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1989 37: 961J Histochem CytochemY Horiguchi, J R Couchman, A V Ljubimov, H Yamasaki and J D Fine

proteoglycan in human skin and other basement membranes.Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate

  

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961

0022-1554/89/$3.30

The Journal of Histochemistry and Cytochemistry

Copyright © 1989 by The Histochemical Society. Inc.

Vol. 37, No. 7, pp. 961-970, 1989

Printed in USA.

Original Article

Distribution, Ultrastructural Localization, and Ontogeny

of the Core Protein of a Heparan Sulfate Proteoglycan in

Human Skin and Other Basement Membranes”2

YUJI HORIGUCHI, JOHN R. COUCHMAN, ALEXANDER V. LJUBIMOV,

HIROSHI YAMASAKI, and JO-DAVID FINE3

Departments ofDermatology (YH, JDF) and Cell Biology and Anatomy (JRC), University ofA/abama at Birmingham

School of Medicine, Birmingham, Alabama; All- Union Cancer Research Center of the USSR Academy of

Medical Sciences, Moscow, USSR (AVL); International Agency for Research on Cancer� Lyon, France (HY); and

Dermatology Section, Medical Service, Birmingham Veterans Administration Medical Center, Birmingham, Alabama (JDF).

Received for publication September 7, 1988 and in revised form January 24, 1989; accepted January 31, 1989 (8A1480).

A variety ofheparan sulfate proteoglycans (HSPG) have been

identified on cell surfaces and in basement membrane (BM).To more fully characterize HSPG in human skin BM, we used

two monodonal antibodies (MAb) directed against epitopesof the core protein of a high molecular weight HSPG iso-lated from murine EHS tumor. Indirect immunofluorescencerevealed linear distribution ofHSPG within all skin BM, and

within BM ofall other human organs investigated. In a study

of the ontogeny of HSPG in human skin BM, HSPG wasdetectable as early as 54 gestational days, comparable withother ubiquitous BM components, such as laminin and typeIv collagen. J.mmunoelectron microscopy on adult skin and

neonatal foreskin showed staining primarily within the lam-

Introduction

Basement membranes (BM), specialized pericellular extracellular

matrices, are believed to influence many aspects oftissue behavior,

such as growth, migration, and differentiation (1-3). In addition,

defects in this matrix can be associated with cancer invasion (4,5)

and certain other diseases, most notably the various types of in-

herited and acquired epidermolysis bullosa (6-14) and bullous pem-

phigoid (15-17). In addition to type IV collagen (18-20), fibronec-

tin (20-23), and the glycoproteins laminin and entactin/nidogen

i Supported in part by the Medical Research Service of the Veterans

Administration (JDF) and the National Institutes of Health (NIAMS

AR34861 and AR36629, JDF; NIAMS AR36457, JRC).2 Presented in part at the annual national meeting of the Society for

Investigative Dermatology, Washington, DC, April 28, 1988, and the Eighth

International Congress of Histochemistry and Cytochemistry, Washington,DC, August 1, 1988.

3 Correspondence to: Jo-David Fine, MD, Department of Dermatol-ogy, Univ. of Alabama Sch. of Med., UAB Station, Box 76, Birmingham,

AL 35294.

ma densa (LD) and sub-lamina densa regions of the der-moepidermal junction (DEJ) and vascular BM. In neonatalforeskin, additional staining was noted of basilar cytoplas.mic membranes ofkeratinocytes, endotheial cells, and pen-

cytes. We conclude that the core protein ofa high molecularweight HSPG is ubiquitous in human BM, appears in fetalskin on or before 54 days, and is present primarily in theregions of the LD and sub-LD. (J Histochem Cytochem

37:961-970, 1989)KEY WORDS: Heparan sulfate proteoglycan; Basement membrane;

Human ontogeny; Immunoelectron microscopy; Immunofluores-

cence; Immunoperoxidase; Human skin; Fetal skin.

(20,24-29), which appear to be ubiquitous basement membrane

zone components, a variety of antigens or antigenic epitopes have

been described through the use ofmonoclonal and polyclonal an-

tibodies, some of which are unique to the dermoepidermal junc-

tion (DEJ) of human skin and related epithelia (30-36). Most of

the antigens defined by the latter, however, are as yet not well charac-

terized.

Proteoglycans are widely distributed in BM, some forms of which

are restricted in their distribution whereas others are widespread

(13,37-42). The murine EHS tumor has been a source of heparan

sulfate proteoglycans (HSPG) (43,44), from which both large and

small forms have been isolated. The large form has a core protein

>400 KD and is approximately 70% protein and 30% carbohydrate.

