lecture 26

Question 1

What are learning objectives?

Answer 1

• what is synovium, and what is normal function?
• what cell types are present in healthy synovium and what are their functions?
• describe how healthy synovial tissue is transformed in RA?
• what are the functions of the cells within the inflamed synovium in RA?
• what are tNF, IL-1 and IL-6 and what do they do?
• why is the use of animal models important in the pre-clinical study of RA pathogenesis?
• identify 2 pre-clinical models of RA and discuss their pros and cons

Question 2

What is the synovium?

Answer 2

• thin membrane that extends from skeletal tissue at interface of cartilage and bone and lines the capsule of diarthrodial joints

Question 3

What is the healthy structure of synovium?

Answer 3

intima (synovial lining layer)

• interface between the joint cavity and the subintima
• 1-3 cell layers deep
• cells = synoviocytes

subintima (sublining layer)

• connective tissues that become denser closer to the joint capsule
• contains blood vessels, lymphatic vessels and nerves

Question 4

What would we see on a healthy biopsy of synovium?

Answer 4

• 3 different types of synovial tissue that differ by histological appearance

areolar

• intima: continuous layer of lining cells
• sub-intima: capillaries immediately below, small arterioles and venules and lymphatic vessels

fibrous

• close to ligaments or tendons, or close to bone/cartilage margins
• layer of cells on ligament or tendon, difficult to distinguish from fibrocartilage

fatty

• found mostly in fat pads
• underlying the intima is superficial net of capillaries amongst the apidocytes (fat cells)

Question 5

What are the functions of a health synovium?

Answer 5

• facilitates movement between non-deformable structures within joint (e.g. bone/cartilage surfaces)
• synovium highly deformable, freely mobile
• non-adherent properties (i.e. not stick to bone and cartilage)
• • slips seemlessly over cartilage/bone matrix
• provision of lubricants that minimuse “wear and tear” on joint tissues
• • hyaluronan (high molecular weight polysaccharide maintains synovial fluid viscosity, effective shock absorption, prevents fluid loss from joint space under loading)
• • lubricin (mucin-like proteoglycan, protects cartilage surfaces from protein deposition and cell adhesion; inhibits synovial cell growth)
• contributes to formation of synovial fluid
• chondrocyte nutrition via solute diffusion from synovial fluid

Question 6

What is hyaluronanan?

Answer 6

high molecular weight polysaccharide maintains synovial fluid viscosity, effective shock absorption, prevents fluid loss from joint space under loading

Question 7

What is lubricin?

Answer 7

mucin-like proteoglycan, protects cartilage surfaces from protein deposition and cell adhesion; inhibits synovial cell growth

Question 8

Is cartilage vascular?

Answer 8

no

Question 9

What are the cell populations in the intima?

Answer 9

2 distinct cell populations

• 20% cells are macrophage-like synoviocytes (type A synoviates
• 80% cells are fibroblast-like synoviocytes (type B synoviocytes)

Question 10

What are macrophage-like synoviocytes?

Answer 10

• type A synoviocytes

cell function

• primarily phagocytic cells
• important for clearance of debris in joint express receptors for Fc domains of IgG (recognition of immune complexes)

Question 11

What are fibroblast-like synoviocytes?

Answer 11

type b

cell function:

• produce lubricin and hyaluronan (joint lubrication)
• produce collagen and fibronectic (extracellular matrices important for cell adherence)

Question 12

What is the target tissue for inflammation in RA?

Answer 12

• synovium
• becomes aggressive like tissue
• infiltration of inflammatory cells
• release of factors that cause destruction of cartilage and bone

Question 13

What characterises RA?

Answer 13

• inflammation and swelling of multiple joints
• synovitis
• e.g. metacarpals
• MRI: healthy joint you see nice clear black spaces in the joints of the hand, RA - bright areas, inflammation tissue

artheroscope:

• jelly like substance
• inflamed synovial tissue

histologic:

• bone and cartilage no longer one intact tissue
• expanded intima layer
• large amounts of inflammatory cells
• formation of new blood vessels

Question 14

What is pannus?

