Volume 58, Number 4Printed in the U.S.A.

INTERNATIONAL JOURNAL OF LEPROSY

Effect of Presensitization with BCG andMycobacterium leprae on Granuloma

Formation to M. leprae'Kona Vijayalakshmi, Jill Curtis, Steve Gschmeissner,

Susan Sibley, and John L. Turk 2

Leprosy is a chronic granulomatous dis-ease involving the intracellular parasitiza-tion of the cells of the mononuclear phago-cyte series (MPS) with Mycobacteriumleprae.

Resistance to intracellular organisms ismediated by a cellular immune reaction in-volving macrophages and specifically sen-sitized T cells (4). Resistance is strongest inthe polar tuberculoid and borderline tuber-culoid forms of the clinical spectrum and isassociated with the formation of epitheli-oid-cell granulomas containing infiltratingT lymphocytes and very few, or no, bacilli.In the lesions of lepromatous leprosy, at theopposite pole, resistance is low and granu-lomas contain foamy macrophages packedwith large numbers of organisms ( 7). Clin-ical manifestations depend upon the strengthof the patient's specific cell-mediated im-mune response to the infecting organism.This also accounts for reversal reactions ob-served along the spectrum which are,due toa rapid development of states of hypersen-sitivity ( 1,10).

A model in the guinea pig has been es-tablished in our department to study gran-uloma induction by mycobacteria ( 5). Intra-dermal injection of BCG vaccine into theear induced a granuloma in the draininglymph node which peaked in 2 weeks andwas characterized by epithelioid-cell infil-tration comparable to granulomas in tuber-culoid leprosy. At the ultrastructural level,

I Received for publication on 15 February 1990; ac-cepted for publication in revised form on 18 May 1990.

K. Vijayalakshmi, M.D.; J. Curtis, Ph.D.; S.Gschmeisser, B.Sc.; S. Sibley, Ph.D., and J. L. Turk,M.D., Department of Pathology, Royal College of Sur-geons of England, 35/43 Lincoln's Inn Fields, LondonWC2A 3PN, U.K.

Reprint requests to Dr. Jill Curtis.

the epithelioid-cell cytoplasm may containstacked rough endoplasmic reticulum withfew other organelles. The nucleus is palewith a prominent nucleolus. When cobalt-irradiated M. leprae organisms were simi-larly injected, granuloma formation wasobserved after 5 weeks. The granulomascontained phagocytosing macrophages, andresembled those in lepromatous leprosy le-sions. A subsequent study revealed thathaptenization of Co-irradiated M. lepraewith fluorescein isothiocyanate (FITC) ledto a granulomatous response similar to thatinduced by BCG ("). It is possible that hap-tenization increased the antigenicity of M.leprae for specific T-cell activation such asoccurs with live BCG organisms.

Vaccination with BCG or BCG plus M.leprae has been used in trials for the im-munoprophylaxis of leprosy. It was the pur-pose of this study to see whether presensi-tization with BCG or M. leprae might inany way alter the granulomatous responseto Al. leprae, particularly, whether a mac-rophage-type granuloma might be convert-ed partly into an epithelioid-cell granulomaby earlier exposure to a vaccine. Acid-fastbacilli (AFB) are rarely detectable in my-cobacteria-induced granulomas in this guin-ea pig model, although the remains ofphagocytosed organisms are seen in electronmicrographs of granulomas formed in re-sponse to dead M. leprae ( 5). It is thus notpossible to assess clearance rates of myco-bacteria in response to vaccination, and ourinterest was focused on the histopatholog-ical evidence of hypersensitivity and tissuedamage.

MATERIALS AND METHODSAnimals. Outbred Hartley strain female

guinea pigs, weighing 250 g-300 g, were ob-tained from David Hall (Newchurch, Staffs,

674

58, 4^Vijayalaksluni, et al.: Granuloma Formation to M. leprae^675

U.K.). They were fed on RGP pelleted diet,supplemented with cabbage.

