Rheumatoid Arthritis

by Alyssa Bennett and Jaclyn Boulais

This website was created for an assignment in the undergraduate course BI 348, Immunobiology, at Skidmore College.

Overview
Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease in which cells of the immune system attack healthy tissues. It is characterized by inflammation of the lining of joints, causing bone and cartilage damage, pain, loss of function, and even disability, although the cause of RA is still unknown. RA affects approximately 2.1 million people or 1% of the population in the United States. About 70% of the people affected are women between the ages of 30-60 years old although anyone can be affected regardless of age or sex. Women are more likely to get rheumatoid arthritis but men are generally more severely affected if they get it (Arthritis Foundation, 2004).
Symptoms
· Effect on Joints
The most common symptom of RA is inflammation and pain in the joints (Ravo, 2002). In most cases, RA initially affects the freely movable joints of the hands and feet and then moves to other joints in the body (Coleman, 1989). The effect on joints is usually symmetrical, causing pain to occur in the same spots on the both the left and right side of the body (Arthritis Foundation, 2004). The ends of the bone of freely movable joints are covered with articular cartilage and held together by a capsule of fibrous tissue called a joint capsule. The joint capsule consists of an outer layer of ligaments and an inner layer of synovial membrane that secretes synovial fluid to act as a joint lubricant. Cells of the immune system can activate a series of steps that initiate and amplify inflammation, causing synovial membrane damage and cell lysis. An acute episode can cause proliferating cells of the synovium to grow in the joint cavity and form what is known as pannus. Pannus is composed of vascularized

fibrous scar tissue that invades the joint cavity and spreads inflammation to articular cartilage, leading to joint destruction and other complications (Coleman, 1989). Because of synovial inflammation and the formation of pannus, people with RA often experience stiffness, which is most evident in the morning or after sitting for long periods of time. Generally, the longer morning stiffness lasts, the more active the disease.
· Advanced Effects
Some advanced symptoms seen in people with RA include damage to cartilage, tendons, ligaments, and bone which cause deformity and instability of the joints. This damage can limit range of motion making everyday tasks such as buttoning a shirt, holding utensils, and brushing hair increasingly difficult (Arthritis Foundation, 2004).
· Other Common Symptoms
Other symptoms include fatigue, weakness, muscle pain, loss of appetite, depression, weight loss, anemia, cold, sweaty hands and feet, and flu-like symptoms including low-grade fever. About one fifth of people with RA experience rheumatoid nodules, which are lumps of tissue under the skin typically found on the elbows (Arthritis Foundation, 2004). Other organs can be affected including the lungs, eyes, skin, and nervous system (Coleman, 1989). The involvement of glands around the mouth and eyes cause decreased production of tears and saliva (Arthritis Foundation, 2004). Rheumatoid arthritis can be characterized by acute phases followed by periods of remission (Coleman, 1989). For example, pregnant women often go into remission but their symptoms intensify after the baby is born. Skin ulcers and a general decline in health may also be evident. Severe cases of RA make people more susceptible to infection which can ultimately lead to death (Arthritis Foundation, 2004).
Diagnostic Tests
Diagnostic tests unveil some of the underlying effects of the disease.
· Blood Cell Counts
People with RA usually have a low red blood cell count suggesting anemia which contributes to feelings of fatigue. White blood cell counts are usually high suggesting infections are present in the body. This increase in white blood cells is due to the body launching an autoimmune response against joint and tissue cells.
· Erythrocyte Sedimentation Rate (ESR) Test
Another common test is an erythrocyte sedimentation rate (ESR) test. This measures how quickly red blood cells fall to the bottom of a test tube. The faster the cells fall, the more inflammation is present in the body. About 60% of people with RA have an elevated ESR.
· C-reactive protein (CRP) Levels
Another lab test measures the amount of C-reactive protein (CRP) in the body, which is elevated when inflammation is present. The higher the level of CRP, the greater the activity of the disease.
· Rheumatoid Factor (RF) Levels
Rheumatoid factor (RF) is an autoantibody that regulates normal antibodies and its level can be measured to determine the severity of RA. Seventy to eighty percent of people with RF also have rheumatoid arthritis. However, not all people with RA have high levels of RFs and not all people with high levels of RF have RA. People with RA who do not have RF in their blood are called "seronegative," while those with RF in their blood are called "seropositive." In addition, an RF test may be negative if performed too early in the course of the disease, making false positives a possibility (Arthritis Foundation, 2004).
The effects of the rheumatoid arthritis are variable in different people and can be controlled by treatment. However, rheumatoid arthritis can lead to death due to infection or complications of therapy (Coleman, 1989).

