Effects Of Cathepsin S In Rheumatoid Arthritis

National Natural Science Foundation of China (Role of cathepsin S in early RA: a new potential treatment target, grant number:81172875).Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the chronic invasive arthritis. The major pathological change of the rheumatoid arthritis includes synovitis as well as the resulting of articular cartilage and bone damage, which will finally lead to the articular deformation. Without systematic treatment, rheumatoid arthritis will develop into disability within10years in about45%of the patients. Rheumatoid arthritis is widely distributed all over the world. Its incidence shows a certain ethnic variation, ranging from0.18%to1.07%in different populations. Rheumatoid arthritis occurs in all ages, with the peak age at30-50years old. With the advent of the aging society of China, the patient population of RA continually increased, which will bring a heavy burden to society and family. So researches on the pathogenesis of RA, finding the new target for early diagnosis and treatment of RA, are the important topics in Rheumatology.Synovial cell hyperplasia and endothelial cell activation are early events in the pathologic process that progresses to uncontrolled inflammation and consequent cartilage and bone destruction. Genetic factors and immune system abnormalities contribute to disease development. TNF-α and IL-1are considered predominant cytokines in the process of human RA, with a claimed cascade of TNF-a inducing most of the IL-1production. In the early stage of RA, synovium is hyperemia and edema, a lot of monocytes, polymorphonuclear cells and lymphocytes infiltrated in the mesenchyma. The typical synovitis of RA is gradually developed in3to6months, and the hyperplasia synovium protrude into articular cavity. Immunocyte proliferated and invade the synovium; mononuclear agglomerated into lymphoid follicle and a few germinal centers are formed. Plasma cells, macrophages and lymphocytes infiltrated around the small vessels and composed the nodular pannus. The pannus continuously expanded over the arthrodial cartilage, blocked the contact of cartilage and synovial fluid, and affected the intake of nutrition. Immunocyte in the pannus released a lot of inflammatory mediators, proteases and collagenases. Ultimately, inflammation and exuberant proliferation of the synovium (ie, pannus) leads to the destruction of various tissues, including cartilage, bone, tendons, ligaments, and blood vessels. As the result, the functions of the joints are completely lost.The pathological mechanism of rheumatoid arthritis is very complex, and involves at least eight types of cells, including mast cells, fibroblasts, macrophages, T cells, B cells, dendritic cells, chondrocytes and osteoclasts. The cells interact with each other under the mediation of the surface receptors, secreted cell factors, chemotactic factors, cathepsins and matrix matalloproteinases, forming a complicated network. Such as the synovial mast cells can express a lot of basic fibroblast growth factor, Leukotriene C4and platelet-derived growth factor or so, which help the synovial fibroblasts hyperplasia and adjust the proinflammatory activity of synovial fibroblasts. The synovial fibroblasts expressed the cathepsins and the matrix metalloproteinases, lead to the destruction of cartilage and bone. The mast cells can express a low level of cathepsins, such as cathepsin G, MMP-2and MMP-9, have a role in the destruction of cartilage and bone. The main pathological change of rheumatoid arthritis is synovitis, which presents as the synovial hyperplasia and inflammatory cell infiltration. Pannus formation is an important pathological feature of synovium in rheumatoid arthritis, and plays an important role in the cartilage and bone damage in the process of rheumatoid arthritis. Rheumatoid arthritis synovial fibroblasts (RASFs; also termed fibroblast-like synoviocytes or type B synoviocytes), together with synovial macrophages, are the two main cell types in the terminal layer of the hyperplastic synovial tissue that invades and degrades adjacent cartilage and bone. Although macrophages probably do not occupy a leading pathogenetic position in RA (except for their potential antigen-presenting capacity), they can produce a broad of pro-inflammatory, destructive, and remodelling potential cytokines and contribute considerably to inflammation and joint destruction in acute and chronic RA. Also, activation of this cell lineage extends to recruiting