INTERNATIONAL. Jot JRNAL OF LEPROSY^ Volume 52, Number 2

Printed in the

In Vivo Effect of Clofazimine in the LysosomalEnzyme Level and Immune Complex Phagocytosis

of Mouse Peritoneal Macrophages'Jorge Sarracent and Carlos M. Finlay

In spite of the fact that clolazimine, 3-(p-chloroaniline)-10-p-chlorophenyl-2,10-dihydro-2-isopropylimine, was synthesizedin the early 1960s ( 2 ) and that its antileproticactivity since then has been well docu-mented ( 3 . 131 , its mode of action remainslargely unknown. We recently reported ( 10 )that this drug, in vitro, at concentrationsnear the scrum therapeutic levels increasedthe synthesis of lysosomal enzymes and theamount of labeled immune complexes (IC)phagocytosed (") by nonstimulated mouseperitoneal macrophages. In this study, wereport the action of the drug on macro-phages obtained from mice fed by gavagewith various drug concentrations. The re-sults obtained further support our hypoth-esis that the beneficial effect of clofhziminein leprosy occurs through its action on themacrophage lysosomal apparatus.

MATERIALS AND METHODSExperimental animals. Male Swiss mice

weighing approximately 25 gm were ob-tained from the mouse colony establishedat the National Center for Scientific Re-search, Havana, Cuba.

Macrophage collection and culture.Groups of mice were fed by gavage duringdifferent time periods with various clofazi-mine concentrations. The clofazimine wasa gift of Ciba Geigy, Ltd., Basel, Switzer-land, and was dissolved in 0.3 ml of sun-flower oil. At a given time, the mice weresacrificed by chloroform inhalation and theperitoneal cell exudate obtained as previ-

' Received for publication on 30 August 1983; ac-cepted for publication on 5 October 1983.

= J. Sarracent, Ph.D., Biochemistry Department, Na-tional Center for Scientific Research (CENIC), Calle158 y Ave. 25, Cubanacan, Apartado 6880, Havana,Cuba. C. M. Finlay, M.D., Ph.D., Senior Research Sci-entist, Pedro Kouri Tropical Medicine Institute, Apar-tado 601, Havana, Cuba.

ously described ( 7 ). Briefly, 5 ml of M 199(Gibco, U.K.) containing 20 U/mI of pre-servative-free heparin (Novo, Denmark),without serum, was injected into the peri-toneal cavity. After gentle massage, theculture medium was aspirated and 5 ml ali-quots of the collected fluid containing 0.5 x10 6 cells/ml were distributed into 50 mmPetri dishes (Culture, grade, Nunc, Den-mark). The plates were incubated 1 hr at37°C in a humidified atmosphere of 5% car-bon dioxide and air to allow the attachmentof adherent cells. Nonadherent cells wereremoved by washing the plates four timeswith warm sterile saline. Morphologicallymore than 90% of the adherent cells weremacrophages and 98% were viable by tryp-an blue exclusion. Fresh medium M 199containing penicillin 100 U/m1 and strep-tomycin 100 pg/m1 supplemented with 10%v/v newborn calf serum (NBCS) was addedto the plates. The plates were incubatedovernight as indicated above. The next daythe culture medium was aspirated, the celllayer washed twice with cold sterile saline,and 2.5 ml of saline containing 0.1% TritonX-100 was added to each plate. The cellswere disrupted by scraping the plate with asilicone rubber policeman.

Enzyme assays. All enzyme substrates andchemicals were purchased from Sigma,London. The assays were performed underconditions giving linear release of productin relation to the amount of sample usedand the time of incubation. 0-Galactosidasewas assayed by the method of Conchie,Findlay, and Levy ( 6). N-Acetyl-13-D-glu-cosaminidase was assayed by the method ofWollen, Heyworth, and Walker ( 14). Ca-thepsin C was assayed as suggested by Bar-rett ('). Substrates, pH, and buffers were usedas previously reported by us ( 10).

Protein assay. The method of Lowry, etC) was employed, using bovine serum

albumin as a standard.

154

52, 2^Sarracent and Finlay: Clgfazimine Effects on Macrophages^155

TABLE 1. LyNOSOMai C/L717//e levels and protein content in peritoneal macrophages ofmice treated with clq1dzimine .Thr 21 days.

Clofazimineconcentration(mg/kg body^glucosaminidaseweight/day)

0-Galactosidase^Cathepsin CProtein

(mg/plate)

0 1536 ± 138 98.2 ± 10.6 345 ± 42 690 ± 150.1 1498 ± 146 102.4 ±^11.5 336 ± 38 681 ± 661 2431 ± 202" 185.7 ± 16.6" 488 ± 57" 677 ± 98

10 2309 ± 219" 176.4 ±^17.3" 495 ± 51" 694 ± 73

Enzyme activities are expressed as nmol of product formed by substrate hydrolysis/mg of protein/hr."Significantly increased compared to untreated controls; p < 0.001. Student's t test.

