Prostaglandins and Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, and complex inflammatory disease leading to bone and cartilage destruction, whose cause remains obscure. Accumulation of genetic susceptibility, environmental factors, and dysregulated immune responses are necessary for mounting this self-reacting disease. Inflamed joints are infiltrated by a heterogeneous population of cellular and soluble mediators of the immune system, such as T cells, B cells, macrophages, cytokines, and prostaglandins (PGs). Prostaglandins are lipid inflammatory mediators derived from the arachidonic acid by multienzymatic reactions. They both sustain homeostatic mechanisms and mediate pathogenic processes, including the inflammatory reaction. They play both beneficial and harmful roles during inflammation, according to their site of action and the etiology of the inflammatory response. With respect to the role of PGs in inflammation, they can be effective mediators in the pathophysiology of RA. Thus the use of agonists or antagonists of PG receptors may be considered as a new therapeutic protocol in RA. In this paper, we try to elucidate the role of PGs in the immunopathology of RA.


Introduction
Rheumatoid arthritis (RA) is a complex autoimmune and progressive inflammatory disease that involves the joints and leads to their destruction. The prevalence of rheumatoid arthritis (RA) is 0.5%-1.0% in the general population worldwide [1,2]. Females are nearly three times more likely than males to develop the disease and can start at any age, although the mean age at the onset is 40 to 60 years [3,4]. The precise cause of rheumatoid arthritis is unknown; like other autoimmune diseases it arises from a variable combination of genetic susceptibility, environmental factors, and the inappropriate activation of the immune responses that eventually result in the clinical signs of arthritis [5]. Multiple genes are associated with disease susceptibility, with the HLA locus accounting for 30% to 50% of the overall genetic risk. Several risk loci have been recognized: HLA-DRB1, PTPN22, STAT4, CTLA4, RAD14 a region in 6q23, and the TRAF1/C5 locus [6][7][8][9]. Similarly, the mouse strains of DBA/1 and B10.Q have the I-Aq and I-Ar haplotypes and are highly susceptible to collagen-induced arthritis (CIA), as experimental models of RA [10,11]. The important role of HLA-DR antigens is to present antigens to T lymphocytes, whereas the PTPN22 protein tyrosine phosphatase appears to have a potential function in the setting of T-cell and B-cell activation [12]. Smoking, the best-known environmental factor, in certain genetic context of HLA-DRB1 can trigger immunity to citrulline-modified proteins and this response, after several years, causes arthritis [13,14]. The adaptive and innate immune responses in the synovial fluid are involved in the pathogenesis of RA. High levels of autoantibodies, including rheumatoid factors and anticitrullinated peptide antibodies, can be diagnosed before the onset of clinical arthritis [15]. Inflamed joint tissues are infiltrated by monocyte/macrophage, rheumatoid arthritis synovial fibroblast (RASF), T cells, and B cells. These cells release proinflammatory cytokines such as interleukin 1(IL-1), IL-17, and tumor necrosis factor α(TNF-α), that 2 Arthritis play important roles in progressive joint destruction and are closely associated with the production of small proinflammatory lipid mediators such as prostaglandins [16,17].

