immunohistological distribution of the tight junction components zo-1 and occludin in regenerating...

Cutaneous Biology

Immunohistological distribution of the tight junctioncomponents ZO-1 and occludin in regenerating humanepidermis

M . M A L M I N E N , * V . K O I V U K A N G A S ,– J . P E L T O N E N , * , ‡, § S - L . K A R V O N E N , * *

A . O I K A R I N E N ‡ A N D S . P E L T O N E N †, *

Departments of *Medical Biochemistry and †Dermatology, University of Turku, Kiinamyllynkatu 4–8, 20520 Turku, Finland

Departments of ‡Dermatology, §Anatomy and Cell Biology and –Surgery, University of Oulu, Oulu, Finland

**Department of Dermatology, University of Helsinki, Helsinki, Finland

Accepted for publication 28 January 2003

Summary Background Molecular characterization of tight junction proteins during the past few years has

provided novel methods for studying these specialized junctions. Tight junctions have recently been

characterized in the granular cell layer of human epidermis, and the role of these junctions in the

epidermal barrier is now being re-evaluated.

Objectives To investigate the expression of tight junction components during the re-

epithelialization of suction blisters and the regeneration of the corneal layer after tape stripping.

Methods Suction blisters were induced in eight healthy volunteers, and skin biopsies were taken 4

or 6 days afterwards. The restoration of epidermal barrier function was evaluated by measuring

water evaporation (WE) from the wound area. Tape stripping was performed on three volunteers to

remove the corneal layer. The tissues were immunolabelled using indirect immunofluorescence or

the avidin–biotin method.

Results Prior to the biopsies, WE from the blister wounds was markedly elevated in comparison

with normal skin. In the epidermis surrounding the blister, occludin and ZO-1 were expressed in the

granular cell layer only. In the hyperproliferative zone adjacent to the border of the blister, the

expression of ZO-1 was redistributed into several spinous cell layers, while occludin expression was

restricted to the upper epidermis. In the leading edge of migrating keratinocytes, both proteins were

expressed exclusively in the most superficial layer of keratinocytes. Double labelling for ZO-1 and

involucrin showed expression of both proteins in the same layers of hyperproliferative

keratinocytes, while the expression patterns were clearly different in the migrating keratinocytes.

Conclusions Tight junctions of regenerating epidermis may provide a functional barrier prior to

regeneration of the corneal layer.

Key words: epidermal diffusion barrier, intercellular junctions, involucrin, tight junction, wound

healing

Tight junctions form a paracellular permeability barrier

that regulates the movement of water, solutes and

immune cells in simple epithelia. Furthermore, tight

junctions divide the plasma membrane into the apical

and the basolateral membrane domains.1–4 For exam-

ple, in the intestinal epithelium, tight junctions (zonula

occludens) form the most apical component of the

lateral junctional complex. Several structural compo-

nents of tight junctions have been characterized during

recent years.Correspondence: Sirkku Peltonen.

E-mail: [email protected]

British Journal of Dermatology 2003; 149: 255–260.

� 2003 British Association of Dermatologists 255

Occludin is a transmembrane protein that only

occurs in tight junctions. Occludin has four hydropho-

bic transmembrane helices, and both the NH2 and the

COOH terminals point towards the cytoplasmic side of

the plasma membrane. ZO-1 is found in the cytoplas-

mic plaques of tight junctions, and also in adherens

type junctions in cells that lack tight junctions, such as

fibroblasts and cardiac myocytes.1,4,5 ZO-1 is a member

of the MAGUK (membrane-associated guanylate kinase

homologue) protein family. ZO-1 is believed to contrib-

ute to the proper organization of proteins within the

tight junction plaque.1,4,5 In addition, the family of

claudins as well as the cytoplasmic plaque proteins

ZO-2, ZO-3, cingulin, symplekin and 7H6 have been

characterized as components of tight junctions.1–5

Structures resembling tight junctions were demon-

strated in the granular cell layer of human epidermis in

the early 1970s using electron microscopy.6 Elias et al.

