nicein, epiligrin, kalinin and other matrix components

Journal of Dermatological Science, 5 (1993) 97-103 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved.

97 0923-181 l/93/$06.00

DESC 00202

Labeling of fractured human skin with antibodies to BM 600/nicein, epiligrin, kalinin and other matrix components Elizabeth Ceilley”, Noriko Watanabe, Dorit Shapiro”, Patrick Verrandob, Eugene Bauera, Robert BurgesonC, Robert A. Briggamand and David T. Woodleya

A.

“Department of Dermatology, Stanford University School of Medicine. Stanford. CA, USA, hLaboratoire de Recherches Dermatologiques. Universite de Nice-Sophia Antipolis, Nice, France, ‘Cutaneous Biology Research Center. Massachusetts General Hospital East, Charleston, MA. USA and dDepartment of Dermatology, University of North Carolina, Chapel Hill, NC, USA (Received

Key words:

25 May 1992; accepted

Anchoring

filament

protein;

25 December

1992)

Dermal-epidennal

junction

Abstract A variety of methods

were used to fracture

the dermal-epidermal

phate buffered

saline, trypsin,

ity of basement

membrane

(EBA antigen)

and linkin) were determined

zone proteins,

BM 600/nicein,

by cold salt, phosphate with trypsin.

components

laminin,

pemphigoid

glycoprotein

in a significantly

shorter

Medicine.

Tarry

7-411, Northwestern

303 East Superior,

Chicago,

M.D.,

in the sera of patients period

Illinois,

of

School of

60611, USA.

Fax: (312) 908-0664. Abbreviations: epidennal

KBr, potassium phate buffered bullosa.

BP,

junction;

bullous EBA,

bromide;

pemphigoid; epidennolysis

NaCl,

sodium

DEJ, bullosa

chloride;

saline; RJEB, recessive junctional

dermalacquisita;

PBS, phosepidermolysis

kalinin,

We found

laminin,

epiligrin

warm and hot phoscollagens

that the basement

side of the DEJ fractured

through

and sensitivIV and VII membrane

the lamina

lucida

and kalinin were not detected after treatment to all of the procedures,

of the DEJ with brief exposure with epidermolysis

of time than salt-separated

Department

University

epiligrin,

by each method.

BM 600/nicein,

that separation

The dermal-epidermal junction (DEJ) of human skin is a complex zone consisting of a Correspondence to: David T. Woodley,

skin. These included

with dispase. The localization

in the lamina lucida. was insensitive

Introduction

Dermatology,

digestion

BM 600/nicein,

with the dermal

bromide.

skin. We also found

for testing the autoantibodies

skin can be prepared

antigen.

remained

(DEJ) of human

and proteolytic

after the DEJ was fractured

and kalinin

another

junction

bromide

saline and potassium

side of DEJ-fractured

a useful substrate

(bullous

epiligrin

buffered

In contrast,

to the dennal

cold 1 M salt, potassium

bullosa

acquisita

but co-localized

of skin to 56°C provided (EBA). Heat-separated

skin.

number of connective tissue components organized into a structural entity [l]. CB3 monoclonal antibody defines a component of the basement membrane zone of human skin [2,3] that is depressed or absent in patients with severe recessive junctional epidermolysis bullosa (RJEB). In RJEB, a hereditary bullous disease of the skin, separation occurs through the lamina lucida of the basement membrane zone. The target for GB3 antibody is a keratinocyte-derived protein with a molecular mass of - 600 kDa. When subjected to SDS-PAGE under reduced conditions, the

98

-600 kDa specie is revealed as four major polypeptides of approximately 100, 125, 146 and 150 kDa [2-41. Recently, the initial cloning of the human gene for one of the components (-600 kDa) has been accomplished 141.