Effect on Immune System at Cellular Level

· Immune System Overview
Although the specific cause of rheumatoid arthritis (RA) is unknown, the body's immune system is an important factor in the disease. RA is an autoimmune disease in which the body produces an abnormal immune system response. In a healthy individual, the immune system produces antibodies which kill foreign substances known as pathogens. In a person with RA, antibodies mistake healthy tissue for a pathogen and attack it (Arthritis Foundation, 2004).
· Rheumatoid Factor (RF)
Rheumatoid factor (RF) is an anti-?-globulin autoantibody whose activity directly relates to RA in some people (Coleman, 1989). Rheumatoid factor regulates normal antibodies produced by the immune system and is effective when present in small amounts. However, the immune system functions abnormally in people with high levels of RF. Typically the higher level of RF in the body, the greater the severity of the disease activity. Although a rheumatoid factor test can be used as a method of diagnosis, it is not completely accurate (see Diagnostic Tests) (Arthritis Foundation, 2004).
· Immune Complex IgM-IgG
A common rheumatoid factor is IgM antibody which reacts with IgG to form IgM-IgG complexes which are found in the joints (Ravo, 2002). Such immune complexes become wedged in small capillary beds and accumulate in the joints of the skeletal system of people with RA. These immune complexes cause an inflammatory response in which synovial (lubricating fluid secreted by joints) membrane damage and cell lysis occurs (Coleman, 1989).
Studies show that some infections may trigger a synovial B lymphocyte to produce an abnormal IgG antibody. The immune system may respond to the Fc region of the IgG by producing RFs which would cause immune complexes (IgM-IgG) to form in the synovial fluid (Coleman, 1989). This explains the increase in RF commonly associated with people with RA.
· Complement Factors C3a and C5a
The complement factors C3a and C5a are also found in the inflamed tissues of people with RA. They cause monocytes and mast cells to release histamine in localized regions which results in the common symptoms of joint swelling, redness, and pain. The complement fragments also cause an influx of phagocytes to the region. The phagocytes release lysozymal enzymes that hydrolyze cell walls which elevates the inflammatory and proliferative responses of the synovium. At this point in the disease, T and B cells can be detected in the synovium and their interaction may allow the immune system to continually produce immunoglobulins and extend the cycle of the immune complex (Coleman, 1989).
· Cytokines TNF-a and Interleukin (IL)
Proteins called cytokines also promote the inflammatory response by activating the inflammatory cells of the synovium. Tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), IL-1, and IL-10 have been shown to play important roles in joint inflammation (Hata et al, 2004) (see Related Research). Studies of cytokines such as (TNF-a) and interleukin-1 (IL-1) have led to the potential development of biological agents that target the cytokines to reduce the inflammation and damage of healthy joints (Arthritis Foundation, 2004).
Treatment
· Overview
Early diagnosis and close interaction with a physician are essential in treating rheumatoid arthritis. The primary doctor for RA patients is a rheumatologist, a physician who has special training in arthritis and other conditions involving diseases of the bone, muscles, and joints. Treatment has become more aggressive early on to prevent severe deformity and joint erosion. Treatment methods vary depending on the severity of the disease and on the individual. However, there is no current cure for RA. The most current treatments focus on pain relief, reducing inflammation, slowing joint damage, and improving well-being. (Arthritis Foundation, 2004).
· Medications
Proper medication is essential in controlling RA. The main categories of drugs used to treat RA are NSAIDs, analgesic drugs, glucocorticoids and prednisone, DMARDs, biologic response modifiers, and protein-A immunoadsorption therapy. NSAIDs (nonsteroidal anti-inflammatory drugs) are used to reduce inflammation and relieve pain. Some common examples of NSAIDs are aspirin and ibuprofen. Analgesic drugs such as acetaminophen and morphine relieve pain but do not have any effect on inflammation. Glucocorticoids and prednisone are used to prevent joint damage. DMARDs (disease modifying antirheumatic drugs) are used in combination with NSAIDs and/or prednisone to slow joint destruction caused over time (Arthritis