Imbalanced expression of RANKL and osteoprotegerin mRNA in pannus tissue of rheumatoid arthritis.

OBJECTIVE: To test if the pannus tissue is characterized by a high receptor activator of nuclear factor kappaB ligand to osteoprotegerin (RANKL:OPG) ratio, which could explain local osteoclastogenesis and formation of bony erosions. METHODS: Messenger RNA and protein expressions of RANKL and OPG in rheumatoid and osteoarthritic tissue samples were measured using quantitative real-time RT-PCR and Western blot/densitometry. Pannus and synovitis fibroblasts explanted from tissue samples were cultured in vitro without and with TNF-alpha, IL-1Beta or IL-17 and analyzed quantitatively for RANKL expression. The ability of pannus fibroblasts to induce formation of multinuclear osteoclast-like cells from human monocytes, with macrophage-colony stimulating factor (M-CSF) but without RANKL added, was tested. Histochemical staining was used to assess the eventual presence of RANKL and tartrate resistant acid phosphatase positive osteoclast-like cells at the pannus-bone interface. RESULTS: RANKL:OPG ratios of messenger RNA (p<0.05) and protein level were high in pannus (2.06+/-0.73 and 2.2+/-0.65) compared to rheumatoid (0.62+/-0.13 and 1.31+/-0.69) and osteoarthritis (0.62+/-0.32 and 0.52+/-0.16) synovial membranes. Resting and stimulated (p dependent on the cytokine used) pannus fibroblasts produced RANKL in excess (p=0.0005) and unstimulated pannus fibroblasts also effectively induced osteoclast-like cell formation from monocytes in vitro without any exogenous RANKL added. Compatible with these findings, multinuclear osteoclasts-like cells were frequent in the fibroblast- and macrophage-rich pannus tissue at the soft tissue-to-bone interface. CONCLUSION: The high RANKL:OPG ratio, together with close fibroblast-to-monocyte contacts in pannus tissue, probably favor local generation of bone resorbing osteoclasts at the site of erosion in rheumatoid arthritis.

Expression of ADAM9 (meltrin-gamma) around aseptically loosened total hip replacement implants.

OBJECTIVE: To investigate the involvement of a disintegrin and the metalloproteinase ADAM9 (meltrin-gamma) in the formation of multinuclear giant cells and osteoclasts in aseptic loosening of hip replacement implants. METHODS: We used in situ hybridization, immunohistochemical staining and western blotting of interface membrane surrounding loosened hip implants, macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) costimulation and polymethyl methacrylate (PMMA) particle stimulation of human monocytes followed by immunofluorescence staining and flow cytometric analysis. RESULTS: Morphometric analysis revealed that the ADAM9+ area in the revision total hip replacement (THR) interface was larger than in primary THR samples (37.6+/-5.1 vs 5.2+/-0.8%, P=0.002). Double immunofluorescence staining showed that CD68+ interface tissue macrophages and multinuclear giant cells were ADAM9+. ADAM9 mRNA containing mononuclear and multinuclear cells was often seen in a close spatial relationship with other ADAM9+ cells. Western blotting disclosed a 50 kDa ADAM9 band in tissue extracts. Upon M-CSF and RANKL costimulation of human monocytes, the ADAM9 staining pattern changed over time and ADAM9+ cells formed bi- and multinuclear cells. Flow cytometry disclosed that cells of the monocyte/macrophage lineage changed from ADAM9-negative cells into strongly positive cells during a 3-day culture. CONCLUSION: ADAM9 is expressed in interface tissues around aseptically loosened THR implants. ADAM9 may play a role as a fusion molecule in the formation of multinuclear giant cells and osteoclasts from mononuclear precursors in diseases characterized by bone tissue destruction.

Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta.

OBJECTIVE: Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. METHODS: Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. RESULTS: MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. CONCLUSION: This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

Neuromagnetic localization of rhythmic activity in the human brain: a comparison of three methods.

