Increased basal phosphorylation of mitogen-activated protein kinases and reduced responsiveness to inflammatory cytokines in neutrophils from patients with rheumatoid arthritis.

OBJECTIVE: We studied the functions of peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA), focusing the molecular basis for the activated state and the functional responsiveness of RA neutrophils to inflammatory cytokines. METHODS: Paired samples of PB neutrophils and SF neutrophils from the inflamed knee joint were obtained from 18 RA patients (5 males and 13 females). RESULTS: RA neutrophils exhibited increased spontaneous superoxide (O2-) release and adherence, increased basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase, accelerated spontaneous apoptosis, and enhanced O2- release in response to N-formyl-methionyl-leucyl-phenylalanine as compared with healthy normal PB neutrophils. When challenged with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or tumor necrosis factor alpha (TNF-alpha), RA neutrophils exhibited reduced responses to these cytokines, which included O2- release, adherence, priming for enhanced O2- release, and phosphorylation of ERK and p38. The functional alterations were greater in SF neutrophils than in PB neutrophils from RA. Reduced responsiveness to cytokines in RA neutrophils was closely associated with increased serum and SF levels of GM-CSF and TNF-alpha. RF and RAHA titers were closely correlated with increased TNF-alpha level in SF. CONCLUSION: These findings indicate that RA neutrophils are in the activated state with increased basal phosphorylation of ERK and p38, and exhibit reduced responsiveness to inflammatory cytokines (G-CSF, GM-CSF and TNF-alpha) and accelerated spontaneous apoptosis.

Time-sequential changes in biomechanical and morphological properties of articular cartilage in cryopreserved osteochondral allografting.

This study examined time-sequential changes in the biomechanical and morphological properties of articular cartilage that had received cryopreserved osteochondral allografting. Osteochondral blocks obtained from the femurs of 18 rabbits were cryopreserved with dimethylsulfoxide (DMSO), using a two-step freezing method, and allografted to the femurs of another 18 rabbits. Specimens for biomechanical and morphological examinations were prepared at the second, fourth, and twelfth weeks after allografting (n = 18). In 12 allografted rabbits, biomechanical features were examined with an indentation test apparatus, and histological changes were studied with a light microscope (second week, n = 4; fourth week, n = 4; twelfth week, n = 4). In the other 6 allografted rabbits, cartilage surfaces were studied with a scanning electron microscope (second week, n = 2; fourth week, n = 2; twelfth week, n = 2). For controls, fresh, DMSO-treated, or DMSO-treated + cryopreserved specimens were examined biomechanically and morphologically. In the time-sequential examination of biomechanical features, both the parameter for elasticity (i.e., ratio of instant elastic strain to maximum strain) and the parameter for viscosity (i.e., average retardation time) significantly changed. Light microscopy showed chronological decreases in safranin-O staining intensity in the matrix, and progression of degeneration. On scanning electron microscopy, disruption of the cartilage surface was also recognized. Therefore, changes in biomechanical properties due to cryopreservation could cause irreversible changes in the cartilage in cryopreserved osteochondral allografting.

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes.

We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNFalpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNFalpha-treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFalpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis.

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Expression of differentiated phenotype in the pseudo-tendon sheath formed by a silicon rod.

We examined the cartilaginous gene expression in the inner layer of the pseudo-tendon sheath formed by an silicon rod. While cartilaginous gene expression was not detected in the outer layer of the tissue, gene expressions of aggrecan and type II collagen were detected in the inner layer of the the newly formed pseudo-tendon sheath around the silicon rod. Relative expression of aggrecan and Type II collagen were 0.15 and 0.28, respectively, compared to that of GAPDH. The expression of type II collagen was 0.57-folds of that of type I collagen. In these tissues, a sliding surface was formed by a silicon rod and the surrounding tissues, and its mechanical stress may induce cartilaginous gene expression. Mechanical stress together with various growth factors and cytokines may be critically important for the formation of more physiological tendon sheath structures. Therefore, we will further examine the changes detected in the tissues and evaluate mechanical stress for formation of the tendon sheath.

Effects of mechanical stress on expression of differentiated phenotypes of chondrocytes.

In the patients with dislocated hip arthropathy, cartilage gene expression was confirmed in weight-bearing inner layer tissues of the joint capsule. Because these inner layer tissues of the joint capsule formed joint-like structures with the femoral head for a long period, cartilaginous genes may have been expressed in the weight-bearing inner surface of the joint capsule. There was a difference in expression of the genes between weight-bearing and non-weight-bearing parts. From a quantitative comparison between GAPDH and aggrecan gene expression, aggrecan gene expression was 30-fold higher in the weight-bearing part than in the non-weight-bearing part. Aggrecan gene expression was not detected in outer layer tissues of the joint capsule. Type II collagen and TGF-beta genes were also detected, and both genes showed differences in expression between the weight-bearing and non-weight-bearing parts like the aggrecan gene. This may have been because mechanical stress caused cartilaginous differentiation in undifferentiated mesenchymal tissues in the inner layer of the joint capsule. Cell differentiation and proliferation caused by mechanical stress are indicate key role to osteoarticular tissues, and it is considered important for orthopedic treatment to evaluate the process in detail.