This keratinocyte-derived - 600 kDa protein has been called ‘BM 600’, and, more recently, ‘nicein’ [4]. A second protein, kalinin, is an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments [5]. Kalinin is synthesized by keratinocytes and consists of chains ranging from 105 to 165 kDa. Antibodies to kalinin specifically label anchoring filaments beneath hemidesmosomes within the lamina lucida region of the basement membrane zone [5]. It appears that BM 600/ nicein and kalinin are related (vide infra) and that both proteins are components of the anchoring filaments. However, the localization by immunoelectron microscopy of kalinin to anchoring filaments within the basement membrane zone of skin is definitive, whereas the ultrastructural localization of BM 6001nicein is less clear. It is also possible that another recently described protein called epiligrin [6,7] is an anchoring filament protein and is related to or identical to nicein and kalinin. Recent studies by Dr. Robert Burgeson and co-workers* have demonstrated that BM 600/nicein, epiligrin and kalinin are very similar if not identical proteins. (Dr. Robert Burgeson, address to the Society for Investigative Dermatology, May 2, 1992, Baltimore, MD, unpublished observation; see footnote below). Further, recent studies by Domloge-Hultsch et al. [g,9] have demonstrated the identity between BM 600/nicein and epiligrin. *Marinkovich Lunstrum

brane proteins logically

PM,

Verrando

GP, Ortonne

P, Keene

JP, Burgeson

DR,

kalinin and nicein are structurally

homologous.

Submitted

Meneguzzi

RE: The basement

for publication.

G,

mem-

and immuno-

A monoclonal antibody developed by Fine et al. [lo] called 19-DEJ-1 also defines an anchoring filament protein of 165 kDa. It is conceivable that it also is related to the other anchoring filament proteins. It has been shown that when the cutaneous basement zone is fractured by trypsin, suction blister induction, heated phosphate-buffered saline or cold 1 M salt, the separation occurs through the lamina lucida [l l-141. Regardless of the method to fracture the basement membrane zone, all of the basement membrane zone components examined to date (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, type VII collagen) remain with the dermal side of the separation except for the bullous pemphigoid antigen which remains with the epidermis [l l-141. The purpose of this study was to fracture the cutaneous basement membrane zone by a variety of methods and determine the localization of putative anchoring filament proteins using GB3 kalinin and epiligrin antibodies. Methods Fracturing the dermal-epidermal junction

Human skin was sliced with a Padgett dermatome set at 0.4 mm depth. The skin was then cut with a scalpel into identical pieces 2 x 3 cm in size. The sliced skin was fractured through the DEJ by the following methods. Cold 1 A4 NaCl. The slices of human skin were incubated in 1 M NaCl at 4°C for 72 to 96 h as previously described [I 1,141. Phosphate buffered saline. The skin slices were immersed in phosphate buffered saline heated to 56°C for 30 s or 37°C for 72 h as described by Regnier et al. [15]. Cold potassium bromide. The skin slices were immersed in 2 M KBr at 4°C for 15 h. Trypsin. The skin slices were incubated overnight in 0.05% trypsin (Gibco, Grand Island, NY) as previously described [9]. Dispase. Slices of skin were floated over-

99

night at 4°C in a solution of dispase (2 units/ml in phosphate buffered saline). After the skin slices were incubated as described above, each specimen was placed on a cork board and the epidermis and dermis were separated manually using fine forceps. Immunofluorescence

The specimens were placed into OCT cryogel (Napersville, IN) and snap frozen in liquid nitrogen. Sections (4 pm) were cut on a cryostat microtome and either used without fixation or subjected to 2% formaldehyde in 0.1 M sodium cacodylate (pH 7.2) in 0.1 M sucrose for 20 min, then permiabilized with 1% Triton X-100 for 15 min at room temperature. Sections cut for L3d antibody labeling were either unfixed or fixed with 100°/o ethanol (-20°C) for 10 min. Sections were stained using indirect immunofluorescent methods with antibodies to kalinin [5], nicein (GB3) [3], epiligrin [6] (a generous gift from Dr. Jonathan Jones, Department of Cellular, Molecular and Structural Biology, Northwestern University School of Medicine), laminin [16], type IV collagen [16], type VII collagen [17,18], and linkin, a non-collagenous protein within the high papillary dermis associated with microthread-like fibers [ 191. In addition, sera from eight patients with bullous pemphigoid and nine patients with epidermolysis bullosa acquisita (EBA) were used in the immunofluorescent staining procedure at 1:10 dilutions. These sera all gave +2 to +4 staining of the basement membrane zone of normal, intact human skin. Five normal human sera were used at 1: 10 dilutions for negative controls.

