Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model.

Transforming growth factor beta is a potent immunomodulator with both pro- and antiinflammatory activities. Based on its immunosuppressive actions, exogenous TGF-beta has been shown to inhibit autoimmune and chronic inflammatory diseases. To further explore the potential therapeutic role of TGF-beta, we administered a plasmid DNA encoding human TGF-beta1 intramuscularly to rats with streptococcal cell wall-induced arthritis. A single dose of 300 microg plasmid DNA encoding TGF-beta1, but not vector DNA, administered at the peak of the acute phase profoundly suppressed the subsequent evolution of chronic erosive disease typified by disabling joint swelling and deformity (articular index = 8.17+/-0. 17 vs. 1.25+/-0.76, n = 6, day 26, P < 0.01). Moreover, delivery of the TGF-beta1 DNA even as the chronic phase commenced virtually eliminated subsequent inflammation and arthritis. Both radiologic and histopathologic as well as molecular evidence supported the marked inhibitory effect of TGF-beta1 DNA on synovial pathology, with decreases in the inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and the expression of proinflammatory cytokines that characterize this model. Increases in TGF-beta1 protein were detected in the circulation of TGF-beta1 DNA-treated animals, consistent with the observed therapeutic effects being TGF-beta1 dependent. These observations provide the first evidence that gene transfer of plasmid DNA encoding TGF-beta1 provides a mechanism to deliver this potent cytokine that effectively suppresses ongoing inflammatory pathology in arthritis.     

Reversal of acute and chronic synovial inflammation by anti- transforming growth factor beta

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.     

Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor beta 1 gene (TGF beta 1).

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement membrane thickening which characterize this disease.     

Severe inflammatory arthritis and lymphadenopathy in the absence of TNF.

It has been postulated that TNF has a pivotal role in a cytokine cascade that results in joint inflammation and destruction in rheumatoid arthritis (RA). To evaluate this, we examined the response of TNF-deficient (Tnf(-/-)) mice in two models of RA. Collagen-induced arthritis (CIA) was induced by injection of chick type II collagen (CII) in CFA. Tnf(-/-) mice had some reduction in the clinical parameters of CIA and, on histology, significantly more normal joints. However, severe disease was evident in 54% of arthritic Tnf(-/-) joints. Tnf(-/-) mice had impaired Ig class switching, but preserved T cell proliferative responses to CII and enhanced IFN-gamma production. Interestingly, CII-immunized Tnf(-/-) mice developed lymphadenopathy and splenomegaly associated with increased memory CD4(+) T cells and activated lymph node B cells. Acute inflammatory arthritis was also reduced in Tnf(-/-) mice, although again some mice exhibited severe disease. We conclude that TNF is important but not essential for inflammatory arthritis; in each model, severe arthritis could proceed even in the complete absence of TNF. These results call into doubt the concept that TNF is obligatory for chronic autoimmune and acute inflammatory arthritis and provide a rationale for further studies into TNF-independent cytokine pathways in arthritis.     

Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.     

Therapeutic effect of long‐interval repeated intravenous administration of human umbilical cord blood‐derived mesenchymal stem cells in DBA/1 mice with collagen‐induced arthritis

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen‐induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long‐interval repeated intravenous administration of human umbilical cord blood‐derived (hUCB)‐MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long‐interval repeated intravenous administration of hUCB‐MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB‐MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin‐O staining, and toluidine blue staining. We used real‐time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB‐MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB‐MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB‐MSCs also significantly decreased IL‐1 beta protein levels in multimedium and multihigh groups and IL‐6 protein levels in all hUCB‐MSCs‐treated groups. Furthermore, mRNA levels of IL‐1 beta and IL‐6 were decreased significantly in all hUCB‐MSCs‐treated groups, whereas the expression of anti‐inflammatory cytokine IL‐10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long‐interval repeated intravenous administration of hUCB‐MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose‐dependent manner.     

Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation

Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of cytokine signaling. To investigate the role of SOCS-1 in regulating inflammatory and immune responses in disease, acute inflammatory arthritis was induced in mice lacking SOCS-1. Expression of SOCS-1 protein was detected within synovial granulomas and pannus tissue of WT mice by day 7 following induction of acute arthritis. The severity of synovial inflammation and joint destruction at the peak of disease was greater in the absence of SOCS-1, although disease resolution occurred normally. There was an increased percentage of myeloid cells infiltrating the synovium in mice lacking SOCS-1, and SOCS-1 promoter activity was present in synovial macrophages, lymphocytes, and fibroblasts, but not granulocytes. The T cell response in draining LNs was also dysregulated, as popliteal LNs from mice lacking SOCS-1 contained approximately fivefold more cells at the peak of acute arthritis. These cells were hyperproliferative on exposure to antigen in vitro, and purified splenic CD4(+) T cells from mice lacking SOCS-1 proliferated more strongly in response to stimulation with anti-CD3. Reporter gene expression was detected in CD4(+) T cells bearing the activation markers CD25, CD44, and CD69. SOCS-1 is therefore expressed in hematopoietic and nonhematopoietic cell types in vivo and is an important regulator of acute inflammatory arthritis and of CD4(+) T cell activation.     

Leukemia inhibitory factor is expressed in cartilage and synovium and can contribute to the pathogenesis of arthritis.

This study reports on leukemia inhibitory factor (LIF) in human articular connective tissues. Biologically active LIF is present in synovial fluids from patients with osteoarthritis and at higher titers in samples from patients with rheumatoid arthritis. Cultured human synoviocytes and articular chondrocytes produced biologically active LIF and synthesized and secreted LIF proteins that migrated in SDS PAGE at approximately 43 kD. This was increased after stimulation with IL-1 beta. Chondrocytes in serum-containing cultures expressed the 4.2-kb LIF mRNA. IL-1 beta, LPS, and to a lesser extent tumor necrosis factor-alpha induced LIF gene expression. LIF autoinduced its mRNA and this provides evidence for an effect of this cytokine on function of joint tissue cells. Among a series of growth factors tested, transforming growth factor (TGF beta), including the isoforms TGF-beta1, TGF-beta 2, and TGF-beta 3, platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor induced this cytokine gene but differed with respect to the duration of their effects. Cultured synoviocytes expressed the LIF gene in response to the same set of peptide regulatory factors. Analysis of signal transduction pathways showed that PMA increased LIF mRNA, whereas calcium ionophore and cAMP had no detectable effects. Cycloheximide was a potent LIF mRNA inducer and dexamethasone inhibited LIF induced by PMA or IL-1 beta. Cartilage organ cultures and synovial tissues stimulated with IL-1 expressed high levels of LIF mRNA as demonstrated by in situ hybridization. These results identify LIF as a new cytokine that is produced by joint tissue cells and is overexpressed in arthritis. The induction of this cytokine by factors that are present during joint inflammation and the effects of LIF on connective tissue cells suggest that LIF is a mediator that can contribute to the pathogenesis of arthritis.     

Tumour necrosis factor as a therapeutic target in rheumatoid arthritis and other chronic inflammatory diseases: the clinical experience with infliximab (REMICADE)

Therapeutic strategies that aim to neutralise the important pro-inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have gained considerable prominence in the therapy of chronic inflammatory diseases, notably rheumatoid arthritis and Crohn's disease. This drug focus review will concentrate on the antitumour necrosis factor antibody infliximab (Remicade), which has been approved for the treatment of rheumatoid arthritis and Crohn's disease in both the US and Europe. In addition, infliximab is under investigation for several other indications, mainly inflammatory rheumatic diseases. Clinical trials have been persuasive that infliximab is both safe and effective, and it has been proven to be far superior to the conventional drug therapy in both rheumatoid arthritis and Crohn's disease. Remarkably, infliximab in combination with methotrexate controls both the inflammatory joint symptoms and the progression of joint damage, which renders it a very attractive therapeutic option in moderate to severe, therapy-resistant rheumatoid arthritis.     

Interleukin 1 receptor dependence of serum transferred arthritis can be circumvented by toll-like receptor 4 signaling

Inflammatory arthritis is associated with the release of a network of key cytokines. In T cell receptor transgenic K/BxN mice interleukin (IL)-1 plays a key role in joint swelling and destruction, as suggested by the ability of anti-IL-1receptor (IL-1R) antibody treatment to delay the onset and slow the progression of this disease. This mechanism is dependent on the signaling pathway intermediary myeloid differentiation factor 88 (MyD88), such that neither IL-1R nor MyD88-deficient mice developed visually detectable synovitis after transfer of arthritogenic sera. The Toll-like receptors (TLRs) share the same signaling pathway through MyD88 as the IL-1R. The administration of a TLR-4 ligand, lipopolysaccharide, concomitant with arthritogenic serum in IL-1 receptor-deficient mice resulted in acute paw swelling, but not in MyD88-deficient mice. Also, serum transferred arthritis was not sustained in TLR-4 mutant mice compared with controls. These results suggest that innate immune functions via TLR-4 might perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation.     

