Collagenase and Connective Tissue Metabolism in Epidermolysis Bullosa* Gerald S

COLLAGENASE AND CONNECTIVE TISSUE METABOLISM IN EPIDERMOLYSIS BULLOSA* GERALD S. LAZARUS M.D. t

ABSTRACT N inteen patients with various types of epidermolysis bullosa were studied for eviden ce of in creased collagen catabolis m. Cultures of cl inically normal but pathologically blisterable skin from nine patients with dermolytic bullous dermatoses, fo ur patients with epidermol ysis bull osa simplex a nd two patients with junctional bull ous dermatoses did not diffe r significant ly in collagenase production. In two patients with dermolytic bullous dermatoses cultures of skin lesions produced three times more coll agenase a nd had twice the DNA content of adjacent normal appearing but pathologically blisterable skin. Two of the nine patients with dermolytic bullous dermatoses had minimally decreased l evels of serum col lagenase inhibitor. None of the patients had increased hyd roxyproline excretion in the urine. Four of six patients with d ermolytic bullous dermatoses a nd one of two patients with junctional bull ous dermatoses had anti-denatured collagen a n t ibodies. It appears t hat in creased local l evels of coll agenase a re a secondary tissue reaction to chronic injury.

Epidermolysis bullosa designates a group of more severe; it is present at birth and there are genetic diseases which are characterized by t he numerous erosions and blisters. There is often formation of blisters upon minor mechanical severe scarring a nd fusion of the ha nds a nd feet trauma to the skin . These mechanobullous dis a nd esophageal stenosis is not uncommon. ' eases have been shown to have distinctive cl inical The connective tissue abnormali ties described presentations and ultrastructural pathology (1) . by Pearson in the dermolytic type of disease sug Pearson has shown that epidermolysis bullosa gest a p rimary abnorma li ty of connective tissue. s implex is a dominantly inherited syndrome c har Recently Eisen and co-workers (4, 5) and Lazarus acterized by the dissolution of the basal epi and Full mer (6) have defined and characterized a dermal ce lls with mechanical stress (2). T here is human skin collagenase. This enzyme is capable little if any dermal component and scarring is of degrad ing collagen fibrils under physiological qu ite uncommon . Junctional bullous dermatosis conditions of temperature a nd pH. The colla (epidermolysis bullosa hereditaria letalis) demon genase is produced primarily in the papillar strates cleavage between the basement membrane dermis (5, 6) and it is inhibited by a compon ent a nd the plasma membran e of the basal cells (3). of normal human serum (7). T his condition is usually quite severe a nd it is Eisen has shown in a study of skin biopsies manifest at birth. C linically there are distinctive from 5 patients with dermolytic bullous derma erosions, without milia formation , and there is toses that there is increased coll agenase produc sparing of the palms and soles. Dermolytic bul tion at the site of bullae. In cultures of normal lous dermatoses (dystrophic epidermolysis bul skin fro m three of these patients there were losa) has a sharp separation below the basement modest increases in coll agenase production as membra ne; this is associated with phagocytosis of we ll. H e wondered from these data whether over collagen fibrils by macrophages and degeneration production of collagenase or decreased inhibition of connective tissue (3). An choring fibrils are of this e nzym e by serum might be of importance characteristically absent (3). T he domina nt ly in in the formation of bullae in this disease (8). herited form of this disease is frequently mild a nd This study evaluates collagenase activity and there are bull ae, and milia over the extensor acral connective t issue metabolism in nineteen patient areas. The recessive form of the disease is often with various forms of epidermolysis bullosa.

