Br. J. Exp. Path. (I988) 69, 605-6I9

Biochemical nature and cellular origin of amyloidenhancing factor (AEF) as determined by anti-AEF antibody

K. Alizadeh-Khiavi and Z. Ali-KhanDepartment of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec,

Canada

Received for publication 2 5 November I 98 7Accepted for publication i March I988

Summary. Low ionic strength acidic buffer, Sephadex G-200 and Benzamidine-Sepharose (BZ)gel chromatography, have been used for the partial purification of alveolar hydatid cyst (AHC)induced amyloid enhancing factor (AEF). BZ-gel bound AEF (AEF-BZ) demonstrated AEFactivity in the mouse bioassay, proteolytic activity against Hide powder azure showed twomajor and three minor peptides on SDS-PAGE. Pretreatment of AEF-BZ with io mmphenylmethylsulphonyl fluoride or 20 mM p-chloromercuribenzoic acid completely abolishedits bioactivity in vivo and proteolytic activity in vitro. Polyclonal anti-AEF antibody (AAA) wasgenerated which on passive transfer into mice completely abolished the bioactivity of bothcasein-induced, or AHC-induced AEF. The AAA absorbed on Sepharose gel conjugated tonormal mouse serum developed one common precipitin band between AE and AEF-positivesera from AHC-infected and old retired mice and in immunostaining it bound to thecytoplasmic granular components ofa majority of splenic and peritoneal leucocytes from AHC-infected mice. In contrast, only a few normal mouse leucocytes showed positive staining. Wesuggest that AEF, in all probability, is a serine/thiol protease of leucocyte origin whoseintracellular and humoral concentrations increase significantly during amyloidosis. The roleof lysosomal proteases and anti-AEF antibody which has been successfully generated for thefirst time is discussed with reference to the origin ofAEF and its presumed biological function inamyloidogenesis.

Keywords: alveolar hydatid cyst, amyloid enhancing factor, anti-amyloid enhancing factorantibody, macrophage, neutrophil, phenylmethylsulphonyl fluoride, p-chloromercuribenzoicacid, amyloidosis

Although the concept of amyloid enhancing It was shown that a single injection of AEFfactor (AEF) and some of its biological activi- and AgNO3, the latter as an inflammatoryties in the induction ofrapid amyloidogenesis agent, remarkably shortened the preamyloi-in experimental animals were initially de- dotic phase to 24 h in the recipient mice andscribed almost two decades ago (Werdelin & the amount of splenic amyloid depositionRanlov I 966; Hardt & Ranlov I 9 76), much correlated positively with the amount ofAEFofour present understanding ofAEF emerged administered (Axelrad & Kisilvesky I980;from the work of Kisilevsky et al. (I9 77), Axeirad et al. I982). These studies alsoAxelrad et al. (I982) and Kisilevsky (I983). demonstrated that AEF existed at low levelsCorrespondence: Dr Z. Ali-Khan, Department of Microbiology and Immunology, McGill University,

3775 University Street, Montreal, Quebec, Canada H3A 2B4.605

K. Alizadeh-Khiavi & Z. Ali-Khan

in normal mouse spleen extracts but its tissue'concentration increased significantly 24-48h prior to amyloid deposition and continuedto increase progressively during the amyloiddeposition phase.AEF activity was also demonstrated in the

amyloidotic mouse sera (janigan & DruetI968; Abankwa & Ali-Khan I988b). Basedon these observations, AEF was suggested tobe an essential pathogenic factor requiredduring amyloidogenesis (Kisilevsky I983).Recent demonstration of AEF in amyloidoticspleen extracts from patients with primary,secondary and multiple-myeloma associatedamyloidosis and in the brain extracts fromAlzheimer's disease patients further re-inforces the importance ofAEF as a constitu-tive component of amyloidotic tissues and acommon link between major clinical andchemical forms of human amyloidosis(Varga et al. I986; Ali-Khan et al. I988b).However, as yet neither the precise bio-chemical nature of the AEF molecule nor itsbiological function in amyloidogenesis hasbeen identified or an antibody against AEFhas been generated to explore its cellularorigin, tissue distribution or diagnostic valuein amyloidosis.

Investigations from this laboratory onalveolar hydatid cyst (AHC; a metazoanparasite) infected mouse model of inflamma-tion-associated amyloidosis have shown thatdespite fundamental differences betweencasein and AHC as amyloidogens, the bio-logical activity of AEF induced by either ofthese two agents is analogous (Ali-Khan etal. I983; Alkarmi & Ali-Khan I984;Alkarmi I 98 5; Abankwa & Ali-Khan i 988a,b). Significant clues to the cell source of AEFand its protease nature were ascertainedusing extracts prepared from fractionatedleucocytes and pretreatment of AEF withvarious protease inhibitors; phenyhnethyl-sulphonyl fluoride treatment totally abo-lished the in vivo bioactivity of AEF, whereasapproximately 90% inhibition was inducedby e-aminocaproic acid, aprotinin and iodoa-

cetic acid treatments. These observationsindicated that AEF might be a lysosomalserine or thiol protease of leucocyte origin(Abankwa & Ali-Khan I988b).The data presented in this communication

confirm and extend these initial observa-tions. Using a modified purification pro-cedure, we have been able to: (i) partiallypurify AEF; (2) establish its proteolytic acti-vity in vitro; (3) abrogate its amyloidogenicactivity in vivo and proteolytic activity in vitroby treatment with synthetic enzyme inhibi-tors; (4) generate anti-AEF antibody whichon passive transfer into mice totally abro-gates the bioactivity of both casein-, or AHC-induced AEF and on immunostaining bindsto the cytoplasmic granular components ofmainly monocytoid cells and granulocytes.

