Pinocytosis in Human Synovial Cells In Vitro

EVIDENCEFORENHANCEDACTIVITY IN RHEUMATOIDARTHRITIS

KATHRIN A. KRAKAUERand ROBERTB. ZURIER, Division of Rheumatic Diseases,Department of Medicine, University of Connecticut Health Center,Farmington, Connecticut 06032

A B S T RA C T Human synovial tissue cells in mono-layer can be shown to take up and digest a soluble pro-tein, horseradish peroxidase (HRP). Uptake of HRPwas linear with increasing concentrations of substrateand cell protein and with time for up to 4 h. Lowtemperature (4°C), and sodium fluoride, an inhibitor ofglycolysis were the most effective metabolic inhibitorsof endocytosis. In addition, colchicine, an inhibitor ofmicrotubule assembly, and yeast mannan, an inhibitorof mannose-specific receptors, reduced HRPuptake.Synovial cells from patients with rheumatoid arthritis(RSC) demonstrated a statistically significantly higherrate of endocytosis (247+107 ng HRP/100 ,ug cellprotein per 2 h.) than cells from control, nonrheumatoidpatients (NSC) (100+80 ng HRP/100 ,ug cell protein per2 h.). Thus, it is possible to discriminate RSCfrom NSCby their quantitatively different rates of endocytosis.

Digestion of HRPby synovial cells is statisticallysignificant by 6 h after uptake. A faster initial rate ofdigestion was seen in RSC. Over the first 6-8 h ofincubation 42% of the endocytosed HRPwas still cell-associated in RSCand 67% remained in NSCcultures.However, by 24 h 20-30% of endocytosed HRPwasfound in both types of cultures. These results indicatethat endocytosed molecules may accumulate morerapidly in RSCand persist within their lysosomes for alonger time than in NSC.

The quantitative determination of enhanced endo-cytosis by RSCcompared with NSCsuggests that thisincreased activity may have a role in the pathologicalfunction of synovial tissue in rheumatoid arthritis.

INTRODUCTIONMorphologic (1, 2) and biochemical (3) evidence in-dicate that endocytosis and lysosomal enzyme activity

Dr. Krakauer is a postdoctoral fellow of the ArthritisFoundation. Address reprint requests to Dr. Krakauer, Hos-pital of the University of Pennsylvania, Philadelphia.

Received for publication 29 January 1980 and in revisedform 7 April 1980.

592

of synovial tissue from individuals with rheumatoidarthritis are enhanced when compared with controlsynovium. However, these differences have not beenfunctionally quantitated. Endocytosis and subsequentintra- and extracellular digestion by synovial cells aremost likely responsible, at least in part, for removal ofmicroorganisms and erythrocytes, and for metabolismof connective tissue and synovial fluid components.In addition, these processes in synovial cells maymediate the destruction of articular tissue observedin rheumatoid arthritis (4).

Studies of synovial tissue in monolayer cultures havesuggested that synovial cells from individuals withrheumatoid arthritis (RSC)1 may be in a state of"activa-tion" compared with nonrheumatoid synovial cellsfrom individuals with other joint diseases or traumaticinjury (NSC). Thus, RSCspontaneously secrete largequantities of collagenase (5), plasminogen activator(6), neutral proteinase (7), and prostaglandin E2 (5).RSCcultures, therefore, resemble elicited peritonealmacrophages that release similar enzymes spon-taneously (8-11) and are considered activated (12)compared with resident peritoneal macrophages. In ad-dition, thioglycolate-elicited macrophages have higherrates of pinocytosis than resident peritoneal macro-phages (13). Thus, stimulated endocytosis and subse-quent intralysosomal digestive activity may be part of astate of activation of RSCand may contribute to thepathologic role of synovial cells in rheumatoid arthri-tis (4).

Very few studies have compared nonrheumatoidwith rheumatoid synovial cells. The experimentsreported here were performed in an effort to identifyquantitative differences between RSC and NSC inmonolayer cultures in terms of pinocytic activity andsubsequent intralysosomal proteolytic digestion. Our

I Abbreviations used in this paper: EMEM,Eagle's mini-mumessential medium; HRP, horseradish peroxidase; LDH,lactate dehydrogenase; NSC, nonrheumatoid synovial cells;RSC, rheumatoid synovial cell.

