). This may indicate stabilization of LILRA5 surface expression by IL-10 and IFN-g, or as the increase in mRNA expression suggests, de-novo synthesis of LILRA5 protein to replace receptors at the cell surface that may be shed during in vitro culture. In contrast, TNF-a significantly down-regulated both constitutive and IL-10-induced LILRA5 mRNA and surface protein expression, whereas levels of soluble protein in culture supernatants increased (Fig. 7). Thus, TNF-a may regulate LILRA5 expression by simultaneously increasing cleavage from the cell surface and inhibiting its de-novo synthesis. The down-regulation of LILRA5 by TNF-a, an important proinflammatory cytokine, combined with the strong increase by key immuno-regulatory cytokines (IL-10 and IFN-g) indicates that the expression of this receptor is tightly regulated. Interestingly, production of both cytokines was increased in response to LILRA5


cross-linking, with a relatively early induction of TNF-a at 12 h compared with the more-delayed induction of IL-10 apparent at 48 h (Fig. 4). Thus, LILRA5 may be regulated by two feedback mechanisms whereby an early increase in TNF-a leads to downregulation of LILRA5 to prevent excessive TNF-a production. On the other hand, the delayed production of IL-10-upregulated LILRA5 expression may restore its activation function, thereby counteracting an exaggerated immunosuppressive effect of IL-10. Although generally considered as an inhibitor of pro-inflammatory cytokine production by monocytes/macrophages [11, 12], IL-10 can enhance the phagocytic function of monocytes by upregulating FcgRI [23], FcgRIII [24] and scavenger receptor (CD163) [25] and is increasingly recognized as having multiple immunomodulatory functions [11, 12]. However, the molecular mechanisms of IL-10-mediated immune-regulation are not fully elucidated. Here, for the first time, we show that IL-10 significantly regulated the expression of a potent novel immune-regulatory receptor that may explain the regulatory role of this cytokine. A recent study using gene array of peripheral blood monocytes from patients with psoriasis showed strong time-dependent upregulation of a number of activating and inhibitory LILR mRNAs after IL-10 treatment [26]. Furthermore, IL-10 caused strong upregulation of LILRB2 on the surface of dendritic cells in vitro with functional consequences [27].

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C

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Figure 7. Time- and dose-dependent up-regulation of membrane LILRA5 mRNA on monocytes by IL-10 and IFN-g but not by TNF-a (A). Treatment of cells with optimal concentration of IL-10 or IFN-g (25 ng/mL) for 12 h caused significant up-regulation of membrane LILRA5-encoding mRNA (B). TNF-a, however, led to down-regulation of constitutively expressed and IL-10-induced membrane LILRA5 encoding mRNA. Data are presented as fold changes relative to mRNA expressed by untreated cells. po0.05; po0.01 compared with fresh monocytes (0 h); po0.01 compared with mRNA induced by IL-10 (n 5 5). Representative flow cytometric analysis of three independent experiments of LILRA5 on monocytes after treatment with optimal concentrations of cytokines for 24 h showing strong up-regulation by IL-10 (plot number 5), slight increase in response to IFN-g (plot number 3) but down-regulation in response to TNF-a (plot number 2) compared with untreated cells (plot number 4) (C). The dotted histogram (plot number 1) shows IL-10-treated cells stained with isotype-matched negative control mAb. Representative Western blot analysis of culture supernatants from monocytes treated with various cytokines for 24 h (upper panel); average densitometry of three independent experiments (7 SEM) shows increased release of LILRA5 in supernatants from cells treated with IL-10 (lane 2), TNF-a (lane 3) or IFN-g (lane 4) compared with untreated cells (lane 1) (D). po0.05; po0.01 compared with untreated cells.