Purified IgG, antiserum and immunecomplexes. Rabbit IgG was obtained by themethod of Chersi and Mage ( 4). Goat anti-rabbit IgG was obtained by repeated sub-cutaneous injections of 1 mg purified IgGin Freund's complete adjuvant. The ani-mals were bled one week after the last in-jection. Antigen-antibody complexes wereprepared at the equivalence zone. The pre-cipitate was thoroughly washed with coldsaline. The protein content of the immunecomplex (IC) was determined by the meth-od of Lowry, et a!. ( 8 ). Solutions were storedat 4°C for no longer than 3 days.

Iodination. Purified IgG was labeled withcarrier-free '"I (Radiochemical Centre,Amersham, U.K.) by the method of Mac-Conahey and Dixon ( 9 ). Unbound ' 2 '1 wasremoved by means of gel filtration on Seph-adex G-25, and IgG aggregates were elim-inated by centrifugation at 15,000 g for 90min. The specific activity of the IgG was 60kiCi/mg of protein. Each batch of radioactiveIC had a specific activity of approximately0.2 Less than 2.5% of the radio-activity was soluble in cold 10% trichloro-acetic acid (TCA). The labeled IgG was fro-zen in 1 ml aliquots until used. Each batchwas used while the IC radioactivity wasenough for the measurement of phagocy-tosis.

Phagocytosis assay. Culture medium wasremoved from the plates and fresh mediumcontaining 20 µg/ml of ' 25 1-IC, supple-mented with 10% NBCS, was added. Twohours later the medium was removed andthe cell layer washed four times with warmsaline. Two plates per clofazimine concen-tration were treated in the same manner ex-cept that they were kept at 4°C during thelength of the experiment and washed with

cold saline. In those plates, cell associated' 25 I-IC was less than 2% of that phagocy-tosed in the experiment. Cells were re-moved from the plates as indicated above.Radioactivity was measured in a well-typescintillation counter.

Statistical tests. Quadruplicate cultureswere done for each drug concentration inevery experiment. Each experimental seriesdescribed in this paper was repeated at leastthree times with the same results. Meansand standard deviations were calculated af-ter samples were shown to be homogeneousby calculations of coefficients of variance.The significance of differences was estab-lished by Student's t test.

RESULTSWe decided to test three clofazimine con-

centrations-0.1 mg, 1 nig, and 10 nig/kgbody weight/day—since Shepard and Chang( I ') reported that the two higher drug con-centrations, when given to animals inocu-lated with M. leprcie in the foot pad, wereeffective in preventing bacterial multipli-cation. Groups of mice were daily given thedrug, dissolved in 0.3 ml of sunflower oil,by gavage. A control group received onlythe sunflower oil. At the beginning of theexperiment and every week for three weeks,mice were killed by chloroform inhalation,the peritoneal cells obtained, and culturedas described above.

Drug effect on lysosomal enzyme level.Nonstimulated peritoneal macrophages ob-tained from mice that had received 1 mgand 10 mg clofazimine/kg body weight for21 days showed a significant increase (p <0.001) of the three lysosomal enzymes tested(Table 1). There was no difference in theprotein concentration. Since N-acetyl-(3-D-

25

0

-, • 200E

15

1000

Clofazimineconcentration(mg/kg bodyweight/day)

00.11

10

0 7

5148 ± 623 5135 -± 4875332 ± 538 5276 ± 4095298 ± 582 5289 ± 4155303 ± 611 5197 ± 493

14 21

4780 ± 496 4878 ± 4754978 ± 454 4675 ± 4054897 ± 472 8398 ± 417'8089 ± 631' 7781 ± 496'

Cell radioactivity cpm/mg of protein(treatment time in days)

1 56^ International Journal of Leprosy^ 1984

21 Days

THE FIGURE. Time-dependent effect of clofaziminetreatment on cellular N-acetyl-0-D-glucosaminidaselevels in macrophages of mice treated for different pe-riods of time with the drug. Each value represents themeans of four determinations. Enzyme activity is ex-pressed as nmol of product formed by substrate hy-drolysis/mg of protein/hr.

•—• = 0.1 mg/kg body weight/day; x x = 1 mg/kg body weight/day; 0-0 = 10 mg/kg body weight/day; L—L= controls.

glucosaminidase is an efficient lysosomalenzyme marker, time dependence of the in-crease was studied with this enzyme. TheN-acetyl-/3-D-glucosaminidase activity wasstudied weekly (The Figure). It was foundthat enzyme activity increased in macro-phages obtained from animals that had re-ceived 1 mg and 10 mg clofazimine/kg bodyweight at 14 days, and at 21 days there wasa further increase of the enzyme activity inboth groups. Neither control groups northose that had received 0.1 mg clofazimine/kg body weight showed an increase of thelysosomal enzyme activity.

Drug effect on immune complex phago-eytosis. There was no difference in the

amount of 125 1-IC phagocytosed by the con-trol group and by those groups receivingvarious clofazimine concentrations duringthe first week. At the end of the second week,macrophages obtained from mice that hadreceived 10 mg clofazimine/kg body weightphagocytosed more (p < 0.001) IC thanthose from other groups (Table 2). At theend of the experiment (three weeks), mac-rophages from mice that had received dailydoses of 1 mg and 10 mg clolazimine/kgbody weight showed a significant increaseof ' 25 I-IC phagocytosed over the othergroups.