J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. * 0021-9738180/09/0592/07 $1.00Volume 66 September 1980 592-598

studies show that, although the kinetics of endocytosisare similar in NSC and RSC, differences in rates ofpinocytosis make it possible to discriminate betweenthese cultures. The increased rate of endocytosisevident in RSCis additional evidence that rheumatoidsynovial tissue is in a state of activation. Furthermore,endocytosis appears due to a unique synovial cellrather than infiltrating cells or fibroblasts.

METHODS

Synovial cell cultures. For all experiments, synovial tissuespecimens were treated identically. Tissue was obtained dur-ing surgery, after appropriate informed consent. Monolayerswere prepared by the method of Dayer et al. (5). Briefly,tissue was rinsed with Eagle's minimum essential medium(EMEM) (Gibco Laboratories, Grand Island Biological Co.,Grand Island, N. Y.) containing antibiotics and placed in apetri dish containing 10 ml of 4 mg/ml collagenase (type II,Sigma Chemical Co., St. Louis, Mo.) in EMEM, and thencarefully minced into small fragments with a scalpel blade.The tissue was incubated for 2 h at 37°C with frequent pipet-ting to breakup clumps of cells. An equal volume of trypsin(0.25% in versene, 1:5,000) was added and incubation con-tinued for 30 min. The suspension was then centrifuged (1,000rpm, 10 min), washed once with trypsin versene solution,twice with Hanks' balanced salt solution and once with 10%fetal calf serum (Associated Biomedic Systems, Inc., Buffalo,N. Y.) in EMEMbefore being counted in an improvedNeubauer hemocytometer. Cells were plated in 35-mm plasticdishes (Lux Scientific Corp., Newbury Park, Calif.) at a con-centration of 0.5-1 x 106 cells/dish. After ovemight incuba-tion, the cultures were washed well with 10% fetal calfserum/EMEM and fresh medium added. Cells were routinelygrown in 10% heat inactivated fetal calf serum/EMEM con-taining 100 U penicillin and 100 ,ug streptomycin/ml. Primarycultures were used throughout and cells were allowed to growfor 4-40 d before being used.

Measurement of endocytosis. The rate of endocytosis wasmeasured by the method of Steinmann and Cohn (14) usinghorseradish peroxidase (HRP) as a substrate at a concentrationof 1 mg/ml in 10% fetal calf serum/EMEM. After incubationof HRPwith cells, culture dishes were carefully washed 10times over a period of 1 h with 2-4 ml phosphate-bufferedsaline. Cell lysates were prepared in 0.1% Triton X-100 (wt/vol in water) (Rohm and Haas Co., Philadelphia, Pa.) andassayed for peroxidase activity (14) and for protein (15). For theperoxidase assay, 0.05 ml 1%0-dianisidine (Sigma ChemicalCo.), 0.06 ml 0.3% hydrogen peroxide, and 6.0 ml phosphatebuffer, pH 5.0 were mixed just before use. Change in ab-sorbance over 3 min of 1 ml of this reaction mixture contain-ing cell lysate was measured at 460 nm in a Beckman (Beck-man Instruments, Inc., Fullerton, Calif.) 24 spectrophoto-meter. Endogenous peroxidase activity was similar for bothRSCand NSCand never accounted for more than a few nano-grams of activity.

Protein and viability determinations. Protein was de-termined according to the method of Lowry et al. (15). Allsamples were centrifuged at 3,000 rpm for 5 min before readingto eliminate the flocculant caused by Triton. Bovine serumalbumin was the standard protein.

Cell viability was determined by measuring the proportionof cytoplasmic lactate dehydrogenase (LDH) that leaked intothe medium. LDH assays were performed by the method ofBergmeyer (16) within 24 h after termination of an experiment.

Metabolic inhibitors, lectins, and charged polymers. Cells

were preincubated for 1 h with each agent and then 1 mg/mlHRPadded for an additional 1 h. DEAEdextran (5 x 105 molwt) and dextran sulphate (5 x 105 mol wt) were purchasedfrom Pharmacia Fine Chemicals, Uppsala, Sweden. Allmetabolic inhibitors and other polymers were purchasedfrom Sigma Chemical Co.

