ENZYMATIC VESICATION IN VIVO

I. EFFECTS OF PAPAIN IN HUMAN SKIN*

ROLF F. MILLER, M.D. AND RICHARD B. STOUGHTON, M.D.

Recent work has indicated that acantholysiscan be induced by enzymatic factors present inhuman skin (1, 2, 3). Mild thermal injury andcantharidin application to human skin in vitrowill induce an enzymatic breakdown of epidermalcells as well as dermal-epiderinal separation.Fairly recent reviews cover current knowledgeof blister formation and cutaneous proteolyticenzymes (4, 5, 6, 7), so the subject will not be re-viewed in detail here.

It has recently been shown that acantholysiscan be induced in vitro in human skin by crystal-line papain and elastase (1) whereas trypsin, ly-sozyme, and a large group of other crystallineenzymes did not induce acantholysis in vitroBecause of the in vitro effects of papain (1) it wasdecided to observe skin of human volunteers in-jected with crystalline papain.

This report concerns the fact that one can in-duce in vivo the following changes by intradermalinjection of the single crystalline enzyme, papain:a) spongiosis and spongiotic vesication, b) acan-tholysis and c) dermal-epidermal separation.

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Papain can be obtained as a crystalline prepa-ration derived from papaya latex. It has beenknown for a long time that partially purified aswell as crystalline derivatives of papaya latexwill hydrolyze many types of synthetic peptidesubstrates (8). Crystalline derivatives of papayalatex also behave as powerful esterases and there-by resemble the animal proteinases trypsin andchymotrypsin, but differ in their requirement ofsulfhydryl groups for activity and in their highresistance to inactivation by di-isopropyl-fluorophosphate (9).

Detailed biochemical studies of crystallinepapain including methods for preparation, activa-tion and inactivation, synthetic substrates, arid

* From the Department of Dermatology, (Dr.RoIf F. Miller, Clinical Instructor of Derma-tology, and Dr. Richard B. Stoughton, Directorof Dermatology), Western Reserve UniversityMedical School, Cleveland, Ohio.

Supported by U.S. Dept. Public Health andWelfare Grant No. A2196

Received for publication February 2, 1960141

composition have been recently published (10).A large number of synthetic substrates for crystal-line papain have been reported. These includea) carbobenzoxyl-L-glutamyl-L-tyrosine, b) car-bobenzoxyglycyl-L-tryptophan, c) benzylarginineamide, d) acetyl-L-tyrosinamide, and e) L-leu-cinamide. The first four of the above are specificsubstrates for papain, carboxypeptidase, trypsin,and chymotrypsin, respectively. Papain will alsohydrolyze other synthetic substrates andobviously has a wide range of peptidase activityas well as esterase activity.

A most interesting biologic effect of papain wasrecorded by Thomas after he observed the collapseof rabbit ears after intravenous injection ofpapain (11). Further experiments (12) revealedthat papain is deposited in the rabbit ear andapparently hydrolyzes the matrix of cartilage andreleases chrondroitin sulfate (13).

This activity of papain is dependent upon freesulfhydryl groups, although crystalline papaincontaining free sulfhydryl groups is inactive whengiven intravenously. Presumably the sulfhydrylpapain is bound before it can be deposited in therabbit ear. Papain can be inactivated in situ byadding sulfhydryl binding agents and it can thenbe reactivated by adding cysteine to the in-activated preparation (12).

Fm. 1. Dermal-epidermal separation one hourfollowing intracutaneous injection of a 1:10 papainsolution. Mild dermal, chiefly perivascular in-filtrate. Hematoxylin and Eosin. X 86.

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142 THE JOuRNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 2. Subepidermal vesicle two hours after intracutaneous injection of a 1:10papain solution. H.and E. X 150.

FIG. 3 Subepidermal vesicle four hours after intracutaneous injection of a 1:10 papain solution H.and E. X 100.

Other than the recent report of acantholysisinduced in human skin in vitro (1) by papain weknow of no reports concerning its direct actionon human skin.

