INTERNATIONAL JOURNAL OF LEPROSY^ Volume 52, Number I

Printed in the U.S.A.

Free Subepidermal Grenz Zone (Band of Unna) inLepromatous Leprosy. Histological and

Ultrastructural Findings'Ulrich Martens and Georg Klingm011er 2

When lepromatous leprosy found its wayinto the histopathological textbooks on skindiseases, the term "band of Unna" or "freegrenz zone" was mentioned for the first timein connection with the disease ( 7 ). The grenzzone appeared to be "free" from leprosybacilli under the light microscope, and itwas for this reason that this zone was as-signed the name free grenz zone. Generally,the term is used in literature as a synonymfor the subepidermal layer of collagen andconnective tissue fibers in lepromatous lep-rosy. However, no comprehensive study ofthe histology and ultrastructure of this areahas been undertaken until now.

The purpose of this paper is to describethe morphology of the grenz zone in lep-romatous leprosy, to compare it with thesubepidermal tissue in tuberculoid and bor-derline leprosy, and to discuss some factorswhich may explain the mechanism of itsformation. Our own histological studies alsoconfirmed the existence of subepidermalzones which are free of the characteristicalterations in the following diseases: lym-phocytoma cutis, granuloma eosinophili-cum faciei, lobomycosis, acrodermatitischronica atrophicans, and lichen amyloid-osis.

MATERIALS AND METHODSClinical material. Twenty skin samples

were taken from 6 patients: 4 were sufferingfrom lepromatous, 1 from tuberculoid, and1 from borderline leprosy. The tuberculoidcase was a low-resistant form and the bor-derline case belonged to the borderline lep-romatous type. Two of the lepromatous lep-

' Received for publication on 23 August 1983; ac-cepted for publication in revised form on 9 September1983.

2 U. Martens, M.D., and G. Klingmiiller, M.D., Pro-fessor of Dermatology, University-Skin Hospital,D-5300 Bonn I, West Germany.

rosy patients were treated for many yearsby the Department of Dermatology, Uni-versity-Skin Hospital, Bonn, Germany, sothat different stages of the disease could beobserved with light and electron micros-copy.

Light microscopy. Hematoxylin and eo-sin (H&E), the Fite-Faraco method for acid-fast bacilli ('), and the modified method withsirius red from Junqueira, cat al. ( 3 ) were usedfor staining. The birefringency of the col-lagen was enhanced with the sirius red dye.When the slide was then placed under apolarization microscope with a bright lamp,the collagen was observed to be surroundedby a dark background.

Scanning electron microscopy. The re-maining histological sections had a thick-ness of 5 pm and were not stained, but re-moved of paraffin and sputtered with gold.The sections were observed under the Hi-tachi scanning electron microscope; pho-tographs were taken with Polaroid instantfilm.

Transmission electron microscopy. Partsof each biopsy were fixed in two parts col-lidine buffer and one part 4% osmium te-troxide for 2 hr. Block staining was per-formed in 1% phosphotungstic acid and0.5% uranyl acetate. The material wasembedded in Epon 812, sectioned in a LKBultratome with Balzer diamond knives, anddouble stained in uranyl acetate and leadcitrate. Micrographs were taken with a ZeissEM 9 S-2 electron microscope.

RESULTSLight microscopy. The grenz zone ap-

peared as a homogeneous layer of connec-tive tissue. It contained flattened nuclei offibroblasts and was always uniformly at-tached to the epidermis without any retepegs (Fig. I). Below the stretched collagenband lay the confluent leproma in which

55

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International Journal of Leprosy^ 1984

FRi. I. A skin cross-section from the finger of alepromatous patient showing at the top a thick stratumcorneum (st c) and a stretched epidermis (e) with agrenz zone (g z) lying directly below it. In the bottom,the foamy leproma (le) is shown to consist of only afew connective tissue strands (1 -1&E x 335).

FIG. 2. Under polarized light, the grenz zone (g z)appears as a bright contrasting band. In the leproma(lc) only the thin and short connective tissue fibers arevisible. A thick collagen bundle (co) is situated in themiddle of the corium (bottom right) (sirius red underpolarized light x 310).

only short and thin connective tissue fiberswere visible (Fig. 2). The capillary vesselswere observed to be extremely diminishedin the grenz zone. Venules and arteriolescould be found between the grenz zone andthe leproma.

The ratio of the widths of grenz zone toepidermis—without the stratum corneum-was measured at 84 different positions onthe skin samples. The average width of thegrenz zone was 35.5 ± 5.6 pm in the initialstage of lepromatous leprosy and 23.7 ± 3.1pm in progressive lepromatous leprosy.Likewise the average width of the epidermiswas greater in the initial stage of leproma-tous leprosy than in the progressive stage.This implied that the width of the grenzzone decreased simultaneously with the epi-dermis in the progressive stage of the dis-ease. However, the epidermis always re-mained thicker than the grenz zone. Theabsolute value of the width of the grenz zonealso depended on the width of the epidermisand where the sample was taken. For in-stance, both the grenz zone and the epider-mis from the thigh were obviously thickerthan those from the ear of the same patient.

