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Pinocytosis in Human Synovial Cells in Vitro. Evidence for Enhanced Activity in Rheumatoid Arthritis

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Pinocytosis in Human Synovial Cells in Vitro. Evidence for Enhanced Activity in Rheumatoid Arthritis Pinocytosis in human synovial cells in vitro. Evidence for enhanced activity in rheumatoid arthritis. K A Krakauer, R B Zurier J Clin Invest. 1980;66(3):592-598. https://doi.org/10.1172/JCI109891. Research Article Human synovial tissue cells in monolayer can be shown to take up and digest a soluble protein, horseradish peroxidase (HRP). Uptake of HRP was linear with increasing concentrations of substrate and cell protein and with time for up to 4 h. Low temperature (4 degrees C), and sodium fluoride, an inhibitor of glycolysis were the most effective metabolic inhibitors of endocytosis. In addition, colchicine, an inhibitor of microtubule assembly, and yeast mannan, an inhibitor of mannose- specific receptors, reduced HRP uptake. Synovial cells from patients with rheumatoid arthritis (RSC) demonstrated a statistically significantly higher rate of endocytosis (247 +/- 107 ng HRP/100 micrograms cell protein per 2 h.) than cells from control, nonrheumatoid patients (NSC) (100 +/- 80 ng HRP/100 micrograms cell protein per 2 h). Thus, it is possible to discriminate RSC from NSC by their quantitatively different rates of endocytosis. Digestion of HRP by synovial cells is statistically significant by 6 h after uptake. A faster initial rate of digestion was seen in RSC. Over the first 6--8 h of incubation 42% of the endocytosed HRP was still cell-associated in RSC and 67% remained in NSC cultures. However, by 24 h 20--30% of endocytosed HRP was found in both types of cultures. These results indicate that endocytosed molecules may accumulate more rapidly in RSC and persist within their lysosomes […] Find the latest version: https://jci.me/109891/pdf Pinocytosis in Human Synovial Cells In Vitro EVIDENCE FOR ENHANCED ACTIVITY IN RHEUMATOID ARTHRITIS KATHRIN A. KRAKAUER and ROBERT B. ZURIER, Division of Rheumatic Diseases, Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032 A B S T R A C T Human synovial tissue cells in mono- of synovial tissue from individuals with rheumatoid layer can be shown to take up and digest a soluble pro- arthritis are enhanced when compared with control tein, horseradish peroxidase (HRP). Uptake of HRP synovium. However, these differences have not been was linear with increasing concentrations of substrate functionally quantitated. Endocytosis and subsequent and cell protein and with time for up to 4 h. Low intra- and extracellular digestion by synovial cells are temperature (4°C), and sodium fluoride, an inhibitor of most likely responsible, at least in part, for removal of glycolysis were the most effective metabolic inhibitors microorganisms and erythrocytes, and for metabolism of endocytosis. In addition, colchicine, an inhibitor of of connective tissue and synovial fluid components. microtubule assembly, and yeast mannan, an inhibitor In addition, these processes in synovial cells may of mannose-specific receptors, reduced HRP uptake. mediate the destruction of articular tissue observed Synovial cells from patients with rheumatoid arthritis in rheumatoid arthritis (4). (RSC) demonstrated a statistically significantly higher Studies of synovial tissue in monolayer cultures have rate of endocytosis (247+107 ng HRP/100 ,ug cell suggested that synovial cells from individuals with protein per 2 h.) than cells from control, nonrheumatoid rheumatoid arthritis (RSC)1 may be in a state of"activa- patients (NSC) (100+80 ng HRP/100 ,ug cell protein per tion" compared with nonrheumatoid synovial cells 2 h.). Thus, it is possible to discriminate RSC from NSC from individuals with other joint diseases or traumatic by their quantitatively different rates of endocytosis. injury (NSC). Thus, RSC spontaneously secrete large Digestion of HRP by synovial cells is statistically quantities of collagenase (5), plasminogen activator significant by 6 h after uptake. A faster initial rate of (6), neutral proteinase (7), and prostaglandin E2 (5). digestion was seen in RSC. Over the first 6-8 h of RSC cultures, therefore, resemble elicited peritoneal incubation 42% of the endocytosed HRP was still cell- macrophages that release similar enzymes spon- associated in RSC and 67% remained in NSC cultures. taneously (8-11) and are considered activated (12) However, by 24 h 20-30% of endocytosed HRP was compared with resident peritoneal macrophages. In ad- found in both types of cultures. These results indicate dition, thioglycolate-elicited macrophages have higher that endocytosed molecules may accumulate more rates of pinocytosis than resident peritoneal macro- rapidly in RSC and persist within their lysosomes for a phages (13). Thus, stimulated endocytosis and subse- longer time than in NSC. quent intralysosomal digestive activity may be part of a The quantitative determination of enhanced endo- state of activation of RSC and may contribute to the cytosis by RSC compared with NSC suggests that this pathologic role of synovial cells in rheumatoid arthri- increased