CLIN. CHEM. 30/9, 1517-1522(1984)

CLINICALCHEMISTRY, Vol.30,No.9,1984 1517

Detectionof a2-Macroglobulin-AssociatedProteasesin the PlasmaofPatientswith RheumatoidArthritisAlexandre Gaspar,1’3John L Skosey,2 Winston Sequeira,4 and Marius Teodorescu1’5

In previouswork a polyclonalB cellactivatorhas beendetectedinthe serum of patientswith rheumatoidarthritis(RA). This activatoris associatedwith a2-macroglobulin(a2M)and itsactivityisblockedby lOWMr trypsininhibitors,which suggests thatitmay be a protease-a2M complex.Here we determinedthe possibilityof developinga routineclinical chemistry test for detection of this complex in pa-tients’ blood. We measured with chromogenic substrates thetotal proteolytic activity of citrated plasma and of the a2Mimmunoabsorbed from plasma. Low-Mr substratescontain-ing Arg were degraded much better by plasma from RApatientsthan by plasma from patientswith other arthritides.LOWMr substrates containing Leu, Lys, or Gly or the large-MrsubstrateAzocoll were not degraded by RA patients’ plasma.a2M from RA patients’plasma attached to a solid-phaseimmunoabsorbent degraded an Arg-containing tripeptidemuch betterthan did the a2M from normal donors,frompatientswithsystemiclupuserythematosus,or from thosewithjointinflammationof other,“non-autoimmune” origin.Althoughthe enzyme associatedwitha2M in the plasma fromRA patients appeared to be similar to trypsin, the differencesin optimal pH, cation concentration, degradation of Lys-containing substrates, and biological activity suggest other-wise. We speculate that the a2M-protease complexes aregenerated in the immune system and contribute to theinflammatory and autoimmune phenomena in RA.

AddItIonal Keyphrases: studies with immunoabsorbent-linked pro-tein trypsin-like enzyme autoimmune disorders . B cells

synthetic substrates . systemic lupus erythematosuslymphokines

Polyclonal gammopathy and production of autoantibodiesare frequent findings in patients with rheumatoid arthritis(RA), systemic lupus erythematosus, and other autoimmunediseases.6 In previous work we detected a factor in patients’blood that caused polyclonal B cell activation (1-4). Thisfactor is associated with a2-macroglobulin (a2M) and isblocked by low-Mr protease inhibitors, which suggests thatit may be a protease (4).

Departments of 1 Microbiology-Immunology and 2Medicine,University of illinois at Chicago, Chicago, IL 60612.On leavefrom Pasteur Institute, Lyon, France.4Department of Medicine, Cook County Hospital, Chicago, IL

60612.5Address correspondence to thisauthor.6 Nonstandard abbreviations: RA, rheumatoid arthritis; a2M, a2-

macroglobulin; PBS, phosphate-buffered isotonic saline; LPN, L-

Leu-p-nitroanilide; BAPNA, benzoyl-Arg-p-nitroanilide; BANA, N-a-benzoyl-Arg-/3-naphthylaniide; TAME N-a-p-toluene sulfonyl-L-Arg-methyl ester.

Received May 3, 1984; accepted June 9, 1984.

Other experiments have suggested that a2M-proteasecomplexes may play a role in the polyclonal B cell activationand in the inflammatory process in RA. These types ofcomplexes have been found not only in the supernates ofhigh-density lymphoid cell cultures (5) but also, in largeamounts, in the sera of rabbits inoculated intravenouslywith allogeneic lymph node cells (6). Moreover, the a2Mfrom RA patients’ serum (2,3) or from the supernate of thelymphoblastoid cell lines RPMI-1788 (Mclntire and Paper-master, personal communication) produces inflammationwhen injected intradermally in guinea pigs. Finally, repeat-ed intra-articular injections of a2M-trypsin complexes inrabbits cause an inflammatory process resembling that seenin RA (7).

On the basis of these observations we investigated thepossibility of using a chromogenic substrate to detect in RApatients any increased protease activity in plasma or anyincreasein the proportionofa2M molecules in plasmas thatcontain proteases.The resultspresented here strongly sug-gest that RA patients had a greater concentration of prote-ase activity and of a2M-protease complexes than did normaldonors or patients with other arthritides.

