In vitro and in vivo efficacy of a recombinant immunotoxin against folate receptor beta on the activation and proliferation of rheumatoid arthritis synovial cells

OBJECTIVE: To investigate the effects of the recombinant immunotoxin dsFv anti-FRbeta-PE38, which consists of the disulfide-stabilized Fv fragment (dsFv) of the anti-folate receptor beta (anti-FRbeta) antibody and the 38-kd portion of Pseudomonas exotoxin A (PE38), on the activation and proliferation of cells that function in inflammatory and degradative processes in rheumatoid arthritis (RA) synovial tissue. METHODS: The Ig VH-PE38 fusion protein and the Ig VL protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins. The effects of dsFv anti-FRbeta-PE38 on the activation and proliferation of cells in RA synovial tissue were investigated by immunohistochemistry; the numbers of cells expressing CD68, vascular cell adhesion molecule 1, angiopoietin 1, CD34, proliferating cell nuclear antigen, and interleukin-6 and the numbers of apoptotic cells were counted in RA synovial tissue engrafted into SCID mice treated or not treated with dsFv anti-FRbeta-PE38. The effects of dsFv anti-FRbeta-PE38 on the generation of osteoclasts from RA adherent synovial mononuclear cells in vitro was investigated by counting the number of resorption pits on dentin slices treated or not treated with dsFv anti-FRbeta-PE38. RESULTS: Administration of dsFv anti-FRbeta-PE38 reduced the numbers of macrophages, activated fibroblast-like cells, endothelial cells, and proliferating cells and increased the numbers of apoptotic cells in RA synovial tissue engrafted into SCID mice. In vitro, the generation of osteoclasts from RA adherent synovial mononuclear cells was largely suppressed by treatment with dsFv anti-FRbeta-PE38. CONCLUSION: Our findings show that dsFv anti-FRbeta-PE38 immunotoxin would be a promising tool for the treatment of RA synovitis, especially when administered intraarticularly.     

Effectiveness of anti–folate receptor β antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages

Objective

To define the distribution of folate receptor β (FRβ)–expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti‐FRβ monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor α (TNFα) production by adherent synovial mononuclear cells from RA patients.

Methods

Anti‐FRβ mAb were produced by immunizing mice with FRβ‐transfected murine pre–B cells. The distribution of the FRβ antigen was examined by immunohistochemical analysis using anti‐FRβ mAb and macrophage‐specific anti‐CD163 mAb. Anti‐FRβ mAb was chemically crosslinked with truncated PEA. FRβ‐expressing macrophages were produced by the transfection of adenovirus vector containing the FRβ gene. Apoptotic cells were detected by staining with propidium iodide. TNFα was measured by enzyme‐linked immunosorbent assay.

Results

FRβ‐expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRβ‐expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRβ‐transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFα production by adherent RA synovial mononuclear cells.

Conclusion

We demonstrated the limited distribution of FRβ‐expressing cells in various tissues. The immunotoxin targeting FRβ‐expressing cells will provide a therapeutic tool for rheumatoid synovitis.

     

Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fcγ receptor I–directed immunotoxin

Objective

To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin‐conjugated antibody CD64–ricin A (CD64‐RiA) directed toward the high‐affinity receptor for IgG (FcγRI), exploiting the capacity of FcγRI to efficiently endocytose antibody which it has bound.

Methods

Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64‐RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light‐scatter patterns and CD14 and FcγRI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64‐RiA–induced cell death of macrophages affected their capacity to stimulate antigen‐induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64‐RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated.

Results

Inflammatory macrophages from RA SF expressed elevated levels of FcγRI and were selectively eliminated by CD64‐RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of FcγRI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen‐induced lymphocyte proliferation and a reduction in tumor necrosis factor α (TNFα) release. Consistent with these effects on SF macrophages, CD64‐RiA also inhibited TNFα production, interleukin‐1β production, and cartilage‐degrading activity of RA synovial tissue explants.

Conclusion

Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through FcγRI‐directed immunotoxins could be a novel approach to the treatment of RA.

     

Structure of t cell antigen receptor β chain in synovial fluid cells from patients with rheumatoid arthritis

We attempted to identify a clonal proliferation of T cells from synovial fluid samples from patients with rheumatoid arthritis, using techniques of restriction fragment length polymorphism. We used probes for the β chain of the T cell receptor to analyze restriction fragments prepared from the genomic DNA of synovial fluid mononuclear cells from 10 patients and synovial fluid T cell preparations from 5 additional patients. The results demonstrated unarranged (germline) T cell receptor gene fragments of DNA in all cell preparations, indicating the lack of clonality of rheumatoid arthritis synovial fluid T cells.     

