Reelin levels are increased in synovial fluid of patients with rheumatoid arthritis

BRIEF PAPER

Reelin levels are increased in synovial ?uid of patients with rheumatoid arthritis A. Magnani1, L. Pattacini1,

L. Boiardi2, B. Casali1,

C. Salvarani2

1Laboratorio di Biologia Molecolare and 2Unità Operativa di Reumatologia, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.

Alberto Magnani, BD

Laura Pattacini, BD

Luigi Boiardi, MD, PhD

Bruno Casali, MD

Carlo Salvarani, MD

Please address correspondence

and reprint requests to:

Dr Carlo Salvarani,

Unità Operativa di Reumatologia, Arcispedale S. Maria Nuova,

Viale Risorgimento 80,

42100, Reggio Emilia, Italy.

E-mail: salvarani.carlo@asmn.re.it Received on February 24, 2009; accepted in revised form on March 22, 2010.

? Copyright C LINICAL AND

E XPERIMENTAL R HEUMATOLOGY 2010. Key words: Fibroblast-like synoviocytes, rheumatoid arthritis, reelin

Competing interests: none declared.ABSTRACT

Objectives.To evaluate the presence

and the glycosylation pattern of reelin

in synovial ?uid and serum of patients

affected by different rheumatic pathol-

ogies.

Methods.Reelin levels were evaluated

in patients affected by rheumatoid ar-

thritis (RA), psoriatic arthritis (PsA),

spondyloarthritis (SpA) and osteoar-

thritis (OA). Reelin semi-quantitative

assays were performed by western blot.

The glycosylation pattern was evalu-

ated by immunoblotting performed by

sepharose conjugated lectins. RT-PCR

was used to detect the presence of

mRNA encoding for reelin and its re-

ceptors.

Results.Reelin is detectable in both

sinovial ?uids and sera and its levels

are more elevated in patients affected

by RA with respect to those affected by

other in?ammatory and non in?amma-

tory joint diseases. The glycosylation

pattern of the protein differs in syno-

vial ?uid and serum. Fibroblast-like

synoviocytes (FLS) express the mRNAs

encoding for reelin and its receptors.

Conclusions.Since its levels are higher

in RA then in the other analysed pathol-

ogies, reelin can represent a candidate

suitable for the differential diagnosis

of this pathology. Moreover, the obser-

vation that this protein is encoded by

FLS and differentially glycosylated in

blood and synovial ?uid supports the

hypothesis that it is locally produced

in the joints, where it could play an

important role in RA development and

maintenance.

Introduction

Rheumatoid arthritis (RA) is a chronic

in?ammatory disease of the joints, af-

fecting 1% of the western population.

The rheumatoid synovium includes a

special cell population, constituted by

activated ?broblast-like synoviocytes

(FLS) that distinguish RA from other

in?ammatory disorders of the joints

(1). FLS produce a variety of cytokines

and metalloproteases, providing for

cartilage degradation. Different stud-

ies have been focused on the role of

metalloproteases in the pathogenesis of

in?ammatory arthritis and on their pos-

sible use as markers allowing to differ-

entiate RA from other pathologies. So

far, none of them has resulted suitable

to this aim (2).

Reelin is a 410kDa protein with the

double function of serine protease (3)

and ligand of the two receptors Apo-

ER2 and VLDLR (4). LDL family of

receptors are not only involved in cho-

lesterol transport and metabolism, but

also in modulation of hippocampal

synaptic plasticity and normal learning

and memory development. Moreover,

the ability to endocytose cellular nu-

trients, clear extracellular matrix pro-

teins, transduce signals from multiple

ligands and activate numerous signal

transduction pathways truly places this

family of receptors in a very exclusive

class of multifunctional receptor pro-

teins (3-5). Reelin is expressed in the

central nervous system, liver, adrenal

chromaf?n cells and pituitary pars in-

termedia; moreover, its presence can be

detected in the blood (5).

Reelin signalling is impaired in au-

tism (6), whereas its downmodulation

has been found in the brain of patients

with schizophrenia (7). In other disor-

ders, such as Alzheimer’s disease, ree-

lin levels in the cerebrospinal ?uid are

increased. Moreover, in these patients,

the reelin glycosylation pattern differs

in cerebrospinal ?uid and blood, sup-

porting the hypothesis that the mol-

ecule is locally produced in the central

nervous system (8).

In our study, we evaluated the levels

and glycosylation pattern of reelin in

synovial ?uid and serum obtained from

patients affected by different joint dis-

eases.

Methods

Sample collection and storage

Synovial ?uids obtained by arthrocen-

tesis from 2 patients affected by RA,

spondyloarthritis (SpA), psoriatic ar-

thritis (PsA) and osteoarthritis (OA)

were collected and centrifuged to elim-

inate the cellular content. Table I shows

the drug treatment of the patients of the

study. After centrifugation, the super-

natants were aliquoted and stored at

-80°C. Blood samples were collected at

the same time of arthrocentesis. All pa-

tients ful?lled the American College of

Rheumatology revised criteria for RA

Clinical and Experimental Rheumatology 2010; 28: 546-548.

and OA (9, 10), the European Study group for spondyloarthropathy criteria for SpA (11) and the CASPER criteria for PsA (12) and gave their informed consent prior to study entry. The eth-ics committee of the Arcispedale Santa Maria Nuova approved all the experi-ments on human cells and tissues.Western and immunoblot

Western blots were performed as previ-ously described (13) using the antibody 142 (kindly donated by Dr Lugli Gio-vanni). As a positive control, recom-binant reelin (kindly donated by Dr Lugli Giovanni) was loaded. Immuno-blots were performed by conjugating the three lectins (EY Laboratories, San Mateo, CA, USA) with sepharose 4B (Amersham Biosciences, Amersham, UK). Equal amount of proteins were then pulled down using the conjugated lectins. The detection was performed by using the anti-reelin antibody. The equal loading was proved by Red Pon-ceau (Sigma-Aldrich, St. Louis, MO, USA) staining of the membrane.