Materials and methods

Chemicals

The following chemicals were used: 2-mer-captoethanol from Kodak (USA); phenyl-methylsulphonyl fluoride (PMSF), and ultra-pure urea from Schwartz/Mann (Cleveland,Ohio, USA); acrylamide, N, N' methylenebisacrylamide, sodium dodecyl sulphate(SDS), Bio-Gel P-6o, Coomassie blue R-250,and silver staining kit from Bio-Rad (Missis-sauga, Ontario, Canada); Sephadex G-200,and Benzamidine-Sepharose 6B from Phar-macia Fine Chemicals (Dorval, Quebec,Canada); Hanks' balanced salt, normalmouse whole serum-agarose, goat anti-rab-bit IgG [F(ab')2 fragment] conjugated tofluorescein-isothiocyanate, polyethyleneglycol compound (mol. wt I 5 000-20 ooo),Congo red, Hide powder-azure, Freund'scomplete and incomplete adjuvant, and dia-minobenzidine-tetrahydrochloride (DAB)from Sigma Chemical Company (St Louis,Missouri, USA); Heparin sodium salt solutionfrom NB Company Biochemicals (Cleveland,Ohio, USA); Vectastatin avidin-biotinimmune-staining kit (ABC) from DimensionLaboratories (Missisauga, Ontario, Canada).

6o6

Characterization of AEF

Infection and source ofamyloid enhancingfactor

Six- to eight-week-old male Cs 7 BL/6J miceUackson Laboratories, Bar Harbor, Maine,USA) were infected intraperitoneally (i.p.)with 50-100 alveolar hydatid cysts (AHC) asdescribed previously (Ali-Khan 1978). Micewere sacrificed between 8 and 12 weekspostinfection (p.i.) and their amyloidoticspleens and livers (Ali-Khan et al. I983;Alkarmi & Ali-Khan I984) were used forextraction of amyloid enhancing factor(AEF).

Crude preparation of amyloid enhancing factorAmyloidotic livers and spleens were homo-genized sequentially using a Waring blender(5 min; i g tissue/io ml of chilled O.OI Msodium phosphate buffer containing 0.3 MKCI, pH 8.o; PBK-8) and a Polytron tissuehomogenizer (Brinkmann Instrument, Rex-dale, Ontario, Canada) at amplitude 7 for 45s. The slurry was centrifuged (27 000 g, ih,

40C), and the supernatant was exhaustivelydialysed (Spectrapore dialysis bag, mol. wt.cut off 3500) against 400 volumes of o.oi Msodium phospate buffer pH 6.o (PB-6; 40C).After five daily changes of the buffer, theturbid suspension was centrifuged (27 000g, i h, 40C) and the sediment was solubilizedin O.OI M sodium phosphate containing 0.3M NaCl, pH 8'o (PBS-8). Both the superna-tant and the solubilized sediment, each at adosage of i mg protein, were tested for AEFactivity in the mouse bioassay (Axelrad et al1982) and then further purified.

Gel filtrationThe solubilized sediment was centrifuged(27 000 g, i h, 40C), passed through aMillipore filter (o 22 um pore size) andapplied (I5 ml; io mg of protein/ml) to aSephadex G-200 or Bio-Gel P-6o gel column(93 X 2.5 cm, flow rate io ml/h) equilibratedwith PBS-8. The two resolved peaks (Fig. i)from several separate runs were pooled

Fl

E

C:0oo

6

6

0.2

0.15

0.1

F2

100 200 300 400 500Elution volume (ml)

Fig. i. Sephadex G-200 chromatography profile of resolubilized aggregates obtained after exhaustivedialysis of amyloidotic liver and spleen extract against O.OI M phosphate buffer, pH6 (column 93 X 2.5cm; flow rate io ml/h; elution buffer O.OI M phosphate buffer containing 0.3 M Nacl, pH 8).

607

K. Alizadeh-Khiavi & Z. Ali-Khan

separately in Spectrapor dialysis bags, con-centrated against polyethylene glycol (PEG)(mol. wt. 15 000-20 ooo) and assayed forAEF activity. The retarded peak containedgreater AEF activity than the void volumefraction. The PB-6 supernatant, althoughless potent than the aggregated AEF, wasalso concentrated and fractionated on aSephadex G-200 gel column.

