INTERNATIONAL JOURNAL OF LEPROSY^ Volume 54, Number 2

Printed in the U.S.A.

In vitro Studies on Dermal Granulomas ofHuman Leprosy: Characterization of Cells

Using Monoclonal Antibodies'R. B. Narayanan, Bhawneshwar K. Girdhar,Ravinder K. Lavania, and Utpal Sengupta 2

Leprosy is a chronic granulomatous dis-ease caused by Mycobacterium leprac. Thelesions in tuberculoid leprosy are charac-terized by an epithelioid cell granuloma withabundant lymphocytes forming dense col-lections around the epithelioid cells. On theother hand, lepromatous leprosy is char-acterized by a granuloma composed of sheetsof macrophages loaded with M. leprae alongwith plasma cells and a few lymphocytesdiffusely distributed in the granuloma. Mostof the studies made to understand the im-munological mechanisms in leprosy havebeen mainly carried out using in vitro testson peripheral blood-derived lymphocytesand monocytes ('). Monoclonal antibodiesagainst cell-surface antigens have been usedto delineate the immunopathological mech-anisms involved in the granulomas of lep-rosy (4, 5, 9, 10 , .) A better understanding of theimmunological mechanisms involved ingranuloma formation could be made by iso-lating the cells from these granulomas.

Recently, we have standardized a methodfor obtaining single cell suspensions fromleprosy granulomas. The suspensions con-tained lymphocytes and "large cells." Thenumbers of lymphocytes were higher in thesuspensions of tuberculoid granulomas incomparison to those in suspensions of le-promatous granulomas. A higher percent-age of lymphocytes from the tuberculoidgranulomas formed rosettes with sheeperythrocytes and also showed the presenceof esterase dots in the cytoplasm. However,they did not form rosettes with antibody

' Received for publication on 22 August 1985; ac-cepted for publication on 20 November 1985.

R. B. Narayanan, Ph.D., Research Officer; B. K.Girdhar, M.D., Assistant Director; R. K. Lavania, M.D.,Research Officer; U. Sengupta, Ph.D., Assistant Di-rector, Central JALMA Institute for Leprosy, Taj Ganj,Agra 282001, India.

and complement-coated erythrocytes (EAC).Most of the "large cells" from both of thegranulomas were esterase positive, exhib-ited peroxidase activity, and did not carryreceptors for the C3 component of comple-ment (6).

In this study, monoclonal antibodiesagainst T-cell subsets and Ia-like antigenshave been used for further characterizationof the cells in the suspensions from leprosygranulomas.

MATERIALS AND METHODSRPM! 1640 (GIBCO Laboratories, U.K.)

was used containing 100 pg/m1 of strepto-mycin and 100 U of penicillin (both fromIndian Drugs and Pharmaceuticals Ltd.) perml. Collagenase type I (from Clostridiumhistolyticum) was obtained from SigmaChemical Company, St. Louis, Missouri,U.S.A.

Monoclonal antibodies. The followingmonoclonal antibodies were obtained fromOrtho Pharmaceutical Corporation, U.S.A.:OKT11 (a pan T-cell marker), OKT8 (sup-pressor/cytotoxic T marker), OKIa (recog-nizing activated T cells, macrophages, andB cells). Leu3a (recognizing inducer/helperT cells) was obtained from Becton Dickin-son Monoclonal Center, U.S.A. Fluorescein(FITC) conjugated sheep anti-mouse IgF(ab) 2 was obtained from New England Nu-clear, Boston, Massachusetts, U.S.A.).Fluorescein-conjugated rabbit anti-humanIgM (recognizing B cells) was from Dako-patts A/S, Denmark.

Skin biopsies. Skin biopsies (15 X 5 mmin size) were taken from 21 untreated lep-rosy patients attending the outpatient clinicof the Central JALMA Institute for Leprosy,Agra, India. Each biopsy was bisected onremoval; one half was fixed in Formol-Zenker's fluid and processed for histology

268

54, 2^Narayanan, et al.: Cells in Dermal Granulomas^269

THE TABLE. Characteristics of the cells in the suspensions from leprosy granulomas.