used freeze-fracture scanning electron microscopy to

study human and mouse stratified epithelia and

concluded that tight-junctional elements were either

fragmentary or absent.7 Thus, the epidermal diffusion

barrier has been considered not to consist of tight

junctions, but of epidermal lipids, especially the lipid

bilayers present in the cornified layer.8 However, the

characterization of the structural components of tight

junctions has provided a new means to study tight

junctions, and the debate on the presence and function

of tight junctions in epidermis has resumed.9,10 Recent

work on claudin-1 knockout mice strongly suggested

that tight junctions function as components of the

diffusion barrier.11 We have previously shown that

occludin and ZO-1 are located in the granular cell layer

of adult human epidermis, while tight junctions have

been localized to the intercellular contacts of perider-

mal cells in fetal skin.10 Pathological conditions affect-

ing the stratum corneum, such as psoriasis, lichen

planus and ichthyosis vulgaris, showed altered expres-

sion of ZO-1 and occludin.10,12 Thus, tight junctions

may play a more important role in the formation of the

epidermal diffusion barrier if the epidermal lipid barrier

is abnormal or absent.

Wound healing provides a model for studying

epidermal keratinocytes in the proliferating, migrating

and differentiating phases.13,14 The present study was

designed to investigate the expression of the tight

junction components ZO-1 and occludin during the re-

epithelization of skin after the induction of suction

blisters and the regeneration of the corneal layer after

tape stripping.

Materials and methods

Suction blisters

Eight healthy medical students aged 23–26 years

(median 24; six men and two women) participated in

the study. All gave informed consent. Suction blisters

were induced on clinically normal abdominal skin

according to the method of Kiistala.15 Prolonged

suction of the skin leads to a detachment of the

dermoepidermal junction. The roof of the blister

consists of the epidermis and the blister floor the

dermis, while the basal lamina remains principally

intact. In the present study, a 150–250 mmHg

negative pressure was applied for 1–2 h.15–17 Dispo-

sable plastic suction devices (Dermovac; Ventipress,

Lappeenranta, Finland) with five pores were used.

The blister roofs were removed after the blister

induction, and the wounds were left open to heal.

The healing process was observed by measuring

water evaporation (WE) from the blister area. WE

was measured using an Evaporimeter EP1 (Servomed,

Stockholm, Sweden).18 This instrument records the

humidity of air in an open cylinder probe of 12-mm

diameter.

Four biopsies were taken on the fourth day and four

biopsies on the sixth day of healing. The WE measure-

ments were performed both after the blister induction

and before the biopsy. The value of daily evaporation

from healthy skin was subtracted from the wound area

values, because the diameter of the cylindrical probe

was larger than that of the wound. The measurements

were done in a supine position in a room with standard

temperature. The biopsies were taken under local

anaesthesia (1% lidocaine) in an operating room. A

small scalpel was used, and the wound was closed

using interrupted sutures. The sutures were removed

after 1 week. There were no complications in the

healing of the biopsy wounds.

Tape stripping

Tape stripping was performed on three volunteers. To

remove the corneal layer,19 cellophane tape was

applied 90 times repeatedly to an area of normal arm

skin. Three-millimetre punch biopsies were taken

immediately after the tape stripping and 1, 2, 3 and

5 days later. Part of the tape-stripped skin area of two

subjects was covered with cotton swabs soaked in

distilled deionized water and occlusive tape for 24 h.

Biopsies were taken after 24 h of water occlusion.

� 2003 British Association of Dermatologists, British Journal of Dermatology, 149, 255–260

2 5 6 M . M A L M I N E N et al.

Control samples were taken from normal skin adjacent

to the treated area.