Although HSPG is known to be present in a number of rodent

tissues (37,40,41,45), its distribution in the human has not been

well defined, and to our knowledge nothing has been published

regarding its ontogeny. Here we describe the distribution and ul-

trastructural localization ofa large HSPG in human adult, neona-

tal, and fetal skin, using two core protein-specific monoclonal an-

tibodies (MAb). In addition, we have investigated the distribution

962 HORIGUCHI, COUCHMAN, LJUBIMOV, YAMASAKI, FINE

Sodium Chloride-split Skin Studies. Human foreskin and adult hu-

man arm skin were chemically separated at the level of the lamina lucida

of this particular HSPG in a wide variety of other normal human

tissues.

Materials and Methods

Source of Immunoreagents. Fluorescein- and rhodamine-conjugatedgoat anti-rat lgG, horseradish peroxidase-conjugated goat anti-rat lgG, and

normal goat serum were obtained from Cappel Laboratories (West Chester,PA). High-titer polyclonal antibodies reactive against human laminin and

type IV collagen were the gift of Dr. Stephen I. Katz (National Cancer In-

stitute, NIH; Bethesda, MD). Unless otherwise specified, all dilutions were

prepared in 0.0067 M PBS, pH 7.4.

Source of Anti-HSPG Monoclonal Antibodies. Two MAb-producingrat x mouse hybridoma cell lines. Cl 1L1 and A7L6, were generated after

immunization of Fisher rats with a murine EHS tumor laminin prepara.

tion containing entactin and HSPG. Details of the protocol have been

documented previously (46,47). Both of these IgG2� i MAb, obtainedfrom hybridoma culture supernatants, were shown to have binding speci-

ficity for the core protein ofa high molecular weight (>400 KD), low buoy-

ant density HSPG present in EHS tumor and cultured human endothelium

U R Couchman et al., submitted).

Source of Tissues Examined. Normal human skin was obtained from

adult volunteers, surgical amputations, or cadavers, and neonatal foreskin

was obtained from elective circumcision. The following normal or mor-

phologically unaltered human organ specimens were obtained at the time

of either autopsy or elective surgery via the Tissue Procurement Service at

the University of Alabama Medical Center, or from the Department of

Pathology. All-Union Cancer Research Center, USSR Academy of Medical

Sciences: buccal mucosa, tongue, upper and lower esophagus, stomach,

duodenum, jejunum, ileum, large intestine. anal canal, kidney, ureter, un-nary bladder, urethra, Fallopian tube. uterus, cervix, vagina, larynx. tra-

chea, bronchus, lung, aorta, skeletal muscle, spleen, lymph node, salivarygland, mammary gland, liver, gallbladder, and placenta.

Sixteen skin specimens from electively aborted normal human fetuses,

ofestimated gestational ages ranging from 54-142 days, were kindly provided

by Dr. Karen Holbrook and the Central Laboratory for Human Embryol-

ogy at the University ofWashington (Seattle, WA). These same fetal skin

specimens were previously used and were shown to express laminin, typeIV collagen, LDA-l (a novel ubiquitous collagenase-resistant lamina densa

antigen). and chondroitin 6-sulfate-containing proteoglycan over this en-

tire range of gestational ages (48-50).

Indirect Immunofluorescence Studies. Indirect immunofluorescence was

performed on each of the previously noted adult, neonatal, and fetal hu-

man tissues, following embedding in OCT compound (Lab-Tek Products;Naperville, IL) and freezing in liquid nitrogen. In brief, 6-8-�tm cryostat

sections were prepared from each specimen and placed on albuminized

glass slides. Each specimen was first exposed to one of the two anti-I-ISPG

core protein MAb (undiluted; 30 mm; room temperature; humidified cham-

ber), rinsed extensively with PBS, and then incubated in the presence of

either fluorescein- or rhodamine-conjugated goat anti-rat lgG (1 20, 30 mm;

room temperature). After further rinsing with PBS, 50% glycerol in PBS

was placed over each tissue section, each slide overlaid with a coverslip, and

the tissue sections examined with a Leitz Laborlux 12 immunofluorescence

microscope by epi-illumination.

In those experiments involving human fetal skin, as an additional posi-

tive control for proper tissue orientation and the presence of intact base-

ment membrane in each specimen. the normal expression of laminin andtype IV collagen was concurrently demonstrated by indirect immunofluo-rescence using two well-characterized polyclonal antibodies.

ofthe DEJ, using a technique previously described (51). In brief, fresh in-

tact neonatal human foreskin was surgically stripped of subcutaneous fat

and excess dermis and then incubated on the surface ofa 1 M sodium chlo-

ride solution (4’C; 72 hr). After gentle rinsing in PBS, the epidermis was

easily separated mechanically from the dermis as an intact sheet. Both epider-

mal and dermal portions were individually embedded in OCT and snap-

frozen in liquid nitrogen. Cryostat sections (6-8 �tm) were then prepared

and indirect immunofluorescence performed with each anti-HSPG core pro-

tein MAb as previously described. As an additional control for uniform

intra-lamina lucida cleavage in this tissue preparation, indirect immuno-

fluorescence was also performed using auto-antibodies from patients pre-

viously well characterized in our laboratory (by direct and indirect immu-

nofluorescence; direct immunoelectron microscopy) as having bullous

pemphigoid and epidermolysis bullosa acquisita.

