Chemokines and Chemokine Receptors as Novel Therapeutic Targets in Rheumatoid Arthritis （RA）： Inhibitory Effects of Traditional Chinese Medicinal Components

Chemokines belong to a large family of inflammatory cytokines responsible for migration and accumulation ofleukocytes at inflammatory sites.Over the past decade,accumulating evidence indicated a crucial role forchemokines and chemokine receptors in the pathophysiology of rheumatoid arthritis(RA).RA is a chronicautoimmune disease in which the synovial tissue is heavily infiltrated by leukocytes.Chemokines play animportant role in the infiltration,localization,retention of infiltrating leukocytes and generation of ectopicgerminal centers in the inflamed synovium.Recent evidence also suggests that identification of inhibitorsdirectly targeting chemokines or their receptors may provide a novel therapeutic strategy in RA.TraditionalChinese medicines(TCMs) have a long history in the treatment of inflammatory joint disease.The basis for theclinical benefits of TCM remains largely unclear.Our studies have led to the identification of numerous novelchemokine/chemokine receptor inhibitors present in anti-inflammatory TCMs.All of these inhibitors werepreviously reported by other researchers to have anti-arthritic effect,which may be attributable,at least inpart,to their inhibitory effect on chemokine and/or chemokine receptor.Therefore,identification of agentscapable of targeting chemokine/chemokine receptor interactions has suggested a mechanism of action forseveral TCM components and provided a means of identifying additional anti-RA TCM.Thus,this approachmay lead to the discovery of new inhibitors of chemokines or chemokine receptors that can be used to treatdiseases associated with inappropriately overactive chemokine mediated inflammatory reactions.Cellular &Molecular Immunology.2004;1(5):336-342.     

Expression of Platelet-Derived Growth Factor Receptors is Induced on Connective Tissue Cells During Chronic Synovial Inflammation

The tissue distribution of the receptor for platelet-derived growth factor (PDGF) was investigated by immunohistochemistry on frozen sections from normal and inflamed synovial tissue using monoclonal antibodies to the receptor. Non-inflamed synovial tissue showed no staining, indicating that PDGF receptor expression is low or absent in normal tissue. In contrast, tissue from synovitis with prominent neovascularization showed a strong staining in the tunica media of the proliferating blood vessels as well as on connective tissue cells in the stroma. Tissue from synovitis with prominent proliferation of synovial lining showed intense staining for PDGF receptors in fibroblast-like cells of the lining and a less intense staining on vascular and connective tissue cells deeper in the stroma. Staining for PDGF receptors was also intense in the pannus tissue close to infiltrated bone and cartilage. In all these forms of synovitis, PDGF receptor staining was associated with increased HLA-DR staining and infiltration of macrophages and T lymphocytes. The finding that PDGF receptor expression is induced in conjunction with the chronic synovial inflammation associated with rheumatoid arthritis and some other forms of arthritides suggests that PDGF may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory disease.     

Differential production of RANTES and MCP-1 in synovial fluid from the inflamed human knee.

Synovial production of chemokines may play an important role in the recruitment of phagocytic leukocytes during inflammation. MCP-1, as well as RANTES mediate many different inflammatory diseases and are important in the recruitment of diverse leukocytes. We set out to study the different production of MCP-1 and RANTES in three different inflammatory conditions of the knee: arthrosynovitis, mechanical trauma, and hyperuricemia. In this study we evaluated if in each pathological condition mentioned above, there was a prevalence in production of one chemokine over the other. ELISA method was used to determine base production of the chemokines in the synovial fluid, serum and in supernatants from activated inflammatory cells. RANTES and MCP-1 messenger RNA (mRNA) was measured by semi-quantitative RT-PCR. Protein expression was detected by Western blot analysis. The synovial fluid cells from the knee of patients affected with arthrosynovitis, trauma, and hyperuricemia, expressed RANTES and MCP-1 and RANTES was produced in higher quantities than MCP-1 in all three pathological conditions. In patients treated with non-steroidal antiinflammatory drugs (NSAD) and dexamethasone, the levels of the two chemokines was reduced in serum and in synovial fluid. In addition, the synovial fluid cells from these patients released less RANTES and MCP-1 when compared to untreated patients. We conclude that in arthrosynovitis, trauma and hyperuricemia, RANTES and MCP-1 are both expressed and RANTES is produced in higher quantities. The fact that these chemokines are found in the three inflammatory diseases suggests that RANTES and MCP-1 are not specific to these inflammatory diseases, however they play a key role in inflammation by recruiting mononuclear leukocytes in the inflamed knee joint.     