activity may have a role in the pathological tis (4). function of synovial tissue in rheumatoid arthritis. Very few studies have compared nonrheumatoid with rheumatoid synovial cells. The experiments INTRODUCTION reported here were performed in an effort to identify Morphologic (1, 2) and biochemical (3) evidence in- quantitative differences between RSC and NSC in dicate that endocytosis and lysosomal enzyme activity monolayer cultures in terms of pinocytic activity and subsequent intralysosomal proteolytic digestion. Our Dr. Krakauer is a postdoctoral fellow of the Arthritis Foundation. Address reprint requests to Dr. Krakauer, Hos- I Abbreviations used in this paper: EMEM, Eagle's mini- pital of the University of Pennsylvania, Philadelphia. mum essential medium; HRP, horseradish peroxidase; LDH, Received for publication 29 January 1980 and in revised lactate dehydrogenase; NSC, nonrheumatoid synovial cells; form 7 April 1980. RSC, rheumatoid synovial cell. 592 J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. * 0021-9738180/09/0592/07 $1.00 Volume 66 September 1980 592-598 studies show that, although the kinetics of endocytosis were preincubated for 1 h with each agent and then 1 mg/ml are similar in NSC and RSC, differences in rates of HRP added for an additional 1 h. DEAE dextran (5 x 105 mol wt) and dextran sulphate (5 x 105 mol wt) were purchased pinocytosis make it possible to discriminate between from Pharmacia Fine Chemicals, Uppsala, Sweden. All these cultures. The increased rate of endocytosis metabolic inhibitors and other polymers were purchased evident in RSC is additional evidence that rheumatoid from Sigma Chemical Co. synovial tissue is in a state of activation. Furthermore, Digestion of HRP. After uptake and thorough washing endocytosis appears due to a unique synovial cell (eight times) in sterile phosphate-buffered saline, digestion of HRP was followed by the loss of peroxidatic activity from rather than infiltrating cells or fibroblasts. cells over time. Media and cells were monitored for ac- tivity. It remains to be investigated whether this loss of ac- METHODS tivity reflects enzymatic degradation of HRP or denaturation of the active site. Synovial cell cultures. For all experiments, synovial tissue Light and electron microscopy. Following uptake of HRP specimens were treated identically. Tissue was obtained dur- by synovial cells, cultures were washed in phosphate- ing surgery, after appropriate informed consent. Monolayers buffered saline and fixed in 2.5% glutaraldehyde in 0.1 M were prepared by the method of Dayer et al. (5). Briefly, sodium cacodylate buffer, pH 7.4 for 5 min at room tempera- tissue was rinsed with Eagle's minimum essential medium ture (14). These cells and control (unincubated) cells were (EMEM) (Gibco Laboratories, Grand Island Biological Co., then stained for peroxidase by incubation for 30 min with 50 Grand Island, N. Y.) containing antibiotics and placed in a mg/100 ml diaminobenzidine in 0.05 ml Tris-HCl buffer, pH petri dish containing 10 ml of 4 mg/ml collagenase (type II, 7.6, and 0.01% hydrogen peroxide. Light microscopy was Sigma Chemical Co., St. Louis, Mo.) in EMEM, and then performed using a Nikon inverted phase contrast microscope carefully minced into small fragments with a scalpel blade. (Nikon Inc., Instrument Div., Garden City, N. Y.). For elec- The tissue was incubated for 2 h at 37°C with frequent pipet- tron microscopy, cells were postfixed in 2% osmium tetroxide ting to breakup clumps of cells. An equal volume of trypsin for 60 min, washed and scraped off the dishes. After dehydra- (0.25% in versene, 1:5,000) was added and incubation con- tion in graded alcohols and embedding in epon-araldite tinued for 30 min. The suspension was then centrifuged (1,000 mixture, cells were examined with a Philips 300 electron rpm, 10 min), washed once with trypsin versene solution, microscope (Philips Electronic, Mahwah, N. J.) by Mrs. twice with Hanks' balanced salt solution and once with 10% Constance Gillies (Department of Pathology, University of fetal calf serum (Associated Biomedic Systems, Inc., Buffalo, Connecticut Medical School). N. Y.) in EMEM before being counted in an improved Neubauer hemocytometer. Cells were plated in 35-mm plastic RESULTS dishes (Lux Scientific Corp., Newbury Park, Calif.) at a con- centration of 0.5-1 x 106 cells/dish. After ovemight incuba- Uptake of HRP over increasing HRP concentrations. tion, the cultures were washed well with 10% fetal calf of HRP was linear over a serum/EMEM and fresh medium added. Cells were routinely Uptake by synoviocytes grown in 10% heat inactivated fetal calf serum/EMEM con- large concentration range. Linearity of uptake by NSC taining 100 U penicillin and 100 ,ug streptomycin/ml. Primary over 1 and 2 h at concentrations ranging from 0.1 to cultures were used throughout and cells were allowed to grow 10 mg/ml is demonstrated in Fig. 1. Similar kinetics for 4-40 d before being used. were seen for RSC. A load of 1 mg/ml HRP was there- Measurement of endocytosis. The rate of endocytosis was measured by the method of Steinmann and Cohn (14) using fore used routinely in all subsequent experiments.
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