Autoantibodies to myeloperoxidase in systemic sclerosis.

OBJECTIVE: Although antimyeloperoxidase (MPO) antibodies provide a sensitive serological test for vasculitis, their significance in sera from a small proportion (about 10%) of patients with autoimmune rheumatic disease is controversial. Our aim was to determine the incidence of anti-MPO antibodies in sera from patients with systemic sclerosis (SSc) and their relation to renal disease. METHODS: Thirty-eight patients had limited cutaneous SSc (lSSc) and 43 diffuse cutaneous SSc, and within each group, 24 and 27 patients, respectively, had renal impairment (defined as stable creatinine clearance less than 60 ml/min). Six patients previously had had a scleroderma renal crisis. After screening for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence, the levels of IgM and IgG anti-MPO antibodies in 8 patients with SSc was determined by ELISA, with human MPO as antigen. RESULTS: Sera from 2 patients, both with lSSc and renal impairment, were perinuclear p-ANCA positive and had significant levels of circulating IgM and IgG anti-MPO antibodies. In one patient, anti-MPO antibodies appeared in the serum only after D-penicillamine was introduced, continued to rise after withdrawal of the drug, and fell only after immunosuppressive therapy. Renal biopsy confirmed vasculitis. The 2nd patient died of unrelated disease before further investigations could be performed. CONCLUSION: We suggest that circulating anti-MPO antibodies are not a feature of SSc per se and, if found, may indicate the presence of an unrelated pathology, such as idiopathic or drug induced vasculitis.

Expression of CD44 in normal and rheumatoid synovium and cultured synovial fibroblasts.

OBJECTIVE: To determine if expression of CD44, the principal receptor for hyaluronan, was altered in rheumatoid (RA) synovium and cultured rheumatoid synovial fibroblasts. METHODS: Synovium was obtained from normal adult human joints (n = 4) and from joints of patients with RA (n = 5). Specific monoclonal antibodies to CD44 were used in immunofluorescence of whole synovium and cultured synovial fibroblasts and in quantitative Western blotting and ELISA of CD44 in cultured synovial fibroblasts. RESULTS: CD44 was restricted to the lining layer in normal synovium but present, in reduced concentrations, throughout rheumatoid synovium. Cultured rheumatoid cells were 19% larger in area and showed far fewer and less extensive CD44-positive cytoplasmic extensions, together with reduced staining intensity compared with normal. Quantitative Western blotting normalised for cell protein showed a 75% reduction (normal = 1754 (835), rheumatoid = 409 (84) mean (SD) arbitrary units) in the amount of CD44 in rheumatoid cells compared with normal, and enzyme linked immunosorbent assay (ELISA) of cultured cell monolayers normalised for cell number indicated a 29% reduction (normal = 0.707 (0.110), rheumatoid = 0.504 (0.103), mean (SD) optical density at 405 nm). CONCLUSIONS: Rheumatoid synovial cells showed altered morphology and reduced CD44 expression compared with normal cells. CD44, by means of modulated associations with the cytoskeleton, may be involved in cell shape change.

The formation of human synovial joint cavities: a possible role for hyaluronan and CD44 in altered interzone cohesion.