Radial-sequence magnetic resonance imaging in evaluation of acetabular labrum.

We investigated the usefulness of a radial-sequence magnetic resonance imaging (MRI) technique in the visualization of the acetabular labrum, which surrounds the acetabulum. In 22 hip joints of 12 volunteers, T2-weighted images were obtained on 24 radial planes of the acetabular rim, set at 15 degrees -intervals, using the small tip angle gradient echo method. We examined 7 planes in the weight-bearing portion. The acetabular labrum in the weight-bearing portion was depicted in good contrast to the surrounding tissues. The shape of the labrum differed among individuals and also in the anterior and posterior portions of the labrum. The signal intensity of the labrum was low or partially moderate. There was a high signal intensity band on the base of the acetabular labrum in several portions, which should be carefully interpreted to avoid confusion with abnormality. We concluded that radial-sequence MRI could be a useful technique for evaluation of the condition of the acetabular labrum in the weight-bearing portion.

Cartilaginous differentiation in the joint capsule.

The proliferation and differentiation of cells are greatly influenced by their environment. Many growth factors and cytokines are reported to be environmental factors that affect the proliferation and differentiation of cells. Mechanical stress is also considered to influence these physiological reactions. The joint capsule, which is a part of the joint tissue, plays a very important role in the stability of the joint and in maintaining the intracapsular phenomenon. In patients with dislocated hip arthropathy, this capsule is involved in the weightbearing function by forming a sliding surface between the capsule and the femoral head articular cartilage. The surface of the tissue macroscopically shows cartilaginous change, which indicates cartilaginous differentiation caused by mechanical stress. We examined the cartilage-specific proteoglycan component, which is composed of cartilaginou matrix at the differentiation site. We investigated proteoglycan production, molecular size, and the gene expression of cartilaginous substrate. At the inner layer of the weightbearing area of the joint capsule, proteoglycan production was significantly higher than that of other noncartilaginous tissue. We also identified the gene expression of cartilaginous proteoglycan using the reverse transcription polymerase chain reaction (RT-PCR) method.

Expression of cartilage specific genes by human capsular cartilaginous cells.

The amount of proteoglycan production in tissue that was considered to have cartilaginously differentiated due to mechanical stress and cartilage specific gene expression were investigated. The inner layer of the joint capsule which forms a sliding surface with the femoral head in the case of dislocated hip arthoropathy showed higher proteoglycan production compared to that in the surrounding non-cartilaginous tissue. On examining gene expression, although large cartilaginous proteoglycan is originally absent in this region, gene expression of aggrecan and versican which are able to bind to hyaluronan was observed. Further, gene expression of decorin and link protein was also examined. These findings in the inner layer of joint capsule forming sliding surface with the femoral head suggested the importance of mechanical stress in cartilaginous differentiatiog of mesenchymal tissue.

Local application of basic fibroblast growth factor minipellet induces the healing of segmental bony defects in rabbits.

Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 microgram or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 microgram of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice.

Measurements of zeta potentials of particulate biomaterials in protein-rich hyaluronan solution with changes in pH and protein constituents.

This study was undertaken to determine the zeta potentials of particulate biomaterials in three types of protein-rich hyaluronan solution with changes in pH; a microelectrophoretic method was used. For the purpose of determining the pH value of synovial fluid in various inflammatory conditions, we collected synovial fluid samples from joints with osteoarthritis (OA), rheumatoid arthritis (RA), and those undergoing revisions arthroplasties. The mean values of the pH in the synovial fluid from joints with OA, RA, and revision arthroplasty were shown to be 7.9, 7.5, and 8.1, respectively. The pH-zeta potential curves obtained differed, depending on the biomaterial and the medium. Addition of gamma-globulin to the medium reduced the absolute value of the zeta potentials of some of the biomaterials. The findings of this study suggest that the electrophoretic behaviors of the particulate biomaterials tested in this study are affected by the protein constituents of and pH changes in protein-rich synovial fluid. The values we obtained will be useful as reference standards and will also aid in the study of the surface phenomena of biomaterials.

Isolation and characterization of a rheumatoid arthritis-specific antigen (RA-A47) from a human chondrocytic cell line (HCS-2/8).

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA.

Alteration in the differentiated phenotypes of cultured chondrocytes from human articular cartilage under various culture conditions.

Chondrocytes produce proteoglycans and type II collagen as their main matrix components, which can also be considered to be indices of their differentiated phenotype. Each differentiation step occurs in accordance with the environment of each cell. The analysis of the properties of chondrocytes has mostly been performed in cultured cells. In this report, we examined the effects of the culture media and fetal calf serum on cultured human articular chondrocytes. It was confirmed that the quantity of proteoglycan produced by the chondrocytes changed according to the different kinds of media used, and that the molecular size of the proteoglycans showed a tendency towards de-differentiation with decreasing fetal calf serum concentrations. We conclude that our experimental results which are obtained in cultured human articular chondrocytes should be discussed with consideration to the finding of this report.

