1NTERNAI IONAL JOURNAL OF LEPROSY^ Volume 50, Number 1Printed in the U.S.A.

Ultrastructural Features of the Multiplication ofHuman and Murine Leprosy Bacilli in

Macrophages of Nude Mice'Yukiko Fukunishi, Seitaro Okada, Mitsugu Nishiura, and Kenji Kohsaka"

The main purpose of the present experi-ment was to study the ultrastructural fea-tures of the growth of M. leprae and M.lepraemarium in macrophages of nudemice. When the lesions of human and mu-rine leprosy are examined by electron mi-croscopy using ultrathin sections ("•"), itbecomes clear that the two species of my-cobacteria produce slightly different elec-tron-transparent zones around the bacillarybodies in their respective host cells. In thecase of M. leprae (s). the electron-trans-parent zones have a tendency to coalescewith each other and to form distinct intra-cytoplasmic foamy structures when the le-sion becomes old. On the contrary, in thecase of M. /epraetmtrium, an electron-transparent zone is formed around each ba-cillus, and the typical foamy structure likethat of human lepra cells is never formed.

In 1970 Draper and Rees (') reported rib-bon-like structures in murine leprosy bacilliobserved by negative staining. In 1972 ( ')and 1977 ( 5 ) Nishiura, et al. reported thedifferences in peribacillary substances inhuman and murine leprosy bacilli observedby the freeze-etching technique. Manysmall spherical droplets accumulated aroundM. leprae in phagolysosomes in human lep-ra cells. They corresponded to the electron-transparent zone and foamy structuresaround 44. leprae. In the case of M. lep-

' Received for publication on 5 January 1981; ac-cepted for publication in revised form on 27 August1981.

Y. Fukunishi, M.D.. Ph.D., Medical Officer, Na-tional Sanatorium Oshima Seisho-en, Kida-gun, Kagawa Pref. 761-02, Japan; S. Okada, M.D.,Ph.D., Associate Professor; M. Nishiura, M.D.,Ph.D., Professor, Leprosy Research Laboratory,Kyoto University School of Medicine, Sakyo-Ku,Kyoto 606, Japan; K. Kohsaka, Ph.D., Assistant Pro-fessor, Department of Leprology, Research Institutefor Microbial Diseases, Osaka University, 3-I Yama-da-oka, Suita City, Osaka, Japan.

raemu•ium growing in murine lepra cells,a peculiar membranous or crystalline sub-stance was found around their bacillarybodies. In 1973 Draper, et al. ( 2 ) reportedfreeze-etching findings of the peribacillarysubstance of M. lepraeararium in the C3Hstrain mouse and identified this substanceas mycoside C by detailed biochemicalanalysis. From the above studies, it is clearthat the peribacillary substances around M.leprae and those around M. lepraemu•iumdiffer very much from each other.

In 1960 Shepard ( 7) described the mul-tiplication of M. leprae in mouse foot pads,and made a breakthrough in animal exper-iments on leprosy. In 1976, Kohsaka, et al.( 1) reported the formation of lepromatoidlesions in the foot pads of nude mice inoc-ulated with M. /eprae and bred under spe-cific pathogen free (SPF) conditions in avinyl isolator. They inoculated 10 .1 M. lep-rae into each foot pad and confirmed thegrowth of lepromata after a year and a fewmonths.

In 1977, when we compared the ultra-structures of human lepra cells containingM. leprae and murine lepra cells of C3Hstrain mice caused by M. lepraemurium, itwas difficult to decide whether the ultra-structural differences observed were due tothe difference of mycobacteria or due to thedifference of macrophages.

When it became possible to grow M. lep-rae in nude mouse foot pads, we decidedto compare the ultrastructural morphologyof the peribacillary substances of these twospecies of mycobacteria in the same kindof host cell, namely the macrophage of thenude mouse.

MATERIALS AND METHODSInoculation of M. leprae. A suspension of

M. leprae was prepared from a lepromafrom a patient with lepromatous leprosyprovided by the Leprosy Research Labo-

68

50, I^Fukunishi, et al.: Ultrastructure of M. I. & M. Im.^69

ratory, Kyoto University School of Medi-cine. After mincing the leproma with scis-sors, a bacillary suspension was made withFlanks' solution by grinding in a mortar,and 10 7 M. leprae in a volume of 0.03 mlwere inoculated into the right hind footpads of 30 each of nude mice (nu/nu) bredoff a BALB/c background and bred underSPF conditions using a vinyl isolator.Food and water were given after completesterilizing by autoclaving.