Answer 14

• inflamed synovial tissue next to cartilage/bone - pannus (latin for “cloth” or “covering”) - inflamed synovial tissue that “creeps” over the cartilage and bone tissue of the joint
• i.e. the synovial tissue ‘really’ adjacent to cartilage and bone
• differs histologically from inflamed synovial tissues away from bone/cartilage
• rich in fibroblast-like cells
• contains macrophages
• contains fewer immune cells (B, T, sometimes mast) than the “peripheral” inflamed synovial tissue
• creates hypoxic micro-environment
• cells within the pannus release factors –> destroys articular cartilage and bone

Question 15

What is inflammation of the synovium?

Answer 15

• occurs very early in the disease
• synovial lining (intima) expands –> up to 12 cells
• cell proliferation (hyperplasia)
• infiltration of inflammatory cells into the sublining (subintima): macrophages, T cells, B cells, neutrophils (less common)
• formation of new blood vessels (neovascularisation)
• ectopic lymphoid neogenesis
• deposition of fibrin in active disease (long standing or severe disease)

Question 16

Do you see neutrophils?

Answer 16

• not so common in synovium but yes in synovial fluid

Question 17

How are cells recruited into the RA synovium?

Answer 17

In RA synovium:

• cells present within the inflamed synovial tissue secrete factors:
  1) attract inflammatory cells into the joint (chemokines)
  2) retain the cells there (adhesion molecules)

Question 18

What are cell types important in the pathogenesis of RA?

Answer 18

• fibroblast
• macrophage
• T cell
• B cell

Question 19

What is the role of type A synoviocytes in RA inflammation?

Answer 19

• outnumber type B synoviocytes (fibroblast-like cells) in RA synovium
• activated phenotype:
• expression of phagocytic markers
• high expression of major histocompatibility class II molecules (required for presentation of peptide antigens to CD4+ T cells)
• significant sources of:
• pro-inflammatory cytokines e.g. TNF, IL-1, IL-6
• chemokines
• synovial macrophages may “trans-differentiate” to a bone-resorbing osteoclast?

Question 20

What is the role of fibroblast-like synoviocutes in RA?

Answer 20

• prevalent mediators of inflammation within the RA joint
• respond to inflammation by producing:
• pro-inflammatory cytokines including IL-1, TNF and IL-6
• chemokines to attract inflammatory cells
• matrix degrading enzymes such as matrix-metalloproteinases (MMPs) which lead to cartilage degradation
• factors that promote local bone destruction (TNF, RANKL)
• factors that inhibit bone formation activity (TNF, DKKs, sFRPs)

interact with mac-like synoviocytes –> production of TNF and IL-1 from these cells

interact with T cells and B cells activating those cells
cytokines like IL-6
promote endothelial cell activation through provision of VEGF, IL-6
release MMPs

Question 21

What is the role of T cells in RA?

Answer 21

• T cells are predominant lymphocyte in RA synovium
• CD4+ (helper) T cells are most prevalent
• TH17 cells: subset of CD4+ T cells that express IL-17
• recruitment and differentiation induced by IL-6
• express RANKL, the essential differentiation factor for osteoclasts (the bone resorbing cell); promotes bone erosion
• IL-17 induces expression of other pro-inflammatory cytokines and also RANKL in other cell types; promotes inflammatory response
• Treg cells (subset of T cells identified by CD25+, FoxP3+)
• normally act to suppress immune/inflammatory response are present
• in RA: cellsa re present within synovial tissues but are not functional (e.g. reduced expression of IL-10, IL-4)

Question 22

What is the roel of B cells in RA inflammation?

Answer 22

• presence and distribution of B cells is variable among RA patients
• depends on stage of disease?
• responsible for antibody production in response to T cell activation by antigen presenting cells
• local production of autoantibodies (e.g. rheumatoid factor, anti-citrulline containing peptide (anti-CCP), anti-collagen type II)
• present antigen to CD4+ T cell and secrete cytokines
• source of cytokine RANKL required for osteoclast differentiation –> contributing to bone resorption

Question 23

How do cells communicated in RA?