Mycobacteria. Live Pasteur strain M.bolls BCG, as a suspension in saline, wasprovided by the Pasteur Institut, Paris,France (s). Cobalt-irradiated (2.5 Mrad), ar-madillo-derived M. leprae was provided byDr. R. J. W. Rees of the National Institutefor Medical Research, Mill Hill, London.

Presensitization. Guinea pigs were pre-sensitized by injecting 1 x 10 7 live BCG or1 x 10 9 cobalt-irradiated M. leprae intra-dermally into the right flank.

Induction of granuloma. Co-irradiatedM. leprae organisms (1 x 109) were injectedintradermally into the dorsum of the rightear 2 weeks after presensitization with BCG.Guinea pigs presensitized with M. lepraewere similarly injected 5 weeks after pre-sensitization. Control guinea pigs were in-jected with 1 x 10' cobalt-irradiated M.leprae intradermally into the right ear only.

Draining post-auricular lymph nodes werecollected for study 2 weeks and 5 weeks afterthe injection of M. leprae into the ear forthe induction of granulomas. Lymph nodeswere weighed individually on a torsion bal-ance, and a portion of each node was fixedin Carnoy's fluid, processed, and stainedwith hematoxylin and eosin (H&E) for light-microscopy. Samples for electron-micros-copy were cut into small pieces and fixed in4% glutaraldehyde containing 0.05 M cac-odylate buffer, pH 7.4, for 24 hr at 4°C.Samples were cut and processed as de-scribed elsewhere ( 6).

Histological study. Granuloma forma-tion was noted by the presence of infiltrationby cells of the mononuclear-phagocyte se-ries. Areas of infiltration from randomtransverse sections were traced on white pa-per from a projection microscope image( x 60 magnification). Measurements wereperformed using a planimeter (1 rev = 100cm'; constant = 18,728). The area of infil-tration was expressed as a percentage of thetotal area of the section.

Statistical analysis. Where appropriate,results were compared by Student's t test.

RESULTSA granulomatous response to cobalt-ir-

radiated M. leprae occurred at 2 weeks inanimals presensitized with either BCG orM. leprae, whereas nonpresensitized guinea

pigs did not show a response at this time(Tables 1 and 2). Histologically, cells of themononuclear-phagocyte series (MPS)formed the infiltrates in all groups. In BCG-presensitized animals, the lymph nodeweights and areas of infiltration at 2 weekswere comparable to those in control animalsat 5 weeks. By 5 weeks there was a signifi-cant (p < 0.05) decrease in lymph nodeweight, but there was considerable variationin weights and areas of infiltration. At thistime, there was histological evidence of thepresence of fibrosis. In M. leprae-presensi-tized animals, the lymph node weights at 2weeks were comparable to those in the con-trol group at 5 weeks. The areas of infiltra-tion, however, were less. The lymph nodeweights at 5 weeks were similar to those at2 weeks, but there was a significant increase(p < 0.01) in infiltration. Histological ex-amination revealed areas of necrosis.

Ultrastructure. Ultrastructural observa-tions revealed morphologic heterogeneity ofthe MPS cells in the granulomatous infil-trates at 2 weeks after granuloma inductionin BCG-presensitized animals. A majorityof the cells had the appearance of epithelioidcells ( 5). They had round or oval nuclei withfinely marginated heterochromatin and aprominent nucleolus. Rough endoplasmicreticulum (RER) was present as stacks orsingle strands. Golgi apparatus was distinctin many cells. Mitochondria and other cy-toplasmic organelles were present in vary-ing proportions in some of the cells. A fre-quently observed cell was one whichcontained RER as well as numerous, well-formed, mitochondria suggestive of earlystages of epithelioid differentiation. Com-plex interdigitations between epithelioidcells with fimbriated borders were often seen(Fig. 1). Some cells showed a paucity of or-ganelles, with scanty or no RER, and weresuggestive of a lack of activity.