Cortical rhythmic activity is increasingly employed for characterizing human brain function. Using MEG, it is possible to localize the generators of these rhythms. Traditionally, the source locations have been estimated using sequential dipole modeling. Recently, two new methods for localizing rhythmic activity have been developed, Dynamic Imaging of Coherent Sources (DICS) and Frequency-Domain Minimum Current Estimation (MCE(FD)). With new analysis methods emerging, the researcher faces the problem of choosing an appropriate strategy. The aim of this study was to compare the performance and reliability of these three methods. The evaluation was performed using measured data from four healthy subjects, as well as with simulations of rhythmic activity. We found that the methods gave comparable results, and that all three approaches localized the principal sources of oscillatory activity very well. Dipole modeling is a very powerful tool once appropriate subsets of sensors have been selected. MCE(FD) provides simultaneous localization of sources and was found to give a good overview of the data. With DICS, it was possible to separate close-by sources that were not retrieved by the other two methods.

Anaemia of chronic disease in AA amyloidosis is associated with allele 2 of the interleukin-1beta-511 promoter gene and raised levels of interleukin-1beta and interleukin-18.

OBJECTIVE: In amyloid A (AA) amyloidosis the receptor for advanced glycation end products is a target for the circulating amyloid precursor protein (SAA) resulting in upregulation of the proinflammatory cytokine pathway. Besides inducing hepatic SAA synthesis the interleukin-1 cytokine family is involved in the regulation of haematopoiesis. We therefore studied the relationship between the circulating levels of interleukin-1beta (IL-1beta) and interleukin-18 (IL-18), a new member of the IL-1 complex, as well as polymorphisms within the IL-1 cluster with the occurrence of anaemia in patients with AA amyloidosis. DESIGN, SETTING AND SUBJECTS: The study included 54 adult patients with biopsy-proven reactive amyloidosis allocated into three groups on the basis of haemoglobin (Hb) level: group I included all patients with Hb < 110 g L(-1) (n = 16); group II patients (Hb > 110 g L(-1), n = 16) were selected to match group I patients with respect to sex, age, underlying disease (seropositive, erosive rheumatoid arthritis) and renal function; and group III patients (n = 38) represented all patients (unselected) with Hb > or = 110 g L(-1). Gene polymorphisms were studied by polymerase chain reaction restriction length assay and included the base exchange at position-889 of the IL-1alpha gene, the polymorphic region at position-511 and the polymorphic locus at exon 5, position +3954 of the IL-1beta gene, as well as the IL-1 receptor antagonist (IL-1Ra) exon 2 polymorphism caused by the 86-bp tandem repeats. Plasma IL-1beta, IL-1alpha, IL-18, IL-1 Ra, SAA, ferritin, soluble transferrin receptor and erythropoietin levels were studied by enzyme immunoassays. RESULTS: Circulating IL-beta and IL-18 were significantly raised in the anaemic patients with AA amyloidosis when compared with group II patients (matched, Hb > 110 g L(-1)) as well as group III patients (nonmatched, Hb > or = 110 g L(-1)). A significant inverse relationship was found between IL-1beta and haemoglobin levels, as well as between IL-18 and haemoglobin levels. The frequency of allele 2 (T) of the IL-1beta-511 promoter gene was significantly increased and that of allele 1 (C) decreased in anaemic amyloid patients (group I) when compared with group II and III patients. Circulating IL-1beta levels tended to be higher amongst the IL-1beta-511 allele 2 carriers than amongst the noncarriers, as well as amongst the anaemic amyloid patients filling all criteria of anaemia of chronic disease. CONCLUSION: The occurrence of anaemia in patients with AA amyloidosis is associated with allele 2 (T) of the IL-1beta-511 promoter gene and elevated levels of circulating IL-1beta and IL-18. In AA amyloidosis the raised cytokine levels may generate a vicious cycle leading to accelerated amyloidogenesis, suppression of erythropoiesis and aggravation of the underlying inflammatory disorder.

Imbalance of RANKL/RANK/OPG system in interface tissue in loosening of total hip replacement.

In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-kappaB (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The mRNA levels of both RANKL (p < 0.01) and RANK (p < 0.05) were high in peri-implant tissue and RANKL+ and RANK+ cells were found in such tissue. Double labelling also disclosed soluble RANKL bound to RANK+ cells. We were unable to stimulate fibroblasts to express RANKL in vitro, but monocyte activation with LPS gave a fivefold increase in RANK mRNA levels. In contrast to RANKL and RANK expression in peri-implant tissue, expression of OPG was restricted to vascular endothelium. Endothelial cell OPG mRNA levels were regulated by TNF-alpha and VEGF, but not by hypoxia. It is concluded that activated cells in the interface tissue overproduce both RANKL and RANK and they can interact without interference by OPG.