Indirect Immunoelectron Microscopy. Indirect immunoelectron micros-

copy was performed with each anti-HSPG core protein, using 10 �tm cryosec-

tions of 3 % paraformaldehyde-fixed human adult skin and neonatal fore-

skin as tissue substrates. Both intact and 1 M NaCl-split tissues were examined

by this technique. Tissue sections were serially incubated with normal goat

serum (1:10, 20 mm; room temperature), anti-HSPG core protein mono-

clonal antibody (1:3-1:12; 18 hr; 4’C), and horseradish peroaldase-conjugated

goat anti-rat IgG (U20-180; 2 hr; room temperature). As a negative con-

trol, normal rat serum (1 l0-m : 100) WA� substituted for each of the two MAb.After re-fixation with 1% glutaraldehyde, tissue sections were then incubated

with diaminobenzidine and hydrogen peroxide, post-fixed with 1% osmium

tetroxide, dehydrated with a gradient alcohol series, and embedded in Spurr’sresin. Ultra-thin sections were counterstained with lead citrate and then

examined under a Phillips 300 electron microscope.

ResultsAs shown in Figure 1, both of the anti-HSPG core protein MAb

bound to all BM (DEJ, dermal vasculature, appendages) in adult

human skin and neonatal foreskin. Staining was linear and uniform,

identical to that observed with other ubiquitous skin BM antigens,

including laminin (25), type IV collagen (19), and LDA-1 (35).

Similarly, continuous linear staining was noted with each anti-

HSPG MAb antibody along all BM in each of the adult human

Figure 1 . Indirect immunofluorescence of adult human skin demonstrating thepresence of HSPG core protein within BM of the DEJ (single arrows), vascula-ture (double arrows), and glandular structures (not shown). Asterisk denoteslocation of epidermis. Original magnification x 368. Bar = 100 pm.

Figure 2. Indirect immunofluorescence of human oral cavity and gastrointesti-nal tract demonstrating the presence of HSPG core protein within BM. (a) Buc-cal mucosa; (b) esophagus; (C) duodenum; (d) jejunum; (e) anal canal. Singlearrows denote epithelial-connective tissue interface; double arrows, vascularbasement membranes. Original magnification x 230. Bars = 50 pm.

organs examined (Figures 2-4). BM staining was ofapparent equal within the mesangial matrix. In the liver, the sinusoids were out-

LOCALIZATION OF SKIN HEPARAN SULFATE PROTEOGLYCAN 963

intensity along epithelial-connective tissue junctions and around

vasculature, muscles, and nerves. In the kidney, HSPG core pro-

tein was detectable within both glomerular and tubular BM, and

lined.

When indirect immunofluorescence was performed with each

anti-HSPG MAb on intra-lamina lucida-separated human neona-

,�. ‘1’s-�:

3.

..- �, .,- ‘.�,.-,. ,,

964 HORIGUCHI, COUCHMAN, LJUBIMOV, YAMASAKI, FINE

Figure 4. Indirect immunofluorescence of human trachea (a), skeletal muscle(b), and kidney (C); HSPG core protein is detectable in all BM within these tis-sues. (a) Single arrow denotes tracheal epithelial-connective tissue interface;double arrows, vascular basement membranes. (b) HSPG core protein-con-taming basement membranes surround skeletal muscle (single arrow) and ad-jacent vasculature (double arrows). (C) Single, double, and triple arrows denotebasement membranes surrounding glomerulus, Bowman’s capsule, and renaltubules, respectively. Original magnifications: a, b x 230; C x 286. Bars =

50 pm.

LOCALIZATION OF SKIN HEPARAN SULFATE PROTEOGLYCAN 965

tal foreskin, staining was noted only along the dermal portion of

the DEJ and around dermal vasculature (Figure 5), suggesting the

predominant localization of HSPG core protein to be at or beneath

the level of the lamina densa. On simultaneously stained sections,

bullous pemphigoid and epidermolysis bullosa acquisita antigens

were present on the epidermal and dermal portions of the DEJ,

respectively, confirming intra-lamina lucida cleavage in this sub-

strate (51).