In this study we immunoprecipitated LILRA5 membrane protein from the surface of monocytes from patients with RA and control subjects. The size of the precipitated protein was larger than unprocessed LILRA5 protein (40 kDa instead of 32 kDa) indicating putative post-translational modifications such as glycosylation and/or citrullination. Furthermore, LILRA5 associated with the ITAM-containing common g chain of Fc receptors in PBMC from patients and controls (Fig. 3). Similar association of the Fc common g chain was previously shown for other activating LILR expressed on monocytes [28] and dendritic cells [29]. Cross-linking of LILRA5 caused strong phospohorylation of the Src family kinases and Syk (Fig. 5), the two most proximal (important) signaling molecules in the tyrosine kinase-mediated activation cascades [3]. These results suggest that Syk recruitment


and activation most likely triggered several downstream signaling events including activation of phospholipase C-g(PLC-g) and MAPkinases, leading to effector functions such as Ca21 influx and cytokine production [3, 30]. Indeed, in vitro cross-linking of LILRA5 on the surface of monocytes induced pro-inflammatory cytokines that play key roles in the pathogenesis of RA (Fig. 4 and Table 3) confirming previous observation by Borges et al. [4]. Abrogation of TNF-a production using pharmacological inhibitors of PTK and MAP-kinases confirmed that responses to LILRA5 crosslinking were dependent on these pathways (Fig. 5). In vitro crosslinking with anti-LILRA5 mAb on monocytes from patients with active and inactive RA induced TNF-a production comparable to control subjects (Fig. 4C), suggesting that the magnitude of responses in vivo may depend on the availability of LILRA5 ligand(s) as well as on LILRA5 expression levels.

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In addition to the induction of pro-inflammatory cytokines, LILRA5 caused selective production of a key immune-regulatory cytokine (IL-10) (Fig. 4) and inhibited constitutive IL-12 and IL-13 production (Table 3), indicating that beyond the early induction of pro-inflammatory mediators, LILRA5 may influence the quality (type) of subsequent immune responses in favour of either cellular or humoral profiles. The latter proposal is supported by our recent observation in which cross-linking of LILRA2 in eosinophils selectively up-regulated IL-12 but not IL-4 [31]. This LILRA2-mediated response is unlike most of the classical eosinophil activators that predominantly induce IL-4 production leading to Th2-like responses [32]. Furthermore, LILRA2 mRNA was highly expressed in the skin of patients with lepromatous leprosy (primarily a Th2 response) but not in tuberculoid leprosy (Th1 response) [13]. Interestingly, LILRA2 cross-linking inhibited LPS-mediated IL-12 production by monocytes while significantly increasing the production of IL-10 [13]. It is possible that IL-10 produced in response to LILRA2 or A5 cross-linking might be responsible for the suppression of IL-12. IL-10 promotes the development of Th2 responses by inhibiting IFN-g production by T cells and suppressing IL-12 synthesis by accessory cells [33]. Our data show that LILRA5 is a potent macrophage activator, and potentially a key immune regulatory receptor, which is tightly regulated by pro-inflammatory and immunosuppressive cytokines. Its wide expression in synovial tissue of patients with active RA suggests that LILRA5 may contribute to the pathogenesis of this disease and could be a potential therapeutic target.

Materials and methods Subjects Synovial tissue was obtained from 14 patients with RA, 10 with OA and 6 individuals with traumatic meniscus rupture undergoing re-constructive knee surgery (Table 1). Synovial fluid was obtained from 5 patients with active RA and 2 patients with OA and peripheral blood was obtained from 15 patients with RA (10 active, 5 in remission). All RA patients fulfilled the 1987 ACR criteria for RA [34]. Clinical assessment of RA patients included the DAS28 (Disease Activity Score assessing 28 joints for pain and tenderness, c-reactive protein (CRP) (420 mg/L), and visual analogue score for disease activity) [35] and a Modified Health Assessment Questionnaire [36]. Low disease activity was defined as a DASo2.6 as defined by EULAR criteria [37]. The institutional ethics committees approved this study and informed consent was obtained from each patient.

