An ultrastructural study of transcutaneous migration of polymorphonuclear leukocytes following application of leukotriene B4.

Skin Pharmacol 1990;3:236-247

© 1990 S. Karger A G . Basel 1011-0283/90/0034-0236S2.75/0

An Ultrastructural Study of Transcutaneous Migration of Polymorphonuclear Leukocytes following Application of Leukotriene B4 M. Paulissen a, J.H.J. Copius Peereboom-Stegemana, P.C.M. van de Kerkhop Departments of aToxicology and bDermatology, University of Nijmegen. The Netherlands

Key Words. Endothelium • Inflammation • Leukotrienes • Polymorphonuclear leukocytes • Psoriasis

The migration o f polymorphonuclear leu kocytes (PMN) into the epidermis is a char acteristic feature o f the psoriatic lesion [1] and classical antipsoriatic therapies such as dithranol, topical corticosteroids, pholo(chemo)therapy, methotrexate and retinoids have been shown to inhibit this process [26]. The production o f chemoattractants is increased in lesional skin o f patients with psoriasis, whereas the symptomless skin of these patients is characterized by an in creased activity o f phospholipase A 2 - the rate-limiting enzyme for the release o f in

flammatory eicosanoids - and an increased formation rate o f the eicosanoid leukotriene B4 (LTB4), which is a potent chemoattrac tant [7, 8]. However, the appearance o f PMN occurs at random, which excludes the possi bility o f a systematic sequentional study of the different stages o f the passage o f these cells into psoriatic skin. Compared to the procedure o f skin cham bers on abraded skin [2] the epicutaneous application o f LTB4 is a noninvasive method to induce PMN accumulation in the epider mis [9], The accumulation of PMN resulting

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Abstract. In the present study the ultrastructural aspects o f the migration o f polymorpho nuclear leukocytes (PMN) in apparently normal skin following the epicutaneous application of leukotriene B4 (LTB4) were studied and it was investigated whether this model is repre sentative o f the migration o f PMN in psoriatic skin. At the ultrastructural level the following features were observed: degranulation o f PMN, formation o f fenestrations and gaps of the endothelium, a multilayered basal lamina and hyperactive endothelial cells with protrusions and focal necrosis o f these cells. Epicutaneous application o f LTB4 is a practical approach to study the dynamics o f the interaction between PMN and the endothelium in vivo.

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Transcutaneous Migration of Leukocytes

Materials and Methods LTBi Application and Skin Sampling Three males and 1 female, between 21 and 44 years old, without signs or history of skin diseases, participated in the investigation. In each of these vol unteers a rectangular lest area (10 X 10 cm2) was delineated on the upper arm. Before LTBj applica tions a biopsy was taken from the corners of the test areas, to assess the ultrastructural appearance of the unchallenged skin. Biopsies (3 mm in diameter) were taken, using a razor blade in conjunction with a metal guard. On 5 test sites within the test areas LTB4 in etha nol was applied and on one test site ethanol only was applied as a control. The application procedure was carried out as described previously [3]. In brief, 50-ng aliquots of LTB4 (Paesel GmbH, Frankfurt, FRG) dissolved in 10 pi ethanol and 10 pi of ethanol only were applied through glass cylinders onto the lest sites (5.5 mm in diameter). Previous studies have shown that this dose results in the extravasation of PMN and intraepidermal pustule formation approaching a max imum 24 h after application [3], Application sites were marked with eosin and covered with impermeable dressings (Silverpatch,

Van der Bend, Brielle, The Netherlands). Razor blade biopsies were taken 1, 2. 4. 8 and 16 h after the application of LTB4 and 16 h after the applica tion of ethanol only. From each application site one biopsy was taken and processed for routine electron microscopy. Fixation Procedures Biopsies were fixed in McDowell (1 % glutaraldehyde, 4% formaldehyde in a phosphate buffer, pH 7.4). The specimens were dissected in about ten smaller parts and fixed in fresh medium during 24 h. The samples were then washed in a phosphate buffer and postfixed in OsOj during I h. Block stain was performed with 0.5% uranylacetate in Palade buffer (pH 7.4. 135 mmol) during 2 h. Specimens were dehy drated in graded alcohols, and embedded in Epon 812 using standard embedding procedures. Semithin sections were double-stained with toluidine blue and basic fuchsin. Ultrathin sections were cut on a Reichert OM U3 ultratome, mounted on 50-mesh grids, stained with uranyl and lead citrate and studied in a Philips 301 at 60 kV. From every individual 2 semithin sections and at least 4 ultra-thin sections were investigated.

Results

Appearance o f the Unchallanged Skin Comparing the 4 biopsy sites within the test area in each volunteer, no significant dif ference was observed with respect to epider mal thickness, number o f microvessels and perivascular mast cells. These features were similar comparing the 4 volunteers, although the female subject showed a relatively thin epidermis. No PMN and no degranulation of mast cells were observed in any o f these biopsies.