Digestion of HRP. After uptake and thorough washing(eight times) in sterile phosphate-buffered saline, digestionof HRPwas followed by the loss of peroxidatic activity fromcells over time. Media and cells were monitored for ac-tivity. It remains to be investigated whether this loss of ac-tivity reflects enzymatic degradation of HRPor denaturationof the active site.

Light and electron microscopy. Following uptake of HRPby synovial cells, cultures were washed in phosphate-buffered saline and fixed in 2.5% glutaraldehyde in 0.1 Msodium cacodylate buffer, pH 7.4 for 5 min at room tempera-ture (14). These cells and control (unincubated) cells werethen stained for peroxidase by incubation for 30 min with 50mg/100 ml diaminobenzidine in 0.05 ml Tris-HCl buffer, pH7.6, and 0.01% hydrogen peroxide. Light microscopy wasperformed using a Nikon inverted phase contrast microscope(Nikon Inc., Instrument Div., Garden City, N. Y.). For elec-tron microscopy, cells were postfixed in 2%osmium tetroxidefor 60 min, washed and scraped off the dishes. After dehydra-tion in graded alcohols and embedding in epon-aralditemixture, cells were examined with a Philips 300 electronmicroscope (Philips Electronic, Mahwah, N. J.) by Mrs.Constance Gillies (Department of Pathology, University ofConnecticut Medical School).

RESULTS

Uptake of HRPover increasing HRPconcentrations.Uptake of HRP by synoviocytes was linear over alarge concentration range. Linearity of uptake by NSCover 1 and 2 h at concentrations ranging from 0.1 to10 mg/ml is demonstrated in Fig. 1. Similar kineticswere seen for RSC. A load of 1 mg/ml HRPwas there-fore used routinely in all subsequent experiments.

z

ir-0

m

I

CL200 -

cc

/~~~~~~~~~~~~~~~~

1 2 3 4 5 6 7 8 9 10

mg HRP

FIGURE 1 Uptake of HRPby NSCwith increasing HRPcon-centrations. Cultures of NSCwere incubated for 1 (-) or 2 (0)h at 37°C with concentrations of HRPfrom 0.1 to 10 mg/ml.

Synovial Cell Pinocytosis 593

Uptake of HRPover time. Uptake of HRPby RSCand NSCwas linear for the first 2-4 h of incubation.Representative experiments for NSCand RSCculturesare shown in Fig. 2. Significant uptake was detectablein both RSCand NSConly after 15 min incubation. Insome RSCcultures the rate of uptake appeared to de-crease between 2 and 4 h. This may reflect digestion ofHRPwhich could be detected by 2 h after HRPuptake(Fig. 5). The rate of uptake of HRP/100 ,ug cell pro-tein per min (calculated from Fig. 2) for RSC de-creased from 30 min to 4 h (2.66 ng HRP/100 ,ug cellprotein per min at 30 min and 1.35 ng HRP/100 ligcell protein per min at 240 min). For NSCthe rate ofuptake remained fairly constant (0.93 ng/100 ug cellprotein per min for the first 30 min and 0.89 ng/100,.g protein per min by 240 min). These results suggestthat an initial rapid rate of removal of HRPby RSCde-creased with time and was completely absent in NSC.These results may also indicate that whereas NSCare ahomogeneous population with respect to pinocytic ac-tivity, RSCare heterogeneous. RSCcultures appear toconsist of a population of cells that has a rapid initialrate of uptake and others that have slower rates andmore closely resemble NSC.

Linearity of HRPuptake with increasing cell pro-tein. HRPaccumulation per culture was linear withincreasing cell protein (Fig. 3; r2 = 0.94). The specificactivity of HRPuptake by RSC (Fig. 3) and NSC(notshown) increased modestly with increased cell density.In Fig. 3 cell concentration is expressed as cell proteinper 60-mm culture dishes. The data for RSC indicatethat at a protein density >30 jug/cm2 (or - 1 x 105 cells/cm2) the specific activity was significantly greater (P< 0.025) than if below this value. NSCbehaved in asimilar manner.

The data for rates of HRPuptake by synovial cells

zu0

mtL,0 200

0

0.

Io 0o

60 120

MINUTES180

FIGuRE 2 Time-course for HRPuptake. RSC (-) and NSC(-) were incubated for 30-240 min at 37°C with 1 mg/ml HRP.The lines were plotted by the method of least squares (RSC,r2= 0.91; NSC, r2 = 0.99).