SOCS-3 negatively regulates innate and adaptive immune mechanisms in acute IL-1-dependent inflammatory arthritis

RA is an autoimmune disease characterized by sustained imbalance between pro- and antiinflammatory immune mechanisms. The SOCS proteins are negative regulators of cytokine signaling, but to date there has been little information on their function in disease. The generation of Socs3(-/Delta vav) mice, which lack SOCS-3 in the hematopoietic and endothelial cell compartment, allowed us to explore the role of endogenous SOCS-3 during acute inflammatory arthritis. Joint inflammation in Socs3(-/Delta vav) mice was particularly severe and was characterized by increased numbers of neutrophils in the inflamed synovium, bone marrow, peripheral blood, and spleen. These features were most likely due to increased production of and enhanced responsiveness to G-CSF and IL-6 during arthritis in these mice. Local osteoclast generation and bone destruction were also dramatically increased in the absence of SOCS-3, as was macrophage activation. Finally, SOCS-3 was found to negatively regulate CD4+ T lymphocyte activation, including production of the pleiotropic cytokine IL-17. The absence of SOCS-3 therefore had dramatic effects in this disease model, with a broader impact on cellular responses than SOCS-1 deficiency. These findings provide direct in vivo evidence that endogenous SOCS-3 is a critical negative regulator of multiple cell types orchestrating inflammatory joint disease.     

SKL-2841, a dual antagonist of MCP-1 and MIP-1 beta, prevents bleomycin-induced skin sclerosis in mice.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis and excessive collagen deposition in the skin and various internal organs. In early stages of SSc, the dermis reveals infiltration of inflammatory cells associated with increased collagen synthesis. SKL-2841 was initially synthesized as a novel small molecule antagonist of MCP-1. In this study, we indicated that SKL-2841 also exerts anti-chemotactic activity for MIP-1 beta in mouse spleen cells. In the early stages of bleomycin-induced skin lesions, immunohistochemical analysis showed the expression of both MCP-1 and MIP-1 beta in dermal inflammatory cells. Moreover, intraperitoneal administration of SKL-2841 suppressed the infiltration of inflammatory mononuclear cells and polymorphonuclear cells in the acute phase and also significantly suppressed fibrillization in the chronic phase in bleomycin-induced scleroderma, compared with PBS treatment. These findings suggest that SKL-2841 has potential as a compound for the treatment of conditions associated with skin fibrosis such as SSc.     

Interleukin-23: a key cytokine in inflammatory diseases

Interleukin-23 (IL-23) is a pro-inflammatory cytokine composed of two subunits, p19 and p40. The p40 subunit is shared with IL-12. IL-23 and IL-12 have different receptors and different effects. Whereas IL-12 induces development of Th1 cells, which produce interferon-γ, IL-23 is involved in differentiation of Th17 cells in a pro-inflammatory context and especially in the presence of TGF-β and IL-6. Activated Th17 cells produce IL-17A, IL-17F, IL-6, IL-22, TNF-α, and GM-CSF. Inflammatory macrophages express IL-23R and are activated by IL-23 to produce IL-1, TNF-α, and IL-23 itself. These effects identify IL-23 as a central cytokine in autoimmunity and a highly promising treatment target for inflammatory diseases. IL-23 is found in the skin of patients with psoriasis, in the bowel wall of patients with chronic inflammatory bowel disease, and in synovial membrane of patients with rheumatoid arthritis. IL-23 is involved in osteoclastogenesis, independently from IL-17, via induction of RANKL expression. Debate continues to surround the role for IL-23 in the pathophysiology of inflammatory joint diseases (rheumatoid arthritis and spondyloarthritis). Ustekinumab, which inhibits IL-12 and IL-23 by blocking p40, has been found effective in cutaneous psoriasis and psoriatic arthritis, as well as in Crohn's disease. Treatments that specifically target IL-23 (antibodies to p19) are being developed.     

Therapeutic potential of targeting IL-1 and IL-18 in inflammation

Interleukin (IL)-1 and IL-18 are cytokines that play a major role in autoimmune and inflammatory human disease. Both cytokines drive a wide range of pro-inflammatory effector networks in many cell types and use common signal transduction cascades. IL-1, IL-18 and other members of the IL-1 superfamily are expressed at elevated levels in tissue and fluid samples isolated from patients with many chronic inflammatory diseases. These cytokines are primary drivers in acute and chronic animal models of inflammation and their blockade has been shown to ameliorate disease in preclinical studies. Biological agents that target IL-1 have demonstrated efficacy in patients with rheumatoid arthritis, and further agents targeting IL-1 or IL-18 neutralisation are in clinical development. The potential for such agents spans human disease where tissue destruction is a primary end point of cytokine action.     

