1. The Biochemistry of Rheumatoid Arthritis
PHM 142
Yunfan Zhang, Jingyi Qiu, Jeffrey Lai, Yifan Zhou
PHM Fall 2016
Coordinator: Dr. Jeffrey Henderson
Instructor: Dr. David Hampson

2. Clinical representation of rheumatoid arthritis
Incidence of RA is 0.05% per year and prevalence is 1% of the adult population
Woman 3x > man in frequency
Onset: 40-50yrs
Systemic autoimmune disease
Number of actively inflamed/swollen joints
Estrogen effect on bones development
Onset can also be any age
stiffness
Ref:

3. Diagnosis
Swelling and stiffness of small joints -- refer to specialist within 3 months
Raynaud’s phenomenon -- burning feel when exposed to cold environment (possible)
Swan-neck deformity of fingers, fixation of joints
International used classification: 1987 ACR (American Rheumatism Association)
Morning stiffness
Arthritis (swelling)
Symmetric arthritis
Arthritis of Hand joints (wrists)
Rheumatoid nodules
Rheumatoid factor
Radiographic changes
If 4/7 are met, the diagnosis can be made
Serum rheumatoid factor test (ELISA)
Take a venous blood sample
Not usually seen in normal individuals
AutoAntibody against IgG
The more positive, the worse the joint destruction and systemic involvement
False positive can occur in 5% healthy individuals

4. Rheumatoid Factors Rheumatoid Factors (RFs)
RFs are autoimmune antibodies produced by the activated B cells (plasma cells)
They are directed against the Fc domain on the immunoglobulins G (IgG)
They are present in 75% of rheumatoid arthritis patients
Anti-Citrullinated Peptide Antibody (ACPA)
ACPAs are a special type of RF that recognize citrullinated proteins
They are more specific in diagnosing rheumatoid arthritis

5. Pathogenesis Overview
In normal joint,
Synovium → synovial membrane and loose connective tissue
The lining cells are either Type A (macrophages- like synoviocytes) or Type B (fibroblast-like synoviocytes)
In early rheumatoid arthritis,
Thickened synovial membrane
Formation of blood vessel network in synovium
T cells and B cells into synovial membrane and present in synovial fluid with neutrophils
Synovial membrane invades cartilage
In established rheumatoid arthritis,
Synovial membrane becomes the pannus (an inflammatory tissue)
Pannus continues invading and destroying cartilage and bone
Ref: Ernest H. et al (2001)

6. Rheumatoid Arthritis Pathways
CD4+ T cells activate monocytes, macrophages, synovial fibroblasts, chondrocytes, and osteoclasts
Secretion of interleukin-1(IL-1), and interleukin-6 (IL-6), Tumor Necrosis Factor (TNF-α),and matrix metalloproteinases (MMPs)
Stimulation of angiogenesis, forming blood vessels in synovium
Synovium endothelial cells express adhesion molecules, recruiting inflammatory cells
Activated CD4+ T cells induce B cells to produce immunoglobulin, including rheumatoid factor which involves in the immune complexes formation.
CD4+ T cells express osteoprotegerin ligands to stimulate osteoclastogenesis
Synovial inflammation caused by TNF-α, IL-1 and IL-6. Joint damage caused by MMPs and osteoclastogenesis.
Ref: Ernest H. et al (2001)

7. Key Mediators Interleukin-1
Produced mostly by monocytes and macrophages, but also by endothelial cell, B cell, and activated T cells
In normal conditions, two mechanisms to regulate interleukin-1 activities:
a. Compete for Type I and Type II receptors
b. Natural occurring antagonist for receptors
Activate leukocytes, endothelial cells and synovial fibroblasts.
Induce matrix-enzyme production by chondrocytes
Interleukin-6
Produced by T cells, monocytes, macrophages, and synovial fibroblasts
Induce the B cell differentiation and maturation
Activate T cells, leukocytes and osteoclasts
Involve in acute phase response
Stimulate the proliferation of synovial fibroblasts
Tumor Necrosis Factor (TNF-α)
Promote inflammation by inducing inflammatory cytokines and fibroblast
Suppress functions of regulatory T-cell
Activate resorption of bone and cartilage by osteoclasts
TNF-α is the main cytokine involved in the inflammatory response of RA
Cartilage and bone destruction is mostly directed by IL1
This cytokine, produced by a variety of cells, mainly activated macrophages and FLS, has both pro- and anti-inflammatory properties. High levels of IL6 have been detected both in the circulation and SF of RA patients
Eck, M.J., Sprang, S.R. (1989)
Protein Data Bank (2008)
Somers, W., Stahl, M., Seehra, J.S. (1997)

