Rheumatoid Arthritis is an advanced inflammatory disease. This disorder shows physical characteristics such as flared up synovium and continuous inflammation; which leads to chronic polyarthritis. Both the knee and finger joints of a diagnosed patient display pathological features of Rheumatoid Arthritis. Most of the inflammatory events that occur within the synovial fluid such as cell death and cytokine release can become life threatening. This disorder affects both men and women of various countries in the world. RA is an autoimmune disorder with 1% prevalence in industrialized countries such as the United Kingdom being an example (Mcinnes and Brennan, 2008). It is more common in women than in men although the difference between genders is not consistent and changes with increase in age (Hochberg et al, 2009).
The above mentioned immune-mediated inflammatory changes observed paves a way for the discovery of many clinical findings associated with Rheumatoid Arthritis (Cush et al, 2010). Both PAR-2 and AHR expression are important factors in the inflamed joints of RA patients. These findings have progressed over the years with new results being discovered (Ferrell et al, 2006: Kobayashi et al, 2008).
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Existence of a link between the AHR and PAR-2 on expressed macrophages is unknown. However, research experiments are being carried out in the laboratory to solve this unexplained scientific theory. There will be a breakthrough both in macrophage inflammatory response and autoimmune diseases if a link exist between Aryl hydrocarbon receptor and PAR-2.
Rheumatoid Arthritis (Pathology and Cytokines)
RA is a systemic disorder caused by various factors such as genetic susceptibility, defective immune reactions and environmental factors (Huber et al, 2006).
RA is composed of various components, which exhibit different functions and structure. Examples include the synovial membrane, plasma cell rich and fibroblasts. The synovial membrane in the joint of an arthritic patient is composed of both type A (macrophage-derived) and type B (fibroblast derived), which increase in number as synovial membrane enlarges. The synovium also containing plasma cell rich areas are responsible for immunoglobulin and rheumatoid factor (RF) production. Local production of large quantities of RF and anti -CCP antibodies are also suggestive factors of Rheumatoid Arthritis (Cush et al, 2010).
Cytokines are of major importance and also plays a key role in Rheumatoid Arthritis. Cytokines are secreted peptide products of activated cells capable of mediating self, local and systemic effects in both the rheumatoid synovium and synovial fluid. Examples of such cytokines involved in the immune response of rheumatoid arthritis are IL-6, IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF), TNF-Î±, IL-1 and interferon-Î³ (IFN-y). Functions of these cytokines include cellular regulation, stimulation of cytokine production and adhesion molecule activation. Previous experiments have shown that these cytokines are involved in the processes associated with the cause of inflammation and joint destruction (Mcinnes & Brennan, 2008).
According to the above diagram which summarises the role of cytokines in RA. Cytokines target cells in RA through cellular interaction. Examples of such cells are macrophages, t-cells, b-cells and fibroblasts. These interactions are enabled by actions of cytokines released from activated cells, which stimulates the production of other cytokines. This immunological mechanism contributes to the pathogenesis of disease (Mcinnes & Brennan, 2008).
Rheumatoid Arthritis & Fibroblast.
Scientific evidence has shown that the structure of activated synovial fibroblasts plays a key role in the pathogenesis of RA. Fibroblasts are flat spindle-shaped cell of mesenchymal origin whose primary role is to produce a wide variety of extracellular matrix components including collagens, proteoglycans, growth factors, proteolytic enzymes and small signalling molecules (Koh et al, 2011).
Rheumatoid Arthritis Synovial Fibroblast (RA-SFs) consists of a cell type that differentiates RA from other inflammatory conditions such as osteoarthritis of the joint. RA-SFs are able to display change in morphology and behavioural abilities including molecular changes in signalling cascades, cell-death response, matrix degrading enzymes and expression of adhesion molecules (Huber et al, 2006:Pap et al, 2000).
Macrophages and lymphocyte secreting factors also contribute to the activation of RA-SF. This activation process is characterized by both a change in expression of regulatory genes and signalling cascades of the immune system leading to an up-regulation of adhesion molecules. The end process enables the attachment of RA-SFs to extracellular matrix and overexpression of matrix degrading enzymes, which leads to continuous destruction of joints. Activated RA-SF can exert specific effects on both macrophages and lymphocytes (Pap et al, 2000).