Affinity chromatography

The lyophilized Sephadex G-200 second peak(20 mg) was dissolved in 3.0 ml of 0.05 MTris/HCl buffer containing 0.5M NaCl, pH.8,dialysed overnight at 40C against the samebuffer and applied to a Benzamidine-Sephar-ose 6B gel column (I 5 x I * 5 cm) equilibratedwith the above buffer at room temperature.Four hours after the sample application theunbound protein was washed out with atleast io bed volumes of Tris/HCl and thebound protein was eluted with O.OI M HCIpH 2.0, containing 0.5 M NaCl at the flowrate of io ml/h; the elution profile of thefractions was monitored at 280 nm. Thebound protein, designated as AEF-BZ, wascollected in a flask containing 20-30 ml of0.5 M Tris/HCl buffer, pH 8.o with conti-nuous stirring. The bound and unboundfractions were concentrated as above, dia-lysed against PBS-8 and tested for AEFactivity. The AEF-positive Sephadex G-200first fraction was also fractionated as de-scribed above.

Protein determination

Protein concentrations were determinedusing the Bio-Rad protein assay kit andbovine serum albumin as the standard.

Mouse bioassay for AEF activity

Crude AEF or its various fractions wereassayed for AEF as described previously(Axelrad et al. I982; Abankwa & Ali-KhanI988a). Control mice received either AgNO3or different AEF preparations only.

Assay for proteolytic and amyloidogenic activi-ties of AEF-BZ

Proteolytic activity of AEF-BZ was measuredwith or without enzyme inhibitors usingHide powder azure as the substrate (Rinderk-necht et al. I968). Briefly, i mg of AEF-BZwas dissolved in O.OI M sodium phosphatebuffer, pH 8.o, and added to various concen-trations of phenylmethylsulphonyl fluoride(PMSF; 20, 10, 2 mM) or p-chloromercuri-benzoic acid (p-CMB; 20, 2, 0.2 mM), bothdissolved in dimethylsulphoxide (DMSO) andincubated for 2 h at 3 70C with end-over-endmixing. After microcentrifugation, the opti-cal density of the supernatant was readagainst the blank (io mg Hide powder azurein I.5 m of o.oi M sodium phosphate buffer,pH 8.o), at 595 nm using Perkin Elmerspectrophotometer. Control tubes containeddifferent amounts of AEF-BZ (62-500 ug)and equivalent amounts of DMSO and thesubstrate. In a previous study we showedthat crude AEF pretreated with io mM PMSFcompletely lost its amyloidogenic bioactivityin vivo; PMSF injected alone did not inhibitthe bioactivity ofAEF (Abankwa & Ali-Khan198 8b). In the present experiment, i mg ofpartially purified AEF-BZ was incubated withIo mM PMSF for 2 h and then injected intomice; control mice received AEF-BZ only.Each mouse concomitantly received subcu-taneously (s.c.) 0.5 ml of 2% AgNO3; themouse spleens were obtained 72 h after theinjections and processed as described above.

Anti-AEF antibody

A rabbit was injected s.c. at multiple siteswith 2 ml ofSephadex G-200 second fraction(0.5 mg protein) emulsified with an equalvolume of Freund's complete adjuvant. Therabbit was boosted three times (total proteinI.5 mg in Freund's incomplete adjuvant) atweekly intervals and then allowed to rest forthree weeks. One week after the fourthbooster injection the rabbit was bled. The Igswere affinity purified from the serum bythree times precipitation with saturated

6o8

Characterization of AEF

(NH4)2SO4 solution (I:i,v/v) and fractiona-tion through a column packed with agaroseconjugated to whole normal mouse serum.The flow through fraction (anti-AEF Igs) wasconcentrated, dialysed against PBS and usedfor immunostaining, immunodiffusion andpassive transfer studies.

Assay for biological activity of anti-AEF serum

First the dose-response curve (o. I, 0.25, 0.5,I.0, 2.5, 5.0, 15 mg protein) was establishedfor both the casein-induced, and AHC-induced AEF as described above. The formerwas prepared in Cs 7BL/6J mice by daily s.c.injections of o.5 ml of io% casein (Hammar-sten, BDH Chemicals, Montreal, Canada) for35 days. The 0.25 mg dosage induced amy-loid (AA) deposition in at least 50% of thefollicles. For passive transfer experiments,mice were injected i.p. with i ml of eitherunabosrbed anti-AEF serum or O.I, 0.5 andI.0 ml of the affinity purified anti-AEF Igs atday - i. On day o each mouse received s.c.0.5 ml of 2% AgNO3 and i.p. 0.25 mg ofeither casein-induced, or AHC-induced AEF.After 72 h mouse spleens were sectioned forAA detection. Control mice received similarvolumes of DEAB-cellulose gel purified nor-mal rabbit IgG (9.o mg protein/ml).