Received October 4, 1971 ; accepted for publication, MATERIALS AND METHODS December 14, 1971. Presented, in part, at the meetin g of The American Patients. Nin eteen patients with epid ermolys is bul Federation for Clini cal Research, Atlantic City, N. J ., losa and ten control patients were studied. N in e of the May 2, 1971. epid ermolys is bullosa patients were admitted to the This wo rk was supported in part by the National In metabolic ward of The Massachusetts General Hospital stitutes of Health Train ing Grant No. 5-70-l-AM-05297 for complete evaluation. These patients had two, twenty an d Research Grants AM-3564 and AM-4501. four hou r urine coll ections for hyd roxyprolin * From the Department of e while on a De rmatology, Harvard ge latin free diet (9). The Medical School and the Department of Dermatology, others were evaluated on an Massachusettes General Hospital, Boston, Mass. outpatient basis. t At prese nt: Ca rl Herzog Fellow of the American Epidermolysis bullosa simplex. Four patients, a Dermatologica l Association and Research Fell ow of The mother and her eight year old daughter, and a mother Arthritis Foundation. Strangeways Research Laborato and her 9 year old son were studied. The eight year old ries, Wort's Ca useway, Ca mbridge, England . female child was hos pi tali zed for co mplete evaluation. 242 COLLAGENASE IN EPIDERMOLYSIS BULLOSA 243

Junctional bullous dermatosis. A seventeen year old and 100 microliters of the solu t ion to be assayed. The sister a nd fifteen year old brother were hos pitali zed for mixture was incubated for 18 hours at 37° C and then study. A younger brother h ad died at age 3 with the dis centri fuged in a Spinco Microfuge for ten minutes. The ease; there was one sibling who died with pneumonia at pellet contained insoluble co llagen fibrils. One hundred age six months a nd another who died b ecause of prema microli ters of the supernatant solution containing intact turity. There are 4 other h ealthy s iblings and there was coll agen molecules and peptide reaction products was no history of consanguinity. Both patients had l a rge counted in a liquid scint ill ation spectrometer. All assays erythematous denuded plaques over the s hins a nd ex were performed in duplicate and included buffer blanks tensor aspects of the a rms; there were no milia or l e and serial dilut ions of hi gh ly purified Clostridial co lla sions of the pa lms or soles. Both children h ad oral ulcer genase (Collagenase P urified, Worthington Biochemical ation s a nd abnormal teeth. The fifteen year old boy had Corp., Freehold, N. J.). Trypsin (Trypsin 2x Crystal recurrent episodes of acute urinary retention secondary li zed, Worthington Biochemical Co rp., Freehold, N. J.) to urethral scarring. controls containing 25 Jig per ml in the incubation mix Dermolytic bullous dermatoses. Eleven patients were ture were used to indicate the extent of non-spec ifi c studied of which 4 demonstrated d ominant inheri tance. proteolytic breakdown of the collagen ge l. Co mparison Ali the patients with dominant inheritance had mil d of the counts lib erated by the Clostridial collagena e acra l scarring disease. A father and daughter were eval standa rds for the experiment allowed determination of uated on an in -patient basis a nd a ll 4 of these patients equivalent Clostridial coll agenase of the sample. The had coll agenase studies. There were six c hildren and reasons for expressin g activity in this manner are dis on e adul t male without a pos itive fa mily history. The cussed elsewhere (14) . severity of the disease varied from quite mild to very Serum was co ll ected from patients in a fasting state severe. T here was adequate t issue for coll agenase and was stored at - 20° C until used. A pool of human studies o n five a nd four were studied in -hospital. skin coll agenase was made from the c ultures of t issue Pathology. All patients had biopsies of freshl y in obtained from patients with dermolytic b ull ous derma duced lesions w hich were studied by li ght and electron toses. To an aliquot of this pool were added the indi microscopy. E it her normal appearing skin or l esions vidual sera so as to make a f inal concentration of serum devoid of bullae were gently