INTERNATIONAL JOURNAL OF LEPROSY^ Volume 61, Number 2

Printed in the U.S.A.

Effect of Recombinant Interferon GammaAdministration on Lesional Monocytes/Macrophages in

Lepromatous Leprosy Patients'Krovvidi S. R. SivaSai, Hanumanthappa K. Prasad,

Radhey S. Misra, Venkatesh Ramesh, Daphne Wilfred, andIndira Nath2

Lepromatous leprosy (BL/LL) is a dis-seminated disease characterized by granu-lomas wherein foamy macrophages areloaded with the pathogen Mycobacteriumleprae. The localized tuberculoid leprosy,on the other hand, rarely shows bacilli with-in the epithelioid cell granulomas (31). Themechanisms by which mycobactcria arekilled or resist intracellular destruction arenot clear. Bactericidal activity is thought tobe mediated by reactive oxygen interme-diates (ROI), such as superoxide anion(0), hydrogen peroxide (H20,), and hy-droxyl radical (OFF) produced during therespiratory burst following phagocytosis (2).Macrophage activation by lymphokincs,particularly interferon gamma (IFN--y) en-hances the production of these radicals bothin vitro and in vivo (21, 22, 24

). Such enhance-

ment has been reported to be associated withthe killing of various intracellular patho-gens (3, 6, 20, 24, 32, 33) Lepromatous leprosypatients appeared to have defective pro-duction of IFN--y when peripheral-blood-derived lymphocytes were stimulated withM. leprae (8, 18, 27s.) In addition, monocytesof lepromatous patients were shown to havereduced release of ROI both before and afterin vitro phagocytosis of the leprosy bacilli

' Received for publication on 28 October 1992; ac-cepted for publication in revised form on 5 January1993.

= K. S. R. SivaSai, M.Sc., Research Fellow; H. K.Prasad, Ph.D., Associate Professor; I. Nath, M.D.,F.R.C.Path., F.N.A., F.A.Sc., F.N.A.Sc., F.A.M.S.,Professor and Head, Department of Biotechnology, AllIndia Institute of Medical Sciences, Ansari Nagar, NewDelhi 110029, India. R. S. Misra, M.D., Senior Der-matologist; V. Ramesh, M.D., Consultant, Depart-ment of Dermatology, Safdarjung Hospital, New Delhi110029, India. D. Wilfred, M.D., Medical Superinten-dent, Leprosy Mission Hospital, Shahadra, India.

Reprint requests to Dr. Nath.

(7. D. '4). Moreover, in the experimental lep-rosy model using nude mice it was observedthat tissue macrophages laden with Al. lep-rae were unable to produce 0;, and wereresistant to activation by IFN--y (". 36). Sub-sequently, monocytes and in-vitro-cultivat-ed macrophages from peripheral blood ofman also were shown to be affected nega-tively when Al. leprae components, such asphenolic glycolipid-I (PGL-I), were used to

‘.pretreat the cells (26,39,40 ) Further evidenceindicated that microbial glycolipids of in-tracellular pathogens such as Al. leprae act-ed as scavengers of oxygen radicals, andthereby contributed to the persistence ofthese pathogens within the macrophages(5. 26).

Of recent interest are studies wherein re-combinant human IFN--y (rIFN--y) injec-tions into bacilli-laden lesions of leproma-tous leprosy patients showed local bacillaryclearance (10). There is scant information onthe functional status of macrophages con-stituting human granulomas with regard toreactive oxygen radical release. The abilityof rIFN--y to activate such well-differenti-ated, bacilliferous lesional macrophages alsois not known. Since such information is re-quired in order to design better strategiesfor bacterial killing, the present study wasundertaken to evaluate the status of lesionalmacrophages in the course of the naturaldisease in man. Our study indicates thatrIFN--y administered into lepromatousgranulomas leads to multiple effects. Manypatients showed enhanced levels of H2O,and 0-7 release as well as an increased abilityof lesional macrophages to be stimulated invitro to release ROI. In conformity with ear-lier studies erythema/induration and bacil-lary clearance were noted also at the injectedsites. However, in a given individual these

259

260^ International Journal of Leprosy^ 1993

features were observed independently ofeach other, and no stringent relationship wasobservable between the diverse local effectsof rIFN--y.