Percentage positive cells'

Tuberculoid leprosy Lepromatous leprosy

Epithelioid cell granuloma(N= 11)

Macrophage granuloma(N= 10)

LymphocytesNo. viable cells/biopsy 0.90 (±0.14) x 106 0.30 (±0.08) x 106

OKT I I 69.9^± 3.65 <2Leu3a 61.5^± 4.12 <2OKT8 26.7^± 3.70 <2OKIa 72.7^± 7.5B cells <2 <2

Ratio Leu3a+ :OKT8+ cells 2.79^± 0.61 —

MacrophagesNo. viable cells/biopsy 0.39 (±0.05) x 106 0.37 (±0.09) x 10 6

la 90-100 90-100

Values are mean ± S.E.M. of percentage of positive-staining cells unless otherwise noted.

by paraffin-embedded blocks; the other halfwas used for obtaining cell suspensions. Thebiopsies were graded histologically accord-ing to the criteria of Ridley and Jopling ( 8).Patients classified as BT and TT weregrouped as tuberculoid, and those classifiedas BL and LL were grouped as lepromatouscases.

Preparation of single cell suspensionsfrom the granulomas. Pilot experimentswere done by incubating the biopsies a) withdifferent concentrations of collagenase andb) for different intervals of time. From theseexperiments, the optimal conditions for theisolation of cells from the biopsies werestandardized.

The biopsies were collected in RPMI 1640containing antibiotics, and cleaned free offat and connective tissues. They were thensliced into small pieces, suspended in 1 mlof RPMI 1640, and incubated with 1 mg ofcollagenase for 4 hr at 37°C, with intermit-tent shaking every 1 hr. The supernatantswere collected and centrifuged at 200 x gfor 10 min. The pellet was then washed oncewith medium and resuspended. The num-ber of viable lymphocytes and viable "largecells" were quantitated using the trypan blueexclusion (0.2% w/v phosphate buffered sa-line) test in a hemocytometer.

Immunofluorescence. A smear of the cellsuspension (25 Al) was made and fixed inacetone-chloroform (1:1) for 20 min. Thesmears were layered with 25 pl of a 1:20dilution of the monoclonal antibodies at

room temperature for 45 min. Smears lay-ered with phosphate buffered saline (PBS)served as controls. Subsequently, the smearswere washed in 0.85% w/v saline for 10 min.They were then incubated with 25 Ail of a1:80 dilution of fluorescein (FITC)-conju-gated sheep anti-mouse Ig F(ab), for 30 min,and washed in 0.85% saline for 15 min. Thesmears were mounted in 90% glycerol-PBSand viewed by epi-illumination using anHBO 50 mercury lamp and a Leitz invertedmicroscope. B cells were defined by directimmunofluorescent staining with FITC-conjugated rabbit anti-human IgM.

Smears of Ficoll-Hypaque-purifiedmononuclear cells from the peripheral bloodof normal individuals and skin lesions fromleprosy patients were used as controls to testthe efficacy of the monoclonal antibodies.

The nature of the cells in suspensionshowing positive staining was based onmorphological assessment in a) smearsstained by immunofluorescence and b) par-allel smears stained with Geimsa stain.Quantitation of the positive cells was madeand expressed as a percentage. A minimumof 100-200 cells were counted (particularlyin the suspensions of tuberculoid granu-lomas), depending on the concentration ofthe cells.

RESULTSSkin biopsies from 21 untreated leprosy

patients were studied-11 tuberculoid (BT/

270^ International Journal of Leprosy^ 1986

TT) and 10 lepromatous (BL/LL) leprosycases confirmed histologically.

Preliminary experiments were carried outon the cryostat sections of leprosy lesionsto assess the optimal dilution of antibodiesrequired for staining. The antibodies werealso used by indirect immunofluorescenceon peripheral blood mononuclear cells.OKT11, Leu3a, and OKT8 antibodiesstained 70-80%, 40-50%, 15-20% of pe-ripheral blood mononuclear cells, respec-tively.

Characteristics of cells in the suspensionsfrom the granulomas. Single cell suspen-sions prepared from the dermal granulomasshowed the presence of lymphocytes andmacrophages. The numbers of lymphocyteswere significantly higher in the granulomasof tuberculoid lesions in comparison to thosein the granulomas of lepromatous lesions.The viability of the cells in the suspensionswas 70-80%.

Lymphocytes. The Table shows that 70%of the lymphocytes in tuberculoid granu-lomas expressed OKT11 and Ia-like anti-gens. At the same time, 61% of the lym-phocytes were Leu3a+, while only 27%expressed OKT8 antigens. The ratio ofLeu3a + :OKT8 + cells in the tuberculoid le-sions was 2.79 ± 0.61. In contrast, the le-promatous granulomas contained few pos-itive lymphocytes (<2%) expressing OKT1 1and Leu3a or OKT8 antigens. OccasionalB cells (<2%) were noticed in both types oflesions.

Thus, it would appear that the predom-inant lymphocytes in the leprosy lesion areactivated T lymphocytes expressing Ia-likeantigens. Leu3a+ (helper/inducer) cells ap-pear to be in higher proportions in the tu-berculoid lesions.