Indirect immunofluorescence and avidin-biotin-peroxidase complex immunostaining

Cytocentrifuged spleen and peritoneal cellsfrom normal and AHC-infected amyloidoticmice were used for immunostaining. Indirectimmunofluorescence staining was carriedout as described by Werb & Chin (I983) andavidin-biotin-peroxidase complex (ABC)staining following the manufacturer'sinstruction. Rabbit anti-AEF Igs was used asthe primary antibody in IFA (I:5) and ABC(I:80) assays; for immunofluorescence mic-roscopy fluoresceinated goat anti-rabbit IgGF(ab'2) fragment was used as the immuno-conjugate.

Urea-SDS-PAGE

Urea-sodium dodecyl sulphate-polyacryla-mide gel electrophoresis was carried outaccording to Laemmli (1970) with minormodifications. The sample treatment buffercontained urea (6 M deionized urea), i% SDS,and I% 2-mercaptoethanol. The gel wasstained with either O.I25% Coomassie blueR-250 or silver staining kit.

Immunodiffusion

Ouchterlony test was carried out using I%agarose in barbital buffer, pH 8.6.

Normal and alveolar hydatid cyst-infectedmouse sera

CS7BL/6J mice, infected i.p. with 50 or 250cysts develop splenic amyloidosis at 6 weeksand 6 days p.i. Sera from 250 cyst-infectedmice were obtained at i and 4 weeks p.i. andat 8 and I2 weeks p.i. from the 50 cyst-infected group. These sera were used for thedetection ofcirculating AEF (Abankwa & Ali-Khan I988b). Sera from I0 to I2-months-old retired female mice were used as normalcontrols.

Results

Extraction and purification of AEF

In a previous report, we suggested O.OI Mphosphate buffer containing 0.5 M NaCl, pH7.8 to be an acceptable buffer for the extrac-tion and purification ofAHC-induced hepaticand splenic AEF (Abankwa & Ali-Khan198 8b). The present procedure utilized 0.3 MKCI (PBK-8) in the extraction buffer followedby exhaustive dialysis of the crude AEFagainst PB-6. This latter step had two mainadvantages. First, most but not all of the AEF(extracted in PBK-8) aggregated on dialysisagainst PB-6 and separated from most of thesoluble mainly non-AEF proteins in the

6og

K. Alizadeh-Khiavi & Z. Ali-Khan

crude AEF (Table i). Secondly, the aggre-gated protein retained its AEF activity andwas readily soluble in a smaller volume ofPBS-8 so as to be suitabe for further purifica-tion.

Both the PB-6 aggregated AEF and itssupernatant were fractionated on a Sepha-dex G-200 gel column. The solubilized aggre-gated AEF resolved into two distinct fractions(Fig. i). Both the Vo fraction and theretarded fraction (Vo x 2.2) showed equallypotent AEF activity in the mouse bioassay(Table i). The Vo fraction contained approxi-mately 75% of the eluted protein. As indi-cated above the PB-6 supernatant hadretained some AEF activity. On fractionationonly the retarded fraction (Vo X 2.2), as

suspected, showed some residual AEF acti-vity (Table i). This indicated that most of theAEF had aggreated after PB-6 dialysis. On a

BioGel P-6o gel column, the solubilizedaggregated AEF showed a similar elutionprofile as in Fig. i and after concentrationboth these fractions showed AEF activity(results not shown). Further purification ofthe lyophilized Sephadex G-200 first (AEF-i)and second (AEF-2) fractions was carried outon a Benzamidine-Sepharose gel column;approximately 15% of the total protein pre-

sent in AEF-i (30 mg) and AEF-2 (40 mg)bound to the gel. Both the unbound andbound (AEF-BZ) fractions contained AEFactivity. Although i mg of AEF-BZ proteininduced relatively less splenic amyloid thanthe crude AEF, three of three mice respondedto AEF-BZ induced rapid amyloid deposition(Table i). Based on protein estimation, theAEF-BZ fraction containined approximately0.25% of the total protein present in thecrude AEF (Table i).

Table i. Summary of purification of amyloid enhancing factor (AEF) fromamyloidotic livers and spleens obtained from alveolar hydatid cyst (AHC)-infectedC5 7BL/6J mice. AEF preparations at each step were tested for their bioactivity;mice were injected with i mg ofprotein i.p. and 0.5 ml of2% AgNO3 s.c. and 72 hafter their Congo red stained spleen sections were graded for amyloid deposition

Amount of proteininjected to mouse Number of mice Microscopic

Step Procedure (mg) positive for AA/total gradingt

I Crude extract I.0 3/3 3 +2 Supernatant I.0 3/3 I +

from low-saltprecipitation

3 Solubilized I.0 3/3 3 +low-saltsediment (SLSS)

4 ist pk. from I.0 3/3 3+SLSS SephadexG-200

5 2nd pk. from I.0 3/3 2+SLSS SephadexG-200

6 Benzamidine- I.0 3/3 I +Sepharose 6Beluate

* Approximate circumferential area offollicle containing amyloid deposits: i +,I0-25%; 2 +, 25-50%; 3 +, 50-I00%.