The interaction between CXCL10 and cytokines in chronic inflammatory arthritis

There are several chemokines and their receptors involved in the pathogenesis of chronic inflammatory arthritis. Of those, CXCL10 and its receptor, CXCR3, are increased in many kinds of chronic inflammatory arthritis, especially in rheumatoid arthritis (RA). CXCL10 and CXCR3 play important roles in leukocyte homing to inflamed tissues and in the perpetuation of inflammation, and therefore, tissue damage. In addition to its chemotactic effect, CXCL10 may have pleiotropic functions. Our recent studies show that the crosstalk between CXCL10 and receptor activator of NF-κB ligand (RANKL) in inflamed synovial tissue may induce and perpetuate bone destruction in RA. The interaction between CXCL10 and tumor necrosis factor-α (TNF-α) can also contribute to sustained inflammation in RA. One human trial with anti-CXCL10 monoclonal antibody showed therapeutic potential of blocking CXCL10 in RA treatment. Understanding the novel interaction between this chemokine and other chemokines or cytokines may add possible therapeutic applications in inflammatory arthritis.     

PECAM-1 and leukosialin (CD43) expression correlate with heightened inflammation in rat adjuvant-induced arthritis.

A hallmark of both adjuvant-induced arthritis (AIA) and rheumatoid arthritis is chronic joint inflammation characterized by ingress of leukocytes into the inflamed synovial tissue. The timing of expression of adhesion molecules, which govern the ingress of leukocytes, is important in the orchestration of an inflammatory response. We examined the expression of vascular cell adhesion molecule-1 (VCAM-1), sialo adhesin, platelet and endothelial cell adhesion molecule-1 (PECAM-1), and leukosialin (CD43) in AIA, starting at adjuvant injection (day 0), through the peak of inflammation (day 18 postadjuvant injection), until day 54. VCAM-1 is constitutively expressed on the lining layer and ECs and its expression levels do not change throughout the progression of AIA. Sialoadhesin synovial tissue lining cell expression is decreased after adjuvant injection. In contrast, PECAM-1 expression is increased on synovial tissue lining cells on day 7 and is elevated through day 54 (peaking on day 54 with six-fold more cells expressing PECAM-1). PECAM-1 expression on endothelial cells peaks on day 7 with three-fold more cells expressing it, while on macrophages expression maximizes on day 25 with six-fold more cells expressing PECAM-1. CD43 expression is increased on synovial tissue lining cells, macrophages, neutrophils, and lymphocytes on days 18 and 25, before going back to basal levels. The increased expression of PECAM-1 and CD43 on leukocytes at the height of inflammation in AIA suggests important roles for these adhesion molecules in potentially binding their EC ligands resulting in leukocyte ingress into the synovial tissue.     

Compartmentalization of human natural killer cells

Human natural killer (NK) cells are bone marrow-derived cells that are found in the bloodstream, but can extravasate into various tissue sites upon inflammation. NK cells that migrate toward inflamed sites must be activated prior to their extravasation. However, the factors responsible for NK cell compartmentalization are not clearly defined. Resting human NK cells (CD16(-) and CD16(+)) express constitutive chemokine receptors, as well as receptors that have both constitutive and inflammatory functions. Upon activation, NK cells up-regulate the expression of the inflammatory chemokine receptors which facilitate their distribution into inflammatory sites. However, chemokines are not expected to play any role in maintaining resting NK cells in the blood circulation. In contrast, members of the lysolipids which are abundant in the bloodstream may be the major factors responsible for maintaining resting NK cells in the bloodstream, and also for facilitating their extravasation into inflamed tissues. Both resting and activated NK cells express receptors for various lysolipids. Hence, chemoattractants which include chemokines and lysolipids have important roles in determining the compartmentalization of NK cells where resting NK cells are found in the blood circulation, and activated NK cells extravasate into inflamed sites.     

Analysis of homing receptor expression on infiltrating leukocytes in disease states

Chemokines represent a large family of polypeptides that signal through G-protein-coupled receptors and have a role in chemotaxis, leukocyte homing, inflammation, hematopoiesis, angiogenesis and tumor growth. The chemokine/chemokine receptor system acts in coordination with a complex cytokine network to elicit and direct leukocyte infiltration into the inflamed tissue. In addition to promoting movement into the inflamed tissue, the chemokine/chemokine receptor system may also activate infiltrating cells, such as neutrophils and eosinophils, and induce local damage. In recent years, the elucidation of intricate chemokine networks has led to the identification of potential target molecules for therapeutic intervention. Of considerable interest has been the role of chemokine/chemokine receptors in regulating allergic lung inflammation. In this review, techniques to study in situ expression of chemokine receptors in inflamed tissues are presented and discussed.     

Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation

Fibroblasts are important sentinel cells in the immune system and, here, it is proposed that these cells play a critical role in the switch from acute inflammation to adaptive immunity and tissue repair. It is suggested that chronic inflammation occurs because of disordered fibroblast behaviour in which failure to switch off their inflammatory programme leads to the inappropriate survival and retention of leukocytes within inflamed tissue.     

Targeting the chemokine network in renal inflammation

Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocyte recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocyte subpopulations contribute to renal damage by releasing inflammatory and profibrotic cytokines. All intrinsic renal cells are capable of chemokine secretion on stimulation in vitro. Expression of inflammatory chemokines correlates with renal damage and local accumulation of chemokine receptor-bearing leukocytes in a variety of animal models of renal diseases as well as in human biopsy studies. Chemokines and their respective receptors could represent new targets for therapeutic intervention in renal inflammatory disease states that often tend to progress to end-stage renal disease. This article summarizes the present data on the role of chemokines and their receptors in renal inflammation with special emphasis on our efforts to identify the chemokine receptors CCR1 and CCR2 as promising targets for therapeutic intervention.     

Angiogenesis in rheumatoid arthritis: a disease specific process or a common response to chronic inflammation?

Angiogenesis is the formation of new blood vessels from existing vessels. During RA new blood vessels can maintain the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen to the proliferating inflamed tissue. The increased endothelial surface area also creates an enormous capacity for the production of cytokines, adhesion molecules, and other inflammatory stimuli, simultaneously the propagation of new vessels in the synovial membrane allows the invasion of this tissue supporting the active infiltration of synovial membrane into cartilage and resulting in erosions and destruction of the cartilage. This angiogenic phenotype is promoted by several pro-angiogenic molecules, the most potent of which is vascular endothelial growth factor (VEGF). Although angiogenesis is recognized as a key event in the formation and maintenance the infiltration of synovial membrane during RA, it is unclear whether angiogenesis should be considered a specific feature of the disease or a common inflammation driven process. However the emergence of biological therapies, such as anti TNF blockade, has suggested that there are features of the inflammatory response that are not general but contextual to the specificity of the tissue where inflammation occurs, and point out the relevant role of tissue-resident stromal cells in determining the site at which inflammation occurs and the specific features of chronic inflammation such as that occurs in RA.     

Tissue stroma as a regulator of leukocyte recruitment in inflammation

The stromal milieu (cellular and matrix components) helps establish tissue "address-codes" that direct leukocyte behavior in inflamed tissue. Coordinated interactions among the stroma, leukocytes, and ECs dictate which leukocytes are recruited, whether they are retained within the inflamed site, and how long they survive. Herein, we discuss how the stromal milieu influences the leukocyte recruitment cascade. Moreover, we explore how corruption of the stromal phenotype in chronic inflammatory diseases contributes to undesired, continuous recruitment of leukocytes. Emerging complex, multicellular, multilayered (co-)culture models are now addressing the molecular circuitry involved in regulating stromal organization during inflammation. Understanding context-specific changes in pro- or anti-inflammatory agents derived from the stroma, such as IL-6 (and its cofactors), is important for the generation of therapeutic strategies that restore the balance between recruitment and clearance of the inflammatory infiltrate in chronic disease.     

Macrophage inflammatory protein 3alpha-CC chemokine receptor 6 interactions play an important role in CD4+ T-cell accumulation in periodontal diseased tissue

The regulatory role of chemokines and chemokine receptors on specific lymphocyte recruitment into periodontal diseased tissue is poorly characterized. We observed that lymphocytes infiltrating inflamed gingival tissue expressed marked levels of CCR6. In periodontal diseased tissue, the expression of MIP-3alpha mRNA was detected by RT-PCR and further, MIP-3alpha was distributed in the basal layer of gingival epithelial cells, microvascular endothelial cells and the areas of inflammatory cells as shown by immunohistochemistry. Moreover, CCR6-expressing cells infiltrated into periodontal diseased tissue, and the proportion of CCR6-positive CD4+ T cells was significantly elevated in periodontal diseased tissue compared with peripheral blood in the same patients. Furthermore, gingival lymphocytes isolated from patients showed migration toward MIP-3alpha in an in vitro chemotaxis assay in which migration was abrogated by specific antibody to CCR6. Thus, these findings suggested that CCR6 and the corresponding chemokine, MIP-3alpha may have an important regulatory role in specific lymphocyte migration into inflamed periodontal tissue.     

