Noradrenergic and peptidergic nerves in the synovial membrane of the Sprague-Dawley rat.

This immunohistochemical study of nerves in the synovial tissue of Sprague-Dawley rats demonstrated the occurrence of 4 neuropeptides and 2 enzymes that are involved in the synthesis of catecholamines. Substance P and calcitonin gene-related peptide were colocalized in fibers that terminated as varicosal endings in the synoviocyte layer. Similarly, tyrosine hydroxylase and dopamine beta-hydroxylase, which reflect the presence of noradrenaline, were colocalized with neuropeptide Y. These fibers were predominantly found adjacent to and within blood vessel walls. Immunoreactivity to vasoactive intestinal polypeptide was seen in varicose nerve terminals in the synoviocyte layer. Many were localized in vessel walls. There is accumulating evidence of an involvement of substance P and noradrenaline in the pathogenesis of inflammatory joint disease and nociception. The role of these colocalized neuropeptides, namely, calcitonin gene-related peptide and neuropeptide Y, in the pathophysiology of such conditions warrants further analysis.

Cathepsin B and cysteine protease inhibitors in human osteoarthritis.

The aim of this study was to determine the involvement of cathepsin B and its inhibitors in the proteolytic degradation of human osteoarthritic (OA) tissue. The characteristics of the cathepsin B found in both normal and OA cartilage and synovium were similar to those of the lysosomal cathepsin B. Two inhibitors of cysteine proteases were found with a molecular weight of 67,000 and 16,000 Da. The cartilage cathepsin B level of OA specimens (54.8 +/- 7.3 units/micrograms of DNA) was greater than the controls (39.8 +/- 3.2 units/micrograms of DNA). Mild-moderate graded samples (78.1 +/- 12.0 units/micrograms of DNA) had significantly higher levels of enzyme activity than the severely graded ones (31.4 +/- 3.9 units/micrograms of DNA, p less than 0.001) and controls (p less than 0.01). Compared to controls (2.3 +/- 0.4 units/mg of tissue w.w.), cysteine protease inhibitory activity in OA cartilage was decreased in specimens with severe lesions (1.5 +/- 0.2 units/mg of tissue). This was particularly noted in patients who had not received steroid injections (1.2 +/- 0.3 units/mg of tissue, p less than 0.05). In OA synovia, the cathepsin B level was greater (40.7 +/- 7.4 units/mg of tissue w.w., p less than 0.02) than in the controls (13.6 +/- 3.7 units/mg of tissue). The cysteine protease inhibitory activity was similar in OA synovium (1.7 +/- 0.2 units/mg of tissue w.w.) and in controls (1.5 +/- 0.3 units/mg of tissue). This data demonstrated an imbalance between the levels of cathepsin B and cysteine protease inhibitors in OA tissue. A decrease of specific inhibitors could be an important contributing factor, particularly in more severe lesions.

Cellular fibronectin in rheumatoid synovium and synovial fluid: a possible factor contributing to lymphocytic infiltration.

Mouse monoclonal antibodies against ED sequence-containing cellular fibronectin (cFn) were used to show that Fn in the inflamed synovium is distinct from the major form of plasma Fn (pFn). An accumulation of cFn was seen at sites of hyperplasia of the rheumatoid synovial membrane and in the walls of small vessels in the synovium by immunofluorescence microscopy. cFn was also found in rheumatoid synovial fluid by immunoblotting. Approximately one-fifth of the T lymphocytes from rheumatoid synovial fluid bound to Fn. The binding of synovial fluid T cells was always higher than that from peripheral blood. These results have two implications. On the one hand, the cellular type of Fn may be an indicator of synovial inflammation. On the other hand, the deposition of Fn may be a factor contributing to the infiltration of mononuclear cells into the synovium.

Salmonella lipopolysaccharide in synovial cells from patients with reactive arthritis.

Synovial cells from nine patients with reactive arthritis following Salmonella enteritidis or Salmonella typhimurium infection were examined for salmonella antigens. Extensive bacterial cultures of the synovial fluid were negative. Eight synovial-fluid cell samples stained positively on immunofluorescence with rabbit antisera against heat-killed S enteritidis or S typhimurium or with monoclonal antibodies specific for the causative salmonella lipopolysaccharide (LPS). Synovial tissue from the ninth patient stained positively in the avidin-biotin-peroxidase complex method with the monoclonal antibody. Control samples (synovial-fluid cells from thirteen patients with other rheumatic diseases and synovial tissue from two) were negative. Synovial cells from eight patients and five controls were studied by western blotting with the same monoclonal antibodies. Four of the eight patients but no controls had blots indicating salmonella LPS in the synovial cells. The presence of bacterial LPS in the joint is a common and pathogenetically important feature of reactive arthritis.

