The pathogenesis of vascular changes in erythema nodosum-like lesions of Behçet's syndrome: An electron microscopic study

The Pathogenesis of Vascular Changes in Erythema Nodosum-like lesions of Beh~etJs Syndrome: An Electron Microscopic Study DONGSIK BANG, MD, PHD," TAKAYOSHI HONMA, DDS,t TAIICHI SAITO, MD,t SHOJIRO NAKAGAWA, MD,+ HIROAKI UEKI, MD,+AND SUNGNACK LEE, MD* The probable sequence of microvascular changes affecting endothelial cells in erythema nodosum-Iike lesions was examined by electron microscopy in 18 patients with typical Behcet's syndrome. Dark endothelial cells in the superficial and deep dermis exhibited degeneration with contraction changes. Consequently, degenerated endothelial cells showed nuclear and cytoplasmic condensation, vacuolization in the cytoplasm, and rich ribosomes. Moreover, blood vessels which were enveloped by cuffs of lymphocytes simultaneously exhibited both endothelial cell hypertrophy and endothelial cell necrosis. The implied associations between perivascular lymphocytic cuffs and blood vessels exhibiting endothelial cell alterations indicate that microvascular changes in erythema nodosum-like lesions may be closely associated with the delayed-type hypersensitivity reaction. HUM PATHOL 18:1172-1179, 1987.

The pathogenesis of Behcet's syndrome has not yet been clarified. However, lymphocytic infiltration has been observed in mucocutaneous lesions during ~he early stage of t.he disease in the following areas: 1!1 the basal and prickle-cell layers of the oral epitheIiurn and around small blood vessels in the corium in aphthous lesions.l <' and in lobules of subcutaneous fat and around small blood vessels in the superficial and deep dermis in erythema nodosum-like Iesions.v" In erythema nodosum-like lesions, the pathogenetic focus may be in the subcutaneous fat accompanied by cellular infiltrates in the histologic for.m ?f a delayed-type hypersensitivity reaction, ,~hICh IS characterized by the perivascular accumulanon of lymphocytes or lymphoid-mononuclear cells. Recently, the details of microvascular endothelial cell injury in delayed-type hypersensitivity reactions have been elucidated by morphologic studies of cellmediated immunologic reactions such as allergic contact dermatitis," first-set skin allograft rejection in humans.f heart allograft rejection in rats.? and the rejection of vascularized syngeneic tumors.I? Because erythema nodosum-like lesions are considered to involve a delayed-type hypersensitivity reaction.P-" some of the microvascular changes at the site of lym-

From the *Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea, and Departments of j Pharmacology and :l:Dermatology, Kawasaki Medical School, Kurashiki, Japan. Revision accepted for publication 22 January 1987. Address correspondence and reprint requests LO Dr. Honma: Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0I,Japan. 0046-8177/87 SO.OO

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phocytic infiltration may represent immunologic damage that has induced endothelial cell injury. In this study, biopsy specimens from 18 patients with Behcet's syndrome were examined by electron mic~oscopy. The appearance of degenerated endothelial cells and focal sloughing of entire endothelial cells is reported. These findings might be evidence for immunologic injury in erythema nodosum-like lesions.

MATERIALS AND METHODS . Biopsy examination was performed in 18 panents with Behcet's syndrome. The criteria for diagnosis of Behcet's syndrome were the presence of two or more of the cardinal manifestations of this disease (aphthous stomatitis, genital ulcerations, uveitis, and skin lesions). All patients had the mucocutaneous type disease with skin manifestations, and a male patient had symptoms of neurologic Behcet's syndrome. The specimens excised were the most recently developed lesions among the erythematous tender nodules on the lower extremities. The specimens were fixed for 2 hours in a chilled solution of 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The prefixed tissues were washed in 0.1 1\1 phosphate buffer (pH 7.4) and s~bdivided into ab~>Ut ~-mm-thick slices cut perpendicularly to the epithelial surface. These slices were postfixed at 4°C for 2 hours in 1 per cent osmic acid buffer solution (pH 7.4). Following dehydration in ethanol, the slices were flatly oriented and embedded in Epon 812. From all slices, l-p.m sernithin sections werecut and stained with toluidine blue. The blocks were trimmed to retain the subcutaneous blood vessels with the perivascular infiltrates. Ultrathin sections were cut and stairied with uranyl acetate-lead citrate. The sections were examined under an electron microscope.

RESULTS . Light microscopy revealed progressive infiltranon of the deep dermis and interlobules in the subcutaneous fat by dominant lymphoid-mononuclear cells in the erythema nodosurn-like lesions of 18 patients. Cellular infiltrates were also occasionally ob- .