The role of the tec kinase Bruton's tyrosine kinase (Btk) in leukocyte recruitment

Recruitment of leukocytes into inflamed tissue is a key component of the immune system. The activation of integrins on leukocytes is required for their recruitment into the inflamed tissue. Btk is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec-kinase family. It plays a key role in B-cell development and function, and recently published studies revealed important roles of Btk in myeloid cells. Btk might be activated through a variety of receptors leading to activation of integrins as the pivotal element in leukocyte recruitment. This review focuses on the role of Btk in B-lymphocyte homing and in neutrophil recruitment.     

Rheumatoid arthritis and interleukin-32]

Inflammatory cytokine cascade plays a pivotal role in the pathogenesis of rheumatoid arthritis. Recently, a novel human cytokine, interleukin-32, was reported to induce TNF-alpha. Interleukin-32 is expressed mainly in lymphoid tissues and leukocytes, but also in stimulated epithelial cells and synovial fibroblasts. Although the interleukin-32 receptor has not been reported, interleukin-32 can induce other inflammatory cytokines, such as TNF-alpha, interleukin-1beta, and interleukin-6 from monocytes/macrophages in vitro and in vivo, and synergizes with signals from pattern-recognition receptors. Notably, in the inflamed synovial tissues from rheumatoid arthritis patients, interleukin-32 is prominently expressed and correlates with the severity of arthritis and the expression of other cytokines, including TNF-alpha and interleukin-1. In experimental mice models of arthritis, joint injection of interleukin-32 induces joint inflammation, and overexpression of interleukin-32beta in haematopoietic cells exacerbates collagen-induced arthritis. Herein, interleukin-32 plays an important role in the pathogenesis of rheumatoid arthritis.     

The Role of the Tec Kinase Bruton's Tyrosine Kinase (Btk) in Leukocyte Recruitment

Recruitment of leukocytes into inflamed tissue is a key component of the immune system. The activation of integrins on leukocytes is required for their recruitment into the inflamed tissue. Btk is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec-kinase family. It plays a key role in B-cell development and function, and recently published studies revealed important roles of Btk in myeloid cells. Btk might be activated through a variety of receptors leading to activation of integrins as the pivotal element in leukocyte recruitment. This review focuses on the role of Btk in B-lymphocyte homing and in neutrophil recruitment.     

Chemokine receptors in inflammation: an overview

Chemokine receptors play a key role in directing the migration of inflammatory cells into various injured or infected organs. However, migration of inflammatory cells into tissues can in itself be a cause and amplifier of tissue damage and disease, particularly in chronic autoimmune or allergic disorders. On this basis, much effort is currently devoted at the identification of molecular signals regulating the recruitment of inflammatory cells into tissues and at developing novel strategies to inhibit discrete pathways in this process. Great progress has recently been made in identification of a number of chemokine receptors involved in the process of leukocyte migration. The challenge is now to elucidate the specific contribution and involvement of the different receptors in distinct inflammatory processes and diseases and to prove that interference with any of these pathways may lead to development of novel therapeutics.     

The effect of sulfasalazine on rheumatoid arthritic synovial tissue chemokine production

Rheumatoid arthritis (RA) is an aggressive inflammatory disease in which chemokines are thought to recruit leukocytes and induce angiogenesis. The aim of this study was to investigate the effects of sulfasalazine (SASP) and its metabolites, sulfapyridine (SP), and 5-aminosalicylic acid (5ASA) on chemokine production by RA synovial tissue explants and interleukin (IL)-1beta-stimulated RA synovial tissue fibroblasts using enzyme-linked immunosorbent assays and flow cytometry. Synovial tissue explants from RA patients secreted a decreased amount of the chemokines IL-8 and growth-related gene product alpha (GROalpha) when treated with SASP over a broad range of concentrations based on the typical clinical dosage of 2 g/day. SP had a significant effect in that it decreased RA synovial tissue explant secretion of IL-8 (22%), GROalpha (55%), and monocyte chemotactic protein-1 (MCP-1) (42%) (P < 0.05). 5ASA had no effect on RA synovial tissue explant production of IL-8 and MCP-1, while increasing GROalpha production. In IL-1beta-stimulated RA synovial tissue fibroblasts, SASP significantly increased chemokine secretion, while SP significantly decreased IL-8 (24%) and GROalpha (21%) secretion (P < 0.05). Flow cytometry showed that the number of IL-8 expressing RA synovial tissue fibroblasts did not significantly change following SP treatment. These data suggest that SASP may function to reduce inflammation in RA through the effects of its metabolite SP to reduce the secretion of the inflammatory chemokines IL-8, GROalpha, and MCP-1.