400 600

jug CELL PROTEIN

FIGURE3 Effect of increasing cell concentration on HRPuptake. RSC were incubated for 1 h with 1 mg/mI HRP at37°C. Cell concentration is expressed as cellular protein per60-mm dish where 100 ,ug of protein is equivalent to 3.4 x 105cells. HRP uptake is expressed as nanograms per dish perhour (V) and as nanograms per 100 jig cell protein per hour.(A) The lines were plotted by the method of least squares(V, r2 = 0.94; A, r2 = 0.64).

are summarized in Table I, and indicate that the rates ofendocytosis for RSC were statistically significantlygreater (P < 0.005) than for NSC. RSC exhibited-2.5 times the specific activity of uptake comparedwith NSCover 2 h incubation when expressed per 100

:,g of protein. For every 100 ,ug of cell protein, how-ever, there were 1.5-fold the number of cells presentin RSC as in NSC cultures. Despite this difference,when specific activity is expressed per 105 cells, themeans of the uptake in these cultures remain statis-tically significantly different (P < 0.025).

Light microscopy and cytochemistry. Primarycultures of adherent synovial cells were morpho-logically heterogeneous. The cells present included(a) fibroblastic cells, derived from B type synovialcells and contaminating subsynovial connective tissue;(b) small, macrophagelike round cells; (c) morpho-logically intermediate type cells similar to type Csynovial cells; (d) stellate or dendritic cells whichcan be immunochemically identified as cells contain-ing collagenase (17); (e) multinucleated cells; and (f)some T lymphocytes. Fc receptor bearing cells (or thereceptors per se) and T lymphocytes were not detect-able in cultures older than 7-10 d (5). The proportionsof cells present in different cultures varied from speci-men to specimen and this variability did not seem torelate to the source of the tissue. Some cultures ofNSC and RSC had as much as 80% stellate cellswhereas others had a majority of fibroblastic and small,round macrophagelike cells. As the cultures aged, fibro-blasts began to predominate. Interestingly, there wasno significant difference between the rates of pinocyto-sis for either RSC or NSC when cells cultured for1-7 d were compared with cells grown for 8-40 d.

594 K. A. Krakauer and R. B. Zurier

F

F

TABLE ISummary of Specific Activities for HRPUptake*

RSC n NSC n RSCvs. NSC

P

247+107 ng HRP/100 ,ug protein per 2 h 11 100±80 ng HRP/100 ,ug protein per 2 h 10 <0.0053.4±+1.3 x 105 cells/100 ,ug protein 20 2.2+0.8 x 105 cells/100 ,ug protein 27 <0.00166.27±49.2 ng HRP/105 cells per 2 h 7 30.62±13.3 ng HRP/105 cells per 2 h 11 <0.025

* Results are expressed as mean±SD.n, numbers of different cultures examined, each run in duplicate.

WhenRSCwere stained for peroxidase activity afteruptake of this enzyme, the endocytosed substratewas demonstrated in a variety of cell types. Macro-phagelike cells were usually strongly positive as weresome fibroblasts and stellate cells. Multinucleated cellsand cells present in clumps were frequently positive.In most cases, however, no cell type could be singledout as the endocytic cell in either RSCor NSCcultures.RSC cultures always contained a greater number ofcells that stained positively and these cells stainedmore intensely than those found in NSCcultures.

Electron microscopy. A macrophagelike RSC isillustrated in Fig. 4. The electron dense reaction

FIGuRE 4 Ultrastructural localization of HRP. RSC wereincubated for 30 min with 1 mg/ml HRPand then stainedwith diaminobenzidine. A macrophagelike cell is illustrated.Reaction product (H) is visible in recently formed endocyticvacuoles where it forms a lining on the inner aspect of thelimiting membrance. A later stage of uptake is visible as adense reaction product which fills the vacuoles (H'). Someendogeneous peroxidatic activity (E) (as determined by con-trol cells that were not given HRP) is also visible. Elaboratefilopodia (F) are evident on the nonadhered side of this cell.x 12,950.

product of HRP-H202-diaminobenzidine is evident invacuoles throughout the cytoplasm. Staining was notevident on the external surface of the plasma membrane.