monocytes and other cells of the mononuclear phagocyte system, including bone marrow precursors of the myelomonocytic lineage and osteoclasts. TNF-α and IL-1are considered master cytokines in the process of RA, produced mainly by macrophages. Macrophages are considered the local and system inflammation amplifier. Since the first description of an altered RASF phenotype by Fassbender in1983, data have been gathered that allow us to understand the transition from an innocent mesenchymal cell to a destructive cell that plays a leading role in established RA. Early studies showed that synovial cells proliferate, attach and invade joint structures even before inflammatory cells migrate into the synovium. Apart from secretion of chemotactic proteins, RASFs produce a wide range of proinflammatory cytokines and effector molecules. Being the source of cyclo-oxygenase (COX)-2in the synovial lining, RASFs are linked to a currently intensively discussed system that is involved in regulation of synovial inflammatory pathways, namely the COX-1/COX-2system. Functional disability of the joints through progressive degradation of cartilage and bone is a hallmark of RA. Known effector molecules in the destruction of articular cartilage and bone are matrix metalloproteinases (MMPs) and cathepsins. RASFs at sites of invasion or within the synovial lining layer are a major source of MMPs and cathepsins, and drive RA joint destruction via these enzymes.Mast cells have long been recognized to reside in the normal human joint, where they constitute almost3%of nucleated cells residing within70μm of the synovial surface; but increase strikingly in number in rheumatoid arthritis. Rheumatoid joints exhibit a6-to25-fold increase in the number of mast cells compared with normal synovium obtained at orthopedic surgery or autopsy. Within the rheumatoid synovium, mast cells expand dramatically, with a particular predominance in the sublining tissue. In addition, they populate the pannus, with localization at the junction of pannus and cartilage and in areas where pannus is invading the cortical bone. Given the broad spectrum of activity of this lineage, it has for decades been considered probable that mast cells are involved in the pathophysiology of synovitis. A hallmark of mast cell activation is degranulation, the release of granules contents either by rapid exocytosis of the majority of granules (anaphylactic degranulation) or by the more measured release of a subset of granules (piecemeal degranulation). Boseila reported that the mast cells might have a role in the pathology of rheumatoid arthritis in1961. Not until2002, David M. Lee found that the serum of K/B×N could not induce the arthritis in W/WV and Sl/Sld mice which have little mast cells, but the arthritis could be induced after the implantation of wild type mast cells.Cathepsin S is a lysosomal enzyme that belongs to the papain family of cysteine proteases. This protein is expressed by antigen presenting cells including macrophages, B-lymphocytes, dendritic cells and microglia. This enzyme has a critical role in antigen presentation. In general, major histocompatibility complex, class Ⅱ selects small peptide fragments for presentation on the surface of the antigen-presenting immune cells. Particularly, cathepsin S participates in a degradation of polypeptide Li that prevents loading the antigen into the complex. The secreted cathepsin S cleaves some extracellular matrix (ECM) proteins. The list of proposed cathepsin S substrates includes laminin, fibronectin elastin, osteocalcin and some collagens. It has been proved that Cathepsin S was involved in many diseases, such as Alzheimer’s disease, atherosclerosis, asthma, chronic obstructive pulmonary disease, tumor, osteoarthritis, neuralgia and several autoimmune diseases. Decades ago, it was showed that the mice knocked out Cathepsin S had a low sensitivity to CIA. Since then, inhibition of Cathepsin S was tried to treat rheumatoid arthritis. Cathepsin S was mainly expressed in macrophage, little in fibroblasts. The level of Cathepsin S in synovial fluid is highly increased in RA patients. Clik60, a specific inhibitor of Cathepsin S, could reduce the infiltration of lymphocytes in the mouse model of primary Sjogren syndrome, the autoantibody inhibited, the symptom relived.At present, the research on Cathepsin S is still less. Most of the research is focus on the Cathepsin S inhibitor, chronic neuralgia and tumor. A hypothesis was proposed on the characters of Cathepsin S that Cathepsin