Antibodies

The following antibodies were used: mouse monoclonal

antibodies to human ZO-1 (33–9100) and occludin

(33–1500), polyclonal rabbit antibodies to human

ZO-1 (61–7300) and occludin (71–1500) (all were

from Zymed Laboratories Inc., San Francisco, CA,

U.S.A.), and mouse monoclonal antibody to human

involucrin (Ab-1, MS-126-P0; NeoMarkers, Fremont,

CA, U.S.A.). The secondary antibody for indirect

immunofluorescence was tetramethylrhodamine isoth-

iocyanate (TRITC)-conjugated rabbit antimouse IgG

(R0270; Dako A ⁄ S, Glostrup, Denmark). In double

labellings, TRITC-conjugated swine antirabbit IgG

(R0156; Dako A ⁄ S) was mixed with Alexa FluorTM

488-conjugated goat antimouse IgG (Molecular Probes,

Eugene, OR, U.S.A.).

Avidin–biotin immunolabelling

To reveal the tight junction antigens from formalin-

fixed and paraffin-embedded tissue, the sections were

deparaffinized and then exposed to microwaves at

560 W in a water solution containing 0Æ1 mmol L)1

sodium citrate (pH 6) for a total of 10 min. Formalin-

fixed and paraffin-embedded specimens were immuno-

labelled with the avidin–biotin method using a

Histostain-Plus Kit (Zymed Laboratories Inc.) according

to the protocol supplied with the kit by the manufac-

turer. The antibody localization was visualized using

3,3¢-diaminobenzidine tetrahydrochloride (DAB-Plus

Kit; Zymed Laboratories Inc.). The slides were count-

erstained with Mayer’s haematoxylin (Oy Reagena Ltd,

Kuopio, Finland).

Indirect immunofluorescence labelling

Frozen sections of suction blisters were cut on silanated

glass slides. The samples were fixed with 100% methyl

alcohol at )20 �C for 10 min.10 To prevent non-

specific binding, the samples were preincubated in

phosphate-buffered saline (PBS) supplemented with 1%

bovine serum albumin (BSA) for 10 min. The primary

antibodies were diluted in 1% BSA–PBS and incubated

on the samples at 4 �C for 20 h. Following five 10-min

washes in PBS, the slides were incubated with secon-

dary antibodies at 20 �C for 1 h. After the incubation,

the samples were washed five times in PBS and

mounted with Glycergel (Dako A ⁄ S). In the control

immunoreactions, the primary antibody was replaced

with 1% BSA–PBS. In addition, the secondary and

primary antibodies used in double labellings were

tested for cross-reactions. In all controls, only faint

uniform background fluorescence was observed.

Confocal laser scanning microscopy

Confocal laser scanning microscopy was carried out

using a Leica TCS SP spectral confocal laser scanning

microscope equipped with an air-cooled argon–krypton

ion-laser system (Leica Microsystems Heidelberg

GmbH, Heidelberg, Germany) and Leica TCS NT

software (version 1.6.551). The final images were

saved in a tagged image file format.

Results

Water evaporation from suction blisters

Based on previous experiments on healing suction

blisters, the time-points of 4 and 6 days after the

induction of blisters were chosen for tissue sampling.20

The healing process was evaluated by measuring WE

from the wound area. WE in the skin indicates the

epidermal barrier function.18,20–23 In the normal skin

of the eight volunteers, WE ranged from 6 to

25 g m)2 h)1 (median 17Æ5). After the blister induc-

tion, WE ranged from 96 to 130 g m)2 h)1 (median

113Æ5). On the fourth day, the WE values from blister

wounds were 9, 25, 18 and 29 g m)2 h)1 higher than

the normal skin values, and on the sixth day, the WE

values were 7, 12, 12 and 13 g m)2 h)1 above the

baseline. The data show barrier function restoration at

these time points. However, the barrier was not yet

complete.

Avidin–biotin immunolabelling for ZO-1 and occludin

A histological analysis of all samples showed that

keratinocytes were migrating on the wound bed, but

that the wound was not fully covered by epithelium.