Inoculation of M. lepraemurium. A bacil-lary suspension was prepared from a C3Hstrain mouse inoculated with M. leprae-muriuon by the same method as for that ofM. leprac. M. lepraentrium, 10 7 in a vol-ume of 0.03 ml, were inoculated into righthind foot pads of each of 10 nude mice bredunder conventional conditions.

Electron microscopy. The tissues of sac-rificed nude mice inoculated with Al. /epraeor Al. lepraenutrium were fixed with 3%glutaraldehyde in 0.06 M phosphate buffer(pH 7.4) for 24 to 48 hr for ultrathin sectionstudy, washed three times with the buffer,and post-fixed with 2% 050 4 in distilled wa-ter for 14 to 24 hr at 4°C. After dehydrationwith a graded ethanol series, the tissueswere embedded in methacrylate, cut by anultra-microtome with glass knives, and thenstained with uranyl acetate and lead citrate.

The tissues for study by freeze-etchingtechniques were fixed with 3% glutaralde-hyde under the same conditions as those forultrathin sectioning, and then immersed in20 to 40% glycerol for I to 2 days at 4°C.Other procedures were the same as de-scribed previously (a).

Other examinations. M. leprae obtainedfrom human lepra cells and from experi-mental nude mouse lepromata and M. lep-raemu•atm from the C3H murine leprosylesions and from the experimental nudemouse lesions caused by Al. /my/eau/raw/inoculation were examined by the dopa-ox-idase test ('"• ") and the pyridine extractiontest (n).

RESULTSMacroscopic findings.a) Lesions in nude mice caused by M.

/eprae inoculation. Foot pads, spleen, liverand skin were examined. Specimens weretaken at 24 hr and at 2, 3, 5, 6, 10, 14 and18 months after inoculation respectively.

FIG. I. Arrow indicates a swollen right hind foot

pad of a nude mouse inoculated with M. leprue.

FIG. 2. Arrows indicate lepromata in the spleen ofa nude mouse inoculated with Al. ((pray.

FIG. 3. Arrows indicate two lepromata originating

from the lymph nodes of a nude mouse inoculated withM. /eprae.

FIG. 4. Arrows indicate lepromata of a nudemouse inoculated with M. leprae.

FIG. 5. Arrow indicates a leproma of a nude mouseinoculated with M. leprae.

No lesions were found up to 8 months afterinoculation, but almost all inoculated righthind food pads became swollen about 10months after inoculation. Twelve monthsafter inoculation, the inoculated right hindfoot pads, the spleen and some lymphnodes showed distinct lepromata (Figs. 1,2, 3. 4. and 5). When the lesions of thisstage were examined by electron micros-copy, a large number of bacilli inside pha-golysosomes of macrophages were ob-served.

Lesions in nude mice caused by Al. lep-raenutrium inoculation. Foot pads, spleen,liver and skin were studied at 24 hr, 25, 30,and 35 days and 2, 3, 5, and 6 months afterinoculation.

Macroscopic signs appeared by I month

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after inoculation, when the inoculated righthind foot pads became swollen. When thelesions were examined hy electron micros-copy, a large number of bacilli were seeninside macrophages. Two months after in-oculation, the tails and backs of sonic ofthese nude mice also showed lepromata andthey soon became ulcerated. The growth ofmurine leprosy lesions in nude mice wasmuch faster than that seen in C3H strainmice.

Ultrastructural findings.a) Ultrastructural features of the multi-

plication of Al. Ieproe inside nude mousemacrophages. M. leprae multiply in pha-golysosomes of macrophages of nude micein essentially the same fashion as they do inhuman lepra cells. In ultrathin sections,electron-transparent zones or intracyto-plasmic foamy structures were seen aroundthe growing leprosy bacilli (Fig. 6), butopaque droplets (lysosomes), which can beseen in human lepra cells, were less fre-quently encountered in nude mouse mac-rophages. The findings with freeze-etchingof the nude mouse lesions were strikinglysimilar to those in human lepra cells. Al-most all of the bacilli in these nude micelesions were long and slender, and hadhand structures on smooth cell wall sur-

faces like those in human lepra cells (Fig.7). Distinct accumulations of small spheri-cal droplets were observed inside phago-lysosomes (Fig. 8).