METHODS

The experiments were performed on two maleindividuals 37 and 68 years of age with healthy,morphologieally normal appearing skin. Crystal-line papain with 0.03% cysteine in saline solution

was used. f The enzyme was diluted 1: 10 and 1:3in trihydroxymethylaminomethane buffer, pH7.4. One-tenth (0.1) ml. of the solution was in-jected intradermally with a 26-gauge needle assuperficially as possible into the skin of the back.For controls papain solution of similar concen-trations were used, but were previously heated to1000 C. for 10 minutes.

t Supplied by Worthington Biochemical Labo-ratories, New Jersey

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ENZYMATIC VESICATION mr vivo 143

Papain in 1:10 dilution was injected at fourdifferent sites on the back of one of the two indi-viduals. For histopathologic examination a punchbiopsy under local procaine anesthesia was doneon each site, one lesion being removed each hourcommencing one hour after the injection. Carewas exercised to remove the central portion ofthe lesion with the vesicle intact. Similarly atfour different sites on the back the control solu-

tion was injected. Punch biopsy specimens werealso takeii from the central portions of these de-veloping lesions, at one hour intervals.

The back of the other individual was injectedwith the 1:3 papain solution at three differentsites, and a punch biopsy of one of these threeareas was performed every hour, starting one hourafter injection. The heated papain 1:3 controlsolution was injected at three different sites, and

FIG. 4. Beginning spongiosis throughout the epidermis two hours after intracutaneous injection of a1:10 papain solution. II. and E. X 275.

Fin. 5. Beginning spongiosis within the epidermis, two hours after injection H. and E. X 1100.

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FIG. 6. Marked spongiosis with intraepidermal vesicle formation four hoors followiog intracutaneousinjection of a 1:10 papain solution. H. and E. X 200.

FIG. 7. Three hours following injection of a 1:10 papain solution intracutaneously changes resemblingliquefaction degeneration of the basal cell layer can be seen. H. and E. X 275.

FIG. 8. Beginning subepidermal vesiculation is demonstrated in this section, removed four hoursfollowing injection of a 1:10 papain solution. H. and E. >< 180.

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ENZYMATIC VESICATION IN VIVO 145

these were subsequently biopsied at one hour in-tervals. All specimens from the control and ex-perimental sites were sectioned after 10% forma-lin fixation, dehydration, and paraffin imhcddiog.The sections were stained with hematoxylin andeosin.

RESULTS

A. Papain 1:10 Dilution

All injections produced wheals of approxi-mately 5 mm. in diameter at which site the skinwas blanched. One hour after injection an erythem-atous, slightly elevated wheal of 1j. cm. indiameter had developed surrounding the site atwhich the needle had been inserted. Two hourslater a small vesicle measuring 2 mm. was visiblein the central portion of the wheals. It was notedthat during the observed period of time (up toseven hours) both wheals and vesieles were per-sistent and showed no tendency to fade.

Injections with the heated 1:10 papain solutionwere followed by the appearance of erythematouswheals, which were smaller than those producedby the unheated active enzyme solution and aver-aged em. in diameter. In no instance was avesiele observed. Some of these wheals had al-most completely faded four hours following in-jection.

Although macroseopically a vesicular lesionwas not recognizable on the specimen removedone hour following injection, under the micro-scope a small area of dermal-epidermal separa-tion was visible (Fig. 1). The specimens removed

Fia. 10. Suhepidermal blister, containing fluid and numerous erythroeytes, two hours following in-traeutaneous injection of a 1:3 papain solution. H. and E. x80.

FIG. 9. Wheal on left upper hack with centrallylocated hemorrhagic vesiele 4 mm in diameter.Photograph was taken one hour following in-traeutancous injection of a 1:3 papain solution.

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FIG. 11. Adjacent area to blister shown in fig. 10 with intraepidermal disruption of collagen fibers, H.and E. x 80.

Fjo. 12. Large aeantholytie vesiele four hours following injection (papain 1:3). Epidermis in dissolu-tion. H. and E. X 80.

FIG. 13. Subepidermal separation and beginning acantholysis of epidermis adjacent to vesiele shownin Fig. 12. In addition spongiosis can be seen on the right. H. and E. X 80.