Table I summarizes the results. The GB3 antibody to BM 600/nicein, anti-epiligrin and the anti-kalinin antibodies all labeled the derma1 side of the human skin fractured through

the lamina lucida by 37°C PBS, 56°C PBS, cold 1 M NaCl, dispase or cold KBr. Trypsin separation of skin induced degradation of the GB3, epiligrin and anti-kalinin target antigens and a consequent lack of antibody labeling was observed. Note that labeling of all of the substrates with these three antibodies labeled the six substrates identically. Laminin remained on the dermal side of the separation, regardless of the method used, and was insensitive to degradation by all of the methods. Separation of the skin by cold KBr was associated with weaker and discontinuous labeling of laminin by the antibody when compared with the other five methods of DEJ fracturing. Type IV collagen remained with the dermal side of all separations. The immunofluorescent staining with anti-type IV collagen antibodies consistently gave bright staining after all fracturing procedures except dispase. No type IV collagen labeling was observed after fracturing the DEJ by dispase. In contrast, dispase-separated skin readily labeled with anti-laminin antibodies. Similar to the staining with anti-type IV collagen antibodies, dispase-separated skin labeled with antibodies to type VII collagen [EBA sera (n = 9)] and two monoclonal antibodies [15,16] gave weak and/or discontinuous staining compared to the other separation methods. It appears that dispase either degrades these basement membranespecific collagens or disrupts intermolecular interactions with other proteins within the DEJ. This inhibition of antibody labeling or protein degradation by dispase was not specific for collagens, since diminished labeling also occurred with BP antibodies and antibodies to linkin, a non-collagenous protein associated with microthread-like filaments. However, laminin was readily labeled by antilaminin antibodies after the skin was separated by dispase. All of the EBA sera (9/9) labeled the dermal

1

number

of sera tested.

+, very bright, continuous,

oLinkin

EBA sera

(n = 2)

-

linear staining;

+

-

+

+(9/9)

, depressed and discontinuous

-

+ -

-

curnAb Type VII

-

+

-

arType IV toll. +

+

-

+

+

aLaminin

but detectable

+

+

+

+

+

+

+

oKalinin

-

+

-

_

+ +

Derm.

Epid.

31°C PBS

with DEJ separation

-

+

+(418) -

-

Derm.

skin substrates

+

+

Epid.

Derm.

Epid.

of human

56OC PBS

or sera labeling

Trypsin

of the antibody.

cvEpiligrin

crNicein

BP sera

Antibodies

A summary

TABLE

staining:

-

_

_

Epid.

KBr

induced

0, number

_

_

_

_

+

Epid.

1 M NaCl

methods

-. no staining;

+

+

+

+

+

+

+

Derm.

by different

of positively

+

+

+

+

+

+

+

+

_

Derm.

labeling

_

_

Epid.

Dispase

sera divided by

+

-

Derm.

8

side of human skin separated through the DEJ by a brief treatment with PBS heated to 56°C. Half of the BP sera (4/8) labeled the epidermal side of human skin treated the same way. Discussion In this study, we fractured the DEJ through the lamina lucida by a variety of methods. The putative anchoring filament proteins defined by the antibodies GB3, anti-kalinin and antiepiligrin were uniformly detected on the dermal-side of the DEJ when fractured by cold salt, warm and hot PBS and KBr. These targets were never detected on the epidermal side of the skin separations by any of the methods used. This suggests that at least some components of anchoring filaments remain with the dermis when the DEJ of human skin is fractured through the lamina lucida. However, it does not rule out the possibility that other components or domains of anchoring filaments (i.e. those domains that are not labeled with the antibodies used here) could remain with the epidermis after fracturing the junction by the methods used herein. We believe that it is likely that the anchoring tilaments within the lamina lucida are fractured by the methods used here, and the domains of the anchoring filaments defined by these antibodies remain adherent to the lamina densa and are, therefore, detected on the dermal floor of skin split through the lamina lucida. Recently, Domloge-Hultsch et al. [8,9] have shown that epiligrin and BM 600/nicein are identical proteins. Moreover, they have shown that a subset of patients with cicatricial pemphigoid have circulating anti-basement membrane autoantibodies that bind to the dermal side of salt-split, human skin substrate and that the target for these antibodies is BM 600/nicein-epiligrin. Our results here are in accordance with those of Domloge-Hultsch et al. [9,10] and explain why these autoan-