MATERIALS AND METHODSPatients. Sixteen, newly diagnosed, un-

treated borderline (BL) and polar lepro-matous (LL) patients attending the derma-tology clinics of Safdarjung Hospital andLeprosy Mission Hospital, Shahadra, NewDelhi, India, were included in the study.They were classified on the clinicopatho-logical criteria of Ridley and Jopling (1').The bacterial index (BI) was scored on alogarithmic scale by slit-smear examinationfor acid-fast bacilli (AFB) from six sites (10).None of the patients had reactions at thetime or examination. Multidrug therapyconsisting of 600 mg rifampin monthly, 100mg of clofazimine on alternate days, anddapsone 100 mg daily was instituted on thetermination of rIFN--y injections.

rIFN-7 injection and study design. Ly-ophilized rIFN--y with a specific activity of2 x 107 units/mg of protein was kindly pro-vided by Dr. Rosenkaimer of BochringerIngelheim, West Germany. After obtainingclearance from the Institutional EthicalCommittee and the Drug Controller of In-dia and informed consent from the patients,10 Ag or 20 pg of excipient reconstitutedrIFN--y was injected intradermally into threewell-characterized lepromatous lesions con-secutively for 3 days. Clinically similar,neighboring or contralateral lesions used ascontrols were injected with the same vol-ume of excipient. The study design was asfollows.

On day 0, a biopsy for histopathologicaldiagnosis and a slit-skin smear examinationfor the BI were undertaken. On days 1, 2,and 3 the diameter of the local erythma/induration at the injected sites was record-ed. On day 4, two 4-mm punch biopsies ofuninjected and injected sites were taken forisolation of lesional macrophages. The BIwas repeated on the same nonbiopsied in-jected sites on day 4 and at monthly inter-vals up to 1 year, except where stated oth-erwise. Biopsies of both types of lesions wererepeated for histopathological examinationbetween 9 and 10 months.

Isolation of peripheral blood monocytes.Venous blood (10 ml) was aseptically drawnfrom patients in heparinized syringes.Mononuclear cells were isolated by the den-sity gradient method (4). After washing thecells two times in RPMI 1640 (SigmaChemical Co., St. Louis, Missouri, U.S.A.),the cell pellet was resuspended in RPMI1640 containing 10% normal AB serum ata concentration of 4 x 10 cells/well. Thecells were layered in 96-well, flat-bottomplates (Nunc, Roskilde, Denmark), and in-cubated for 2 hr at 37°C in a 5% CO, in-cubator. The nonadherent cells were re-moved by washing twice, and only theadherent cells were used for functional as-says.

Extraction of lesional macrophages.Punch biopsies (4-mm) from control andrIFN-7-injected sites were washed withRPMI 1640 (Sigma) containing 10% AB se-rum to remove traces of blood. The epi-dermis was discarded, and the dermis wasmechanically teased, suspended in RPMI1640 with 0.1% collagenase (Type IV; Sig-ma) and incubated at 37°C for 14-16 hrwith gentle shaking. Subsequently, the tis-sue debris was removed by slow-speed cen-trifugation (Sorvall RT 6000; DuPont Com-pany, Willmington, Delaware, U.S.A.) at 50x g for 5 min. The supernatant consistingof lesional cells was centrifuged at 250 x gfor 10 min. The pellet consisting of lesionalcells was washed twice with phenol-red-freeHanks' balanced salt solution (HBSS; Sig-ma) and delivered into 96-well, flat-bottomplates (Nunc) at a concentration of 6 x 104cells/well. Aliquots of lesional cells weretested for viability by trypan blue dye ex-clusion which ranged from 85% to 95%. Eachsample also was stained for nonspecific es-terase (NSE), as described below, to distin-guish the monocyte/macrophage popula-tion from other cells. The yield of cells from4-mm biopsies ranged from 5 x 10' to 1.8x 106, of which 60%-70% consisted of NSE-positive cells. Care was taken to use phenol-red-free medium to avoid interference inthe H2O, and CO, assay.

NSE assay. The lesional cells/mono-cytes were tested for NSE activity (4'). Inbrief, the cells were spun onto slides (Cy-tospin 2; Shandon, U.K.), fixed for 60 sec,and incubated at 37°C for 60 min in a stain

61, 2^SivaSai, et al.: Effect of rIFN-y on Lesional Macrophages^261

containing pararosaniline (Sigma) and al-pha-naphthyl acetate as a substrate (Sigma).The slides were washed with distilled water,air dried, and scored for NSE positivity.

Extraction of M. leprae. M. leprae wereextracted from the cryopreserved spleen ofan infected armadillo (29) (kindly providedby Dr. R. J. W. Rees, National Institute forMedical Research, London). Care was takento ensure sterility, and phenol-red-free HBSS(Sigma) was used to suspend the bacilli.