Effect on HRP uptake of metabolic inhibitors,charged polymers, lectins, and colchicine. The mosteffective metabolic inhibitors of HRPuptake were (1)the glycolytic inhibitor fluoride and (2) the nonspecificdepressant 4°C (Table II). Differences between NSCand RSC in susceptibility to the metabolic inhibitorsstudied were not detected; therefore, results for bothcell types are combined in Table II. Greater than 85%of the cells remained viable (LDH determination)after incubation with all agents listed in Table II.

Charged polymers, the lectin concanavalin A (ConA), colchicine, and yeast mannan were examined fortheir abilities to influence synovial cell endocytosis.

TABLE IIEffect of Metabolic Inhibitors on HRPUptake

by Synovial Cells

Percent inhibitionInhibitor Concentration of HRPuptake*

miM

GlycolyticSodium fluoride 10 84+7 (6)

1 45+5 (4)0.1 7 (1)

2-Deoxyglucose 10 10±31 (2)

RespiratorySodium azide 10 8±56 (3)

1 3±48 (7)

Oxidative phosphorylation2,4-Dinitrophenol 1 11±15 (2)

0.1 0 (1)Oligomycin 2 ,ug/ml 41 (1)

10j±g/ml 46 (1)General

40C 96±4 (4)

* Results are expressed as mean SD. Numbers in parenthesesrepresent numbers of different cultures examined, each runin duplicate.

Syntovial Cell Pinocytosis 595

Cationic polymers DEAE-dextran (1-10 ,ug/ml), poly-L-ornithine (1-0.01 Zg/ml), and poly-D-lysine (1-0.1,ug/ml) all had no effect on HRPuptake at concentra-tions that did not reduce viability. The anionic poly-mer dextran sulfate (100-10 ,g/ml) had variable effectseither not influencing or inhibiting uptake slightly.Hyaluronic acid (1-0.1 mg/ml), poly-D-glutamic acid(1-0.01 ,ug/ml), and heparin (10-1.0 ,ug/ml) all had noinfluence on HRPuptake. After 30 min simultaneousincubation Con A (100 ,ug/ml) stimulated HRPuptake15-fold in both NSCand RSC. This stimulation of pino-cytosis increased to 30-fold by 1 h.

Table III demonstrates that colchicine, a compoundthat inhibits microtubule assembly, significantly in-hibited pinocytosis in both NSC and RSC at 1 ,uM.In addition, yeast mannan, an inhibitor of mannose-specific cell surface receptors (18), produced a dose-dependent inhibition of HRP uptake. Neither cellviability nor HRP activity was affected by either ofthese agents.

Digestion of HRP. In experiments where digestionof HRP was observed, as much as 15% of the cell-associated activity was detected in the medium over24 h, suggesting that either some exocytosis of un-digested HRPdid occur or that some membrane- ordish-bound HRPwas further eluted into the mediumon incubation at 37°. Beyond that, however, furtherloss of activity appears to be the result of digestion.The rate of loss of cell-associated HRP activity wasbiphasic with a very rapid initial loss of activity duringthe first 6 h of incubation, and a much slower loss overthe next 20 h (Fig. 5). These results may be furtherevidence for heterogeneous functioning of the endo-cytic lysosomal process in synovial cell cultures. Thedecrease in cell-associated HRP activity was sta-tistically significant by 6 h. RSC had a significantlylower percentage of endocytosed HRP remaining inthe cultures by 6-8 h when compared with NSC(42.33±21.37%, n = 7 vs. 61.14+23.60%, n = 7; P< 0.025 in RSCand NSC, respectively). The percent-age of total HRP remaining at 24 h was similar for

TABLE IIIEffect of Inhibitors of Pinocytosis on HRPUptake by RSC

Percent inhibitionAgent Concentration of HRPuptake*

Colchicine 10 /AM 43+14% (3)1 AM 39+12% (4)0.1 ,uM 16-+17%o (4)

Yeast mannan 500 jug/ml 70%100 ,ug/ml 50%50,ug/ml 42%10 ,ug/ml 11%

* Results are expressed as mean±+SD. Numbers in parenthesesrepresent numbers of different cultures examined, each runin duplicate.

nO

co

10- XI

1-

0 4 8 12 16 20 24 28HOURS

FIGURE 5 Time-course for digestion of HRP. Cells (RSC l;NSC0) were incubated for 2 h with 1 mg/ml HRP, washedthoroughly and harvested (zero time), or reincubated inHRP-free medium for up to 28 h. Results represent nanogramsrecovered HRP in both cells and medium. The t1/2 for RSCwas 5 h and that for NSCwas 8 h. For RSC(n = 4) the nano-grams HRPremaining at 6 h (P < 0.005) and 24 h (P < 0.01)were significantly less than that present at zero time.

