INTERNATIONAL JOURNAL OF LEPROSY^ Volume 56, Number 3

Printed in the U.S.A.

Transitory Macrophage Activation in the GranulomatousLesions of Mycobacterium lepraemurium-induced

Lepromatoid Leprosy in the Mouse'Oscar Rojas-Espinosa, Rosario Vega, Antonina Oltra,

Patricia Arce, and Augustin Nunez2

Mycobacterium lepraemurium (M LM),like other pathogenic mycobacteria, de-pends on the intracellular milieu of certainphagocytic cells (free and tissue-fixed mac-rophages) to survive and to propagate in themurine host. Paradoxically, macrophages inresistant animals, or in the case of abortedinfection, are the cells responsible for theeventual killing of the microorganism.However, for this to happen the macro-phages need the concourse of T lympho-cytes (") with a helper/inducer phenotype(Th Lc). MLM-reactive Th Lc in the pres-ence of antigen and other soluble mediatorsproduce lymphokines able to activate mac-rophages and, in so doing, increase theiroverall metabolism and their cidal capabil-ities ( 6). In the absence of MLM-reactive Tcells or in the presence of excessive sup-pressive activity, the macrophages do notbecome activated, thus allowing the intra-cellular proliferation of the microorganismwhich first saturates the cell and then thetissues of the host.

Since lepromatoid leprosy in the mouseproceeds in a manner suggestive of defectivecell-mediated immunity (CMI), and consid-ering the link between CMI and macro-phage activation ( 13), we decided to look forevidence of macrophage activation precise-ly at the site of the lepromatoid granuloma.Preliminary observations indicate that apositive biochemical activation occurs in theperitoneal mouse macrophages as a conse-quence of the infection with MLM ( 8 . ' 5 . ' 6 ).This biochemical activation, however, is

' Received for publication on 15 June 1987; acceptedfor publication in revised form on 19 May 1988.

= 0. Rojas-Espinosa, Q.B.P., Sc.Dr.; R. Vega, Q.B.P.;A. Oltra, Q.B.P.; A. Nunez, Q.B.P., Department ofImmunology and P. Arce, I.B.Q., Department of Bio-chemical Engineering, National School of BiologicalSciences, National Polytechnic Institute, Carpio y Plande Ayala, 11340 Mexico, D.F., Mexico.

transitory and not sufficient to arrest theprogress of the disease. Other workers ( 9 )have also found evidence of the early andtransient enhancement of antigen- and lee-tin-induced mitogenic responses of lymph-oid cells from MLM-infected mice and ofthe generation of suppressive activity ( 10 . 17 ),which, very likely, abrogates the incipientimmunity.

In this communication, we report the re-sults of a kinetic study on the onset andevolution of lepromatoid granulomas in themouse liver (a target organ), the process ofmacrophage biochemical activation withinthe granulomas, and the fate of MLM dur-ing the entire period of infection.

MATERIALS AND METHODSAnimals and their inoculation. Sixty al-

bino, NIH, male mice weighing between 18and 20 g were inoculated with MLM bacilliby the intraperitoneal (i.p.) route. A similarnumber of noninoculated animals servedas a control group. The mice were main-tained under a standard Purina chow dietand water ad libitum until completion ofthe experiments. MLM (Hawaiian strain)were separated from the lepromas of pre-viously infected mice following Prabhak-aran's procedure ( 14).

Sampling. Every 2 weeks following in-oculation, three control and three infectedmice were anesthetized by chloroform in-halation and then exsanguinated by cardiacpuncture (blood was saved for serologicaltests). The spleen and the largest liver lobulewere carefully excised, trimmed, and cutalong the middle horizontal axis. A portionof tissue (always from the same region) wasremoved, embedded in O.C.T. compound(Miles Laboratories, Elkhart, Indiana,U.S.A.), frozen on dry ice, and stored at—70°C in a Revco freezer until used.

428

56, 3^Rojas-Espinosa, et al.: Transitory Macrophage Activation^429

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FIG. 1. Numbers and sizes of cell infiltrates in livers of mice injected with 10" MLM i.p. At differentpostinoculation times shown, three animals were sacrificed, their livers excised and processed as described underMaterials and Methods. (•) = average number of cell infiltrates in 30 microscopic (12.5 x 40 x ) fields; (0) =average number of cells per area of infiltrate. Thirty cell infiltrates (or maximum number possible) were analyzedat a 12.5 x 40x magnification. Counts were made in triplicate tissue sections. Bars are corresponding standarddeviations.