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ERYTHEMA NODOSUM-liKE LESIONS OF BEH<;ErS SYNDROME (Bang et al.)

FIGURE 1. Electron micrograph of a lymphocyte collection beside a venule in the upper dermis of an erythema nodosum-Iike lesion. a, All endothelial cells (E) viewed in the long itudinal section of the vessel seem to be activating with the presence of well-developed organelles and show no evidenc e of contact with lymphocytes (Lc). A peripheral monocyte (Mo) and erythrocytes ('" are seen in the lumen. Several macrophages (Mp) are also located around the venule. ( x 3.750.) Insets, Detail of the portions of a lymphocyte -lymphocyte cluster (panel b. x 4.500) and a lymphocyte-macrophage cluster (panel c . x 9.000) surrounding venules is shown.A pericyte (Pc) isalso separated from an enveloping macrophage and lymphocyte.

served around blood vessels of the superficial and deep dermis. Electron microscopy of these infiltrates confirmed the presence of lymphocyte cuffs enveloping these vessels (fig. 1). l\facrophages were also included in the perivascular collection of lympho-

Lc

cytes. The blood vessels that were surrounded by such cellul ar infiltrates exhibited activation and en largement of their endothelial cells, displaying welldeveloped cytoplasmic organelles and cytoplasmic fibrils. 1173

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FIGURE 2. Electron micrograph of a venule with dark endothelial ce lls In the deep dermis of the skin lesion. Some endothelial cells (D£tD£2) show the dark configuration with contraction changes. and others (£1-£3) show the normal configuration with hypertrophic changes. Dark cellular debris (arrows) and nucleus fragments (open arrow) are observed inside the lumen. Lymphocytic infiltrate is rather scanty in this perivascular area. Only one lymphocyte (Lc) and a proliferative pericyte and/or smooth muscle cell (PclSfv1) are observed outside the lumen. [ x 8.000.)

On the other hand, in blood vessels with mild perivascular [yrnphocytic infiltration, dark endothelial cells were readily distinguishable from the adjacent activated endothelial cells by their higher electron density (fig. 2). These dark cells exhibited contraction changes and showed cellular lobation. Blood vessels with markedly narrowed or obliterated lu-

mina were surrounded by irregular basal lamina and prominent pericytes. The progression of cellular contraction consequently caused endothelial cell degeneration and necrosis. Each degenerating endothelial cell showed nuclear and cytoplasmic condensation, vacuolization in cytoplasm, and rich ribosomes. Sometimes degen-

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ERYTHEMA NODOSUM-L1KE LESIONS OF BEH<;ITS SYNDROME (Bang et al.)

o

FIGURE 3. Electron micrograph of a degenerating dark endothelial cell protruding into the lumen of a venule in the deep dermis of the skin lesion. Five intact endothelial cells (E1-E5) construct the lumen (Lu). and one cell (DE) lies in the lumen. The protruding cell (DE) shows condensation of the nucleus and cytoplasm. Large smooth muscle cells (SM) are observed outside the lumen. [x 10.000).Inset, The position of the star corresponds to that in figure 3. Such a DEforms adhesive junctions with E5. (x 37.000.)

erated dark endothelial cells lying wholly within the lumen of a blood vessel showed adherens-type junctions with the luminal surfaces of other endothelial cells (fig. 3). The extent of individual cell degeneration varied considerably and, when slight, consisted only of nuclear and cytoplasmic condensation with a tendency towards vacuolization. Such degenerated endothelial cells still formed integral parts of the wall

of the blood vessel with the interdigitated junctions between the adjacent intact endothelial cells (fig. 4). Blood vessels whose endothelium contained such degenerated dark cells showed dark cellular fragments both inside and outside the vascular lumen. Sometimes the structure of basal lamina was noticed along the adjacent dark cellular fragments, which showed rich ribosomes (fig. 4). When endothelial cell degen-

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FIGURE 4. Electron micrograph of a capillary with a degenerated dark endothelial cell in the deep dermis of the skin lesion. Among five endothelial cells (E1-E4 and DE). one cell (DE) shows degenerated changes including nuclear and cytoplasmic condensation. On the left side of DE. an accumulation of dark cellular fragments is observed (open star). Pericytes (Pc) are observed outside the lumen. lymphocytes (Lc) and fibroblasts (Fb) are also observed in the perivascular area . [ x 4.000.) Inset,ln a high magnification of dark cellular fragments (open star). the structure of basal lamina is observed (arrOWhead) adjacent to a DE. [x 11.000.)

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ERYTHEMA NODOSUM-liKE lESIONS OF BEH<;EfS SYNDROME (Bang et al.)