Other such transitional areas between theconnective tissue and epithelium in lepro-matous leprosy, such as below the epithe-lium of the tongue, had no grenz zone.

Scanning electron microscopy. When theconnective tissue fibers of the grenz zonewere magnified by the scanning electron mi-

croscope, bundles of fibrils were observed(Figs. 3 and 4). These bundles had a band-like form and had their flat sides lying onhorizontal planes below the stretched epi-dermis (Fig. 5). This was not seen in thedeeper layers of the corium or in normalskin. Only a few short, extremely thin bun-dles were observed to be perpendicular to

FIG. 3. The epidermis (e) lies beneath the brightstatum corneum (st c). The remains of the nuclei of itsbasal cells are found in the cavities formed by theirremoval (arrow). Sectioned lepra cells form membra-neous cavities containing lepra bacteria (arrows)( x 1200).

52, 1^Martens and Klinsunidler: Gren: Zone in Lepromatous Leprosy^

57

FIG. 4. The cavities in the grenz zone (g z), whichwere formed as a result of the removal of cells and cell

extensions, are found to be thinner and stretched (ar-row) compared with the cavities left by lepra cells

( x 840).

the epidermis. Each of these bundles had adiameter of 0.2-0.3 pm, thus consisting of5-10 elementary fibrils. The diameter of thecollagen bundles increased in the grenz zonefrom top to bottom. But in comparison withthe middle and deep corium, the band ofUnna contained relatively small bundles offibrils (0.7-1.2 pm). It was often possible tofind bundles of fibrils with a width of 20 pmin the lower third of the corium outside theleproma. This was nearly as thick as thewhole grenz zone of progressive leproma-tous leprosy. The gap-like cavities in theconnective tissue of the grenz zone were onlylarge enough for flattened cells. The shapeof the connective tissue in the grenz zoneallowed maximum filling by the collagen.Examinations of the borderline leprosy caseconfirmed the existence of a grenz zoneequivalent; this was not true in our case oftuberculoid leprosy. When the grenz zoneequivalents were found to be present in oursamples of borderline leprosy, they differedfrom the grenz zone in lepromatous leprosyin the following ways: 1) they had a largerwidth; 2) the transitional area between theleproma and the grenz zone was poorly de-fined; and 3) their widths depended on thenumber of epithelioid cells to macrophagesin the borderline lesion, i.e., the greater the

FIG. 5. The upper third of the picture shows an

empty space, which contained the nucleus (nu) of abasal cell of the epidermis (e). The band-like collagen

bundles (co) of the grenz zone lie horizontally with theirflat sides facing towards the basal side of the epidermis.

A fibroblast could have been originally situated in the

small space (indicated by the arrow). The large lepra

cell (lc) is found below the grenz zone ( x4100).

number of epithelioid cells, the wider werethe grenz zone analogues.

Transmission electron microscopy. Thebasement lamina of the dermal-epidermaljunction above the grenz zone showed nopathological changes. Most of the cells sit-uated in the grenz zone in early and pro-gressive lepromatous leprosy were fibro-blasts. These fibroblasts were together withcertain macrophages only slightly infestedwith leprosy bacilli. None of these cells con-tained giant phagolysosomes, which werecharacteristic of the typical lepra cells withtheir foamy intracytoplasmic structure. Inmost cases the leprosy bacilli were foundalone inside the phagosomcs of the grenzzone cells.

These slightly infested cells could also beseen directly below the basement lamina ofthe dermal-epidermal junction (Figs. 7 and8). More cells were found in the grenz zoneof the progressive stage of lepromatous lep-rosy than in the early stage. The ultrastruc-

58^ International ,lournal of Lelvosj'^ 1984

Fui. 6. The basement lamina of the dermal-epidermal junction is clearly visible (arrow). One can see length-wise stretched thin fibroblast-extensions (ti) between the collagen bundles (possibly type III and type I). Thediameter of these bundles increases with increasing depth. Infested macrophages can be seen; these form theupper edge of the leproma ( x 7500).

• • _

52, 1^Martens and Klingtniiller: Grenz Zone in l_epronuttous Leprosy^59

Fu;. 7. Two engulfed lepra bacteria (arrows) areshown in a grenz zone cell lying directly below theepidermis (e) ( x 6860).

FIG. 8. An enlarged section showing the extensionof a grenz zone cell with two lepra bacilli (arrows), eachsituated peripherally in different lysosomes (1y)( x 15, 680).

tural appearance of each grenz zone cell de-pended on how progressive the lepromatousform of the disease was. It was, however.never identical with the ultrastructure of thecells seen in the leproma.

Leprosy bacilli were found to be rare inthe lumina and in the endothelial cells ofthe vessels.

More than 330 measurements of collagenfibrils of the grenz zone were taken. Fibrilswith a diameter of 370-420 A appeared inthe whole narrow grenz zone of patients withprogressive lepromatous leprosy and in theupper third or upper half of the large grenzzone in the early stage of lepromatous lep-rosy. The lower part contained collagen fi-brils with a diameter ranging from 600-800 A.

