Identification of synovium-specific homing peptides by in vivo phage display selection

OBJECTIVE: To identify homing peptides specific for human synovium that could be used as targeting devices for delivering therapeutic/diagnostic agents to human joints. METHODS: Human synovium and skin were transplanted into SCID mice. A disulfide-constrained 7-amino acid peptide phage display library was injected intravenously into the animals and synovial homing phage recovered from synovial grafts. Following 3-4 cycles of enrichment, DNA sequencing of homing phage clones allowed the identification of specific peptides that were synthesized by a-fluorenylmethyloxycarbonyl chemistry and used in competitive in vivo assays and immunohistochemistry analyses. RESULTS: We isolated synovial homing phages displaying specific peptides that distinctively bound to synovial but not skin or mouse microvascular endothelium (MVE). They retained their tissue homing specificity in vivo, independently from the phage component, the original pathology of the transplanted tissue, and the degree of human/murine graft vascularization. One such peptide (CKSTHDRLC) maintained synovial homing specificity both when presented by the phage and as a free synthetic peptide. The synthetic peptide also competed with and inhibited in vivo the binding of the parent phage to the cognate synovial MVE ligand. CONCLUSION: This is the first report describing peptides with homing properties specific for human synovial MVE. This was demonstrated using a novel approach targeting human tissues, transplanted into SCID mice, directly by in vivo phage display selection. The identification of such peptides opens the possibility of using these sequences to construct joint-specific drug delivery systems that may have considerable impact in the treatment of arthritic conditions.     

Mechanisms of Disease: the link between RANKL and arthritic bone disease

Chronic inflammation and bone loss are closely linked pathophysiologic events. The most typical example of inflammatory bone loss is seen in patients with rheumatoid arthritis who develop systemic osteopenia as well as local breakdown of bone in the direct vicinity of inflamed joints. Understanding the mechanisms of arthritic bone degradation is crucial for designing therapies that can specifically protect joints from structural damage. Since osteoclast differentiation and activity are key events in arthritic bone damage, the signals that trigger osteoclastogenesis are potential therapeutic targets. Receptor activator of nuclear factor-kappaB (RANK) is activated by its ligand, RANKL, an essential molecule for osteoclast development: in the absence of RANKL or RANK, osteoclast differentiation from monocyte precursors does not occur. RANKL is expressed on T cells and fibroblasts within the synovial inflammatory tissue of patients with RA and its expression is regulated by proinflammatory cytokines. In animal models of arthritis, blockade of RANKL-RANK interactions, or a genetic absence of RANKL or RANK, protects against joint damage despite the presence of joint inflammation. Therefore, inhibition of RANKL is regarded as a promising future strategy for inhibiting inflammatory bone loss in patients with chronic inflammatory arthritis.     

Identification of synovium‐specific homing peptides by in vivo phage display selection

Objective

To identify homing peptides specific for human synovium that could be used as targeting devices for delivering therapeutic/diagnostic agents to human joints.

Methods

Human synovium and skin were transplanted into SCID mice. A disulfide‐constrained 7–amino acid peptide phage display library was injected intravenously into the animals and synovial homing phage recovered from synovial grafts. Following 3–4 cycles of enrichment, DNA sequencing of homing phage clones allowed the identification of specific peptides that were synthesized by a‐fluorenylmethyloxycarbonyl chemistry and used in competitive in vivo assays and immunohistochemistry analyses.

Results

We isolated synovial homing phages displaying specific peptides that distinctively bound to synovial but not skin or mouse microvascular endothelium (MVE). They retained their tissue homing specificity in vivo, independently from the phage component, the original pathology of the transplanted tissue, and the degree of human/murine graft vascularization. One such peptide (CKSTHDRLC) maintained synovial homing specificity both when presented by the phage and as a free synthetic peptide. The synthetic peptide also competed with and inhibited in vivo the binding of the parent phage to the cognate synovial MVE ligand.