Histochemical studies. The frozen tissuespecimens were cut at —20°C in a TissueTek II cryostat to produce 6 pm-thick sec-tions. The sections were collected on stan-dard glass slides, fixed for 5 min with 1.25%glutaraldehyde made in 0.15 M phosphatebuffer, pH 7.2. The slides were washed threetimes for 5 min with 0.1 M phosphate —0.15M NaC1 (PBS), pH 7.4, and sequentiallyreacted with a) a substrate for 0-galactosid-ase activity ((3-Gal), b) hot phenol-fuchsinfor acid-fast bacilli (AFB), and c) Harris'hematoxylin for counterstaining.

fl-Galactosidase staining. (I') The fixedand washed tissue sections were incubatedovernight at 37°C in a mixture containing2 mg of 5-bromo-4-chloroindoxyl-(3 -D -ga-lactoside dissolved in 0.5 ml of dimethylformamide, 31 ml of 0.1 m acetate buffer,pH 5.4, 0.5 ml of 0.85% NaC1, 3.0 ml of0.05 M potassium ferro-fcrricyanide, and8.0 mg of spermadine•3HCI.

Acid-fast staining. The slides from theprevious step were gently rinsed in waterand immediately stained for 10 min withthe phenol-fuchsin reagent [25 ml of heat-melted phenol, 5 g of basic fuchsin, 50 mlof 95% ethanol, and distilled water (DW)to make 500 ml]. The best results were ob-tained when the slides were placed on a hotplate, covered with phenol-fuchsin, andheated until steam began to appear; the slideswere then allowed to cool somewhat andthen they were heated once more until steambegan to appear, all within the 10-min stain-ing period. After washing out the excess stainwith DW, the slides were dipped for 20 secin 1% HCI-70% ethanol to decolorize thetissue, washed again with DW, and thencounterstained.

Counterstaining. The tissue sections werestained for 5 min at room temperature withHarris' hematoxylin with a 5 sec acid de-coloration (1% HCI-70% ethanol), a 30 sec

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430^ International Journal of Leprosy^ 1988

DAYS OF INFECTION

FIG. 2. Cell activation in the cell infiltrates. (•, scale 0-50%) = percent of total cells that were positive for0-Gal activity; (0, scale 0-100%) = relative percent of mononuclear phagocytes (MN) positive for fl-Gal activity;(■) = relative percent of polymorphonuclear cells (PMN) positive for 0-Gal activity. Cells were counted in 30(or maximum number possible) cell infiltrates ;under oil (12.5 x 100 x ).

alkaline bluing performed by dipping slidesinto solution containing 2 mg MgSO 4 /m1 of0.35% NaHCO 3 in water (a 0.2% MgSO 4solution in 0.35% NaHCO 3), washing be-tween steps with DW. The stained prepa-rations were mounted with synthetic resinfor microscopic examination.

Microscopic examination. Three slides,each containing 4 to 6 tissue slices, wereprepared from each tissue fragment andstained by the process indicated above.Those preparations showing the neatest sec-tions and staining were further analyzed,and those showing progressive pathologicchanges were selected for detailed micro-scopic examination and description.

Cell infiltrates. The tissue sections wereexamined at a 40 x 12.5 magnification, andthe number of discernible cell infiltratescounted in 30 microscopic fields. The sizeof such infiltrates was assessed both by mi-crometry (two perpendicular diameters) atthe above magnification and by counting

the number of cells in the infiltrates at a 40x 12.5 magnification.

Cell types, bacilli, and enzyme activity.The cell types in the infiltrates and the pres-ence and number of bacilli per area of in-filtrate were determined by examining theinfiltrates found in at least 30 immersionfields. Those cells showing 0-Gal activitywere identified and recorded. A scale from0 to 4+ activity was arbitrarily establishedand used to assess the degree of biochemicalactivation per cell.

