Epidermal-Dermal Separation with Proteolytic Enzymes*

Vol. 46, No. 5

THE JOURNAL OF INVESTIOATiVE DERMATOLOGY

Copyright

Printed in U.S.A.

1966 by The Williams & Wilkins Co.

EPIDERMAL-DERMAL SEPARATION WITH PROTEOLYTIC ENZYMES* JULIA M. EINBINDER, MS., RICHARD A. WALZER, M.D. AND INES MANDL, ThuD.

3. Preparation and Examination

There have been many reports on the effectiveness of proteolytic enzymes in producing separation at the dermal-cpidermal junction

of Sections After incubation with enzyme solutions, skin

digesting different structures of the skin. This paper considers an additional enzyme, pronase, a broad spectrum proteinase, and compares the relative digestive actions on the skin of five other proteolytic enzymes, namely "keratinase" derived from Streptomyces fradiae, pancreatic

and processed by the usual paraffin histological technic. The following stains were used: (a) Hematoxylin and eosin, routine method, (b) Periodic Acid Schiff (samples predigested with diastase) and counterstained with Light Green, (c) Van Gieson's Collagen Stain, (d) Weigcrt's ResorcinFuchsin Elastica stain, (a) Alcian blue, (f) Azure

and on the specificity of those enzymes for was fixed in 10 per cent neutral aqueous formalin,

elastase, fungal elastase, eollagenase, and

A, (g) Gram stain.t

trypsin. The possible relationship of proteolytio

4. Experimental Methods (a) Relationship of Pronose Concentration and

enzymes to several pathologic states is indicated.

Time of Incubation to Mouse Skin Changes.

Pieces of depilated mouse skin (approximately 10 mm x 5 mm) were incubated in pronase solution

MATERIALS AND METHODS

1. Source of Material Human skin was obtained either at autopsy (done within 12 hours after death) or from surgical specimens. Mouse skin was taken from the abdomens of Swiss albino animals immediately after sacrifice with ether. Prior to removal of the skin it was depilated with Zip (Jordeau Ives). Experiments with rabbits were done in vito on shnven paraspinal skin.

2. Preporation of Enzymes and Conditions of Incubation All enzymes were dissolved in Tris buffer (0.05M) at a pH at which optimal activity could

of the following concentrations: 1, 0.5, 02, 0.1, and 0.01 mg/3 ml. Samples were removed at 1, 2, 4, and 24 hr. intervals, and immediately fixed in formalin.

(b) Relationship of Time to Digestive Action

of Pronase on Human Skin. Fresh as well as frozen (—25°C) specimens of autopsy skin were

incubated in a pronase solution (1 mg/3 ml tomyccs fradiac, kindly supplied by Drs. A. L. Everett and T. C. Cordon, 4.0 units. (c) Fungal elastase, Actinomyces species, supplied by Agric.

Biol. Corp., Lynbrook, LI., 2.7 units. (d) Pan-

creatic elastase, crude powder, 4.4 units. (a) Trypsin, Difco 1:250, 3.2 units.

The non-specific proteolytic activity of these enzymes tested against azocoll was as follows: Collagenase 3300 Q, Pancreatic elastase 3100 Q, Fungal elastase 2600 Q, Pronase 2700 Q, Kera-

be expected. Keratinase, pancreatic elastase, fungal elastase, and trypsin were prepared at pH 8.8; tinase 3500 Q. The total proteolytic activity of all collagenase and pronase at pH 7.2.t All in vitro enzymes used was of the same order of magniexperiments were incubated at 37°C in small vol- tude, elastolytic activity of pronase was highest, but not significantly so.

umes of enzyme solution.

Trypsin was used at pH 8.8 as this is the opti-

This work was supported by Public Health mum for elastolytic enzymes. The trypsin prepService Research grant AM 07241-02 from the National Institute of Allergy and Infectious Diseases. Received for publication July 9, 1965.