6I0

Characterization of AEFSDS-PAGE analysis

On SDS-PAGE, AEF-i and AEF-2 resolvedinto 33 to 35 and at least io peptide bands,respectively. AEF-BZ showed two prominentpeptide bands between 43 and 66 kD andthree minor low molecular weight peptides(Fig. 2) suggesting that Benzamidine-Sepharose gel binds to non-AEF proteins aswell.

Immunodiffusion test

The unabsorbed anti-AEF Igs developed 3-4precipitin bands against AEF-2, sera from the

Mol. wt

66 200 -

43 000-

AHC-infected mice (I, 4, 8 and 12 weeks p.i.)and old retired normal mice (NMS). Afteraffinity purification on agarose gel conju-gated to normal mouse serum the anti-AEFIgs showed only one common precipitinband between AEF-2 and the sera from NMSand AHC-induced mice (Fig. 3). Since we andothers have shown that sera from old andamyloidotic mice contain AEF activity (jani-gan & Druet i968; Abankwa & Ali-KhanI988b), the common precipitin bandbetween these sera and AEF-2, appears torepresent monospecific antibody againstAEF; this was further confirmed by passivetransfer experiments.

-43 000

-25 700

-18400

6 5 4 3 2 1

Fig. 2. Two different runs of amyloid enhancing factor (AEF) factors on 12% (lanes i and 2) and IO%(lanes 3-6) polyacrylamide gels: (i) Sephadex G-200 first fraction; (2) Sephadex G-200 second fraction;(3,4) Benzamidine-Sepharose bound Sephadex G-200 second fraction; (5,6) Benzamidine-Sepharosebound Sephadex G-200 first fraction. Note the two prominent peptides between mol. wts 43 ooo and66 ooo.

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K. Alizadeh-Khiavi & Z. Ali-Khan

Fig. 3. Coomassie blue-stained immunodiffusion plates showing the common precipitin band developedbetween the normal mouse serum (I); sera from alveolar hydatid cyst infected mice at one (2), four (3),eight (4) and twelve (5) weeks. Sephadex G-200 second AEF fraction (6); see Fig. i. The central wellcontained: a, unabsorbed rabbit-antiamyloid enhancing factor Igs or; b affinity purified monospecificrabbit anti-AEF Igs.

Biological activity of anti-AEF serum

Table 2 shows the results of passive transferof anti-AEF Igs on the amyloidogenic activityof casein- and AHC-induced AEF. Based ondose-response studies (see Materials andmethods), 0.2 5 mg ofprotein in both the AEFpreparations was chosen for passive transferexperiments; this dosage induced amyloiddeposition in at least 50% of the splenicfollicles. Passive transfer of o. i ml of anti-AEF Igs did not inhibit splenic amyloiddeposition against either AEF preparation;the o.5 ml dosage induced 80% reductionand i ml, similar to i ml of unabsorbed anti-AEF serum, completely abolished the acce-lerated amyloid deposition bioactivity ofboththe AEF preparations. Passively transferrednormal rabbit IgG (o.9-9 mg protein) did notaffect the biological activity of either AEFpreparation.

Immunofluorescence and ABC assays

Cytocentrifuged spleen and peritoneal cellsfrom normal and AHC-infected amyloidoticmice were used in immunofluorescence(IFA) and ABC assays using affinity purified

anti-AEF Igs as the primary serum. Fourdistinct cell types from both normal andamyloidotic mice: monocytoid cells, granulo-cytes (most probably neutrophils), mast cellsand a small number of lymphocytes, showedcytoplasmic granular staining (Fig. 4).Figure 4a and b show typical staining reac-tion of leucocytes from amyloidotic mice inIFA and ABC assays. Both the number ofpositive cells and the intensity of stainingreaction were unmistakeably and signifi-cantly higher in the spleen and peritonealcells from amyloidotic mice; approximatelyioo% of the monocytoid cells and granulo-cytes gave an intense staining reaction.Between 5 and io% of the monocytoid cells,especially the large macrophages, from nor-mal mice stained as intensely as those fromthe amyloidotic mice (Fig. 4).

Protease activity ofAEF-BZ and effect ofenzymeinhibitors

Figure 5 and Table 3 show the in vitroproteolytic activity of AEF-BZ against Hidepowder azure with or without PMSF or p-CMB in the incubation mixture. AEF-BZ

612

Characterization of AEFTable 2. Amyloid inhibitory activity of passively transferred affinitypurified rabbit anti-AEF Igs on the bioactivity of alveolar hydatid cyst(AHC)-induced and casein-induced amyloid enhancing factor (AEF).Mice received i.p. various amounts of anti-AEF Igs on day - i; on day othe mice were challenged i.p. with 0.25 mg AEF and 0.5 ml of 2%AgNO3 s.c. and sacrificed after 72 h. Congo red stained spleen sectionswere used for grading AA deposition

0.2 5 mg of AHC- induced 0.2 5 mg of casein-inducedAEF AEF

number of Number ofmice positive Microscopic mice positive Microscopicfor AA/total gradingt for AA/total grading

Affinity o. I 2/2 3 + 2/2 3 +purified 0.5 2/3 1+ 2/2 1+anti-AEF I.0 0/3 0/3Igs (ml)