Quantitation of H202 and 4:i) release bylesional monocytes/macrophages. H202release was estimated essentially by themethod of Pick and Mizel (28) based on thehorseradish-peroxidase (HRP0)-mediatedoxidation of phenol red by H20„ which re-sults in a compound with increased absor-bance at 610 nm. Briefly, 100 pl of the re-action mixture containing 0.028 M phenolred (Sigma), 50 pg/m1 of HRPO (Type II;Sigma) was added to duplicate wells. Theoptical density (OD) was recorded in anELISA reader (Titertek Multiskan; FlowLabs, Scotland, U.K.) immediately and af-ter 60 min. The differences in the absorbancevalues were converted into nanomoles ofH2O, released by extrapolating on a stan-dard graph set up with each set of tests.

0-T release was determined by quantify-ing the superoxide dismutase (SOD) inhib-itable reduction of the ferricytochrome c asdescribed by Johnston (9). Briefly, 100 pl ofthe reaction mixture containing ferricyto-chrome c (4 mg/ml, Sigma) alone or withSOD (100 pg/ml; Sigma) were added to du-plicate wells. Absorbance changes were not-ed at 0 and 60 min after addition of thereaction mixture in an ELISA reader usinga 550-nm filter. The difference in values wasconverted to nanomoles of 07 using theextinction coefficient of ferricytochrome c(9). All values were expressed as nano-moles/1 x 106 NSE-positive cells/60 min.NSE positivity was used instead of proteinconcentration to normalize the data sinceother lesional cells were present in the le-sions. Preliminary time kinetics for H2O,and 0-T over 30-120 min had indicated 60min as the peak period.

Stimulants for in vitro 1120, and 0.c- re-lease. To evaluate the potential of lesionalmonocytes/macrophages to be stimulatedin vitro, duplicate wells were set up as before

with freshly extracted M. leprae at a ratioof 50:1 (bacilli : monocyte/macrophage).After a 1-hr incubation at 37°C in a 95% airand 5% CO, incubator, H2O, and 0-7 wereestimated.

Bacterial index (BI). The bacterial loadin the patients was evaluated by slit-skinsmears prepared from six sites and scoredon a logarithmic scale for AFB (29) usingZiehl-Neelsen staining prior to the start andat the termination or the study. A monthlyBI was done for both control and rIFN--y-injected sites.

Histopathology. On day 4 as well as atthe termination of the study (9-10 months),the injected sites were biopsied for histo-pathological examination and BI scoring.Biopsies were fixed in 10% buffered for-malin and stained with hematoxylin and eo-sin (H&E) as well as Ziehl-Neelsen stain.

Statistical analysis. The p values werecalculated by the Student's t test, Wilcoxonsigned rank test, and rank correlation anal-ysis by the Spearman method (").

RESULTSClinical status. Table 1 gives the clinical

details of the lepromatous leprosy patientsincluded in the study. Care was taken toselect clinically similar, contralateral lesionsfor injections with excipient (control) andrIFN--y. Patients were injected intrader-malty with 100 pl of excipient or 10/20 pgof rIFN-7 into three lesions for 3 consecu-tive days, except where stated otherwise. Nountoward systemic symptoms were report-ed, except for patient 12 who complainedof malaise, headache and fever (38°C) afterone injection of 20 ptg, of rIFN-y.

Skin reaction to rIFN-7 injections. Us-ing a 5-mm induration diameter as the cut-off point, it was observed that none of thesix patients receiving 10 pg, rIFN-7 respond-ed; whereas all responded to the higher dose.Ten of 16 borderline and polar lepromatouspatients showed erythema and indurationof the injected lesions, with the average di-ameter ranging from 5 to 25 mm (The Fig-ure). In conformity with our earlier studies(10) maximal responses were observable by48 hr in 6 of 10 patients; the others showedno change or a mild increase at 72 hr. How-ever, patient 12 showed an 18-mm indu-ration after one dose of 20 pg and was not

PatientAge

4 506 609 28

14 7.)

15 /016 /6

3035

3 305 357 358 22

10 15111"/ 3413 45

Diagnosis ill'

rIFN-yc

Dose(mg)

Erythema"/Injections induration

(days)

13L 2.3 10 3I3L 2.5 10 3LL 3.3 10 3BL 1.5 10 3

1313/BL 1.9 10 3BL 2.5 10 3LL 4.3 3LL 2 . 3 20 3BL 3.7 20 3LL 3.3 /0 3LL 3.7 /0 2LL 4.0 20 2BL 3.3 /0 3LL 3.5 20 3LL 2.3 20 1I3L 3.2 20

Sex

7.1 13.2 12.230 2.1^2.3^1.8

72(7)

24^48(10)^(9)

(Hours)

15

10

262^ International Journal of Leprosy^ 1993

TABLE 1. Clinical profile of borderline (BL) and polar lepromatous (LL) patients.