* From the Departments of Dermatology and

Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N. Y.

t (1) Cl. histolyticum Collagcnasc, crude (NH4)2S04 precipitate prepared in one of our laboratories (I.M.) and assayed against CbGlyProGlyGlyProAla. Activity of preparation used was 42.4 units. (2) Elastolytic enzymes assayed against orcein elastin: (a) Pronase B, Streptomyces griseus, crude commercial preparation from

aration used is known to contain elastasc activity previously reported as being the specific agent responsible for dermal-epidarmal separation.

Procedures for PAS, Van Gieson's Collagen Stain, Weigert's Resorcin-Fuchsin Elastica stain and Alcian blue were those described in the Manual of Histologic and Special Staining Tachnics, Armed Forces Inst. of Path., p. 195—7, 66, 80, 134—5, 143, Washington, D.C., 1957. Method for Azure A is given in Kramer, H. and Windrum, G. M.: The metachromatic staimng reaction, J. Histochem. and Cytocbem. 3: 227, 1955, and directions for the Gram stain in Krajian, A. A. and Gradwohl, R. B. H.: Histopathologic Technic,

Cal. Corp. for Biochem. Res., Los Angeles, 4.7 elastolytic units. (b) "Kcratinase" Merck, Strep- 2nd ed., 200-1 p., St. Louis, C. V. Mosby Co., 1952. 492

EPIDERMAL-DERMAL SEPARATION WITH PEOTEOLYTIC ENZYMES

I

493

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Fin. 1. Human skin (Weigert's Resorcin-Fuchsin Elastica Stain) 4 hr. incubation in

pronase solution (0.1%). All nucleated epidermal layers lost. Separation is at the dermalepidermal junction. Dermal digestion apparent. (LP X 93)

buffer). Samples were removed at 2, 3, and 24 hr. intervals and immediately fixed in formalin.

human Skin. Human skin taken at autopsy (one source) was cut into small pieces and suspended in the following buffered solutions: (1) pronase 10 mg/10 ml, (2) collagenase 10 mg/10 ml, (3) trypsin 2.5 mg/b ml, (4) pancreatic elastase 5

(c) Effect of Pronose Applied Topically to Human Skin. Skin strips were placed on gauze moistened with saline in petri dishes. Circular wells (approximately 10 mm in diameter x 3 mm mg/b ml, (5) fungal elastase 10 mg/b ml, (6) depth) were drawn with a 1:1 vaseline-paraffin keratinase 10 mg/b ml, and (7) pH 72 or pH 8.8 mixture on the skin surface. Wells were filled with either buffer or pronase. Samples were incuhated for 16 hrs. In some experiments a common straight pin was used gently to prick the skin before buffer or enzyme solutions were applied.

Tri buffer 0.05M. One specimen was immediately fixed in formalin. Skin samples were removed at 30 mm., 90 mm., and 4 hr. intervals and fixed in formalin. These specimens were stained by all 7 staining teehnics and examined and compared to

(d) Effect of Intradermal and Topical Appli- normal farmalin fixed tissue.

Formalin treated sections were exposed to a cation of Pronase Solutions to Rabbit Skin. Paraspinal areas of 2 rabbits were shaved and cleansed pronase solution (1 mg/10 ml) at 37°C. Slides with alcohol. Five intradermal injections of en- were removed every 15 mm. far 3 hrs. and then zyme were made in a line and were paralleled by routinely stained with H & E. 5 topical applications of enzyme made by soaking RESULTS AND OBSERVATIONS cotton pledgets in appropriate solutions and taping them in place. The following concentrations 1. Relationship of Pronase Concentration were used moving caudally: (1) 0.5 ml of a pro-

nase solution 1 mg/3 ml, (2) 0.5 ml of a Seitzand Time of Incubation to Changes filtered pronase solution 1 mg/3 ml, (3) 0.5 ml in Mouse Skin of a pronase solution 0.1 mg/3 ml, (4) 0.5 ml of The only abnormal finding noted after incua pronase solution 0.01 mg/3 ml, (5) 0.5 ml of a Tris buffer solution pH 72. Rabbits were sacri- bation in Tris buffer pH 7.2 was occasional pen-

ficed by air embolism; one 3 hrs. after the experi- nuclear halo_formation* in epidermal cells. After ment started, the other after 24 hrs. had elapsed. * The great majority of cells in the basal and (a) Comparison of Relative Action of Several

