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Monocyte-macrophage polypeptides (monokines) cause synovial cells to increase the levels of putative mediators of destruction and inflammation. This interaction may account for some of the properties of rheumatoid pannus. We report here that samples of purified human interleukin-1 beta (IL-1 beta) and recombinant IL-1 alpha stimulate both the plasminogen activator activity and prostaglandin E2 levels of human synovial fibroblast-like cells. The same holds true for purified pig IL-1 (catabolin) and recombinant murine IL-1. The elevation in plasminogen activator activity was inhibited by indomethacin, and this suggests that endogenous prostanoids are important in the IL-1-mediated stimulation of proteinase activity.

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The properties of synovial cells are altered in vitro by monocyte-macrophage polypeptides (monokines), and these changes could explain some of the properties of the inflamed synovium in rheumatoid disease. Purified monokines have become available only recently for testing on the target synovial cells. We report here that purified human interleukin (IL)-1 beta and recombinant human IL-1 alpha stimulate the extracellular activity of the lysosomal hydrolase, N-acetyl-beta-glucosaminidase (NAG), of human synovial fibroblast-like cells. In contrast, another monokine, synovial activator, does not increase the NAG activity. Thus NAG is another cellular activity which can be modulated by interleukin-1.

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Supernatant media from cultured human mononuclear blood leukocytes (MCCM) induced morphological changes in normal human synovial fibroblasts in culture, including the formation of cells with a dendritic or stellate morphology and, less frequently, cells with a striking fenestrated appearance. These changes were fully reversed within 1 h of removing the MCCM. They were inhibited by indomethacin, the glucocorticoids hydrocortisone, prednisolone and dexamethasone, and by colcemid, but not by actinomycin D and only weakly by cycloheximide. The morphological responses to MCCM could be reproduced by MCCM fractions containing interleukin 1-like activity and by purified forms of human interleukin 1 (IL-1), including monocyte-derived IL-1 beta and recombinant IL-1 alpha. These responses were also inhibited by indomethacin, indicating a link with prostanoid production. However, the morphological responses were not related to the stimulation of plasminogen activator activity due to MCCM, MCCM fractions, or IL-1.

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Fibroblast-like synovial cells, isolated from intact joints of non-arthritic human donors and from explants of rheumatoid and non-rheumatoid synovial tissue, released prostacyclin (PGI2) when incubated in conditioned medium from human peripheral blood mononuclear cells (MCCM). The effect of MCCM on the rate of PGI2 synthesis (measured by radioimmunoassay as the stable product, 6-keto-PGF1 alpha) was clearly established within 2 h and appeared to require RNA and protein synthesis as judged by inhibition with actinomycin D and cycloheximide, respectively. Low concentrations of dexamethasone suppressed the increase in PGI2 levels. Prostaglandin E2 (PGE2) levels were also raised by the MCCM and reduced by dexamethasone. All-trans retinoic acid did not stimulate the levels of either prostanoid. These findings offer an explanation for some of the inflammatory events occurring in rheumatoid lesions.

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Fibroblast-like synovial cells isolated from intact joints of non-arthritic human donors released up to nine-times higher activity of the lysosomal acid hydrolase N-acetyl-beta-glucosaminidase (NAG) than controls when incubated in conditioned medium from homologous peripheral blood mononuclear cells (MCCM). This increase occurred without decrease in cell numbers or other evidence of cytotoxicity. An increase in cell-associated NAG activity was also suggested, but this was not statistically significant. Indomethacin present during production of MCCM or added with MCCM to fibroblast cultures did not alter the response to MCCM, indicating that the effect of MCCM was not due to the presence of products from the cyclo-oxygenase pathway. At a concentration known to block protein synthesis in most cells (10(-5) M), cycloheximide markedly suppressed the NAG releasing response to MCCM. The secretion of NAG due to MCCM was not affected by all-trans retinoic acid (10(-6) M) but was suppressed by the corticosteroid, dexamethasone.

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Glycosaminoglycan production, acid hydrolase activity, proliferation, and morphology were examined in human synovial cells subjected to low environmental pH. The amount and the molecular size of newly synthesised glycosaminoglycan (GAG) were increased without significant change in the rate of cell proliferation. Lowered pH produced an increase in the size of cytoplasmic organelles. Some of these possessed ultrastructural features of lysosomes, but others were clearly nonlysosomal and were of uncertain identity. Intracellular activity of the lysosomal acid hydrolase N-acetyl-beta-glucosaminidase (NAG) was not altered by low pH, but a marked increase occurred in extracellular NAG activity, indicating enhanced release.

