INTERNAIIONAI .1()VRNAI ()I I iransi^ Volume 46. Number IPrinted iii the

Mycobacterium leprae Found in EpidermalCells by Electron Microscopy'

S. Okada, J. Komura and M. Nishiura

A figure of a leproma stained by the Ziehl-Neelsen method in the hook entitled DieLepra, written by Klingmuller ( 3 ), showsleprosy bacilli in the epidermis. Sato (I ), inhis hook Rai (frpro.9), wrote that the leprosybacillus invades the epidermal cells. Ilarada(I) reported that leprosy bacilli could hefound in the epidermis with his modified al-lochrome staining method.

We have noticed leprosy bacilli in parts ofthe epidermis in some cases utilising theoptical microscope. However, the histopath-ologic sections for optical microscopy arerelatively thick. Therefore, it could not heconcluded with the optical microscopewhether the bacilli are certainly located inthe epidermal cells or whether they are at-tached to the surface of the epidermal tissuesection. In the latter case, the bacilli and theepidermis are seen one above the other as ifthe bacilli are present in the epidermal cells.Leprosy bacilli attached to the sectioningknife can he transferred and adhere to otherplaces. In fact, it can he seen in some casesthat the leprosy bacilli are attached to placeswithout any tissue or nearby epidermis (Fig.I ).

Recently we have found by electron mi-croscopy that leprosy bacilli may he en-gulfed within epidermal cells. The sectionfor electron microscopic observation is verythin. Therefore, it can he readily determinedwhether or not the leprosy bacilli are piledup on the epidermis.

MATERIALS AND METHODSA leproma from a male patient, aged 65

years, having lepromatous leprosy, was ex-cised for the study of the cultivation of lep-rosy bacilli. A part of the leproma was fixedwith formalin without separating its epider-mis and histopathologic sections were made

'Received for publication 23 September 1977.=S. Okada. M.D., Associate Professor, Leprosy Re-

search Laboratory; .1. Komura, M.D., Lecturer. Depart-ment of Dermatology: and M. Nishiura, M.D.,Professor. Leprosy Research Laboratory, Kyoto Uni-versity School of Medicine, Kyoto, Japan.

for optical microscopic study. The epidermisof the remainder was cut off with scissors inorder to facilitate the grinding of the lepro-ma. In order to observe the condition of theleprosy bacilli used for cultivation, a smallpart of the leproma from which the epider-mis had been cut off was fixed with lc; Os-mium tetroxide in M/ 15 phosphate buffer(pH 7.4), dehydrated in graded ethanol andembedded in methacrylate for electron mi-croscopy. Because the purpose of the prep-aration of the specimen was the electronmicroscopic observation of leprosy bacilli,methacrylate which can, among the variousresins, best permeate leprosy bacilli infoamy cells was used. 1.1Itrathin sections ofthe embedded specimen were stained with

FIG. I. The leprosy bacilli (M) are found notonly at the part occupied by the epidermal tissue,but also at the part not occupied by any tissue.

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46, I^Okada et al: M. leprae in Epidermal Cells^ 3I

FIG. 2. The leprosy bacilli (M) are found in an epidermal cell. D:desmo-some.

FIG. 3. Higher magnification of a portion of Figure 2. D:desmosome, whichfairly loses its normal figure by being pulled by scissors: K:keratohyalin gran-ule: M:leprosy bacilli; O:Odland body; T:tonotilament.

uranvl acetate and lead citrate and observedwith an Akashi AEM-80 electron micro-scope.

RESULTS AND DISCUSSION

A portion of the epidermis was found stillpresent in some specimens under electronmicroscopic observation. The bacilli, over

twenty of which were grouped together werefound in an epidermal cell (Figs. 2, 3). Trans-parent tones are observed around the bacillibut there is no membrane surrounding thegroup of bacilli. The cell which contains thebacilli and the other cells near it are epider-mal cells because they have desmosomesand tonofilaments.

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International Journal of Leprosy^ 1978

FIG. 4. A foamy cell which contains leprosybacilli (M) is observed just under the basal celllayer of the epidermis.