Response to LTB4 Application Light microscopical studies on semithin sections revealed a considerable intersubject variation. In 2 o f the 4 volunteers (2 males,


from this application is well reproducible as to magnitude and dynamics [3, 10]. There fore the LTB4-induced accumulation of PMN is an attractive approach to study the migration o f PMN in vivo, using dermis and epidermis as a natural diffusion chamber. In the present investigation the ultra structure o f the transcutaneous migration of the PMN was studied at intervals following the epicutaneous application o f LTB4 in or der to answer the following questions: (i) What is the sequence o f events after LTB4 application with respect to ultrastruc tural changes o f small blood vessels, PMN extravasation and migration o f PMN? (ii) To what extent are these changes rep resentative o f the established ultrastructural changes in psoriasis?


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21 and 36 years old) the first extravasation o f PMN was seen 4 h after LTB4 application. In the remaining 2 subjects (1 male and 1 female, 26 and 44 years old) no extravasa tion at all was observed at different time intervals. Electron microscopical investigations in the ‘nonresponders’ revealed a few PMN sporadically distributed over the epidermis, whereas the ‘responders’ showed pro

F ig .1. Degenerating PMN within the vessel wall 4 h after the application of LTBj. The cytoplasm is heavily vacuolated (asterisks). Several endothelial cells show dis integration (D). X 5,650. Fig. 2. Detail of PMN just out side the lumen of a vessel close to the basal lamina of the dermoepidermal junction 16 h after the ap plication of LTBj. The endothelial cell neighboring the PMN is necrot ic. X 13,100. Fig. 3. Blebbing of the endothe lium (E) 4 h after the application of LTBj. X 11,600.

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nounced intraepidermal micropustules 8 and 16 h after LTB4 application. In the re sponders ultrastructural characterization of biopsies at 4, 8 and 16 h after LTB4 applica tion revealed a consistent and pronounced accumulation o f PMN. After 1 and 2 h no ultrastructural changes at all were observed. At 4 h PMN in close vicinity to the vessels and endothelial changes dominated the pic ture; only a mild accumulation o f PMN oc-



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curred in the epidermis. At 16 h the forma tion o f micropustules in the epidermis was the major observation; a relatively modest extravasation o f PMN and mild changes of the endothelium were seen. At 8 h the ap pearance had an intermediate position. The earliest event observed following the application o f LTB4 was the accumulation of PMN within the vessels. The relatively older PMN, possessing a nucleus with 4 or 5 lo bules, showed high numbers o f relatively

large and irregularly shaped electron-lucent areas indicating degeneration o f these cells (fig. 1). Some PMN showed rounded va cuoles indicative o f degranulation. The en dothelium was essentially normal outside ar eas where PMN attached to the endothelial wall. In close vicinity to PMN or remnants o f degenerated PMN, the endothelial cells showed a number of changes. At some sites the endothelium demonstrated signs o f de generation (fig. 2). A frequent event was the


Fig. 4. Remnants of PMN in close vicinity to the endothelium 8 h after the application of LTB4. The endothelium shows many fe nestrations (arrows). The basal lam ina is multilaminaied (arrowheads). X 15,500.


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formation o f protrusions o f the endothelium at the sites o f interaction (fig. 3). A rather conspicuous feature was an increased num ber o f fenestrations and gap formation in the endothelium at places where there was close contact to a PMN (fig. 4, 5). At the same time duplications o f basal lamina, transuda tion and the occurrence o f PMN granules between the layers o f the basal lamina were observed (fig. 6). PMN were seen in close vicinity to endothelial gaps (fig. 7a, b). In the

nonresponders no abnormalities o f the endo thelium were observed. After extravasation some PMN showed signs of degranulation on their way to the epi dermis. After passing the dermoepidermal junction, formation o f sharply demarcated micropustules was seen (fig. 8). No ultrastruc tural changes o f epidermal cells surrounding the micropustules were observed. Remark ably, the PMN at the intraepidermal localiza tion still possessed a large number of granules.


Fig. 5. Detail of a PMN in close vicinity to the vessel wall 4 h after the application of LTB4. The endo thelium shows gaps (arrow) and transudate is coagulated outside the vessel (asterisks). X 44.500.

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Discussion By ultrastructural assessment, the first ex travasation o f PMN was shown 4 h after LTB4 challenge. Using light microscopy, the accumulation o f PMN has been reported to commence after 6 h with the same micromorphological characteristics and subse quent dynamics as has been observed in the present investigation [9, 10].

The present study and previous investiga tions [3, 9, 10] demonstrated that the re sponsiveness to epicutaneous applications of LTB4 is well reproducible within the same individual. However, intersubject variation is substantial. Both the structure o f the stra tum corneum and the production rate o f arachidonic acid metabolites by the skin itself are o f relevance in this respect. The penetra tion o f epicutaneously applied substances is


Fig. 6. Free granules situated between the layers of the basal lam ina of the endothelium 8 h after the application of LTB4. The endothe lium shows many protrusions (P) and fenestrations (arrows). The transudate is coagulated in the stroma adjacent to the vessel. X 95.000.