During fetal development, cavitation occurs within the primitive skeleton along planes destined to become the articular surfaces of synovial joints. A histochemical study of human fetal limbs was undertaken to identify the cell types involved in this cavitation and the possible role of interactions between cells and extracellular matrix. Cryostat sections were stained with antibodies to CD68, factor VIII related antigen, prolyl hydroxylase, beta 1 integrin, VCAM-1, proliferating cell nuclear antigen, chondroitin-4 sulphate, chondroitin-6-sulphate, hyaluronan synthase and CD44. Similar sections were reacted for uridine diphosphoglucose dehydrogenase (UDPGD) and acid phosphatase activity. Hyaluronan was demonstrated using an aggrecan core protein hyaluronan binding region probe. Macrophages were present prior to cavitation in the periphery of joint interzones but not at the presumptive joint line in the central interzone. Fibroblastic cells were present throughout. Absence of local VCAM-1 expression indicated that cavitation was temporally distinct from full fibroblast-like synoviocyte differentiation. CD44 was expressed by interzone cells at all stages. Staining for hyaluronan and hyaluronan synthase, but not chondroitin sulphates was present in the interzone before and at the time of cavitation. UDPGD activity was increased in a narrow band of cells at the presumptive joint line prior to cavitation. These findings suggest that joint cavitation is dependent on the behaviour of fibroblastic cells and/or adjacent chondrocytes, rather than macrophages. Since UDPGD activity is involved in hyaluronan synthesis, it is proposed that joint cavitation is facilitated by a rise in local hyaluronan concentration in an area of tissue where cohesion is dependent on the interaction between cellular CD44 and extracellular hyaluronan. As proposed by Toole et al. (1984) such a local rise in hyaluronan concentration may lead to a switch from intercellular cohesion to dissociation, leading to tissue cavitation.

Zonal distribution of chondroitin-4-sulphate/dermatan sulphate and chondroitin-6-sulphate in normal and diseased human synovium.

OBJECTIVES: Chondroitin sulphate is the major sulphated glycosaminoglycan present in the extracellular matrix of soft connective tissues and the aim of this study was to investigate the distribution of chondroitin sulphate species in normal and diseased synovium. METHODS: Distribution of chondroitin-4-sulphate/dermatan sulphate (Ch4S/DS) and chondroitin-6-sulphate in normal (n = 6), osteoarthritic (n = 4) and rheumatoid (n = 10) synovium was determined using an immunoperoxidase technique and specific monoclonal antibodies to chondroitinase ABC-digested preparations. RESULTS: Ch4S/DS was expressed throughout the interstitium of all tissues and was also present on blood vessels in rheumatoid samples only. Ch6S was expressed in the lining layer of normal synovium but was absent from this site in osteoarthritic and rheumatoid tissues. Ch6S was also present on all blood vessels in all tissues. CONCLUSIONS: The distinct zonal distributions of Ch4S/DS and Ch6S and their alteration in disease suggest these molecules have different and specific functions in normal and diseased synovium.

Hyaluronan concentration in non-inflamed and rheumatoid synovium.

The concentration of hyaluronan was measured by a novel application of an ELISA technique, using biotinylated hyaluronan binding-region (HABr) derived from cartilage proteoglycan core-protein, to digested frozen sections of synovium. The relative extractability of hyaluronan, from sections of synovium by short-term washes in buffer, was assessed by the same method. The distribution of hyaluronan in adjacent sections was assessed histochemically using the biotinylated HABr and alkaline phosphatase-conjugated streptavidin. Hyaluronan concentrations were lower in rheumatoid synovium (0.71 +/- 0.10 mg/cm3; mean +/- S.E.M.) than in non-inflamed synovium (1.07 +/- 0.16 mg/cm3). However, the ratio of extractable or 'free' hyaluronan to non-extractable or 'bound' hyaluronan, was greatly increased in rheumatoid synovium, being 4.53 +/- 0.40 (mean +/- S.E.M.) compared with 1.87 +/- 0.42 in non-inflamed synovium. Histochemical staining showed hyaluronan to be concentrated in the lining layer of non-inflamed samples, whereas in rheumatoid synovium the stain was more uniformly distributed throughout the tissue. Although the total concentration of hyaluronan was not increased in rheumatoid synovium, the increased proportion of 'free', and therefore presumably mobile, hyaluronan molecules together with increased synovial bulk may contribute to the known increases in serum levels of hyaluronan in patients with RA.

The development of monoclonal antibodies to the human mitochondrial 60-kd heat-shock protein, and their use in studying the expression of the protein in rheumatoid arthritis.

