Control of fibroblast‐like synoviocyte proliferation by macrophage migration inhibitory factor

Objective

The hyperplasia of fibroblast‐like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation.

Methods

Human RA FLS were treated with recombinant MIF, interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and/or anti‐MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor κB (NF‐κB) and mitogen‐activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively.

Results

FLS proliferation was significantly increased by MIF. IL‐1β and TNFα also induced proliferation, but these effects were prevented by neutralization with anti‐MIF mAb. Activation of NF‐κB was induced by IL‐1β, but not by MIF. Anti‐MIF mAb had no effect on IL‐1β–induced NF‐κB nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal–regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF‐κB antagonism, prevented the effect of MIF on FLS proliferation.

Conclusion

These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL‐1β and TNFα. In addition, the effects of MIF on FLS activation are independent of NF‐κB, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

Interleukin‐18 enhances monocyte tumor necrosis factor α and interleukin‐1β production induced by direct contact with T lymphocytes: Implications in rheumatoid arthritis

Objective

At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up‐regulation of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL‐18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA).

Methods

Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL‐1β, IL‐10, and IL‐18 were measured by enzyme‐linked immunosorbent assay. Expression of adhesion molecules, IL‐18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF‐κB p65, phosphorylated IκBα, and phosphatidylinositol 3‐kinase (PI 3‐kinase) p110 was analyzed by Western blotting.

Results

IL‐18 dose‐dependently enhanced the production of IL‐1β and TNFα, but not IL‐10, by monocytes following contact with RA synovial T cells or PHA‐prestimulated T cells. NF‐κB inhibitors N‐acetyl‐L ‐cysteine and Bay 11‐7085 and PI 3‐kinase inhibitor LY294002 inhibited the enhancing effects of IL‐18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF‐κB in the nucleus, phosphorylated IκB, and PI 3‐kinase were confirmed in monocytes cocultured with PHA‐prestimulated T cells, and the levels were further increased by stimulation with IL‐18. Neutralizing antibody to IL‐18 inhibited monocyte activation induced by direct contact with PHA‐prestimulated T cells. Via cell–cell contact, PHA‐prestimulated T cells increased autocrine production of IL‐18 by monocytes, which was mediated by activation of the NF‐κB and PI 3‐kinase pathways, and up‐regulated the expression of the IL‐18 receptor in monocytes. IL‐18 up‐regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) on monocytes. Blocking the binding of the TNF receptors VCAM‐1 or ICAM‐1 on monocytes to their ligands on stimulated T cells suppressed the IL‐18–enhanced production of TNFα and IL‐1β in monocytes induced by contact with PHA‐prestimulated T cells.

Conclusion

IL‐18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF‐κB and PI 3‐kinase pathways. IL‐18 up‐regulates the expression of the TNF receptors VCAM‐1 and ICAM‐1 on monocytes, which mediate the enhancing effects of IL‐18 on T cell–monocyte contact.

     

Bradykinin potentiates cytokine‐induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression

Objective

Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin‐1 (IL‐1)–induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation‐induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL‐1 and tumor necrosis factor α (TNFα), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine‐induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation).

Methods

Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG‐63 and in mouse calvarial bones. The role of NF‐κB and MAP kinases was studied using pharmacologic inhibitors.

Results

PGE₂ formation and cyclooxygenase 2 (COX‐2) protein expression were induced by IL‐1β and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE₂‐dependent enhancement of RANKL. The enhancements of PGE₂ formation and COX‐2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF‐κB resulted in abolishment of the PGE₂ response with only slight inhibition of COX‐2.

Conclusion

Kinin B1 and B2 receptors synergistically potentiate IL‐1– and TNFα‐induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX‐2, resulting in increased RANKL. The synergistic stimulation is dependent on NF‐κB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.

     

Outside‐to‐inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin‐1β production and deficient apoptosis of rheumatoid arthritis monocytes

Objective

Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and inhibitors of monocytic cytokines are highly efficient agents for treatment of the disease. The aim of this study was to analyze the effects of a therapeutic anti–tumor necrosis factor α (anti‐TNFα) antibody on monocytes from patients with RA and healthy control subjects.

Methods

Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti‐TNFα antibody or IgG. Annexin V staining, caspase activation, poly(ADP‐ribose) polymerase cleavage, and DNA staining with propidium iodide were used to analyze apoptosis. The signaling events elicited in monocytes by infliximab were analyzed by Western blotting and electromobility shift assay.

Results

Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNFα, spontaneous in vitro production of interleukin‐1β (IL‐1β), and a decreased rate of spontaneous ex vivo apoptosis. Incubation with infliximab induced significantly increased apoptosis in monocytes from patients with RA but not in monocytes from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after ligation by infliximab and was independent of caspase activation. Instead, transmembrane TNF reverse signaling inhibited the constitutive NF‐κB activation in RA monocytes, suppressed IL‐1β secretion, and normalized spontaneous in vitro apoptosis. This normalization was reversible by the addition of exogenous IL‐1β.