RESULTSNumber and size of cell infiltrates. Figure

1 shows distinct cell infiltrates in the liverparenchyma shortly after the i.p. inocula-tion of bacilli. The number of cell infiltrateswas, however, small during the first 2.5months postinfection but grew steadilythereafter. Beyond 195 days postinocula-lion, most of the cell infiltrates were con-fluent.

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56, 3^Rojas-Espinosa, et al.: Transitory Macrophage Activation^431

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Fin. 3. Acid-fast bacilli per cell infiltrate in relation to degree of macrophage activation. (0) = average numberof bacilli counted in 30 (or maximum number possible) cell infiltrates at 12.5 x 100 x magnification. (---) between75 and 90 days postinoculation = granular-staining bacilli were particularly predominant; (•) = percent of cellsper infiltrate that were biochemically activated (0-Gal-positive macrophages).

Not only did the number of cell infiltratesgrow in parallel to the time of infection buttheir size also increased proportionally (Fig.1). During the early stages of the infection,the granulomas were predominantly madeup of macrophages and a few scattered poly-morphonuclear leukocytes (PMN). As thenumber and size of granulomas increased,the PMN became more abundant but theyhad an exclusively peripheral localization,indicating their recent arrival from the cir-culation.

Number of bacilli per average infiltratearea. The number of AFB per area of cellinfiltrate ranged from 1 to 5 within the first30 days postinoculation to "too many to becounted" at the end of the fourth month ofinfection. Since the count of AFB was madedirectly in the cell infiltrates, subtle changescould not be noticed. The AFB growth curveshowed a bimodal profile: a small peak 60days after inoculation that decreased to a

minimal value 30 days later, and a secondincrement (this a very marked one) thatraised the number of bacilli to unassessablelevels from day 120 postinoculation on-ward. Concomitant to the diminution in thenumber of bacilli within the 60-to-90-daypostinoculation period was the predomi-nance of bacterial bodies with granularstaining, this suggesting nonviable bacilli(Figs. 3 and 4).

Biochemical activation of macrophages.Macrophage activation within the granu-lomas was deduced from the presence andlevel of the hydrolitic lysosomal enzyme0-Gal ( 2 . 5 ). Figures 2 and 4 show the earlygranulomas seen 15 days postinoculationwhich contained macrophages with no evi-dence of biochemical activation (0-Gal-neg-ative). Some 0-Gal-positive macrophageswere found 15 days later. Their number in-creased, reaching a peak between 45 to 60days postinoculation, then decreasing and

432^ International Journal of Leprosy^ 1988

FIG. 4. Histochemical assessment of macrophage activation and bacillary multiplication.a. A 30-day-old granuloma showing infiltrating macrophages, some PMNs (arrowhead), and several MLMs

within cytoplasm of one of the macrophages (arrow). Macrophages in this early granuloma show a "clean"cytoplasm with no evidence of i3-Gal activity.

b. A mature, activated granuloma, 60 days old, showing intense macrophage /-Gal activity (13) which totallyor partially masks the several bacilli present (arrows).

c. A 75-day-old granuloma, showing less and more condensed /3-Gal activity and several bacilli with granularstaining (arrows).

d. A fully consolidated lepromatoid granuloma, 120 days old, showing macrophages heavily loaded withbacilli. Insert is a higher magnification of a similar granuloma showing presence of bacilli and absolute absenceof /3-Gal activity in the macrophages' cytoplasm.

56, 3^Rojas-Espinosa, et al.: Transitory Macrophage Activation^433

practically disappearing by clay 105 after in-oculation. At any time investigated withinthe peak period, there were some (less than10%) PMNs showing some 13-Gal activity(Fig. 2, The Table).

Other histologic changes included somevacuolation of the hepatocytes noticeablefrom day 45 postinoculation but no inflam-matory reaction (cell infiltration in the nor-mal liver tissue), the presence in the gran-ulomas of lymphocytes, some epithelioidand giant cells between 30 and 75 days post-infection, and a predominance of histio-cytes thereafter. As mentioned above, PMNswere always present, scarce in the early le-sions but abundant in the well-conformedgranulomas where they showed a peripherallocalization. Despite the fact that PMNs arehighly phagocytic and bactericidal cells, theydo not seem to efficiently participate in thecontrol of the lepromatoid disease causedby MLM in the mouse. Perhaps this is dueto the following circumstances: a) the PMNsthat circulate and enter the tissues are ma-ture cells with a relatively short life span (24to 42 hr '), and b) MLM has a long repli-cation time (9 ± 1 days) and a very complexlipidic envelope ( 7) which makes it partic-ularly resistant to intracellular digestion.