Normal* I.0 3/3 3 + 3/3 3 +rabbitIgG (ml)

6I3

* DEAE-Cellulose purified normal rabbit IgG containing 9 mg ofprotein/ml.

t Approximate circumferential areas of follicle containing amyloiddeposits: I+, I0-25%; 3+, 50-I00%.

b

Fig. 4. Cytocentrifuged peritoneal cells from alveolar hydatid cyst infected mouse (a) and (b) and normalmouse (c). Affinity purified monospecific (see Fig. 3) rabbit anti-amyloid enhancing factor Igs was used asthe primary serum and fluoresceinated goat anti-rabbit F(ab')2 was used for staining (a) and (c) andavidin-biotin-peroxidase complex for (b). Note intense cytoplasmic granular straining of macrophages,monocytes, granulocytes and a small number of lymphocytes from the infected mouse; much reducedstaining activity is present in (c).

K. Alizadeh-Khiavi & Z. Ali-Khan0.9

0.8 -

0.7 -

EE 0.6 -

LO)

a)e 0.5 -

CD0~~~~D0.4 -

0

0.3 -

0.2-

0.1ls

100 200 300 400 500

jig AEF-BZ/ml in incubation mixture

Fig. 5. In-vitro protelytic activity of Benzamidine-Sepharose gel eluted amyloid enhancing factor (AEF-BZ) against Hide powder azure. Note absence of proteolytic activity in the presence of io mM ofphenylmethylsulphonyl fluoride (.) or 20 mM p-chloromercuribenzoic acid (--).

Table 3. In vitro proteolytic activity of Benzamidine-Sepharose geleluted amyloid enhancing factor (AEF-BZ) on Hide powder azure andinhibition of its proteolytic activity after incubation with variousconcentrations of phenylmethylsulphonyl fluoride (PMSF) and p-

chloromercuribenzoic acid (p-CMB); the activity ofdimethylsulphox-ide (DMS0) treated AEF-BZ against the substrate was taken as I00%

Concentration AEF-BZTreatment (mM) (mg) O.D. Percentage inhibition*

PMSF 20 0.5 o.oI 99.98I0 0.5 0.02 97.952 0.5 o.658 32.52

p-CMB 20 0.5 0.046 95.292 0.5 0.549 43.70

0.2 0-5 o.699 28.3 I

DMSO 0.5 0.975 0

* Percentage inhibition of AEF activity was calculated from theO.D. of reaction product generated with DMSO treated AEF againstHide powder.

proteolysed the Hide powder azure in a dose- complete abolition of its proteolytic potential.dependent manner; pretreatment of variable Similar results were obtained when variableamounts of AEF-BZ (60-500 Yg) with either amounts of the enzyme inhibitors were usedI0 mM PMSF or 20 mM p-CMB resulted in against a constant amount of AEF-BZ (Table

6I4

Characterization of AEF3). The percentage inhibition of proteolysiswas calculated against the O.D. of the reac-

tion product generated by AEF-BZ againstthe substrate in the presence of DMSO only.

Since p-CMB is toxic to mice, only PMSF-treated AEF-BZ was used in the mousebioassay to assess the effect of PMSF on theamyloidogenic potential of AEF-BZ. Asshown in Table 4, I0 mM PMSF treated AEF-BZ failed to induce splenic amyloid deposi-tion in mice; AEF-BZ treated with DMSO onlyretained its amyloidogenic potential.

Discussion

Experimental secondary amyloidosis is a

biphasic process and involves a primary anda secondary phase (Sipe et al. I978). Theprimary phase is characterized by increasedsynthesis and secretion of interleukin-i, pri-marily by activated macrophages, rapidelevation of hepatocyte synthesized serum

amyloid A protein (SAA) and hyperplasticchanges in the reticuloendothelial cells insplenic marginal sinsuses, hepatic sinusoidand renal glomeruli (Hardt & Ranlov I976;Vogel & Sipe I982). Despite intense inflam-mation and significantly elevated levels ofSAA, amyloid deposition does not occur intissues during the primary phase (KisilevskyI983; Ali-Khan et al. i988a). Amyloid (AA-protein) deposition occurs, either slowly or

rapidly, during the secondary phase (Kisi-levsky et al. I983).However, the precise pathogenetic mecha-

nism that leads to the generation of AA-likepeptides in vivo, presumably by proteolyticcleavage of the carboxy terminus of SAA, isunknown (Levin et al. I972; Ericksen et al.I976). It is believed that under normalconditions the interstitially deposited parentprotein of amyloid is catabolized and clearedfrom the tissue by proteases released fromendothelial cells. In vitro evidence for such a

mechanism has been provided by incubatingnormal blood monocytes or Kupffer cellswith purified SAA (Lavie et al. I978; Skogenet al. I980; Fuks & Zuker-Franklin I985).