Conclusion

This is the first report describing peptides with homing properties specific for human synovial MVE. This was demonstrated using a novel approach targeting human tissues, transplanted into SCID mice, directly by in vivo phage display selection. The identification of such peptides opens the possibility of using these sequences to construct joint‐specific drug delivery systems that may have considerable impact in the treatment of arthritic conditions.

     

Yersinia enterocolitica in the synovial membrane of patients with Yersinia-induced arthritis

Using a monospecific rabbit antibody against Yersinia enterocolitica outer membrane protein 1, we examined synovial biopsy specimens from 7 patients with Yersinia-induced arthritis. Yersinia were demonstrated in the synovial membrane by indirect immunofluorescence in 4 patients with Yersinia-induced arthritis, but not in 6 control patients with Salmonella-induced arthritis or with rheumatoid arthritis. These findings suggest the persistence of Yersinia in the joints of patients with Yersinia-induced arthritis.     

Secretion of antibodies to types I and II collagen by synovial tissue cells in patients with rheumatoid arthritis

Production of antibodies to IgG and to type I and type II collagen (CI and CII) was analyzed by enzyme-linked immunospot assay in patients with rheumatoid arthritis (RA) and patients with other inflammatory or degenerative joint diseases. Anti-CII-secreting cells, generally in high numbers, were found among mononuclear cells eluted from inflamed synovial tissue in 12 of 13 patients with seropositive RA and 9 of 14 patients with seronegative RA or with undetermined serum rheumatoid factor levels. In contrast, no anti-CII-producing B cells were present among synoviocytes from 4 patients with other joint diseases. In none of 7 RA sera did we find significant levels of anti-CII. Synovial B cells secreting antibodies specific for CI were observed less frequently in patients with RA. These results indicate that measurement of serum antibody levels is not adequate to assess actual autoantibody production in rheumatoid joints and that local autoimmune reactions to CII are common in RA, which implies that collagen-reactive T cells are present within the inflamed joints of RA patients. The possible role of a local collagen autoimmunity in RA is discussed, particularly in relation to its putative role in rheumatoid factor production.     

Morphometric analysis of blood vessels in synovial membranes obtained from clinically affected and unaffected knee joints of patients with rheumatoid arthritis.

Synovial tissues from inflamed and noninflamed knee joints of 13 patients with untreated rheumatoid arthritis were examined for vascular proliferation and morphological alteration of endothelial cells. Perivascular mononuclear cell infiltration and increased thickness of the synovial lining layer were noted in tissues from inflamed and noninflamed joints of patients with rheumatoid arthritis; vascular proliferation and morphological alteration of endothelial cells to resemble high endothelial venules were seen only in tissues from inflamed joints of patients with rheumatoid arthritis. These observations suggest that the migration of mononuclear cells from the peripheral blood to the perivascular areas and lining layer occurs before vascular proliferation and morphological alteration of endothelial cells.     

Receptor expression in synovial fluid neutrophils from patients with rheumatoid arthritis.

OBJECTIVES--The aim of this study was to determine if neutrophils isolated from the blood and synovial fluid of patients with rheumatoid arthritis had patterns of receptor expression resembling those of blood neutrophils from controls which had been activated and primed in vitro. METHODS--Fluorescence activated cell sorting was used to measure receptor expression in paired blood and synovial fluid neutrophils from patients and in control neutrophils exposed to phorbol myristate acetate and granulocyte-macrophage colony stimulating factor. RESULTS--There was no significant difference in the patterns of receptor expression in blood neutrophils from patients and healthy controls, but neutrophils in the synovial fluid had been primed and activated within the joint. About 50% of rheumatoid synovial fluid neutrophil samples expressed Fc gamma RI, a high affinity receptor for monomeric IgG, which is only expressed in neutrophils exposed to cytokines. CONCLUSIONS--Synovial fluid neutrophils are activated and primed within the inflamed joint and hence their ability to respond to activating factors such as immune complexes will be modulated. As the expression of Fc gamma RI requires active biosynthesis, this work indicates that selective gene activation occurs when neutrophils are recruited into rheumatoid joints.     