Ultrastructural features of the multi-plication of Al. Icpracmurium inside nudemouse macrophages. The fundamental ultra-structural features of the multiplication of41. /epraenwrium inside the macrophagesof nude mice (I ig. 9 and 10) were identicalto those seen in the macrophages of C3Hstrain mice (Fig. I I). In ultrathin sections,distinct electron-transparent zones wereobserved around each bacillus (Fig. 9), butintracytoplasmic foamy structures were notseen. In freeze-etching pictures, crystallinematerial was seen around each bacillusgrowing in the phagosomes of nude mice(Fig. 10). Contrary to the findings in humanlepra cells and in nude mouse macrophagesinfected with AL leprae, no spherical drop-lets were observed in the lesions of nudemice caused by M, lepraemurium. M. lep-racmurium in nude mouse macrophages aresurrounded by membranous or crystallinematerial just as they are in murine lepracells in C3H strain mice. Almost all of thebacilli in these nude mice inoculated withM. Iepraeinuriuiu were long and slender,and hand structures were seen on them.

FIG. 6. Electron-microscopic finding of intracytoplasmic foamy structures inside the macrophages of nudemouse inoculated with M. leprae by ultrathin sectioning. N = nucleus of macrophages. (Scale 1 p.m, Magni-fication x8500.)

FIG. 7. Electron microscopic finding of many spherical droplets (S) around leprosy bacilli (B) inside amacrophage of a nude mouse inoculated with M. leproe by freeze-etching. Arrows indicate band structures onthe cell wall of leprosy bacilli. (Scale I p.m, Magnification x39,000.)

FIG. 8. Electron microscopic finding of intracytoplasmic foamy structures inside the phagolysosome of amacrophage of a nude mouse inoculated with M. leprae by freeze-etching. A large amount of spherical droplets(S) are observed around the leprosy bacilli (B) inside the phagolysosome. (Scale Magnification x 15,500.)

FIG. 9. Electron microscopic findings of murine leprosy bacilli inside the macrophage of a nude mouseinoculated with M. hpraemurium by ultrathin sectioning. Arrows indicate electron-transparent zones (E) aroundmurine leprosy bacilli (B). (Scale I p.m, Magnification x22,000.)

FIG. 10. Electron microscopic finding of murine leprosy bacilli inside the macrophage of a C3H strain mouseinoculated with M. lepraemarium by freeze-etching. Thick arrows indicate crystalline materials (C) around themurine leprosy bacilli (B). Thin arrows indicate band structures on the cell wall of the murine leprosy bacilli.(Scale I p.m, Magnification x36,500.)

FIG. I I. Electron microscopic finding of murine leprosy bacilli inside the macrophage of a nude mouseinoculated with M. lepraemurium by freeze-etching. Thick arrows indicate crystalline materials (C) around themurine leprosy bacilli (B). Thin arrows indicate band structures on the cell wall of the murine leprosy bacilli.(Scale I p.m, Magnification x51,0(().)

50. I^Fukunishi, et al.: Ultrastructure of M. I. & M. Im.^71

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THE TABLE. A summary of '&1 -dm -mita -al findings with the multiplication of M. lepraeand M. lepraemuriam in different host cells.

humanlepra cell

Nude miceinoculated

with^leprae

C'311 Miceinoculatedwith 11.

lepraenturiam

Nude miceinoculatedwith M.

/epracimiriam

Spherical droplets Positive Positive Negative NegativeCrystalline material Negative Negative Positive PositiveBand structure Positive Positive Positive PositiveForm of bacilli Long and slender Long and slender Long and slender Long and slenderDopa-oxidase test Positive Positive Negative NegativePyridine extraction test Positive Positive Negative Negative

These findings and the results of thedopa-oxidase tests and the pyridine extrac-tion tests are shown on The Table.