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ENZYMATIC VESICATION IN VIVO 147

2, 3, and 4 hours following injection morpholog-ically presented a vesiele. Again, under the micro-scope the vesiele was seen to he at the dermal-epidernial junction (Figs. 2 and 3). The maximumdegree of dermal—epidermal separation apieare Iover the site of injection. Various degrees of spon-giosis can be seen witlnn the epidermis adaeentto the area of dermal-epidermal separation (Figs.4, 5, and 6). Spongiosis is most marked iii the

fonr-hour specimen. In addition very earlychanges involving the basal cell layer of the epi-dermis can he seen, resembling liquefaction de-generation (Figs. 7 and 8). Dermal changes arecharacterized by slight disruption of collagenfibers and by the presence of a moderate degree ofcellular infiltration consisting mainly of poly-morphonuclear cells, round cells, and many eo-sinophils. In part the infiltrate is perivasenlar in

FIG. 14. Acantholytic epidermal and peculiar vacuolated cells, probably representing sebaceous cellswithin acantholytie blister. H. and E. X 440.

FIG. 15. Acantholytic prickle cells freely floating in blister fluid. H. and E. X 882.

arrangement and this is most marked in an areabelow the epidermal changes. The later the speci-men was removed, the denser the cellular accu-mulation within the dermis. Only minimal de-grees of acantholysis of epidermal cells were seen.

Histologically, the control areas did not showepidermal changes. There was a mild dermal in-filtrate, chiefly perivascular, consisting of roundcells, polymorphonuclcar cells, histiocytes, and avery occasional cosinophil.

B. Papain 1:3 Dilution

One hour following injection of the active en-zyme solution crythematous and markedly ele-vated wheals measuring 2j cm. in diameter haddeveloped and vcsicles were already visible inthe central portions of the lesions at that time.These vcsicles were larger than those producedby the 1:10 papain solution, measuring between3 and 4 mm. in diameter. The lesions were some-what reddish in color and the vesicular fluidseemed to contain fresh blood (Fig. 9).

On histologic examination the macroscopicallyvisible vesiclc is seen to be subcpidermal in na-ture (Fig. 10). Dermal-epidermal separation ap-pears to occur cspecial]y at the site of injection.The dermis is the site of mild infiltration withsome perivascular arrangement, composed chiefly

of polymorphonuclear cells and round cells. Thevcsicle fluid contains numerous erythrocytes.Spoagiosis within the epidermis is seen in areasadjacent to the vesicle (Fig. 11). The specimenremoved four hours following injection showshistologically clear-cut acantholysis (Fig. 12). Inthis section the epidermis is partially dissolved inits structure and acantholytic cpidermal cells arcfreely floating in the vesicle, the roof of which isformed by the preserved stratum corneum. Bothsub-epidermal separation and severe spongiosisare seen in epidermal regions adjacent to theacantholytic area (Fig. 13). Peculiarly shapedlarge vacuolatcd cells with a small nucleus areseen floating free in the blister fluid together withcpidermal cells (Figs. 14 and 15). It is believedthat these cells represent either degeneratedcpidcrmal cells or sebaceous cells. The latterinterpretation is more probable since the processof enzymatic cellular disruption is seen extendinginto the hair follicles and adncxal structures. Inareas, chiefly above the injection site, the firststages of acantholysis can be observed, showingcpidcrinal cells in their normal situation, clearlyseparated from each other by cleft-like spacesdevoid of intercellular bridges (Fig. 16).

The heated control papain solution, 1:3 dilu-tion, also produced a wheal at the site of injec-tion. These wheals were equal in size to the ones

148 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Fin. 16. Acantholytic prickle cells in situ, separated from each other by cleft-like spaces. H. and E.X 840.

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ENZYMATIC VESICAT1ON iw vivo 149

which developed following injection of the 1:10papain solution, and no vesicles were observed.The microscopic picture of the specimens fromthe control areas correspond with those observedfollowing injection of the heated 1:10 papainsolution. No epidermal changes were seen.

The only subjective symptom following theseinjections was pruritus. In neither instance wasnecrotic tissue breakdown at the sites of theinjections observed. Without the biopsy thehealing process was uneventful and required twoto three days.

DISCUssION

The most interesting observation is that asingle crystalline enzyme is capable of inducingin vivo in perfectly normal skin not only all themorphological changes of the eczematous reac-tion, but dermal-epidermal separation, and, withhigher concentrations, definite acantholysis aswell.