stroked with a pencil eraser of 2.5%, 1% and 0.5% in the incubation mixture. These until the s kin was fP. lt to separate or until thirty strokes mixtures were then incubated with radioactive, recon had been made. T issue for light microscopy was stained sti-tuted collagen fibrils and the results expressed as re with hematoxylin and eosin, periodic acid Schiff, tolui sidual co llagenase activity. Aliquots of serum were also dine blue, and Verhoeffs elastic t issue stain . The elec assayed for antibodies to both native and denatured tron microscopic studies will be reported in detail later. human co llagen by the passive hemagglu tination tech Tissue culture. Specimens of skin weighing in excess nique of Michaeli (15). of 600 mg were obtained by excisional biopsies under sterile conditions from the buttocks. These areas were RESULTS nstrated to be abnormal in their response to trauma demo C ult ures of tissue from 25 patients were studied eit h e r clinica lly o r hi stologically. In several cases sepa llagenase production in vitro (Fig. 1.). Coll a rate skin biopsies, weighing at least one gram, were ob for co tained directly from a lesion a nd its normal appearing gen ase production by normal appearing buttock margin. Ten bi opsies of normal buttock skin served as a s kin was similar in a ll grou ps studied. It is impor control group. tan t to emphasize t hat a lt hough t he buttock skin Immediately after excision, the t issue was placed in a ppeared clinically norm a l in th e epidermolysis Dulbecco's modification of Eagle's tissue c ulture me dium with penicillin and streptomycin . After the tissue was trimmed of fat it was weighed and diced in to 1- 2 3·5 mrn cubes. The tissue was then cultured in Petri dishes ~ in 5 per cent carbon dioxide in air at 37° C at a ratio of ~ 3·0 1 rnl of cul ture media per 200 mg o f t issue. T he c ul ture media was harvested d aily and frozen and the c ul ture was then replenished with an equal volume of m edia. At the end of the seven d ay culture period the tissue but ton was lyophilized and stored at - 20° C for subsequent DNA assay (10) . The daily harvests were individua lly assayed for collagenolytic activity a nd the total enzyme production was calculated by summing t he individual collagenase va lu es. Collagenase assays. Two methods of detection of col lagenolytic activity were used: 1) release of radioactive degradation products from reconstituted "C-labelled collagen fibrils a nd, 2) acryla mide gel electrophoresis (11- 13). 0 Simplex Junctional Dermolytic Dermolytic The reconstituted "C-collagen fibril assay has been control skin skin lesion in detail elsewhere and was used with the fol described l. Total coll agenase production by seven day microliters FIG. lowing modifications (11, 12). One hundred cultures of: normal appearing s kin from patients with of the dialyzed, centrifuged 0.2% collagen solut ion w ere epidermolysis bullosa simplex, junctional bu ll ous der pipetted into microfuge tubes and allowed to ge l at 37° matoses, and dermolytic bullous dermatoses a nd by C overnight. To the opalescent gel, which consisted of skin lesions from two patients with dermolytic derma organized co ll agen fibrils, was added 100 microliters of toses. T he cross hatched area is the mean ± l. S.E. of co l 0.001 M calci um chl oride in 0.05 M Tris buffer, pH 7.6 lagenase production by 10 normal skin biopsies. · 244 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY bullosa patients, it behaved abnormally when 600 exposed to mechanical stress. The epidermolysis - ~ Clinical bullosa s implex_patients demonstrated character 550 - lesion istic histological cha nges with trauma and devel oped blistering at the suture sites. The junctional Normal oappearing bullous dermatoses patients demonstrated typical 500 1- tearing of the skin with t rauma and total intoler skin ance of any form of tape. The severe dermolytic 450 1- bullous dermatoses pat ients developed frank bullae with trauma. The less severe dermolytic 400 bul lous dermatoses patients had histological evi dence of dermoepidermal separation. :l 350 - In two adult male dermolytic dermatoses pa c tients, biopsies were obtained of clinically abnor .E mal, non-bullous, erythematous, scarred, exten ";;)..... 