S might has an important role in the pannus formation, in the degradation of extracellular matrix, besides the antigen presentation; and the functions of Cathepsin S might effect mainly in the early stage of rheumatoid arthritis. To test this hypothesis, a clinical trial was experiment first.29patients of rheumatoid arthritis were enrolled. It is proved that Cathepsin S is increased in the blood samples of RA patients, and the level of Cathepsin S is linear correlated with the score of DAS-28and the level of rheumatoid factor. Then mice peritoneal macrophages, synovial fibroblasts, articular chondrocytes and bone morrow-derived mast cells were primary cultured, the expression of Cathepsin S and collagen Ⅱ in culture supernatant were detected.In this research,29patients of rheumatoid arthritis were enrolled,5of them had7times of follow-up, so there were36samples at last. The expression of Cathepsin S in the samples was much higher than that of the control. The level of Cathepsin S was linear correlated with the disease activity of rheumatoid arthritis and the level of rheumatoid factor, but was not correlated with the level of anti-CCP. On the contrary, the level of collagen Ⅱ was not correlated with the disease activity of rheumatoid arthritis, the level of rheumatoid factor or the level of anti-CCP, but was negative correlated with the level of Cathepsin S. On the basis of these results,18patients of rheumatoid arthritis,7patients of knee osteoarthritis and10patients of gout were enrolled and the synovial fluid of the knee were collected. We found that the level of Cathepsin S in the rheumatoid arthritis patients and the gout was higher than that of the knee osteoarthritis patients. IL-17A was expressed in all synovial fluid of the rheumatoid arthritis patients, but few patients of osteoarthritis and gout, in a low level. The level of collagen Ⅱ was usually thought an indication of cartilage destruction. But in our research, collagen Ⅱ was not detected in the synovial fluid of osteoarthritis patients, but a high level of the rheumatoid arthritis patients.It was shown that a high expression of Cathepsin S existed in the serum and the synovial fluid of the rheumatoid arthritis patients, the level of Cathepsin S was linear correlated with the level of the disease activity and the rheumatoid factor, but not the basic level of anti-CCP. It could also propose that Cathepsin S might take apart in the inflammatory arthritis.Four types cells were primary cultured in this research, including synovial fibroblasts, peritoneal macrophages, articular chondrocytes and bone marrow-derived mast cells. We improved the primary culture method of synovial fibroblasts, abandon the synovium in the knees, but choose the lager size one surrounding the hip joints, and get a higher purity. It was proved that these cells are synovial fibroblasts by the flow cytometry of Vimentin, CD90.2and CD54. The commercially available recombinant mouse IL-3was fail to effect on the primary culture of bone marrow-derived mast cells. We change it with purified IL-3protein by the bacterial strain and get a satisfied result.When articluar chondrocytes co-cultured with synovial fibroblasts, the secretion of collagen Ⅱ could be restrained by the synovial fibroblasts, or the collagen Ⅱ could be destroyed by the synovial fibroblasts. When articular chondrocyts co-cultured with bone marrow-derived mast cells, the collagen Ⅱ in the supernatant could be enhanced by APC366, a kind of tryptase inhibitor, but restrained by C48/80, an activator of mast cells. It was shown that activated mast cells could decrease the secretion of collagen Ⅱ by articular chondrocytes, or activated mast cells destroy the collagen Ⅱ. Cathepsin S could be detected only in the supernatant of macrophages, and the expression effected by TNF-α. When mast cells co-cultured with macrophages, the expression of Cathepsin S in macrophages could be increased by mast cells, and the expression could be further increased by APC366, a kind of tryptase inhibitor. But Cathepsin S might be inhibited by completely activated mast cells.Even if we put a great deal of energy in this research, but we got a lot of negative results, such as the level of IL-17or IL-1β in the supernatant could not be detected. The band of Cathepsin G, C, K or S could not be found in Western Blot electrophoresis in the protein extraction of mast cells, synovial fibroblasts or articular chondrocyts, only a weak band of Cathepsin S could be found in the protein extraction of macrophages.