It seems that the lower the concentration ofcrystalline papain in a given area the more likelyis there to be spongiosis rather than dermal-epidermal separation or ncantholysis.

Naturally one must speculate as to the exist-ence of such an enzymatic mechanism in humanskin which might be a responsible agent in thepathogenesis of cczematous responses, dermal-epidermal separation, and ncantholysis. Thiscan be compatible with the obviously differentetiologic agents, as such an enzyme would be amediator of the pathologic response rather thanan inducing agent. Again, previous work hasshown that acantholysis dependent upon enzy-matic factors within human skin can be inducedby mild thermal injury, and cantharidin (1, 2).Evidence seems to be accumulating to supportthe theory of enzymatic mediators responsiblefor vesicant diseases.

Potassium chloride extracts of skin have beenshown to contain many protenses, but injectionof these extracts into human skin does not inducespongiosis, acantholysis, or dermal-epidennalseparation (14).

It is most probable that spontaneous formationof blisters in disease is not as simple as one mightbe tempted to conclude from our experiments.Whether one single enzyme or several enzymes orinactivators of physiologic inhibitors (5) are re-sponsible for blister formation in disease remainsto be shown.

SUMMARY

1) There is evidence that one or severalenzymatic factors are related to the patho-physio-logic mechanism of blister formation in the hu-man epidermis.

2) Crystalline papnin was employed in vivo toelicit vesiculation of the human epidermis.Spongiosis, dermal-epidermal separation, andncantholysis of epidermal cells are demonstratedin the histopathological picture.

3) The higher the concentration of pnpain themore likely will ncautholysis develop, while areasof less concentrated papain are more likely toshow spongiosis and/or dermal-epidermal separa-tion. The most fascinating observation is that asingle enzyme preparation will induce in vivo allthree of these morphologic changes.

REFERENCES1. STOUGHTOx, R. B. AND I3AGATELL, F.: The

nature of eantharidin acantholysis. J.Invest. Dermat., 33: 287, 1959.

2. STouonTox, R. B.: Disruption of epithelialcells by heat and specific chemical agents.J. Invest. Dermat., 27: 395—403, 1956.

3. Srouowrox, H. B. AND NOVAK, N.: Disruptionof tonofibrils and intercellular bridges bydisulphide splitting agents. J. Invest.Dermat., 26: 127—135, 1956.

4. STOUGRTON, H. B.: Mechanisms of blisterformation. Arch. Dermat. & Syph. 76:584—590, 1957.

5. ROTHMAN, S.: Pathophysiology of blisterformation. Chapt. 28, pages 699—705 in S.Rothman's Physiology and Biochemistryof the Skin. Chicago, University of ChicagoPress, 1954.

6. BURHAcTI, J. P. E.: Experiments on blisterformation. Dermatologica, 118: 379, 1959.

7. HEHzBERG, J. J. AND HORDE, B.: Uber denMechanismus der Blasenbildung. Dermato-logica, 6: 396, 1959.

8. BEnGMAN, M. AND FEnTON, J. S.: Specificityof proteinases. Advances Enzymol., 1:63—98, 1941.

9. K1MMEL, J. H. AND SMITH, E. L.: Crystallinepapain. I. Preparation, specificity andactivation. J. Biol. Chem., 207: 515, 1954.

10. KTMMEL, J. H. AND SMITH, E. L.: The prop-erties of papain. Advances Enzymol., 19:267—334, 1957.

11. THOMAS, L.: Reversible collapse of rabbit earsafter intravenous papain and prevention ofrecovery by cortisone. J. Exper. Med., 104:245—252, 1956.

12. McCLUsKEY, H. T. AND THOMAS, L.: Theremoval of cartilage matrix in vivo bypapain. J. Exper. Med., 108: 371—383, 1958.

13. BRYANT, J. H., LEDEE, I. G. AND STEETTEN,1).: The release of ehondroitiu sulfate fromrabbit cartilage following the intravenousinjection of crude papain. Arch. Biochem.,76: 122—130, 1958.

14. ST0UGHT0N, H. B.: Personal observation.