Brit. J. Ophthal. (I975) 59, 670

Mooren's ulcerHistopathology and proteolytic enzymes of adjacent conjunctiva

STUART I. BROWNFrom the Department of Ophthalmology, University of Pittsburgh School of Medicine andEye and Ear Hospital, Pittsburgh, Pennsylvania

It was reported in 1971 that the conjunctivaadjacent to Mooren's ulcers exhibited collageno-lytic activity (Brown, 1972). More recently, bothnormal and rabbit conjunctiva inflamed by Freundadjuvant was shown to produce a collagenase thatwas typical of earlier reported mammalian collage-nase (Bloomfield and Brown, 1975).The limbal conjunctiva adjacent to Mooren's

ulcers has been excised, both as a treatment and inorder to examine the histopathology, ultrastructure,and proteolytic enzymes of the tissue (Brown,1975). The results of the pathological examinationare presented in this paper.

Methods

FIXATION FOR EXAMINATION WITH LIGHT AND

ELECTRON MICROSCOPE

The comeal and conjunctival specimens were firstfixed in 3 per cent glutaraldehyde in 0-2M sodiumcacodylate buffer (pH 7.4) and then fixed with 2 per centosmium tetroxide in 0-2M sodium cacodylate and em-

bedded in Epon. Thick sections of the epoxy-embeddedtissue were stained with basic fuchsin and alkalinizedmethylene blue and then examined under the lightmicroscope. Thin sections were cut with an LKB-3microtome using a diamond knife, mounted on coatedcopper grids, stained with uranyl acetate and leadcitrate, and examined with an AEI EM-6B at 6o kV.Excised conjunctiva from three additional eyes fromtwo patients were also examined.

HARVEST OF PROTEOLYTIC ENZYMES

The excised conjunctiva was cut into approximately2 X 2 mm pieces, washed with a mammalian Tyrode'sstreptomycin solution, and placed in sterile organ culturedishes. The culture media were prepared from equalamounts of Tyrode's solution and Dulbecco's modifiedEagle's medium with ioo mg/l streptomycin sulphate,

Supported in part by National Eye Institute Grant No. EYoI490

Address for reprints: S. I. Brown, MD, Department of Ophthal-mology, Eye and Ear Hospital, Pittsburgh, Pennsylvania 15213, USA

and were equilibrated with 95 per cent oxygen and 5 percent carbon dioxide before use. The tissues were incu-bated at 370C in a humidified chamber for 6 days with areplacement of medium every 2 days. The unconcen-trated, harvested media were frozen and stored.

RADIOACTIVE COLLAGEN PREPARATION

Radioactive collagen was prepared from the skin ofguinea-pigs after intraperitoneal injection of Ioo ItCiglycine ,_C14, 6 and 12 h before death (Gross, 1958).Solubilized collagen was eluted from the skin witho04M NaCl and purified by repeated salt precipitation.After dialysis, the collagen was lyophilized and stored at-20oC. The specific activity of the labelled collagen was20 000 disintegrations per min/mg protein.

TISSUE CULTURE ASSAY FOR COLLAGENOLYTICACTIVITY

The excised pieces of conjunctiva and cornea werecut into pieces approximately 2 X 2 mm. They were thendipped in a tissue culture medium and placed on rigidopalescent collagen gels in tissue culture plates with aninside diameter of 3-5 cm. Eight similarly preparedpieces of normal human conjunctiva were removed withdonor eyes from deceased humans within 4 h of death.The plates were incubated at 370 C. The method andpreparation of the collagen has previously been des-cribed (Brown, Weller, and Wasserman, i969).