RSCand NSC(20-30%). From Fig. 5 the tl2 for loss ofHRP within RSC was - 5 h, whereas that for NSCwas 8 h.

Digestion did not occur in all cultures. In theseexperiments as much as 50%of the total cell-associatedHRP could be found in the medium after 24 h ofincubation.

DISCUSSIONThis study demonstrates that primary cultures ofsynovial cells from individuals with RSC exhibit asignificantly higher rate of uptake of a soluble antigen(HRP) than synovial cells from individuals with otherjoint diseases (NSC). These results also indicate thatRSC and NSC have rates of pinocytosis that moreclosely resemble those of macrophages (13) than offibroblasts (19). When uptake studies were performedon passaged cultures of synovial cells, a decreasein the primary rate of uptake to that resembling fibro-blasts was found in most subcultures. Thus, for thestudies presented in this paper primary cultures wereused throughout.

Increases in cell density stimulate HRPuptake insynovial cells, L cells (19), and epithelial cells (20).

596 K. A. Krakauer and R. B. Zurier

In addition, we have noted that some RSC cultures,like L cells (21), fail to demonstrate contact inhibitionof growth on reaching confluence. Thus, cultures ofRSCcould achieve higher cell densities than those ofNSC during a comparable culture period, and itmay be this phenomenon that is at least partiallyresponsible for the enhanced pinocytosis found inRSC on a per cell basis. Enhanced endocytosis maybe the result in part, of as yet unknown eventsinduced by high cell density. Some of these may in-clude cell surface phenomena (20), cell-cell interac-tions, metabolic alterations, and alteratiQns in con-centrations of and/or responses to such regulatory mole-cules as cyclic nucleotides and prostaglandins. Asproliferative synovium in vivo is the "sine qua non"of rheumatoid arthritis, these events may be importantto the pathological process in rheumatoid arthritis.

The mechanism of uptake of HRPby synovial cellsprobably involves both macro- and micropinocytosis.With the electron microscope many micropinocyticvesicles are observed in the cytoplasm, and freesurface of many cells is covered with an elaborateconfiguration of microvilli. These microvilli appear tofuse with each other to form endocytic vacuoles.Furthermore, from our studies we can conclude thatuptake of HRPappears to be both by fluid phase (21)and adsorptive pinocytosis. Linearity of uptake isdemonstrable over a wide range of substrate concentra-tions (0.10-10 mg/ml) and adsorption of HRPat 4°Cnever exceeds 10% of the activity found at 37°C. Thiscompares well with the amount of HRPremoved bytrypsinization after 2 h of uptake.

Con A is thought to stimulate endocytosis by form-ing bridges between cell surface glycoproteins and"low uptake" or poorly endocytosed forms of solubleglycoproteins such as f3-glucuronidase (22). In ourexperiments Con A may be forming aggregates witha low uptake form of HRPand thus enhancing endo-cytosis of a portion of this preparation. Although wehave not investigated this material, Steinman andCohn (14) have reported that HRPfrom the same source(Sigma Chemical Co.) as ours was composed of fiveseparate enzymatic bands on agar and agarose electro-phoresis. Each of these forms may be taken up withdifferent avidity. The heterogeneity of this prepara-tion may also help to explain the conflict betweenlinearity of uptake over a large HRP concentrationrange and the 70% inhibition of uptake we find withmannan (Table III). Mannan is a specific inhibitor ofmannose receptor-dependent uptake (18). Addition tocells of mannan, either simultaneously with or beforeHRP, may inhibit the "high uptake" form(s) of HRPthat is rapidly interiorized and is sensitive to inhibitionat 4°C. Interiorization of the low uptake form(s) maybe by fluid phase and thus would be concentrationdependent. These speculations are supported by ourobservation that maximum inhibition of uptake (67-

70%) is achieved with mannan to HRPratios of 10:1,2:1, 1:1, and 1:2. Mannan is not a nonspecific inhibitorof pinocytosis as uptake of [3H]sucrose was unaffectedby its presence (unpublished observations).