Glycosaminoglycans of the hemodialysis-associated carpal synovial amyloid and of amyloid-rich tissues and fibrils of heart, liver, and spleen.

Significant amounts of glycosaminoglycans (GAGs) were found in amyloid fibril preparations. Using two-dimensional electrophoresis to fractionate GAG mixtures, we quantified and identified for the first time the GAGs of the fibrils from carpal synovium of patients with amyloid associated with chronic hemodialysis. The total GAG content was small, but the GAG distribution (high relative content of chondroitin sulfate and hyaluronic acid and lack of the other GAGs) was unique, unlike that for the other amyloid fibril preparations. The amyloid-rich heart, liver, and spleen tissues, as well as the fibrils isolated from these tissues of patients with systemic forms (primary amyloid and secondary amyloid) of amyloid disease, were also analyzed for GAGs. Fibrils from heart tissue of a patient with primary amyloidosis, now examined for the first time, contained four major GAGs (chondroitin sulfate, dermatan sulfate, hyaluronic acid, and heparan sulfate).

Immunohistopathologic findings in synovial biopsies before and after synovectomy in patients with chronic inflammatory joint diseases and their relation to clinical evaluation. A prospective study of biopsies taken from areas selected by arthroscopy.

Synovial biopsies were obtained from 28 patients with various kinds of chronic arthritis, at synovectomy and 6 and 12 months later. The tissues were examined by immunofluorescence technique, recording the quantities of cells and extracellular deposits staining with polyclonal antisera to IgG, IgA, IgM, C3c, fibrinogen, and chi and lambda light chains, and monoclonal antibodies to CD3, CD5, CD11b, HLA DR, and TCC (Terminal Complement Complex). These parameters were compared with scores obtained by arthroscopy and clinical evaluation (Colorado Knee Score) performed at the same time. Taken as a group, the immunological parameters showed reduction in activity 6 months after synovectomy (p less than 0.01), and a tendency to revert to base-line values after 12 months. A similar reduction in activity after 6 months was also found by arthroscopic and clinical evaluation. Thus, this longitudinal study demonstrated a relationship between changes in immunologic activity, arthroscopic findings and clinical activity after synovectomy in patients with chronic arthritis. This is consistent with an immunological pathogenesis for the inflammation in these joints.

Localisation of lysozyme mRNA in rheumatoid synovial membrane by in situ hybridisation.

Type A synovial lining cells have been shown to contain lysozyme in their lysosomes. This might be phagocytosed because synovial fluid contains lysozyme originating from tissue macrophages and articular cartilage but in arthritides, in particular, from neutrophils. In situ hybridisation with 35S labelled cDNA was used to detect mRNA for lysozyme over synovial lining in patients with rheumatoid arthritis. No hybridisation was found with lactoferrin cDNA, which was used as a negative control. Computer search against the EMBL gene bank (release 14) did not show any significant cross hybridisation to a known sequence. In cytological specimens 35S-cDNA:mRNA hybrids were observed in positive but not in negative control cells. The presence of lysozyme and its mRNA suggests that type A synovial lining cells are of mononuclear phagocyte lineage.

Human epidermal growth factor for the stratification of synovial lining layer and neovascularisation in rheumatoid arthritis.

Immunohistochemical study showed selective localisation of human epidermal growth factor (hEGF) to the synovial lining layer. Although the synovial lining layer of the rheumatoid, osteoarthritic, and traumatic joints was hEGF positive, hEGF staining was especially dense at the rheumatoid synovial lining layer; the staining increasing linearly according to the degree of stratification of the lining layer (r = 1). Human epidermal growth factor was ultrastructurally localised to cytoplasm, especially to rough endoplasmic reticulum, of the synovial lining fibroblast-like (type B) cell. Only the cell surface of macrophage-like (type A) cells was hEGF positive. When different histological variables were compared with each other a positive correlation was found between hEGF staining of the synovial lining layer and the degree of neovascularisation of rheumatoid synovium (r = 0.72). Although some lymphocytes were weakly hEGF positive, neovascularisation did not correlate with the extent of lymphocyte infiltration or of hEGF staining of lymphocytes. Lymphocyte infiltration or hEGF staining of lymphocytes did not correlate with hEGF staining of the synovial lining layer, whereas the lymphocyte infiltration correlated positively with the extent of perivascular accumulation of lymphocytes (r = 0.89). These findings suggest that (a) hEGF is synthesised by and secreted through endoplasmic reticulum and Golgi apparatus from the synovial lining type B cell; (b) hEGF is at least partially responsible for the pathogenesis of stratification of the rheumatoid synovial lining layer, and perhaps of neovascularisation of the rheumatoid synovium, whereas it is not responsible for lymphocyte accumulation to the rheumatoid synovium.