Sixteen human fetal skin specimens ofestirnated gestational ages

ranging from 54-142 days were similarly examined by indirect im-

munofluorescence with each of the two anti-HSPG core protein

MAb (Figure 6). Intact DEJ staining was seen in all fetal specimens

with each antibody, consistent with previously published data from

our laboratory with antibodies to laminin, type IV collagen, WA-i,

and chondroitin 6-sulfate-containing proteoglycan (38,48-50) and

coinciding approximately with the time offirst visualization of lam-

ma densa in intact human skin (52).

Indirect immunoelectron microscopy was performed on both

intact and 1 M NaCI-separated adult skin and neonatal foreskin,

using an immunoperoxidase staining technique; findings were iden-

tical for both MAb. In intact neonatal foreskin, reaction products

were primarily detectable along the lamina densa and along the

basilar cytoplasmic membrane of basal keratinocytes (Figures 7a

and 8a). At higher magnification, these immunoreactants were also

shown to be associated with hemidesmosomes, as well as being pres-

ent to a lesser extent within the lamina lucida, on occasional colla-

gen fibers, and on anchoring fibrils adjacent to the lamina densa.

In adult arm skin, more diffuse immune deposits were noted in

the region of the lamina densa and upper dermis (Figures 7b and

8b), whereas basal keratinocyte membrane staining was weak and

focal, or absent. At higher magnification, some deposits were also

noted within the lamina lucida and on hemidesmosomes. In both

adult skin and neonatal foreskin, dense deposits of immunoreac-

tants were also noted along and surrounding the basement mem-

branes ofcapillaries, as well as the basilar cytoplasmic membranes

ofendothelial cells and pericytes ofdermal blood vessels (Figure 9).

When indirect immunoelectron microscopy was similarly per-

formed on 1 M NaCI-split neonatal foreskin and adult arm skin,

basal keratinocyte cell membrane staining was noted in areas of

partial but not complete separation of epidermis from the dermis

Figure 5. Epidermal (a) and dermal (b) portions of human skin separated with 1 M sodium chloride; in completely separated skin, HSPG core protein localizesto the dermal portion of the DEJ (solid arrows) and adjacent dermal vasculature (double arrows). Open arrows in a depict absence of staining by anti-HSPG coreprotein MAb along the epidermal side of the DEJ. Asterisk denotes unstained epidermis. Original magnification x 230. Bars = 50 pm.

Figure 6. Indirect immunofluorescence microscopy demonstrating the distri-bution of HSPG core protein in intact human fetal skin (142 gestational days).HSPG core protein is present along the DEJ (single arrow) and in basementmembranes surrounding dermal vasculature (double arrows), tangentially sec-tioned hair follicles (open arrows), arrector pili muscles, and glandular struc-tures (neither of the latter two present in this particular field). Original magnifi-cation x 219. Bar - 50 pm.

966 HORIGUCHI, COUCHMAN, LJUBIMOV, YAMASAKI, FINE

(Figure lOa). At higher magnification (Figure lOb), such deposits

were present just beneath hemidesmosomes, and decorated occa-

sional fine filaments which traversed the lamina lucida.

Discussion

To date, at least 17 antigens or antigenic epitopes have been de-

tected in human skin BM using immunohistochemical techniques

(53). Despite this, however, only a few have been well character-

ized immunologically and biochemically. One such component,

heparan sulfate proteoglycan (HSPG), has been recently identified

within the lamina densa of human skin DEJ by a polyclonal anti-

body (13). We have recently produced two MAb with proven bind-

ing specificity for the core protein ofa high molecular weight HSPG

from EHS mouse tumor, crossreacting with human high molecu-

lar weight HSPG. These immunoreagents have been used here to

further address the issues ofskin BMZ ontogeny, human organ dis-

tribution, and age-dependent ultrastructural localization of HSPG

in the BM of both DEJ and dermal vasculature in human skin.

Using both MAb, we have confirmed that high molecular weight

HSPG core protein is present in all BM of human skin and have

further demonstrated that HSPG core protein is present in the BM

of all other human organs we have surveyed. Such ubiquitous cx-

pression in tissues with otherwise marked differences in structure,

location, and function suggests that HSPG must serve one or more

critical roles in all basement membranes. Additional evidence of

the basic importance ofhigh molecular weight HSPG core protein

is its detection in human skin BM as early as 54 estimated gesta-

tional days, coinciding with the approximate time of first detec-

tion ofan intact lamina densa in this tissue (52), a finding shared

with few other BM antigens (laminin, type IV collagen, fibronec-

tin, LDA-i, and chondroitin 6-sulfate-containing proteoglycan)

(38,48-50). Since others have shown that the large EHS tumor HSPG

can interact with laminin and type IV collagen (45,46) and may

also self-associate (54), it is possible that this proteoglycan has a

structural role in basement membrane integrity. This would be con-

sistent with its ubiquitous distribution, unlike that ofa small HSPG

detected recently by a polyclonal antibody (37).

