Expression . of Anionic Sites at the Dertnoepibolic Junction

Motomu M anabe, M .D. , Shigaku Ikeda, M .D ., T sutol11u ' Muramatsu, M .D., and Hidcoki Oga wa, M.D. , Ph. D. Departm cn ts of Derm ato logy, Jun tendo Un ivers ity School o f Medi cine (MM , SI, HO), Tokyo; and N ara Medica l Uni ve rsity (TM) , NaTa, J apan

T he emcrgence of anioni c sites during basement m em brane zone forma tion was studied usin g migratin g epidermis in organ cui tu re as a m odel systcm .

U lt rast ructu ra l in ves tigations Ll sing a strongly ca tionized

Considerable ad vances have bee, n achieved in th e anal­ys is of the loca li za tio n of co ll ageno us and no n co l­lageno us componen ts o n sk in base m ent mem brane zone (sB M Z) (reviewed in 11 -3]). H eparan sul fa te proteoglyca n (H SPG) is a peri cellui;ir m acro m o le­

cule consisting of a co re pro tein bea rin g hepa ran sul fa te g lycos­am inog lycan chains, Jnd was fo und in m an y tiss ll es as a com­po nen t of the base m ent m em brane as well as of the cell surf:tce (rev iewcd in 14J). An tibody prepared aga inst H SPG isolated fro m the Engelb reth- H olm-Swarm sa rco m a m atri x was fo und b y illl­lll unoRu o rescence to react w ith sB M Z 15]. T his antigenic distri­b utio n was co nside red to correspo nd w ith ul tras tructurall y delll­o nstrated anio ni c sites alo ng the epiderm al and derm al edges o f the basa l lamina (BL) [6]. In this locati on , H SPG played a m aj or ro le in th e reg ula tion of permeabili ty b y crea tio n o f a charge­se lective ba rri er (rev iewed in [7]).

Recen tly, it has been ~epo rted th at Sch wa nn cells synthes ize 2 major H SPG that diffe r in size and appa rent distribu tio n. Fur­therJ1lore, the large r (B L-associated) pro teoglyca n accu mul ates o nl y w hen Sch wa nn cells are acti vely sy nth esizin g B L and the accuillula tio n of the sm aller (m embrane-associated) proteog lycan is independen t of B L produ ctio n [S]. N evertheless, detail ed in­formation relat in g to th e synthes is of H SPG d urin g sB M Z fo r­mation has been rather sca rce to date. In the present stud y, the exp ression of an io ni c sites, parti cul arl y in relatio n to that o f th e B L, was in vestigated usin g mi g rating epidermis in o rga n culture (epiboly).

M ATE RI ALS AN D M ETH O DS

Orga n Cul t ure System O rga n culture of adul t human skin was carried o ut accord in g to the m ethod of Hintner et al [91. T he specim ens were kept at 37°C in a humid atmos phere containing 5'1'0 CO2 in air fo r 7 days.

Mallllscript received April 8, 1986; accepted for publica tio n Jul y 8, 1986.

Rep rint rcq ues ts to: Hideoki Ogawa. M .D. , Depa rtmc.1t of Dermatology, Jun tendo Unive rsity School of Medicinc, Hongo 2-1 - 1, 13un kyo-k u , Tokyo 11 3, J apan.

Abbrev iations: BL: basa l lami lla H SPG: hepara n sul fate proteoglyca n (s) PEl: polycth yicncim ine s13MZ: ski n basement mem bra ne zonc

probc revea led that the heparitinase-s1ensiti ve, anionic sites w ere fo rmed syn chronously with the newly built basal lamin a after 7 days in culture. J II/ ves t D erlll atof 88:94-96, 1987

Ultrastructural and Tracer Studies The specim ens were re­m o ved frOIll th e culture afte r 7 days and were stained acco rdin g to th e m eth od repo rted pre vio usly usin g strong ly ca ti oni zed po lyethy leneimine (PE l) as a tracer 16]. Fo r enzy m e di ges tion stud y, th e specimens were di ges ted by 50 units/ IllI heparitin 3se in 0. 1 M sodiulll acetate bu ffe r (pH 7.0) at 43°C fo r 2 h and were sta ined b y th e sa m e m eth od. After ro utin e processin g, ultrathin sectio ns w ere o bserved in a J E M 1200EX electro n microscope.

R ES ULTS

T he BL fo rm atio n alo ng th e derm oepibo li c juncti on lagged far behind the advancin g tip of the mig rating epidermis.