The glycosylation pattern of reelin was analysed by pulling down reelin by the sepharose conjugated lectins. Galan-thus nivalis (GNA), maackia amurensis (MAA) and lotus tetragonolobus (LT) were used to detect mannose, alpha (2-3) linked sialic acid and fucose resi-dues, respectively. Then, the primary anti-reelin antibody was used for the detection.

Semi-quantitative analysis

For semi-quantitative studies, the inten-sity of bands detected by Western blot-ting was measured by densitometry us-ing x-ray ?lms exposed to ECL reagents and the Scion Image software (14).Cell culture

FLS cultures were set up as previously described (15, 16). For FLS obtained from synovial ?uid, the positive stain-ing with the anti-?broblast antibody and the lack of staining with CD68 antibody were proved by immunocyto-chemistry.

RT-PCR

Retrotranscription and PCR were per-formed following standard procedures

Table I. Demographic, ERS and therapy of patients studied.

Gender Age ESR Treatment PsA Female 56 10 MTX PsA Male 51 25 AINS RA Female 73 36 Etanercept RA Female 75 120 MTX RA Female 72 53 MTX SpA Male 49 3 No therapy SpA Female 30 18 No therapy OA Female 75 28 Steroid OA

Female

57

9

No therapy

Fig. 1. Serum and synovial ?uid from RA patients are characterized by higher reelin levels respect to

other in?ammatory and non in?ammatory pathologies. Equal volumes of synovial ?uid (A ) or serum (B ) obtained from patients affected by RA, PsA and SpA were loaded, together with recombinant reelin used as a positive control. Then, a western blot was performed by using the antibody 142. Synovial ?uid (C ) and serum from patients with OA and RA were run. The western blots were performed using the same antibody.

Fig. 2. Reelin is highly glycosylated in serum, but not in synovial ?uid. Sepharose conjugated lectins

GNA, MAA and LT were used to pull down the glycosylated fraction of proteins in serum and synovial ?uids of patients affected by SpA, PsA and RA. Then, the western blots were performed by using the

antibody 142.

(17).To detect reelin, VLDLR and Apo-ER2 the following primers were used: CCTCACCAACACAACTCGACT-TCG (forward), TGGTGGGTTGT-GAAGCCACTTCTT (reverse) for ree-lin; CTGGATAGATGGGGAAAAT-GA (forward), TTTGACAGTCTCG-GCCATTT (reverse) for VLDLR and GATTGCGAAAAGGACCAATT (for-ward), TAGCACAGCCGGCCTCAT (reverse) for ApoER2.

Results

Reelin was detected by Western blot in synovial ?uids from patients affected by RA, PsA and SpA. The densitomet-ric quanti?cation showed that the pro-tein levels in RA patients were 5-fold higher with respect to PsA patients and 10-fold with respect to SpA patients (Fig. 1A). A similar trend of reelin lev-els was found in sera obtained from the same patients. In these samples, a three- and a six-fold decrease were found respectively in sera from PsA and SpA with respect to RA patients (Fig. 1B). Moreover, a three-fold in-crease was found in both synovial ?uid (Fig. 1C) and serum (Fig. 1D) of RA with respect to OA patients.

We then studied the glycosylation pat-tern of reelin. In the serum, the 160kDa form contained mannose and sialic acid residues, whereas all the three forms were fucosylated. On the contrary, syn-ovial ?uid reelin did not contain any of these residues (Fig. 2).

Reelin mRNA was detected in ?brob-last-like synoviocytes obtained from patients with the four analysed pathol-ogies. Moreover, in the same cells, also the mRNA encoding for the two reelin receptors, ApoER2 and VLDLR, were identi?ed.

Discussion

In our study we showed, to our knowl-edge, for the ?rst time that reelin con-centration is higher in synovial ?uids and sera of patients with RA with re-spect to the other studied pathologies. Our ?nding could be important for a differential diagnosis, since so far it has not been found any other protease

increased speci?cally in RA patients.

A higher number of samples though

would be needed to support our pre-

liminary results. Nevertheless, the dif-

ference we have detected is striking.

Then we evaluated the possibility that

reelin could be produced in the synovi-

al environment. To this aim, we studied

its glycosylation pattern and showed

that it differs in serum with respect to

synovial ?uid. This difference could

be due to the presence of different gly-

cosidases in the synovial environment.

We cannot exclude that the changes in

either level or glycosylation of reelin

are due to different treatment that pa-

tients with distinct pathologies have

received.

We have shown the presence of mRNA

coding for reelin and its receptors in

FLS. This ?nding supports the possi-

bility that reelin is produced by FLS in

the synovia and the increased levels in

RA synovial ?uid could be due to the

increased number of these cells. Ree-

lin binding to its receptors induces the

activation of PI3K and src kinase path-

ways and the cytoskeletal reorganisa-

tion leading to neuronal migration (18),

which can at least in part explain its

activity in neuronal migration. In the

same way, it could also participate in

FLS migration. Since reelin is involved

in neuron migration, it could also play

a role in FLS migration.

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