By indirect immunofluorescence using NaCl-split skin as a tis-

sue substrate, it appears that the majority of HSPG core protein

in neonatal human skin DEJ is present within and/or beneath the

level of the lamina densa. This coincides with recently reported

data with a polyclonal antibody to HSPG (13). When the ultra-

structural localization ofeach anti-HSPG core protein MAb is more

precisely examined by indirect immunoelectron microscopy, how-

ever, it is clear that the localization of this antigen is far more corn-

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Figure 8. Infrequently in human foreskin (a), HSPG core protein was almostexclusively associated with the inferior portion of basal cell (BC) membranes(arrows), although focal sparse upper dermal deposits (star) were still detect-able. In contrast, diffuse deposits (stars) were usually visible in the upper dermis(De) in adult human skin (b) but infrequently associated, except focally, withbasal cell membranes. Original magnification x 15,500. Bars = 1.0 pm.

LOCALIZATION OF SKIN HEPARAN SULFATE PROTEOGLYCAN 967

Figure 7. Indirect immunoelectron microscopic localization of HSPG core protein in the DEJ of neonatal foreskin (a) and adult arm skin (b). (a) In neonatal foreskin,reaction products are distributed along the lamina densa (arrowheads), dermal surface of the basal cells (BC) (arrows), and only minimally within the uppermostdermis (De) (star). (Inset) Enlarged rectangular area showing reaction products associated with the lamina densa (large arrowhead), anchoring fibrils (small arrow-heads), collagen fibers (double arrowhead), hemidesmosomes (arrows), and even sparsely within the lamina lucida (small arrows). (b) In adult skin, reaction prod-ucts are distributed along the lamina densa (arrowhead), much more diffusely present within the upper dermis (stars), and only focally and weakly along basalcell membranes (arrows). (Inset) Enlarged rectangular area showing reaction products within the lamina densa (arrowhead), lamina lucida (small arrow), hemides-mosomes (arrows), and along collagen fibers (double arrowheads), as well as more diffusely in the sublamina densa region. Original magnifications: a, b x12,500; insets x 53,500. Bars = 1.0 pm.

� plex than that recently reported, and appears to be dependent on

the age of the individual examined. Although we have detected

some HSPG core protein within the lamina densa of the DEJ and

dermal vasculature, consistent with recent work with a polyclonal

antibody (13), in adult skin this antigen is even more densely pres-

ent within the adjacent dermal connective tissue (i.e. , sub-lamina

densa region) and to a much lesser extent is also detectable within

the lamina lucida. The presence ofsub-lamina densa deposits may

be analogous to the recent findings oftype VII collagen in aggregates

within the upper dermis (“anchoring plaques”) (55 ) as well as within

anchoring fibrils (33). In neonatal foreskin, HSPG core protein is

also readily detectable at the level of the basilar cytoplasmic cell

membrane of basal keratinocytes, suggesting a possible cellular

source for HSPG in skin during early development.

Apparent discrepancies in basal keratinocyte membrane stain-

ing in neonatal foreskin by indirect immunofluorescence and in-

direct immunoelectron microscopy can be explained when the lat-

.o ter technique is applied to 1 M NaCI-separated tissue. As illustrated

. in Figure 9, whereas basilar cytoplasmic membrane staining is

detectable in unseparated areas, HSPG core protein appears to be-

End -a

Lu

V. i�I1b ‘�.T.1

1-

4.,

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I

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.,i

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968 HORIGUCHI, COUCHMAN, LJUBIMOV, YAMASAKI, FINE

./ Pc

i�t ;�:.

�. �

‘� � �

� ,,.:�

t. � �FV�i/’

�t: � ‘�

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Figure 9. Indirect immunoelectron micro-scopic localization of HSPG core proteinaround adult dermal blood vessels. Reac-tion products are diffusely distributed alongthe basement membrane zone of a capillaryendothelialcell(End)and a pericyte(Pc). Lu,lumen. (Inset) Enlarged rectangular areashowing reaction products along the cyto-plasmic membrane of the endothelial cell(arrow), within the lamina lucida (small ar-row), and within and below the lamina densa(arrowhead). Original magnification x 8500;inset x 31,000. Bar = 1.0 pm.

.s

LOCALIZATION OF SKIN HEPARAN SULFATE PROTEOGLYCAN 969

come dissociated and then lost from basal cell membranes as intra-

lamina lucida cleavage becomes more complete. At higher mag-

nification, some ofthese immunoreactants appear to be associated

with anchoring filaments present within portions of the lamina

lucida directly underneath hemidesmosomes.

Several conclusions can be made on the basis of our findings.