8. Inflammatory Mediators
Activated T cells and macrophages produce important inflammatory mediators
Tumour necrosis factor alpha (TNF-α)
Interleukin-1 (IL-1)
IL-6
IL-17
Highlights of IL-6, IL-1 and TNF-α functions
IL-6 and TNF-α stimulate macrophage-like synoviocytes to produce metalloproteinases (MMPs) which degrades proteins within synovium → destruction
TNF-α binds to TNF-α receptor on the osteoclast precursor cells and stimulate their differentiation.
IL-1 induces accumulation of leukocytes in the synovium, causing inflammation and increases collagenases in extracellular space, damaging the cartilage
IL-1, 6, 17 and TNF-α induce osteoclast precursor cells to differentiate into osteoclasts

9. Osteoclastogenesis mechanism
Osteoclast is bone resorptive cell
Three main regulators
RANK, RANKL, OPG
RANK is expressed on osteoclast precursor and mature osteoclast
RANKL binds to RANK leading to osteoclast maturation and activation
Cytokines e.g. IL1 and TNFα induces the expression of RANKL
RANKL binds to RANK leading to osteoclasts maturation and activation
OPG (osteoprotegerin) is competitive inhibitor of RANKL
Osteoclasts are multinucleated bone resorptive cells deriving from the hematopoietic progenitors of the monocytemacrophage lineage.
osteoclastic cells appear on the bone surface and resorb the mineral components of bone by an acid extracellular mechanism
RANK, RANKL and OPD are three main factors that modulates osteoclasts
under control by cytokines, humoral factors and hormones e.g. IL1, IL6
OPG and RANKL are synthesized by stromal cells/osteoblasts
RANK is on the cell surface of mature osteoclasts and osteoclastic precursors
RANK is expressed on osteoclast precursors and mature osteoclast
RANKL is the ligand of RANK
Binding of RANKL to RANK stimulates osteoclastic precursors to differentiate into mature osteoclasts
Binding also activates of mature osteoclast
OPG is a competitive inhibitor of RANKL

10. Chronic Inflammation and Citrullinated Proteins
Initially
Initial inflammatory response of the body brings in inflammatory cells (granulocytes, monocytes, T cells, B cells etc) into the synovial joint.
Most of these inflammatory cells have PAD (protein arginine deaminases) enzymes that converts the arginine on a protein into another a citrulline.
When these cells die, and there is insufficient phagocytosis removal, they release citrullinated proteins and activated-PADs
Citrullinated Proteins and MHC
Specific MHC (major histocompatibility complex) such as HLA-DR4 recognizes the citrullinated proteins and present them
This elicits a citrulline-specific T cell response, which in turn activates B cells
Activated B cells differentiate into plasma cells, producing autoantibodies, anti-citrullinated protein antibodies (ACPAs), that can form immune complexes against the joint, causing chronic inflammation

11. Inflammation potentiates osteoclastogenesis
TNF∂, IL1, IL6 are inflammatory cytokines
RANKL–RANK signalling pathway is not the only pathway leading to osteoclastogenesis
Ref: Steeve K.T. (2004)

12. DKK1 expression modulates bone formation and resorption
Bone formation is uncoupled from bone resorption in RA
TNFα is an inducer of DKK1
DKK1 is the major contributor to bone loss in inflammatory tissue, by impairing bone formation
DKK1 interferes Wnt signalling
Wnt induces OPG expression
Bone formation and resorption usually happen simultaneously, but TNF∂ induces DKK1 protein
DKK1 directly impairs bone formation and indirectly inhibits OPG expression
Figure A: normal tissue, bone formation is balanced with bone resorption
Figure B: arthritic tissue, bone resorption is uncontrolled, due to a change DKK1 and wnt signalling
Ref: Diarra D. (2007)

13. In summary, Diagnosis: 1987 ACR is the most commonly used criteria
Diagnostic test: ELISA for serum RF or ACPA: positive = systemic involvement
Rheumatoid arthritis is a systemic autoimmune disease that is characterized by chronic inflammation of the synovial joint and progressive joint destruction.
Activated T cells stimulate inflammatory cells such as macrophages to produce IL-1, IL-6, and TNF-α
They lead to increased osteoclasts and MMP production, causing joint inflammation.
RFs are autoantibodies targeting IgG; ACPAs are a specific type that target citrullinated proteins.
ACPAs are more specific in diagnosing rheumatoid arthritis.