Phagocytosing cells with cytoplasmic ex-tensions were present. The nuclei were roundor indented and contained heterochroma-tin. The cytoplasm contained mitochondriaand other organelles. The presence of en-doplasmic reticulum with cisternal dilata-tions was also a feature in some of thesecells (Fig. 2). In many areas, the cells formeda syncytium and giant cell formation re-sulted from the fusion of phagocytic cells.

Five weeks after granuloma induction in

194324208323

Mean ± S.E.^262 ± 35

55%81%56%85%

69% ± 8%

5 weeks195 100%202 97%355 76%161 81%

228 ± 43^

89% ± 6%

676^ International Journal of Leprosy^ 1990

TABLE 1. Effect of presensitization with BCG on granuloma formation to M. leprae.

Controls Presensitization with BCG

Lymph node wt (mg) Area of infiltration^Lymph node wt (mg) Area of infiltration

2 weeks62 0 387 62%78 0 387 78%38 0 169 56%15 0 176 63%65 0 294 69%

Mcan S.E. 52± 11 0 283 ± 48 66% ± 4%

5 weeks258 74% 128 68%254 53% 177 63%144 77% 124 58%433 70% 92 23%244 44% 201 37%

281 85%87 87%

Mean ± S.E.^267 ± 47^

64% ± 6%^

156 ± 26^

60% ± 9%

BCG-presensitized animals, infiltrates con-tained epithelioid-type cells. Cells of theMPS showing early epithelioid differentia-tion with RER and mitochondria were oftenobserved (Fig. 3). Langhans' giant cells wereoccasionally present. A few of the cellsshowed vesicular cytoplasm. A noteworthyfeature was the presence of collagen as finestrands or bundles in the intercellular ma-trix (Fig. 4). However, fibroblasts were notidentified.

In M. /eprae-presensitized animals thegranulomas at 2 and 5 weeks containedmainly macrophages, and resembled thoseseen in unsensitized animals at 5 weeks.Phagocytosis was seen in some cells.. At 5

weeks there was evidence of cell degenera-tion and cell death as shown by the presenceof swollen mitochondria, shrinking or dis-appearance of nuclei and dissolution of cellborders.

In the guinea pigs injected with M. lepraealone, without presensitization, most of thecells were macrophages as described pre-viously ( 5). Phagocytosis was seen in someof these cells (Fig. 5).

DISCUSSIONThe present study shows that presensiti-

zation with BCG or cobalt-irradiated M.leprae has accelerated the onset of granu-

TABLE 2. Effect of presensitization with M. leprae on granuloma formation to M.leprae.

Controls^ Presensitization with Al. leprae

Lymph node wt (mg) Area of infiltration Lymph node wt (mg) Area of infiltration

2 weeks50 52%

234 40%149 70%334 35%367 38%

Mean ± S.E.^56 ± 10^

0^

227 ± 58^

47% ± 6%

34^

038^

049^

079^

083^

0

58, 4^Vijayalakshmi, et al.: Granuloma Formation to M. leprae^677

FIG. 1. Electronmicrograph of interdigitating epi-thelioid cells in 2-wk M. leprae granuloma in the lymphnode after BCG presensitization ( x 4500). Note palenucleus with prominent nucleolus and RER.

loma formation in the lymph node drainingthe site of the subsequent intradermal in-jection of M. leprae into the ear. No gran-uloma formation to M. leprae was found at2 weeks in control animals that had not beenpresensitized, although granulomas werefully developed by 5 weeks. However, inpresensitized animals, granulomas werefound at 2 weeks, a similar time course tothat of granuloma formation in response toBCG vaccine (5). At 2 weeks, the BCG-pre-sensitized animals showed infiltration whichconsisted of an ultrastructurally heteroge-nous population of cells of the mononuclearphagocyte series (MPS). Langhans' giant cellformation and cells with complex interdigi-tations were present. Phagocytosing cellswhich also contained endoplasmic reticu-lum were observed. However, a majority ofthe MPS cells showed epithelioid differen-tiation of various stages. Epithelioid cellscontaining RER and well-developed mito-chondria were frequently observed; thesecells were also seen in the 5-week granulo-mas of the BCG-presensitized animals inwhich collagen formation was present.Phagocytic cells containing M. leprae, butwith abundant endoplasmic reticulum, havebeen described in a Mitsuda granulomatoushypersensitivity reaction in borderline tu-berculoid (BT) leprosy (6). In the same study,