Primary antibodies Specific mouse IgG1 mAb against LILRA5 was generated in BALB/c mice by immunization with Fc fusion protein containing the


Immunomodulation

extracellular domain of LILRA5 as described previously [4, 38, 39]. This mAb was screened for binding specificity by ELISA against a panel of LILR–Fc fusion proteins and by FACS analysis using COS-1 cells transfected with full-length LIR cDNAs [38, 39]. Irrelevant mouse IgG 1 control and anti-MHC class I IgG1 mAb were purchased from Sigma (Australia) and Pharmingen (Mountain View, CA), respectively. Antibodies to detect tissue macrophages (mouse IgG1 anti-CD68), T cells (rabbit polyclonal anti-CD3), B cells (mouse IgG1 anti-CD20), endothelial cells (mouse IgG1 antiVon-Willebrand factor), neutrophil cathepsin G (rabbit polyclonal) and mast cell tryptase (mouse IgG1, AA1) were purchased from DAKO (Glostrup, Denmark). Flurochrome-conjugated primary monoclonal antibodies against CD14-PE, CD56-PE, CD3-Percp and CD19-Percp and corresponding isotype- and Flurochromematched IgG controls were purchased from Pharmingen. Zenon mouse IgG1 labeling kit was used to directly conjugate anti-LILRA5 and anti-CD68 mAb with Alexa 488 or Alexa 568 (Molecular Probes).

Immunohistochemical studies Synovial tissue was embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Miles, Elkhart, IN), snap-frozen in liquid nitrogen, sectioned at 2–4 mm and used for immunohistochemical staining as described previously [7, 8]. In brief, acetonefixed sections were equilibrated with TBS and blocked with neat goat serum for 20 min at room temperature. Sections were then incubated with 5 mg/mL of primary antibodies overnight at 41C. After four washes with TBS, sections were incubated with 2.5 mg/mL of biotinylated goat anti-mouse IgG (Vector Laboratories, Burlingame, CA) for 1 h at room temperature, washed four times and then incubated with streptavidin-alkaline phosphatase conjugate (Vector Laboratories) for 45 min at room temperature. Immunoreactivity was detected using alkaline-phosphatase substrate for 10 min (Vector Red, Vector Laboratories). Double immunofluorescence staining of frozen tissue sections using Alexa 488-conjugated anti-CD68 and Alexa 568-conjugated antiLILRA5 mAb was performed to confirm the identity of LILRA5 expressing cells [16]. Standard haematoxylin and eosin staining was also used to evaluate histopathological changes by an independent pathologist. Sections from immunohistochemical studies were evaluated semi-quantitatively by counting contiguous fields across the whole section as described previously [7, 8]. Although regional variation in staining was observed, median count of the entire section is reported as a conservative measure of staining. To determine the proportions of LILRA51 macrophages, overlapping images, each spanning an area of approximately 1.5 mm2 was captured from sections and multiple images stitched into single continuous picture as described previously [40]. Adjacent sections stained with anti-CD68 and LILRA5 were then aligned, and CD681 and LILRA5/CD6821 cells counted [40]. An area of 1.9–2.2 mm2 was sufficient to count over 100 macrophages in RA synovium. However, more than 5 mm2 was required

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to acquire approximately 100 macrophages in synovial tissue from OA.

for another 2 days in culture medium without GM-CSF as described previously [41].

Western blotting of synovial fluid and culture supernatants

Cell surface expression of LILRA5 by flow cytometry

Synovial fluid from patients with RA and OA (2 mL) was treated with bovine hyaluronidase as per the manufacturer’s instruction (Sigma, Synovial fluid clarification protocol) and cells and debri removed by centrifugation at 2000 rpm for 15 min at room temperature. Cell-free fluid was then pre-cleared with protein G Sepharose-conjugated goat anti-mouse Ab overnight at 41C (Zymed). Supernatants (250 mL) from monocytes treated with IL-10, IFN-g, TNF-a or medium alone were concentrated 10 fold using 10 kDa cutoff 1.5 mL Centricon concentrators (Millipore, Australia). Forty microliters of pre-cleared synovial fluid or 25 mL of concentrated macrophage supernatants were then separated by SDS-PAGE under reducing conditions. Proteins were transferred to PolyScreen polyvinylidene difluoride membranes (Millipore) and then blocked in 5% skim milk powder in TBST at 41C overnight. Membranes were washed briefly in TBST before incubation with 1 mg/mL of anti-LILRA5 mAb diluted in 2% BSA/ TBST for 2 h at room temperature and washed thoroughly in TBST (4 ). Membranes were then incubated with HRPconjugated goat anti-mouse Ab (BioRAD) for 1 h at room temperature, washed in TBST and reactivity detected by chemiluminescence (Pierce, Rockland, IL) and protein visualized by CCD camera.