DISCUSSIONThis histochemical and bacteriologic

study of the granulomas that appear in theliver of mice infected with MLM revealsthat, despite the large number of bacilli usedfor infection, each lesion begins with thearrival of isolated phagocytic cells (namely,macrophages) containing from 1 to 5 bacilli.These moderately parasitized phagocytesreach the liver parenchyma through theblood circulation so that the small granu-lomas that appear early in the infection havea predominantly perivascular distribution.Bacilli within the phagocytic cells, or theparasitized phagocytes themselves, mustproduce potent chemotactic factors that at-tract fresh phagocytes to the site of infectionwith the kinetics depicted in Figure 1. Mac-rophages within the granulomas becomebiochemically activated due to their normalmaturational process, to the presence andendocytosis of bacilli, or in response to mac-rophage-activating lymphokines generatedduring the early stages of the infection. On

the average, the biochemical activation ofmacrophages in the liver granulomas, as as-sessed by their levels of 13-Gal, an enzymewhose activity increases proportionally tothe degree of cell activation ( 4 ' 5 ), becomesevident around 30 days after inoculation. Itthen increases relatively rapidly to reach apeak between 45 and 60 days, decreasingthereafter and completely disappearing byday 150 postinfection (Figs. 2 and 4). With-in the granulomas the bacilli seem to mul-tiply steadily during the first 60 days of in-fection, then their growth seems to bearrested and granular bacilli become evi-dent. This period of stasis and/or killing ofbacilli lasts for some days (around 2 weeks),and it is followed by a period of logarithmicbacterial growth that continues until satu-ration of the tissue occurs (Figs. 3 and 4).Within this period of logarithmic growth,the mean generation time was 9 ± 1 days.

These findings on the in situ macrophageinactivation that ensues and vanishes dur-ing the course of the lepromatoid infectionof the mouse support and complement thosefrom a previous study ( 15) in which the levelsof several lysosomal enzymes were quan-titated in the peritoneal cell population ofmice infected with MLM. In that study, itwas observed that most of the assayed en-zyme activities showed a progressive ele-vation that peaked between 40 and 75 dayspostinfection. There was a tendency for theseenzyme activities to decrease thereafter un-til the end of the experiment (over 6 monthspostinoculation), when most animalsshowed obvious signs of the disease thateventually killed them.

There is the possibility that the increasedgrowth rate of bacilli in the liver might bedue to an overflow (by redistribution) ofbacilli from other compartments ( 2), and thatwe might be confronted with immunologi-cal exhaustion rather than active suppres-sion. We favor the latter possibility because:a) several groups have found evidence insupport of the generation of a cell-mediatedimmune response following the inoculationof mice of several strains (CB6, C57BL/6,CBA, and BALB/c) with MLM ( 2 ' 9 ' I" ' 12, 17 );b) in most of these studies, the MLM-elic-ited CMI has also been found to be tran-sitory (C.". 10 . ' 7 ); and c) in some cases, Lyt1+2— and Lyt 2+ T cells ('") or nylon-wooladherent spleen cells ( 2 . ' 7 ) have been found

434^ International Journal of Leprosy^ 1988

THE TABLE. Some histologic, bacteriologic, and biochemical changes in the livers ofmice infected with Mycobacterium lepraemurium."

Days of infection

15 30 45 60

0.2' 1.2 1.5 1.4

75^90^105^120^135^150^165^180

2.0^4.9^5.0^9.9^9.1^9.3^16^28

16^39^85^86^114^102^161^196^218^263^292^339

0^0^46^47^14^8^I^4^3^0.5^0^0

0^0^94^95^92^93^100^100^100^100^0^00^0^6^5^8^7^0^0^0^0^0^0

I^5 13 21^11^8^99 TMC TMC TMC TMC TMC

No. cell infiltratesper field"

No. infiltrating cellsper area of infiltrated

To 0-Gal(+)" total cellsper area of infiltrate

Relative To of 0-Gal(+)"MN cellsPMN cells

No. bacilli per area ofinfiltrate'

" Each animal received 1.0 x 10" bacilli i.p.'Thirty microscopic (12.5 x 40x) fields in triplicate tissue sections were investigated.