First, high molecular weight HSPG core protein is a ubiquitous

component of human BM. As such, it can be concluded that it

serves some integral function(s) in maintenance of homeostasis in

a variety ofotherwise disparate organs. Similarly, HSPG is present

in all skin BM and at the earliest time at which BM is formed, fur-

ther supporting the hypothesis that high molecular weight HSPG

core protein contributes to the early structural integrity ofthe epider-

mal-connective tissue interface. Second, the detection of HSPG

core protein beneath as well as within the lamina densa suggests

the likelihood of its physical interaction with other extracellular

matrix components present both within the BM itself and within

the upper dermis. As evidence for the latter, these anti-HSPG core

protein MAb were noted to decorate occasional anchoring fibrils

and collagen fibers within the uppermost portion of the dermis.

Furthermore, the apparent association ofHSPG core protein to an-

choring filaments in NaCI-split skin, as well as its sparse presence

within the lamina lucida in intact skin, suggests that a portion of

high molecular weight HSPG core protein is capable of spanning

the basement membrane zone, thereby providing additional in-

teraction within the overlying epidermis. Finally, the differences

noted in the ultrastructural localization of HSPG core protein in

adult and neonatal skin specimens, with regard to the presence

or absence of basilar keratinocyte cell membrane staining as well

as the extent ofsub-lamina densa deposition, suggest possibly differ-

ent functional roles or interactions ofthis antigen at different ages,

as well as significant antigenic reorganization or redistribution within

human skin BM as a function of age.

Acknowledgments

We gratefully acknowledge the excellent technicalassistance ofMs Barbara

Stewart, Mr Ken Bynum, and Ms Lisa Rodgers during the course of these

studies. We thank Proffury M. Vasiliev (All-Union Cancer Research Cen-

ter� USSR Academy ofMedical Sciences, Moscow) for helpful comments

andcritical reading ofthe manuscript, Dr Stephen I. Katz (National Can-

cer Institute, Bethesda, MD)for the generous gift ofanti-laminin and anti-

type IVcollagen antibodies, andDrKaren Holbrook (University of iVashing-

ton, Seattle, WA) for the generous gift ofhuman fetal skin specimens.

Literature Cited1. Bissell MJ, Hall HG, Parry G. How doesthe extracellular matrix direct

gene expression’ J Theor Biol 1982;99:31

2. Bernfield M, Banerjee SD, Koda JE, Rapraeger AC. Remodelling of

basement membrane as a mechanism ofmorphogenetic tissue interac-

tion. In Treistad RC, ed. The role of extracellular matrix in develop-

ment. New York: Alan R Liss, 1987:545

3. Hay ED. Cell-matrix interaction in the embryo: cell shape, cell sur-

face, cell skeletons, and their role in differentiation. In Trelstad RC,

ed. The role ofextracellular matrix in development. New York: Alan RLiss, 1984:1

4. Liotta LA. Tumor invasion and metastasis- role ofthe extracellular ma-

trix: Rhoads Memorial Award Lecture. Cancer Res 1986;46:1

5. Martinez-Hernandez A, Amenta PS. Basement membrane in pathol.

ogy. Lab Invest 1983;48:656

6. Goldsmith LA, Briggaman RA. Monoclonal antibodies to anchoringfibrils for the diagnosis of epidermolysis bullosa. J Invest Dermatol

1983;81:464

7. FineJD, Breathnach SM, Hintner H, Katz SI. KF.1 monoclonal anti-

body defines a specific basement membrane antigenic defect in dys-

trophic forms of epidermolysis bullosa. J Invest Dermatol 1984;82:35

8. FineJD. Epidermolysis bullosa: variability of expression of cicatricial

pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquis.

ita antigens. J Invest Dermatol 1985;85:47

9. Heagerty AHM, Kennedy AR, Leigh IM, Purkis P. Eady RA. Identifi.

cation ofan epidermal basement membrane defect in recessive forms

ofdystrophic epidermolysis bullosa by LH 72 monoclonal antibody:

use in diagnosis. BrJ Dermatol 1986;115:125

10. Heagerty AHM, Kennedy AR, Eady RAJ, Hsi BL, Verrando P. Yeh CJ,

Ortonne JP. GB3 monoclonal antibody for diagnosis of junctional

epidermolysis bullosa. Lancet 1986;i:860

11. FineJD, Gay S. LDA-1 monoclonal antibody: an excellent reagent for

immunofluorescence mapping studies in patients with epidermolysis

bullosa. Arch Dermatol 1986;122:48

12. FineJD. Altered skin basement membrane antigenicity in epidermol-

ysis bullosa. Curr Prob Dermatol 1987;17:lll

13. Caughman SW, Krieg T, Timpi R, Hinter H, Katz SI. Nidogen andheparan sulfate proteoglycan: detection of newly isolated basement

membrane components in normal and epidermolysis bullosa skin. JInvest Dermatol 1987;89:547