" Same patient number has been used in the text and in all tables.131 = Bacterial index, mean of acid-fast bacilli of six sites scored as 1 to 5 on a logarithmic scale (30).rIEN-y injections were given on consecutive days intradermally into well-characterized lesions.+ and — indicate > 5 mm and < 5 mm erythema/induration, respectively.

given additional injections of rIFN-y (TheFigure).

Twenty-four hours after the last injection(i.e., day 4), two each of control and rIFN-y-injected lesions from subjects showing er-ythema/induration were biopsied at previ-

THE FIGURE. Time kinetics of local response toHEN-7 injections into lesions of lepromatous leprosypatients who showed > 5-mm induration. Numbersabove the graph at each time point depict mean di-ameter + S.E. of patients whose numbers are given inparentheses on the X-axis.

ously marked sites. The extracted lesionalcells were checked for nonspecific esterase(NSE) positivity, and all estimations wereexpressed in terms of 106 NSE-positive cells.In general, the number of lesional cells ob-tained from the same size biopsies of in-jected sites were 1.5- to twofold higher thanthose from control sites.

Effect of rIFN-'y on ROI release by le-sional macrophages. 11,0,. Lesional NSE-positive cells from 10 individuals were eval-uated for baseline H2O, release in controland rIFN--y-injected sites (Table 2). Therewas individual variation in both sites in themean nmoles of I-1,03 released per 106 le-sional cells. However, when the two sites ofthe same individual were compared, the cy-tokine-injected site showed a net incre-ment/change of H20, release in 8 of 10 sub-jects. When the two groups of control andtest sites were compared by Student's t test,this increment was of significance (p <0.05).However, when the nonparametric Wilcox-on signed rank test was used, no statisticalsignificance was obtained. To ascertainwhether the lesional macrophages could beinduced further to release H202, aliquots ofcells from control and rIFN-y-injected siteswere exposed in vitro to freshly extractedM. leprae (Table 3). Cells from both controland rIFINT-y-injected lesions of 3 of 4 pa-

61, 2^SivaSai, et al.: Effect of rIFN-y on Lesional Macrophages^263

TABLE 2. HD, release (nmoles/1 x 106 NSE-positive cells/60 min) by lesional mac-rophages of control and rIFN--y-injected lesions of lepromatotts patients.

Lesions

Netchange

Patient Diagnosis Control' rIFN--y"no. Duplicate

values Mean Duplicate Meanvalues

1^LL 4.0 4.2 5.8 6.3 2.14.4 6.8

LL 1.8 2. 1 3.8 3.9 1.84.0

3^BL 8.3 8.8 12.2 12.5 3.79.3 12.8

5^LL 10.0 10.5 13.2 13.5 3.010.1 13.9

7^LL 7.5 7.8 10.0 9.7 1.98.1 9.4

8^LL 3.0 3.1 3.0 3.2 0.13.2 3.4

10^BL 8.8 8.8 13.2 13.8 5.0ND 14.4

II^LL 16.0 16.3 11.8 12.5 -3.816.6 13.2

1/^LL 6.0 6.3 7.5 8.3 2.06.6 9.1

13^LL 5.0 5.1 8.0 7.8 2.75.2 7.6

Mean ± S.D. 7.3 + 4.2 9.2 ± 3.9

p value by Student's t test; a vs b = < 0.05."p value by Wilcoxon signed rank test; a vs b = < 0.06.

ND = Not done.

tients showed a negligible-to-low increasein H,02 after treatment with bacilli. Patient11, however, showed marked improvementafter three injections of 20 tg rIFN--y.

0 - 7 . Due to the paucity of cells only fivepatients were tested for a7 release. In gen-eral, 02- release was low in lesional mac-rophages. However, cells from rIFN--y sitesshowed higher levels (p < 0.05) as com-pared to control lesional cells (Table 4). Invitro exposure to M. leprae showed a de-crease in O release which was reversed in4 of 5 rIFN--y-injected lesions (Table 5).

Peripheral blood monocytes. Monocytesisolated from blood before and after the ter-mination of rIFN--y injections showed nostatistically significant increases in H2O, orO either at the basal level or after in vitroexposure to M. leprae. H20, released (mean

S.D. nmoles/106 NSE-positive cells) byNSE-positive monocytes from 10 patientsbefore and after three rIFN--y injections was16.5 ± 6.5 and 20.5 ± 10.2, respectively.After in vitro exposure to M. leprae, the 1ey-

els of showed mild but insignificantdifferences before (14.6 ± 5.3) and after(18.5 8.9) intralesional cytokine admin-istration. Similar results were obtained for0-T, where mean S.D. nmoles/106 NSE-pos-itive cells before and after treatment were4.4 1.3 and 4.8 ± 2.8, respectively. Afterin vitro M. leprae exposure, the respectivevalues were 3.4 + 0.6 and 2.7 ± 1.0 nmoles/106 NSE-positive cells.