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The acid hydrolase N-acetyl-beta-glucosaminidase (NAG) was used to examine the effects of prostaglandins E1 (PGE1), E2 (PGE2), and F2 alpha (PGF2 alpha) on the lysosomal system of human synovial cells in vitro. A spontaneous release of the enzyme occurred from control cultures, which was accelerated by each of the prostaglandins in a concentration-dependent manner, within the range of 10(-8)-10(-6) moles per litre (M). No clear order of potency could be established. The effects of the prostaglandins on release of NAG were less consistent and of smaller magnitude when human serum was replaced by bovine serum albumin in the medium. In the presence of serum small increases also occurred in intracellular NAG activity, but only the effect of PGE1 was statistically significant. The prostaglandins did not appreciably affect the previously established pattern of increased intracellular activity of NAG and reduced release produced by sucrose.

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Synovial tissue from patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and having menisectomies was examined by immunofluorescence for deposits of alpha-2-macroglobulin (alpha 2M). In inflammed tissues, alpha 2M was found in the synovial lining cells and in perivascular cells. The amount of alpha 2M correlated with the degree of inflammation. Similarly, free lining cells obtained by trypsination of the intact synovial membrane contained identical inclusions. alpha 2M was not detected in the menisectomy cases and in the less inflammatory osteoarthritic specimens. In-vitro studies demonstrated uptake of alpha 2M-trypsin complexes but not of native alpha 2M by most of the cultured synovial cells whether they came from rheumatoid patients or controls. The internalised complexes disappeared within 12 hours of culture. The results suggest that alpha 2M-proteinase complexes formed in the joint are taken up by phagocytic and perivascular cells in a similar way to immune complexes.

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Previously described morphological changes in human synovial cell cultures due to cholera enterotoxin (CT) were studied in relation to activation of adenylate cyclase. A single pulse of CT at nanomolar concentration or less induced at least two-fold activation of adenylate cyclase, which persisted for 7 days or more. The enzyme hyaluronidase was found to cause a rapid reversal of the morphological effects of CT. There was also a reduction in adenylate cyclase activity but only with hyaluronidase concentrations greater than those required to produce maximum reversal of the CT-induced morphological changes. Removal of hyaluronidase was followed by reappearance of the CT-associated morphological effects and a slower reactivation of adenylate cyclase. The mechanism by which hyaluronidase produces the observed changes in synovial cells is not known, but might be related to the dispersal of hyaluronic acid gels bound to the surface of these cells.

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Life spans, growth rate, glucose utilisation, response to hydrocortisone, and intracellular activity of lysosomal N-acetyl-beta-glucosaminidase of rheumatoid synovial cells in culture were compared with these properties in nonrheumatoid synovial cells. Except for a small group of RA cells derived from tissue explants, the cells were all isolated by trypsinisation of synovial tissue, either within intact joints or after synovectomy. Cell lines were established by passaging with trypsin. In a study of 56 nonrheumatoid and 24 rheumatoid synovial lines isolated during a 7-year period the latter were found to have a shortened mean life expectancy in culture, though there was wide variation between individual lines. This is in agreement with reported findings from untrypsinised explant-derived synovial lines. However, in the present study mean multiplication rates were identical for nonrheumatoid and rheumatoid synovial cells, and on clear differences could be demonstrated for the other properties studied. No correlation could be found between the life spans of synovial cell lines and the age of the cell donors, whether from rheumatoid or nonrheumatoid sources. Rheumatoid synovial cells isolated from intact joints were notable for especially high proportions of macrophage-like cells and suppression of fibroblasts. In most cases cell lines could not be established from these rheumatoid primary cultures, and in others the lines were short-lived. Early association with relatively high proportions of macrophage-like cells in rheumatoid cultures might thus be important in influencing the establishment and behaviour of synovial cell lines.

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Examination of synovial fluid in eight patients with epidemic polyarthritis following Ross River virus infection showed cell counts ranging from 1,500 to 13,800 per mm3. Stained smears were notable for a paucity of neutrophils and high proportions of monocytes and vacuolated macrophages, which were further characterised by light and electron microscopy. Ross River virus antigen was detected by specific immunofluorescence in monocytes and macrophages of four cases early in the course of the illness, but intact virus was not identified by electron microscopy or cell culture. No cell-associated C3 component of complement or immunoglobulin of IgM or IgG class was detected in any cell-type. These findings indicate that cell counts and simple smears may be useful in the early diagnosis of suspected epidemic polyarthritis, and provide further information pertinent to the pathogenesis of this disease.