14. Woodley Dl’, Briggaman RA, O’Keefe EJ, Inman AO, Queen LL, Gam-

mon WR. Identification ofthe skin basement-membrane autoantigen

in epidermolysis bullosa acquisita. N EngI J Med 1984;310:1007

15. Diaz L, Calvanico N, Tomasi T, Jordon R. Bullous pemphigoid anti-

gen: isolation from normal human skin. J Immunol 1977;118:45

16. StanleyJR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI: Char-

acterization of bullous pemphigoid antigen: a unique basement mem-

brane protein of stratified squamous epithelia. Cell 1981;24:897

17. StanleyJR, Woodley DT, Katz SI. Identification and partial character-

ization of pemphigoid antigen extracted from normal human skin. JInvest Dermatol 1984;82:108

18. Miller EJ, Gay S. The multiple types and forms ofcollagen. Meth En-

zymol 1982;82A:3

19. Yaoita H, FoidartJ-M, Katz SI. Localization of the collagenous com-

ponent in skin basement membrane. J Invest Dermatol 1978;70:191

20. Laurie GW, Leblond CP, Martin GR. Localization of type IV collagen,

laminin, heparan sulfate proteoglycan, and fibronectin to the basal

Figure 10. Indirect immunoelectron micrograph of 1 M NaCI-split foreskin (a) and adult skin (b) incubated with anti-HSPG core protein MAb. (a) Reaction productsare located in the lamina lucida, lamina densa (arrowhead), and the dermal portion of basal cells (BC)(large arrow) in unseparated (left margin offield) and partiallyseparated (double arrowhead) skin. Apparent reaction products (small arrows) are located between the dermis (De) and separated epidermis and along the laminadensa (triple arrowheads), but not along the epidermal roof (double arrows), in completely separated areas (asterisk). (b) Reaction products are distributed inthe sub-lamina densa, lamina densa (arrowheads), and beneath the hemidesmosomes (arrow). In the enlarged Iamina Iucida, reaction products are associatedwith occasional fine filaments (small arrows) connecting epidermis and dermis; note reaction products (empty arrowhead) detached from a hemidesmosome (dou-ble arrows). Original magnifications: a x 26,500; b x 22,000. Bars = 1.0 pm.

970 HORIGUCHI, COUCHMAN, LJUBIMOV, YAMASAKI, FINE

lamina of basement membrane. J Cell Biol 1982;95:340

21. Couchman J, Gibson W, Thom D, Weaver A, Dees D, Parish W.Fibronectin distribution in epithelial and associated tissues in the rat.

Arch Dermatol Res 1979;266:296

22. Gibson WT, CouchmanJR, Weaver AC. Fibronectin distribution dur-

ing the development of fetal rat skin. J Invest Dermatol 1983;81:480

23. Clark RAE Fibronectin in the skin. J Invest Dermatol 1983;81:475

24. Timpl R, Rohde M, Robey PG, Rennard SI, FoidartJ-M, Martin GR.

Laminin: a glycoprotein from basement membranes. J Biol Cheml979;254:9133

25. FoidartJ-M, Bere EW, Yaar M, Rennard ST, Gullino M, Martin GR,

Katz SI. Distribution and immunoelectron microscopic localization of

laminin, a non-collagenous basement membrane glycoprotein. Lab In-

vest 1980;42:336

26. Chung AE, Freeman IL, Braginski JE, Carlin B. A novel extracellular

membrane elaborated by a mouse embryonal carcinoma-derived cellline. Biochem Biophys Res Commun 1977;79:859

27. Carlin Bjaffe R, Bender B, Chung AE. Entactin, a novel basal lamina-

associated sulfated glycoprotein. J Biol Chem 1979;254:9133

28. Hogan BLM, Taylor A, Kurkinen M, CouchmanJR. Synthesis and lo-

calization of two sulfated glycoproteins associated with basement mem-

branes and the extracellular matrix. J Cell Biol 1982;95:197

29. Alstadt SP, Hebda PA, Chung AE, Eaglstein WH. Effect of basement

membrane entactin on epidermal cell attachment and growth. J InvestDermatol 1987;88:55

30. FineJD, Neises GR, Katz SI. Immunofluorescence and immunoelec-tron microscopic studies in cicatricial pemphigoid. J Invest Dermatol1984;82:39

31. FineJD. Cicatricial pcmphigoid, bullous pcmphigoid, and epidermolysisbullosa acquisita antigens: differences in organ and species specifici-ties and localization in chemically-separated human skin of three base-

ment membrane antigens. Coil Relat Res 1985;5:369

32. Woodley lYE Briggaman RA, Scheidt V), Reese MJ, Paller AS, Yoshike

T The localization of type V collagen in human skin. Clin Res 1986;