Monocytes and Macrophages
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Monocyte is a key component of the blood with various functions and fine structure. It is the largest of all normal peripheral blood cells with sizes ranging from 12-20Âµm. It has an uneven nucleus and dense cytoplasm with fine dye granules (Bain, 2006). Monocytes are part of the innate immune system that provide both immediate defence against foreign antigens and also help during the initiation and growth of adaptive immune response (Martinez et al, 2012).
Monocytes originate from the myeloid cells located in the bone marrow. They circulate the blood stream for several days and enter the peripheral tissues such as lymph nodes where it is differentiated into macrophages and dendritic cells. Both these cells exhibit high oxygen consumption, production of cytokine and protein synthesis (Martinez et al, 2012).
Macrophages have various functions such as engulfing of foreign antigens during inflammation and wound healing. Response to macrophage activation varies depending on extracellular environment factors and they possess specialized receptors and mechanism in identifying and destroying infected cells (Schull et al, 2010).
Macrophages are divided into two types. The polarized activated M1, which is induced by IFN-Î³ alone with microbial stimuli as LPS. They are shown to have an increase levels of IL-12, IL-23, reduced levels of IL-10 phenotype and also secrete inflammatory cytokines such as IL-1B, TNF-Î± and IL-6. This activated M1 act as both an inducer and effector cells in cellular Th1 response whereas the other macrophage, which is the M2 is an activated macrophage resulting from cell exposure to cytokines, immune complexes and vitamin D3. They show decreased levels of IL-12 and IL-23 phenotype (Martinez et al, 2012).
Role of Macrophage in Rheumatoid Arthritis
Macrophages are quite abundant in the inflamed region of synovium. They express clear signs of activation such as overexpression of class II MHC molecules, growth factors and regulatory cytokines. They also show broad destructive abilities, which contribute to inflammation and joint destruction in both phases of Rheumatoid Arthritis (Kinne et al, 2012).
Macrophages initiate various processes when activated in the immune system of a rheumatoid arthritis patient. Macrophages switch to an anti-inflammatory phenotype when they come across an apoptotic cell (Schull et al, 2010). This degree of activation correlates not only with joint pain but also with radiological advancement of continuous joint damage (Kinne et al, 2012).
Toll like receptors such as TLR-2 and TLR-4 increase activation of RA synovial macrophages compared with patients with other forms of arthritis (Huang et al, 2007).
PAR (Protease Activated Receptors) is a membrane cell surface protein discovered in 1990 and belongs to the family of G-protein coupled receptor (GPCR). PARS act as cell surface sensors and proteases, which contribute, to homeostatic regulation when activated (Adams et al, 2011).
Main feature of PARS is their irreversible activation by serine proteases. Recent studies have shown PAR activation has effects on leukocyte activity, cytokine production, adhesion molecule expression and patho-physiological functions. The crucial role of PAR activation during disease progression was observed in various animal models of different pathologies, neuro-inflammatory process, joint inflammation and innate immune system (Adams et al, 2011:Shpacovitch et al, 2008).
PAR can be activated and signalled through a series of irreversible proteolytic mechanisms. Serine Proteases such as thrombin or tryptophan bind and cleave the N-terminal domain of PARS at specific sites to unmask new N-terminus. Additionally, this N-terminus act as a tethered ligand which attaches to the receptor and triggers intracellular signalling. PARS can also be activated without the need of proteolysis by synthetic peptides (so called PAR-APS). (Soh et al, 2010).
There are four PAR family members such as PAR-1, PAR-2, PAR-3 and PAR-4 which belong to the GPCR family (Adams et al 2011). PAR2 will be the main focus of discussion due to its connection to inflammatory response. PAR2 is resistant to the serine protease called thrombin but can be activated by trypsin, mast cell, tryptase, and bacteria derived enzymes. Activated PAR-2 is the only protease-activated receptor shown to bind with Î²-arrestins which facilitates ERK1 to mediate changes in actin cytoskeleton and cell migration. And this discovery can be useful in connection to the pathophysiological response in rheumatoid arthritis (Soh et al, 2010: Shpacovitch et al, 2008).