DISCUSSION

The present findings demonstrate byelectron microscopy that the ultrastructuralfeatures around M. leprae and Al. leprae-minium inside phagolysosomes or phago-somes of nude mouse macrophages showstriking differences from each other. M./eprae growing inside nude mouse macro-phages produce the same ultrastructuralchanges as those in human lepra cells. Onthe other hand, M. lepraemurium producecrystalline material around the bacillarybodies inside the phagosomes of the nudemouse macrophages. This finding is thesame as that in murine lepra cells of theC3H strain mouse. In the present experi-ment, the host cells for both M. /eprae andAl. lepraemurium were the macrophages ofnude mice. Thus, differences produced inthese macrophages by inoculations with M./eprae and Al. lepraenutrium respectivelyseem clearly to be due to the differencesbetween the metabolism of M. /eprae andthat of M. lepraenutrium.

In 1977 Nishiura, et al. ( 5) and Takeo,et al. CI examined the peribacillary sub-stance of various cultivable mycobacteriaby freeze-etching. Of the 14 species of cul-tivable mycobacteria which were exam-ined, none showed the spherical droplet asobserved around M. leprae in both humanand nude mouse macrophages.

On the other hand, peribacillary spheri-cal droplets similar to those found aroundM. /eprae have been seen in experimentalarmadillo (Dasypus novem•inctus) leprosy

lesions ( 12 ) and, in recent studies, in someof the armadillos with naturally acquiredleprosy-like disease (").

The nature of the mycobacteria in natu-rally acquired leprosy-like disease of ar-madillos is not fully clarified as yet. How-ever, when Al. /eprae multiply in varioushost cells (human, nude mouse, and arma-dillo macrophages), spherical droplets al-ways appear around the organisms growingin macrophages. Thus, the spherical drop-lets around M. /eprae are most probablymade up of specific substances produced bythe multiplication of M. /eprae in suitablehost cells.

SUMMARY

Ultrastructural features of the growth ofM. leprae and M. lepraemurium in nudemouse macrophages were studied by ultra-thin sectioning and freeze-etching. In nudemouse macrophages, Al. leprae producedspherical droplets (foamy structures) simi-lar to those in human lepra cells. On theother hand, M. lepraemurium producedtypical crystalline material in nude mousemacrophages, which is quite the same asthat observed in the C3H strain mouse.Spherical droplets in the form of foamystructures seem to be made up of a specificsubstance produced by the multiplication ofAl. /eprae in suitable host cells (human,nude mouse, and armadillo macrophages).

RESUMEN

Se estudiaron las caracteristicas ultraestructurales

del crecimiento del M. leprae y del M. lepraemitrium

en los macrOfagos de los ratones desnudos por las tee-

nicas de microscopia en cortes ultradelgados y deimpresion por congelackin (freeze-etching). En los

50, I^Fukunishi, et al.: Ultrastructure of M. I. & M. Im.^75

macrOfilgos de los ratones desnudos, el M. leprae pro-dujo gotitas esfericas (estructuras espumosas) simi-lares a las observadas en las celulas de Ia lepra hu-mane. Por otro lado, el 3/. lepraenuirium produjo, enlos macnifagos de los animales desnudos, un tipicomaterial cristalino muy similar al observado en Ia cepade ratones C3H. Las gotitas esfericas en forma de es-tructuras espumosas parecen estar hechas de una sub-stancia especifica producida por la multiplicaciOn delAl. leprae en celulas huesped susceptibles (macrOfa-gos humanos, de ratan desnudo, y de armadillos).

RESUMEOn a etudie les caracteristiques de ultrastructure

de M. /eprae et de M. //Tray/mu -hem en wars decroissance, chez des macrophages de souris glabres,au moyen de sections ultra-minces et de cryofractures.Dans les macrophages de la souris glabre, ,If. lepraeproduit des goutelettes spheriques (structures spu-menses) semblables a cellos que I'on observe dans lescellules de la lepre humaine. D'autre part, Al. leprae-murium donne naissance, dans les macrophages desouris glabres, it du materiel cristallin typique, tout itfait semblable it celui que l'on observe chez les sourisde la souche C3H. Les goutelettes spheriques,l'aspect de structures spumeuses, semi -flew constituerune substance specifique qui serail produite par lamultiplication de AL /eprae dans des ccllules-hOtesappropriees (macrophages humains. de souris glabres,et de tatous).

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