类风湿关节炎成纤维样滑膜细胞诱导分化的体外研究

目的 研究类风湿关节炎（RA）成纤维样滑膜细胞（FLS）体外培养时的增生分化特点及细胞分化诱导剂全反式维甲酸（ATRA）、骨化三醇[1，25（OH）2D3]地塞米松（DEX）对RA—FLS增殖分化的影响。方法 关节镜、关节活检针取RA患者滑膜组织或抽取RA患者关节积液分离培养鉴定滑膜细胞．四甲基偶氮唑蓝（MTT）法和流式细胞仪法分别观察ATRA、骨化三醇和地塞米松对FLS细胞存活分数（SF）和细胞周期的影响。结果 RA—FLS在体外无诱导剂作用时呈现良性增殖方式。ATRA、骨化三醇和地塞米松对FLS的细胞标记和免疫染色无明显影响。三种诱导剂对RA—FLS的SF值均有不同程度的抑制（P〈0．05），其中地塞米松抑制作用最明显；骨化三醇和地塞米松对RA—FLS的SF抑制作用呈现剂量依赖性曲线（P〈0．01）。三种诱导剂均促使RA—FLS细胞周期改变即凋亡峰出现、S期缩短。结论 体外培养的RA—FLS生长特性为非恶性无限制增生。ATRA、骨化三醇和地塞米松抑制RA—FLS细胞增殖，促进细胞分化，诱导FLS凋亡，其中地塞米松抗增殖作用最明显；但未发现三者将FLS诱导分化为其他类型细胞。     

Necessary role of phosphatidylinositol 3‐kinase in transforming growth factor β–mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts

Objective

Rheumatoid arthritis is a disease that, pathologically, is characterized by the progressive growth and invasion of the synovial pannus into the surrounding cartilage and bone. Many cytokines, including transforming growth factor β1 (TGFβ1), have been implicated in this process, but their mode of action is incompletely understood. The goal of the present study was to better understand the downstream signaling pathways of TGFβ in fibroblasts.

Methods

The role of phosphatidylinositol 3‐kinase (PI 3‐kinase) was determined by chemical inhibition with LY294002 or wortmannin. Activation of protein kinase B (Akt), c‐Jun N‐terminal kinases (JNKs), and extracellular signal–regulated kinases (ERKs) was evaluated by Western blot analysis using phospho‐specific antibodies.

Results

Exposure of fibroblasts to TGFβ rapidly induced activation of a kinase, Akt, that is known to inhibit apoptosis by a variety of pathways. Activation of Akt was blocked by the specific PI 3‐kinase inhibitor, LY294002, indicating that TGFβ‐mediated phosphorylation of Akt was dependent on PI 3‐kinase activation. This activation pathway was relatively selective for Akt, since inhibition of PI 3‐kinase failed to substantially modify activation of ERKs or JNKs in synovial fibroblasts. Inhibition of the PI 3‐kinase/Akt pathway resulted in impaired proliferation of synovial fibroblasts and partial attenuation of the protective effect of TGFβ on Fas‐mediated apoptosis.

Conclusion

TGFβ exerts its growth and antiapoptotic effects on fibroblasts, at least in part, by activation of the PI 3‐kinase/Akt pathway.

     

Detection of tumor necrosis factor α but not tumor necrosis factor β in rheumatoid arthritis synovial fluid and serum

Synovial fluids from 6 of 12 patients with rheumatoid arthritis (RA) and from 3 of 11 patients with reactive arthritis contained measurable levels of tumor necrosis factor α (TNFα). Seven of 12 sera from RA patients contained TNFα, while only 1 of those from reactive arthritis patients was positive. Gamma-inter-feron was detected in the synovial fluids and sera of only the RA patients. Tumor necrosis factor β was not detected in any sera or synovial fluids. RA patients with detectable TNFα had higher erythrocyte sedimentation rates and synovial fluid leukocyte counts.     