T he ani oni c sites could be vi suali zed in bo th the derm al and epiderm al edges o f th e continu o us area (Fig 1 a) and end point (Fig 1 b) o f the regenerated BL as sm all particl es (approximately 20 nm in di am eter) occwrin g at regul ar di stances fro m each o ther w ith a center-to- cen ter spacin g of approx im ately 60 nm . T hey were rem o ved co mpletel y b y di ges tio n w ith heparitin ase (Fig I e) . T hese results we re identical to that of no rmal sBMZ repo rted previo usly 16]. Ad d itio nall y, th ey were p resen t o n the focal area of regenerated B L subj acent to th e hemid es m oso m e (Fig l li) . Occasio nall y, they could also be detected o n the cell surface of basal cell s th at res ted o n dermal g ro und substan ce and co llagen w itho ut an intervenin g B L stru cture (Fig 1 e). However, the dis­tances between partieles were rather irregular and th e size and electro n density w ere redu ced compared with the continuo us area of regenerated BL, altho ug h it was uncl ea r w hy there were such di fferences.

D ISC U SS IO N

T he distin ct components of sBMZ have been no ted to re-fo rm Lltili zin g organ culture as a m odel system . Hintner et al reported th at bullous pemphigoid anti gens emerged sy nchro no usly w ith th e ad vancing tip o f the mig ratin g hum an epiderm al cells up to 7 days of culture, w hereas type IV collagen and laminin appea red w ith considerable dela y linking to each o ther [9J. Stenl1 et al also repo rted th at in a 4S-h culture o f m ouse skin , mi g ratin g epiderm al cells contained type V co ll agen but no t ty pe IV co ll agen 1'101 ·

In t his stud y, we have sought to electron mi croscopica ll y ex­amin e the fo rm atio n of ani oni c sites in 7-day cultures of no rm al hum an skin , in o rder to bette r understand th eir rel atio nships to the BL fo rm atio n . Ult ras tru ctural studi es usin g a stro ngly ca­tio nized tracer (PE l) dem o nstra ted that the sm all PE l-positi ve

0022-202X/87/l1OJ .SO Copyright © 1987 by T he Socie ty fo r In vestigat ive Der.l1I ato logy . Inc.

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(anionic), heparitinase-sensiti ve particles were present on the co n­tinuous areas , end po int and fo cal area of regenerated BL, and basa l surface w hi ch rested directl y on the dermis without BL. Since it has been reported that mouse mammary epithelial cells deposit at their basa l surf.1ce :111 extra cellubr HSPC that binds to type I coll agen "Il 'j and that type I co ll agen fibril s ca use these cells to accumulate a BL-like la yer 11 2, 13] , our findin gs mi ght suggest that the an ionic sites were form ed synchronously with BL formation , and that th e interaction between the anioni c sites and dermal cO lllponent(s) would be important during sBMZ re­modeling.

There w as ab und ant ev idence that keratin ocy tes synth esized severa l sBMZ components (reviewed in [14]) including sulfated g lycosa minoglycans [1 5, 161. In our present stlld y, howeve r, it remained unclear whether keratinocytes synthes ized BL-associ­ated HSPC or w hat factor(s) were essenti al for HSPG biosyn­thes is. It wou ld be interesti ng in future research to determine w hi ch cell type is producing the anioni c sites, and what other basement membrane components interact fo r sB MZ format ion.

We "'ish ro r!wllk Mr. Mirsllraka Yoshida alld Mr. Karsllhiro Sa'" (Cellrm! EM Lab , Jllllrwdo Urrillc/'siry ) for rlr e;/, rCc/llrien! assisrallcr.

AN ION IC SITES IN E1'I130 Ll C SU IU' ACE 95

Figure 1. E lectron mic rogr~ph s of dcrmoepibolic junction stained with a ca tionic tracer, polyethy lcneimine (PE l). The PEl-positive p~ rtiC\cs were present on the collti nuous :lrc~s (a) and end po int (b) of the newly syn­thes ized ill and were removed after hep~ritina sc diges tion (e). They were also present on the focal are~ of the newly synthes ized Bl subj acent to the hel11ides l11 oso rlIC (d) . Occasio nall y, smaller particles occuring at ir­regular di stan ces with low electron density were present on the cell surface o f basa l cel ls whi ch rested on the dermis w ithout BL (e). Arrowheads indicate PEl = positi ve particles, Bl = basa l lamina . Bm's == 0.2 Mill .

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