Report

Correlation between diffuse pigmentation and keratinocyte-derived endothelin-1 in systemic sclerosis

Hideyuki Tabata, MD, Noriaki Hara, MD, Shun Otsuka, MD, Akio Yamakage, MD,Soji Yamazaki, MD, and Noriyuki Koibuchi, MD

Abstract

Background The precise mechanism of diffuse pigmentation in systemic sclerosis (SSc) is

still unknown. We suspected the participation of endothelin-1 (ET-1), which is produced by

keratinocytes, in the hyperpigmentation in SSc. The aims of this study are to demonstrate the

hyperproductivity of ET-1 from epidermal cells in SSc by in situ hybridization histochemistry,

and to show a correlation between the hyperproductivity of ET-1 in keratinocytes and skin

hyperpigmentation.

Methods In situ hybridization histochemistry was performed on nine SSc specimens (®ve

cases of diffuse scleroderma (dSSc), four cases of acrosclerosis (lSSc)), and compared with

four normal control specimens. We counted the grains on 10 3 10 mm2 of epidermis and

microvessels in each histology and examined the degree of skin pigmentation using the skin

re¯ectance factor (Y).

Results In the specimens of the SSc patients, the number of grains on the epidermis was

remarkably higher than those of the control specimens (P < 0.01). We found a close

correlation between the number of grains and the skin re¯ectance factor in dSSc patients

(P = 0.02). Correlations were not identi®ed between serum ET-1 and skin pigmentation and

between serum ET-1 and the frequency of grains on the epidermis. As for grains on

microvessels, lSSc patients showed a greater frequency than dSSc patients.

Conclusions The ®ndings of this study suggest that an increase in the ET-1 productivity of

keratinocytes is experienced in SSc patients, especially in dSSc patients. The results

suggest a strong correlation between the ET-1 productivity of keratinocytes and skin

pigmentation in severe cases of SSc. We conclude from these results that keratinocyte-

derived ET-1 plays an important role in the pathogenesis of the hyperpigmentation of the skin

in SSc patients.

Introduction

Diffuse pigmentation is one of the main skin signs in

systemic sclerosis (SSc); however, the mechanism of diffuse

pigmentation in SSc is still unknown. Endothelin-1 (ET-1)

is mainly produced in endothelial cells of vessels and has

potent vasospastic activity.1 Recently, some studies have

suggested that ET-1 is also produced in keratinocytes,2 and

promotes the melanogenesis of melanocytes in the epider-

mis.3±6 An increased plasma level of ET-1 in patients with

SSc has also been reported.7,8 From these facts, we

suspected the participation of ET-1, produced by kerati-

nocytes, in the melanogenesis in SSc. The aims of this study

were to demonstrate the hyperproductivity of ET-1 from

the epidermal cells in SSc by counting the grains observed

in in situ hybridization histochemistry, and to show a

correlation between the hyperproductivity of ET-1 in

keratinocytes and skin hyperpigmentation by a photo-

electric colorimeter.

Materials and methods

Specimens were taken from the extensor side of the forearm of

nine patients with SSc by the biopsy method (eight women, one

man; mean age, 60 years; range, 41±72 years; mean duration of

disease, 11.1 years; range, 1±27 years). According to the

classi®cation by Tuffanelli and Winkelmann9, ®ve cases were

categorized as diffuse scleroderma (dSSc) and four cases as

From the Department of Dermatology

and Physiology, Dokkyo University

School of Medicine, Tochigi, Japan

Correspondence

Hideyuki Tabata, MD

Department of Dermatology

Dokkyo University School of Medicine

880 Kitakobayashi

Mibu, Shimotsugagun

Tochigi 321-0293

Japan

ã 2000 Blackwell Science Ltd International Journal of Dermatology 2000, 39, 899±902

899

acrosclerosis (lSSc). A group of four cases (dSSc, three cases;

lSSc, one case) showed diffuse pigmentation on a clinical basis

(Fig. 1). The plasma ET-1 level was higher in all cases. The

clinical data are summarized in Table 1.