Materials and Methods

Patients and Sample Collection

We collected blood from patients with classical “seroposi-tive” RA, systemic lupus erythematosus, or other arthritides(i.e., psoriatic arthritis, gout, osteoarthritis) and from nor-mal donors. The blood was collected in Vacutainer Tubescontaining no anticoagulant or either sodium citrate (3.8mg/mL of blood), EDTA (2 mg/mL of blood), or heparin (0.2mg/mL of blood) (Becton-Dickinson and Co., Rutherford,NJ). The plasma, collected by centrifugation for 10 miii at3000 x g, was either tested immediately or frozen andstored at -25 #{176}C.To collect serum, we incubated the wholeblood at 37 #{176}Cfor 1 h, centrifuged, then froze the serum andstored it at -25 #{176}C.Serum was also obtained from plasmacontaining sodium citrate by adding CaC12 to a final concen-tration of 0.2 moltL, and incubating at 37 #{176}Cfor 1 h.

Reagents

Enzymes and substrates. We prepared a stock solution ofbovine trypsin (EC 3.4.21.4; from Sigma Chemical Co., St.Louis, MO), 1 mg/mL in 2 mmol/L HC1, and stored it inaliquots at -25 #{176}C.We used the following protease sub-strates. Carbobenzoxy-Val-Gly-Arg-p-nitroanilide (Chromo-zym Try; Boehringer Mannheim, Indianapolis, IN) wasdissolved in ethanol (20 mg/mL) and then diluted to 1mg/mL with distilled water; L-Leu-p-rntroamlide (LPN;Boehringer Mannheim), 1 mg/mL, was prepared similarly.For separate solutions of benzoyl-Arg-p-nitroanilide

A ,,,,, x iO’

Plasma

1518 CLINICALCHEMISTRY,Vol.30,No.9,1984

(BAPNA; Boehringer Mannheim) and N-a-benzoyl-Arg--naphthylamide (BANA; Sigma) we dissolved 43.5 mg of

either in 1 mL of dimethyl sulfoxide and diluted 100-foldwith distilled water. N-a-p-Toluene su1fonyl-i-Arg-methylester (TAME; Sigma) was dissolved directly in distilledwater to 1 mg/mL. We suspended 50 mg of Azocoll (Calbio-chem Behring, Lajolla, CA) in 5 mL of phosphate buffer, 0.1mol/L, pH 7.0. All substrates were stored in the dark at 4#{176}C.

Protease inhibitors. The stock solution of phenylmethyl-sulfonyl fluoride (Sigma) was 0.1 mol/L in ethanol. Thestock solution of soybean trypsin inhibitor (Sigma), 20mg/mL of phosphate-buffered saline (PBS) at pH 7.2, wasdialyzed overnight against PBS in a dialysis tubing with aMr cutoff of 10 000 (Spectra Por; Pierce, Rockford, IL). Theaprotinin (Boehringer Mannheim) stock solution contained10000 kallikrein inhibitory units per milliliter of PBS. Allinhibitor solutions were stored at -25 #{176}C.

Procedures

Determination of protease activity in plasma or serum.After diluting the sample of plasma or serum fourfold withPBS, we mixed 0.1 mL of this with 0.1 mL of ChromozymTry, LPN, TAME, or Azocoll solutions or with 0.2 mL ofBANA or BAPNA solutions. For the reaction mixturescontaining Chromozym Try, LPN, TAME, or BAPNA weadjusted the volume to 0.6 mL with ‘fr HC1 buffer (50 moleach of Tris and NaC1 per liter, pH 8.2). For BANA andAzocoll we adjusted the volume with phosphate buffer (0.1molfL, pH 7.0). The controls were constituted as follows: (a)the plasma or serum was replaced by the buffer; (b) thechromogenic substrate was replaced by buffer; or (c) theplasma or serum was replaced by trypsin solution (stocksolution diluted 300-fold). Each sample was processed intriplicate.

All mixtures except those containing BANA were incu-bated at 20#{176}Cfor 2 h, then diluted to 1.2 mL with H20, afterwhich we measured their absorbances with a Beckmanspectrophotometer (Model 25) and semimicro cuvettes. Theabsorbance for TAME was determined at 247 nm, forAzocoll at 520 nm, and for all the other substrates, whichrelease p-mtroaniline, at 380 nm.

When BANA was used, we incubated the mixture at 20#{176}Cfor 1 h, then added 0.1 mL of a freshly prepared solution ofFast Garnet Salt (Sigma), 3 mg/mL. After another hour ofincubation at 20#{176}Cwe stopped the reaction by adding 0.5mL of acetate buffer (1 molJL, pH 4.2, containing 100 g ofTween 20 per liter), according to Hopsu and Glenner (8) anddetermined the absorbance at 525 nm.