OBJECTIVE: To assess the claim that the human 60-kd heat-shock protein (HSP) is highly expressed in the joints of patients with rheumatoid arthritis (RA), but is not readily detected in normal tissues. METHODS: Monoclonal antibodies were raised against the human 60-kd mitochondrial heat-shock protein (P1 protein; hsp60), and their specificity was established. They were then applied to synovial tissue. RESULTS: HSP was expressed similarly in normal, osteoarthritic, and RA synovium. Low levels of hsp60 were detected in synovial fluid by immunoprecipitation. CONCLUSION: Minor differences in the distribution of hsp60 in synovium from RA joints were attributable to increased cellularity and to the disorganization of the tissue architecture.

Light microscopic characterization of the fibroblast-like synovial intimal cell (synoviocyte).

OBJECTIVE: To reassess synovial intimal cell populations by light microscopy. METHODS: Non-inflamed, rheumatoid and osteoarthritic synovia were analyzed as tissue sections and cytospin preparations by a series of combined immunohistochemical and cytochemical staining techniques. RESULTS: Two populations of intimal cells were identified. The first carried macrophage markers. The second showed high uridine diphosphoglucose dehydrogenase (UDPGD) activity, minimal cytoplasmic CD68, absent non-specific esterase (NSE) activity, and absent leukocyte and endothelial antigens. The majority of these cells showed a high content of prolyl hydroxylase. CONCLUSION: Combined cytochemical staining for NSE and UDPGD activity allows effective separation of intimal cell populations. We suggest that the cells of high UDPGD activity are the fibroblast-like or type B synovial intimal cells defined by electron microscopy. High UDPGD activity probably reflects a preferential ability to synthesize glycosaminoglycans, including hyaluronan.

Morphological localization of hyaluronan in normal and diseased synovium.

The morphological distribution of hyaluronan in normal and diseased synovium has been determined using a probe derived from the hyaluronan binding region of cartilage proteoglycan core protein. Normal synovium showed hyaluronan surrounding the lining layer cells with little in deeper layers. Rheumatoid synovium showed intense staining for hyaluronan throughout the tissue, notably associated with blood vessels and areas of dense cellular infiltration. Osteoarthritic tissues varied according to the degree of infiltration present, with inflamed specimens closely resembling rheumatoid tissue. The distribution of hyaluronan in diseased synovium suggests a role in aspects of the inflammatory process such as angiogenesis and cell traffic.

Racial variation in serum creatine kinase unrelated to lean body mass.

In a group of 30 black and 30 white healthy workers, matched for age, sex and body weight, serum creatine kinase was significantly higher in black males than in white males (P less than 0.01). Seventeen blacks but only four whites had levels above the accepted upper limit of normal of 195 IU/l. There was no correlation with lean body mass. Elevation of serum creatine kinase need not signify disease in blacks, for whom a separate reference range should be established. Two cases are reported of physically healthy black men subjected to unnecessary investigation on the basis of persistently elevated serum creatine kinase.

Immunohistological reassessment of accessory cell populations in normal and diseased human synovium.

Accessory cell populations in normal and diseased synovial tissue have been reanalyzed following the development of the monoclonal antibody, EBM11, directed against the CD68 epitope which is expressed by macrophages in all locations so far examined. Previous studies used as a macrophage marker the monoclonal antibody RFD7 which binds only a proportion of (mature) macrophages. Using double indirect immunofluorescence, normal and rheumatoid synovial samples were examined for the presence of cells which bind the putative dendritic cell marker, RFD1, in conjunction with either RFD7 or EBM11. RFD1 positive cells were found in five of 40 normal synovia. Of these cells, 30-35% were negative for RFD7 or EBM11 and, when closely apposed to T-lymphocytes, showed a typical interdigitating morphology. In contrast, all of ten rheumatoid synovia contained RFD1 positive cells; the extent of double labelling with macrophage markers varied with the position of the cells in the tissue. As expected, EBM11 stained a larger number of cells with macrophage morphology than RFD7. The combination of RFD1 and EBM11 appears to be a useful method for identifying interdigitating dendritic cells in connective tissue, these cells being characterized by positive RFD1 and negative EBM11 binding. On this criterion, interdigitating dendritic cells were plentiful in rheumatoid synovium and present, albeit infrequently, in normal synovium.