Isolation of PBMC Buffy coats (100 mL) obtained from healthy donors through the Australian Blood Services (Australian Red Cross, Sydney) or 50 mL of peripheral blood from healthy volunteers or patients with RA were used to isolate PBMC using density gradient centrifugation (Ficoll-Paque Plus, Amersham Biosciences) [40]. PBMC washed twice with PBS were suspended at 1 107/mL in RPMI 1640 containing 2 mM L-glutamine, 10 U/mL penicillin and 100 mg/mL streptomycin (all from Invitrogen Life Technologies) and 10% autologous serum. Cells were incubated at 371C and 5% CO2 in 24-well Costar plates (Corning, NY). Non-adherent cells were removed after 1.5–2 h and adherent cells washed twice with PBS yielding 2–3 106 monocytes per mL (490% as confirmed by CD14 staining) per well. For some experiments, monocytes were negatively selected from freshly isolated PBMC using a monocyte isolation kit with 495% purity (Miltenyi Biotec) or positively selected using dynal beads conjugated anti-CD14 mAb with 498% purity (Dynal). To differentiate monocytes to macrophages, purified cells from healthy subjects were cultured in 24-well plates at 2 106/mL in RPMI 1640 complete media containing 10% autologous serum supplemented with 25 ng/mL GM-CSF (Biosource) for 3 days, then washed twice with PBS and cultured


Flow cytometric studies using freshly isolated PBMC from patients with RA and age- and sex-matched controls were performed as described previously [8, 16]. In brief, cells were washed with cold PBS containing 0.05% NaN3 and 1% BSA (PAB buffer) and used for two-step single colour staining using primary mAb against LILRA5 or control mouse IgG1 (each at 5 mg/mL) followed by 10 mg/mL of FITC-conjugated F(ab0 )2 goat antimouse (F(ab0 )2-specific) secondary Ab (Jackson ImmunoResearch Laboratories). In some experiments, LILRA5-expressing cells were characterized by co-staining with Alexa Fluor-488conjugated anti-LILRA5 mAb (Zenon labeling technology, Molecular probes) and a combination of saturating amounts of antiCD14-PE and CD3-Percp or CD56-PE and CD19-Percp. Isotype and flurochrome-matched negative control antibodies were added to cells stained with Alexa Fluor-488 conjugated IgG1 control mAb. Cells were fixed in 1% paraformaldehyde and analyzed by a three-colour FACScan Flow cytometer (BD Biosciences).

