Necrobiosis Lipoidica: Ultrastructural and Biochemical Demonstration of a Collagen Defect Aarne Oikarinen, M .D ., Ph. D., Minna Mo rtenhul11 cr, M .D ., M atti Kallioincn, M .D., Ph.D., and Eeva-Riitta Savolainen, M. D., Ph.D. Coll agen Resea rch Unit, Unive rsity of O Ulll , Departmcnts of Dc n1l3tology (AO, MM ), Anatomy (AO), Pathology (MK), and Medi ca l Biochcmistry (AO, E-RS), Uni ve rsity of O ulu , O Ulll , Finl and T en pati ents with necrobiosis lipoidica lesions were stud­ lagen was unchanged in the affected skin . Fibrobl asts ied. Five pati ents had diabetes mellitus. The age of the es tablished fr o m affected skin synthesized less coll agen than patients vari ed from 15 to 73 yea rs and the durati on o f the ce ll s deri ved fr om hea lth y-looking skin. T he decreased col­ skin lesions w as fro m 2 to 20 yea rs. Histologica ll y, the lagen synthesis was due to a decreased amount of m essen­ lesions w ere characterized by degeneration of coll agen and ger RN A fo r type I procoll agen, m easured by hybridization elas tin. In som e lesions el as tin fibers could be seen in areas w ith a specific human cDN A cl one. T he producti on of devoid of normal-looking coll agen. Electron microscopy colla genase by these fib robl as ts was not in creased. O ur revea led loss of cross-striation of coHa gen fibrils and a marked res ults thus indica te that in necro bi osis lipoidica lesions, vari atio n in the diameter o f individual coll agen fibrils. T he coll agen fi brils are defecti ve and the amount of coll agen is concentrati on o f co ll agen, m easured by assay of hydroxy­ reduced, probabl y due to decreased synthesis of coll agen proline, a coll agen-specifi c amino acid, was markedl y de­ by affected fibrobl as ts. J f' lllesl Del'lll(1/o/ 88:227-232, 1987 creased in the lesional skin , but the ratio of type lIIll co1- ecrobiosis lipo idica is a chronic skin disease that is lesio ns fro m 2 to 20 yeJrs. Nine of 10 were fe m ales and 5 had o ften associated w ith diabetes mellitus. Typica l le­ d iabetes mellitus. sio ns are irregul arl y delnarca ted , yell owish lesions on the shins. O n histologic examination there is Skin Biopsies Contro l sa m plcs (s ite-matchcd) were taken dur­ degenerati o n o r necro bi osis o f coll agen and poly­ ing therapeutic operati ons on 3ge-matched patients in the Der­ Nm o rp hi c cellular infiltrates composed o f ly mphoid ce ll s, fibro­ n13to logiea l C linic, O ulu University Central Hospi ta l, Finland . bl as ts, and histi ocytes [11 . Sometimes, the dermis contains gran­ All the sa mples were taken in accordance w ith the Decl aration of ul o m atous foci composed of epithclo id cell s and giant cell s. T he Helsinki. degeneratio n and hyaliniza ti o n o f coll agen bundles adj acent to the Skin samples fro m necrobio ti c lesions of 10 patien ts wcre cx­ g ranulo m as are va ri able. The bas ic eti o logy of necrobi osis li ­ cised fo r li ght microscopy, and, in 4 patients, fo r electro n mi­ po idica is unknown. H owever, focal degeneratio n o f coll agen has croscopy. been suggested to have a central role in its pathogenesis II]. The Sa mples fo r li g ht microscopy w ere fi xed in 10% phosphate­ purpose o f the present study was to examine coll agen by ultra­ buffered fo rmalin , processed ro uti nely, and embedded in paraffi n. structural and biochemica l m eans in necro biosis Iipo idica les io ns. Secti o ns were cu t at 5 j.Lm and stai ned w ith H & E and Verhoeff­ van Gieson stains. Specimens fo r electron microscopy were fixed PATIE NTS AND M ETH O D S in 4% g lutarald ehyde, postfixed in 1 % osm ium tetroxide, and embedded in E pon. Ultrathin secti ons were stained w ith uran yl The bi o psy sa mples w ere taken fro m affected and nonaffected acetate and lea d citrate and examined in a Philips 410 LS trans­ (s ite-matched) skin of l O patients. T he clinica l chara cteri zation of mission electro n microscopc. the patients g iven in T able I dem onstrates heterogeneity in the Primary cell cultures were established by routine m ethods, and ages o f t he patients and the durati on of disease. T he age of the subcul tivated on plasti c cul ture dishes in D ulbccco's m odified pati ents va ri ed fro m 15 to 73 yea rs, and durati on of necrobio ti c Eagle 's medium (DMEM ) supplemented with 10% fetal ca lf serum, 50 j.L g/ml of ascorbate, 290 j.L g/ml L-glu ta m ine, penicillin (100 Manu sc ript recc iv cd Ma y 28, 1986; acce pted for publication August 22, U / ml), and strepto m ycin (100 j.L g/ml). Analyses of fib ro blast 1986. cul tures were ca rried out at 4-8 passages of subculti va ti o n. Supported in part by a grant from the Medica l Resca rch Coun cil of th e Acade my of Finland. Collagen Biosynthesis Studies Fibro blasts at confluence were Reprint req ues ts to: Aarn c O ik arin cn, M . D., Dc partmcnt of De rm a­ in cubated fo r 24 h in D ME M supplcmented as above, except that to logy, O ulu Unive rsity Ccntral Hos