In situ hybridization histochemistry was performed on nine SSc

specimens and four normal control specimens. cDNA against

human ET-1 mRNA was kindly provided by Dr Tomoh Masaki,

University of Kyoto, Kyoto, Japan. 35S-UTP was incorporated to

label the probes for In situ hybridization. The riboprobes were

hydrolyzed to approximately 250 bases according to the

procedure described by Cox et al.10 A detailed description is

given in Koibuchi et al.11.

The hybridization signal consists of dark silver grains overlying

sections. We counted the grains on 10 3 10 mm2 of epidermis and

microvessels in the upper dermis for each histology, 10 times at

random, and calculated the average with an image analyzing

computing system (Win roof, Mitsuya Co, Tokyo, Japan). We

examined the degree of skin pigmentation on the extensor side of

the forearms by reading the CIE (Commission Internationale de

l'Eclairage) skin re¯ectance factor (Y)12 with a photoelectric

colorimeter (Minolta CR-300, Tokyo, Japan). These

measurements were performed at the points adjacent to the

biopsied sites, just before they were taken, and were carried out

from December to April to avoid the effect of ultraviolet radiation.

Figure 1 Clinical diffuse pigmentation in a diffuse

scleroderma patient (Case 4)

Table 1 Tissue specimens

Grains/10 3 10 mm2

Period from Skin Serum ET-1

Case Age/sex onset (years) pigmentation Epidermis Microvessels (1.1±1.9 pg/mL) Y*

SSc patients

dSSc

1 61M 1 (±) 1.7 3.1 10.0 32.0

2 59F 19 (+) 3.7 1.5 3.0 24.6

3 72F 2 (±) 2.5 1.4 37.5 30.9

4 71F 1 (+) 2.2 1.8 3.6 ND

5 41F 10 (+) 3.3 1.7 2.1 26.3

(mean 2.7) (mean 1.9) (mean 11.2)

lSSc

1 67F 27 (+) 1.6 2.4 3.0 24.8

2 55F 9 (±) 1.6 6.5 9.2 26.2

3 51F 11 (±) 3.9 4.3 2.8 26.3

4 64F 20 (±) 2.9 4.8 2.2 ND

(mean 2.5) (mean 4.5) (mean 4.3)

Grains/10 3 10 mm2

Case Age/sex Diagnosis Epidermis Microvessels

Control group²

1 74F Malignant lymphoma 0.9 1.2

2 82F Parapsoriasis en plaque 1.6 1.8

3 65F Epidermal cyst 0.7 1.7

4 57F Epidermal cyst 0.7 0.9

(mean 1.0) (mean 1.4)

*Skin reflectance factor (smaller figure means more pigmented skin).²Normal skin adjacent to lesional areas was collected.ND, not measured; dSSc, diffuse scleroderma; lSSc, acrosclerosis.

Report Diffuse pigmentation in systemic sclerosis Tabata et al.

International Journal of Dermatology 2000, 39, 899±902 ã 2000 Blackwell Science Ltd

900

Results

Sections that were treated with the antisense probe showed

that the grains were concentrated exclusively over the

epidermis in the skin of SSc patients (Figs 2A,B). In the

specimens of the SSc patients, the numbers of grains on the

epidermis were clearly increased, compared with those of

the control specimens (P < 0.01). We could not ®nd clear

concentrations of grains on dermal microvessels in dSSc

patients (Fig. 2A); however, concentrations of grains on

dermal microvessels were found in lSSc patients (Fig. 2C).

With regard to the correlation between the numbers of

grains and skin pigmentation, a close correlation was

found only in dSSc patients (n = 4, R = 0.981, P = 0.02).

Correlations were not found between serum ET-1 and skin

pigmentation and between serum ET-1 and the numbers of

grains on the epidermis. The results of the study indicate an

increased level of ET-1 productivity in keratinocytes of SSc

patients and a correlation between ET-1 productivity in

keratinocytes and skin pigmentation in dSSc patients.