Three morphologically different zones could be detec-

ted: (i) the normal epidermis surrounding the blister;

(ii) the hyperproliferative zone at the border of the

blister; and (iii) the leading edge of migrating keratino-

cytes (Fig. 1A,B). Avidin–biotin immunolabelling

showed that, in the normal epidermis, ZO-1 and

occludin were expressed in the granular cell layer, as

reported earlier.10,24 The hyperproliferative zone


Z O - 1 A N D O C C L U D I N I N R E G E N E R A T I N G H U M A N E P I D E R M I S 2 5 7

consisted of an increased number of spinous cells,

which expressed ZO-1 (Fig. 1A). Occludin was, how-

ever, detected only in the upper spinous cell layers of

the hyperproliferative zone (Fig. 1B). ZO-1 was ex-

pressed in the superficial layers of migrating keratino-

cytes. In contrast, the avidin–biotin technique

visualized occludin only at the tip of the leading edge

on some but not all samples.

Indirect immunofluorescence labelling for ZO1, occludin

and involucrin

To study the expression patterns of ZO-1 and occludin

in more detail, indirect immunofluorescence labelling

was performed on frozen sections and analysed using

confocal microscopy. In normal epidermis and in the

hypertrophic zone, the expression patterns of ZO-1 and

occludin were essentially the same as described above

for Figure 1. The normal epidermis displayed occludin

and ZO-1 in the granular cell layer (Fig. 2A,D), while

occludin and ZO-1 labelling in the hypertrophic zone

was more intense and diffuse than in normal skin

(Fig. 2B,E). In the leading edge, ZO-1 and occludin

were localized to the intercellular junctions of the most

superficial keratinocyte layers (Fig. 2C,F).

In order to study the colocalization of ZO-1 and

occludin with the differentiation marker involucrin,

double immunolabelling for tight junction proteins and

involucrin was carried out (Fig. 2).25 In the normal

skin adjacent to the blister, both occludin and ZO-1

showed a high degree of colocalization with involucrin

in the granular cell layer (Fig. 2A,D). In the hyper-

trophic zone, the expression of ZO-1, occludin and

involucrin extended from the granular cell layer to the

spinous cell layers (Fig. 2B,E). In the leading edge,

involucrin was present in essentially all cell layers,

while ZO-1 concentrated to the intercellular contacts of

the uppermost cell layers (Fig. 2F).

Tape stripping was applied to normal skin in order to

evaluate the effect of the lipid barrier disruption on the

expression of ZO-1 and occludin. Tape stripping had no

effect on the expression of tight junction components,

as estimated by immunofluorescence labelling. How-

ever, during the regeneration of the corneal layer after

tape stripping, some expression of involucrin was also

detected in the spinous cell layers (not shown).

Discussion

The suction blister model enables studies on epidermal

keratinocytes in three different phases in a single tissue

sample: (i) in normal epidermis surrounding the blister;

(ii) in the hyperproliferative zone; and (iii) in cells

migrating over the wound bed. In the present study the

epidermal barrier function was assessed by measuring

WE from healing suction blisters.21–23,26 WE was about

10-fold higher after the removal of the blister roof

compared with the WE values measured 6 days later.

Once keratinocytes started to migrate across the

wound, WE decreased. After 4 and 6 days, the WE

value of the wound was clearly reduced but was still

above baseline. This indicates that the epidermal

barrier function was being restored but was not yet

complete. In the suction blister wound, the clot and the

migrating epidermis are the only new constituents seen

during healing. The fibrin clot covers the suction blister

wound during the first week, contributing to barrier

restoration. The clot then gradually disappears. Before

epidermal regrowth, the influence of the clot is prob-

ably relatively greater. Because WE continues to

Figure 1. Avidin–biotin immunolabelling of a healing suction blister

6 days after induction. Normal skin is seen on the left, the hyper-

trophic zone in the middle, and the leading edge on the right, under

the partly detached crust. (A) ZO-1 is present in the granular cell

layer of the normal epidermis, in several suprabasal layers in the

hypertrophic zone, and in the superficial cell layers of the leading

edge of migrating keratinocytes. (B) Occludin is detectable after

avidin–biotin labelling in the granular cell layer of the normal

epidermis, and the most superficial cell layers of the migrating

keratinocytes. Scale bars ¼ 100 lm.



decrease, it is likely that the migrating epidermis and its

evaporation-controlling elements, tight junctions, cre-

ate a new barrier. As the stratum corneum does not

exist in the new epidermis20,23 on the fourth or sixth

days, the lipid barrier has no effect on evaporation at

that time.