Answer 23

• cytokines
• cellular communication mediated by large array of soluble and membrane bound factors that can induce response in that cell, neighbouring cells, or cells that are distant to cellular source (in case of soluble only)

3 categories

1) cytokines
2) chemokines (attracts cells)
3) growth factors

• molecules belonging to all 3 categories are released in RA synovium –> contribute to RA pathogenesis

Question 24

What are three cytokines known to play a critical role in RA pathogenesis?

Answer 24

• TNF
• IL-1
• IL-6

Question 25

What is TNF?

Answer 25

- TNF belongs to a large family of cytokines that share structural features - major cytokine in RA pathogenesis - intially membrane-bound protein, subsequently cleaved to form a soluble form by TNF-alpha converting enzyme (TACE) - both membrane bound and soluble forms are active - 2 receptors for TNF: - - TNFRI: expressed constitutively - - TNFRII: expression is induced

Question 26

What are cellular sources of TNF in RA synovium?

Answer 26

- activatedd macrophages predominantly - activated fibroblast-like synoviocytes and other cell types e.g. T cells also contribute - found at elevated levels in synovial fluid, also in periphery (serum): local and possible systemic actions

Question 27

What are the roles of TNF in RA?

Answer 27

proinflammatory cytokine release - including IL-1,6, 23 and GM-CSF chemokine release - RANTES, MCP-1, IL-8, and SDF-1 - leukocyte accumulation endothelial cell activation - upregulation of E-selectin and VCAM-1 (adhesion molecules) - leukocyte accumulation ``` leukocyte accumulation - induction/maintenance of HLA class II expression ``` angiogenesis chondrocyte activation - MMP production - cartilage destruction osteoclast activation - bone resorption PGE2 production - local inflammation Hepcidin induction - acute-phase response (systemic) --> several TNF inhibitors have been developed for use in RA

Question 28

What is IL-1?

Answer 28

- also belongs to large family of proteins that share structural features - there are two isoforms of IL-1 - alpha: cytosolic form, stored in cytoplasm in some cells - beta: inducible form, secreted and then cleaved into its active form by IL-1 converting enzyme (ICE) - both act by binding a receptor complex made up of IL-1RI and IL-1RAcP - IL-1 activity is tightly regulated by endogenous inhibitors: - soluble IL-1 receptors that can act as decoy receptors - IL-1 receptor antagonist (IL-1ra) competes with IL-1 for binding to IL-1 receptor (competitive inhibitor) - it is balance between cytokine and inhibitor that determines the effect on IL-1 signalling

Question 29

What are the major cell sources of IL-1 in RA?

Answer 29

- MACROPHAGES - synovial fibroblast-like synoviocytes - endothelial cells - neutrophils - lymphocytes - found at elevated levels in RA synovial fluid and serum

Question 30

What is the role of IL-1 in RA?

Answer 30

- activates leukocytes, endothelial cells, and synovial fibroblasts - induce expression of chemokines, cytokines - induce metalloproteinase production by chondrocytes; synovial fibroblasts --> cartilage destruction - induce expression of factors required for osteoclast differentiation --> bone resorption

Question 31

What is ANAKINRA?

Answer 31

- IL-1R antagonist like molecule which was trialled in RA | - not very successful

Question 32

What is Canakinumab?

Answer 32

- monoclonal antibody against IL-1beta trialled in RA

Question 33

What is IL-6?

Answer 33

- binds either a membrane bound IL-6R or soluble IL-6R and interacts with homodimer of gp130 to elicit its effects

Question 34

What are cell sources of IL-6 in RA?

Answer 34

- macrophages - SYNOVIAL FIBROBLAST-LIKE SYNOVIOCYTES - t cells - found at high levels in RA synovial fluid and serum

Question 35

What is the role of IL-6 in RA?

Answer 35

- increases acute phase response in liver (systemic inflammation) (CRP levels?) - induces immunoglobulin production by B cells - promotes differentiation of TH-17 cells - induces cytokine production by sunocial fibroblasts and macrophages - promotes osteoclast differentiation via induction of RANKL

Question 36

What is Tocilizumab?