Foundation, 2004). An example of a DMARD is slow acting gold salt injections, which reduce the release of lysosomal enzymes in the joint (Coleman 1989). Other DMARDs include methotrexate, penicillamine, hydroxychloroquine, and sulfasalazine. Biologic response modifiers are drugs that directly modify the immune system by inhibiting cytokines which contribute to inflammation. Some examples of this are etanercept, infliximab, and anakinra. Protein-A immunoadsorption therapy is a process that filters blood to remove antibodies and immune complexes that promote inflammation (Arthritis Foundation, 2004). In some severe cases immunosuppressive agents are used but this is avoided because they generally depress the entire immune system, enhancing the risk of infection (Coleman, 1989).
· Surgery
Another treatment option for people with RA is surgery, which can improve a patient's quality of life. There are four main types of surgical options used to treat RA. Synovectomy is used when one or two joints are affected more severely than others. It is a procedure to remove the diseased synovium or lining of the joint to reduce swelling and pain. It also slows and prevents further joint damage. Arthroscopic surgery is a procedure in which a small tube with a light is connected to a camera and inserted through a small incision to allow the surgeon to see the damage in the joint. Tissue samples can be taken, loose cartilage removed, tears repaired, rough surfaces smoothed, and diseased synovial tissue removed. This surgery is mostly performed on the knee and shoulder. Joint replacement surgery or arthroplasty is the reconstruction or replacement of a joint. This involves removing the joint, resurfacing or relining the ends of bones, and inserting a manmade joint. This is usually used in people over 50 years old who have severe disease progression. Arthrodesis, or fusion, is the process of fusing two bones together. This procedure limits movement but decreases pain and increases stability in certain joints such as ankles, wrists, fingers, toes, and spine (Arthritis Foundation, 2004).
· Other Treatment Options
Patients with RA often use alternative therapy and complementary therapies. Massage therapy can ease pain and stiffness, decrease stress hormones and depression, and increase the production of natural pain killing endorphins by the body. Acupuncture and acupressure are suggested to release endorphins and have may have anti-inflammatory properties. Diet can become an important factor for people with RA. Some people with arthritis have certain food sensitivities that can trigger symptoms or cause them to worsen. Also, a diet high in saturated fat can increase the inflammatory response which contributes to joint and tissue inflammation. A proper balance of diet, exercise, and rest can be very beneficial for people affected by RA. Other alternative treatments are taking herbs and supplements and participating in prayer and spirituality.
Related Research
· Distinct contribution of IL-6, TNF-a, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice. Hata, Sakaguchi, Yoshitomi, et al.
Researchers used SKG mice as a new model of rheumatoid arthritis in humans. SKG mice spontaneously develop T cell-mediated chronic autoimmune arthritis due to a genetic mutation that affects the signal transduction of T cells. The mutation affects both positive and negative selection of T cells in the thymus, thereby causing the production of arthritogenic T cells. Studies show that CD4+ T cells can transfer arthritis in SKG mice to T cell-deficient BALB/c nude or T cell/B cell-deficient SCID mice. This indicates that RA is a T cell-mediated autoimmune disease which may or may not be dependent on the presence of rheumatoid factors (RF). Rheumatoid factors are autoantibodies that regulate normal antibodies and are found as complexes in the joints of many RA patients.
In this research article, the authors studied how synoviocytes (specialized cells of the synovium) stimulated by arthritogenic CD4+ T cells affect arthritis in SKG mice as well as how cytokines produced by synoviocytes or CD4+ T cells contribute to the development and progression of the disease.
The researchers examined the histology of swollen joints in SKG mice and found a proliferation of synovial cells which caused mononuclear cells and neutrophils to travel to the site. The immunohistochemistry of the SKG synovial cells revealed many CD4+ T cells that stimulated B cells to aggregate. There was also an abundance of granulocytes and macrophages whose distribution corresponded to that of class II MHC-expressing cells.