Inhibition of fas antigen-mediated apoptosis of rheumatoid synovial cells in vitro by transforming growth factor β1

Objective. To investigate the mitogenic and anti-apoptotic effects of transforming growth factor β1 (TGFβ) on rheumatoid synovial cells in vitro. Methods. Synovial cells were cultured with or without TGFβ1. After incubation, the proliferative response of synovial cells and the expression of Fas antigen and bcl-2 on synovial cells were examined. Finally, Fas antigen-mediated apoptosis of synovial cells was investigated by the addition of anti-Fas antibody. Results. TGFβ1 enhanced the proliferation of synovial cells in a dose-dependent manner. In addition, Fas antigen expression on synovial cells was inhibited by the addition of TGFβ1 with up-regulation of bcl-2 expression. The addition of anti-Fas antibody induced synovial cell apoptosis. However, stimulation of synovial cells with TGFβ1 became markedly resistant to Fas antigen-mediated apoptosis. The results were not affected by the addition of a neutralizing antibody to platelet-derived growth factor type AA (PDGF-AA), which suggests that the effect of TGFβ1 on synovial cells was promoted via PDGF-AA-independent mechanisms. Conclusion. Our results suggest that TGFβ1 promotes synovial cell proliferation through its mitogenic effect on synovial cells and interference with the apoptotic process mediated by the Fas antigen, resulting in the perpetuation of the synovial hyperplasia in patients with rheumatoid arthritis.     

Regulation of interleukin‐1β–induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin‐3‐gallate in rheumatoid arthritis synovial fibroblasts

Objective

To evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin‐1β (IL‐1β)–induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP‐1/CCL2), epithelial neutrophil–activating peptide 78 (ENA‐78/CXCL5), growth‐regulated oncogene α (GROα/CXCL1), and matrix metalloproteinase 2 (MMP‐2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

Methods

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP‐1, ENA‐78, and GROα produced in culture supernatants were measured by enzyme‐linked immunosorbent assay. MMP‐2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF‐κB.

Results

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 μM or 20 μM significantly inhibited IL‐1β–induced ENA‐78, RANTES, and GROα, but not MCP‐1 production in a concentration‐dependent manner. EGCG at 50 μM caused a complete block of IL‐1β–induced production of RANTES, ENA‐78, and GROα, and reduced production of MCP‐1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL‐1β–induced, and chemokine‐mediated MMP‐2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCδ and inhibited the activation and nuclear translocation of NF‐κB in IL‐1β–treated RA synovial fibroblasts.

Conclusion

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

     

Differential tissue expression and activation of p38 MAPK α, β, γ, and δ isoforms in rheumatoid arthritis

Objective

Activation of p38 MAPK is a key signaling step in chronic inflammation. Inhibition of p38 MAPK is considered to be a promising future strategy to control inflammatory diseases, but studies of compounds to inhibit this kinase have so far been limited to investigation of their side effects. We undertook the present study to investigate which specific molecule, among 4 different isoforms of p38 MAPK (α, β, γ, and δ), is predominantly expressed and activated in inflammation. Such knowledge could allow more specific targeting of p38 MAPK in inflammatory disease.

Methods

Studies were performed on inflamed tissue from patients with rheumatoid arthritis, as a prototype of inflammatory disease. The expression and activation of the α, β, γ, and δ isoforms of p38 MAPK were examined by immunoblotting, immunoprecipitation, and immunohistochemistry.

Results

Immunoblot analysis revealed that α and γ were the predominantly expressed p38 MAPK isoforms, whereas the other 2 isoforms were less frequently present. By immunohistochemistry, the expression of all p38 MAPK isoforms was localized to the synovial lining layer as well as to blood vessels. Colabeling with cell‐specific markers revealed that macrophages expressed the α and γ isoforms, synovial fibroblasts the β and γ isoforms, and granulocytes the δ isoform, whereas T lymphocytes were rarely positive for any p38 MAPK isoform. Double‐labeling with isoform‐specific antibody and pan‐p38 antibody against the phosphorylated form of p38 MAPK showed activation of the α and γ isoforms. Occasional activation of the β isoform was also noted in the synovial lining and the endothelium, whereas the δ isoform, although expressed in pericytes around blood vessels, was not phosphorylated. This phosphorylation pattern was confirmed in immunoprecipitation studies in which activated p38 MAPK from synovial tissue extracts was identified as p38 MAPKα and ‐γ but not p38 MAPKβ or ‐δ.

Conclusion

These data show that the α and γ isoforms of p38 MAPK dominate in chronic inflammation. Effective strategies to inhibit p38 MAPK should therefore aim to specifically target either or both of these isoforms.