Conversely, defective monocytoid cellfunction was suggested to be involved in the'faulty processing' of SAA, that is, in thegeneration of AA-like peptides, instead of itscomplete degradation. Membrane bound ser-

ine esterases (Lavie et al. I978; Fuks &Zucker-Franklin I 98 5), cell free supernatantof cultured monocytes (Skogen et al. I980),different serum serine proteases (kallikreinand thrombin) (Skogen & Natvig I98 I), andenzymes released by zymogen-activated gra-nulocytes which were sensitive to specificinhibitors of serine and acid proteases (Sil-verman et al. I982), have been implicated inthe generation of AA-like peptides. These invitro observations raise a number of funda-

Table 4. In vivo inhibition of the bioactivity of Benzamidine-Sepharose gel elutedamyloid enhancing factor (AEF-BZ) after incubation with dimethylsulphoxidesolubilized phenylmethylsuphonyl fluoride (PMSF). Mice were injected i.p. withthe PMSF-treated ABF-BZ and s.c. with 0.5 ml of 2% AgNO3. After 72 h, Congored spleen sections were graded for amyloid (AA deposition)

AEF- Number ofioMm DMSO BZ mice positive Percentage of follicle Microscopic

Group PMSF (M1) (mg) for AA/total positive for AA grading*

I PMSF 8o I 0/3 0 02 8o I 3/3 10.5 I +

* Approximate circumferential area of follicle containing amyloid deposits:I +, I0-2 5%.

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K. Alizadeh-Khiavi & Z. Ali-Khan

mental questions about the role of leucocyteor endothelial cell derived proteases in thepathogenesis of amyloidosis. Does 'faultyprocessing' of the parent protein indicate thepresence of a site specific protease in theinterstitial milieu which cleaves the SAAmolecule to generate amyloidogenic AA-likepeptide? If so, then such a protease; (a) ismost likely to appear at the interphasebetween the preamyloidic and amyloidoticphases; (b) should be present in sufficientamounts to commence amyloidotic phasethroughout the target organ and; (c) finally,its tissue concentration should progressivelyincrease in amyloidotic organs so as tooutpace its neutralization by natural pro-

tease inhibitors (Fritz et a). I986). AEFappears to fulfil all of the above criteria(Kisilevsky I983; Abankwa & Ali-KhanI988a, b). Kisilevsky (I983) in discussingthe pathogenesis of amyloidosis, deductivelyargued that macrophage or reticuloendothe-lial cell (RE cell) aberration, probably at thesite of SAA clearance, and the presence ofREcell derived glycoprotein in preamyloidotictissues, termed AEF, were at least the twomain prerequisites for amyloid depositionduring persistent inflammation.

Based on the available information on

AEF, i.e. its presumed cellular origin, pro-gressive increase in tissue and body fluidsduring chronic inflammation and its acceler-ated amyloid enhancing potential across

species (Varga et a]. I986; Ali-Khan et al.I 988b), we postulated that AEF might repre-sent lysosomal proteases derived fromincreased numbers of activated inflamma-tory or RE cells (Abankwa & Ali-KhanI988a, b). Amyloidogens such as casein,AgNO3 or AHC induce intense and persistentinflammation and hyperplasia of RE cells inthe host (Kazimierczak I969; Hardt & Ran-lyv 1976; Kisilevsky et al. 1977; Ali-Khan1978; Ali-Khan & Siboo I980; Treves & Ali-Khan I984; Fuks & Zucker-Franklin I985).

In addition, there is mounting evidence inthe literature which indicates that vigorousinflammatory reaction induced either by

chemical stimulators, immune complexes orpathogens radically alters the biochemicaland functional parameters of activatedmonocytoid cells; accelerated extracellularsecretion of lysosomal proteases and othercellular proteins by such cells may measureup to I5% of their total protein synthesis(Schnyder & Baggiolini 1978; Ragsdale &Arend I979; Werb I983; Werb & ChinI983). These observations along with ourprevious (Abankwa & Ali-Khan I988a, b)and current findings reinforce the hypothesisthat AEF may indeed be a lysosomal proteaserelease from degenerating and/or activatedinflammatory and RE cells. This, however, isnot a novel concept; releasates from RE cells,macrophages and neutrophils have pre-viously been either suspected or associatedwith AEF activity (Kedar & Ravid I980;Kisilevsky I98 3).

Using the AHC-infected mouse model ofinflammation-associated amyloidosis wehave shown AEF activity in: (a) sera fromAHC-infected mice; (b) extracts preparedfrom whole spleen cells or fractionated peri-toneal macrophages and; (c) culture super-natants from spleen cells stimulated withlipopolysaccharide, Concanavalin A or AHC-antigen (Abankwa & Ali-Khan I988a, b).Furthermore, phenylmethylsulphonyl fluor-ide (PMSF) treatment of AEF completelyabolished its bioactivity in vivo and partialinhibition was induced by e-aminocaproicacid, aprotinin and iodoacetic acid.We suggested that AEF might be an

inflammatory cell derived serine or a thiolprotease. The current data confirms andextends these observations and describethree major steps in the characterization ofAEF: (a) partial purification of AEF; (b)evidence for its enzymatic nature and; (c)generation of biologically active antibodyagainst AEF, in order to determine its cellularorigin. Until this study, none of these objec-tives, especially the latter, had beenachieved. Initial extraction of AEF in 0.3 MKCI (PBK-8) followed by exhaustive dialysiseither in PB-6 or distilled water (data not