Fibrillogenesis in gelsolin-related familial amyloidosis.

To clarify the mechanisms involved in amyloid formation in Finnish-type familial amyloidosis (FAF), we have tested the in vitro fibrillogenicity of synthetic wild-type and mutated gelsolin peptide analogs and studied the fragmentation patterns of gelsolin in the circulation of FAF patients with the Asn-187 or Tyr-187 gelsolin mutation. Fibril formation of synthetic peptides having sequence homology with wild-type or mutant gelsolins was monitored by Congo-red staining and polarization microscopy, negative staining electron microscopy and quantitative thioflavine-T fluorometry. Immunoblotting with anti-gelsolin and amyloid-specific antibodies and sequence analyses were used to study the fragmentation pattern of gelsolin. Ultrastructurally amyloid-like fibrils were formed from mutant Asn-187 and Tyr-187 gelsolin peptides. Fluorometric analysis revealed highly accelerated fibril formation from the mutant peptides as compared with the corresponding wild-type peptides. Addition of mercaptoethanol alone or in combination with dithiotreitol tended to enhance fibril formation of the 9-mer and 11-mer Asn peptides. Blocking of the C-terminal carboxyl of the mutant Asn-187 gelsolin182-192 peptide by amidation increased amyloidogenicity. The Tyr-187 gelsolin mutation, corresponding to the naturally occurring mutation in the Danish subtype of FAF, required acidic conditions to form fibrils meeting the criteria of amyloid. In FAF patients, in addition to the full-sized gelsolin, a series of lower-molecular mass C-terminal fragments of gelsolin (70,000-45,000 Da) was found in the circulation. In homozygous FAF(Asn-187) the 65-kDa fragment containing the amyloid forming region and the 55-kDa fragment, devoid of that region, was the major gelsolin species in the plasma. The results indicate that the 65-kDa gelsolin fragment derived by alpha-gelsolinase cleavage at the mutation-induced novel proteolysis site Arg172-Ala173 represents the putative circulating precursor protein of tissue amyloid in FAF and that the Asp187Asn/Tyr substitution in gelsolin creates a conformation that is highly fibrillogenic.

Tumor necrosis factor alpha, its soluble receptor I, and -308 gene promoter polymorphism in patients with rheumatoid arthritis with or without amyloidosis: implications for the pathogenesis of nephropathy and anemia of chronic disease in reactive amyloidosis.

OBJECTIVE: To study tumor necrosis factor alpha (TNFalpha) -308 gene promoter polymorphism and circulating levels of TNFalpha and soluble TNF receptor type I (sTNFRI) in rheumatoid arthritis (RA) patients with and without reactive amyloidosis. METHODS: In a retrospective study, we examined 55 RA patients with biopsy-proven reactive amyloidosis and 55 control RA patients without amyloidosis (matched for age, sex, rheumatoid factor titer, and RA duration). Inflammatory activity was assessed by measuring the erythrocyte sedimentation rate and C-reactive protein level. TNFalpha gene promoter polymorphism was studied using polymerase chain reaction-restriction fragment length polymorphism assay. Cytokine and receptor levels were measured by enzyme-linked immunoassays. RESULTS: Patients with RA and amyloidosis had significantly higher TNFalpha and sTNFRI levels than did the control RA patients. The increased circulating levels of TNFalpha correlated with interleukin-18 levels, but not with the serum amyloid A protein levels or with TNFalpha -308 gene promoter polymorphism (reported to be associated with high TNFalpha levels and certain disease susceptibilities). In the patients with RA and amyloidosis, those with anemia had significantly higher TNFalpha and sTNFRI levels than did those without anemia, and circulating TNFalpha and sTNFRI levels correlated negatively with hemoglobin concentrations. In the patients with RA and amyloidosis, those with nephropathy had significantly higher TNFalpha and sTNFRI levels than did those without nephropathy; in patients with isolated proteinuria (but no creatinine elevation) the TNFalpha level was also significantly increased, indicating that the TNFalpha elevation was not merely a consequence of impaired renal function. CONCLUSION: This study shows that circulating levels of TNFalpha and sTNFRI are significantly increased in RA patients with amyloidosis as compared with control RA patients without amyloidosis and that the increased levels may be implicated in the pathogenesis of certain disease manifestations, including anemia of chronic disease and renal pathology in reactive amyloidosis.