     

α2-macroglobulin-proteinase complexes as correlated with α1-proteinase inhibitor-elastase complexes in synovial fluids of rheumatoid arthritis patients

The levels of α₁-proteinase inhibitor-elastase and α₂-macroglobulin (α₂M)-proteinase complexes were measured in synovial fluids from arthritis patients by use of specific immunosorbent assays. Both types of proteinase inhibitor-proteinase complexes were significantly correlated with each other as well as with the total neutrophil count in synovial fluids of rheumatoid arthritis patients but were discordant in synovial fluids of patients with osteoarthritis. One synovial fluid sample showed active (inhibitory) α₂M as well as active collagenase. We purified α₂M from pooled synovial fluids obtained from patients with rheumatoid arthritis. This α₂M retained approximately 90% of its proteinase binding (inhibiting) capacity, compared with that of normal plasma α₂M. We found no evidence that α₂M was inactivated by means other than proteinases.     

Double-blind trial of recombinant γ-interferon versus placebo in the treatment of rheumatoid arthritis

One hundred five patients were enrolled in a 12-week, randomized, prospective, double-blind, placebo-controlled trial of recombinant human γ-interferon (rHuγ-IFN) for the treatment of rheumatoid arthritis. Fifty-four patients received rHuγ-IFN and 51 received placebo. Forty-two patients in each group completed the 12-week trial. Some clinical improvement occurred in both groups of patients. Although the improvement with rHuγ-IFN was greater than that with placebo, the differences were generally not statistically significant.     

Divergent T cell receptor γ repertoires in rheumatoid arthritis monozygotic twins

Objective. To determine if the expressed T cell receptor (TCR) γ repertoire is altered in rheumatoid arthritis (RA). Methods. Peripheral blood lymphocytes were collected from monozygotic twins who were either concordant or discordant for RA, or from a normal twin pair. TCR γ-specific complementary DNA libraries were constructed using the anchored polymerase chain reaction. Gene usage was analyzed by plaque hybridization and sequencing. Results. The expressed TCR VΓ repertoires both in RA patients and normal subjects were extremely diverse. Monozygotic twins who were concordant for RA expressed very different frequencies of TCR VΓ genes. Conclusion. RA does not lead to a specific clonal expansion or deletion of TCR VΓ genes in peripheral blood.     

Increased usage of vβ2 and vβ6 in rheumatoid synovial fluid t cells

Objective. To determine if the T cell antigen receptor V_β usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). Methods. Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V_β gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V_β primers with a constant region C_β primer, and 2 C_α primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. Results. There were consistent differences between the V_β gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA–DR haplotype. In all synovial specimens, V_β2 was increased relative to the peripheral blood, while V_β13.1 and V_β13.2 were decreased. V_β6 and V_β21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V_β bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V_β gene usage of separated CD4+ and CD8+ synovial T cells showed that V_β2 and V_β6 were more highly expressed on CD4 cells. Conclusion. Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V_β gene usage compared with PB T cells. V_β2 and V_β6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V_β bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.     

Activation of human synovial mast cells from rheumatoid arthritis or osteoarthritis patients in response to aggregated IgG through Fcγ receptor I and Fcγ receptor II

Objective

Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). A plausible pathway for the activation of synovial MCs is through IgG receptors, given the prevalence of circulating IgG isotype autoantibodies and synovial immune complexes in patients with RA. However, IgG receptor expression on human synovial MCs remains uncharacterized. The aim of this study was to identify which IgG receptor(s) on synovial MCs are responsible for MC activation in immune complexes.

Methods

Synovial tissue specimens were obtained from patients with RA or patients with osteoarthritis (OA) who were undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Cultured synovium‐derived MCs were generated by culturing synovial cells with stem cell factor, and receptor expression was analyzed using fluorescence‐activated cell sorting. Mediators released from MCs were measured using enzyme immunoassays or enzyme‐linked immunosorbent assays.

Results

Primary synovial MCs and cultured synovium‐derived MCs obtained from both patients with RA and patients with OA expressed Fcε receptor I (FcεRI), FcγRI, and FcγRII but not FcγRIII. Cultured synovium‐derived MCs induced degranulation and the production of prostaglandin D₂ and tumor necrosis factor α (TNFα) through FcγRI. The aggregation of FcγRII caused histamine release from cultured MCs but not from primary MCs. Histamine release induced by aggregated IgG was significantly inhibited by neutralizing anti‐FcγRI monoclonal antibody and anti‐FcγRII monoclonal antibody.

Conclusion

With regard to the FcR expression profile, synovial MCs from patients with RA and patients with OA were similar. FcγRI was responsible for producing abundant TNFα from synovial MCs in response to aggregated IgG. Immune complexes may activate synovial MCs through FcγRI and FcγRII.