Mean values, standard deviations not given but are shown in the figures.d Cells in 30 cell infiltrates were counted; when infiltrates were scarce all of them were counted in triplicate

tissue sections (12.5 x 40x).Cells that were positive (1 + to 4+) for 0-gal activity were counted in 30 (or maximum number possible)

cell infiltrates under oil (12.5 x 10()x).'Acid-fast bacilli were counted in 30 (or maximum number possible) cell infiltrates (12.5 x 100 x ); TMC =

too many to be counted.

to exert the suppressive activity on theMLM-induced cellular immunity.

The presence and initial proliferation ofbacilli in the host tissues very likely stim-ulates MLM-reactive, T lymphocytes (Th)which, through lymphokines, activate themacrophages of the incipient granulomas,increasing their metabolic and microbicidalfunctions. Theoretically, this activationshould be enough to render the macro-phages able to destroy the still scant localbacilli. However, when activation vanishes(whatever the reason might be), the few ba-cilli surviving in the lesions, or those bacilliarriving from infected sites lacking lym-phoid tissue (bacilli that have not be sub-jected to the CMI-dependent microbicidaleffects), now in the absence of macrophagicresistance, start growing within the granu-loma-resident macrophages and within therecently arrived histiocytes. The macro-phagic granulomas extend their volume andfuse with each other to eventually replacethe otherwise normal tissue in the targetorgans which, for this reason, become non-functional (Fig. 4). This can account for theimmunological (and other) disturbancesfrequently reported in advanced murine andhuman leprosy.

Proof that activated (0-Gal-positive)macrophages have increased microbicidaland digestive power comes from Dannen-berg's group (4 5 ). By autoradiography, theyfound that intact ' 4C-labeled tubercle bacilliwere very rare in macrophages staining 4+for 0-Gal yet these cells frequently con-tained ' 4C-associated bacillary breakdownproducts. The macrophages staining 1+,2+, and even 3+ for 0-Gal frequently con-tained both intact bacilli and, ofcourse, ' 4C-labeled bacillary fragments.

SUMMARYA kinetic study on the evolution of gran-

ulomas that appear in the liver of NIH miceinoculated with 10 8 Mycobacterium leprae-min•um by the intraperitoneal route hasbeen performed. The liver was chosen be-cause of its nonlymphoid histology whichallowed us to visualize the appearance andmaturation of the cell infiltrates generatedas a consequence of the mycobacterial in-fection. The study analyzed both the mac-rophage activation within the granulomasand the fate of bacilli within the macro-phage. The results showed that this myco-bacteriosis induces a relatively early mac-rophage activation (a very likely result of a

56, 3^Rojas-Espinosa, et al.: Transitory Macrophage Activation^435

cell-mediated immune response triggeredby the bacilli) that peaks between 45 and 60days postinoculation, fades thereafter, andpractically disappears several days later. Ba-cilli are susceptible to the microbicidal ef-fects of activated macrophages, but whenthe macrophages arc turned off (probablydue to active suppressive mechanisms), thesurviving bacilli reinitiate the infection withno further macrophage opposition. As a re-sult, more phagocytes are attracted to theinfection sites and the cell infiltrates growsteadily to become confluent, increasing thegranuloma fraction and eventually replac-ing the liver parenchyma. The findings sug-gest that in murine "leprosy" infection, ear-ly immunological changes occur that enablethe macrophages present in the granulomasto kill the infecting Al. lepraetintrium re-gardless of the eventual lepromatoid evo-lution of the granulomas. Lepromatoidgranulomas in the mouse and lepromatousgranulomas in man are equivalent struc-tures in regard to their histology and bac-teriology.