Local clearance of M. leprae and granu-loma histology. The patients' BIs were takenfrom the same predetermined lesions in-jected or not injected with rIFN--y at month-ly intervals for 1 year (Table 6). In addition,the BI was taken from histopathological ex-aminations of the biopsies on day 4 and 9to 10 months after injections. Table 6 givesthe mean BI of two lesions each. Using thecriteria of change in the BI of at least 2consecutive months and also comparing thecontrol values with the cytokine-treatedsites, it was observed that only 4 of 9 sub-jects showed a decrease in their Bls attrib-

264^ International Journal of Leprosy^ 1993

TABLE 3. H2O, release by lesional macrophages derived from control and rIFN-y-injected lesions of lepromatous patients with and without (baseline) in vitro exposure toM. leprae.

H20, (nmoles/1 x 10' NSE-positive cells/60 min)

Patientno.

Diagnosis

Control

Netchange

rl FN-ry

Net'.change

Baseline tI. leprae Baseline Al. leprae

Dupli-Mean' cateDupli-Mean" cate

Dupli-Mean" cateDupli-Mean' cate

3 BL 8.8 8.3 19.4 19.2 10.6 12.5 12.2 24.5 24.0 12.09.3 19.6 12.8 25.0

5 LL 10.0 10.0 13.4 13.0 3.4 13.5 13.2 17.5 17.2 4.010.1 13.8 13.9 17.8

10 BL 8.8 8.8 14.2 14.1 5.4 13.8 13.2 22.2 22.1 8.4ND" 14.3 14.4 22.3

11 LL 16.3 16.6 15.8 15.4 -0.5 12.5 11.8 20.0 19.8 7.516.0 16.2 13.2 20.2

Mean ± S.D. 5.0 ± 4.3 8.0 ± 3.3

p values: a vs b = NS; d vs e = < 0.08; c vs f = < 0.05." ND = Not done.

utable to rIFN-y. This decrease occurred asearly as 4 to 8 weeks and ranged from 0.5to 3 logs. Histopathological examination ofthe biopsied site at 9 months revealed twoadditional patients with a higher bacillaryclearance at the cytokine-administered sitewhich was missed by slit-skin smear ex-amination. Thus, in total, 6 of 10 patientsshowed bacillary clearance. The BIs of thosepatients who had received the lower doseof the cytokine did not show any change(data not shown).

In addition, granuloma size was moder-ately reduced at the rIFN-y sites in five sub-jects; one showed an ill-formed granulomawith epithelioid cells and another had pen-vascular cuffing and increased cellularitywith mononuclear cells.

DISCUSSIONThe reversibility of immune defects in

disease states has become possible with therecent availability of recombinant lym-phokines. Interleukin-2 and IFN-y are of

TABLE 4. Oi release by lesional macrophages derived from control and rIFN-y-injectedleprotnatous leprosy lesions.

Patientno.

.Diagnosis

02^(nmoles/1 x 10' NSE-positive cells/60 min)

Control rl FN--yNet

changeDuplicatevalues Mean" Duplicate

values Mean"

3 BL 1.0 1.3 2.2 2.6 1.31.6 3.0

7 LL 1.7 1.8 3.4 3.8 2.01.9 4.?

10 BL 3.6 3.8 5.4 5.6 1.84.0 5.8

11 LL 4.8 5.0 5.8 6.3 1.35.2 6.8

13 BL 2.1 2.4 4.3 4.4 2.02.7 4.5

Mean ± S.D.^ 2.9 ± 1.5^

4.5 ± 1.5^

1.7 ± 0.4

p value: a vs b = < 0.05.

6 1 , 2^SivaSai, et al.: Effect of rIFNy on Lesional Macrophages^265

TABLE 5. 0 -T release by lesional macrophages derived from control and rIFN--y-injectedlesions of lepromatous patients with and without (baseline) in vitro exposure to M. leprae.

0, (nmoles/l x 10' NSE-positive cells/60 min)

Patientno.