Discussion

The present study illustrates an increased in situ hybridiza-

tion signal over the keratinocytes of the epidermis in skin

biopsied from SSc patients. These ®ndings indicate that the

ET-1 mRNA level is elevated in these cells, due to the fact

that the subcloned antisense probe speci®cally hybridizes

with ET-1 mRNA. This study therefore shows increased

production of ET-1 in these cells at a molecular level. ET-1

is mainly produced in endothelial cells of various kinds of

vessels, responding to various mediators, such as trans-

forming growth factor-b (TGF-b)13 and interleukin-1 (IL-

1).14 ET-1 also takes part in the regulation of vascular tone

as paracrine and autocrine elements.15 We have previously

demonstrated the cutaneous localization of ET-1 in SSc

patients by immuno-electron microscopy.16 High density

deposits suggest that ET-1 coincides with ribosomes on the

endoplasmic reticulum in keratinocytes, endothelial cells of

microvessels, and ®broblasts. In this study, grains were not

clearly concentrated on the dermal microvessels in dSSc

patients. This may be due to microvessel damage and

capillary loss in dSSc patients. It is suspected that ET-1 is

produced in greater amounts in keratinocyes than in the

endothelial cells of microvessels in dSSc patients. In

contrast, in lSSc patients, ET-1 from the endothelia of

blood vessels may have a direct effect on the serum level of

ET-1.

Recently, there has been increasing evidence that ET-1

has certain other trophic effects in human skin.17 It has

been reported that ET-1 is involved in both the multi-

plication of melanocytes and melanin synthesis in human

melanocytes.5 Normal human keratinocytes express ET-1

mRNA in normal cultured conditions in vivo, and

ultraviolet B (UVB) exposure highly stimulates the para-

crine linkage of endothelins between keratinocytes and

melanocytes.4 Also, in pathologic conditions, such as

acquired dermal melanocytosis, ET-1 which is produced

Figure 2 In situ hybridization histochemistry. (A) Epidermis

and microvessels (arrows) of Case 2 in diffuse scleroderma

group. No concentration of grains on microvessels. (B)

Epidermis of Case 4 in control group. (C) Microvessels of

Case 4 in acrosclerosis group (magni®cation, 3 400)

Tabata et al. Diffuse pigmentation in systemic sclerosis Report

ã 2000 Blackwell Science Ltd International Journal of Dermatology 2000, 39, 899±902

901

and secreted by keratinocytes after UV irradiation affects

melanocytes and accelerates melanogenesis.18

Diffuse pigmentation of the skin is one of the major

clinical signs of SSc, especially in severe cases. In about

30% of SSc patients, abnormalities of skin color have been

identi®ed.19 The pathogenesis of this pigmentation of the

skin has not been elucidated. The pigmentation may at

times predate the sclerotic changes and is not associated

with elevated levels of plasma b-melanocyte-stimulating

hormone (b-MSH).20

In this study, we showed increased ET-1 productivity in

keratinocytes of SSc patients and a correlation between ET-

1 productivity in keratinocytes and skin pigmentation in

severe cases of SSc. We suspect that keratinocyte-derived

ET-1 has an important role in the pathogenesis of the

hyperpigmentation of the skin in SSc. It is not apparent

whether ET-1 works alone or with other cofactors to

induce the hyperpigmentation seen in SSc. In SSc patients,

it is dif®cult to identify a de®nite participation of UV

irradiation in the pathogenesis of hyperpigmentation of the

skin. Some cytokines, such as IL-1a, may be overproduced

in SSc patients, and therefore may promote ET-1 produc-

tivity from keratinocytes.4 In future studies, the mechanism

of the increase in ET-1 from keratinocytes and cofactors,

leading to skin pigmentation in SSc, needs to be clari®ed.

Acknowledgments

Prof. Tomoh Masaki, University of Kyoto, Kyoto, Japan,

provided the ET-1 cDNA.

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