Figure 2B: Expression of cytokines at mRNA and protein levels. (A) Quantitative RT-PCR for indicated genes was performed with ankle joints from 8- or 24-week-old SKG or BALB/c mice. Bars show the means ± SD. See Methods for the definition of units. (B) Concentrations of indicated cytokines, assessed by ELISA, in the joint fluid of 32-week-old SKG and BALB/c mice (n = 10 each). Bars show the means ± SD. (C-F) Immunohistochemical staining of the synovial tissue of a finger joint of a 4-month-old SKG mouse for IL-1ß (C), TNF- (D), or IL-6 (E), with staining control (F) (original magnification, x40). Insets show higher magnifications of a part of synovium.

The cytokines IL-1ß, IL-6, and TNF-a were found in the inflamed joints. There was a high level expression of IL-1ß and IL-6 mRNA in the swollen joints, while insignificant amounts of the cytokines were expressed in mice without joint swelling. All three cytokines were present in joint fluid removed from swollen joints. The results indicate that inflamed synovial tissue of SKG mice actively produces IL-1ß, IL-6, and TNF-a. It seems that the same cells produce IL-1ß and TNF-a while different cells produce IL-6. Further study revealed that IL-6 is probably produced by CD4+ T cells and subsynovial stromal cells such as macrophages and dendritic cells, while IL-1 and TNF-a are produced by superficial lining cells facing the joint cavity.
The article also studied the roles of proinflammatory cytokines such as IL-1a, IL-1ß, TNF-a, and IL-6 in the development of SKG arthritis. Researchers prepared three groups of female SKG mice with varying expression of the cytokine genes IL-6, IL-1, TNF-a, IFN-?, IL-4, and IL-10. The groups were homozygous cytokine gene-deficient (-/-), heterozygous cytokine gene-deficient (+/-), and cytokine gene-intact (+/+). As expected, the majority of cytokine gene-intact (+/+) SKG mice showed significant joint swelling, severe synovitis, and cartilage and bone destruction. IL-6(-/-) SKG mice did not exhibit these effects. Some IL-1a/ß(-/-) began to develop arthritis but the severity was low, the swelling was minimal, and the destruction of cartilage and bone was less prominent in comparison to the cytokine-intact (+/+) SKG mice. The IL-1a/ß(-/-) also had a lower incidence of arthritis than the (+/+) group and their results were similar to that of TNF-a(-/-) SKG mice. IL-10(-/-) SKG mice exhibited a much higher incidence of disease than either its (+/-) or (+/+) groups which included severe synovitis and joint destruction. Therefore, IL-10 suppresses the development of SKG arthritis. All of the SKG mice with heterozygous deficiency (-/-) had intermediate results in comparison to homozygous deficient (+/-) and cytokine-intact (+/+) mice. Therefore, the amount or dose of cytokine affects the severity of the disease.

Figure 3A: Development of arthritis in cytokine-deficient SKG mice. (A) Incidence and severity of arthritis in female SKG mice homozygously (-/-) (filled circles) or heterozygously (+/-) (open squares) deficient in indicated cytokines or cytokine-intact (+/+) (open triangles). Vertical bars represent the means ± SD of the whole group of mice. Arthritis scores are significantly different (P < 0.01) between IL-6+/+ and IL-6+/- mice at 19-32 weeks; between IL-6+/- and IL-6-/- mice at 16-32 weeks; between IL-1+/+ and IL-1+/- mice at 17-32 weeks; between IL-1-/- and IL-1+/- mice at 19-32 weeks; between TNF- +/+ and TNF- +/- mice at 15-32 weeks; between TNF- -/- and TNF- +/- mice at 21-32 weeks; between IL-10+/+ and IL-10+/- mice at 18-24 weeks; and between IL-10-/- and IL-10+/- mice at 12-24 weeks. (B-D) Histology of finger joints of a 32-week-old wild-type (B) or IL-6-deficient (C) or a 24-week-old IL-10-deficient SKG mouse (D) (original magnification, x10).

Researchers also discovered a high level of IgM-type rheumatoid factor in the groups of SKG mice even when there was no joint inflammation. This proves that IgM rheumatoid factor development is independent of joint inflammation.
The major findings of the article show that a deficiency in either IL-1, TNF-a, or IL-6 cytokines and an increase in IL-10 can inhibit the development and progression of SKG arthritis whether or not rheumatoid factor develops. Nonetheless, immunologists are still uncertain about how these cytokines are controlled at the molecular and cellular levels. The research also demonstrated that rheumatoid factor is independent of joint inflammation in SKG mice. The successful use of SKG mice will be important to further study the role of cytokines and arthritogenic T cells as well as formulate effective treatments of RA.