Desmosomes are particularly present inthe stratified squamous epithelium, especial-ly of the epidermis, but they can he foundalso in the other types of epithelium. In theskin, the sweat glands, sebaceous glands andepithelial hair follicles have the other typesof epithelium. Nevertheless, it can he con-cluded from the electron microscopic find-ings that the epithelial tissue around the ob-served bacilli is not of the latter types ofepithelium but is characteristic of the epi-dermis. The cell containing the bacilli comesin contact with adjacent cells by desmo-somes, though these desmosomes are dis-torted by being pulled by scissors. The inter-cellular space between the cell containingthe bacilli and the adjacent cell is widenedexcept for bridging by the desmosomes. It isthought that the cell having the bacilli waspulled by the external force at the time ofcutting off the epidermis from the lepromawith scissors. The presence of desmosomesin the cell haying bacilli indicates that thiscell is an epidermal cell and, furthermore,

that it is not a dendritic cell such as amelanocyte or a Langerhans cell, but that itis a keratinocvte. It is also evident that thecell is not a nerve fiber extending into theepidermis. The cell having the bacilli haskeratohyaline granules and the organelleswhich are regarded as Odland bodies. Thetonofilaments in the cell are found in ir-regular groups. Judging from these findings,the cell containing the bacilli can be con-sidered as a cell of the granular layer. A partof the horny layer was seen near the cell,though this is not contained in the figurespresented.

Homogeneous bodies are seen in severalbacilli. The bacilli found in the cell are re-garded as acid-fast bacilli because all the ba-cilli found in the epidermis of the specimenstained with the Fite-Faraco method for op-tical microscopic observation were acid-fastbacilli. The epidermis was not cut off fromthe leproma used for the preparation of sec-tions for optical microscopic observation. Asdescribed below, the foamy cells can be seenin contact with the epidermal basal.cell layerat some places, and acid-fast bacilli are con-tained in these foamy structures (Fig. 4).Therefore, these acid-fast bacilli are con-sidered to he leprosy bacilli. Since these ba-cilli are arranged in accordance with thecontour of the foam vacuole, it cannot beheld that bacilli attached to the leproma sec-tioning knife were transferred for attach-ment to this locale. Instead it is thought thatleprosy bacilli in contact with the basal cellswere engulfed into the keratinocyte havingphagocytic activity and that the keratinocyteshifted to the upper layer as now observed.We regret that no attempt was made to cul-tivate the acid-fast bacilli from the epidermisin order to confirm that they were not cul-tivable acid-fast bacilli. This was not donebecause the EM finding was totally unex-pected.

Generally speaking, bacteria are notfound in epidermal cells. Montes and Wil-horn ( 7 ) examined by optical and electronmicroscopy normal skin from various loca-tions, the skin after occlusion, and the skin ofcomedones and intertrigo. They could notfind bacteria in the epidermal cells. Lever( 4 ) described that cocci lay extracellularly orwithin neutrophils in the lesion of impetigo.Pillsbury ( 10 ) wrote that the resident organ-isms did not occur in or between living cellsof the epidermis and its appendages. Lovell

46, 1^ Okada et al: M. leprae in Epidermal Cells^ 33

( 5 ) reported that small numbers of skin bac-teria were found between the most super-ficial cells of the horny layer, but bacteriawere not present in or between the livingcells of the epidermis. Montes ( 6 ) noted thatCorynebacterium mintissimum could hefound in the intercellular space of the stra-tum corneum of patients suffering from ery-th ra s ma.

the presence of pathogens in the epider-mal cell may he a specific phenomenon inleprosy in which numerous bacilli are pres-ent just below the basal epidermal layer,and the young basal cells having phagocyticactivity can come in contact with the patho-genic microorganisms.

The problem of how these leprosy bacilliwere ingested into the epidermal cell mustbe considered. In lepromatous skin there is aclear sub-epidermal zone below the epider-mis and this usually separates the epidermisfrom the leprosy bacilli. However, foamycells having leprosy bacilli can sometimes befound just under the basal layer. Therefore,if the keratin ocyte of the basal layer hasphagocytic activity, it is possible that theleprosy' bacilli are transferred into kerati-nocytes.