300 sor, acral skin . A specimen of normal appearing c tissue from within 5 em of the clinical lesion was 6 250 re moved at the same time and cultured for com u parison. Significant increases in coll agenase pro 200 duction were ev ident in the clinically diseased skin (Figs. 2 and 3). T he cultured tissue from the lesion had two to three times the DNA content of 150 the normal appearing surrounding skin. Histological evaluation of the lesion revealed 100 dermal-e pidermal separation, new vessel proli fer ation, a loss of elastic tissue in the upper papil 50 lary dermis, and an increase in the number of I I I 600 2 3 4 5 6 7 I Clinical Days in Culture lesion FIG. 3. Coll age nase r- activity, expressed as CPM solu 550 bilized from "C labelled reconstituted collage n fibrils produced by cultures of lesion and normal appearin ~ oNormal skin 500 I- in patient A.D. with dermolytic bullous d e rm a~ appearing toses. skin 450 mast cells in the dermis (Figs. 4- 6). These find in gs were not specific for dermolytic bullous der 400 - matoses and occured in the lesions of junctional bullous dermatoses as well. Two of seven patients with dermolytic derma 35 0- ..... toses had dimini :::l shed serum levels of collagenase c inhibitor (Table I). The rest of the patients h ad ·E 30 Or- inhibitor levels within the normal range ...... (f) Urinary hydroxyproline determinations on 5 c 25 0 - patients with dermolytic bullous dermatose, in :::J 0 cluding one very severely affected child, both Pa u 20 Or- tients with junctional bullous dermatoses, and one patient with epidermolysis bullosa simplex 15 01- when corrected for age and body surface ac: cording to the method of Kivirikko (16) were within normal limits. 10 0- None of the patients had unequivocal titers of antibody to native collagen (Table II) . Four of the 5 0- s ix patients with dermolytic disease and one of the two patients with junctional disease had a n ti I I I denatured human co ll agen antibodies. 2 3 4 5 6 7 Patients hospitalized on the metabolic ward Days in culture had numerous blood studies including immuno electrophoresis, rheumatoid and anti-nuclear fac FIG. 2. Coll agenase activity, expressed as CPM solu tor determinations, Coombs tests, and creatinine bilized from "C labell ed r econstituted co ll age n fibril s, produced by cu ltures of les ion and normal appea rin g clearances. Other than hypochromic microcytic skin in patient G.D. with derm olytic bullous derma anemia, some mild non-specific diffuse elevations toses. in gam rna globulins a nd an occasional minimally ... FIG. 4. Photomicrograph of a skin lesion of d ermolytic bullous dermatoses. There is dermal epidermal separa tion, vascul a r proli fe ration and infilt ration b y endothelial cells a nd round cell s. H a nd E ; x 480.

F IG. 5. Photomi crogra ph of a skin lesion of d ermolytic bullous dermatoses stained f or elastic t issue. There is dermal epiderm al separation, vascula r proliferation, a nd lack of stainabl e e lastic t issue in the pa pillary dermis. N umerous elastic fibres, designated b y a rrows, are seen coursing t hrough the reticular d ermis. Verh oeffs Stain ; x 350. 245 246 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 6. Photomicrograph of a s kin lesion of dermolytic bullous dermatoses demonstrating proliferation of mast ce ll s in the papillary derm is . Arrows indicate individual mast ce ll s. Toluidene Blue; X 480.

TABLE I TABLE II Individual sera from patients were incubated wi th a Antibody titers to native and denatured co llagen in the pool of skin co llagenase from cultures of patients with sera of patients with various types of epidermolysis dermolytic bullous dermatoses bullosa The results are expressed as per cent of ac tivity re Titers of 1: 2 or greater, are considered significant. maining when co mpared with an uninhibited sa mpl e of enzy me. The normal serum is a Diagnosis An ti -co llngen Anli-dcnature d pool of 25 normal se ra. titer coll agen tile r Final conCe ntrution of Epidermolys is bullo serum in incubation sa 0 0- 1:2 Diagnosis sim plex 0 0 2. !"