Preparation of anti-a2M-coated beads. We coated Affigel10 beads (Biorad, Richmond, CA) with goat anti-humana2M antibodies (Cappel Lab Industries, Westchester, PA).For controls, we also coated beads with normal goat IgG(Cappel Lab Industries) and used other beads uncoated. To 1mL of Affigel 10 beads we added 20mg of antibodies in 1 mLof NaHCO3 buffer (0.1 mol/L, pH 8.5). After gentle rockingat 4#{176}Cfor 4 h we washed the beads with more NaIICO3buffer. The remaining active esters on the beads wereblocked by incubation for 1 h at 4#{176}Cwith 0.2 mol/L glycinebuffered with 0.1 molJL Tris (pH 8.2). The uncoated beadswere also treated with glycine-Tris buffer. We then washedall the beads with borate saline buffer (0.16 mol/L, pH 8.2)and stored them at 4#{176}Cin the same buffer.

Assay of a2M-associatedproteolytic activity. We mixed 0.1mL of plasma (diluted fourfold with PBS) with another 0.1mL of PBS and 50 ,uL of anti-a2M-coated beads washed inPBS. As a control, we mixed the diluted plasma with normalgoat IgG-coated beads or with uncoated beads. Each samplewas prepared in triplicate.

After incubating the mixtures at 37#{176}Con a rockingplatform (Lab Industries, Berkeley, CA) for 2 h, we washedthe beads three times with PBS by centrifugationat 750 x gfor 5 mm. After the finalwash each tube contained thebeads in 0.2 mL of PBS; to each we added 0.1 mL ofChromozym Try and adjusted the final,volume to 0.6 mLwith Tris . HC1 buffer (pH 8.2). We incubated the mixtureagain for 2 h at 20#{176}Cwith continuous rocking, then added0.6 mL of distilled water and centrifuged the tubes at 750 xg for 10 mm before measuring the absorbance of eachsample at 380 nm.

Inhibition of proteases. To inhibit proteases in wholeplasma, we incubated equal volumes of fourfold-dilutedplasma and inhibitor or solvent at 37 #{176}Cfor 1 h. After thisstep, we tested for protease activity as described above.

To inhibit proteases associated with a2M, we first incubat-ed theplasma with the beads coated with anti-a2M antibod-ies, as described above. The beads coated with a2M frompatients’ plasma in PBS were mixed with an equal volumeof inhibitor solution and incubated at 37 #{176}Cfor 1 h withcontinuous rocking. The degradation of the substrate wasdetermined as described above.

Results

Collection and Storage of Plasma

Our previous work suggested that a trypsin-like proteaseassociated with a2M was present at higher activities in theplasma of patients with RA than in normal individuals (4).Therefore, we attempted first to use Chromozym Try (atrypsin substrate) to measure the total proteolytic activityin plasma of three normal donors and one patient with RA.The lowest value of proteolytic activity toward ChromozymTry was seen in plasma containing citrate and the highestwas in serum (Table 1). EDTA-containing plasma gavevalues slightly higher than those obtained with citrate (datanot shown). The differences between the enzyme activity ofthe plasma of the three normal donors and that of the RApatient was clearly higher with citrated plasma than withheparinized plasma. Conversion of plasma to serum causeda substantial increase in the enzyme activity and obscuredany difference between normal donors and the patient. Thisincrease strongly suggested that in the serum, and verylikely in the plasma containing heparmn, most of the activitywas due to enzymes of coagulation.

To determine whether the enzyme activity of citratedplasma changes with time, we incubated the samples atroom temperature for various periods before testing fordegradation of Chromozym Try. For up to 2 h of storage atroom temperature the proteolytic activity was unchanged(Figure 1), but increased significantly at 3 h and 4 h.However, adding soybean trypsin inhibitor at time 0 keptthe activity unchanged, even after 4 h of incubation.

To determine whether citrated plasma could be stored

Table 1. Effect of Anticoagulants on EnzymeActivityof Whole Plasma and Serum

Source Citrated Heparinized Serum

Normaldonor 160 ± 2 906 ± 16Normaldonor 175 ± 7 891 ± 10Normaldonor 154±4 1004±16RA pationt 544 ± 9 1106 ± 27

1225 ± 70951 ± 56

1151±681275 ± 90

Absorbance (mean ± SE) of p-nitroanhlinereleasedfromChincubatedwithplasma or serum for2 h at 20#{176}C.n = 3 each.

romozymTry

oa-

‘C

EC

0I’)

. 100

Citrate

ratSBT I

0 2 3

?70

64?

0

4 4

C

0

B

25

4

3

2

4

35

24 ‘C

2

a

4

0

4

4 5

5

#{174}c0

24

&24 E’4D?