COLLAGENASE ASSAY

Plastic conical tubes containing 0-2 ml 0-2 per centglycine C14 collagen solution which was trypsin resistantpH 7-6, were incubated at 370 C for IS h to facilitategelling. The fractions to be assayed for collagenaseactivity were applied to the gels. The tubes were thenincubated for 24 or 48 h in a moist atmosphere at 370C.After incubation, o-i ml distilled water was added andthe tubes were centrifuged at 44000 g for io min.Aliquots of the supematant (o025 ml) were pipetted intovials containing glass filter papers and dried ovemight.The radioactivity was counted in a liquid scintillationcounter in I-O ml POPOP-I, 4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene, PPO-2, 5-di-phenyloxazole,and toluene mixture.

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Mooren's ulcer 671

INHIBITION OF THE HARVESTED COLLAGENASE

Cysteine io-2M, Na2EDTA io-3M, and freshly pre-pared rabbit serum I:0OO were separately added to thecrude collagenase harvest preparation and assayed forcollagenase activity.

PROTEOGLYCAN PREPARATION

Proteoglycans were extracted from rabbit corneas with4-oM guanidine chloride. (Fractions high in uronic acidcontent were obtained through the courtesy of Dr JohnD. Gregory of Rockefeller University, New York, NewYork.)

VISCOMETRY

Viscometry was used to indicate both collagen andproteoglycan breakdown. The viscometry measure-

ments were made in Ostwald viscometers with waterflow time of 49 and 53 S at 370 C.The preparation of proteoglycan and the viscosity

measurements were carried out as previously described(Brown, Hook, and Tragakis, 1972; Brown, Bloomfield,and Tam, 1974). In brief, the proteoglycan substratewas prepared by adding o0osM acetic acid containing0oI5M NaCl, pH 3.5, or mixed with o0osM Tris-HClbuffer, with o0IsM NaCl, pH 7-4. Measurements ofproteoglycan breakdown from crude and NH4SO4precipitated harvest media were made over a 3-hperiod. The initial viscosity (Nsp) was approximately4-0.

In order to relate the breakdown of proteoglycan withchanges in viscosity, a diffusion apparatus was construc-ted consisting of two plastic chambers separated by a

millipore filter of 0-2 micron size. This is similar torecently reported diffusion vessels which permitteddiffusion of chondroitin sulphate but not the largerproteoglycan across the millipore filter (Brown andothers, 1974; Weissmann and Spilberg, I968). Measure-rments of uronic acid (hexuronate) in the 'output'chamber indicated the amounts of proteoglycan de-graded (Brown and others, 1972; I974).Comeal proteoglycan o-9 per cent in 2 ml of o0osM

Tris-HCl with o IsM NaCl and 5 [tg of the harvestmedia were placed in the input chamber. In the outputchamber were placed 7 ml of o-05M Tris-HCl witho0 IM NaCl. The diffusion vessel was then incubatedat 370 C for4 h. The uronic acid content of the outputchamber was estimated hourly by the carbozole reaction.The controls for this were two additional diffusion vesselswith the same contents except that one contained onlyTyrode's medium and was dialysed against o-05M Tris-HCI, pH 7.I, with 5mN CaC12, and in the other, theharvest media were added after it had been warmed at800 C for I5 min. This technique was identical to thatused in an earlier study of the enzyme of Pseudomonasaeruginosa (Brown and others, 1974).The collagen substrate was prepared and optical

rotation and viscosity measurements carried out as

previously reported (Brown and others, 1972; Hook,Brown, Iwanij, and Nakanishi, 1971). Control viscosityof only the collagen solution was run simultaneously

with the viscosity of the reaction mixture. The collagenbreakdown products were analysed by polyacrylamidegel electrophoresis (Nagai, Gross, and Piez, 1969).

Results

LIGHT AND ELECTRON MICROSCOPY

Histological examination of the host's excised,ulcerated comea revealed many lymphocytes andpolymorphonuclear leucocytes. The conjunctivalepithelium was intact in most sections while thesubepithelial tissues were packed with plasmacells and only an occasional polymorphonuclearleucocyte or monocyte (Fig. I). Examination ofthe conjunctiva with the electron microscopecorroborated the cell types and showed substantialamounts of endoplasmic reticulum (Fig. 2).Histological examination of the remaining threespecimens of conjunctiva showed similar findings-that is, concentrations of plasma cells and varyingamounts of lymphocytes. The specimens examinedfrom the eye that had multiple excisions of con-junctiva showed relatively fewer cells.