The distribution of differentiated phenotypes of chondrocytes in osteoarthritic knee.

Articular cartilage consists of chondrocytes and a cartilagenous matrix produced by these cells. The cartilaginous matrix is considered to undergo various changes in response to the surrounding environment. In this study, cartilaginous changes related to osteoarthritis of the knee were investigated. Histological changes were apparent in medical joint lesions, and changes typical of osteoarthritis, such as cell clustering and disruption of the matrix, were always observed. Degeneration was less severe in the lateral joint lesions. A biochemical analysis was undertaken and the molecular size of the proteoglycan produced in the matrix was measured by glycerol density gradient ultracentrifugation method, following pulse labeling with 35S-Sulfuric acid. The proteoglycan produced in the histologically altered regions tended to be of low molecular size. However, the size was still significantly larger than that of proteoglycans located in the nonarticular tissues such as synovia membrane, so the differentiated phenotype of chondrocytes persisted.

The role of physicochemical properties of biomaterials and bacterial cell adhesion in vitro.

This study was undertaken to investigate the physicochemical aspects of the interaction between the surface of biomaterials and bacterial cell membranes in vitro, aimed at studying the mechanisms of bacterial adhesion to biomaterials. Correlations were made between the number of adherent bacterial cells (S. aureus) and each of the calculated components of surface free energy (i.e., dispersion, polarity and hydrogen bond) of biomaterials. The effect of antibodies to cell-adhesion molecules on bacterial adhesion was also studied using monoclonal antibodies to vitronectin receptor, fibronectin receptor and CD44. This study indicates the polarity component of surface free energy plays a dominant role in the process of bacterial adhesion at least in vitro. The number of cells adherent to materials decreased to 44-73% of the control value in the presence of antibodies tested, showing that cell adhesion molecules affect adherence to biomaterials. Moreover, the results suggested that bacterial adhesion was prevented by specific blockade of cell adhesion molecule receptors.

Clinical effect of etidronate disodium (EHDP) on heterotopic ossification following total hip arthroplasty.

We investigated the status of the development of heterotopic ossification and compared the frequency of heterotopic ossification in the EHDP group with that in the untreated (control) group to evaluate the efficacy and safety of EHDP. Seventy-seven patients who had undergone total hip arthroplasty or femoral head prosthesis for the treatment of osteoarthritis and femoral neck fracture were enrolled in the study. Heterotopic ossification surrounding the hip joint was evaluated according to the roentgenographic classification. In the control group, heterotopic ossification was found in 10 patients (20%) during the period, while in the EHDP group, it was found in one patient (3.7%) during the treatment period after the operation. Gastrointestinal disorder, in one case only was a side effect observed during EHDP treatment. It was revealed that EHDP inhibits heterotopic ossification following total hip replacement in patients with osteoarthritis.

A rare case of sarcoidosis with rheumatoid arthritis.

Sarcoidosis shows many of the clinical and immunological abnormalities and is a multi-organ granulomatous disease of unknown cause which has histological features of non-caseous epithelioid granuloma formation. Sarcoidosis is rarely coexisted rheumatoid arthritis. Although such arthropathy if occur is thought to manifest rheumatoid changes, the current presence of sarcoidosis and rheumatoid arthropathy has been reported by very few histological studies. We experienced a case of combination of sarcoidosis and rheumatoid arthritis both of which were histologically confirmed. This patient initially developed small papules on the face and was diagnosed as having sarcoidosis after one year. Later, she showed symptoms and signs of polyarthritis and was confirmed to have sarcoidosis and rheumatoid arthritis in combination by histological analysis. We present this case with some reference to literatures.

Protective effects of neutral amino acids against amphipathic drug-induced hemolysis.

Some neutral amino acids were compared for their anti-hemolytic effects with sugars which are well-known colloid-osmotic protectants. The kinetic studies in isotonic suspensions of erythrocytes indicated that the hemolysis induced by the amphipathic drug chlorpromazine (CPZ) or flufenamic acid (FA) was retarded by addition of sugars, and the degree of the anti-hemolytic effect increased with increases in molecular size. Phenylalanine (Phe), the largest among the amino acids tested, showed the greatest inhibitory effect on CPZ-induced hemolysis, but not on FA-induced hemolysis. This demonstrated that the anti-hemolytic effects of amino acids were not the result of colloid-osmotic protection. Hemolytic actions of amino acids were also examined to determine their interaction with the erythrocyte membrane, and the mechanism of their inhibitory effects against amphipathic drug-induced hemolysis was discussed.

Radiological evaluation of disease progression in rheumatoid arthritis.

X-ray photographs of the wrist joints in 210 cases of Rheumatoid Arthritis (RA), who visited an outpatient clinic for RA in Orthopedic Department of the Osaka City Medical School were investigated using Carpo-Metacarpal Ratio (CMR) and Shinomiya's criteria, more detailed classification system based on the staging criteria of Steinbrocker. Radiological staging of the wrist joints revealed a correlation with the duration of RA and was considered to be a useful parameter in disease assessment. Radiological alterations in the wrist joints were progressive, but with great individual differences.