Arthritis of the large joints - in particular, the knee - at first presentation is predictive for a high level of radiological destruction of the small joints in rheumatoid arthritis

OBJECTIVE: To investigate the predictive value of the distribution of inflamed joints at first presentation for the severity of the disease course in rheumatoid arthritis (RA). METHODS: Of the 1009 consecutive patients included in the Leiden Early Arthritis Clinic (Leiden, The Netherlands), 285 patients fulfilled the American College of Rheumatology criteria for RA within 1 year of follow-up. Of these, 28 patients achieved remission. Radiographs of hands and feet were scored according to the Sharp-van der Heijde method, and the 28 patients with the most destructive disease were selected. The distribution of inflamed joints of the patients with the extreme disease courses was compared. The association between the distribution of inflamed joints and the level of destruction of the joints of hands and feet in the whole group of patients with RA was assessed using regression analysis. RESULTS: Comparison of patients with extreme disease courses using univariate and logistic regression analyses showed that arthritis of the large joints - in particular, the knee - was associated with severe RA. In the whole group of patients with RA, the total number of swollen joints and the presence of knee arthritis were associated independently with the level of destruction of the small joints. Patients with RA with knee arthritis had higher C reactive protein (CRP) levels than patients without knee arthritis, and investigating the distribution of inflamed joints together with other variables yielded the number of swollen joints, CRP, presence of anti-cyclic citrullinated peptide antibodies and symptom duration as predictors for severity of RA. CONCLUSION: Arthritis of large joints - in particular, the knee - at first presentation is associated with a destructive course of RA.     

Histopathology of the synovial membrane of peripheral joints in ankylosing spondylitis.

The histological features of the synovial membrane of peripheral joints in ankylosing spondylitis are similar to those seen in rheumatoid arthritis. There is intimal cell hyperplasia, a diffuse lymphocyte and plasma cell infiltrate, and formation of lymphoid follicles. Peroxidase-antiperoxidase staining shows the presence of IgG-, IgA-, and IgM-containing plasma cells in ankylosing spondylitis. The percentage of IgM-containing cells is significantly lower in ankylosing spondylitis than in rheumatoid arthritis.     

Identification of three novel peptides that inhibit CD40–CD154 interaction

Abstract

The CD40–CD154 interaction is an attractive target for therapeutic intervention in various autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and myasthenia gravis. In this study, to develop a new disruption strategy of the CD40–CD154 interaction, we screened for peptides with inhibitory effects on such ligation. 2 × 10¹¹ phage display libraries displaying liner peptides of 12-mer amino acids were screened by CD40-Ig binding assay and eight phages which expressed a different respective peptide (40BP-1 to -8) were able to specifically bind to CD40. Competitive inhibition analyses showed that 3 of the 8 peptides (40BP-N1-1 – APELPNMTPSWT; 40BP-N1-2 – APRPHTSYSPLP; and 40BP-N1-3 – GMTAPPPPRLTQ) blocked CD40–CD154 interaction when used at high concentrations. A consensus sequence (APxPPxxT) was conserved in these three peptides. These peptides may constitute a useful and novel strategy for the inhibition of the interaction between CD40 and CD154 molecules.     

A型滑膜细胞与类风湿关节炎血管新生

类风湿关节炎(RA)是一种常见的多系统性炎症性自身免疫性疾病.RA最主要病理特征是关节滑膜血管新生和对关节破坏力极强的血管翳形成.血管的新生是个复杂的过程,多种细胞及其所分泌的细胞因子参与了此过程.近来大量的研究表明,A型滑膜细胞即滑膜巨噬细胞在RA病理过程中起着非常重要的作用,其参与了炎症和血管新生的整个过程.A型滑膜细胞表达黏附分子、趋化因子受体和多种表面抗原物质,还能分泌产生大量的细胞因子、生长因子和其它化学活性物质.它们在RA滑膜病理过程中起着关键性作用.因此,A型滑膜细胞可能将成为RA治疗很好的靶点.     