14. References
Silman AJ. The 1987 Revised American Rheumatism Association Criteria For Rheumatoid Arthritis. Rheumatology. 1988;27(5):341–3.
Aletaha D , Neogi T , Silman AJ , et al: 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62(9):
Choy, Ernest H. S., and Gabriel S. Panayi. "Cytokine Pathways and Joint Inflammation in Rheumatoid Arthritis." N Engl J Med (2001): Massachusetts Medical Society.
Alam, Javaid, et al. “Rheumatoid arthritis: Recent advances on its etiology, role of cytokines and pharmacotherapy.” Biomedicine & Pharmacotherapy, vol. 92, 2017, pp. 615–633., doi: /j.biopha
Steeve, Kwan Tat, et al. “IL-6, RANKL, TNF-Alpha/IL-1: interrelations in bone resorption pathophysiology.” Cytokine & Growth Factor Reviews, vol. 15, no. 1, 2004, pp. 49–60., doi: /j.cytogfr
Diarra, Danielle, et al. “Dickkopf-1 is a master regulator of joint remodeling.” Nature Medicine, vol. 13, no. 2, 2007, pp. 156–163., doi: /nm1538.
Karsdal, Morten A., et al. "Biochemical markers of ongoing joint damage in rheumatoid arthritis-current and future applications, limitations and opportunities." Arthritis research & therapy 13.2 (2011): 215.
Nijenhuis, Suzanne, et al. "Autoantibodies to citrullinated proteins in rheumatoid arthritis: clinical performance and biochemical aspects of an RA- specific marker." Clinica Chimica Acta (2004):
Stanaszek, Walter F., and Bruce C. Carlstedt. "Rheumatoid Arthritis: Pathophysiology." Journal of Pharmacy Practice 12.4 (1999):
De Clerck, L. S. "B lymphocytes and humoral immune responses in rheumatoid arthritis." Clinical rheumatology 14.2 (1995):
Fournier, Catherine. "Where do T cells stand in rheumatoid arthritis?." Joint Bone Spine 72.6 (2005):
Aletaha, Daniel, et al. "2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative." Arthritis & Rheumatology 62.9 (2010):
Cantagrel, Alain, et al. "Le TNF-α, l’interleukine-6 et l’interleukine-1: trois cytokines centrales de la polyarthrite rhumatoïde." Revue du Rhumatisme Monographies 84.4 (2017):

15. Karsdal, Morten A., et al. "Biochemical markers of ongoing joint damage in rheumatoid arthritis-current and future applications, limitations and opportunities." Arthritis research & therapy 13.2 (2011): 215.
Nijenhuis, Suzanne, et al. "Autoantibodies to citrullinated proteins in rheumatoid arthritis: clinical performance and biochemical aspects of an RA-specific marker." Clinica Chimica Acta (2004):
Stanaszek, Walter F., and Bruce C. Carlstedt. "Rheumatoid Arthritis: Pathophysiology." Journal of Pharmacy Practice 12.4 (1999):
De Clerck, L. S. "B lymphocytes and humoral immune responses in rheumatoid arthritis." Clinical rheumatology 14.2 (1995):
Fournier, Catherine. "Where do T cells stand in rheumatoid arthritis?." Joint Bone Spine 72.6 (2005):
Aletaha, Daniel, et al. "2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative." Arthritis & Rheumatology 62.9 (2010):
Cantagrel, Alain, et al. "Le TNF-α, l’interleukine-6 et l’interleukine-1: trois cytokines centrales de la polyarthrite rhumatoïde." Revue du Rhumatisme Monographies 84.4 (2017):