Characteristics of tumor necrosis factor production in rheumatoid arthritis.

The biological effects of tumor necrosis factor (TNF) include the enhancement of fibroblast proliferation, the secretion of collagenase and prostaglandin E2 (PGE2) by fibroblasts, and the resorption of bone and cartilage, suggesting a role for this cytokine in arthritic conditions. To investigate this, we measured the levels of TNF in synovial fluids and evaluated its secretion by synovial fluid mononuclear cells and tissues from patients with rheumatoid arthritis, osteoarthritis, and seronegative arthritis and normals. TNF was found to be secreted in all arthritic conditions but not in normals. The levels of TNF were highest in synovial fluid and correlated with interferon-gamma (IFN-gamma) levels but not PGE2. The production of TNF was stable in a single joint for 3 to 6 months. Using immunohistochemical staining, TNF was localized to mononuclear cells in the lining layer, sublining, and perivascular areas of synovial tissue. The secretion of TNF by rheumatoid synovial fluid mononuclear cells was inhibited by PGE2, while IFN-gamma enhanced its production in those cells which were spontaneously secreting TNF. Our data suggest that TNF may play a role in various arthritic diseases.

Leukocyte migration in synovial tissue. Leukocyte distribution, orientation, and migratory pattern after immune complex deposition in rabbit knee joints.

Complement-activating bovine serum albumin (BSA)-anti-BSA immune complexes (ICs) were injected into rabbit knee joint cavities; the contralateral control joint was injected with BSA together with normal rabbit serum. The migration of leukocytes from the synovial venules into the joint cavity was analyzed with light microscopy (LM), scanning (SEM) and transmission (TEM) electron microscopy. EM autoradiography was used to study the endocytosis of ICs by leukocytes. The shape, orientation, and distribution of migrating polymorphonuclear granulocytes (PMNGs) were analyzed by LM morphometry. PMNGs accumulated in the joints injected with ICs. The peak of the number of PMNGs in the synovial tissue was reached after 4 hours, in the joint cavity after 6 hours. PMNGs in the synovial tissue were concentrated in the intimal layer. Migrating PMNGs were polarized, as judged by the ratio between the long (D max) and short (D min) axes of the cells. There was a close association between the migrating PMNGs and the collagen fibers. The morphometric data showed that the nonflattened, cylindrically-shaped PMNGs were oriented along the collagen bundles, running parallel to the synovial surface, and did not migrate in the straight direction of a theoretic leukotactic gradient originating in the joint cavity after IC deposition. SEM and TEM showed that the PMNGs were aligned along the collagen fibers and interacted activity with the collagen by pseudopods and cytoplasmic projections. EM autoradiography showed that the PMNGs in the joint cavity had ingested 125I-labeled ICs and were degranulated. In contrast, the PMNGs within the synovial membrane did not show any signs of IC endocytosis or any apparent degranulation. Synovial type A cells were found to contain ICs. This study indicates that the response of PMNGs in IC-induced synovitis consists of two distinct phases: an initial, mainly migratory phase in the synovial membrane where the PMNGs appear to use the collagen fibres as a climbing framework, and a second phase, in the joint cavity, characterized by PMNG metabolic activation, endocytosis of ICs, and degranulation. The apparent inability of PMNGs in the synovial membrane to ingest ICs and become degranulated might be due to not only concentration differences of ICs and leukotactic factors between the joint cavity and the synovial tissue but also might be related to the apparently active interaction with collagen.

Presence of platelet glycoproteins Ib and IIb-IIIa in inflammatory and noninflammatory synovium.