Proteolytic Enzymes with that of Pronase on malpighian layer showing perinuclear haloing were

494

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Fm. 2. Human skin (Van Gieson's Collagen Stain) 30 mm. incubation in collagenase

solution (0.1%). Note haloing of nuclei and dermal-epidermal separation. (LP >< 100)

1 hr. of enzyme action (1 mg/3 ml) there was complete dcrmal-epidermal separation. After 2 hrs. acantholysis was evident and after an incubation of 4 hrs. all cpidermal cells had rounded

cellular elements was present; only renmants of swollen, frayed collagen fibers remained.

After incubation for 24 firs, in dilutions of pronase solutions (0.5, 0.2, 0.1 mg/3 ml), epi-

up with no two cells adhering to each other. dermal changes were similar to those seen with (See Figures 1 and 10.) After 24 hrs. of incuba- undiluted enzyme. The dermis, however, tion no evidence of dermal appendages or showed less destruction of fibrous material. Inprobably not clear cells, primarily because of the number of cells involved, their location above the basal layer and the appearance of the nucleus itself.

cubation in 0.01 mg/3 ml of enzyme revealed

complete dermal-epidermal separation and pronounced acantholysis.

495

EPIDERMAL-DERMAL SEPARATION WITH PROTEOLYTIC ENZYMES

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FIG. 3. Human skin (H & E) 90 mill, incubation in collagenase solution (0.1%). Compare to Figure 2 dermal-epidermal separation is almost complete. (LP >< 93) FIG. 4. Human skin (PAS) 4 hr. incubation in collagenase solution (0.1%). Basement membrane well defined. No PAS positive material associated with remaining collagen. Stratum corneum is intact. Marked dermal digestion with loss of skin appendages is apparent. (LP X 93)

496

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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FIG. 5. Human skin (PAS) Collagenase digested. Note basement membrane structures

of hair follicle and sebaceous gland. Also note hair shaft. (HP >K 430)

2. Relationship of Time to Digestive Action of Human Skin

all epidermal cells. Results were similar with a

Epidermal changes are summarized briefly as greater details will be given in a subsequent section. After 2 hrs. of incubation, microscopic

3. Effect of Topical Application of Pronase to Human Skin (in Vitro)

dermal-epidermal separation was apparent.

prc-frozen sample of skin.

Changes occurring after topical application of enzyme were minor and consisted of minisive and marked acantholysis had occurred. mal microscopic cpidermal-dermal separation. After 24 hrs. there was complete separation of There was no evidence of acantholysis. In the After 3 hrs., separation was much more exten-

497

EPIDERMAL-DERMAL SEPARATION WITH PROTEOLYTIC ENZYMES

S

I FIG. 6. Human skin (Van Gieson's) 90 mm. incubation in trypsin (0.025%). Separation

is beginning at the dermal-epidermal junction. (HP X 400)

experiments where the skin was pre-injured with

(predominantly eosinophils) in the dermis. All

a straight pin microscopic examination re- concentrations of enzyme, 0.01—1.0 mg/3 ml, vealed that surface application of buffer (0.05M sterile and non-sterile, produced similar reacTris pH 7.2) was sufficient to induce some tions. The findings consisted of areas of intact separation and acantholysis. Application of en- epidermis alternating with other areas where zyme resulted in the loss of all epidermis, skin necrosis and hemorrhage were apparent. The dermis showed marked changes: edema, round appendages and dermal cellular elements. cell infiltration, massive hemorrhage, and de4. Effect of Intradermal and Topical struction and liquefaction of tissue. After 24 Application of a Pronase Solution hrs. the changes were similar to those observed to Rabbit Skin (in vivo) after 3 hrs. but more marked. Results were relatively non-specific and may be summarized as follows: Pathologic alterations 5. Comparison of Relative Action of of patch testing sites (0.01—1.0 mg/3 ml) varied Several Proteases with that of from an intact epidermis and normal appearing Prona.se on Human Skin

dermis to patchy necrosis of epidermis with cellular necrosis, some dermal-epidermal separa-

tion, and an infiltrate in the upper dermis consisting of round cells and polymorphonuclear leukocytes. Damage did not correspond to enzyme concentration or to period of application. Pathologic alterations of intradermally injected sites were marked. After 3 hrs. buffer