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A case of herpes zoster complicated by acute arthritis with effusions is described. The white cell count in the synovial fluid was low, with a predominance of neutrophils. The synovium showed superficial deposits of fibrin, slight intimal hyperplasia, and subintimal polymorph infiltration. Varicella virus was not detected by culture or electron microscopy, but varicella antigen was demonstrated in the cytoplasm of macrophages in the effusion. Other findings did not determine whether the antigen had appeared in the joint from circulating immune complexes, or following synovial phagocytosis or proliferation of virus.

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In contrast with newly isolated cells or early primary cultures, synovial cell lines in standardised growth conditions assume a rather uniform fibroblast-like appearance. However, 2 distinct variations in the cytological pattern can be induced at this stage. The first is characterised primarily by increased numbers of small phase-dense organelles that show the distinctive fluorescence of lysosomes after supravital staining, and are interspersed with vacuoles. The associated functional changes include increased enzyme activity and decreased net synthesis of hyaluronic acid. This variation can be induced by exposure to indigestible neutral sugars, adenosine, or its 5' nucleotides. The second variation consists of a striking reorganisation of cytoplasm by condensation into dense ridges or a dendritic network of processes. It is accompanied by increased hyaluronic acid secretion and is induced by agents that enhance intracellular activity of cyclic adenosine monophosphate, such as dibutyryl cyclic adenosine monophosphate and cholera enterotoxin. It appears possible to direct differentiation in synovial cell lines to correspond at least in part with the presumed functions of the different cell types in the parent tissue. The 2 patterns may be useful markers to correlate with other aspects of synovial cell function in vitro.

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Some in-vitro effects of the arthritogenic polysaccharide carrageenin were studied on cells from human synovium. Synovial cells were isolated from intact human knee joints, and cell lines were developed by passaging with trypsin. Carrageenin was ingested by the cells but did not significantly affect cell growth, numbers of lysosomes, intracellular lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase), or release of lysosomal enzyme from cells. Carrageenin produced a reduction in net hyaluronic acid synthesis. It also induced a striking morphological change in a high proportion of synovial cells, characterised by increased spreading over the culture surface and apparent condensation of the cytoplasm into a pattern of ridges. Nonrheumatoid and rheumatoid synovial cells behaved similarly to one another.

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Synovial cell lines were isolated by instillation of trypsin or chymotrypsin into intact knee joints of patients with persistent rheumatoid effusions resistant to conventional therapy. Morphology and growth in the primary phase were compared with rheumatoid cells isolated from excised synovium and nonrheumatoid synovial cells obtained from intact joints of cadavers or amputated limbs. Cell populations from all sources included varying proportions of macrophage-like and fibroblast-like cells, with only 1-3% multinucleated cells. In medium supplemented with calf serum alone, rheumatoid cells from intact joints showed negligible changes in morphology. However, in the presence of nonrheumatoid, autologous rheumatoid or homologous rheumatoid serum a rapid increase occurred in size of the macrophage-like cells and numbers of polykaryocytes, including some giant syncytial cells. These effects were directly proportional to serum concentration and were identical in fresh or heat-inactivated serum. In most of these rheumatoid cell lines no multiplication occurred, regardless of serum type or concentration. In rheumatoid synovial cells from excised synovium, human serum induced both polykaryocytosis and rapid growth of fibroblasts. Nonrheumatoid synovial cells grew rapidly but few polykaryocytes developed, mostly with less than 6 nuclei. Evidence of viral infection in rheumatoid synovial cells was sought by electron microscopy after stimulation of polykaryocytosis by human serum. In one of the cultures many cells were found with intranuclear particles possessing characteristics of the adenovirus group.

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A patient with epidemic polyarthritis was studied within 48 hours of onset when specific serum antibodies were still low. The synovial fluid showed a fall in hyaluronic acid level and rise in protein levels though the immune globulins, and especially IgM, rose to a lesser degree and remained well below the levels in the serum. Attempts to grow virus from synovial fluid or blood lymphocytes failed despite the use of several new techniques. The complement components C'3 and C'4 were not depleted in serum or synovial fluid. The synovial fluid was devoid of neutrophil leucocytes, and contained predominantly monocytes and macrophages which were remarkable for mitotic and for enhanced and indiscriminate phagocytic activity. From this and other evidence, an explanation is proposed for the cytological response and difficulties in recovering infective virions in virus-induced arthritis. No virus antigen was detected in the supernatant synovial fluid and electron microscopy showed virus-like particles only in cytolysosomes.

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