Prostaglandins
Prostaglandins are small potent inflammatory mediators that are generated by the release of arachidonic acid (AA) from the membrane phospholipids by the phospholipase A2 (plpA2) family. Subsequently, cyclooxygenase (COX; prostaglandin endoperoxide H synthase; PGHS) and Prostaglandin synthase enzymes metabolize AA to prostaglandins including PGE 2 , PGF 2α , PGD 2 , PGI 2 (prostacyclin), and TXA 2 (thromboxane), that play pivotal roles in the modulation of physiological systems, such as CNS, and the inflammatory and immune responses [18,19]. The cyclooxygenases are heme containing enzymes that have two major isoforms in mammals named COX-1 and COX-2. Although COX-1 and COX-2 have about 60% homology at the amino acid level and catalyze the same reactions, they have different patterns of expression and are encoded by different genes [20,21]. COX-1 is constitutively expressed in many tissues and is responsible for the physiological function of PGs and thus is known as a "housekeeping" enzyme whereas COX-2 expression is induced by inflammatory mediators like cytokines, growth factors, and bacterial endotoxins [21,22]. Traditional NSAIDs (nonsteroidal anti-inflammatory drugs), that have antipyretic, analgesic, and anti-inflammatory properties, inhibit both COX-1 and COX-2 and are associated with side effects such as gastrointestinal bleeding due to the suppression of both COX isozymes. The recently developed COX-2-selective inhibitors retain effectiveness in reducing inflammation and pain in rheumatoid and osteoarthritis but have a lower incidence of gastrointestinal side effects [23]. Prostaglandins use G-protein coupled receptors (GPCRs) for exerting their functions. The prostaglandins receptors subfamilies include DP, EP1-4, FP, IP, and TP which bind to PGD 2 , PGE 2 , PGF 2α , PGI 2 , and TXA 2 , respectively [24]. Recently, another PGD 2 receptor, the chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), was identified [25]. The prostaglandins (PGs) found at elevated levels in the synovial fluid and the synovial membrane are considered to play a pivotal role in the development of vasodilatation, fluid extravasation, and pain in synovial tissues. Moreover, there is an increasing evidence that PGs (especially prostaglandin E 2 ) are mediators involved in complex interactions leading to the development of erosions of the articular cartilage and the juxta-articular bone [26]. PG synthesis inhibitors (COX-2 inhibitors and NSAIDs) are widely used in the treatment of RA [27].

Prostaglandins and Rheumatoid Arthritis
3.1. PGD 2 and RA. Prostaglandin D synthase is responsible for the generation of the PGD 2 and J series. This enzyme has two isoforms including the lipocalin (brain) type PGDS (L-PGDS) which is responsible for PGD 2 biosynthesis in the CNS where it plays a central role in the regulation of sleep and the hematopoietic PGDS (h-PGDS) which is also known as "spleen type" PGD 2 synthase [28,29]. PGD 2 regulates multiple physiologic and pathologic processes, such as sleep, nociception, vasodilation, bronchoconstriction, and bone metabolism. PGD 2 has anti-inflammatory effects in several models of inflammation [30,31]. It exerts its function by binding two receptors, DP1 and CRTH2. DP1 activation by PDG 2 causes the elevation of intracellular cAMP level which is typically associated with damping of the cellular effector function, whereas the CRTH2/DP2 receptor stimulation leads to the elevation of intracellular Ca +2 . DP2 activation by PGD 2 induces TH2 cytokines production, their migration, and enhancing their adhesiveness to endothelial surfaces [29]. On the other hand, when PGD 2 is produced in a large amount for activating PPAR-γ, it will be able to inhibit T-lymphocyte proliferation and, consequently, the inflammatory response. In contrast, if only nanomolar concentrations of PGD 2 and its metabolites are produced, the PGs may be expected to activate T lymphocytes [25,32,33]. In synovial fluid, PGD 2 abundantly is released by chondrocytes [33], osteoclasts [34], synovial fibroblast, and mast