Diag-nosis

Control

Net'change

r1FN-7

Net'change

Baseline ti. leprae Baseline M. leprae

MeanDupli-catc

valuesMeant'

Dupli-cate

valuesMeand

Dupli-cate

valuesMean'

Dupli-catc

values

3 BL 1.3 1.0 3.4 3.7 2.1 2.6 2.2 4.6 4.4 2.01.6 3.6 3.0 4.8

7 LL 1.8 1.7 1.5 1.1 -0.3 3.8 3.4 6.4 6.3 2.61.9 1.9 4.2 6.5

10 LL 3.8 3.6 0.0 0.0 -3.8 5.6 5.4 6.8 6.1 1.24.0 5.8 7.5

11 LL 5.0 4.8 1.7 1.1 -3.8 6.3 5.8 4.4 4.2 -1.95.2 1.3 6.8 4.6

13 BL 2.4 7.1 2.0 1.8 -0.4 4.4 4.3 5.8 5.2 1.42.7 2.2 4.5 6.4

Mean ± S.D.^ -1.2 ± 2.5^ 1.1 ± 1.7

p values: a vs b = Not significant (NS); d vs e = NS; c vs f = < 0.05.

particular interest in leprosy since they arerequired for T-cell proliferation (19' 37) andmacrophage activation ('' 23), respectively,and have, moreover, been shown to be lowin lepromatous leprosy (8'27). Earlier studieson Indian patients (10) had shown that localinjection of rIFN--y led to an influx of CD4-positive T cells and monocytes as well asbacillary clearance (10' '7). The present studyon a different ethnic population in Indiaconfirms the development of lymphokine-induced dermal reaction similar to a de-layed-type reaction in bacilliferous lesionsof lepromatous leprosy patients.

To evaluate the effect of rIFN-y on ox-ygen intermediates produced by lesional ba-cilli-laden macrophages, the H2O, and 0-;release were studied. Of interest was the en-hanced induction of H,0-, in lesional cellsof rIFN--y-injected sites as compared to con-trol sites in 8 of 10 patients. Although therewas considerable variability in H70-, pro-duction in the two sites among the lepro-matous leprosy patients, the increment wassignificant when the control and rIFN-y-injected lesions of the same individual werecompared with each other. Although the0-T levels were generally low in lesional cells,there was a consistent increase in rIFN-y-injected sites compared to the control con-tralateral lesions. On in vitro exposure to M.leprae, lesional cells from both sites showedstatistically insignificant and low increases

in H2O, as compared to the equivalentunexposed cells. The rIFN--y-injected sitesdid not show any further change. O-T levelswere inhibited by the in vitro addition of M.leprae in 3 of 4 subjects. This inhibition wasnot observed in rIFN-y-injected lesions ofthe same individual.

Previously reported studies on granulo-mas of experimental models (35' 36) andblood-derived monocytes and macrophages(14, 39, 40\) showed that O-T production notonly was low but also unresponsive to theactivating effects of rIFN--y. Using pureproducts of M. leprae, such as phenolic gly-colipid and lipoarabinomannan, inhibitionof Oy generation was observed (38,39). IFN-yhas been reported to reverse the loss of func-tional activity that occurs in aged humanmonocytes cultivated in vitro (1). Our stud-ies indicate that cells from rIFN--y-injectedlesions are able to withstand the inhibitoryeffects of Al. leprae in vitro. Although, his-tologically, the granulomas in our studiesconsisted mainly of well-differentiated mac-rophages, the role of newly migrating mono-cytes (10, 11,22,34) in contributing to the ox-ygen radical release cannot be stringentlyexcluded. As far as we are aware, studiessimilar to ours on macrophages of humangranulomas have not been reported.

In the present study, peripheral bloodmonocytes showed a mild but not statisti-cally significant increase in ROI production

266^ International Journal of Leprosy^ 1993

TABLE 6. Monthly, mean bacterial index (BI) of 2 each of control and rIFN-y-injectedlesions of BL/LL patients followed up for 6-12 months and scored on a scale of 1 to 5+(30) (BI scored on the skin biopsies taken on day 4 and 9-10 months are also depicted).

Pa-^.Diag-tient^ihnossno.a

Ini-tialBI

Lesiontype'