Alternative for Anti-TNF Antibodies for Arthritis Treatment

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-α antibodies has shown its efficacy in rheumatoid arthritis (RA) and is now widely used. Nevertheless, some patients currently treated with anti-TNF-α remain refractory or become nonresponder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. In this study, we investigated in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α triplex-forming oligonucleotide (TFO), as judged from effects on two rat arthritis models. The inhibitory activity of this TFO on articular cells (synoviocytes and chondrocytes) was verified and compared to that of small interfering RNA (siRNA) in vitro. The use of the anti-TNF-α TFO as a preventive and local treatment in both acute and chronic arthritis models significantly reduced disease development. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-α TFO modulates arthritis in vivo, thus providing proof-of-concept that it could be used as therapeutic tool for TNF-α-dependent inflammatory disorders.     

Attenuation of colitis in the cotton-top tamarin by anti-alpha 4 integrin monoclonal antibody.

Recent studies have demonstrated the induced expression of endothelial adhesion molecules including E-selectin (also called endothelial leukocyte adhesion molecule-1), vascular cell adhesion molecule and intercellular adhesion molecule in actively involved mucosa of patients with ulcerative colitis and Crohn's disease. Similar induction has been demonstrated in the colon of the Cotton-top tamarin (CTT), a New World primate that experiences a spontaneous acute and chronic colitis resembling ulcerative colitis. To assess the potential importance of leukocyte adhesion as a necessary step in acute colitis, the effect of parenteral mAb directed against adhesion molecules on CTT colitis was evaluated in placebo-controlled blinded trials. Serial administration of either of two anti-E-selectin mAb designated BB11 and EH8 effectively coated endothelial surfaces expressing this vascular adhesion molecule. Although colitis activity was slightly diminished after the 10-d treatment period in CTT receiving either BB11 or EH8, this reduction was not significantly different than that seen in animals given a placebo control when assessed by a previously validated standardized scale of inflammatory activity: mean histologic activity grade 2.2 +/- 0.2 pretreatment vs 1.5 +/- 0.5 posttreatment in group receiving mAb and 2.1 +/- 0.1 pretreatment vs 1.3 +/- 0.5 posttreatment in the placebo group (P > 0.2). In contrast, administration of an anti-alpha 4 integrin mAb designated HP1/2 that binds VLA4 (alpha 4 beta 1) and presumably alpha 4 beta 7 integrins resulted in significant attenuation of acute colitis when compared to both pretreatment activity index (P = 0.005) and the placebo control group (P < 0.01): mean histologic activity grade 1.6 +/- 0.3 pretreatment vs 0.2 +/- 0.1 posttreatment in the group receiving HP1/2 and 1.8 +/- 0.5 pretreatment and 1.2 +/- 0.2 posttreatment in the placebo control group. These studies using a model of spontaneous colitis in the CTT demonstrate the feasibility of modulation of leukocyte-vascular adhesion and/or other integrin-mediated events possibly including T cell aggregation and T cell-stromal interactions, as well as lymphocyte homing. These results suggest both that these processes are important and possibly essential elements in sustaining acute colitis and that their disruption may result in therapeutic benefit.     

Inhibition of spinal p38 MAPK prevents articular neutrophil infiltration in experimental arthritis via sympathetic activation

The central nervous system controls the innate immunity by modulating efferent neuronal networks. Recently, we have reported that central brain stimulation inhibits inflammatory responses. In the present study, we investigate whether spinal p38 mitogen‐activated protein kinase (MAPK) affects joint inflammation in experimental arthritis. Firstly, we observed that intra‐articular administration of zymosan in mice induces the phosphorylation of the spinal cord p38 MAPK. In addition, we demonstrated that spinal p38 MAPK inhibition with intrathecal injection of SB203580, a conventional and well‐characterized inhibitor, prevents knee joint neutrophil recruitment, edema formation, experimental score and cytokine production. This local anti‐inflammatory effect was completely abolished with chemical sympathectomy (guanethidine) and beta‐adrenergic receptors blockade (nadolol). In conclusion, our results suggest that pharmacological strategies involving the modulation of spinal p38 MAPK circuit can prevent joint inflammation via sympathetic networks and beta‐adrenoceptors activation.