The phagocytic activity of keratinocyteshas been fairly well studied. First, studiesusing tracers are briefly noted. As tracers,Nordquist et al ( 9 ) used ferritin, and Wolffet al ( 12 . 14 ) used peroxidase and thorotrast.The tracers were injected intradermally andobserved under the electron microscope.They penetrated into the intercellular spaceof the epidermis and were ingested by thekeratinocytes. Wolff and his associates ob-served that thorotrast was stored in the vac-uoles of keratinocytes and eliminated fromthe epidermis during the keratinization pro-cess. Next, studies on the process of transferof melanosomes from the melanocyte whichproduced them to keratinocytes should henoted. Mottaz et al ("), studying the humanhair, observed that melanosomes were trans-ferred from melanocyte to keratinocyte bythe phagocytic activity of the keratinocyte.Klaus ( 2 ) observed that melanosomes wereingested by keratinocytes from a projectionof the melanocyte by keratinocyte hetero-phagy. In addition to these studies, Wolff etal ( 13 ) injected latex beads, having a diam-eter of 0.109p or 0.81p, into suction blistersand observed electron microscopically thatthe beads were engulfed into epidermal

cells. Their study also revealed that the latexheads were eliminated from the epidermisby desquamation in conjunction with thekeratinized cells. 13ecause the cell describedas having leprosy bacilli is not a dendriticcell but a keratinocyte, it is held that the ba-cilli were transferred to the upper layer ofthe epidermis together with the cell andwould finally have been eliminated from theepidermis. Therefore, the possibility of dis-charge of leprosy bacilli from the skin with-out ulceration should he considered.

SUMMARYLeprosy bacilli were found in a keratino-

cyte of the epidermis by the electron micro-scopic observation of the ultrathin section ofa leproma. The possibility of discharge ofleprosy bacilli from the skin should he con-sidered even if the lepromatous patient doesnot have any ulceration.

RESUMENPor microscopia electrdnica de una seccidn ul-

tradelgada de un leproma se encontraron hacilosde Ia lepra en un keratinocito de la epidt!rmis.Dehe tomarse en cuenta la posihilidad de Ia des-carga de los hacilos do la lepra a partir de la piel,attn si el paciente lepromatoso no presentaguna ulceracidn.

RESUM EOn a dt!couvert des hacilles de la 14we dans tin

keratinocyte de Npiderme, en utilisant des mMt-odes de microscopic tflectronique stir section ultramince de 10romes. On envisage Ia possibility clueles hacilles de Ia liTre pourraient etre libc!rtS de Iapeau, memo lorsqu'un malade ldpromateux neprdsente aucune ulcdration.

Acknowledgment. This work was supported inpart by a grant from the U.S.-Japan CooperativeMedical Science Program.

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2. KLAUS, S. N. Pigment transfer in mamma-lian epidermis. Arch. Dermatol. 100 (1969)756-762.

3. KLINGMULLER, V. Die Lepra, Berlin: JuliusSpringer, 1930, p 526.

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international Journal of Leprosy 1978

4. LEVER, \V. F. Ihstopathology of the Skin,3rd edit., Montreal: J. B. Lippincott Co.,1961, p 217.

5. LovEt.i, D. L. Skin bacteria. Their locationwith reference to skin sterilization. Surg.Gynecol. Obstet. 80 (1945) 174-177.

6. MoNTEs, L. F., McBRIDE, M. E., JoliNsoN,W. P., OWENS, D. W. and KNox„1. M. Ultra-structural study of the host-bacterium rela-tionship in erythrasma. J. Bacteriol. 90(1965) 1489-1491.

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9. NORDQUIST, R. E., OLSON, R. L. and EVERETT,M. A. The transfer, uptake, and storage offerritin in human epidermis. Arch. Dermatol.

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H. SALO, S. Rai (leprosy), Tokyo: KaneharaPublishing Co., 1954, p 75.

12. Wom, , K. and IIONIGSMAN, H. Permeabilityof the epidermis and the phagocytic activityof keratinocytes. Ultrastructural studies withthorotrast as a marker. J. Ultrastruct. Res.36 (1971) 176-190.

13. Wol.rr, K. and KONRAD, K. Phagocytosis oflatex beads by epidermal keratinocytes invivo. J. Ultrastruct. Res. 39 (1972) 262-280.

14. Wot.rr, K. and SCHREINER, E. Aufnahme, in-tracellularer Transport and Abbau exogenenProteins in Keratinocyten. Eine elekronemi-kroskopischcytochemische Studie mit Perox-idase als Maskierungssubstanz. Arch. Klin.Exp. Dermatol. 235 (1969) 203-220.

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