pital, K'\iaanif1(ie 50, SF-90220 O ulll , the serum w as repla ced w ith 2% dialyzed feta l calf serum, and Finl and . [I 4C]proline (2 j.L C i/ ml) w as added . After the labeling peri od the Abbrcv iati ons: mcdium was coll ected and pro teinase inhibitors were added to DMEM: Dul becco's modificd Eagle's medium give fin al con centrati ons of 25 111 M N a2EDT A, 10 m M N-cthyl­ GGT: gal actosy lh yd roxylysy l glu cosyltransferase PH: prolyl 4-hydroxyla sc m aleil11i dc, I m M phen ylmerhylsul fo nyl fl uoride, and I mM SDS-PA GE: sodium dodecy l sul fa te-polyac rylamide gel paraa mino benzam id ine. T he m ediul1l p roteins wen: then precip­ el ec trophores is itated by add ing ammoniul11 sul fa te to a fi nal concentratioll of 0022-202X/87/S03.50 Copyri ght © 1987 by T hc Socicty fo r In ves ti gati ve DC r11l3 tology, In c. 227 228 O IK A IUNEN lOT AL THE JOURNAL O F INV ESTIGATIV E DERMATOLOGY Table I. C linica l Data on Patients With N ecrobiosis Lipoidica Age Diabetes, D uration Code Sex (years) (yea rs) C linica l Findings 1 F 69 Yes, 17 O n the anteri o r site of legs, lesio ns fo r 10 yea rs 2 F 73 N o O n the left leg, Ilumero us les ions fo r 8 years 3 F 56 No O n th e left leg, some les io ns for 3 yea rs 4 F 18 Yes, 6 O n the lower legs, lesions for 5 yea rs 5 F 65 N o Above the ankles, numerous les ions for 20 years 6 F 50 Yes, 10 O n th e legs, les ions fo r 2 yea rs 7 F 15 Yes , 14 O n the lower legs, lesions for 8 yea rs 8 F 49 N o O n the legs, lesions for 7 yea rs 9 F 53 N o O n ri ght legs, 2 les ions fo r 5 yea rs 10 M 20 Yes, 13 O n legs, some lesio ns 290 mg/ml, and th e precipitates were coll ected by centrifuga ti on by addin g soya-bea n trypsin inhibito r (50 /-L g/ml) , and coll agen ase for 30 min at 1 0,000,~ after stirring overnight at 4°C. This material activity was assayed by incubatin g samples with radioactive type was used fo r the assays of 1"'C lh yd roxypro lin e and total in cor­ I coll agen, as described previo usly [1 2], The coll agenase activity po ration of I'IC radioactivity by a specifi c radi ochemi ca l method was expressed as degradation of 31-:1-labeled coll agen, dpm X 12], h - '/ mg D NA, The ce ll layer was rinsed with phosphate-buffered saline and Analyses of Total Collagen and Collagen Types The amount the ce ll s scraped w ith a rubber poli ce lllan into 2 ml of 0.4 M of hydroxyprolin e, a measure of colla gen, was determined by a N aCI, 0, 1 M Tris-HC I, pH 7,5, contain ing th e proteinase inhib­ specific colorimetric assay [1 3], itors described above, T he ce ll s were soni cated at 60 Hz for 30 For determination of genetica ll y distinct coll agen types, ti ssu e s, T hese sa mples were used for assays o f total ce ll layer protein specimens were ho mogenized in 0,5 M acetic acid and submitted [31 and DNA 141, to limited proteolysis by pepsin (Worthington, 2 X crystalli zed), Part of the am 111 0 niull1 sulfate precipitate of the culture medium at a fin al concentration of 300 /-L g pepsin per mL The sa mples was used for 6% sodium dodecyl sulfate-polyacrylamide gel elec­ were in cubated for 3 h at 24°C, fo ll owe'd by 16 h at 4°C The trophoresis (SDS-PAGE) after reducti on 151 and radioactive pep­ pepsin -solu bilized m aterial was recovered by centrifugation for tides were visualized by flu orography 16J, 60 min at 37,000g at 4°C, and the insoluble material was subjected Assay of Type I Procol\agen mRNA Steady-State Level to further pepsinization as above, The supernatants ' containing Total RNA was isolated as described previo usly 17] and used for th e pepsin -solubilized materi al were combined, and protease in­ dot-blot hybridization assay [8J, The RNA sa mples were dotted hibitors at the concentrations indicated above were added, The (0,3-0,9 /-L g of total RNA) onto nitrocellulose paper, and the pH o f the sa mples was adjusted to 8,5 by adding 1 M Tris, and fi lters were hybridized w ith recombinant plas mid Hf677 con­ th e sa mples were in cubated for 60 min at 4°C to in activate pepsin, taining cDNA for human proal (I ) coll agen mRNA [9], The re­ T he sa mples were then dia lyzed ,against 0.4 M NaC l, 10 mM Tris­ combinant pl asmid was labeled w ith 132 P lnucleotides to a specifi c HC I, pH 7,5, containing the proteinase inhibitors, Coll agen was acti vity of5-8 x 10M cpm/ /-Lg by ni ck-translati on [1 0], T he amount precipitated by adding N aCI to a fin al concentration of 4.4 M of recom binan t 32 p_la beled plasmid hybrid ized to III RNA was and the precipitate was coll ected by centrifugation, visuali zed by autoradiograph y usin g Kodak X-Omat film and The SDS-PAGE was performed Ll sin g 8% polyacrylamide gels, cassettes w ith intensifyin g screens, T he autoradiograms were w ith and without delayed reduction w ith 2-mercaptoethanol [14) , quantitated by sca nning w ith a Kontes K 495000 densito meter The colla gen polypeptid es were visuali zed by stai'ling with connected to a Spectra-Physics SP4 \ 00 computing integrator.