Report

Cutaneous localization of endothelin-1 in patients withsystemic sclerosis: immunoelectron microscopic studyHideyuki Tabata, MD, Akio Yamakage, MD, and Soji Yamazaki, MD

From the Department of Dermatology,Dokkyo University School ofMedicine, Mibu, Tochigi, Japan

' . . • • ' . '

Correspondence

Hideyuki Tabata, MDDepartment of DermatologyDokkyo University School of MedicineMibuTochigi321-02 Japan

AbstractBackground Endothelin-1 (ET-1) has some relation to the pathogenesis of systemic

sclerosis (SSc) and Raynaud's phenomenon. This study was performed to determine the

localization of ET-1 in patients with SSc.

Methods The localization of ET-1 on the specimen by skin biopsies from nine patients

with SSc, \/vas observed with immunoelectron microscopic techniques.

Results High-density deposits existed on the ribosomes and on the rough endoplasmic

retlculum in the endothelial cells of microvessels of the upper dermis. The same findings

were also seen in the fibroblasts of the dermis, but not found in the skin of normal

controls. The level of deposits in the endothelial cells and dermal fibroblasts seemed to

have a positive correlation with the serum levels of ET-1 of patients with SSc.

Conclusions From these results, it can be seen that ET-1 is produced much more from

the endothelial cells and fibroblasts of the dermis in the skin of SSc patients than from the

normal controls. It is suspected that ET-1 is one of the pathogenetic factors of SSc.

272

Endothelin (ET) is an endothelial cell-induced peptide,discovered by Masaki in 1988.^ In the ET family, ET-i haspotent vasospastic activity, and it is reported that the serumlevel of ET-i increases in patients with SSc and patientswho show Raynaud's phenomenon.̂ ~5 It is suspected thatET-I has some relation to the pathogenesis of SSc andRaynaud's phenomenon.

We observed the locations of ET-i in the skin of SScpatients by immunoelectron microscopic study. ,,! •

M a t e r i a l s a n d m e t h o d s ' '

Tissue specimens

Nine patients with SSc were randomly selected as an "SSc

group" from the out-patient files of our department from / •

January to March 1995. Biopsies were performed on the

extensor side of their forearms. In order to compare and

contrast the histologic findings of the SSc group with those of

normal skin, we prepared normal skins which were picked from

a control group of six patients of other diseases without

exanthema. Details are shown in Table 1. In the SSc group

there were eight women and one man, with an age range of

41-72 years (mean 60 years). According to the classification of

Tuffanelli and Winkelmann,® five patients were diffuse

scleroderma type, and four patients were acrosclerotic type.

Serum ET-1 levels were higher than normal in all SSc group

patients.

The avidin-biotin-peroxidase method was used for

immunoelectron microscopy, we referred to the method of

Nagura and Komatsu.'' Half of the specimens were cut into

smaller sizes (about 5 x 5 x 2 mm) and fixed in Zamboni

solution^ (saturated picric acid in distilled water and 20%

paraformaldehyde) soon after the biopsies were performed;

they were then washed in 10, 15, 20% saccharose in

phosphate-buffered saline (PBS) overnight, and dipped in 10%

goat serum at 4 °C. The specimens were incubated in ET-1

antiserum (Peptide Institute Inc., Osaka) overnight at a dilution

of 1:100, then incubated in biotin-labeled antirabbit

immunoglobulin goat serum for 12 h, and then reacted in

streptavidin-peroxidase complex for 24 h. The specimens were

washed in 10% saccharose in PBS three times for 30 min

each, re-fixed in 1% glutaraldehyde (diluted with PBS) for 3 h,

and washed in PBS for 6 h at 4 °C. Specimens were incubated

in 1% 3,3'-diaminobenzidine, tetrahydrochloride (DAB), and

International Journai of Dermatoiogy 1997, 36, 272-275 © 1997 Blackweii Science Ltd

Tabata. Yamai<age, and Yamazai<i Cutaneous localization of ET-1 Report 273

Figure 1 High-density minute granules (arrowheads) werelined in the endothelial cells (N, nucleus) of microvessels(X 13,000)

Table 1 Tissue specimens

Case Age/sex Period from Type Raynaud's Serum ET-1onset (years) phenomenon (1.1-1.9 pg/

mL)

SSc group123456789

61/M59/W72/W71/W41/W67/W55W51AW64/W

Control group'

123456

74/W65/W48/M50/W57/W82/W

11921

1027

91120

Disease

D (+)D (+)D ( + )D (+)D ( + )A (+)A ( + )A ( + )A (+)

malignant lymphomadrug eruptionburnpsoriasis vulgarisdrug eruptionparapsoriasis en plaque

10.03.0

37.53,62.13.09,22.82.2

D, diffuse scleroderma; A, acrosclerosis; M, man;W, woman."•Normal skins were used.