Histologic changes after incubation in pH 7.2 buffer were minimal and were limited to microscopic areas of dermal-epidermal separation and occasional perinuclear halo-formation. Changes after incubation in pH 8.8 buffer were similar but more marked and were accompanied by vacuolization of the cytoplasm, distortion of

alone produced moderate cellular reactions the cell walls of the sweat glands, and an in-

498

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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FIG. 7. Human skin (Gram) 4 hr. incubation in pancreatic elastase (0.05%). There is loss of much of the gram positive material associated with the stratum corneum, however

such positive material is still found coating collagen bundles. There is no staining of basement membranes. (LP >< 93)

FIG. 8. Human skin (PAS) 4 hr. incubation in fungal elastase (0.1%). Note marked digestion of sweat glands, however, basement membrane intact. PAS positive material still present in association with collagen bundles. (HP X 400)

*

t.

t FIG. 9. Human skin (Weigert's Resorcin-Fuchsin Elastica Stain) 30 mm. incubation, in keratinase (0.1%). Note characteristic vacuole formation in basal epidermal cells which precedes dermal-epidermal separation. (HP X 400)

FIG. 10. Human skin (H & E) 90 mm. incubation in pronase solution (0.1%). Note

nuclear halos and acantholytic cells. (HP X 400)

499

tion

(1) Dermal-epidermal separa-

Dermis

changes

epithelial

(1) Sweat

None

Tris buffer, pH 8.8 None

Keratinase, lOmg/ 10 cc, pH 8.8

None

Epidermis

30 mill.

Tris buffer, pH 7.2 None

Enzyme

gland

at basal layer

Complete dermal-epidermal sepparation with loss of epidermal cells

(1)

None

None

Epidermis

dular epithelium (2) Swelling of epithelial cells of hair follicle (3) Fraying of collagen bundles

Dermis

(1) Loss of all glan-

None

None

90 mill.

Time of observation

TABLE I The relative effectiveness of proteinases on skin

rial

(1)

gram

+

Dermis

mate-

mate-

fibers

(2)

mast cells Loss of all elastic

lar elements except

(1) Loss of all cellu-

material

+

(2) 1 metachromatic

rial

(3)

Fraying of collagen bundles (4) No gram + material (5) No PAS staining material in papillary area Basement membranes and mature keratin structures intact

dermal cells lost

(1) All nucleated epi-

mal-epidermalseparation

(2) Microscopic der-

ing

(1) Perinuclear halo- (1) 1 PAS

(2) Microscopic der-

(2) 1 PAS + material mal-epidermalseparation (3) 1 metachromatic material

Perinuclear haloing

(1)

Epidermis

4 hrs.

swollen

None

None

Fungal elastase, 10 (1) Minimal der- None mg/b cc, pH 8.8 mal-epidermal separation, no acantholysis (2) Epidermal cells

pH 8.8

Pancreatic elastase, 5 mg/10 cc,

tion Perinuclearhalo-

tholysis

ing (3) Minimal aean-

(2)

(1) Minimal dermalepidermal separa-

epidermal separation and acantholysis

(1) Minimal dermal-

Glandular epithelium shows evidence of digestion

(1)

(1) Glandular epithelium gone

fibers (2) Loss of all PAS

elastic and collagen

(1) Some digestion of

fibers (3) PAS

uefaction and loss of intercellular ccment (2) Marked reduction in number of elastic

Collagenand mus-

dc fibers show liq-

(1)

+ material still evident in dermis (4) 1 (increased) metachromasia of ground substance and associated with digested skin appendages Basement membranes and mature keratin structures undigested

Complete dermal-epidermalseparation (2) All epithelial cells gone (1)