Lesional 111 slit-smear techniqueHisto-

pathology

Day Months Day Months

4 I 2 4 6 8 10 12 4 9-10

1 LL 4.3 5 5 4.5 4 4 4 3 3 5 4IFN 5 4.5 3.5 3.5 4 4 3 3 5 3

2 LL 2.3 4 4 2.5 NA" 2 / / 2 4 4IFN 4 4 2.5 NA 2 / 1.5 2 4 3

3 BL 3.7 5 3 3 3 1.5 3 3 2 5 5IFN 5 4 4 3 1.5 3 3 2 5 3

5 LL 3.3 3 4 5 4 4 3 3.5 4 5 5IFN 3 3 5 4 4.5 4.5 3.5 2 5 5

7 LL 3.7 4 5 5 5 5 NA 5 NA 4 5IFN 4 5 2 3 3 NA 4 NA 4 3

8 LL 4.0 4 5 5 5 4 3 2 3 5 4IFN 3 3 4 3 4 3 3 4 5 4

10 BL 3.3 3 1 1 1 —ve' — ye —ye —ye 3 3IFN 3 1 —ye —ve — ye — ye —ye —ye 3 1

II BL 3.5 5 5 5 5 4 4 4 3 4 4IFN / / 1 2 2 1 1 3

12 LL 1.0 1 1 —ye —ve —ve —ye —ye —ve 3 2IFN 1 0.5 —ve — ye —ye —ve —ve —ve 1 1

Patients 1, 7, 10, and 11 showed BI decreases in slit-skin smears; patients 2 and 3 showed BI decreases inbiopsies.

LL = Lepromatous; BL = borderline lepromatous leprosy.' C = Control lesion; IFN = rIFN-7-injected lesion.d NA = Not available.' —ve = Negative.

after intralesional rIFN--y. No consistentpattern was discernable since individualswithin the lepromatous group showed vari-able levels of ROI both before and aftercytokine administration. Using a differentmethodology, Nathan, et al. (22) earlier hadreported augmentation of H2O, levels aftera single intradermal rIFN--y injection.

That rIFN--y can alter lesional macro-phages in lepromatous leprosy is supportedfurther by the early but persistent clearanceof bacilli in many lepromatous patients.Monthly conventional slit-skin smear ex-amination of the injected site showed a 0.5-to 3-log decrease in the BI compared to clin-ically similar lesions in the same patients.In conformity with previous studies (10), thisdecrease was observed by 4 to 8 weeks. Che-motherapy had been instituted on day 4,i.e., after the completion of rIFN--y admin-istration. Such therapy is known to showonly a 1-log reduction of bacilli as late as12 months. Although the intracellular re-

moval and clearance of M. leprae from thedermal macrophages is hastened in manypatients, in the absence of viability studiesit is not possible for us to comment onwhether this clearance reflects microbicidalactivity or the removal of previously killedbacilli. Nor can we stringently rule out themigration of bacilli-containing cells, alter-ing the BI status of the lesions. We earlierhad reported that patients with nodular le-sions showed lower clearance of bacilli (10),and the present study had included mostlysuch patients. Further supporting this ob-servation is patient 5, who had histoid lep-rosy and failed to clear bacilli from the in-jected nodule.

Although oxygen radicals have been im-plicated in bacterial killing (16), the possi-bility of other microbicidal pathways play-ing a role in the killing of M. leprae cannotbe ruled out (12. '5), particularly since strictcorrelation was not observed between bacil-lary clearance and the levels of H-,02 or

61, 2^SivaSai, et al.: Effect of rIFNy on Lesional Macrophages^267

0-7 released by the cells of the same lesion.A stringent relationship also could not beestablished in individual cases between theinitial bacterial load of the patient, BI ofinjected sites, erythema/induration andbacillary clearance. Taken together, the ob-servations in the present study indicate thatmultiple events may be initiated by rIFN-7in the bacilli-laden granulomas of humanlepromatous leprosy. Some of these relateto direct activation events in lesional mac-rophages such as increased H-,07 and 0,-levels and clearance of intracellular M. lep-rae; whereas others, such as erythema andinduration of the skin, are more complexand may involve recruitment of other cellsand cell products. At the individual level,all of the effects are not observed concom-itantly.

Recent data on systemic administrationof rIFN--y have shown encouraging results(25). rIFN-7 may, therefore, have therapeu-tic value in the early clearance of bacillifrom the skin of lepromatous patients andmay act as an adjunct to chemotherapy, par-ticularly in drug-resistant and recalcitrantleprosy.

SUMMARYHydrogen peroxide (H,0-,) and super-

oxide anion (0i) were estimated in lesionalcells from 10 lepromatous leprosy patientsinjected intralesionally with recombinantinterferon-gamma (rIFN-7). Clinically sim-ilar contralateral lesions injected with ex-cipient served as controls. Lesional ester-ase-positive cells (suggestive of monocytes/macrophages) from rIFN-7-injected sites ofmany subjects showed net increments in theH202 and 0-7 levels compared to controls.When these cells were exposed to Myco-bacterium leprae in vitro, there was a down-regulation of C/T in 4 of 5 subjects. Suchinhibition was not observed in rIFN-7-in-jected sites. From the present studies it wasnot possible to determine whether the aboveeffects of rIFN-7 were primarily on lesionalmature macrophages or on newly migratedyoung monocytes. Erythema and indura-tion were observed at the cytokine-injectedsite but not at the control site between 24and 72 hr. A monthly slit-skin smear ex-amination of the former site showed a bac-terial index (BI) reduction compared to thecontrols in 4 of 10 patients, this reduction

occurring as early as 4 to 8 weeks. Histo-pathology of the biopsies of 6 of 10 subjectsbetween 9 and 10 months showed a furtherBI decrease attributable to rIFN--y and notto the subsequently instituted chemother-apy.