> ~ 1% 0.5% 0 0 Epidermolys is bullosa simplex 0 0 14 0 0 0 0 18 Junctional bullous derm a- 0- 1: 2 1: 2 0 0 0 toses 0 0 Junctional bu ll ous dermatoses 0 0 18 Derm olytic bullous derma- 0- 1: 2 1: 4 0 0 24 toses 0 1:4 Dermolytic bu ll ous dermatoses 0 0 0 0 1:2-1:4 0 0 0 0 1:2 0 0 20 0 0- 1:2 0 0 28 0 0 0 0 36 0 0 36 7 19 41 formed on the same patients were normal except 10 40 65 for one child with severe dermolyt ic bullous der matoses who was found to have lysine and cystin Normal se rum 0 0 25 uria. The patient's mother and three sisters had a similar aminoaciduria wi thout evidence of dermo lytic disease. This suggests that the lysine-cys elevated complement level, the results were tinuria in this patient was coincidental. within n ormal limits. Four patients with dermo lytic bullous dermatoses, two patients with junc tional bullous dermatoses and one patient with DISCUSSION epidermolysis bullosa simplex had normal kary Epidermolysis bullosa is a heterogeneous group otypes. Urinary amino acid chromatograms per- of syndromes whose common characteristic is an CO LLAGENASE IN EPIDERMOLYSIS BULLOSA 247

unusual suscept ibili ty to mechanical stress. Only of two patients with junctiona l bullous derma one of these diseases, dermolytic bullous derma toses. toses, has definite derma l connective tissue re It appears that the increased l ocal l evels of col sorption. C ultures o f norma l appearing, but path lagenase in d ermolytic bullous dermatoses are a ologicall y blisterabl e, skin in this co ndit ion did secondary t issue reaction. The increased a mounts not produce increased amounts of collagenase. of collagenase might perpetuate the blistering and Urinary hydroxyproline excretion, a lthough only a scarring by degrading t he dermal connectiv e very crude estimation of collagen m etabolism, tissue a nd thus weakening the mechanical integ was co mpletely normal in our patients. These rity of the s kin. In a ddition, the products of this data support t he notion that a prima ry abnor local collagen degradative phenomenon are prob mality of collagen catabolism secondary to colla ably the a ntigens responsible for the a n ti-dena genase excess is unlikely (7). tured co ll agen ant ibodies found in our patients. Two of seven patients with dermolytic bullous The primary pathophysiological event in this dis dermatoses had dec reased levels of serum coll a ease, however, is probably a geneticall y deter gen ase inhibitor; t he other five patien ts including mined defect in the structure of the papillary one wi t h severe dystrophic disease had levels dermis. within the normal range. These data mediate I would like to acknowledge the support of Dr. Ste against the hy pothesis t hat bullous dermatoses phen Krane who provided me with laboratory faci li ti es are caused by a lack of circulatin g serum colla and encouragement and performed the urinary hydroxy Michaeli of the Depart gen ase inhibitor. The decreased l evel of serum proline determination. Dr. Dov ment of Biochemistry, Uni ve rsity of Ca lifornia Medical collagenase inhibitor found in two of our patients Schoo l, San Francisco, performed the anti-coll agen anti migh t refl ect non -specifi c c ha rges in the levels of body studies and offe red much useful discussion. Drs. certain serum proteins or it might indicate a de Robert Griesemer, Irwin Freedberg, Kenneth Arndt, Rosenbaum protease inhibitors s uch as seen John Fromer, Samuel Moschella, Haskell fect in serum and Howard Baden allowed m e to study t heir patients. w ith alpha-1-antitrypsin deficiency (17) . Repeat The karyotypes were performed in the laboratory of Dr. determination of serum co ll agenase inhibitor con Leonard Atkins and the am ino ac id chromatograms centration over a period of time a nd further eval were performed by Dr. Viv ian Shih . levels s hould clarify uation of serum anti-protease REFERENCES t hese questions. 1. Pearson, R.: The mechanobullous diseases, p. 621 , We are in complete agreement with Eisen in Dermatology in General Medicine. Eds ., Fitzpa fi nding s ignificant increases in co llagenase pro trick, T. B., Arndt, K. A. , Clark Jr. W. H., Eisen , duction in clinical l es ions (8). The lesions a re A. Z. , Van Scott, E. J. and Vaughn, J. H., ch aracteri zed histologicall y by vascular prolifera McGraw Hill , 1971. 