The aminoterminal-type-III procollagen peptide and proteoglycans in serum and synovial fluid of patients with rheumatoid arthritis or reactive arthritis

Summary The concentrations of aminoterminal-type-III procollagen (procollagen N-) peptide, and of proteoglycans were measured in knee-joint synovial fluid and serum from patients with rheumatoid arthritis or reactive arthritis. All synovial fluids contained large amounts of intact propeptide. The synovial fluid : serum propeptide ratios were high, suggesting local propeptide liberation. A correlation was demonstrated between the propeptide concentration in synovial fluid and in serum. In rheumatoid arthritis, the propeptide concentration in synovial fluid was related to local inflammatory activity, and the serum concentration was correlated with the presence of nonspecific markers of inflammation. The presence of smaller propeptide fragments in synovial fluid indicated that some degradation occurred locally. The local metabolic changes were most prominent in patients with joint erosions. Patients with nonerosive rheumatoid arthritis and reactive arthritis had similar synovial fluid propeptide concentrations. The proteoglycan content of synovial fluid was inversely related to the degree of joint destruction, and was highest in patients with reactive arthritis. No correlation was observed between the concentrations of propeptide and proteoglycan in synovial fluid. Intra-articular glucocorticoid injection reduced the levels of propeptide and proteoglycan in synovial fluid.     

Constitutive expression of angiopoietin-1 and -2 and modulation of their expression by inflammatory cytokines in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: Angiopoietin- I (Ang-1) and Ang-2 are ligands for the receptor tyrosine kinase, Tie-2. Ang-1, a Tie-2 agonist, may have a vascular stabilizing role in angiogenesis, while Ang-2, an endogenous antagonist of Tie-2, may have an early role in angiogenesis, destabilizing existing vasculature. We show that these ligands are expressed by rheumatoid synovial fibroblasts (RSF) and investigate whether their expression was modulated by proinflammatory cytokines present in the joint in rheumatoid arthritis (RA). METHODS: Using quantitative PCR we determined the level of expression of these 2 ligands in RSF and chronic inflamed synovial tissue. The level of expression of these ligands after treatment with proinflammatory cytokines and hypoxia was also determined. RESULTS: We observed constitutive expression of Ang-1 and Ang-2 in RSF and chronic inflamed synovial tissue. Ang-1 was the most highly expressed ligand in late stage RA synovial fibroblasts; however, in chronic inflamed synovial tissue, Ang-2 was predominant and was expressed at strikingly high levels (70 to 120-fold increase). We observed that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), but not interleukin 1beta or hypoxia, stimulated Ang-1 gene expression in RSE This was confirmed at the protein level as media from TNF-alpha treated RSF resulted in increased autophosphorylation of Tie-2. In contrast, TNF-alpha and TGF-beta had no effect on Ang-2 expression in RSF, but augmented expression of Ang-2 in normal synovial fibroblasts. CONCLUSION: The angiopoietins are important angiogenic factors constitutively present in RA, and their expression is modulated by certain cytokines. Ang-2 may have an important role in rheumatoid tissue where vigorous angiogenesis is occurring.     

Identification of three novel peptides that inhibit CD40–CD154 interaction

The CD40–CD154 interaction is an attractive target for therapeutic intervention in various autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and myasthenia gravis. In this study, to develop a new disruption strategy of the CD40–CD154 interaction, we screened for peptides with inhibitory effects on such ligation. 2 × 10¹¹ phage display libraries displaying liner peptides of 12-mer amino acids were screened by CD40-Ig binding assay and eight phages which expressed a different respective peptide (40BP-1 to -8) were able to specifically bind to CD40. Competitive inhibition analyses showed that 3 of the 8 peptides (40BP-N1-1 – APELPNMTPSWT; 40BP-N1-2 – APRPHTSYSPLP; and 40BP-N1-3 – GMTAPPPPRLTQ) blocked CD40–CD154 interaction when used at high concentrations. A consensus sequence (APxPPxxT) was conserved in these three peptides. These peptides may constitute a useful and novel strategy for the inhibition of the interaction between CD40 and CD154 molecules.     

Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis.

Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovial tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis.     

Peptide based adsorbers for therapeutic immunoadsorption

Peptides as ligands for immunoadsorption exhibit several potential advantages over native proteins. Two newly developed adsorbers are based on peptides covalently coupled to sepharose CL-4B. Globaffin is capable of binding immunoglobulins independent from their antigen specificity and thus, applicable in transplant recipients and several antibody mediated autoimmune diseases. Among others, the most important disorders suitable for the treatment with Globaffin are rheumatoid arthritis, systemic lupus erythematosus, and acute renal transplant rejection. Coraffin is a specific adsorber using two linear peptide ligands mimicking epitopes of the beta1-adrenergic receptor, that bind corresponding autoantibodies from patients suffering from idiopathic dilated cardiomyopathy. Specific immunoadsorption has been shown to be beneficial for patients with dilated cardiomyopathy. Coraffin can be used as a new therapeutic option for these patients, who get only limited benefit from medical therapy. Both adsorbers may be combined with all approved apheresis control devices available.     

Cytokine Combinations Increase p75 Tumor Necrosis Factor Receptor Binding and Stimulate Receptor Shedding in Rheumatoid Synovial Fibroblasts

Objective. To identify cytokines responsible for the increased levels of tumor necrosis factor receptor (TNFR) in the joints of patients with rheumatoid arthritis. Methods. Antibodies to TNFR types were used both to inhibit ligand cell binding and to quantify released receptors in rheumatoid synovial fibroblasts. Results. Binding by and shedding of the p75 TNFR was affected by interleukin-1 (IL-1), IL-4, and interferon-γ. Conclusion. IL-1 could cause increased TNFα binding and TNFR shedding in inflamed joints.     

Vascular disease in rheumatoid arthritis: From subclinical lesions to cardiovascular risk

Rheumatoid arthritis (RA) is one of the most prevalent and complex inflammatory diseases affecting primarily the joints, but also associating several extra-articular features. The vascular disease in RA encompasses a large spectrum of lesions, from rheumatoid vasculitis to atherosclerotic lesions. During the last years the importance of the vascular disease related to atherosclerosis in terms of cardiovascular morbidity and global mortality became evident in RA. The inflammatory hypothesis of atherosclerosis in RA implies that mediators originating from the inflamed synovial tissue or from the liver may have systemic vascular consequences, leading to endothelial dysfunction and structural abnormalities of the vessels. Hence, the global management of patients with RA must include the improvement of cardiovascular risk in parallel with the management of joint disease.     

Synovial collagenase: Its presence in culture from joint disease of diverse etiology

Closed needle biopsy of synovium from inflamed joints yields enough tissue to detect production and/or release of collagenase from these tissues. Collagenase activity was found in synovium from 8 of 8 patients with probable rheumatoid arthritis, in 6 of 9 patients with nonrheumatoid inflammatory synovitis, and in 1 of 6 patients with degenerative joint disease. One of 2 patients with classic rheumatoid arthritis had activity in synovium regrown after synovectomy. Therefore synovial collagenase is not unique to rheumatoid arthritis.     

Human platelets in synovial fluid. A focus on the effects of growth factors on the inflammatory responses in rheumatoid arthritis.

The alpha granules of platelets are a major storage site for peptide growth factors. The inflamed synovial fluid of rheumatoid arthritis contains a high number of platelets as well as platelet-derived growth factors. These platelets may apparently be acted upon to release their alpha granule-located substances. It can thus be suggested that platelets present in the synovial fluid express growth factors of significance to the local inflammatory responses of rheumatoid arthritis.