C

E

A

so

E

0

A

C

S

C0

AC

B

4

S

3

223

400

300

200

100

CHROM BAPNA BAPIA TAME LPN AZOCOLL

Fig. 2. The degradation of various substratesby plasma from normaldonors (1-5)and from patients with RA (A-E)Procedurewas as describedintext Chrom,ChromozymTry; circledT, trypeincontrols.Absorbancewasmeasuredat thewavelengthsmarkedintheupperpartofthegraph

The optimal pH for trypsin activity is 9.0. Thus, todetermine whether the protease in RA patients’ plasma andserum had the same pH of optimal enzyme activity, weincubated fourfold-diluted plasma or eightfold-diluted se-rum with Chromozym Try at various pH values. Plasmawas incubated in the Tris-saline buffer; serum and trypsinsolutions were incubated in the same buffer but with 50mmol of CaCl2 added per liter. The optimal pH of degrada-tion of Chromozym Try was 9 for trypsin and between 8 and8.5 for the protease in the patients’ plasma and serum(Figure 3). Thus, we performed all other experiments at pH

900

800

700

600S

2‘C 500

EC

400S

300

200

00

5.5 6 6:5 7 7.5 8 8.5 9 9.5 0pH

Fig.3. Effectof pH on the degradationof ChromozymTry by trypsin,plasma,or serumBarsrepresentSEM oftriplicateassays.

Serum

300

CLINICALCHEMISTRY, Vol.30,No.9,1984 1519

HoursFig. 1. The effectof timeof storageof plasmaat 20#{176}Con the enzymeactivityOne tube contained citrate and the other citrate plus soybean tiypsininhibitor(SBTI),20 g/mL. line shownis how longplasma was stored before beingincubatedwiththesubstratefor another2 h.Thesnow showsthemaximum timethebloodwas stored in subsequent experiments.Bars indicateSEMof tiiphcatedeterminations

without affecting the activity we were investigating, wefroze the samples at -25 #{176}Cwithin 2 h of collection, thawedthem within two months, and retested. There was no changein the ability of the plasma to degrade Chromozym Try (datanot shown). Therefore, in subsequent work we routinelyfroze and stored our samples in sodium citrate solutionunder these conditions.

Selection of Substrate and Optimization of Assay

In previous work we had shown that a2M from the serumof patients with RA (4) or from the serum of rabbitsinoculated intravenously with allogemc lymphocytes (6)degrade TAME. However, the values obtained were rela-tively low. To determine which substrate yields the highestvalues with plasma from RA patients, as compared withnormal plasma, and to show the specificity of the enzyme forsubstrate, we performed the following experiment. Plasmasfrom five normal donors and five RA patients were tested forthe ability to degrade several low-Mr substrates containingargimne, lysine, leucine, or glycmne. In addition, we testedAzocoll, a particulate substrate for trypsin and thereforeinaccessible to a2M-associated enzymes.

The highest overall values of degradation were obtainedwith Chromozym Try substrate followed in order by TAME,BANA, and BAPNA (Figure 2). All four substrates showedclear differences in the amount of proteolytic activity be-tween normal and RA patients’ plasma. Moreover, patientE, whose plasma degraded the most Chromozym Try, alsohad the greatest or near-greatest effect on the other threesubstrates, and patient B, with the least effect on Chromo-zym Try, also had the least effect on the other threesubstrates. Thus, all four substrates containing argininewere apparently capable of indicating differences betweenthe proteolytic activity of patients’ plasma and normalplasma. All these substrates were also degraded by trypsin.In fact, the high values of enzyme activity in patients’plasma seemed equivalent to the enzyme activity of about15 tg of trypsin per milliliter (Figure 2).

The substrates containing other amino acids such asleucine (Figure 2), glycine, or lysine (data not shown) werenot significantly degraded and thus showed no differencesbetween plasma from RA patients and that from normals.Azocoll was degraded by trypsin but not by normal plasmaor RA patients’ plasma; presumably, the plasma enzymesdid not have accesstothislargesubstrate,probably becauseof their association with a2M (see below).

1000

800

0‘Co 600aIn

4400

200

600

20#{176}C37#{176}C

0

EC0a

100

0 30 60 90 120 50 80 210

None NaC1

Material added#{176}

CaCI2

Table 2. Effect of Cations on the Proteolytic Activity of Plasma or Serum from RA Patients or of Trypsin

Materialsteated

Plasma 371 ± l85 462 ± 3 460 ± 10 417 ± 5 490 ± 5 310 ± 4Serum 671 ± 11 743 ± 18 830 ± 10 797 ± 10 683 ± 23 579 ± 5Trypsin 880 ± 3 856 ± 6 1,034 ± 78 962 ± 31 969 ± 6 666 ± 25

#{176}All salts were at 50mmol/L inTrisbuffer.‘ The resultsare expressedas Aw,r,,,n (meanoftriplicates± SE).