Rheumatoid synovial tissue and noninflammatory synovial tissue from patients with meniscus lesions were stained using monoclonal antibodies against platelet 150 kDa Ib glycoprotein (gp Ib) and against 140/110 kDa IIb-IIIa glycoprotein complex (gp IIb-IIIa) applied with the avidin-biotin-peroxidase complex method. Gp Ib and gp IIb-IIIa positive intravascular platelet aggregates were not seen, except locally in the capillary blood vessels of one rheumatoid synovial sample. This suggests that the platelets and the clotting sequence are not activated in inflamed synovial tissue. However, in many of the synovial capillaries endothelial immunoreactivity was seen. This reaction could have been due to cross reaction, since the vitronectin receptor beta chain is structurally identical to platelet gp IIIa. The gp IIb-IIIa member of the integrin receptor family plays a role in the transmembrane linkage between its extracellular ligands and intracellular microfibers. Gp IIb-IIIa may thus contribute to normal synovial physiology and to the pathogenesis of chronic synovitis.

Amyloid localized to tenosynovium at carpal tunnel release. Natural history of 124 cases.

One hundred fifty-two patients with amyloid in the tenosynovium who had carpal tunnel release were identified. Twenty-eight patients were excluded because of systemic amyloidosis: primary systemic amyloidosis (AL) in 24, secondary amyloidosis (AA) in 3, and familial amyloidosis (AF) in 1. The remaining 124 patients (82%) had carpal tunnel syndrome with local deposition of amyloid and no evidence of systemic amyloidosis. Median survival of the 124 patients from diagnosis of amyloidosis was 12 years. Only two patients had systemic amyloidosis develop--9 and 10 years after recognition of tenosynovial amyloid. Of particular interest were 12 patients who had an M-protein in the serum or urine. None of the 12 patients have had evidence of systemic amyloidosis or multiple myeloma during the median follow-up of 14 years. The authors conclude that amyloid may be localized to the tenosynovium and that systemic amyloidosis rarely develops during long-term follow-up.

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Beta-2-microglobulin-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome.

The clinical manifestations of beta-2-microglobulin (beta 2M)-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome from a medical center hospital are presented. The predominant morbidity of beta 2M-amyloid was musculoskeletal, with deposits identified in surgical or biopsy specimens from trigger fingers, carpal tunnels, fractures, and radiolucent bone lesions. Lucent bone lesions were the characteristic radiologic finding of beta 2M-amyloidosis and were most commonly found in carpal bones, humeral heads, and femoral heads. Carpal tunnel syndrome occurred in greater than 20% of our chronic hemodialysis patients. The longer the period of time on chronic hemodialysis the greater the morbidity from beta 2M-amyloid. Although significant amounts of beta 2M-amyloid were detected in the perivascular regions of viscera, clinical compromise of internal organs from this type of amyloid was not documented. In acute studies, beta 2M clearance during hemodialysis was markedly increased using the Fresenius polysulfone dialyzers compared to cuprophane dialyzers. In summary, beta 2M-amyloid is common and causes significant morbidity in chronic hemodialysis patients. Long-term dialysis with highly permeable membranes effects greater beta 2M clearance which may result in less tissue deposition of beta 2M-amyloid, and therefore, fewer clinical complications.

Osteoarthrosis and apatite synovitis. Pathological study of a metacarpophalangeal joint.

A case of metacarpophalangeal osteoarthrosis associated with synovial apatite deposits is reported. The size of the crystals indicates that they have been thickened by a recrystallization process; the latter could have been provoked by Ca and Po4 ions released by dissolution of some apatite crystals brought by calcified debris of bone or cartilage coming from the abraded osteoarthrotic surfaces. The role of such thickened crystals in synovial inflammation is discussed as well as their possible diagnostic value in determining origin and pathogenesis of a given synovial apatite deposit.

Epithelial markers in synovial sarcoma. An immunohistochemical study on paraffin embedded tissues.

Immunohistochemical studies on synovial sarcomas have proved the potentiality of these neoplasm for epithelial and mesenchymal differentiation and antibodies detecting epithelial cells have been found to be helpful in determining the histological types. In this study different epithelial markers directed against various cytokeratins, HMFG-2 and EMA were investigated on paraffin embedded tissues of 13 cases of synovial sarcomas, with regard to their reliability in unmasking the epithelial components demonstrable in this type of neoplasm. The results lead to three conclusions: firstly, synovial sarcomas possess the capacity for generating different epithelial cell types with uncommon compositions of intermediate filaments as well as of membrane proteins, secondly, these features may be expressed in a heterogenous pattern even within the same tumour and finally, the use of wide range anti-cytokeratin antibodies covering the spectrum of basic as well as acidic type proteins seems to be necessary for the detection of all epithelial components demonstrable in synovial sarcomas.