Co-precipitation of LILRA5 and ITAM-containing common c chain of the Fc receptors Freshly isolated PBMC (4 107) from patients with RA or healthy controls were first labeled with Sulfo-NHS-LC-biotin according to the manufacturer’s instructions (Pierce). Cells were lysed in 2 mL Western lysis buffer (1% NP-40 in PBS, pH 7.5, 10 mM EDTA, 20 mM Idoacetamide, 1 mM PMSF, 10 mg/mL Leupeptin, 1 mg/mL Pepstatin A, 0.5 mM sodium orthovanadate, 5 mM sodium fluoride) and insoluble components removed by centrifugation at 14 000 rpm for 20 min at 41C. Soluble lysates were first precleared using 5 mg of protein G Sepharose-coupled goat antimouse IgG (Zymed) at 41C and then incubated with either antiLILRA5 (7.5 mg/mL) or IgG1 control mAb overnight at 41C. Next day 5 mg of protein G Sepharose-coupled goat anti-mouse secondary Ab was added and samples were incubated for 3 h at 41C. Sepharose-bound protein was precipitated by 1 min centrifugation at 10 000 rpm followed by two washes of the pellet with Tris buffer (10 mM Tris–HCl, pH 8, 140 mM NaCl and 0.025% NaNa3) containing 0.1% Triton X-100 and 0.1% bovine haemoglobin, two washes with Tris buffer alone and a final wash with 50 mM Tris–HCl, pH 6.8. Samples were separated by 10% SDSPAGE under reducing conditions, transferred onto PVDF membranes and blocked with 5% skim milk12% BSA in TBS for 2 h. Membranes were first blotted with HRP-conjugated streptavidin to detect biotin-labeled immunoprecipitates (LILRA5 protein) and then stripped and immunoblotted with 1 mg/mL rabbit anti-Fc common g chain Ab as per the manufacturer’s protocol (Upstate, Lake Placid, NY) followed by HRP-conjugated

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goat anti-rabbit (BioRAD) and immunoreactive bands detected as described above.

Phosphorylation of Src family PTK and spleen tyrosine kinase (Syk) upon LILRA5 cross-linking Negatively selected primary monocytes (5 106 cells, 95–99% pure) (MACS Human Monocyte Isolation Kit) were re-suspended in 1 mL of RPMI 1640 supplemented with 10 mM HEPES and 0.1% BSA. Five aliquots of cell suspension (200 mL each) were dispensed onto 96-well plates coated with optimal doses of antiLILRA5 as described above. Cells were then incubated at 371C for 3 min and cell pellets immediately lysed with 100 mL/well Western lysis buffer containing fresh sodium pervanadate (0.1 mM). Lysates were then incubated on ice for 10 min and insoluble material was removed by centrifugation (14 000 rpm) at 41C for 15 min. Western blotting using rabbit anti-phosphorSrc family (Tyr416) Ab was performed on 20 mg lysates following manufacturer’s instructions (Cell Signaling Technology, Danvers, MA). To confirm equal loading of protein, the same membrane was later blotted with mouse anti-b-actin mAb according to the manufacturer’s instructions (Sigma). Lysates derived from cells incubated in wells coated with anti-MHC class I mAb, irrelevant isotype-matched IgG or secondary Ab alone were used as controls. Following recruitment of Src family kinases such as Lyn, recruitment and phosphorylation of Syk is one of the most proximal events in the transduction of activating signaling through ITAM-containing receptors in monocytes [3]. To determine whether Syk was phosphorylated in response to LILRA5 cross-linking, 1 106 purified monocytes or PBMC were activated for 2 min in 96-well plates coated with anti-LILRA5 mAb or control antibodies and levels of phosphorylated Syk determined using a Bioplex analysis kit according to the manufacturer’s instructions (Beadlyte, Phosphoprotein Receptor Signalling Kit, Upstate).

Immunomodulation

ted with 10 mM HEPES (Sigma) and 0.1% BSA (Sigma), and 2 105 cells in 200 mL were added to each well. After incubation for 6–48 h at 371C and 5% CO2 in air, cell-free supernatants were collected and stored at 201C for measurement of TNF-a by a DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) or for a multi-cytokine Bioplex assay (BioRad).

Inhibition of TNF-a production by pharmacological inhibitors of PTK and MAPK Signaling of LILRA5-induced TNF-a production through PTK and/or MAPK pathway was determined by pre-treating monocytes (0.5 106/mL) with pharmacological inhibitors (Calbiochem, La Jolla, CA, USA). In brief, cells were pre-incubated for 1 h with 50 mM of Src inhibitor PP2, 1–10 mM of Syk inhibitor II, 10 mM of Erk inhibitor PD98059, 1 mM of p38 inhibitors SB202190 or SB203580. PP3 (50 mM) or DMSO (0.05%) were used as controls for PP2 and other inhibitors, respectively. Cells were then washed in media [29, 42] and plated onto anti-LILRA5 coated plates as described previously. Culture supernatants were collected 12 h after cross-linking and production of TNF-a determined.