DISCUSSIONThe following factors could have been in-

volved in the formation of the free grenzzone: optimal growth temperature, ultra-violet rays, lack of oxygen, mechanicalcompression, and immunologic factors.

Optimal growth temperature of the leprabacilli C) is primarily responsible for thedistribution of the leproma in different skinareas of the body ( 5 ). The fact that the grenzzone has only a minimal Bacterial Index

may be due to a lack of this optimal bac-terial growth temperature.

Also conceivable is the inhibition of bac-terial growth directly below the epidermisby ultraviolet radiation because ultravioletrays in sunlight are found to be able to killMycobacterium leprac in bacillary suspen-sions C).

The fact that only a few lepra macro-phages were present in the grenz zone isdefinitely not due to the lack of oxygen sinceblood supply in the leproma, which is fullof lepra macrophages, is hardly better thanin the grenz zone. The vessels in the lepromaare compressed much faster than the vesselsin the connective tissue of the grenz zone.The lack of oxygen can only accelerate thenarrowing of the grenz zone; if the numberof vessels is diminished, the collagen pro-duction is also reduced.

The pressure exerted by the leproma onthe papillary connective tissue is not re-sponsible for the grenz zone remaining"free" but, rather, for its structure, wherethe parallel layers of fibrils in the grenz zoneare compressed b■,.' the leproma below it.

That the fibroblasts form the main cel-lular fraction of the grenz zone is the expla-nation for the relatively low number of ba-cilli which were seen under the light

60^ International Journal of Leprosy^ 1984

microscope because the fibroblasts have alow capability of phagocytosis.

Fleischmajer, et al. ( 2 ) could label fibrilswhich have the same diameter as the fibrilsin the smaller grenz zone with ferritin-marked, type I II antibodies. Fibrils with di-ameter-range from 600-800 A, which arefound in the lower part of the grenz zone inthe early stage of lepromatous leprosy, havebeen identified by Fleischmajer, ci al. withimmunoelectron microscopy as type 1 col-lagen in normal and scleroderma skin. Inlepromatous leprosy this could mean thatunder the progressive compression of theleproma, only a type III collagen layer re-mains which corresponds to the normalpapillary (fermis since Meigel, et al. ( 4 )showed by immunofluorescence microsco-py that the main part of the papillary dermisconsists of type III collagen.

SUMMARYAlthough the grenz zone is indeed free of

typical leprosy cells with giant lysosomes,it is not free from leprosy bacilli—these arefound in slightly infested macrophages andfibroblasts. For that reason, it would bemuch clearer if we simply called this regionthe subepidermal grenz zone of leproma-tous leprosy. The subepidermal grenz zoneis not pathognomonic of leprosy since grenzzones can be found in other diseases as well.Whereas the grenz zone is typical in lep-romatous leprosy, it is not necessarily char-acteristic of the borderline or the tubercu-loid form of the disease. The light andelectron microscopical structure of the grenzzone shows that the formation of the sub-epidermal grenz zone cannot be explainedby any particular pathogenetic principle onwhich therapy can be based.

RESUMENAunque Ia zona epidermica de grenz es realmente

libre de celulas tipicas do lepra con lysosomas gigan-

tescos, no es fibre del bacilo de la lepra—estos se en-cuentran en los macrofugos y fibroblastos poco infes-

tados. Por esa razon stria much° mas claw si

simplemente le Ilamaramos a esta region la zona sub-epidermica grenz de lepra lepromatosa. La zona epi-

dermica de grenz no es patognomOnica de Ia lepra pues-to que esta zona tambien puede encontrarse en otras

enfermedades. Mientras que la zona de grenz es tipica

en lepra lepromatosa, no es necesariamente caracte-ristica de las formas dimorfa o tuberculoide de Ia en-

fermedad. La estructura microscOpica de la zona de

grenz no sugiere que su formaciOn pueda explicarse por

algOn principio patogenico derivado de la terapia.

RESUMEBien que la zone I imitrophe (grenz zone) soil en clfet

liberee de cellules typique de Ia lepre avec de lysosomes

grant, cc West pas sans de bacilles de la lepre—celles-ci soul un peu ill de les macrophages et fibro-

blasts. Pour cette raison, it serait plus chair si nousappelions cette region la zone limitrophe sub-epider-

mique de la lepre lepromateuse. La zone limitrophe

(grenz zone) sub-epidcrmique West pas pathogno-mique de la lepre, car des zones limitrophes peuvent

etre observees egalement dans d'autres maladies. Alors

que Ia zone limitrophe est typique dans Ia lepre lepro-

mateuse, elle nest pas necessairement caracteristique

dans les lOrmes dimorphs ou tuberculoide. La structurede cette zone limitrophe. telly qu'elle est observer au

microscope optique ou au microscope elcctronique,

niontre que Ia frmation dune zone limitrophe sous-

epidermique ne peut pas etre expliquee sur la base d'un

mecanisme pathogene particulier, qui pourrait servir

a decider la therapeutique.

Acknto Iedgments. The authors wish to thank Mrs.

A. Schneider and Mrs. R. Speck for technical assis-

tance.U. Martens has contributed essential parts of his

dissertation for the degree of Doctor of Medicine for

this article.

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