The purpose of the present study was to map the

distribution of tight junction proteins in the healing

epidermis. Recent studies have emphasized the role of

tight junctions in the regulation of epidermal permeab-

ility. Genetic ablation of the claudin-1 gene in mice

results in neonatal death, probably because of signifi-

cant dehydration.11 Mice overexpressing claudin-6 die

within 2 days of birth, apparently due to barrier

dysfunction and increased water loss.27 Other recent

studies demonstrate that tight junctions are present in

fetal epidermis10,24,27 which lacks the corneal layer.

Thus, it is feasible to speculate that tight junctions form

a functional barrier in premature skin. Regenerating

epidermis is analogous to fetal skin in its lack of a

normal corneal layer. In this study, the two most

superficial layers of migrating keratinocytes on the

wound bed expressed occludin and ZO-1, suggesting

the presence of tight junction proteins between these

cells. The expression of the tight junction proteins

claudin-1, occludin and ZO-1 has recently been studied

in a human skin organ culture model, showing

occludin and ZO-1 in the first ingrowing epithelial

cells early during wound healing.24 The results of

Brandner et al.24 are thus in agreement with the results

of our study on the in vivo wound healing model. Taken

together, these studies indicate that at least three tight

junction components are present in the regenerating

epithelium before the reconstruction of stratum cor-

neum. These observations further emphasize the role of

tight junctions in epidermis, especially in conditions

where no normal lipid barrier is present.

The hyperproliferative zone, which develops adjacent

to the original border of the blister, displayed a unique

labelling pattern for ZO-1 and occludin. The expression

of ZO-1 was redistributed into several spinous cell

layers. Involucrin was also detected in the granular cell

and spinous cell layers to approximately the same level

as ZO-1. In analogy with healing epidermis, ZO-1 and

involucrin are also coexpressed in the granular and

spinous cell layers in psoriasis.10,12 The expression of

ZO-1 may thus be linked with the differentiation of

keratinocytes. Claudin-1 is expressed in the granular

Figure 2. Indirect immunofluorescence

labelling of a healing suction blister for

occludin (Occl, red), and double immuno-

labelling for tight junction proteins and invo-

lucrin (Inv, green). (A,D) In normal epidermis,

double labelling for occludin or ZO-1 (red) and

involucrin reveals colocalization in the gran-

ular cell layer, while the polyclonal antibody

also detects occludin in the uppermost spinous

cell layers (A, arrow). (B,E) In the hyper-

trophic zone, both tight junction proteins and

involucrin are detected in a wider zone

extending to the spinous cell layers. (C,F) In

the leading edge, both ZO-1 and occludin are

present in the most superficial cell layers. The

white dotted line indicates the location of the

dermal–epidermal junction. (F) Involucrin is

expressed in essentially all cell layers, while

ZO-1 is restricted to the intercellular contacts

of the uppermost cell layers. Panel F repre-

sents a sample taken 4 days after the induc-

tion of the blister, while other figures

represent the sixth day of healing. Scale

bars ¼ 20 lm.


Z O - 1 A N D O C C L U D I N I N R E G E N E R A T I N G H U M A N E P I D E R M I S 2 5 9

and spinous cell layers in normal epidermis and at the

border of the wound.24 However, it is currently not

known why the distribution of occludin differs from

that of ZO-1 and claudin-1. To conclude, these

results suggest that tight junctions may have a role

in the development of the diffusion barrier in healing

epidermis.

Acknowledgments

This work was supported by Turku University Foun-

dation, Turku University Central Hospital (grant

13338), Oulu University Hospital, Cancer Societies of

Finland, and Department of Dermatology, University of

Oulu, Oulu, Finland.

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