RESUMENSc estimO la cantidad de perOxido de hidrOgeno

(F120,) y del anion superOxido (0,0 en las celulaslesionales de pacientes lepromatosos inyectados intra-lesionalmente con interferon gamma recombinante(rIFN--y). Como controles Sc incluyeron las lesionescontralesionales inyectadas con excipiente. Compara-das con las celulas de las lesiones control, las celulaslesionales esterasa positivas (sugestivas de monocitos/macrOfagos) de los sitios inyectados con rIEN-y demuchos sujetos mostraron incrementos netos en susniveles de H,0, y . Cuando estas cêlulas se expu-sieron al Mycobacterium leprae in vitro, se observe, unadisminuciOn de la producciOn de 0.-T en 4 de 5 pa-cientes. Tal inhibiciOn no se observó en los sitios in-yectados con rIEN-y. No fue posible determinar silosefectos del rIFN-y fueron primariamente sobre los ma-crOfagos lesionales maduros o sobre los monocitos re-cien Ilegados a la lesion. Los sitios inyectados con ci-tocinas mostraron eritema e induraciOn 24 a 72 horasdespues. El examen mensual de los extendidos de linfacutanea del sitio inyectado con HFN-y, mostrO unareducciOn en el indice bacteriano (BI) en 4 de 10 pa-cientes; esta reducciOn ocurriO tempranamente, entrelas 4 y las 8 semanas. El estudio histopatolOgico rea-lizado entre las 9 y 10 semanas despues de la inyecciOndel rIFN--y, mostrel una disminuciOn adicional del BIen 6 de 10 pacientes. Este efecto se atribuyO al HEN-1,y no a la quimioterapia instituida subsecuentemente.

RÉSUMÉ

Le peroxyde d'hydrogene (F1,02) et l'anion supc-roxyde (0.-T) ont ete doses dans des cellules provenantdes lesions de 10 patients lepromateux chez qui onavail injectê en intralesionnel de l'interferon-gammarecombinant (IFN-yr). Des lesion cliniquement simi-laires situCes sur l'autre moitie du corps et 0 l'interieurdesquelles on avail inject& un excipient ont servi detemoins. Les cellules esterase-positives (suggestivesmonocytes/macrophages) en provenance des sites ocion avait inject& de l'IFN-yr ont montre chez beaucoupde patients de nettes augmentations des taux d'H2O,et d'Oi par rapport aux temoins. Quand ces cellulesont Ote exposêes au Mycobacterium leprae in vitro, ily cut une diminution de l'QT chez 4 des 5 patients.Une telle inhibition n'a pas ête observêe au niveau dessites oa on avait inject& de l'IFN--yr. A partir de cesanalyses, il ne fut pas possible dse determiner si leseffets de l'IFN--yr ci-dessus se rapportaient premiere-ment 0 des macrophages mars ou a des jeunes mono-cytes nouvellement migres. De l'erytheme et une in-duration ont ete observes au site d'injection de cytokine

268^ International Journal of Leprosy^ 1993

mais non au site temoin entre la vingt-quatrieme heureet la septante-deuxieme heure. Un examen mensuel defroths cutane du premier site a montre une diminutiondc l'index bacterien (IB) par rapport aux ternoins pour4 des 10 patients, cette diminution survenant de ma-Mere aussi precoce qu'entre la quatrieme et la huitiemesemaine. L'histopathologie des biopsies de 6 des 10patients entre 9 et 10 mois a montre une poursuitela reduction de 1'113 attribuable FN--yr et non a lachimiotherapie instauree ulterieurement.

Acknowledgment. This study was supported by agrant from the Indian Council for Medical Research.We thank Drs. Z. A. Cohn and G. Kaplan for helpfuldiscussions, Dr. Rosenkaimer for recombinant humaninterferon-gamma, and Dr. R. J. W. Rees for providingAl. /eprae-infected armadillo spleens through theIMMLEP component of the UNDP/World Bank/WHOSpecial Program for Research and Training in TropicalDiseases.

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