Raised circulating interleukin-18 levels in reactive AA-amyloidosis.

OBJECTIVE: To study the circulating levels of interleukin-18 (IL-18), a proinflammtory cytokine implicated in the T helper I response, in patients with rheumatoid arthrtitis (RA) with or without amyloidosis. METHODS: Plasma IL-18 levels were studied by enzyme-linked immusorbent assay in 55 RA patients with reactive amyloidosis and in 55 RA patients without amyloidosis matched with respect to age, sex, seropositivity, disease duration and inflammatory activity, as well as in 55 healthy control subjects. RESULTS: Plasma IL-18 levels were significantly elevated in RA patients as compared with control subjects. Those RA patients who had amyloid had significantly higher circulating level of IL-18 than those without amyloid (418.1 +/- 32.1 ng/l versus 317.0 +/- 21.3 ng/l, P<0.02). This difference was not due to differences in inflammatory activity, nor was it related to renalfunction. CONCLUSION: RA is associated with increased levels of plasma IL-18, the levels being significantly higher in patients with amyloid than in those without amyloid The increased level in the amyloidosis patients may reflect the interaction ofamyloid with cellular meatbolic pathways or, possibly, suggest a direct role of IL-18 in amyloidogenesis.

Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

Apolipoprotein E phenotypes in rheumatoid arthritis with or without amyloidosis.

The role of apolipoprotein (apo) E in the pathogenesis of reactive amyloidosis is unclear. Here we evaluated the apoE phenotype distribution and apolipoprotein e allele frequencies in 55 adult patients with seropositive, erosive RA with amyloid and compared them with 55 matched RA patients without amyloid The apoE isotypes were determined by isoelectric focusing and immunoblotting. RA patients without amyloid had more often the apoE 3/3 phenotype (71%) than the RA+A patients (49%, P<0.05) or Finnish control subjects (47%, P<0.01) and the frequency of the apo epsilon3 allele was significantly higher among the RA patients without amyloid than among RA+A patients (P<0.05) or control subjects (P<0.01). The prevalence of the apoE3/4 phenotype among the RA+A patients, although higher, did not significantly differ from the RA patients without amyloid (40% and 26%, respectively, NS) or Finnish control subjects (40% and 35%, respectively, NS). The frequency of the apo epsilon4 allele among the RA+A patients did not signficantly differ from that of RA patients without amyloid (0.23 and 0.13, respectively, NS) or Finnish control subjects (both 0.23). However, the apo epsilon4 frequency of 0.13 among the RA patients without amyloid was significantly lower than that of Finnish control subjects (0.23, P<0.05). We conclude that the prevalence of the apoE4 isotype is not increased in patients with RA complicated by amyloidosis when compared with Finnish control subjects. Since the frequency of the apo epsilon4 allele is significantly decreased in RA patients without amyloid when compared with Finnish control subjects, the presence of the apoE4 in a patient with RA could, though, represent a relative risk factor for developing reactive amyloidosis.

Danish type gelsolin related amyloidosis: 654G-T mutation is associated with a disease pathogenetically and clinically similar to that caused by the 654G-A mutation (familial amyloidosis of the Finnish type).

BACKGROUND: Familial amyloidosis of the Finnish type (FAF, Finnish hereditary amyloidosis) is caused by a 654G-A mutation in the gelsolin gene on chromosome 9 resulting in the expression of mutant Asn-187 gelsolin which is abnormally proteolytically processed generating amyloidogenic fragments that polymerize into amyloid fibrils. We have recently shown that in a Danish and a Czech family with a clinical syndrome similar to FAF, including corneal lattice dystrophy, cranial neuropathy and skin changes, the disease is caused by another mutation at the same position, namely 654G-T predicting a Try-for-Asp substitution at 187 in secreted gelsolin. AIM: To undertake a closer examination of the Danish subtype of FAF and report immunohistochemical and biochemical findings. RESULTS: Immunostaining of plasma gelsolin isolated from heterozygous FAF of the Danish subtype revealed a pattern similar to that found in FAF-Asn 187. The > 60 kDa gelsolin species contain an epitope characteristic of the amyloid forming region as revealed by an amyloid specific antibody, whereas the approximately 50 kDa fragments are devoid of it. Compared with the wild-type gelsolin peptide (Asp-187), the corresponding mutant peptide (Tyr-187) showed dramatically increased fibrillogenicity as revealed by quantitative thioflavine-T based fluorimetry; ultrastructurally, amyloid-like fibrils were formed by the mutant peptide. Immunohistochemistry showed that antibodies directed against residues 231-242 of secreted gelsolin, representing the carboxy terminus of the sequence forming the amyloid protein (residues 173-243) laid down in the tissues in a fibrillar form in FAF, specifically labelled the amyloid deposited in rectum and skin in the Danish (654G-T) subtype. CONCLUSIONS: The 654G-T mutation in the gelsolin gene gives rise to an amyloid disease clinically and pathogenetically similar to that caused by the 654G-A mutation.