DISCUSSIONWhen studying the mode of action of a

drug sometimes experimental results ob-tained with simple in vitro models cannotbe reproduced when animal models are uti-lized. In those cases, the in vitro results arefrequently not relevant for comprehensionof the drug's mechanism of action. There-fore, with the experimental series reportedin this paper we aimed to evaluate the actionof clofazimine on the macrophage lysosom-al apparatus of mice that received the drugfor up to three weeks. We assumed that ifclofazimine modified the macrophage func-tion in these animals, this time period wouldbe sufficient for the expression of the drug'sactivity.

The results obtained by feeding mice clo-fazimine at concentrations of 1 mg and 10mg/kg body weight/day demonstrated thesame effects on macrophage function asthose found in vitro. How to explain this?We have found no difference in the peri-toneal cell adherence (data not shown) andin cell populations between control and clo-

TABLE 2. Time-dependent effect of clofazimine on the phagocytosis of ' 251 labeledimmune complexes by peritoneal macrophages qf mice treated with dilletvnt levels of thedrug.

Significantly increased compared to untreated controls; p < 0.001, Student's / test.

52, 2^Sarracent and Finlay: Clofazitnine Effects on Macrophages^157

fazimine-treated mice, although peritonealmacrophages obtained from clofazimine-treated mice were bigger and had more densegranules than macrophages obtained fromcontrol mice (data not shown). We have pre-viously reported ( 10) that the drug does nothave direct action on lysosomal enzyme ac-tivity and that the presence in the culturemedia of a protein synthesis inhibitor, cy-cloheximide, inhibits the increase of this ac-tivity. The above-mentioned experimentalobservations allow us to suggest that theincrease of lysosomal enzyme activity is dueto an increase of lysosomal enzyme synthe-sis induced by the drug. In an attempt toexplain the increased phagocytic capacityfound in vitro as well as in vivo, we studiedmacrophage Fc and complement receptors.Macrophages cultured in the presence ofclofazimine or obtained from the peritonealcavity of m ice receiving the drug in the samemanner as reported in this paper have moreof these receptors than control macrophages(data not shown). This may explain the re-sults reported here.

An increase in the time of exposure to ahigher drug concentration does not result ina proportional increase of lysosomal en-zyme level or phagocytic capacity. Resultshave been published demonstrating thatclofazimine accumulates in the macro-phage, and Conalty, et al. ( 5) have shownthat prolonged exposure to clofazimine re-sults in intracellular crystal deposition ofthe drug. Perhaps progressive accumulationof the drug may alter macrophage functionin a manner different from that observed byus. The results obtained suggest that a con-tinuous increase of the drug concentrationover a longer time period does not neces-sarily lead to an enhanced macrophagefunction and could, in fact, be harmful tothe cell.

These results add further experimentalevidence to our suggestion that clofazi-mine's beneficial action on leprosy is at leastpartially mediated through an action of thedrug on macrophage function.

SUMMARYThe effects of clofazimine on macro-

phages obtained from mice fed by gavagewith various drug concentrations were stud-ied. The results obtained demonstrated anincrease in the activity of various lysosomal

enzymes and in the amount of labeled im-mune complexes phagocytosed at drug con-centrations of 1 mg/kg and 10 mg/kg bodyweight. This confirms and extends the ef-fects reported by us of clofazimine's actionon the lysosomal apparatus.

RESUMENSe estudiaron los efectos de la clofazimina sobre los

macrOfagos obtenidos de ratones tratados por via oralcon varias concentraciones de Ia droga. Los resultadosobtenidos demostraron un increment° en la actividadde varias enzimas lisosomales y en la cantidad de corn-plejos inmunes marcados fagocitados cuando la con-centraciOn de la droga se increment° de 1 a 10 mg/kgde peso corporal. Esto confirma y extiende los efectosde la clofazimina sobre el aparato lisosomal que fueronpublicados antes por nosotros.

RESUMEOn a etudie les effets de Ia clofazimine sur des macro-

phages obtenus chez des souris nourrics par gavageavec differentes concentrations de ce medicament. Lesresultats obtenus ont demontre unc augmentation dansFactivite des diverses enzymes lysosomiqucs, de memcque dans Ia quantite de complexes immuns marques,et ccci pour des concentrations de medicaments allantde 1 mg/kg a 10 mg/kg de poids corporel. Ces obser-vations confirment et elargissent la portee des effetsrapportes anterieurement sur Faction de la clofaziminesur l'appareil lysosomique.

Acknowledgments. The authors wish to express theirappreciation to Dr. Marian Ridley for her kind helpand suggestions, to Lic. Ana Valdes-Portela for herstimulating discussion, and to Eslivia Prestamo for typ-ing the manuscript. This investigation received supportfrom the UNDP/World I3ank/WHO Special Pro-gramme for Research and Training in Tropical Dis-eases.

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