6I6

Characterization of AEFshown) was an important step in whichapproximately 90% of the unwanted solubleprotein was separated from the aggregatedAEF (Table i). Reasonably uncontaminatedand probably monomeric AEF which elutedin the retarded peak, AEF-2 (Vo x 2.2) wasthen fractionated on Benzamidine-Sephar-ose gel. The eluted fraction (AEF-BZ) showedAEF activity in vivo and on SDS-PAGEresolved into two prominent peptidesbetween 43 and 66 kD and several minorpeptides of lower molecular weights indicat-ing that AEF is a small molecular weightpeptide of under 66 kD (Fig. 2). Because ofthe tendency of the AEF molecule to aggre-gate under physiological conditions in tissueextracts and bind to particulate componentsreports on its molecular weight have rangedbetween I 5 and over Ioo kD (Keizman et al.I972; Axelrad et al. I982; Hol et al. I985).Furthermore, contrary to our expectation,amyloidogenic potency of i mg of AEF-BZ,which was eluted with o.oi M HCl, pH 2.0,from the Benzamidine-gel, was biologicallyless potent than the crude AEF preparation(Table i). Axelrad et al. (I 982) have reportedabolition of AEF activity after exposure to pH3.0 or 9.5. We suspect that exposure of AEF-2 to 0.01 M HCI might have partially affectedthe bioactivity of AEF-BZ. Benzamidine-gelthus appears to be less than suitable for thepurification of AEF.

Monospecific anti-AEF antibody developedone common precipitin band between AEF-2, and AEF-positive AHC-infected and nor-mal mouse sera (Fig. 3) and on passivetransfer induced, in a dose dependent man-ner, partial to total inhibition of AA deposi-tion against homologous (AHC-induced) orheterologous (casein-induced) AEF (Table2). How this is brought about is a matter ofspeculation at this stage ofour investigation.A simple explanation would be the neutrali-zation ofAEF prior to its biological activity invivo. The passive transfer experiments, how-ever, show unequivocally the presence ofspecific anti-AEF antibody in our antiserumpreparation. The common precipitin banddeveloped between AEF-2 and the AHC-

infected mouse sera (Fig. 3) further confirmsprevious observations of circulating AEF inamyloidotic mice (Janigan & Druet i968;Abankwa & Ali-Khan i 988b). Sera from thenormal retired mice also showed the com-mon band suggesting the presence of circu-lating AEF. As to the cellular origin andbiochemical nature of AEF, results fromimmunostaining (Fig. 4) and in vitro and invivo studies with and without enzyme inhibi-tors (Tables 3 and 4; Fig. 5) clearly suggestthat AEF is an intracellular, probably alysosomal protease, common to at least fourtypes of leucocytes. Its increased tissue andserum AEF 'titre' during chronic inflamma-tion or amyloidosis might thus representincreased intracellular and, as a result ofexocytosis, extracellular concentrations ofthis protease. Nonspecific lysosomal esterasehave been shown in T-lymphocytes, mono-cytoid and RE cells and mast cells (Li et al.19 73; Woodberry et al. I978; Treves & Ali-Khan I983).AEF is found in measurable amounts in

the sera of casein- or AHC-induced amyloi-dotic mice (Janigan & Druet I968; Abankwa& Ali-Khan I988b). Hol et al. (I985) mea-sured lysosomal hydrolases in the sera ofcasein-treated hamsters and found a signifi-cant increase in cathepsin D and ,B-N-gluco-saminidase during the amyloid depositionphase. They correlated these findings withsecretion of lysosomal enzymes from acti-vated macrophages and concluded thatthese enzymes might be involved in extracel-lular degradation of SAA and formation ofAA. Whether AEF-BZ, as shown here,cleaves SAA and generates AA like peptidesin vitro remains to be shown. However, basedon our in vivo studies using various specific(p-CMB,PMSF) and nonspecific protease in-hibitors (aprotinin, iodoacetic acid, s-amino-caproic acid), we tentatively suggest thatAEF might be a trypsin-like serine or a thiolprotease (Gold I965; James I978; WerbI98I). Pretreatment of AEF with PMSF,which is a serine and thiol protease inhibitor,completely blocked its bioactivity in vivo andsimilar results were obtained with PMSF and

6I 7

6I8 K. Alizadeh-Khiavi & Z. Ali-Khan

p-CMB in vitro against Hide powder (Tables 3and 4). p-CMB, however, is a specific inhibi-tor of thiol proteases at an effective concen-tration of i mMi (Werb I98I). Because of thetoxic nature ofp-CMB, we were unable to testthe bioactivity of p-CMB-treated AEF in vivo.Studies are in progress to further purify AEFand classify it using various synthetic pro-tease inhibitors.

Acknowledgement

This work was supported by a grant (MA-92 52) from the Medical Research Council ofCanada.

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