FIG. 2. Electronmicrograph of cell of mononuclearphagocyte series in 2-wk Af. leprae granuloma in thelymph node after BCG presensitization ( x 7500). NoteRER and phagocytosis (1).

epithelioid cells containing RER as well asmitochondria were described in two casesof BT leprosy in reaction.

The type of epithelioid cell which has arough endoplasmic reticulum and is non-phagocytic is thought to be a secretory cell("). Its development at various stages may

FIG. 3. Electronmicrograph of epithelioid cell in 5-wk M. leprae granuloma in the lymph node after BCGpresensitization ( x4500). Note well-formed mito-chondria and RER.

678^ International Journal of Leprosy^ 1990

LS

FIG. 4. Electronmicrograph showing infiltrationwith collagen formation (T) in 5-wk AL leprae granu-loma in the lymph node after BCG presensitization( x 3000). Note absence of typical fibroblasts.

depend upon the nature of the antigenicstimulus. Healing is a feature of epithelioidgranulomas, but it is not established wheth-er the epithelioid cell has a role in promot-ing fibrogenesis (8). It is possible that acuteexacerbations followed by residual nervedamage may arise due to the developmentof the epithelioid cells following furtherstimulation by M. leprae antigens in clinicalreversal reactions. Studies of the lympho-cyte function revealed a correlation with hy-persensitivity reactions that would lead toreversal reactions ( 1 ).

Granulomas in M. leprae-presensitizedanimals were not of the hypersensitivitytype. They contained macrophages, but ep-ithelioid cells were rarely seen. At 5 weeks,cell degeneration and cell death were ob-served both by light- and electron-micros-copy. Cell necrosis as a result of presensi-tization in a model where there is littleevidence of cell-mediated immune activitycould be due to an antibody-mediated event,or could be due to a local accumulation oftumor necrosis factor (TNFa).

Protection against M. leprae is consideredto be a cell-mediated immune mechanism.Macrophage interactions with specificallyelicited T cells are readily demonstrated invitro and well perceived at the histopatho-logical level. Vaccination has been proposed

FIG. 5. Electronmicrograph of a phagocytic mac-rophage in a 5-wk Al. leprae granuloma in the lymphnode of a nonpresensitized animal ( x 2500).

as a means of mounting protection againstM. leprae (2). A number of recently initiatedtrials in northern Malawi, Venezuela, andIndia have been designed using a combi-nation of BCG with killed M. leprae andcomparing this with BCG alone. The eval-uation of the efficacy of a vaccine is ulti-mately the absence of clinical disease.Moreover, it is observed that vaccinationmay not achieve uniform results as far asindividuals at risk are concerned and in dif-ferent endemic areas. Environmental andgenetic factors and prevalent forms of thedisease in a defined geographic area mayinfluence the overall outcome ( 3). The re-sults of immunotherapy in Venezuela, usinga combination of BCG and killed M. leprae,are also based on similar factors ( 2). How-ever, there is a suggestion that vaccinesmight stimulate a state of hypersensitivitywith or without producing a positive stageof protective immunity against M. leprae(9). In this situation, vaccination could makean individual more prone to develop a re-versal reaction. It appears from these stud-ies that BCG vaccination might accelerategranuloma formation and produce a tuber-culoid type of granuloma in circumstanceswhere one would normally expect to find alepromatous lesion.