Answer 36

- monoclonal antibody against IL6-R used in treatment of RA

Question 37

Why are rodent models in RA important?

Answer 37

- scope for studies investigating RA pathogenesis in humans is limited - identification of cellular and moleuclar mechanisms driving initiation and progression of RA - pre-clinical studies prior to proceeding to primate models and human trials

Question 38

What is collagen-induced arthritis (CIA) model?

Answer 38

- a model of RA bone erosion - most commonly used model for screening potention therapeutic compounds - immunisation of foreign source of Type II collegen/ in Freund's complete adjuvant at base of tail - like human RA: genetics contribute to susceptibility to disease - DBA/1 mouse strain most susceptible, although protocols now available to induce CIA in C57Bl6 mice (less robust) - see swelling around ankle/forefoot/toes - histologically: proliferation of synovial tissue, inflammatory infiltrate, fibrotic tissue, destruction of bone

Question 39

How do you induce collegen-induced arthritis?

Answer 39

Day 0 - immunise with foreign source of collagen type II/Freunds complete adjuvant (FCA) at base of tail by intradermal (i.d.) injection activates T and B cells - systemic activation - collagen antibody production Day 21 - booster injection (i.d.) of foreign source of collagen type II/FCA soln at base of tail arthritis onset: - local synovitis - infiltration of synovium w/ inflammatory cells Day 30 - significant local inflammation w/i the affected joint - destruction of cartilage and bone

Question 40

What are pros of rodent CIA?

Answer 40

- symmetrical arthritis affecting knees and paws: synovial inflammation, significant bone and cartilage erosion - dependent on T and B cells, expression of certain MHC class II alleles - TNF and IL-1beta expression is elevated - inhibition of either of these cytokines reduces arhtritis onset and severity - rheumatoid factor reported to be produced

Question 41

What are cons of CIA?

Answer 41

- disease susceptibility is dependent on expression of certain classes of MHC class II molecules - -> only certain mouse strains are susceptible (DBA/1 mice most, although now protocols available to induce CIA in C57Bl6 mice, but less robust) - timing for disease onset and severity of disease can be variable --> requires larger size for experimental groups - unlike many RA patients, antibodies to collagen are produced

Question 42

What are mouse genetic models of RA?

Answer 42

- genetic modification of the expression or activity of factors known to be involved in RA - -> several mouse genetic models of arthritis hTNF.Tg mouse - mouse that has been genetically engineered to over-express human TNF - develop arthritis spontaneously, affecting knees and paws - synovial hyperplasia, inflammatory cell infiltrates from 3-4 weeks of age - full blown arthritis by ~10 weeks of age - inflammation, pannus formation, cartilage destruction and focal bone erosion and systemic bone loss - blocking TNF will reduce/block disease severity - is dependent on the IL-1/IL-1 receptor expression and signalling - used extensively to demonstrate mechanisms mediating bone and cartilage destruction

Question 43

What are pros for hTNF.Tg mouse?

Answer 43

- reliable, robust arthritis - synovial inflammation, cartilage and bone destruction, systemic bone loss - arthritis is chronic - has been useful to assess effect of TNF inhibition in treatment of arthritis

Question 44

What are cons for hTNF.Tg mouse?

Answer 44

- even though IL-1 is essential to arthritis, this is a TNF driven model --> role of other cytokines may not be as important - arthritis not dependent on T or B cells

Question 45

Are rodent models of RA good or bad?

Answer 45

- invaluable for the pre-clinical identification and validation of therapeutic targets for the treatment of RA - cytokine targets (e.g. TNF, IL-1, IL-6, IL-17, RANKL) - mechanism for bone erosion, systemic bone loss in RA + protential treatments - process of inflammation and bone loss is similar with TNF and IL-1 and RANKL playing dominant role one potential limitation: - most animal models are dependent on IL-1 expression - TNF blockade in human RA is very powerful BUT current treatments targeting IL-1 less effective - efficacy issues of targeting strategies?