RESUM ENSc hizo un estudio cinetico sobrc Ia evoluciOn de los

granulomas quc aparecen en el higado de ratones NIHinoculados con 10 8 ,1/3'm/why/um lepraemurium porla via intraperitoneal. Se escogiO el higado porque suhistologia no linfoide nos permits visualizar Ia apari-ciOn y maduraciOn de los infiltrados celulares genera-dos como consecuencia de Ia infecciOn micobacteriana.En el cstudio se analizO tanto Ia activaciOn de macro-fagos dentro de los granulomas como el destino de losbacilos dcntro de los macrOfagos. Los resultados mos-traron quc esta micobacteriosis induce una activaciemde macrOfagos relativamente temprana (muy proba-blemente resultante de una respuesta inmunc celularcontra los bacilos) quc alcanza su pico entre los 45 y60 dias post-inoculaciOn y que disminuyc progresi-vamente hasta practicamente desaparecer varios diasmas tarde. Los bacilos son efectivamente susceptiblesa los efectos microbicides de los macrOfagos activadospero cuando los macrOfagos son desactivados (debiclo,probablemente, a mecanismos supresores activos), losbacilos sobrevivientes rcinician Ia infecciOn sin pos-terior oposicion macrofagica. Como resultado de esto,mas fagocitos son atraidos a los sitios de infecciOn, losinfiltrados cclulares crecen sostenidamente hasta ha-ccrse confluentes, aumcntando la fracciOn granulomay finalmente substituycndo a la mayor parte del pa-renquima hepatic°.

Los hallazgos sugieren quc en Ia infecciOn leprosadel ratOn, ocurren cambios inmunolOgicos tempranosque capacitan a los macrOfagos de los granulomas para

matar al M. lepraemurium aUn cuando la infecciOntcrmine en su forma lepromatoide, una forma cqui-valente a Ia lepromatosa del humane en cuanto a his-tologia y hacteriologia.

RÉSUMÉ

On a mene une etude cinetique de revolution desgranulomes qui apparait dans le foie des souris NIHinoculees par 10 8 Mycobacterium lepraenzurium parvoic intraperitoneale. Le foie a etc choisi you ces etudes,car son histologic non lympholde permet de rendrevisible l'apparence et la maturation des infiltrats cel-lulaires qui se dêveloppent A la suite de l'infection my-cobacterienne. Cette etude a analyse a Ia fois l'acti-vation des macrophages A rinterieur des granulomcs,et le sort des bacilles dans Ics macrophages. Les re-sultats ont montre que cette mycobacteriose entraineune activation relativement pi-Ocoee des macrophages,resultat probable de Ia reponse immunitaire A media-tion cellulaire produite par les bacilles. Les pies se si-tuent entre 45 et 60 jours apres inoculation, s'estom-pent ensuite, et disparaissent pratiquemcnt quelquesjours plus tard. Les bacilles sont vulnerables aux dietsmicrobicides des macrophages actives; neanmoins,lorsque les macrophages ne sont plus actives, proba-bier-tient A la suite de mecanismes actifs de suppression,les bacilles qui survivent reprennent le relais sans au-cune defense ulterieure par Ics macrophages. En con-sequence, davantage de phagocytes sont attires vers lessites d'infection, et les infiltrats cellulaires se develop-pent sans obstacle jusqu'a cc qu'ils deviennentconfluents, ce qui entraine une augmentation de la pro-portion de tissu granulomateux, qui en vient A rem-placer le parenchyme du foie. Ces observations sug-gerent quc dans l'infection par la lepre murine, desmodifications immunologiques surviennent precoce-ment, permettant ainsi aux macrophages presents dansles granulomes de tuer les Al. lep•aemurium respon-sables de l'infection, et ceci sans egard a une evolutionlepromatoIde eventuelle des granulomes. Les granu-lomes lepromatoldes chez la souris, de memo que lesgranulomcs lepromateux chez rhomme, constituent desstructures equivalentes sur le plan histologique et surle plan bacteriologique.

Acknowledgments. This work received financial sup-port from the DirecciOn de Graduados e InvestigaciOn,Institute Politecnico Nacional, Mexico, to the project862193 "Inmunopatologia de la lepra en un modclomurino." We are indebted to Dr. S. Estrada-Parra forhis advice and interest in our work. 0. Rojas-Espinosais a fellow from the COFFA, IPN, and from the SNI,Mexico.

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