Studies of metabolic inhibitors presented here in-dicate that glycolysis is the most important source ofenergy for endocytosis in synovial cells. NSCand RSCare equally susceptible to these inhibitors. Dingle andThomas (23) and Castor (24) have demonstrated in-creased glucose and oxygen metabolism and increasedlactate formation in rheumatoid synovial tissue slicesand cells in culture. This enhanced metabolism maybe partially responsible for the increased rate ofendocytosis seen in rheumatoid arthritis cells. All formsof endocytosis in macrophages (macropinocytosis,micropinocytosis, and phagocytosis) are sensitive to in-hibitors of glycolysis (25). In fibroblasts, micropino-cytosis of HRP is partially inhibited by glycolyticinhibitors and partially by inhibitors of oxidative phos-phorylation and respiration (19). Thus, these data sug-gest that synovial cells resemble macrophages morethan fibroblasts in their sensitivity to these inhibitors.Because there are no differences between NSC andRSCin energy requirements for endocytosis, similarlyderived cells may be responsible for HRPuptake inboth cultures.

Charged polymers stimulate pinocytosis in macro-phages and fibroblasts. Cationic polymers inducevacuole formation in fibroblasts (26) whereas anionicpolymers are effective in macrophages (25). Neitheranionic nor cationic polymers affected HRP uptakeunder the conditions tested. These studies indicate thatsynovial cells are different from both fibroblasts andmacrophages in their sensitivities to charged polymers;and suggest that endocytosis is occurring primarilyin a unique synovial cell rather than contaminatingmacrophages.

Colchicine, a compound that impairs microtubuleintegrity (27), partially inhibited uptake of HRP bysynovial cells indicating that endocytosis of HRPbysynoviocytes depends at least in part, on the presenceof intact microtubules. Similar results have been ob-tained for 131I-albumin uptake by macrophages (28).

Although variable in amount, digestion of HRPbysynovial cells was detected. In some experimentsenzyme was simply released into the medium. Thiswas not the result of cell death or loss of cells becauseperoxidase release was not accompanied by release ofLDH. Thus, in contrast to studies in which exocytosiswas not observed in macrophages (14), fibroblasts (19),and rat yolk sac (29) during periods when digestionof proteins was being detected, exocytosis appearsto occur in synovial cells.

The time it takes for half of the endocytosed HRPto be digested by RSCand NSC(t,/2) is approximatelythe same as it is for fibroblasts (19) and resident peri-toneal macrophages (14).

Synovial Cell Pinocytosis 597

The high rates of endocytosis found in synovial cellsresemble those of professional phagocytes. In normalsynovial cells this function is probably important tothe turnover of joint fluid and synovial tissue con-stituents such as hyaluronic acid, plasma proteins,collagen, fibrin, and proteoglycans. In rheumatoidarthritis, increased rates of endocytosis may occur inattempts to clear such molecules as rheumatoid fac-tors and their immune complexes, altered collagen andcollagen-anti-collagen-immune complexes, immuno-globulins, soluble mediators of inflammation, and/orviral, bacterial, or mycoplasmal antigens.

Because the rate of uptake by RSCis so much greaterthan NSC, but rates of digestion are similar, endo-cytosed molecules may accumulate more rapidly andpersist longer in RSC. Thus, activated and proliferat-ing synovial cells may become pathogenic to articulartissues by endocytosing large amounts of joint consti-tuents which they only slowly digest. This in turn mayinduce the release of degradative enzymes and media-tors of inflammation that may help perpetuate chronicinflammation and joint tissue injury.

ACKNOWLEDGMENTS

Weare very grateful to Dr. Harry Gossling, Dr. Ira Spar, andother members of the orthopedic and operating room teams atJohn Dempsey Hospital, Farmington, Conn., and HartfordHospital, Hartford, Conn. who generously obtained thesurgical specimens used in these experiments. We thankJoan Barger for her diligent technical assistance.

This work was supported by National Institutes of Healthgrants AI 12225 and AM17309, an Arthritis Foundation Centergrant, and a grant from the Kroc Foundation.

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