SLE: a rheumatological view. Analysis of the clinical features, serology and immunogenetics of 100 SLE patients during long-term follow-up.

Clinical features and immunogenetics were assessed in 100 SLE patients attending a rheumatology clinic for periods ranging from six months to 11 years (mean five years). Five-year survival was 88 per cent. Joint problems (94 per cent), rash (90 per cent) and haematological abnormalities (89 per cent) were the most common clinical features; neuropsychiatric disturbance (45 per cent) and renal disease (29 per cent) were seen less frequently. A range of serological abnormalities was found, including antinuclear antibodies (98 per cent) and antibodies to phospholipids (38 per cent). Anti-Sm antibodies (7 per cent) showed a marked ethnic bias. Tissue typing confirmed the importance of genetic factors by demonstrating significant increases in A1, B8 and DR3 in white Caucasians. The composite phenotype A1,B8,DR3 was present in 35 per cent of white Caucasian patients with SLE. The A1,B8 phenotype was associated with a relative risk of 8.0 and B8,DR3 with a relative risk of 8.32.

Terminal N-acetylglucosamine in chronic synovitis.

The distribution of terminal GlcNAc residues in normal and diseased synovial tissue has been studied using a mouse monoclonal antibody (mAb) which binds to terminal N-acetylglucosamine (GlcNAc). Normal human connective tissue, including synovium, showed no staining for terminal GlcNAc. Normal epithelial tissues, including tonsillar epithelium, skin, small intestinal epithelium and salivary epithelium showed cellular staining. Synovium from patients with definite rheumatoid arthritis showed dense granular staining of macrophages. In addition, synovium from 9 of 12 patients with definite rheumatoid arthritis showed reticular extracellular staining indicating deposition of material bearing terminal GlcNAc in the connective tissue stroma. The extracellular staining was not seen in synovium from patients with osteoarthritis. Extracellular material bearing terminal GlcNAc may act as an inflammatory stimulus in rheumatoid arthritis, either by acting as antigen or by interaction with receptors on macrophage membranes which also recognize GlcNAc on bacterial material, thus triggering biochemical pathways normally occurring in response to the presence of micro-organisms.

Persistence of scleroderma-like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells.

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients.

Dermal fibroblast cultures from patients with progressive systemic sclerosis (PSS) synthesize up to 5 times more glycosaminoglycan (GAG) than normal cultures. In an in vitro model of fibroblast-lymphocyte interactions, we show that the supernatants of activated mononuclear cells (MNC) modulate GAG synthesis, as measured by the incorporation of 3H-glucosamine into GAG following incubation of the confluent fibroblast monolayers with active supernatant preparations. GAG accumulation was selectively increased up to 18 times in normal dermal fibroblast cultures. Cell viability was not affected, and 3H-thymidine uptake and cell numbers were depressed in cultures treated with the supernatants. In contrast to normal dermal fibroblast cultures, PSS fibroblasts responded to MNC supernatants by only a 1-2-fold increase in GAG. Supernatants of concanavalin A-activated PSS MNC had higher stimulatory activity than those of normal MNC. Supernatants made with MNC that had been depleted of monocytes on Sephadex G-10 columns were only minimally stimulatory. The GAG-stimulatory supernatants modulated the synthesis, but not the degradation of GAG. Gel filtration on a calibrated Sephadex G-100 column indicated the presence of stimulatory activity in both the 50,000 and 15,000 molecular weight fractions. These activities were trypsin-sensitive, but had different susceptibilities to heat. The active column fractions also contained interleukin-1 activity, as shown in an assay measuring proliferation of mouse thymocytes. Like our factors, interleukin-1 preparations increased GAG in normal and PSS dermal fibroblasts. Products of activated MNC may modulate normal and pathologic processes in human skin.