· Apolipoprotein A-I infiltration in rheumatoid arthritis synovial tissue: a control mechanism of cytokine production? Bresnihan, et al.
Researchers tested apolipoprotein A-I (apoA-I) as a possible control mechanism for the production of inflammation inducing cytokines. Cytokines TNF-a (tumor necrosis factor alpha) and interleukin-1ß (IL-1b) are produced by monocytes; their production is induced by direct contact with stimulated T cells. The first step in RA is believed to be the proliferation of fibroblast-like synoviocytes; the synoviocytes release chemokines that recruit inflammatory cells such as monocytes and lymphocytes. The first cells thought to enter the synovial tissue are T cells, suggesting that they have an important role in the initiation of the disease. T cells directly interact with monocyte-macrophages which causes the abundant and unregulated production of TNF-a and IL-1b. ApoA-I, which is a 'negative acute-phase protein' and is the principal protein in high-density lipoproteins (HDLs), has recently been found to be a specific inhibitor of contact-mediated activation of monocytes. It can inhibit the production of TNF-a and IL-1b in monocytes activated by direct contact with stimulated T cells. In patients with RA the levels of circulating apoA-I and HDL cholesterol are lower than healthy patients while apoA-I levels are increased in the synovial fluid. This article investigated the presence and location of apoA-I in patients with RA.
Synovial biopsies were taken from the knee joints of RA patients following arthroscopy and normal synovium was taken from a non-RA patient. The synovial sections were cut and mounted on slides and stored at -80°C and a three-stage immunoperoxidase technique was used. The samples were thawed to room temp and incubated in a primary antibody and then a secondary antibody for cell specific visibility.
Six of the patients were receiving nonsteroidal anti-inflammatory drugs (NSAIDs) at the time of the biopsy, two were receiving methotrexate, a disease-modifying anti-rheumatic drug (DMARD), and two were receiving prednisolone. Two patients also had what is called quiescent RA meaning they currently had no swollen joints but both were taking methotrexate. One patient was unaffected by arthritis. All of the synovial tissue sections from the 8 patients with RA showed prominent blood vessels and perivascular cellular infiltration. ApoA-I specific staining was present in most blood vessels in all of the samples of RA patients. The blood vessels were surrounded with a localized region of intense staining that was an indication of apoA-I located in the perivascular cell infiltrate. No staining was present in the control tissue. There was only faint vascular and perivascular apoA-I staining in the patients in remission even though the blood vessels were very evident. There was no CRP or A-SAA (another inflammation indicator), which are 'positive acute-phase proteins,' in the perivascular region even though the CRP serum levels were elevated at the time of the biopsy. Faint CRP staining of endothelial cells was observed.

Figure 1: Apolipoprotein A-I (apoA-I) is localized in the perivascular region of the inflamed synovium