PAR Activation leads to the production and release of various chemokine, growth factor; Platelet derived growth factor and cytokines. Activated PAR signal different effector cells by using unique active arrangements and also regulated by different mechanisms. Identification of molecular structure and composition is essential for PAR signalling and regulation because these will encourage development of new options to change receptor signalling (Soh et al, 2010).
Role of PAR-2 in macrophages and Rheumatoid Arthritis
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Macrophages express PAR1, PAR2 and PAR3 at both mRNA and protein levels. Previous experiment carried out showed that human monocytes do not express PAR2. On the other hand, another experiment was carried out which disproved the notion (Colognato et al 2003: Johansson et al 2005). The initial experiment involved the use of buffy coats by a surface adherence protocol whereas the latter experiment used human monocytic cells isolated from healthy adult individuals (Shpacovitch et al, 2008).
According to the results from the isolation of human peripheral blood monocytes. There was a detection of a distinct PAR-2 expression on the cell surface of CD14+ monocytes, which was later on increased after centrifugation. Further experiment involving stimulation of these purified monocytes with PAR-2 resulted to the production of cytokines such as IL-8, IL-6, Il-1Î² but TNF-Î± was not detected (Johansson et al, 2005).
Monocytes are producers of diverse range of inflammatory mediators. Recent findings show that PAR-2 interaction in an inflammatory response is influenced by cytokine release from monocytes. PAR-2 is an up-regulator of pro-inflammatory cytokines supporting previous evidence of the involvement of this receptor as a key regulator in the innate immune system (Kelso et al, 2006: Johansson et al, 2005).
Recent studies have shown PAR-2 and serine proteases inhibitors to be effective anti-inflammatory agents in murine model. Serine protease induced joint swelling appears to be mediated via PAR-2 activation and also the inhibition of PAR-2 up regulation in synovial fluid joint inflammation in mice. (Ferrel et al, 2003: Kelso et al, 2006)
Recent experiment produced a comparative data showing the inflammatory role of PAR-2 in humans diagnosed with rheumatoid arthritis and osteoarthritis infections. This comparative data showed that PAR-2 was highly expressed in RA synovium as compared with control subsets (synovial tissue of an osteoarthritic patient). However, the immune cell type mediating the inflammatory effects which was triggered by PAR-2 activation remains unknown (Busso et al, 2007).
Another study was performed to look at the functional significance of PAR-2 expression in RA derived leukocytes from human blood samples. Results of these experiments showed that patients with RA had elevated surface expression of PAR-2 on monocytic cells compared to the control subsets. This elevated PAR-2 expression in cells was treated with methotrexate treatment, which reduced significantly (Crilly et al, 2011).
All these findings are consistent with the pathological role, which PAR-2 plays in macrophages of a Rheumatoid Arthritis Patient.
Aryl Hydrocarbon receptor (AHR)
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor and member of the per-ant-sim (PAS) superfamily of proteins. Its functions include detection of intracellular or environmental changes. This transcription factor has been a topic of intensive research for over 30 years by various field of science such as toxicology and immunological field.
In the human cell, AHR is located in the cytoplasm and upon agonist binding translocate to the nucleus where it becomes active as a transcription factor (Stockinger et al, 2011).
AHR signalling controls the proteosomal breakdown of AHR/ARNT complex. AHR interacts with ARNt, which binds to specific sequences available in the target promoters named Dioxin Responsive Elements. AHR activation system is involved in various physiological processes such as cell cycle regulation and differentiation. The most key area of AHR system is the presence of polymorphisms, which plays a major role in response ability to AHR ligands (Stockinger et al, 2011).
In a recent experiment performed on AHR deficient murine models, supporting evidence was provided that AHR have central physiological functions. A notion supported by extensive evolutionary conservation (Hahn et al 1997). Although results have suggested that AHR mediated gene expression may not be well considered across species, which could have a significant implication in human risk assessment (Sun et al, 2007).
AHR mediates the toxicity of TCDD (dioxin), which is its major effective ligand. TCDD is a toxic contaminant that has various implications on the human system such as host susceptibility to infections, supressed adaptive immune system and lymphocyte maturation. TCDD is the most stable xenobiotic ligand of the AHR. However, it lacks sensitivity to metabolic enzymes (Stockinger et al, 2011).