KCISodiumcitrate

Ferriccitrate

1520 CLINICALCHEMISTRY, Vol. 30, No. 9, 1984

8.2.The enzyme activity measured in other buffers such asphosphate and borate was generally lower than in Trisbuffer.

To determine which cation best facilitates the expressionof enzyme activity of plasma or serum from RA patients andto determine whether Ca2 affects the expression of thisactivity, we performed the following experiment. The pro-teolytic activity of plasma or serum collected from RApatients was determined with Chromozym Try as a sub-strate in the presence of the following cations, 50 mmol/L inTi-is buffer: Na (Na3C6H507 2H0), Ca2 (CaC12), K(KC1), or Fe3 (C6H5FeO7 . H20). Trypsin solution was usedas a control. The addition of CaCl2 increased the proteolyticactivity of serum, trypsin, and plasma by about 20% (clot-ting did not occur, probably because of the use of dilutedplasma). The addition of Nat, either as NaCl or as Nacitrate, also increased the proteolytic activity of plasma. Theferric citrate reduced the proteolytic activity of all threeenzyme-containing solutions (Table 2).

To determine the optimal molarity of Na in the reactionmixture, we incubated citrated plasma from RA patientswith Chromozym Try in Ti-is buffer in the presence ofincreasing concentrations of NaC1. As a control we alsoadded increasing concentrations of CaC12 and examinedtrypsin and serum samples in addition to plasma. Theproteolytic activity of plasma was optimal at in the presenceof 50 mmol of NaC1 per liter (Figure 4). CaC12 at lowconcentrations increased the proteolytic activity of plasmabut reduced it at high concentrations; however, these highconcentrations of CaCl2 did not reduce the activity oftrypsin.

To determine the optimal time and temperature of incu-bation on the degradation of Chromozym Try by patients’plasma, we incubated the reaction mixtures in disposablecuvettes containing patients’ plasma or normal plasma atroom temperature or at 37 #{176}Cin a waterbath and measuredA3so at 30-mm intervals. The maximum difference in p-nitroaniline release between normal and RA plasma wasobserved after 150-mm incubation. There was practically nodifference between the values obtained at 37 #{176}Cand 20 #{176}C(Figure 5). This lack of difference may be due to the excess ofsubstrate and its interaction with other plasma proteins, orto insufficient substrate. However, there was a large differ-ence between results at the two temperatures when we usedtrypsin solution instead of plasma (data not shown).

Under the conditions described above we tested samplesin no particular order from four groups of patients: normaldonors, patients with other arthritides [osteoarthritis (five),psoriatic arthritis (two), and gout (three)I, 10 patients withRA, and 10 with systemic lupus erythematosus. The proteo-lytic activity for Chromozym Try was significantly greaterin patients with RA than in all other groups (Figure 6).

Association of Proteolytic Activity in Patient’s Plasma

with a2M

As is well known, proteases associated with a2M candegrade low-Mr substrates and are inhibited by low-Mrinhibitors but do not interact with large substrates or

0 0.05 0.1 0.15 0.2 0 0.OS 0.1 0.15 0.2

Co Cl2 NaCI

Fig. 4. Effect of molarity of CaCI2 or NaCI on the degradation ofChromozym Try by plasma(-), serum (-), or trypsin (----)

�F

ig.5 .T hee ffecto ft emperatureo fi ncubationo nt hetimecourseofdegradationo fChromozymT ryb yp lasmaf romanRAp atiento rf romanormald onori

nhibitors.T hus,w ed etermined,i np lasmao ft hes amepatientsa si nF igure6 ,t hed egradationo fC hromozymT rybya 2Ma ttachedt oa ni mmunoabsorbent.I na f irsts erieso fexperimentsw ed eterminedt hec onditionso fs aturationo fthei mmunoabsorbentw ithp lasmaa 2M( datan ots hown).Thea mountd egradedb ya 2Mf romp lasmao fp atientsw ithRAw asm uchg reatert hanb ya 2Mf romn ormald onorso rfromp atientsw ithl upuso ro thera rthritides.T hev aluesobtainedw ithp lasmaf romt heo therp atientsd idn otd ifferfromt hoseo btainedw ithn ormalp lasma.T

od eterminet hati ndeedt hea ssociationo fe nzymeactivityw asw itha 2Ma ndn otw iths omeo there nzymet hatreactedw itht heg oata nti-a2Ma ntibodies,w ep erformeda competitione xperiment.S omea garoseb eadsc oatedw ithgoata nti-a2Ma ntibodyw eref irstt reatedw ithp urifieda 2Mfromn ormals erumb eforeu sei nt hea ssays;t hisn ormala2Mh adb eenp retreatedw ithm ethylaminet op reventa nyadditionalb indingo fp roteases( 9).C ontrolb eadsi nt hisexperimentw eret reatedw ithn ormalh umanI gG.T heabilityo ft hes olidp haset oa ttacht hep roteasef romR Apatients’p lasmaw asc ompletelyb lockedb yp retreatmentwithn ormala 2Mb utw asn ota ffectedb yh umanI gG.F or�

0

aC

0aIn

-.±SE A380,,600

500

400

300

200

00 1-a-

CLINICALCHEMISTRY,Vol. 30, No.9,1984 1521

NORMALS RA SLE

Fig. 6. Degradationof ChromozymTry by plasmaor by a2M-solid-phase immunoabsorbentfromnormalindMduals(normals),frompa-tientswith“non-autoimmune”jointinflammation(patientcontrols),andpatientswith rheumatoidarthritis (RA)or systemiclupuserythematosus(SLE)(#{149})degradationby wholeplasma;(A)degradationby agarosebeads coated withanti-a2Mantibodyand treatedwithplasma,correctedfor thevaluesobtainedwithagarose bead coatedwith normallgG insteadof antibody.The hotizontalbars (±SEM) are on the samescaleof valuesas the ordinate(A,,,,,)

example, for a background degradation value of 0. 190 (A0)the beads pretreated with saline or IgG had values of 0.420and 0.426, respectively; the beads pretreated with a2M hada value of 0.193, i.e., practically equal to the background.

In addition we determined the amount of Chromozym Trythat was degraded by the solid-phaseimmunoabsorbenttreated with patients’ plasma in the presence of proteaseinhibitors of high Mr, [soybean trypsin inhibitor (20 p.g/mL)]and low Mr [phenylmethylsulfonyl fluoride (1 mmol/L) andaprotinin (5000 kallikrein inhibitory units/mL)1. For con-trols, we prepared trypsin-.a2M complexes and used theminstead of patients’ plasma. The two low-Mr iiihibitorsblocked completely the proteolytic activity of both trypsin-a2M complexes and patients’ plasma; soybean trypsin inhib-itor inhibited only 18% (range 7% to 40% in six patients) ofthe activity of patients’ plasma and 8% of that of trypsin-a2M complexes. The slight but significant inhibition bysoybean trypsin inhibitor and the complete inhibitionbyphenylmethylsulfonyl fluoride or aprotinin are well-knowncharacteristics of a2M-trypsin complexes (10). Thus, theincrease in proteolytic activity in RA patients’ plasmaappears to be due to a trypsin-like enzyme associated witha2M.

DiscussionWe showed here that the proteolytic activity of plasma

from patients with RA is higher than that of normal donorsor of patients with other “non-autoimmune” arthritides orwith systemic lupus erythematosus, and we have deter-mined the optimal conditions of pH, time of incubation,substrates, etc. for detecting these differences. This activitycan be measured either in whole citrated plasma or on asolid-phase immunoabsorbent containing anti-a2M antibod-ies.

The higher than normal degradation of Chromozym Try

and of other substrates by plasma from patients with RAappears to be due a trypsin-like enzyme, as indicated by thenecessity for the presence of arginine at the point of attack.However, there are three differences. First, the optimal pHfor patients’ plasma is lower than that for trypsin. Second,asubstrate containing lysmne was degraded by trypsin but notby RA patients’ plasma. Thirdly, Ca2 exceeding 50 mmolJLdoes not affect trypsin but inhibits the proteolytic activity ofpatient’s plasma. However, the exact nature of the enzymeor enzymes responsible for the degradation of ChromozymTry by plasma from RA patients remains to be determined,particularly because some enzymes of coagulation can alsodegrade this substrate.

The enzyme activity primarily was associated with a2M,although some free enzymes probably also contributed tothe degradation of the substrates. These free enzymes mayaccount for the increase in differences between RA patients’plasma and controls when we determined the activityassociated only with anti-a2M bound to immunoabsorbent(Figure6).The primary associationwith a2M was demon-strated on the basis of immunoabsorption with anti-a2Mantibody and on the evidence that normal a2M can effective-ly compete with the enzyme activity attached to the solidphase. Also, when anti-Ig light-chain antibodies were usedinstead of anti-a2M, there was practicallyno increaseinprotease activity (data not shown). Moreover, soybean tryp-sin inhibitor was relatively inefficient in blocking the pro-teolytic activity, but 1OWMr inhibitors, aprotinin and phen-ylmethylsulfonyl fluoride, completely blocked this activity.The proteolytic activity associated with a2M represents onlythe proportion of a2M molecules that carries proteases, theimmunoabsorbent having been used under conditions ofsaturation.

The origin of the increasedenzyme activity in plasmafrom RA patients has not yet been determined. It may begeneratedby the coagulationsystem,because some of theenzymes involved in coagulation such as thrombin or plas-mm (11) can also degrade Chromozym Try and othersubstrates containing arginine; moreover, these coagulationenzymes can bind to a2M (12). Several facts make a primaryrole for coagulation enzymes unlikely, however. First, in thepresenceofcitratethe enzyme activity undergoes practical-ly no increasefor2 h, which suggeststhatthe coagulationenzymes are kept under control for the period of time of theassay. Second, the a2M from normal human serum degradedTAME much less than the a2M from the serum of RApatients (4); third, we have observed similar differencesbetween normal rabbit aM (the rabbits have two a-macro-globulins, a1 and a2) and aM from the serum of rabbitsinoculated with allogeneic lymphoid cells (6). Thus, it ap-pears that the proteolytic activity associated with a2M maybe greater in RA patients than in normals even when thecoagulation system is activated. This aspect requires furtherinvestigation.

Complement system proteases are also an unlikely sourceof this activity because they do not associate with a2M (13).Moreover, the a2M from patients with systemic lupus ery-thematosus had less activity than that from RA patients,although it is well known that complement is more activat-ed in the blood of systemic lupus erythematosuspatientsthan in the blood of RA patients.

Two other possibilities remain to be investigated. One isthe production of enzymes by the inflammatory cells in thesynovium; these enzymes could be produced in largeamounts and could be released in the blood and becomeassociated with a2M. This hypothesis would not explain,however, the experiments in rabbits inoculated with alloge-neic cells (6). Also, we have seen patients with psoriatic

1522 CLINICALCHEMISTRY, Vol.30,No.9,1984

arthritis with substantial synovitis but normal concentra-tions of a2M-protease complexes, and patients with jointscompletely cleared by use of aspirin, who continued to havehigh activities of protease in the blood (unpublished obser-vations). Thus, as has to be determined in longitudinalstudies,the measurement ofa2M-proteasecomplexesmaygive information regarding the underlying chronic inflam-matory process. So far no clinical laboratory test can givesuch indication.

The other possibility, which we favor at this time, is thatthe protease-a2M complexes are generated in the immunesystem. In previous work we have shown that close contactsbetween rabbit lymphocytes in vitro leads to the productionof a factor, recovered in the aM fraction, that activates Bcells polyclonally. The activity of this factor is blocked byaprotiin (5) but not by soybean trypsin inhibitor (3). Thesame type of activity was observed with aM from the serumof rabbits immunologically activated by inoculation of allo-geneic lymphocytes (6). Moreover, a2M purified from theserum of patients with RA also activates B cells polyclonallyand this activity is blocked by aprotinin (3). Taken together,these data suggest that a2M-protease complexes may begeneratedby theimmune system.Infact,activesecretionofa2M by lymphocytes (14) or by macrophages (15) has beendemonstrated.

From the above data we speculate that the proteaseactivity detected in whole plasma or in a2M attached to animmunoabsorbent represents the enzyme activity of a lym-phokine. If this is indeed demonstrated in future studies, itmay lead to routine measurement of this lyinphokine inpatients’blood.

The cause of inflammation in the synovium of HA pa-tients and of the progressively destructive process is stillunknown. The factors of acute inflammation-immune com-plexes, prostaglandins, and enzymes of polymorphonuclearcells-may contribute to a certain extent. However, al-though the nonsteroidal anti-inflammatory drugs can re-duce this inflammation substantially, they do not alter thecourse of this process in HA. Some other, as yet undeter-mined inflammatory agents or chronic-phase reactants maybe responsible. The a2M-protease complexes along withother lymphokines may be candidates for such pathogenicfactors.The roleofa2M-proteasecomplexesinthe inflammatory

process in RA has some experimental basis. For example,intra-articular inoculation of normal a2M in rabbits has noeffect; but inoculation of a2M-trypsin complexes causes aninflammatory synovitis similar to that seen in HA patients(7). Also, a2M from patient’s serum causes inflammation inguinea pigs 6-8 h after intradermal inoculation (2). Thesecomplexes also have other effects: they activate macro-phages to produce neutral proteases (16); are incorporatedfaster than native a2M by cells of the reticuloendothelialsystem (14) and by fibroblasts (9); are suppressive for T cellresponses (18); and are chemotactic for neutrophils (19).

