Collagenase and Connective Tissue Metabolism in Epidermolysis Bullosa* Gerald S
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TilE .JOUHNAI. OF I N\'ESTJGATJV~: OEKMATOLOG\' Vo l. 58, No.4 Copyright © 1972 by The Willioms & Wilkins Co. Pn"nted in U .S.A. 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.