Modulation of LILRA5 expression on monocytes and macrophages by cytokines in vitro Monocytes or GM-CSF differentiated cells were incubated in RPMI complete media containing 10% autologous serum with various doses of TNF-a, IL-10 or IFN-g (R&D Systems) for 6–24 h at 371C in 5% CO2. Cells and culture supernatants were harvested for flow cytometry and Western blot analysis, respectively. In some experiments, RNA was extracted for quantitative RT-PCR (qPCR).

RNA extraction and qPCR Cytokine production by monocytes in response to LILRA5 cross-linking PBMC were activated by cross-linking LILRA5 by anti-LILRA5 mAb bound to a plate as described previously [4, 31]. In brief, wells in a 96-well flat-bottom tissue culture plates (Costars 3596, Corning) were coated with 100 mL (5 mg) of F(ab0 )2 goat anti-mouse IgG, Fc-specific (Jackson ImmunoResearch) overnight at 41C. After aspiration of unbound Ab, 50 mL of anti-LILRA5 mAb was diluted to the desired concentration in PBS containing 2.5% BSA. For a negative control, irrelevant mouse IgG1 and/or isotype-matched anti-MHC Class I mAb were used. Plates were incubated for 2 h at 371C and 5% CO2 and wells were washed twice with 0.9% NaCl before use. PBMC were washed twice with PBS, blocked for 10 min at room temperature with FcR blocking buffer (Miltenyi Biotec), re-suspended in RPMI 1640 supplemen-


Total RNA was extracted using Trizol (Invitrogen) from control and cytokine-stimulated monocytes and macrophages after 2–24 h. Reverse transcription was carried out using SuperScript III First Strand Synthesis Supermix for qRT-PCR (Invitrogen). LILRA5 transcript levels were determined by real-time RT-PCR and b-actin was used as a reference housekeeping gene. Aliquots (5 mL) of cDNA were amplified using SYBR GreenER qPCR SuperMix and 200 nM of primer set that detect both forms of membrane LILRA5 mRNA (forward 50 -TCACGGCTGAGATTCGACAG-30 ; (reverse 50 -GTTTTGTGACGGACTGAGGTTAT-30 ), 200 nM primer set that detect both forms of soluble LILRA5 mRNA (forward 50 -CTCTGCCTCGGGAACCTCT-30 ; reverse 50 -TAACCAGACGGTATTCCTGGG-30 ) or 100 nM of b-actin primers (forward 50 -CATGTACGTTGCTATCCAGGC-30 ; reverse 50 -CTCCTTAATGTCACGCACGAT-30 ). QRT-PCR was performed in an ABI Prism 7700 Sequence detector (Applied Biosystems).

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A 2 min start at 501C and then 2 min at 951C was followed by 45 cycles of PCR (each cycle 951C, 15 s; 601C, 45 s). The reactions finished with 2 min at 251C. The same background subtractions and threshold values for all reactions were used to determine CT values. Fold changes in levels of mRNA expression from control samples were calculated for individual experiments by dividing the CT value of control samples to corresponding experimental samples. These standardized values were then used to obtain mean values of multiple experiments for statistical analysis.

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4 Borges, L., Kubin, M. and Kuhlman, T., LILRA5, an immunoglobulinsuperfamily-activating receptor, is expressed as a transmembrane and as a secreted molecule. Blood. 2003. 101: 1484–1486. 5 Tak, P. P. and Bresnihan, B., The pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis. Arthritis Rheum. 2000. 43: 2619–2633. 6 Bromley, M. and Wooley, D. E., Histopathology of the rheumatoid lesion. Identification of cell types at sites of cartilage erosion. Arthritis Rheum. 1984. 27: 857–863. 7 Tedla, N., Gibson, K., McNeil, H. P., Cosman, D., Borges, L. and Arm, J. P., The co-expression of activating and inhibitory leukocyte immunoglobu-