cells [35,36]. It inhibits chondrocyte apoptosis [37], stimulates chondrogenic differentiation, enhances the expression of collagen type II and aggrecan [38], prevents IL-1-induced generation of MMP-1 (metalloproteinase-1) and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway, indicating that PGD 2 may contribute to the cartilage maintenance and integrity [39,40]. PGD 2 is readily dehydrated and generates PGs of the J series, such as PGJ 2 , δ12-PGJ 2 , and 15-deoxy-δ12,14-PGJ 2 (15d-PGJ 2 ). The 15d-PGJ 2 was identified as a ligand for peroxisome proliferator activated receptor-γ (PPAR-γ) which enhances the differentiation of adipocytes and trophoblasts [41,42] and implicates as a mediator of many anti-inflammatory effects of PGD 2 [43]. 15d-PGJ 2 is released by articular chondrocytes and diagnosed in synovial fluid RA patients. It enhances chondrocyte apoptosis in a dose-and time-dependent manner by a PPARγ-dependent pathway [44]. PGD 2 and its metabolite, 15d-PGJ 2 , may have chondroprotective effects. For instance, they counteract the induction of matrix metalloproteinases in cytokine-activated chondrocytes, which play an important role in cartilage degradation [39,45]. The 15d-PGJ 2 inhibits the production of several inflammatory mediators by monocytes/macrophages. It blocks nitric oxide (NO) production as well as proteoglycan degradation [45][46][47][48]. 15d-PGJ 2 also inhibits apoptosis of human primary chondrocytes induced by the NF-κB inhibitor (Bay 11-7085) [37,49]. 2 and RA. PGE 2 is the major PG that is generated by chondrocytes and synovial fibroblasts; the biosynthesis can be enhanced by proinflammatory cytokines such as IL-1β, TNF-α, and trauma [50]. Prostaglandin E synthase (PGES) converts COX-derived PGH2 to PGE 2 [51], a potent lipid mediator, that regulates a broad range of physiological activities in the immune and the other biological systems such as cardiovascular, endocrine, gastrointestinal, neural, Arthritis 3 pulmonary, reproductive, and visual systems [52]. Three different forms of the PGESs have been identified, microsomal PGES-1,2 (mPGES-1,2) and cytosolic PGES(cPGES, p23). mPGES-1 is preferentially linked with COX-2 and is induced in response to various stimuli [53]. Glutathione-independent mPGES-2 is a unique PGES that is constitutively expressed and coupled with both COXs in the production of the PGE 2 involved in both tissue homeostasis and disease [30,54]. The cPGES is constitutively expressed and to be preferentially coupled to COX-1 than COX-2 and its expression is not affected by proinflammatory stimuli [55]. Of the three PGES isozymes, mPGES-1 is upregulated in synovial fluid in active RA and is minimally expressed in inactive RA [56]. The mPGES-1 induction is coordinated with COX-2 expression under inflammatory conditions in different cells and tissues as well as RA synovium. Some of selective COX-2 inhibitors may cause cardiovascular side effects in RA patients due to simultaneous decrease in production of PGE2 and antithrombotic PGI2. In order to reduce this side effect selective inhibition of mPGES-1 derived PGE2 production will be an desirable therapeutic alternative [57,58]. PGE 2 exerts its diverse roles by acting on a group of rhodopsinlike 7-transmembrane-spanning GPCRs: EP1, EP2, EP3, and EP4. The EP subtypes show differences in binding affinity, signal transduction, tissue localization, and regulation of expression. The Ep3 and EP4 are the most abundant of the EP receptors and their binding affinity to PGE2 is higher than EP1 and EP2 receptors [59,60]. EP receptors link to different intracellular signaling molecules that mediate the effects of receptor activation on cell function. EP2 and EP4 receptors couple to a Gs-type G-protein that activate adenylate cyclase, increasing intracellular cAMP. EP1 links to Gq and activates phosphatidylinositol metabolism leading to mobilization of intracellular free calcium. EP3 receptor can couple to Gi or G12 for elevation of intracellular Ca 2+ , inhibition of cAMP generation, and activation of the small G-protein Rho [19,60].