(3)

staining in dermis metachromatic staining associated with ground substance and glandular remnants Basement membranes and mature keratin structures intact

tion with acantholysis

(1) Completesepara-

C

Epidermis

30 mm. Dermis

10

mg/b

cc, pH 8.8

Trypsin, 2.5 mg/b

cc, pH 7.2

Pronase,

(1) Reticular area —collagen shows fraying and dis-

(1) Some haloing None of nuclei and vacuolization of nuclei and cells

separation solution of inter(2) Acantholysisin basal areas of fibrillar cement (2) Glandular epiepidermis thelinm gone (3) Perinuclear haloing

(1) Complete dcrmal -epidcrmal

very vacuolated

fine fibrils (3) Epithelium cells of sebaceous glands

(1) Complete der- (1) Papillary collaCollagenase, 10 gen frayed and mg/b cc, pH 7.2 mal-epidermal PAS staining separation and diffuse (2) Marked pennuclear haloing (2) Collagen of reticular area sep(3) Acantholysisin arated into very basal layer

Enzyme

Epidermis

4 hrs. Dermis

(1) Swelling and liq-

(2) (3)

Haloing of nuclei Minimal acantholysis

aration

mal-epidermalsep-

(1) Microscopic dcr-

Only remnants of (1) Loss of all nucleated epidermis glandular and vascular elements remain (2) Loss of much of the fibrous elements. Remaining Basement membranes structures intact collagen is swollen and liquefied (1)

(1) Papillary

changed

Collagen bundles frayed and swollen (2) Elastic fibers un(1)

to scattered fibrils and mature kcratin

(2) No evidence of muscle fibers (3) Collagen reduced

cellular elements

(1) Loss of all dermal

area (1) No epidermis left (1) Complete digestion of papillary markedly digested area Reticular area— (2) collagenshows gel(2) Elastic fibers still atinization evident (3) Loss of all glan- Basement membrane and mature keratin dular epithelium structures undigested

Dermis

ucfaction of mustion. No acantholcle ysis (2) Loss of glandular (2) There is characcpithelium teristic vacuolization of basal cells

(1) Minimal dermalepidermal scpara-

Marked aeantholysis, few intact epidermal cells

(1)

Epidermis

90 mm.

Time of observation

TABLE I—Continued

EPIDERMAL-DERMAL SEPARATION WITH PROTEOLYTIC ENZYMES

crease in metachromasia of the dermal ground substance. No acantholysis was noted.

Histologic studies with the 6 proteinases:

503

DISCU55ION

Our studies on the effect of proteinases on

skin extend the observations described in compronase, keratinase, fungal elastase, pancreatic parable studies by other workers (1—12). Both elastase, eollagenase, and trypsin indicated that mouse and human tissue, frozen or recently the digestive action of these enzymes on skin obtained at autopsy or surgery, were suitable tissues was similar, although the rate at which for experimentation. It was necessary to incudigestion took place and the amount of enzyme bate the tissue specimens in solution to effect necessary for a comparable result differed. (See proteolytic changes. Topical application may not Figures 1—10.) The result of a 4 hr. incuba- have been effective because of the inability of tion, in all eases, was dermal-epidermal separa- the enzyme to penetrate or digest the stratum tion and aeantholysis, usually progressing to comeum. When the stratum corneum was predestruction of all epidermal cells. The papillary injured the usual digestion pattern occurred. area of the dermis showed more marked diges- This has been shown by other workers using

tion of fibrillar elements than the retieular other technics for altering skin permeability dermis. Fibrillar elements (muscle, collagen

such as cellophane stripping (5). and elastic tissues) of the retieular area showed Hambrick and Blank (1) applied a battery fraying, separation into fibrils (presumably due of chemical agents including the 4 enzymes colto loss of interfibrillar cement), swelling, gelalagenasc, bacterial proteinase, trypsin, and tinization, and ultimately solubilization. Sweat hyaluronidase topically to the dermal side of and sebaeeous glandular epithelium were skin and obtained successful separation at the quickly destroyed; sweat ducts were more re- dermal-epidermal junction with colla genase and sistant and the hair follicle epithalium was less proteinase. Trypsin produced supra-basal sepalabile than glandular epithelium. (See Figures 5 ration. Trypsin and proteinase produced "aeanand 8.) Hairs were eventually freed and could whereas eollagenase did not. Only trypbe found intact in the incubation media. Con- tholysis" sin attacked sweat duets and pilosebaceous