Conclusion

This study demonstrates that outside‐to‐inside signaling through transmembrane TNF after ligation by infliximab inhibits constitutive NF‐κB activation and suppresses spontaneous IL‐1β production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects observed during treatment with TNFα inhibitors.

     

Inhibition of inflammatory bone erosion by constitutively active STAT‐6 through blockade of JNK and NF‐κB activation

Objective

NF‐κB and JNK signaling pathways play key roles in the pathogenesis of inflammatory arthritis. Both factors are also activated in response to osteoclastogenic factors, such as RANKL and tumor necrosis factor α. Inflammatory arthritis and bone erosion subside in the presence of antiinflammatory cytokines such as interleukin‐4 (IL‐4). We have previously shown that IL‐4 inhibits osteoclastogenesis in vitro through inhibition of NF‐κB and JNK activation in a STAT‐6–dependent manner. This study was undertaken to investigate the potential of constitutively active STAT‐6 to arrest the activation of NF‐κB and JNK and to subsequently ameliorate the bone erosion associated with inflammatory arthritis in mice.

Methods

Inflammatory arthritis was induced in wild‐type and STAT‐6–null mice by intraperitoneal injection of arthritis‐eliciting serum derived from K/B×N mice. Bone erosion was assessed in the joints by histologic and immunostaining techniques. Cell‐permeable Tat‐STAT‐6 fusion proteins were administered intraperitoneally. Cells were isolated from bone marrow and from joints for the JNK assay, the DNA‐binding assays (electrophoretic mobility shift assays), and for in vitro osteoclastogenesis.

Results

Activation of NF‐κB and JNK in vivo was increased in extracts of cells retrieved from the joints of arthritic mice. Cell‐permeable, constitutively active STAT‐6 (i.e., STAT‐6‐VT) was effective in blocking NF‐κB and JNK activation in RANKL‐treated osteoclast progenitors. More importantly, STAT‐6‐VT protein significantly inhibited the in vivo activation of NF‐κB and JNK, attenuated osteoclast recruitment in the inflamed joints, and decreased bone destruction.

Conclusion

Our findings indicate that the administration of STAT‐6‐VT presents a novel approach to the alleviation of bone erosion in inflammatory arthritis.

     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.

     

Thalidomide dramatically improves the symptoms of early‐onset Sarcoidosis/Blau syndrome: Its possible action and mechanism

Objective

Early‐onset sarcoidosis (EOS), which occurs in children younger than 5 years of age, is associated with granulomatous lesions and a sporadic genetic mutation of the nucleotide‐binding oligomerization domain 2 that causes constitutive NF‐κB activation. The symptoms of EOS can be uncontrollable, progressive, and associated with profound complications. However, appropriate therapy is still under investigation. The aim of this study was to assess the efficacy of thalidomide in patients with severe EOS, based on etiology supporting an initial role of NF‐κB in activation of this disease.

Methods

Thalidomide was given to 2 patients with EOS (a 16‐year‐old girl and an 8‐year‐old boy) at an initial dosage of 2 mg/kg/day, and the dosage was increased if necessary. To elucidate the mechanism of the drug, peripheral blood monocytes were isolated from the patients and stimulated with cytokines (macrophage colony‐stimulating factor, tumor necrosis factor α, and interleukin‐4), and their ability to form multinucleated giant cells (MGCs) and osteoclasts was measured.

Results

Both patients showed dramatic improvement of their clinical symptoms (alleviation of fever and optic nerve papillitis, achievement of a response according to the American College of Rheumatology Pediatric 50 and Pediatric 70 criteria) and laboratory findings. Monocytes from patients with EOS had a greater ability to survive and induce MGCs and osteoclasts than those from healthy control subjects. The formation of MGCs and osteoclasts was inhibited by the presence of thalidomide.

Conclusion

The ability of thalidomide to improve clinical symptoms and laboratory findings in patients with EOS indicates a central role for NF‐κB activity in this disorder. Inhibition of IKK might be a pharmacologic action by which thalidomide down‐regulates NF‐κB signaling. Thalidomide may be an effective medication in patients with severe complications of EOS, including ocular involvement.

     

Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative,but independent,signaling via ERK/MAPK and phosphatidylinositol 3‐kinase/Akt

Objective

To determine whether angiopoietin 1 (Ang‐1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell‐signaling mechanisms of Ang‐1 in the rheumatoid joint.

Methods

Expression of Ang‐1, TIE‐2 (a receptor for Ang‐1), and matrix metalloproteinase 3 (MMP‐3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3‐kinase/Akt pathways and of NF‐κB was determined by Western blotting and an NF‐κB p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin‐Rho and p21‐activated kinase 1. Matrix degradation was examined by induction of proMMP‐3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay.

Results

Ang‐1 stimulated the ERK/MAPK and PI 3‐kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang‐1 activated NF‐κB via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang‐1 directly potentiated the extension of synovial cells in an ERK‐ and Akt‐dependent manner by up‐regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang‐1 induced proMMP‐3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix.

Conclusion

Ang‐1 stimulates the ERK/MAPK and PI 3‐kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang‐1/TIE‐2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint.