Annals of the Rheumatic Diseases 1992; 51: 476480

Production of angiotensin converting enzymerheumatoid synovial membrane

Douglas Veale, Ghada Yanni, Barry Bresnihan, Oliver FitzGerald

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Department ofRheumatology,St Vincent's Hospital,Dublin 4, IrelandD VealeG YanniB Bresnihan0 FitzGeraldCorrespondence to:Dr 0 FitzGerald,Department ofRheumatology,St Vincent's Hospital,Elm Park, Dublin 4,Ireland.

Accepted for publication16 July 1991

AbstractVascular proliferation and mononuclear cellinfiltration are prominent changes observed insynovium from actively inflamed joints ofpatients with rheumatoid arthritis. Angio-tensin converting enzyme (ACE) is a halideactivated peptidase produced mainly byendothelial cells and by activated monocytes.It has been proposed that levels of ACEactivity in synovial fluid might reflect changesin membrane vascularity, the degree ofmonocyte infiltration, or the thickness of thelining layer. In this study, ACE activity inserum and synovial fluid samples from 18patients with inflammatory arthritis wasmeasured and compared with levels in 12control subjects with non-inflammatoryarthritis. Although serum levels were similarin the two groups, ACE activity in synovialfluid was significantly increased in the groupwith inflammatory arthritis compared withcontrols (mean (SE) 37 (5) v 19 (3)). Stainingof synovial membranes from patients withrheumatoid arthritis with a monoclonal anti-body to ACE localised ACE to the endotheliumand to mononuclear cells of macrophageorigin. ACE activity was then measured insupernatants of synovial membrane frompatients with rheumatoid arthritis after oneand seven days of culture. A significantincrease in ACE activity was observed afterseven days of culture (mean (SE) day 1, 17 (5)v day 7, 25 (3)). Levels of ACE activity,however, did not correlate with the lininglayer thickness, with the number of macro-phages per square millimetre, nor with thenumber of blood vessels per square millimetreofsynovial tissue. No correlation was observedeither between levels of ACE in the super-natant of synovial membrane and levels ofinterleukin 1 or interleukin 6.

In conclusion, ACE is produced by thesynovial membrane of patients with rheuma-toid arthritis and is localised to monocytesand endothelial cells. Levels of activity donot directly reflect membrane vascularity,monocyte or macrophage number, or thethickness of the lining layer.

Rheumatoid arthritis, is a chronic inflammatorydisease characterised histologically by hyper-plasia of the synovial lining cells and an intensemononuclear cell infiltrate which may formaggregates. The importance of cells of monocyteor macrophage origin in the pathogenesis of thedisease has been highlighted in particular asmost of the cytokines which appear to be of

importance in rheumatoid arthritis are derivedfrom these cells.' More recently, attention hasfocused on vascular changes in the synovium.2In a previous study, the vascular changes werequantified and an increase in vessel numbers aswell as a change in vessel morphology wasobserved in synovial membranes obtained fromclinically affected joints of patients with earlyrheumatoid arthritis compared with synoviumfrom clinically unaffected joints and fromnormal controls at necropsy.3 These findingsconflict with the report by Stevens et al, whichsuggested reduced vascularity with advanceddisease.4 It might be suggested that thesedifferences reflect stages in disease progression.In a further study, however, the number ofvessels and vascular morphology in synovialmembrane obtained from patients with rheuma-toid arthritis, by needle biopsy and at arthro-plasty, were examined and no difference betweenthose with early or late disease was observed.5 Ithas been suggested that some of the diseasemodifying antirheumatic drugs may act byinhibiting macrophage function or by reducingvascular proliferation."8 Thus it would be ofinterest to examine the effects of treatment withthese drugs on the histological features ofsynovial membrane.As the repeated taking of synovial biopsy

samples might prove unacceptable for manypatients, a readily detectable marker ofmonocytefunction and vascularity in synovial fluidsamples would be useful. Angiotensin convertingenzyme (ACE) produced mainly by endothelialcells9 and by activated monocytes,10 might besuch a marker. In this study, ACE activity wasmeasured in serum and synovial fluid samplesobtained from patients with inflammatory andnon-inflammatory joint disease. The localisationof ACE in synovial membranes from patientswith rheumatoid arthritis was also examinedusing a monoclonal antibody to ACE. ACEactivity was also measured in the synovial fluidof patients with rheumatoid arthritis under-going arthroplasty and in the supernatantsderived from their synovial membrane cultures.Levels of ACE activity in the synovial fluid andin the supernatants were correlated with theimmunohistological features of the synovialmembrane and with interleukin 1 and inter-leukin 6 production in the synovial membrane.

Materials and methodsPATIENTSConcomitant samples of serum and synovialfluid were taken from the knees of patients withinflammatory arthritis and from patients with

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Production ofangiotensin converting enzyme by rheumatoid synovial membrane

non-inflammatory knee effusions. The groupwith inflammatory arthritis included patientswith rheumatoid arthritis, as defined by therevised American College of Rheumatologycriteria,"1 reactive arthritis, seronegative rheu-matoid arthritis, gout, and systemic lupuserythematosus. The group with non-inflam-matory arthritis was composed of patientswith osteoarthritis and patients undergoingarthroscopy for traumatic knee injury. In thesecond part of the study, synovial fluid andsynovium samples were obtained at arthroplastyfrom a further group ofpatients with seropositiverheumatoid arthritis.

Blood and synovial fluid samples were takenin plain tubes and centrifuged. All samples ofserum and centrifuged synovial fluid were thenstored at -20°C until analysed. Blood sampleswere also analysed for haemoglobin, erythrocytesedimentation rate (Westergren), and rheuma-toid factor (latex fixation).

SYNOVIAL MEMBRANE CULTURES

Undigested synovial membrane (0-25 g), dis-sected free of tendinous and adipose tissue, wascultured in 1-25 ml of RPMI (Gibco, Paisley,UK) containing 10% fetal calf serum (Panox,Antrim, UK), 1% glutamine (Gibco), 1-8 ,ug/mlamphotericin, and 50 ,ug/ml gentamicin (Gibco)in sterile flat bottomed multiwell tissue cultureplates. Following incubation at 37°C in ahumidified atmosphere with 5% CO2, the tissuesupernatants were harvested at days 1 and 7 forthe determination ofcytokines and ACE activity.Interleukin 1 and ipterleukin 6 were determinedby radioimmunoassay.

STATISTICAL ANALYSISDifferences between paired data were analysedusing the Wilcoxon signed rank test or theMann-Whitney U test. The significance ofcorrelations was determined using the Spearmanrank correlation test.

ACE ASSAYACE activity was measured in all samples usinga two point kinetic assay based on the method ofHolmquist et al.'2 The assay reagents wereavailable in a kit produced by Sigma Diagnostics(Poole, Dorset, UK) and the ACE activity wasdetermined using a fully automated spectro-photometer (Abbott, ABA-100) with detectionat 340 nm. The assay was reproducible with anintra-assay variation of 5 U/ml. One freeze/thawcycle did not alter the ACE activity in the serumor centrifuged synovial fluid samples.

IMMUNOHISTOLOGICAL STUDIESSynovial membrane obtained at arthroplastywas snap frozen in isopentane and liquid nitrogenbefore storage at -70°C until sectioning.Cryostat sections 7 [tm thick were cut at -200C,placed on gelatin coated glass slides and allowedto dry at room temperature. The sections werefixed in acetone for 10 minutes and were thenwashed in phosphate buffered saline beforeincubation with the primary antisera for onehour. The table shows the monoclonal antibodiesused.The sections were stained using a routine

three stage immunoperoxidase technique.'3 14This allowed quantification of the various celltypes (expressed as number of cells per squaremillimetre) including T lymphocyte sub-populations and macrophages as well asmembrane vascularity (expressed as number ofvessels per square millimetre).

Monoclonal antibodies and their specificitiesAntibody Antigen Specificity Source

Leu 1 CD5 T cells Becton DickensonLeu 2a CD8 Suppressor/cytotoxic

T cells Becton DickensonLeu 3a CD4 Helper/inducer T cells Becton DickensonUCHL1 CD45Ro Memory T cells -'Leu 14 CD22 B cells Becton DickensonM02 CD14 Monocyte/macrophages Becton DickensonAntibody to ACE ACE Angiotensin converting

enzyme -tAntibody to FVIII FVIIIRag Endothelium Dako

*A gift from Professor P Beverley, University College Hospital, London, UK.tA gift from Professor R Auerbach, University of Wisconsin, USA.

ResultsCLINICAL FEATURESOf the 18 patients in the group with inflam-matory arthritis, 13 had seropositive rheuma-toid arthritis, one seronegative rheumatoidarthritis, two reactive arthritis, and one eachgout and systemic lupus erythematosus. Thegroup with non-inflammatory arthritis wascomposed of 12 patients, seven with osteo-arthritis, and five with traumatic knee injury.There were 13 women and five men with a meanage of 47 years (range 17-80) in the group withinflammatory arthritis and there were 10 menand two women with a mean age of 48 years(range 24-71) in the control group. The patients

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Figure I Mean (SE) activity ofangiotensin convertingenzyme (ACE) in synovialfluidfrom patients withinflammatory and non-inflammatory arthritis.

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in the two groups were receiving a variety ofnon-steroidal anti-inflammatory drugs and sixof the patients with rheumatoid arthritis werealso receiving disease modifying drugs. Nopatients had received intra-articular cortico-steroids within three months of the study.

ACE IN SERUM AND SYNOVIAL FLUIDAlthough serum ACE levels tended to be higherin the group with inflammatory arthritiscompared with the controls, no statisticallysignificant difference was observed. In contrast,the mean (SE) ACE activity (U/ml) in thesynovial fluid of the group with inflammatoryarthritis was 37 (5) which was significantlygreater than that in the group with non-inflam-matory arthritis (19 (3); p<005) (fig 1). Inaddition, 10 of 18 (55%) of the group withinflammatory arthritis compared with two of 12(16%) of the non-inflammatory controls hadhigher levels of ACE activity in synovial fluidthan in serum samples, although the differencewas not statistically significant. Furthermore,ACE activity in synovial fluid from the groupwith inflammatory arthritis did not correlatewith rheumatoid factor positivity, diseaseactivity, or the use of disease modifying agents.

LOCALISATION OF ACE IN SYNOVIAL MEMBRANEFROM PATIENTS WITH RHEUMATOID ARTHRITISSynovial membrane samples obtained at arthro-plasty from patients with rheumatoid arthritiswere stained with a monoclonal antibody toACE. Analysis of samples showed that the ACEwas localised to the luminal surface of theendothelial cell membrane and to mononuclearcells in the stroma and the lining layer (fig 2).The mononuclear cells were identified asmacrophages by staining serial sections with

* ...............;....W;..~~~~~~~~~~~~~~~~~~~~~~~~~~~~...Figure 2 Rheumatoid synovial membrane stained with a monoclonal antibody toACEshows staining ofthe luminal surface ofthe endothelium (thick arrow) and monocytelmacrophage cells (thin arrow) ofthe stroma. Bar=2-5 mm (immunoperoxidase stain).

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Figure 3 Mean (SE) activity ofangiotensin convertingenzyme (ACE) in supernatants taken on days I and 7fromsynovial membrane cultures.

antibody to ACE and the CD 14 monocytemarker.

SYNOVIAL MEMBRANE CULTURESThe mean (SE) ACE activity in the synovialfluid samples taken at arthroplasty from 12patients with rheumatoid arthritis was 38 (6).The ACE activity in the synovial membraneculture supernatants at day 1 was 17 (5) and atday 7 showed a significant increase to 25 (3)(p=005) (fig 3). A significant correlation wasobserved between the ACE activity in synovialfluid and the increase in ACE activity from days1 to 7 (p=0-001). Levels of ACE activity at days1 and 7 showed a significant correlation (p=0o001).As ACE is a product of endothelial cells and

activated macrophages, attempts were made tocorrelate ACE activity in the synovial fluid orsynovialmembrane supernatants with membranevascularity or macrophage number. No suchcorrelation was observed. The ACE activity didnot correlate either with numbers of T cells, Tcell subsets, B cells, lining layer thickness, orsynovial membrane supernatant levels of inter-leukin 1 or interleukin 6.