tibodies would indeed label the dermal floor of skin split through the lamina lucida. Heretofore, it was believed that labeling of autoantibodies to the dermal floor of salt split skin only occurred when the antibody was directed against type VII collagen, usually in the setting of EBA or bullous SLE. However, it is now clear that labeling the dermal side of skin separated through the lamina lucida (regardless of the method) is not limited to autoantibodies against type VII but also includes autoantibodies in the sera of patients with cicatricial pemphigoid that are directed against BM 600/nicein-epiligrin, non-collagenous matrix components within the lamina lucida [9,10]. These studies also suggest that anchoring filament proteins (or proteins with which they interact in situ) are sensitive to trypsin, a protease that is commonly used to release keratinocytes from tissue culture dishes or to separate human skin through the DEJ. Taken together, these observations suggest that the anchoring filament components defined by GB3 and antibodies to kalinin and epiligrin are critical for epidermal adherence to substrata including constituent components of the cutaneous basement membrane zone. It also appears that the methods used to fracture the DEJ of human skin vary in their ability to leave the immunolabeling of matrix components unperturbed by their respective antibodies. Specifically, fracturing the DEJ with dispase may be the most harsh method, since treatment of skin with this proteolytic enzyme was associated with poor or negative staining with antibodies to BPA, linkin and type IV collagen. Likewise, although type VII was detected by both polyclonal and monoclonal antibodies, the staining was significantly diminished after the skin was separated with dispase. In contrast, laminin, a basement membrane glycoprotein that is known to be sensitive to many different proteolytic enzymes, was readily detected by immunostain-

102

ing after dispase treatment. It is not clear if dispase directly degrades BPA, linkin and type IV collagen. The lack of staining by antibody after dispase treatment could be due to degradation or it could be due to dislodgement of these components from the DEJ by dispase. It is known that DEJ components have specific affinities for one another [ 161. For example, it is conceivable that dispase dislodges BM 600/nicein from the DEJ by perturbing another matrix molecule that serves to anchor it into the DEJ. Linkin is a newly described protein that is associated with micro-thread-like fibers within the papillary dermis [ 191. Therefore, its localization in normal skin is high within the papillary dermis and is not strictly confined to the cutaneous basement membrane zone. We expected linkin to remain with the dermal side of human skin when the DEJ was fractured. Although linkin is non-collagenous in nature, it significantly lost the ability to be immunolabeled by antibody after dispase treatment. Finally, we found that treating human skin for a few seconds with PBS heated to 56°C provides a human skin substrate that is useful for defining EBA antibodies in patients’ sera. Skin prepared in this manner appears to have utility similar to skin substrate separated by a 72-96 h incubation in 1 M NaCl [ 11,121. One advantage of hot PBS is that the skin fracturing procedure takes a matter of seconds as opposed to several days, as is the case with substrate preparation by cold salt. This substrate is excellent for EBA autoantibodies since 100% of the EBA sera tested reacted strongly with this substrate and with the same or greater intensity as intact skin. However, it should be noted that only half of the BP sera gave the expected labeling of the epidermis when heat-separated skin substrate was used, suggesting that the BP antigen is much more heat sensitive than the EBA antigen. We are currently using both salt-split and heatseparated skin substrates for the immuno-

screening of putative EBA antibodies in the sera of patients with subepidermal bullous disorders who have positive routine indirect immunofluorescent staining of standard substrates such as monkey esophagus. Acknowledgements

The authors are grateful to Dr. Paul R. Bergstresser, Department of Dermatology, and the Transplant Services Center at the University of Texas Southwestern Medical Center, Dallas, TX, for providing critical supplies for this study. This work was supported by National Institutes of Health Grants ROlAR33625, R37AR19537, ROlAR41551 and POlAR41045. References Stanley JR, Woodley

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