In conclusion, BCG presensitization in-duced changes in granulomatous responsesto Al. leprae in the guinea pig comparable

58, 4^Vijayalakshmi, et al.: Granuloma Formation to M. leprae^679

to reversal reactions in human leprosy. Theeffect of presensitization with BCG is prob-ably to increase the strength of the cell-me-diated immune response to M. leprae. Theeffect may be comparable to that obtainedby linking a hapten to the surface of M.leprae ("). There is, no doubt, an increasein the strength of the cell-mediated hyper-sensitivity granulomatous reaction. Evi-dence for the protective effect of the cell-mediated protective immune response hasbeen available only in a limited number oftrials in man (3), and these have involvedmainly BCG vaccine alone. Care, therefore,should be taken to avoid boosting the po-tential for the development of reversal re-actions without more evidence of a consid-erable protective function.

SUMMARYGranulomas which develop in draining

lymph nodes, following the intradermal in-jection of cobalt-irradiated Mycobacteriumleprae into the ear of the guinea pig 2 and5 weeks earlier, were studied in animalswhich had been presensitized with BCGvaccine or M. leprae and compared withgranulomas that developed in previouslyunsensitized guinea pigs. Presensitizationwith mycobacteria accelerated the devel-opment of the granulomas. Granulomas inpreviously unsensitized guinea pigs werefound ultrastructurally to contain phago-cytosing macrophages similar to those inlepromatous leprosy, and M. leprae presen-sitization did not alter the type of granu-loma found. Those in BCG-presensitizedguinea pigs contained secretory epithelioidcells with rough endoplasmic reticulumsimilar to those found in borderline tuber-culoid leprosy or reversal reactions. The sig-nificance of these findings in relation to thecurrent use of vaccines in leprosy is dis-cussed.

RESUMENSe inocule• intradermicamente Mycobacterium lep-

rae irradiado con cobalto en la oreja de cobayos. Dosy cinco semanas despues se estudiaron los granulomasdesarrollados en los ganglios linfAticos regionales deanimales que habian sido presensibilizados con BCGo con Al. leprae y se compararon con los granulomasque se desarrollaron en cobayos sin presensibilizar. LapresensibilizaciOn con micobacterias acelerO el desa-

rrollo de granulomas. Los granulomas en cobayos nopresensibilizados contuvieron macraagos fagocitantesultraestructuralmente similares a aquellos de la lepralepromatosa; la presensibilizaciOn con .1f. leprae noaltere• el tipo de granuloma encontrado. Los granulo-mas en los cobayos presensibilizados con BCG con-tuvieron celulas epitelioides con reticulo endoplasmicorugoso similares a aquellas encontradas en la lepra tu-berculoide limitrofe o en las reacciones reversas. Sediscute el significado de estos hallazgos en relaciOn aluse de vacunas en la lepra.

RÉSUMÉ

Les granulomes qui se developpent au niveau desganglions lymphatiques, a la suite de ]'injections in-tradermiquc 12 et 5 semaines auparavant, de Myco-bacterium leprae irradie au Cobalt dans l'oreille decobayes, ont Cie etudies chez des animaux qui avaienteta sensibilises avec le vaccin BCG ou M. leprae, etcompares aux granulomes developpes par des cobayesqui n'avaient pas etc sensibilisês anterieurement. Lasensibilisation anterieure avec des mycobacteries a ac-Mere la developpement des granulomes. On a montreque les granulomes des cobayes non sensibilisês an-terieurement contenaient des macrophages phagocy-teurs semblables a ceux existant dans la lepre lepro-mateuse, et la sensibilisation anterieure par Al. lepraene modifia pas le type de granulomes decouverts. Lesgranulomes des cobayes pr e-sensibilises du BCGcontenaient des cellules secrêtrices epithelioIdes avecun reticulum endoplasmique rugueux, semblables acelles trouvees dans la lepre borderline tuberculokleou dans les reactions d'inversion. La signification desces decouvertes en relation avec ('utilisation actuellede vaccins pour la lepre est discutee.

Acknowledgments. This work was supported by agrant from the British Leprosy Relief Association(LEPRA). The authors wish to thank Panayiotis Pa-pasavva and Francis Schindler for their technical as-sistance and June Saxby for typing the manuscript.

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