Elsevier

International Immunopharmacology

Volume 55, February 2018, Pages 297-305
International Immunopharmacology

Blocking osteopontin-fibronectin interactions reduce extracellular fibronectin deployment and arthritic immunopathology

https://doi.org/10.1016/j.intimp.2017.12.028Get rights and content

Highlights

  • A polypeptide corresponding to thrombin-cleaved osteopontin (OPNT) was expressed.

  • Phage display technology was used to make an scFv antibody (scFv 31) against OPNT.

  • Antibody scFv 31 blocks osteopontin-fibronectin interactions.

  • Antibody scFv 31 alters B cell adhesion to fibroblasts and fibronectin deployment.

  • Antibody scFv 31 reduces arthritic immunopathology.

Abstract

Elevated levels of a thrombin-cleaved fragment of osteopontin (OPNT) are seen in synovial fluid (SF) and tissues of rheumatoid arthritis (RA) patients. OPNT binds to integrins on cell surfaces, inducing adhesion, migration and survival of inflammatory cells in the synovial joints, where OPNT binds to fibronectin to link fibroblast-like synoviocytes (FLS) with B cells, stimulating the latter to produce inflammatory cytokines. Our aim was to block OPNT-fibronectin interactions and examine whether this reduces inflammation. A human antibody (phage displayed) library was used to select scFv antibodies cognate to OPNT, and a particular scFv antibody (scFv 31) was evaluated. Adhesion, migration and fibronectin polymerization of FLS cells derived from RA patients were monitored, in cultures incorporating scFv 31. Also, scFv 31 was used in mice with CAIA (collagen antibody-induced arthritis), subjected to clinical and histological assessment, analysis of fibronectin and cartilage damage and induction of pro-inflammatory cytokines. The scFv antibody, scFv 31, appeared to cause significantly reduced migration of synovial fibroblasts, altered cell morphology, changes in actin stress fiber arrangement, and marked reduction in fibronectin. In CAIA mice, scFv 31 appeared to prevent arthritic changes through inhibition of synovial hypertrophy and loss of articular cartilage, decrease in fibronectin polymerization and expression of pro-inflammatory cytokines implicated in arthritis. Osteopontin-fibronectin interaction(s) appear to play a role in the expression of key inflammatory molecules by B cells infiltrating the synovial joint. The scFv antibody, scFv 31, provides a potential therapeutic lead for inhibition of some processes implicated in rheumatoid arthritis.

Introduction

Rheumatoid arthritis (RA) is an autoimmune, inflammatory disease of unknown etiology which mainly affects the synovial joints of the hands and feet. Some genetic and environmental factors are thought to play a role in RA. Also, interactions of immune cells with synovial fibroblasts have been implicated in the etiology of RA [1], [2], [3], [4]. Resident synovial fibroblasts potentially invade articular cartilage and bone tissue through interactions with the extracellular matrix (ECM), causing damage to the architecture of joints. Cell-ECM interactions are mediated through cell surface receptors known as integrins which also trigger complex intracellular signaling events following such interactions [5], [6].

Osteopontin (OPN) is a matricellular protein, i.e., it is known to exist both as an extracellular matrix component and as a soluble protein, capable of stimulatory pro-inflammatory phenomena [7], [8], [9]. Amino acid sequence motifs within OPN which participate in its (adhesive) interactions with the ECM include (i) the sequence, RGD, and (ii) the sequence, SVVYGLR (SLAYGLR in rat and mouse), lying adjacent to the RGD motif, at its C-terminal end. The RGD motif interacts with integrins αvβ1, αvβ3, αvβ5, αvβ6, α8β1, and α5β1 [10], [11], [12]. Upon cleavage of OPN by thrombin, the motif SVVYGLR is exposed and available to interact with integrins α9β1, α4β1, and α4β7 [13]. These interactions activate the integrins. Activated integrin-mediated signaling further regulates vital cellular processes like cellular adhesion, migration, proliferation and cell survival.

Rheumatoid arthritis patients have been reported to have elevated levels of thrombin-cleaved OPN in the synovial fluid [14]. The importance of the exposure of the SVVYGLR/SLAYGLR motif upon thrombin cleavage of OPN has also been shown in murine and primate models of rheumatoid arthritis [15], [16]. Notably, thrombin-cleaved OPN is present on the surfaces of fibroblast-like synoviocyte (FLS) cells, cross-linked with fibronectin [17]. Thrombin-cleaved OPN (OPNT) aids in the adhesion of B cells to FLS cells, leading to the production of inflammatory cytokines [17].

Given the above, we resolved to develop and test an antibody cognate to OPNT and to examine the effects of blocking its interaction with fibronectin. We report that scFv 31, a single chain variable fragment antibody (scFv) selected through screening of a phage-displayed antibody library, completely abrogates fibronectin polymerization. It also reduces inflammation in a mouse model of RA. The antibody possesses a completely ‘human’ sequence. It can thus be tested in humans.

Section snippets

Ethics statement

Patient material was obtained through written consent of participants. The protocol of the study was approved by the Institutional Ethics Committee of the Postgraduate Institute of Medical Education and Research. The study adhered to the tenets of the Declaration of Helsinki.

Cultures of synovial fibroblasts from synovial fluid

Synovial tissue was obtained from the knees of RA patients undergoing knee replacement surgery (and fulfilling ACR criteria [18]), with informed consent. Finely minced tissue was attached to tissue culture dishes, and grown

Anti-osteopontin scFv antibodies

Using screening of a phage display scFv antibody library (Tomlinson I; MRC, London) and analysis of 96 shortlisted scFv clones through ELISA experiments following multiple rounds of biopanning and amplification, and expression of clones as soluble scFv antibodies in E.coli strain HB2151, one antibody, scFv 31, was selected on the basis of its ELISA result and also its yield after expression.

Binding of scFv 31 to other ECM components

Antibody scFv 31 showed significant binding to OPNT, but not to other ECM components or to the irrelevant

Discussion

In RA pathology, activated synovial fibroblasts play a crucial role in remodeling of the ECM. The synovial fibroblasts are reported to express surface bound fibronectin (Fn) which forms covalent crosslinks with a thrombin-cleaved form of OPN and promotes infiltration of B cells through engagement of VLA4 (very late antigen-4) expressed on B cell surfaces. OPN cross-linked to fibronectin, thus acts as a bridge between fibroblasts and B cells, causing activation of B cells, and secretion of

Funding

This work was supported by the Department of Biotechnology, Government of India (No. 102/IFD/SAN/5131/2011-2012).

Disclosure statement

The authors declare no conflict of interest.

Acknowledgements

MLG thanks the DBT (No. 102/IFD/SAN/5131/2011-2012) for funding; BBM thanks the ICMR for fellowship.

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