Increased expression of extracellular matrix metalloproteinase inducer in rheumatoid synovium.

OBJECTIVE: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in the synovial membrane of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Mouse monoclonal antibody against human EMMPRIN was applied according to an avidin-biotin-peroxidase complex method to reveal EMMPRIN expression. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to check for the presence of EMMPRIN protein and messenger RNA (mRNA). RESULTS: EMMPRIN immunoreactivity was more intense in RA than in OA synovial membrane (P < 0.01). EMMPRIN staining was more widespread in RA than in OA, especially in association with macrophage infiltrates. RT-PCR of synovial membrane samples disclosed the presence of EMMPRIN mRNA. Nucleotide sequencing of the PCR amplification products confirmed the identity of the amplified bands. Immunoblot analysis revealed 55-kd glycosylated EMMPRIN bands, which were particularly prominent in RA samples. CONCLUSION: The expression of EMMPRIN is upregulated in the rheumatoid synovial membrane. EMMPRIN can induce local production of at least MMPs 1, 2, and 3, and can thereby play a role in joint destruction in RA.

Identification of the circulating amyloid precursor and other gelsolin metabolites in patients with G654A mutation in the gelsolin gene (Finnish familial amyloidosis): pathogenetic and diagnostic implications.

Familial amyloidosis of the Finnish type (FAF) is an autosomal dominant type of systemic amyloidosis caused by a G654A (Asn-187) or G654T (Tyr-187) mutation in the gelsolin gene. Herein we show that patients with the Asn-187 gelsolin mutation have, in addition to full-sized gelsolin, a series of lower-Mr C-terminal fragments of gelsolin (Mr of 70,000-45,000) in the circulation, and that a 50 to 55-kd fragment of gelsolin is excreted in the urine. In homozygous FAF (Asn-187), the 65-kd fragment, which contains the amyloid-forming region (Ala173-Met243), and the 55-kd fragment, which is devoid of that region, are the major gelsolin species in plasma; whereas normal gelsolin, as well as a 70-kd fragment identified as the C-terminal portion of gelsolin starting at Glu122, and a 45-kd fragment starting at Ser384, are minor components. In patients heterozygous for the Asn-187 mutation--the usual form of the expression of the dominant disease--normal-sized gelsolin is the major circulating form; the 65- and 55-kd fragments represent minor components. Immunodetection of the plasma 65-kd gelsolin fragment, which is disease-specific, and measurement of the urinary gelsolin fragment provide useful means for diagnosing FAF.

Characterization of adhesion molecule expression in the pathogenesis of homologous collagen-induced arthritis in the DA rat.

We have investigated the inflammatory cell infiltrates and adhesion molecule expression in the synovial fluid (SF) and synovial membranes (SM) of rats with homologous collagen-induced arthritis. Immunohistochemical staining revealed that the majority of the cells in the SF were granulocytes, expressing CD11b and CD11c. In SM, the majority of the cells were monocytes/macrophages. CD49d and CD49f were expressed mainly in the erosion zone in SM, and ICAM-1 was expressed in the lining layer, in the capillaries, and in the erosion zone. In SF 7% of the cells were ICAM-1 positive. CD2 was more abundant in SM than in SF. These findings might explain the difference in granulocyte counts between SF and SM. CD49d and CD49f expression might retain lymphocytes and monocytes in SM, while granulocytes not expressing CD49d and CD49f are not retained.