ROLE OF AHR IN MACROPHAGES
AHR is expressed both in macrophages and dendritic cells based on reaction of TCDD on cell numbers (Kerkvliet, N 2009).
Both cells were analysed in gene array studies. Micro array analysis of the AHR system showed that some macrophages had low AHR expression and differed from other immune cells. Moreover, in matured stage (peritoneal macrophages) AHR expression levels are higher (Frericks et al, 2007:Stockinger et al, 2011).
Recent research has shown the negative aspect of AHR role in inflammatory responses of macrophage induced by LPS. This report states that both IL-6 and TNF had an increased response in macrophages of an AHR deficient murine model (Kimura et al 2007). Additionally, another study showed hyperactive macrophage IL-1 cytokine response to LPS which resulted to an increased susceptibility to septic shock. The later study involved the use of AHR deficient mice expressing a DNA deletion in their myeloid lineage (Sekine et al, 2009).
Early studies have shown that exposure to dioxin (TCDD) displays an increase in cytokine production by human macrophage cell line. It also plays a role in AHR activation and signalling on gene expression of macrophages in vitro. (Cheon et al, 2007).
Previous experiment have also shown an increase in mRNA levels of AHR ligands in human macrophages treated with TCDD and also an increase in gene expression could be blocked by inhibiting calmodulin (CAM)- dependent protein kinase pathway (Monteiro et al, 2008).
The interaction of drugs with the AHR has shown good results. The low molecular weight compound VAF347, and its pro drug version VAG539 interact with the transcription factor aryl hydrocarbon receptor on monocytes to mediate anti-inflammatory activity such as dendritic cell differentiation (Baba et al, 2012)
ROLE OF AHR in RHEUMATOID ARTHRITIS
Environmental factors have been showed to be involved in RA pathogenesis and AHR. An example of such environmental factor is the chemical TCDD.
Smoking of cigarettes is a major environmental factor of Rheumatoid Arthritis. These cigarettes contain TCDD, which is toxic to the human body cells and enters through the human body via lung and skin route (Esser et al 2009).
TCDD alters the immune response by acting on AHR. TCDD releases inflammatory cytokines such IL-1 and IL-8 via interaction with AHR which results to stimulation of DNA signalling pathway and nuclear factor-kB. Exposure to TCDD enhances RA inflammatory processes.
Previous experiments have shown increased AHR expression in both mRNA and protein levels of a Rheumatoid Arthritis synovial tissue than in Osteoarthritis tissue. Another factor is the Overproduction of TNF-Î± which activates AHR expression in RA synovial cells (Kobayashi et al, 2008).
In addition to the review, experiments will be carried out to establish a link between PAR-2 and AHR with the use of isolated human blood monocytes. The hypothesis derived is to stimulate these monocytic cells with PAR-2 and AHR to observe the immunological reactions with the use of various laboratory techniques. The amount of cytokines being produced by the monocytic cells when stimulated by external factors will be observed to see the connection between Rheumatoid Arthritis, PAR-2 expression and Aryl Hydrocarbon Receptor. This will give an insight on both factors in their expression in other autoimmune diseases apart from rheumatoid arthritis.
Autoimmune disease such as rheumatoid arthritis are multifactorial and can be associated with many factors such as smoking, genetics, hormones, age, diet, infections, drugs and exposure to chemicals. The best outcome is to treat patients early, which requires close monitoring to assess the effectiveness of treatments (Edward and Edward, 2010).
However, Drug responses such as the administration of a combination therapy consisting of infliximab with methotrexate to this disease have shown to lessen the inflammation but not cure the disorder entirely. This may be as a result of a genetic or immunological factors associated with rheumatoid arthritis.
Rheumatoid arthritis still remains a challenging scientific setback withstanding the phenomenal discoveries of recent years (Mcinnes and brennan, 2008). There is still an increased prevalence of Rheumatoid arthritis today in industrialized countries.
Research and clinical trials are going on at the moment to understand the physiological function of the AHR and PAR-2 in rheumatoid arthritis. These experiments might provide new information showing the role of these receptors in macrophage driven inflammatory response in Rheumatoid Arthritis.