34:788A

33. Sakai LY, Keene DR. Morris NP, Burgeson RE. Type VII collagen is a

major structural component of anchoring fibrils. J Cell Biol 1986;

103:15 77

34. Breathnach SM, Fox PA, Neises GR, Stanley JR. Katz SI. A unique

squamous epithelial basement membrane antigen defined by a mono-clonal antibody (KF-1). J Invest Dermatol 1983;80:392

35. FineJD, Gay S. WA-i: a ubiquitous noncollagenous lamina densa com-

ponent of basement membrane detected by monoclonal antibody tech-

nique. J Invest Dermatol 1986;86:286

36. Verrando P, OrtonneJ-P, Pautrat G, Hsi B-L, Yeh C-J. Identification

of a 37 kilodalton protein at the epidermal basement membrane by

an anti-serum to human amnion. J Invest Dermatol 1986;86:190

37. CouchmanJR. Heterogeneous distribution ofa basement membrane

heparan sulfate proteoglycan in rat tissues. J Cell Biol 1987;105:1901

38. FineJD, Couchman JR. Chondroitin-6-sulfate-containing proteogly-can: a new component of human skin dermoepidermal junction. 3 In-vest Dermatol l988;90:283

39. HassellJR, Robey PG, Barach H-J, WilczekJ, Rennard SI, Martin GR.

Isolation of heparan-sulfate-containing proteoglycan from basement

membrane. Proc Natl Acad Sci USA 1980;77:4494

40. StowJL, Sawada H, Farquhar MG. Basement membrane heparan sul-

fate proteoglycans are concentrated in the lamina rarae and podocytes

of the rat renal glomerulus. Proc Natl Acad Sci USA 1985;82:3296

41. Kato M, Koike Y, Suzuki 5, Kimata K. Basement membrane proteogly-

can in various tissues: characterization using monoclonal antibodies

to the Engelbreth-HoIm-Swarm mouse tumor low density heparan sul-

fate proteoglycan. J Cell Biol 1988;l06:2203

42. HassellJR, KimuraJH, Hascall VC. Proteoglycan core protein fami-lies. Annu Rev Biochem 1986;55:539

43. Hassell JR. Leyshon WC, Ledbetter SR. Tyree B, Suzuki 5, Kato M,

Kimata K, Kleinman HK. Isolation of two forms of basement mem-

brane proteoglycans. J Biol Chem 1985;260:8098

44. Dziadek M, Fujiwara 5, Paulsson M, Timpl R. Immunological charac-

terization of basement membrane types of heparan sulfate proteogly-

can. EMBO J 1985;4:905

45. Kleinman HK, McGarvey ML, HassellJR, Star VL, Cannon FB, Laurie

GW, Martin GR. Basement membrane complexes with biological ac-tivity. Biochemistry 1986;25:312

46. Fujiwara 5, Wiedemann H, Timpl R, Lustig A, EngelJ. Structure andinteractions ofheparan sulfate proteoglycans from a mouse tumor base-

ment membrane. Eur J Biochem 1984;143:145

47. Ljubimov AV, Afanasjeva AV, Lirvinova LV, Senin VM. Basement mem-

brane components produced by a mouse ascites teratocarcinoma TB24.

Exp Cell Res 1986;165:530

48. FineJD, Smith IT, Holbrook KA, Katz SI. The appearance offour base-

ment membrane zone antigens in developing human fetal skin. J In-vest Dermatol 1984;83:66

49. Lane AT, Helm KF, Goldsmith LA. Identification of bullous pem-

phigoid, pemphigus, laminin, and anchoring fibril antigens in human

fetal skin. J Invest Dermatol 1985;84:27

50. FineJD, Gay S. Characterization ofa new ubiquitous non-collagenous

component of basement membrane as defined by monoclonal anti-body. J Invest Dermatol 1986;86:475

51. Gammon WR, Briggaman RA, Inman AO III, Queen II, Wheeler CE.

Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZantibodies by indirect immunofluorescence on 1.0 M sodium chloride-

separated skin. J Invest Dermatol 1984;82:139

52. Breathnach AS, Robins J. Ultrastructural feature of epidermis of a 14

mm (6 weeks) human embryo. Br J Dermatol 1969;81:504

53. FineJD. The skin basement membrane zone. In Advances in derma-tology. Vol 2. Chicago: Year Book Publishers, 1986:283

54. Yurchenco PD, Cheng Y-S, Ruben GC. Self-assembly ofa high molec-

ular weight basement membrane heparan sulfate proteoglycan intodimers and oligomers. J Biol Chem 1987;262:17668

55. Keene DR. Sakai LY, Lunstrum GP, Morris NP, Burgeson RE. Type VIIcollagen forms as extended network of anchoring fibrils. 3 Cell Biol1987;104:611