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Fig. 7. Extravasation of PMN. a Overview of a small blood vessel and PMN (P) extravasation. At the luminal site endothelium still is showing a gap (arrow). X 23,000. b Detail of extravasating PMN. On the left part of the figure endothe lium is seen (E). Bottom left and right shows parts of two pericytes (P). Basal lamina discontinuous (between arrows). X 65,500.


a

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enhanced if the barrier function o f the stra tum comeum is defective [11]. As the clini cally involved and symptomless skin o f pa tients with atopic dermatitis is relatively permeable [12], subjects with an atopic con stitution might display an increased bio availability o f epicutaneously applied LTB4. The clinically uninvolved skin o f psoriatic patients is characterized by a decreased re sponsiveness to LTB4 [13, 14], which has been explained by the supposition o f a habi

tuation to the increased production rate of LTB4, also in the symptomless skin o f these patients [8]. Subjects with a psoriatic consti tution might have a decreased responsive ness to LTB4. No endothelial abnormalities were shown before the extravasation o f PMN and abnor malities o f the endothelium were restricted to the sites o f attachment o f the PMN. This observation is in line with studies using an in vitro model and animal model: endothelial


Fig. 8. Sharply demarcated mi cropustule in the epidermis 16 h af ter the application of LTB4. The PMN still contain a substantial amount of granules. X 4,200.

cell damage and increased permeability of the endothelium occurred via activated PMN [15, 16]. The limited degree o f degranulation, re stricted to the phase o f extravasation, is compatible with the relatively low capacity o f LTB4 to induce degranulation in vitro compared to other chemoattractants [17, 18]. Using the skin chamber technique on abraded skin - an alternative model for stud ies on the transcutaneous migration o f PMN in vivo - a massive degranulation o f PMN was observed if LTB4 was used as chemoat tractant, indicating the rather unphysiological and nonselective nature o f this approach [19]. Exclusively at sites o f PMN attachment the endothelium showed pronounced changes permitting the extravasation of PMN. Following epicutaneous application o f LTB4 the PMN migrated exclusively via a gap completely lined with an intact plasma membrane (fig. 7). However, the question whether PMN migrated through the gaps at the point o f cell junctions or migrated transcellularly cannot be answered from the present study. At the molecular level the for mation o f fenestrations and gaps at the at tachment points might be explained by the release o f kationic proteins, calcium ions and oxygen radicals from the PMN [15, 20]. The release o f these radicals might explain the sporadically occurring cytolysis o f endo thelial cells. Histamin has been reported to induce fenestrations and gaps in the endo thelium [21]. However, as degranulation of mast cells was not observed in the present study, no ultrastructural evidence is avail able to suggest a role for histamin in the endothelial abnormalities as observed in this investigation. During the process o f degran ulation, elastase is released. This enzyme be

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longs to the group o f neutral protéinases and degrades collagen type IV, which is a major constituent o f the basal lamina [22], Interest ingly, the basal lamina at these sites is multi layered, which might be interpreted as an attempt o f defense against the invasion of PMN. Although a multilayered basal lamina is a common feature in the venous segments o f the capillary loops o f noninflamed human skin, a combination o f increased multilayer ing, the occurrence o f transudate and o f PMN inclusions (fig. 6) was restricted to the LTB4-treated areas only. The transcutaneous migration o f PMN during the pathogenesis o f the psoriatic le sion shares important ultrastructural charac teristics with the migration of PMN follow ing epicutaneous application of LTB4. In the psoriatic lesion degranulation o f PMN, in creased density o f fenestrations and gaps and a multilayered basement membrane are wellestablished features [23-25]. However, in addition to these common features, the pso riatic lesion demonstrates some changes which indicate that LTB4 is not the only determinant. Other factors contribute to psoriatic inflammation. In psoriasis endo thelial gaps are seen also outside attachment sites o f PMN [25]. The early degranulation o f mast cells [26] refers to a role o f histamin in this respect. In psoriasis those PMN which have reached the epidermis are largely degranulated. As LTB4 has a weak degranu lating potential other chemoattractants might play a role. Indeed a range o f chemoat tractants with a more pronounced induction o f degranulation are present in the psoriatic lesion such as platelet-activaling factor and C5a [27, 28]. Epicutaneous application of LTB4 proved to be a useful in vivo model for sequential studies o f the different phases o f the migra-



tion o f PMN in human skin. The extravasa tion of PMN was not preceded by ultrastruc tural changes o f the endothelium. The ultrastructure o f the interacting PMN and endo thelium proved to be representative for this interaction in psoriasis, although other fac tors contribute to the inflammation o f the psoriatic lesion.

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Received: December 28, 1989 Accepted: June 5. 1990 P.C.M. van de Kerkhof, MD, PhD University of Nijmegen Javastraat 104 NL-6524 MJ Nijmegen (The Netherlands)


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Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The effect of topical retinoids on the leukotriene-B4-induced migration of polymorphonuclear leukocytes into human skin.

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

Pasteurella haemolytica leukotoxin enhances production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by bovine polymorphonuclear leukocytes.

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