Lu

b FIGURE 5. Electron micrograph of a venule with a more degenerated endothelial cell in the deep dermis of the skin lesion. a, One endothelial cell (DE) shows severe damage af the contracted cytoplasm. and another cell (E) seems to be intact morphologically. Dense deposits of amorphous material are found a long the reg ion of the basal lamina (arrow). large smooth muscle ce lls (SM) are a lso observed outside the lumen (Luj. ( x 6.000.) b, A higher magnification of DE in panel a. The DE possesses rich ribosome s (arrowheaD). swelling mitochondria (M). and contracted vacuoles (smallarrows) in the cytoplasm. [ x 15.000.)

eration was more extensive, an entire cell or all of the endothelial cells viewed in a vascular cross section exhibited necrosis accompanied by contracted vacuolization and fragmentation of the cells. However, the integrity of the blood vessel wall was maintained by 11n

preservation of the remainder of the cells (figs. 5 and 6). Sometimes, dense amorphous material was deposited along the basal lamina of necrotic endothelial cells (fig. 5). These degenerated dark endothelial cells were observed in the arterioles, capillaries, and/

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FIGURE 6. Electron micrograph of a capillary with endothelial cell necrosis in the deep dermis of the skin lesion. The capillary viewed in the vessel cross section shows d isintegration changes of the endothelial cells accompanied by the thickened basal lamina (arrow). The lumen (Lujis full of cellular debris. An intact pericyte (Pc) is observed outside the lumen. (x15.000.)

or venules of the skin lesions of all 18 patients studied (table 1). DISCUSSION

Electron microscopic examination of endothelial cell damage in the erythema nodosum-like lesions of patients with Behcet's syndrome demonstrated that degenerated dark cells appeared in the endothelium of arterioles, capillaries, and venules in the superficial and deep dermis. These degenerated dark endothelial cells showed nuclear and cytoplasmic condensation and were characterized by rich ribosomes in the cytoplasm and a tendency towards vacuolization. On the other hand, it has been reported that the morphologic features of endothelial cell damage in cell-mediated hypersensitivity reactions are characterized by a perivascular accumulation of lymphocytes and by endothelial cells swelling with cytoplasmic lucency.U Additional features of cell-mediated hypersensitivity reactions including swelling, blebbing, and disruption of cellular and intracellular membranes have also been reported. The endothelial cell damage we describe predominantly consists of contraction changes of the entire cells and clearly differs from the morphologic features of endothelial cells injured by cell-mediated hypersensitivity reactions.Z" '? If, indeed, cytotoxic or activated lymphocytes are responsible for the degen-

eration of dark endothelial cells, a cytotoxic mechanism that does not require direct and persistent anatomic contacts may be implicated, because, except during diapedesis, such endothelial cells were separated from enveloping perivascular lymphocytes by the basal lamina and, at times, by perivascular collagen as well.f Consequently, the contraction changes of endothelial cell degeneration may undergo two morphologically distinct processes of immunologic injury. Moreover, lymphocyte-cuffed blood vessels observed in these skin lesions simultaneously exhibited endothelial cell hypertrophy, luminal obstruction, and proliferation of pericytes, as well as endothelial cell degeneration. The cause of these alterations in the microvasculature is unknown, not only in typical cell-mediated hypersensitivity but also in the erythema nodosum-like lesions in Behcet's syndrome. 11,12 TABLE 1. Appearance of Blood Vessels with Degenerated Dark Endothelial Cells in the Upper and Deep Dermis in Erythema Nodosum-like LesIons of 18 Patients with sehcers Syndrome Classification of Blood Vessels

Per cent of Blood Vessels with Degenerated Dark Endothelial Cells*

Arteriole Venule Capillary

34.0 ± 30.9 43.8 ± 25.9 44.5 ± 20.1

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* Mean

± SD.

ERYTHEMA NODOSUM-L1KE LESIONS OF BEHc;rrS SYNDROME (Bang et 01.)

On the other hand, it has been reported that erythema nodosum-like lesions of Behcet's syndrome have strong associations with the presence of circulating immune complexes and the deposition of immunoglobulins and C3 in the wall of venules and capillaries.P Our electron microscopic observations revealed the deposition of an amorphous material along the adjacent basal lamina of damaged blood vessels. Such an amorphous material may be regarded as perivascular precipitates of immunoglobulins and C3. 14 Subsequently, neutrophilic leukocytes were identified in perivascular collections which extended into the intervascular dermis, although similar cells could also be seen within such vessels. The neutrophilic infiltrates seen in Behcet's syndrome must be secondary to vascular degeneration typified by contraction changes of dark endothelial cells.

6. 7.

8.

9.

10.

Acknowledgments. The authors thank Kenzo Uehira for technical assistance.

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