Function of CXCR2 signalling within the articular cartilage

Cartilage is the tissue that covers the ends of our long bones, allowing the frictionless motion of our joints. This tissue is lost (degraded) during osteoarthritis, leading to joint pain and incapacity to walk or perform simple tasks. Osteoarthritis is the leading cause for disability allowance in the UK; it affects greater than 1 in 3 of the population over the age of 50 and therefore is a major factor affecting well-being, particularly in the elderly.

To-date, we do not have any treatment that stops progression of osteoarthritis and the only remedies are pain killers and joint replacement. Although generally effective, joint replacement does not live up to patients' expectations in ~20% of cases, and has a limited life span often requiring complex and challenging revision surgery (with a poor success rate). These serious limitations of joint replacement are becoming more prominent due to the fact that people live much longer and because more people are developing osteoarthritis at a younger age (e.g. due to obesity, or following sports injuries).

Following injury, cartilage responds to maintain its integrity, however, cartilage breakdown progresses when this response is insufficient. Excitingly, recent data have shown that supporting these intrinsic protective responses can be very successful in animal models, preventing cartilage breakdown and even regenerating damaged tissue.

We have discovered that molecules called ELR+CXC chemokines are involved in these protective responses. Previously known for attracting immune cells to sites of inflammation, we have discovered that, when present within healthy cartilage, these molecules have a totally different role supporting cartilage integrity following injury. As a consequence, mice that have been engineered to lack this protective response are more susceptible to experimental osteoarthritis. We discovered that ELR+CXC chemokines are retained in the cartilage tissue because they are bound to complex sugar molecules called HSPGs. Therefore, ELR+CXC chemokines present in cartilage cannot attract inflammatory cells and, instead, help cartilage to stay healthy and recover from minor injury. When, during osteoarthritis, cartilage becomes badly damaged, ELR+CXC chemokines are lost from the tissue so that they can no longer protect cartilage and they may also contribute directly to inflammation.

In this study we intend to answer the following questions:
1) How exactly do these chemokines protect cartilage during osteoarthritis? To do this we will make a mouse model that will enable us to separate the effects of chemokines on cartilage from those on other aspects of joint function and inflammation. Importantly, it will also provide information on which point during the development of osteoarthritis these molecules function, which is essential if we are to harness this protective activity for therapeutic applications.

2) What is the most potent ELR+CXC chemokine to support cartilage health? There are different ELR+CXC chemokines and there is data to suggest that some may be better than others at supporting cartilage integrity. Identifying the best chemokine is very important for therapeutic applications.

3) Can we modify chemokines so that they become unable of attracting inflammatory cells and yet retain their capacity to protect cartilage? We will achieve this by modifying the way they bind to HSPGs so that they cannot adhere to blood vessels where they would recruit inflammatory cells.

4) We will build a biomaterial containing and retaining ELR+CXC chemokines to test the potential of tissue engineering to repair/protect cartilage without causing inflammation.

This project develops from our discovery of this unexpected function of chemokines and identifies a way we can protect/repair cartilage in osteoarthritis: a leading cause of disability for which we still do not have a cure.

Osteoarthritis (OA) is characterized by cartilage breakdown and is a leading cause of disability for which we do not have a cure. We discovered that ELR+CXC chemokines retained within cartilage through binding to heparan sulphate proteoglycans (HSPGs) have an unsuspected homeostatic function. Disruption of their receptor CXCR2 predisposed mice to experimental OA with chondrocyte dedifferentiation and apoptosis; similar data were obtained in human chondrocytes by blocking both CXCR1 and 2. CXCL6 supported human chondrocyte differentiation in vitro and, during embryogenesis, was expressed in the future permanent articular cartilage. The scope of this application is to understand the mechanism by which ELR+CXC chemokine signalling protects cartilage in osteoarthritis without causing inflammation, with the final goal of developing a therapeutic application.

Aims:

1) To isolate the function of CXCR2 within adult cartilage from developmental or inflammation phenotypes we will induce OA in a cartilage-specific, tamoxifen-inducible CXCR2 knockout mouse.

2) We will compare the cartilage homeostatic potency of different CXCR1/2 ligands.

3) We will study the biochemistry and function of ELR+CXC-HSPGs interactions in the context of cartilage homeostasis by HS digestion, mutagenesis of the HSPG binding residues, and by binding assays. We will identify the chemokine-HSPGs binding partners by co-immunoprecipitation followed by western blotting of candidate HSPG or mass spectrometry.

4) We will test if we can inhibit the capacity of ELR+CXC chemokines to cause inflammation without altering their homeostatic properties by disrupting HSPG binding (necessary for the formation of haptotactic gradients and neutrophil extravasation) and incorporating these mutant molecules in a bioscaffold. The efficacy of this strategy will be tested in an in vivo cartilage formation assay involving the adoptive transplantation of human chondrocytes and neutrophils in nude mice.

Greater knowledge of the homeostatic (and intrinsic protective) mechanisms in adult cartilage will likely facilitate the development of new therapeutics for osteoarthritis with the potential for huge socio-economic impact (i.e. with benefit for the UK pharmaceutical industry, OA sufferers and their carers). One in 3 people over the age of 45 suffer from this debilitating condition with increased incidence in older individuals (e.g. 42% of men and 49% of woman age 75 and over).

The costs to society are estimated over $185 billion yearly in the USA only for medical care (Kotzlar & Rizzo, Arthritis Rheum 2007). In the UK OA caused the loss of 36 million working days in 1999-2000 costing the economy nearly 3.2 billion in lost production (NICE 2012 OA guidance draft).

Therefore, the beneficiaries will include:

1) Pharmaceutical industry active in the field of osteoarthritis and cartilage regeneration for developing on one hand selective chemokine blockade in inflammatory diseases, or, on the other hand, harnessing such homeostatic role to achieve chondroprotection in OA.

2) Biotech companies involved in musculoskeletal tissue engineering. The pathway will be similar to that in point one, but in vitro treatment of cellular products could be made readily possible and available for clinical practice and human trials.

3) The medical and orthopaedic community, and clinical investigators involved in osteoarthritis and cartilage regeneration.

4) Patients with osteoarthritis and cartilage defects.

5) Society at large for the spectacular direct and indirect costs related to osteoarthritis, in terms of medical costs, disability and loss of productivity.

6) veterinary surgeons involved in cartilage repair in race horses.