PGE
The knock-out mouse studies revealed that PGE 2 can exert both proinflammatory and anti-inflammatory responses, depending on receptor subtype, cell population, context of activation, and receptor gene expression in tissues [61]. Using mice deficient in the EP subtypes, Honda et al. found that the simultaneous inhibition of EP2 and EP4 significantly decreased the arthritic score in CIA. Loss or inhibition of a single EP does not affect the extent of CIA [62]. Stock et al. using EP1-deficient(EP1-/-) mice showed a reduced stretching response following administration of acetic acid or 2-phenyl-1 benzoquinone (PBQ) suggesting that the central hyperalgesic effects of PGE 2 are mediated by spinal EP1 receptors [63]. Minami et al. observed that the PGE 2 -induced hyperalgesia is mediated by the EP3 receptor at lower doses and by the EP2 receptor at higher doses [64]. Yao et al. showed that Th1 and IL-23-dependent Th17 differentiation is promoted by PGE 2 -EP4 signaling in DCs and T cells [65]. PGE 2 /EP4 plays a proinflammatory role in the pathogenesis of rheumatoid arthritis as in CIA, the homozygous deletion of EP4(EP4-/-) receptor but not the EP1, EP2, or EP3 receptors, which led to decrease in incidence and severity of disease [66]. The EP1 downregulates the expression of COX-2 in a concentration-dependent approach through a receptor activation-independent pathway. The reduction in COX-2 protein occurs due to the enhancement of substrateindependent COX-2 proteolysis because EP1 facilitates COX-2 ubiquitination via an unknown E3 ligase. This suggests a new role for the EP1 in resolving inflammation [67]. It has been shown that chondrocyte apoptosis is induced by PGE 2 binding to EP2 or EP4 receptors [68]. This is dependent on the activation of the cell, since PGE 2 enhances apoptosis in resting mature T cells, whereas it protects T cells from T-cell receptor mediated activation-induced apoptosis [69,70]. Aoyama et al. found the dominant expression of EP2 receptors in human articular cartilage and cultured chondrocytes, whereas EP1 and EP4 receptor expression was not significantly increased [71]. Otsuka et al. showed that PGE2 signal via EP2 not only has an anti-inflammatory property but also promotes chondrocyte proliferation and the regeneration of articular cartilage. Attur et al. reported that the catabolic activities of PGE 2 is mediated by PGE 2 -EP4 signaling in OA cartilage. PGE 2 -EP4 induces matrix metalloproteinase production and type II collagen degradation. PGE 2 through EP4 receptor shows a potent antianabolic effect on human adult articular cartilage in vitro via the suppression of proteoglycan biosynthesis, which suggests EP4 receptor antagonist could be as a potential therapeutic agent for the treatment of osteoarthritis and RA [72,73]. Clark et al. also found that a selective EP4 antagonist reduces COX-2-dependent arthritic inflammation and pain (NSAID-like activity). This reagent is well tolerated by the gastrointestinal tract and, unlike COX-2 inhibitors, it does not inhibit PGI2 and may be cardioprotective [74]. PGE 2 has also inhibitory effects on NF-κB through ERK-dependent and -independent pathways in RASF, key mediators of RA inflammation, and cartilage erosion. This process may paradoxically inhibit the action of inflammatory cytokines and may participate in the resolution phase of inflammation to prevent cartilage degradation in arthritis [75]. 2 and RA. Prostacyclin (PGI 2 ) the main PG produced by vascular endothelial cells exerts its functions through a seven-transmembrane-spanning GPCR, known as the IP receptor. Both cyclooxygenase enzymes (COX-1/2) convert AA into the prostaglandin precursor PGH 2 , which is subsequently converted into PGI 2 via prostacyclin synthase (PGIS), a member of cytochrome P450 superfamily [76,77]. The IP receptor is coupled predominately to a Gs subunit (and in some circumstances with Gi-and Gq-dependent pathways) and the G-protein leads to an increase in the cAMP level and this signaling pathway is responsible for vasodilatory and antithrombotic effects of prostacyclin [78][79][80]. PGI 2 may also signal through the PPAR-γ pathway [81]. Prostacyclin plays a regulatory role within the cardiovascular system. It has been found that the IP receptor signaling by enhancing Th2-cell production of the anti-inflammatory cytokine IL-10 inhibits Th2 mediated allergic inflammatory responses [19,82]. PGI 2 is the most frequent prostaglandin in synovial fluid of patients with RA [83]. In rheumatoid arthritis PGI 2 acts as a proinflammatory lipid mediator. IP receptor antagonists inhibit experimental hyperalgesia, edema, and osteoarthritis in the rat, indicating that prostacyclin plays an important role in these pathological conditions. In CIA, IP receptor-deficient mice showed a significant decrease in arthritic score in spite of anticollagen antibodies and complement activation similar to wild-type mice. In addition, the administration of the IP antagonist in this model also reduced the symptoms (NSAID-like efficacy) [84,85].