2. Pea rson, R. W. and Spargo, B.: Electron mi cro tion, a peri vascul ar round cell infiltrate a nd de scopic studi es of dermalepidermal se paration in creased amounts of stainable e lastic t issue. In a hum an skin. J. Invest. Derm., 36: 21 3, 1961. previous study, coll agenase production by rheu 3. Pearson, R. W.: Studi es on the pathogenesis of epi matoid synovi al tissue correlated with a simila r dermolysis bullosa. J. Invest. Derm., 39: 551 , 1962. pathological picture (14). This association was 4. Eisen, A. Z., Jeffrey, J. J. and Gross, J.: Human independent of polymorphonuclear leukocyte col sk in collagenase: isolation and mechanism of at lagenase (12). Carrageenin granulomas have a lso tack on the coll agen molecule. Biochim. Biophsy. been found to produce collagenase (18). These Acta, 151: 637, 1968. 5. Eisen, A. Z.: Hum an skin co llagenase: loca li zation data suggest that vascular granulation tissue is and distribution in norma l human skin . J. Invest. capable o f producing collagenase. Whether the Derm., 52: 442, 1969. source of the e nzy me in dermolytic bullous der 6. Laza rus, G. S. and Fullmer, H. M.: Co llagenase uction by human d erm is in vitro. J . Invest. matoses is the granulation tissue itself or its over prod Derm. 52: 545, 1969. lying epidermis remains in question. Norma l epi 7. Eisen, A. Z., Bauer, E. A. and Jeffrey, J . J.: Animal dermis produces little co llagenase in cul ture (5, and human co llagenases. J. Invest. Derm., 55: 19). The ·epidermis over healing skin wounds and 359, 1970. idermis from a patien t with 8. Eisen, A. Z.: Human skin co llagenase: Relationship a culture of pure ep to the pathogenesis of epidermolys is bullosa dys dermolytic disease has been shown to produce trophica. J . In vest. Derm., 52: 449, 1969. signifi cant amounts of collagenase (7, 8). This 9. Prockop, D. J . and Udenfriend, S.: A specific implies that an epid ermal mesenchymal in terac method for the analysis of hydroxyproli ne in tis tion might be operative in collagenase production. ~ u es ann urine. Analyt. Biochem ., 1: 228, 1960. 10. Webb, J. M. an d Levy, H. B.: Sensitive method tor The finding of an t ibodies to denatured collagen determining deoxyribonucleic ac id in tissues and in our patients is of interest. Michaeli has found microorganisms. J. Bioi. Chern ., 2 13: 107, 1955. this antibody in only 8 % of a control population 11. Naga i, Y., Lapiere, C. M. and Gross, J.: Tadpole age nase: preparation and purification. Bio adults (20). By contrast, 60% of 203 sera co ll of 181 chemistry, 5: 3123, 1966. from patients with rheumatoid arthritis had anti 12. Lazarus, G. S., Daniels, J. R. , Brown , R. S., Bladen, denatured coll agen a ntibody. In these patients it H. A. and Fullmer, H. M.: Degradation of co lla was felt that the an tibody was a resul t of exten ge n by a human granulocyte collagenolytic system. of a rticula r J. Clin . Invest., 47: 2622, 1968. . sive damage and denaturation 13. Sakai, T. and Gross, J .: Some properties of the connective tissue. Our findings would support products of reaction of tadpole coll agenase with such an h ypothesis. The antibody was present in collage n. Biochemistry, 6: 518, 1967. two thirds of our dermolytic patien ts and in one 14. Laza rus, G. S., Decker, J. L.. Oliver, C. H., Daniels, 248 THE JOURNAL OF IN VESTIGATIVE DERMATOLOGY

J . R., Multz, C. V. and Fullmer, H. M .: Collagen 17. P ierce, J . A. , E isen, A. Z. and Dhingra, H. K. : R e la olytic activity of synoviu m in rheumatoid ar t ionship of ant itrypsin defi ciency t o t he pathogen thrit is. New-Eng. J . Med., 279: 914, 1968. esis of emphysema. Trans. A ssoc. Amer. Physi 15. Michaeli, D., Ma rtin, G. R. , Kettma n, J ., Benja cia ns, 82: 8, 1969. mini, E., Leing, D. Y. K. and Blatt, B. A.: Locali 18. Perez-Ta mayo; R. : Collagen resorption in carra zation of ant ige nic determinants in the polypep geenin gra nulomas. Lab. Invest., 22: 137, 1970. t ide c hains of collagen. Science, 166: 1522, 1969. 19. Lazarus, G. S.: Unpublished d ata. 16. Ki virikko, K. and Laitinen, 0 .: Clinicial sign ifi 20. Michaeli , D. and Fudenburg, H . H.: Incidence of cance of urinary hydroxyproline determinations in a nt ibodies to denatured collagen in rheumatoid children. Ann Paed. Fenn., II : 148, 1965. arthritis. Arthr. Rheum., /4: 404, 1971.