TISSUE CULTURE ASSAY FOR COLLAGENASE

Growth of the excised conjunctiva from the eyeswith Mooren's ulcers on collagen gels resulted intotal lysis of the whole gel within 48 h in allspecimens. When IO similar sized pieces of humanconjunctiva excised from essentially normal eyeswithin 6 h of death of the donor were grown onsimilar gels, gel lysis of 6Io-Io0o was noted in threespecimens.Growth of the excised conjunctiva from eyes

with Mooren's ulcer on collagen gels containingeither cysteine, NaEDTA, or serum did not pre-vent gel lysis.

COLLAGENASE ASSAY WITH RADIOACTIVE COLLAGEN

The crude, unconcentrated harvest media com-pletely lysed the radioactive collagen gels. Thisgel breakdown was inhibited 50 per cent byNaEDTA Io-3M and not at all by cysteine 10-2Mor serum I:IOO. When the volume of the harvestmedia added to the gels was halved, its collageno-lytic activity was completely inhibited by serumI :IOO, 9I per cent NaEDTA, and 72 per cent bycysteine (Table).

VISCOMETRY

The specific viscosity of a solution of collagen andundiluted crude harvest media was reduced to 6oper cent after 50 min. A control run simultaneouslywithout the harvest media did not show any signifi-

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672 British Journal of Ophthalmology

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FIG.I Histological appearance of excised conjunctiva adjacent to Mooren's ulcer. Stroma almost filled with plasma cells

cant reduction in viscosity for up to go min (Fig. 3).The optical rotation readings were all between-425 and -430.

PROTEOGLYCAN

The viscosity of the reaction mixture of proteo-glycan and the harvest media fell quickly and at auniform rate to almost zero within 30 min.At the end of the 4-h incubation period there

was 7 [±g/ml hexuronate released into the outputchamber of the control diffusion vessel without the

Table Inhibitors of collagenase of Mooren's ulcer

Collagen lysis Inhibition(cpm) (%)

Control 1090±52* 0

Cysteine IO 414±1I5 72Cysteine io-3M 545±30 50Na2EDTA io-3M 51±IO 9ISerum I:Ioo 0 100

*Standard error of mean

active fraction, IO jig/ml hexuronate in the outputchamber of the control vessel in the pre-heatedfraction, and 57 Fg/ml hexuronate released intothe output chamber in the vessel with the activefraction. This shows that comeal proteoglycan isdegraded by the harvest media, that heat inactivates

90-

0.80-

70-

060

50 * Controlo Enzyme

0 1 2 3 45 6Hours

FIG. 3 Viscosity measurements of reaction of mixture ofconjunctival harvest media with solution of collagen

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q

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Mooren's ulcer 673

FIG. 2 Electron microscopic appearance of tissue in Fig. I, showing plasma cells with abundance of endoplasmic reticulum

the enzymatic activity of the media, and sub-stantiates the relationship of fall in viscosity tosubstrate degradation.

ANALYSIS OF COLLAGEN BREAKDOWN PRODUCTS

Polyacrylamide gel electrophoresis of the reactionmixture showed collagen breakdown productsthat were typical of mammalian collagenase (Fig. 4).