Statistical analysis

lin-like receptors in rheumatoid synovium. Am. J. Pathol. 2002. 160: 425–431. 8 Huynh, O. A., Hampartzoumian, T., Arm, J. P., Hunt, J., Borges, L., Ahern,

After determining the median number of macrophages and cells expressing LILRA5 from each individual, mean values were calculated for each group. The mean cell counts in synovial tissue of patients with RA were then compared with patients with OA and control subjects using one-way ANOVA with Dunnett’s multiple comparison post test. Non-parametric correlation analysis (Spearman) was used to assess the relationship between the median LILRA5 count and the number of macrophages or DAS. Significant changes in cytokine production at each time point were compared with the corresponding IgG1 control using paired t-test. One-way ANOVA with Dunnett’s multiple comparison post test was used to evaluate the statistical significance of LILRA5-mediated Syk phosphorylation, the effects of pharmacological inhibitors on LILRA5-mediated TNF-a production and changes in LILRA5 mRNA/protein in culture overtime and after treatment with cytokines. A p value of o0.05 was considered significant.

M., Smith, M. et al., Down-regulation of leucocyte immunoglobulin-like receptor expression in the synovium of rheumatoid arthritis patients after treatment with disease-modifying anti-rheumatic drugs. Rheumatology 2007. 46: 742–751. 9 Choy,

E.

H.

and

Panayi,

G.

S.,

Cytokine pathways

and

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inflammation in rheumatoid arthritis. N Engl. J. Med. 2001. 344: 907–916. 10 Zwerina, J., Redlich, K., Schett, G. and Smolen, J. S., Pathogenesis of rheumatoid arthritis: targeting cytokines. Ann. NY Acad. Sci. 2005. 1051: 716–729. 11 Moore, K. W., de Waal Malefyt, R., Coffman, R. L. and O’Garra, A., Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 2001. 19:683–765. 12 Pestka, S., Krause, C. D., Sarkar, D., Walter, M. R., Shi, Y. and Fisher, P. B., Interleukin-10 and related cytokines and receptors. Annu. Rev. Immunol. 2004. 22: 929–979. 13 Bleharski, J. R., Li, H., Meinken, C., Graeber, T. G., Ochoa, M. T., Yamamura, M., Burdick, A. et al., Use of genetic profiling in leprosy to discriminate clinical forms of the disease. Science 2003. 301:1527–1530. 14 Firestein, G. S. and Zvaifler, N. J., Peripheral blood and synovial fluid monocyte activation in inflammatory arthritis. I. A cytofluorographic

Acknowledgements: This work was supported by grants from the National Health and Medical Research Council (NHMRC: ID 300452) of Australia. We thank Amgen for kindly supplying LILRA5 mAb. We are grateful to Dr Taline Hampartzoumian for performing the Bioplex cytokine assays and for helpful comments.

study of monocyte differentiation antigens and class II antigens and their regulation by gamma-interferon. Arthritis Rheum. 1987. 30: 857–863. 15 Kang, Y. M., Kim, S. Y., Kang, J. H., Han, S. W., Nam, E. J., Kyung, H. S., Park, J. Y. and Kim, I. S., LIGHT up-regulated on B lymphocytes and monocytes in rheumatoid arthritis mediates cellular adhesion and metalloproteinase production by synoviocytes. Arthritis Rheum. 2007. 56:

Conflict of interest: Dr. Luis Borges is an employee of Amgen that provided us with the anti-LILRA5 mAb.

1106–1117. 16 Tedla, N., Lee, C. W., Borges, L., Geczy, C. L. and Arm, J. P., Differential expression of leukocyte immunoglobulin-like receptors on cord bloodderived human mast cell progenitors and mature mast cells. J. Leukoc. Biol. 2008. 83: 334–343.

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Received: 14/4/2008 Revised: 2/9/2008 Accepted: 15/9/2008

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