[Expression of the interleukin-2 receptor in T lymphocytes of the rheumatoid synovial membrane]. / Espressione del recettore per l'interleuchina-2 nei T-linfociti della membrana sinoviale reumatoide.

Indirect immunofluorescence was used to stain DR antigen and interleukin-2 receptor (Tac) of T-lymphocytes (Leu 4+). Tissue samples of synovial membrane were cut from arthroscopic biopsies of inflamed knees in four patients with active rheumatoid arthritis (RA). Consecutive cryostatic sections of rheumatoid sinovium were analysed using monoclonal antibodies. It was found that a high percentage of T-lymphocytes express DR antigen. In contrast the proportion of T cells expressing Tac was small. We conclude that T cell activation in synovial membrane is incomplete, and this disfunction may contribute to the chronic inflammation of RA.

P component in the synovium in rheumatoid and osteoarthritis.

P component is present in amyloid deposits, normal serum, and normal tissues in relation to elastic fibres. Its pathological role in inflammatory synovitis was investigated. Its distribution was determined immunohistologically in 33 synovia: 15 rheumatoid; seven osteoarthritic; seven traumatic controls; and four infected biopsy specimens. P component was present in two circumscribed distributions: extracellular fibrils in dense fibroelastic tissue of the more fibrotic synovia; and in the arterial wall, where it was confined to a single elastic lamina in some cases and in others showed reduplication and fragmentation. These were not related to amyloid material. It shows no disease specificity, but P component categorises the nature of the pathological reaction and is typically in biopsy specimens showing the development of chronic fibrosis. There was close codistribution of P component with elastic tissue, though this was not absolute. P component had a different distribution from C reactive protein (in synovial lining cell layer), and fibronectin, which was absent from fibrotic areas. Understanding the pathological interactions of P component may help elucidate why some synovial reactions remain inflammatory and other progress to chronic fibrosis.

Augmented interleukin-1 production and HLA-DR expression in the synovium of rheumatoid arthritis patients. Possible involvement in joint destruction.

Potent interleukin-1 (IL-1) activity was detected in culture supernatants from synovium, obtained by arthroscopy, from rheumatoid arthritis (RA) patients but not from non-RA patients. Production of IL-1 by RA synovium correlated well with findings of inflammation on arthroscopy and HLA-DR expression in immunohistochemical staining. Furthermore, there was a positive correlation between IL-1 production from RA synovium and joint changes detected on roentgenograms. These findings strongly suggest that IL-1 might play an important role in the joint destruction in RA.

Metallic wear in failed titanium-alloy total hip replacements. A histological and quantitative analysis.

We conducted extensive histological examination of the tissues that were adjacent to the prosthesis in nine hips that had a failed total arthroplasty. The prostheses were composed of titanium alloy (Ti-6Al-4V) and ultra-high molecular weight polyethylene. The average time that the prosthesis had been in place in the tissue was 33.5 months (range, eleven to fifty-seven months). Seven arthroplasties were revised because of aseptic loosening and two, for infection. In eight hips cement had been used and in one (that had a porous-coated implant for fifty-two months) no cement had been utilized. Intense histiocytic and plasma-cell reaction was noted in the pseudocapsular tissue. There was copious metallic staining of the lining cells. Polyethylene debris and particles of cement with concomitant giant-cell reaction were present in five hips. Atomic absorption spectrophotometry revealed values for titanium of fifty-sic to 3700 micrograms per gram of dry tissue (average, 1047 micrograms per gram; normal, zero microgram per gram), for aluminum of 2.1 to 396 micrograms per gram (average, 115 micrograms per gram; normal, zero micrograms per gram), and for vanadium of 2.9 to 220 micrograms per gram (average, sixty-seven micrograms per gram; normal, 1.2 micrograms per gram). The highest values were found in the hip in which surgical revision was performed at fifty-seven months. The concentrations of the three elements in the soft tissues were similar to those in the metal of the prostheses. The factors to which failure was attributed were: vertical orientation of the acetabular component (five hips), poor cementing technique on the femoral side (three hips), infection (two hips), and separation of a sintered pad made of pure titanium (one hip). A femoral component that is made of titanium alloy can undergo severe wear of the surface and on the stem, where it is loose, with liberation of potentially toxic local concentrations of metal debris into the surrounding tissues. It may contribute to infection and loosening.