DiscussionThe results of the investigation of the excisedhuman tissues with Mooren's ulcer showed thattissue culture of the comeal epithelium, theulcerated corneal stroma, and the limbal conjunc-tiva on collagen gels resulted in breakdown of thesegels. The collagenolytic activity by the conjunctiva

was further indicated to be the result of a truecollagenase since the conjunctival harvest mediacaused the characteristic limited reduction inviscosity of a collagen solution without a change inits optical density. The typical collagen breakdownproducts exhibited by polyacrylamide gel electro-phoresis was the final proof for the presence of acollagenase.The collagen gel breakdown by the conjunctiva

surrounding Mooren's ulcer was at least IO timesmore than the breakdown caused by a similarquantity of any of the diseased corneal tissuespreviously investigated by the author. Furtherindications of the relatively large amounts of thecollagenase produced by growth of the conjunctivawas that the crude harvest media did not have to beconcentrated to effect significant collagen break-down. In fact, they had to be diluted before they

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674 British Journal of Ophthalmology

I-Ma ~~~FIG. 4 PolYacrYlamide gelelectrophoretic patterns of

A collagen degraded byp conjunctival collagenase.

A. Collagen controlA B. Effect of conjunctival

collagenase.single polypeptide chain(s);

X cross-linked dimers of chains

A B

could be inhibited. Once diluted, the crude enzymewas completely inhibited by NaEDTA and serum.The enzyme was almost, but not completely in-hibited by cysteine, probably because the enzymewas still too concentrated to be inhibited by cysteine.This hypothesis was not tested because of thelimited supply of the enzyme. For the same reasonthe enzyme was not further characterized and com-pared with rabbit conjunctiva collagenase.The harvest media from Mooren's ulcers also

quickly degraded comeal proteoglycan which is a

major component of the ground substance of thecorneal stroma. This is significant since a recentstudy showed that corneal proteoglycan protectscollagen from breakdown by collagenase indicatingthat, in order for collagenase to attack collagenfibrils in vivo, the proteoglycan must first bedegraded (Nagai and others, 1969). Currently, theonly cell known to contain a protease capable ofdegrading proteoglycan at a neutral pH is the poly-morphonuclear leucocyte PMN (Brown and others,1972; Weissmann and Spilberg, I968). However,there were very few PMN found in the excisedspecimens of conjunctiva.

It was interesting that each of the four specimensof conjunctiva examined histologically had un-usually large numbers of plasma cells. The signifi-cance of this finding is not clear at present. It maybe that these cells are the source of proteolyticenzymes, activators of precursors of enzymes, orfinally and more probably, the source of substanceswhich induce other cells to produce enzymes. Thepresence of these cells in the conjunctiva suggestedthat they were part of an immune or an auto-immune phenomenon. However, repeated attemptsby the author to identify immune globulins in theconjunctiva via fluorescent antibody staining werenegative.

In conclusion, this and the following study,showing that the conjunctiva adjacent to Mooren'sulcers contains large numbers of plasma cells, thatit produces a collagenolytic enzyme and probablya proteoglycanolytic enzyme, and that the ulcersheal after excising this tissue, indicate that theconjunctiva is intimately involved in the patho-genesis of Mooren's ulcers.

References

BLOOMFIELD, S. E., and BROWN, S. I. (1975) Invest. Ophthal., 13, 547BROWN, S. I. (1972) Israel 7. med. Sci., 8, 1537

(1975) Brit. 7. Ophthal., 5g, 675BLOOMFIELD, S. E., and TAM, w. I. (1974) Invest. Ophthal., 12, 174HOOK, C. W., and TRAGAKIS, M. P. (1972) Ibid., iI, 149,WELLER, C. A., and wASSERMAN, H. (I969) Arch. Ophthal., 8I, 370

GROSS, J. (I958) .7. exp. Med., 107, 247HOOK, C. W., BROWN, S. I., IWANIJ, w., and NAKANISHI, I. (1971) Invest. Ophthal., 10, 496NAGAI, Y., GROSS, j., and PIEZ, K. A. (I969) Ann. N.Y. Acad. Sci., 121, 494WEISSMANN, G., and SPILBERG, I. (I968) Arthritis Rheum., iI, I62

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