nective tissue cells were no longer seen, presumably due to the separation from surrounding tissues. (See Figure 4.) Some mast cells with their characteristic metachromatic gran-

ule were still apparent after 4 hours. The

apparatus. Burbach (12) injected trypsin, proteolytic skin extracts and testicular and pneumococcal hyaluronidase intracutaneously and produced acantholysis and separation with only the first two. He was unable to produce separation with this technic in frozen skin.

basement membrane structures associated with the dermal-epidermal junction, sweat glands, We did not find that injection of enzyme sebaceous glands, hair fo]lieles, and blood ves- intracutaneously in vivo produced dermalsels appeared undigested and intact as did the epidermal separation or reproducible acantholystratum eorneum and the hair shaft. (See sis. (This may have been because the injection Figures 4 and 8.) These alterations in treated of enzyme was deep intradermal rather than specimens were accompanied by a loss of gram subepidermal.) The observed epidermal changes positive material (Figure 7), an increase in were presumably due to necrosis of underlying metachromasia, most marked in the area of tissues. In vivo injection of trypsin into human digested skin appendages, and an increase in skin has also been shown not to produce dermalcharacteristic PAS staining of the papillary area epidermal separation (12). Stoughton (13) using of the dermal ground substance and material an in vitro technic obtained dermal-epidermal

associated with collagen bundles (Figure 8). separation after subcutaneous injection with Pronase effectively digested formalin fixed speci- only the first two of the following series of mens. Acantholysis and dermal-epidermal sepa- enzymes: papain, elastase, trypsin, lipase, /3 ration occurred in that order. Aeantholysis glucuronidase, desoxyribonuclease, ribonuelease, appeared to differ from that occurring in un- /3 glucosidase, lysozyme hyaluronidase and carfixed tissue. This difference may tentatively be boxypeptidase. explained by the adhesion of the cells to the Our observations support the findings of Dob-

slide during the histological procedure fol- son (5) that the keratinase derived from lowed.

Streptomyces fradiae is not truly a keratinase.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

In our experiments there was no evidence of clemcnts. Mature keratin structures or basedigestion of either the stratum corneum or the ment membrane structures do not appear to be hair shaft. Moreover, none of the other enzymes studied appears to have keratinolytic activity. The histologic picture produced by protcolytic

altered.

3. Specimens of skin must be incubated in

enzyme solutions for consistent dcrmal and epienzymes differs from that produced by can- dermal changes to occur. Topical application in tharidin. The latter lyscs intercellular bridges, vivo or in vitro, as well as intracutancous injecresulting in the freeing and rounding up of tion in vivo, did not produce consistent results. 4. The mechanism of cantharidin action is not epidermal cells, and leads to the supra-basal

separation of the epidermis from the dermis. similar to that of the proteolytic enzymes The dermis as well as the basement membrane studied. appears unaltered. In the case of proteolytic REFERENCES enzymes the first change noted was vacuolization of cells at the basal layer accompanied by 1. Hambrick, C. \T aod Blank, H. J.: '9/hole prominent perinuclear halo-formation, and fol-

lowed by changes in the fibrillar elements of the dermis. The adhesive material between the basement membrane and the basal cell mem-

brane appears to be the primary site of the enzyme action. There arc other marked differ-

ences in the action of cantharidin and proteinascs. For example, cantharidin is effective topically, and does not cause acantholysis of pre-frozen or formalin fixed skin. The histologi-

cal changes produced by cantharidin are very similar to those of pemphigus vulgaris. On the other hand, pathological alterations produced

by protcascs arc quite similar to those of epidcrmolysis bullosa simplex. CONCLUSIONS

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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