The main discovery of this research was that apoA-I infiltrated into inflamed synovial tissue and was retained in the perivascular regions, which is where T cells and macrophages accumulate. The location of CRP and A-SAA were different from apoA-I in that the CRP was limited to vascular endothelium and A-SAA was found in lining layer cells. The results show that apoA-I infiltrated into the perivascular regions of the synovium where CRP and A-SAA, which can dissociate apoA-I from HDLs, are absent. This location of the apoA-I suggests that it may play an inhibitory role in areas where T cells are in close contact with monocyte-macrophages. Since A-SAA is absent in these areas it cannot restrict the inhibitory action of apoA-I although it would be expected to localize in the same area. Since the apoA-I is basically absent in the synovial tissue of inactive RA subjects, its presence in actively inflamed tissue suggests that the way it infiltrates during flare-ups may be a mechanism that inhibits the proinflammatory cytokines and limits disease reoccurrence. The way apoA-I leaves when the disease calms may be an indication of why RA tends to follow phases of remission and reoccurrence. When RA is the joint damaging phase, the inhibitory effects of apoA-I may not be strong enough.
The article summarizes that apoA-I that binds to the surface of stimulated T cells and is retained in the perivascular regions may limit contact-mediated cytokine production in monocyte-macrophages and also inhibit certain disease pathways that are critical for disease progression. The changes in apoA-I infiltration may explain the relapsing-remitting phases of RA. This finding that apoA-I can infiltrate the inflamed tissue may have implications for new treatments in chronic inflammatory diseases.
Future Directions
Extensive research will continue to develop more effective ways of treating rheumatoid arthritis. Over the past decade there has been a major shift in the way that RA has been approached. Previously, there was an initial stage of conservative treatment in which NSAIDs and DMARDs were not used until there was clear evidence of joint and tissue erosion. This treatment approach has been replaced by the early start of DMARDs and combination DMARD therapy in patients with the potential for progressive disease. Another major step in treatment has been implementing an organized system for measuring the clinical outcomes and disease activity to compare different drugs used to treat RA (Lee and Weinblatt, 2001).
Studies are now being directed at improving methods for early diagnosis and improving understanding of the molecular factors of RA. This information could improve immunotherapy techniques, biotechnology, and rational drug design to develop specific pharmacotherapy for the treatment of RA (Coleman, 1989 and Lee and Weinblatt, 2001).
Many tests are in progress in different facets of RA that will hopefully result in better treatment options. Significant progress has been made in finding ways to locate and destroy only the harmful immune cells without affecting other normal functioning parts of the immune system. This reasoning has the potential to turn off the disease process, which would lead to remission or even a cure. Drugs directed at B cells or T cell-B cell interactions are being tested in clinical trials with the best studied B cell specific agent, rituximab, directed at B cells with a CD20 marker. There are also studies in effect trying to better understand how to switch on regulatory cells that send self-attacking T cells into anergy which will guide drug design to enhance the body's natural self-protective mechanisms (Arthritis Foundation, 2004).
Another method being tested involves the innate immune system. There is a family of proteins called toll-like receptors that are able to recognize markers on foreign organisms and signal other immune cells to attack and kill the foreign invader. Scientists have found that this recognition pathway seems to be involved in RA; certain toll-like receptors appear to help and perpetuate the chronic inflammation. Drugs are being developed to block this innate response pathway or by redirecting the faulty immune response in RA.
Inherited deficiencies in certain complement proteins, which are supposed to work with antibodies to destroy foreign invaders, are associated with a risk of RA. Therapies that block the complement pathway are a new way to inhibit inflammation and tissue damage. Research has also identified certain cytokines that promote inflammation such as tumor necrosis factor alpha (TNF-a) and interleukin-1 (IL-1). These discoveries led to the development of drugs that target TNF and IL-1. Another cytokine called RANKL has been found to activate osteoclasts that play a role in bone destruction in RA. Finding a distinct pathway that contributes to bone loss allows more effective therapeutic approaches for arthritis that target osteoclasts and protect RA patients from bone destruction (Arthritis Foundation, 2004).
The overgrowth of the joint lining seen in RA has been compared to tumor-like growth and has been shown to be fueled by new blood vessels within the joint; this is called angiogenesis. Researchers are working to show the benefit of cancer drugs called anti-angiogenic drugs that "starve" tumors. A new drug called vitaxin has been found to selectively reduce blood vessel formation and limit the growth of inflamed joint tissues (Arthritis Foundation, 2004).
The direction of research of RA has shifted to an attempt to understand more about the underlying causes of the disease such as signaling pathways, specific proteins involved in regulation, the complement pathway, and target cells. A clearer understanding of the specifics of the disease allows for the development of more effective drugs that can be individualized to different patients. These drugs have the potential to make patients go into remission or even to create a cure.

References

Apolipoprotein A-I infiltration in rheumatoid arthritis synovial tissue: a control mechanism of cytokine production? Bresnihan, Gogarty, FitzGerald, Dayer, Burger. Arthritis Res. Ther. 6:563-566 (2004).

Arthritis Foundation, 2004. Rheumatoid Arthritis [Online]. Available: http://www.arthritis.org/ conditions/DiseaseCenter/RA/default.asp [2004, October 10].

Distinct contribution of IL-6, TNF-a, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice. Hata, Sakaguchi, Yoshitomi, Iwakura, Sekikawa, Azuma, Kanai, Moriizumi, Nomura Nakamura, Sakaguchi. J. Clin. Invest. 114:582-588 (2004).

Fundamental Immunology. Coleman, Lombard, Sicard; Wm. C. Brown Publishers: Dubuque, Iowa, 1989; p. 416-418.

Introduction to Immunology, An. Ravo, C.V.; Alpha Science International Ltd.: Pangbourne, UK, 2002; p.436-438.

Image References

· Hand picture- http://medicine.ucsd.edu/clinicalimg/upper-rheumatoid-arthritis.html
· Joint picture-http://www.meritcare.com/hwdb/showtopic.asp?module_abbrev=HWKB4&pd_hwid=tp10169-sec&topic_name=Joints%20typically%20affected%20by%20rheumatoid%20arthritis&sequence=1
·