DAB-hydrogen peroxide solution, adding 10 mM sodium azide

(NaN3) to block endogenous peroxidase, for 2 h each, washed

in cold PBS for 3 h, and then fixed in 2% osmium tetraoxide for

3 h, and dehydrated in ethanol. Sections were stained with

uranyl stain for 3 min only in order to avoid a significant

background by lead stain. A control study used rabbit serum

instead of ET-1 antiserum. We observed endothelial cells of

Figure 2 No granules were found in the endothelial cells ofcontrols: (a) using rabbit serum instead of ET-i antiserum;(b) normal control (L, lumen; N, nucleus of endothelial cell)(X 13,000)

microvessels and fibroblasts In the dermis using a Nihon densi

100-C electron microscope operating at 80 kV.

Results :

In the immutioelectron microscopic findings of the SScgroup, high-density minute granules were found on ribo-somes in the endothelial cells of microvessels in the upperdermis (Fig. i). These granules were lined along the endo-plasmic reticulum mainly near the lumens of the vessels.In a study using rabbit serum instead of ET-i antiserum,and in the skin of normal controls, these granules wereeither not seen at all or only a few granules were found inthe endothelial cells of the microvessels (Eig. 2).

We observed the fibroblasts in the dermis in the SScgroup, and found high-density minute granules coincidingwith ribosomes on the endoplasmic reticulum (Eig. 3). Itiparticular, we found many granules in the fibroblasts ofpatients whose serum ET-i levels were high; these structureswere not found in the control group (Fig. 4).

© 1997 Blackwell Science Ltd InternatiOhai Journal of Dermatology 1997, 36, 272-275

274 Report Cutaneous localization of ET-1 Tabata, Yamakage, and Yamazai<i

Figure 3 High-density minute granules coinciding withribosomes on the endoplasmic reticulum in dermalfibroblasts (X 3400)

Figure 4 No granules were found in dermal Hhroblasts ofcontrols: (a) using rabbit serum instead of ET-i antiserum;(b) nortnal control (X 13,000) . .; _ .. , , .,,

Discussion

Endothelin-1 is produced mainly in the endothelial cellsof the vessels responding to various mediators, such astransforming growth factor-P (TGF-P)? and interleukin-

i,^° and ET-I takes part in the regulation of vascular toneas paracrine and autocrine elements.̂ ^ Recently there hasbeen increasing evidence that ET-i has some other trophiceffects in human skin;̂ ^ it has been reported that ET-i isinvolved in collagen synthesis in dermal fibroblasts"* andmelanin synthesis in human melanocytes.'^ The collagensynthesis and increased serum level of ET-i in the patientswith SSc have led to the suggestion that ET-i may bepathogenic in the fibrosis of SSc. On the other hand,pigmentation is a well-known sign of scleroderma skin.Some studies showed histologic localization of ET-i on thebiopsied skin, cultured endothelial cells, culturedfibroblasts, and cultured keratinocytes, using the methodsof ";M situ hybridization," autoradiographic techniques,and immunocytochemistry."'''t-i7 Some of the results sug-gest that ET-I is the pathogenic factor of SSc.

In this study, we showed cytoplasmic localization ofET-I at the electron microscopic level, and showed quantit-ative predominance of ET-i in the skin of patients in SScrather than in the normal skin of patients with otherdiseases. These findings seem to support the pathogenicrole of ET-I in SSc. Especially in dermal fibroblasts, wefound distinct quantitative differences of ET-i depositionbetween the skin of SSc and the control skin. This suggestsan important role of production of ET-i from dermalfibroblasts to collagen synthesis.

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© 1997 Blackwell Science Ltd InternatiOhai Journai of Dermatology 1997, 36, 272-275