PGI
3.4. PGF 2α and RA. Prostaglandin F 2α (PGF 2α ) is biosynthesized from PGH 2 and other PGs (PGE 2 , PGD 2 ) by three enzymes, PGH 9-, 11-endoperoxide reductase, PGE 9-ketoreductase, and PGD 11-ketoreductase, respectively [86]. It exerts its biological functions by binding to a prostanoid receptor FP which has two differentially spliced variants (FP A , FP B ). The FP receptor couples with the G q protein for increasing the inositol phosphate accumulation, protein kinase C (PKC) activation, and intracellular calcium release [87]. In addition, stimulation of FP receptor leads to activation of G-protein Rho via a Gq-independent process, resulting in cytoskeleton rearrangement [88]. The FP receptor is the least selective of the prostaglandin receptors in binding the principal endogenous prostaglandins, binding both PGD 2 and PGE 2 at nanomolar concentrations [19]. PGF 2α has a pivotal role in the reproductive system, renal function, contraction of arteries, myocardial dysfunction, and regulation of intraocular pressure and pain [89][90][91][92][93]. Basu showed that the oxidative metabolism of arachidonic acid through both enzymatic (cyclooxygenase) and nonenzymatic (free radical) pathways is engaged in endotoxin-induced inflammation in pigs as indicated by the significantly increased formation of F 2 -isoprostane and PGF 2α metabolite in plasma [94]. They also showed that the measurement of F 2 -isoprostanes in body fluids provides a reliable analytical tool to study oxidative stress-related diseases and experimental inflammatory conditions [95]. High levels of both free radical mediated F 2 -isoprostanes and the cyclooxygenase derived PGF 2α metabolite were diagnosed in blood and synovial fluid from patients with various rheumatic diseases such as RA and OA that shows both oxidative injury and inflammation play a role in various degrees in chronic inflammatory conditions [96]. The arising role of PGF 2α in inflammatory reactions opens the unique opportunities for designing the new anti-inflammatory drugs [61].

Conclusion
Elevated levels of prostaglandins have been diagnosed in the synovial fluid and synovial membrane of RA patients. In the inflamed joints PGs play pivotal roles through complex interactions with leukocytes and other cells. They can induce both pro-and anti-inflammatory responses, depending on the receptor subtype, cell population, context of activation, and the receptor gene expression in tissues. The role of prostaglandins in the metabolism of articular cartilage is still controversial. Some studies show that prostaglandins contribute to the destruction of articular cartilage by degrading cartilage ECM, while others found that they induce chondrogenesis and terminal differentiation. The different biological roles attributed to these lipid mediators are a direct indication of the molecular complexity of prostaglandins and their exclusive cognate receptors. Mice deficient in individual PGs receptors and combinations of these receptors will allow the investigation of the role of these receptors and their ligands in various models of inflammatory diseases such as RA. The most important therapies for RA should both inhibit inflammation and activate resolution. A broad spectrum of different enzyme inhibitors and receptor antagonists has been studied, showing a variety of effects on the course of the disease. Thus, it seems that the pharmacological intervention to modulate the release of lipid mediators will be important to improve the patient outcomes. The research efforts of recent years, however, contribute to a better understanding of the pathophysiological impact of lipid mediators in inflammatory disorders and provide new therapeutic approaches.