On the basis of previous work on the generation of a2M-protease complexes and on their biological properties (seeref. 3), we propose the following hypothetical model for thepathogenesis of HA. Because of the action of infectious ormetabolic factors on a genetically predisposed organism, thenormal homing of lymphocytes is altered. Prolonged cell-cell contacts between B and T cells results in their mutualactivation and production of lymphokines, including a2M-protease complexes. These complexes are cleared from theblood up to the point of saturation of the system, when their

concentrations increase and cause inflammation in targetorgans, including the joints. These complexes also activate Bcells polyclonally, producing polyclonal gammopathy anddevelopment of autoantibodies and immune complexes.

Supported by NIH grant AM 28469 and NC! grant 21399.

References1. Teodorescu M, Chang J-L, Skosey JL. PolyclonalB cell activatorassociated with alpha-2-macroglobulin in the serum of patientswith rheumatoid arthritis. mlArch Allergy Appllmmunol 66,1-12(1981).2. Teodorescu M. Characterization and role in autoimmune dis-eases of the polyclonal B cell activator produced by T cells/thehelper factor. Immunol Rev 55, 155-178 (1981).3. Teodorescu M. A polyclonal B cell activating lymphokine and itsnatural inhibitor: Possible role in antibody formation and autoim-mune diseases Lymphokines 8, 81-141 (1983).4. Teodorescu M, Ganea D, Lee ‘F, et al. The effect of proteaseinhibitors on the polyclonal B cell activator from the serum ofpatients with rheumatoid arthritis. mt Immunopharmac 4, 1-7(1982).5. Chang J-L, Ganea D, Dray S, Teodorescu M. Blast transforma-tion of B cell8 induced by an a2-macroglobulin-associated lympho-kine produced in crowded lymphoid cell cultures. J Immunol 130,267-273 (1983).

6. Qanea D, Teodorescu A, Dray S, Teodorescu M. Polyclonal B-cellactivator with esterolytic activity and polyclonal gammopathyinduced by allogeneic cells in rabbits. Immunology 65, 227-237(1982).7. Borth W, Susani M. Rheumatoid-like synovitis in rabbits byintra-articular administration of a2-macroglobulin-trypsin com-plexes and alkylamine-treated a2-macroglobulin. ht Arch Allergy72, 180-183 (1983).8. Hopsu VK, Glenner GO. Histochenucal enzyme kinetics systemapplied to the trypsin-like amidase and esterase activity in humancells. J Cell Biol 17, 503-570 (1963).9. Marynen P, Van Levven F, Cassiman J-,J, Van Den Berghe H. Amonoclonal antibody to a neoantigen on a2-macroglobulin complex-es inhibits receptor mediated endocytosis. J Immunol 127, 1782-1786 (1981).10. Bieth JG. Inhibition of a2-macroglobulin-bound trypein bysoybean trypein inhibitor. J Biol Chem 256, 7954-7957(1981).11. Thplett DA, Harms CS. The use of synthetic substrates in thediagnostic clinical laboratory. In Perspectives in Hemostasi.s, SFareed, HL Messmore, JW Fenton, KM Brinkhous, Eds., PergainonPress, New York, NY, and Oxford, England, 1980, pp 389-404.12. Barrett AS. a2-Macroglobulin. Methods Enzymol 80, 735-757(1981).

13. Muller-Eberhard HJ. Complement reaction pathways.ProgImmunol 4, 1001-1024 (1980).14. ChangJ-L, Ganea D, Dray 5, Teodorescu M. An a2-macroglobu-un-associated factor produced by T lymphocytes which providespolyclonal stimulation of B lymphocytes to maintain the turnover oftheir surface 1g. Immunology 44, 745-754 (1981).15. Hovi T, Mosher D, Vaheri A. Cultured human monocyteesynthesize and secrete a2-macroglobulin. J Exp Med 145, 1580-1989 (1977).16. Visch#{233}rTL, Berger D. Activation of macrophages to produceneutral proteinases by endocyto8is of a2-macroglobulin-trypeincomplexes. J Reticuloendothel Soc 28, 427-435 (1980).17. Kaplan J, Nielsen ML. Analysis of macrophage surface recep-tors 1. Binding of a2-macroglobulins-protease complexes to rabbitalveolar macrophages. J Biol Chem 254, 7323-7328 (1979).

18. Hubbard WJ, Hess AD, Hsia S, Amos DB. The effect ofelectrophoretically “slow” and “fast” alpha-2-macroglobulins andmixed lymphocyte cultures. J Immunol 126, 292-298 (1981).19. Forrester JV, Wilkinson PC, Lackie JM. Effect of modified a2-macroglobulin on leukocyte locomotion and chemotaxis. Immunolo-gy 50, 251-259 (1983).