DiscussionAngiotensin converting enzyme is a halide acti-vated peptidase which catalyses the releaise ofhistidylleucine from the COOH terminus of thedecapeptide angiotensin I to yield a pressorpeptide, angiotensin II.9 In addition, ACEinactivates bradykinin, an important vasodilator,by catalysing the sequential release ofphenylalanine-arginine and serine-proline fromthe COOH terminus.'5 Bradykinin has beenshown to be present in synovial fluid in inflam-matory arthritis,'6 to have proinflammatoryeffects on fibroblasts,'7 and also leads to stimu-lation of pain fibres.'8 This suggests that ACEmay have important regulatory functions in theinflammatory process. Using a fluorescein

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Production ofangiotensin converting enzyme by rheumatoid synovial membrane

labelled antibody, ACE has been localised to thevascular endothelium'9 and the presence ofACE on cultured endothelial cells has been usedas a marker as cultured fibroblasts and smoothmuscle cells do not have it.20 The control ofACE secretion by the vascular endothelium isuncertain-however, it is of interest that hypoxiahas been reported to inhibit ACE and results ina decrease in angiotensin II generation and adecrease in bradykinin degradation.2' 22

Serum levels of ACE have been found to beincreased in a number of diseases, notablysarcoidosis.23 The origin of the ACE activity insarcoidosis is uncertain, but evidence pointstowards cells of the monocyte/macrophagesystem.24 Considerable numbers of macrophagesand monocytes are found within sarcoid granu-lomas,25 and human monocytes in culture can

be induced to produce ACE following exposureto dexamethasone.'0 Serum and synovial fluidlevels of ACE have also been measured pre-viously in patients with polyarthritis.26 27 In onestudy, although serum levels were similar inpatients with rheumatoid arthritis, osteoarthritis,seronegative arthropathy, and in normal con-trols, synovial fluid ACE activity was higherin patients with rheumatoid arthritis than inthose with osteoarthritis, though they weresimilar when corrected for protein levels.26More recently, ACE activity was assayed inserum free conditioned medium from unstimu-lated peripheral blood monocytes from patientswith rheumatoid arthritis, osteoarthritis, andfrom normal controls.27 The results indicatethat monocytes from patients with rheumatoidarthritis, in particular those with early disease,produce more ACE than monocytes from controlsubjects. That study did not examine theproduction of ACE by synovial monocytes anddid not address the possibility that cells otherthan monocytes, such as vascular endothelialcells, might synthesise and release ACE in therheumatoid synovial membrane.

Results from this study confirm that synovialfluid levels of ACE activity are increased inpatients with inflammatory joint disease (mostlyrheumatoid arthritis) compared with controlswith non-inflamnmatory arthritis. That thesynovial membrane actively synthesises ACE isshown by the finding of increased ACE activityin synovial membrane culture supernatantsafter seven days of culture. Although otherfactors may affect the concentration of a

macromolecule such as ACE in synovial fluid inaddition to local production, our results point toan important contribution by synovial membranesecretion. Furthermore, the close correlation(p=O-001) in this study between levels of ACEin synovial fluid and the rate of secretion ofACE by synovial membrane between days1 and7 into the culture supernatant shows that localproduction by the synovial membrane is clearlya major source of ACE in synovial fluid. Using a

monoclonal antibody to ACE, we have for thefirst time localised ACE to the vascular endo-thelium and to some mononuclear cells in thestroma andlining layer of synovium in patientswith rheumatoid arthritis. These cells were

identified as cells of the macrophage/monocytelineage, thus confirming previous observations.

Supernatant levels of ACE, however, do notcorrelate with any of the immunohistologicalfeatures or with supernatant levels of thepredominantly macrophage derived cytokines,interleukin 1 and interleukin 6. Although theseresults indicate that ACE activity in synovialfluid does not directly reflect the lining layerthickness, macrophage number, nor membranevascularity, it could be that ACE production is afunction of cell activation rather than cellnumber.

This is the first study to show the de novoproduction of ACE in the synovial membrane ofpatients with rheumatoid arthritis and thelocalisation of the enzyme to the endotheliumand to monocytes. The potential part played byACE in the inflamed synovial membrane isuncertain, but by increasing the conversion ofangiotensin I to angiotensin II and by inacti-vating bradykinin, ACE would predominantlyhave a vasopressor effect. This might in turnlead to episodes of tissue hypoxia, which whenfollowed by reperfusion may lead to a release ofoxygen free radicals, thought to be importantmediators of cell damage in rheumatoid arth-ritis.28

The authors are grateful for the support of The CharitableInfirmary Charitable Trust in funding this project.

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