Macrophages. Macrophages from theleprosy granulomas had a very granular ap-pearance by light microscopy. It is interest-ing that most of the macrophages from bothtypes of granulomas expressed Ia-like an-tigens.

DISCUSSIONThere are two main advantages of char-

acterization of cells in single cell suspen-sions over in situ studies for understandingthe immunopathological mechanisms in-volved in the granuloma formation: a) It is

easier to characterize and quantitate lym-phocytes and macrophages in the granu-lomas, and b) it can be used as an experi-mental system for functional studies of thelymphocytes and macrophages infiltratingthe granulomas.

We have recently shown that it is possibleto prepare single cell suspensions from thedermal granulomas. The single cell suspen-sion from a tuberculoid granuloma containsa significantly higher number of lympho-cytes in comparison to a single cell suspen-sion from a lepromatous granuloma. A highpercentage of lymphocytes from the tuber-culoid granuloma formed rosettes with sheeperythrocytes. In addition, these cells exhib-ited esterase dots in the cytoplasm which isknown to be a marker of T cells. Most mac-rophages from both granulomas were ester-ase positive and exhibited peroxidase activ-ity (6).

In the present study, we have used mono-clonal antibodies for further characteriza-tion of these cells in suspension. A high per-centage of lymphocytes in suspensions fromtuberculoid granulomas expressed OKT11and Ia-like antigens. The proportion ofLeu3a+ cells was greater in comparison toOKT8 + cells. The ratio of Leu3a + :OKT8+cells was also higher in these granulomas.By contrast, lepromatous granulomas con-tained only a few positive lymphocytes ex-pressing OKT11 or Leu3a or OKT8 anti-gens. The ratio of Leu3a+ : OKT8 + cellscannot be enumerated accurately in thesegranulomas because the patients were un-treated and, as such, the lymphocytes in thegranulomas were very low in number. Theseresults show a good correlation with the insitu characteristics of cells infiltrating theleprosy lesions as reported by us ( 5) and byother workers ( 4. '"). A similar ratio ofLeu3a+ : OKT8 + cells has been recordedin other conditions where epithelioid cellgranulomas are prevalent ( 3 • 4 ).

Most of the macrophages from both typesof granulomas were granular in appearanceand expressed Ia-like antigens. Recent stud-ies suggest that the antigen-presenting ca-pacity of macrophages is related to theexpression of Ia-like antigens on their sur-face ("). It is interesting that the macro-phages from the suspensions of lepromatousgranulomas express Ia-like antigens and

54, 2^Narayanan, et al.: Cells in Dermal Granulomas^271

may, therefore, possess the ability to presentantigen. Incidentally, a high proportion ofmacrophages from lepromatous granu-lomas were found to be adherent to a plasticsurface (6). Adherent cells have been shownto be involved in antigen presentation ( 2 ).An analogous finding of similar Ia expres-sion has been noted in macrophages isolatedfrom vigorous and immunomodulatedschistosome granulomas in mice ( 1 ). This,therefore, raises the question of why thereis a paucity of T lymphocytes or a distur-bance in lymphocyte traffic in the lepro-matous granulomas. Further studies areaimed at delineating the mechanisms oflymphocyte traffic in these granulomas.

It appears from the present study that itis possible to obtain the constituent cells ofthe granulomas in different types of leprosyas single cell suspensions and to characterizethem. This should help in further under-standing the immunological mechanisms ofgranuloma formation in leprosy.

SUMMARYSingle cell suspensions from granulomas

of leprosy cases were prepared to enable anin vitro study on the characteristics of infil-trating cells. In all, biopsies from 21 un-treated cases of tuberculoid leprosy and le-promatous leprosy were analyzed. Thegranulomas were found to contain lympho-cytes and macrophages. The numbers oflymphocytes were higher in the suspensionsof tuberculoid granulomas in comparison tolepromatous granulomas. A high percentageof lymphocytes from tuberculoid granu-lomas expressed OKT11 and Ia-like anti-gens, thereby indicating the presence of ac-tivated T cells. The proportion of Leu3a+cells was greater in comparison to OKT8+cells in these granulomas. In lepromatousgranulomas, only a few positive lympho-cytes expressing OKT II or OKT8 antigenswere observed. The ratio of Leu3a+:OKT8+ cells (2.79 ± 0.61) was higher inthe tuberculoid granulomas than in the le-promatous granulomas. Most macrophagesfrom both types of granulomas expressedIa-like antigens.

RESUMENSe prepararon suspensiones de celulas aisladas a par-

tir de granulomas de casos de lepra para estudiar in

vitro las caracteristicas de las celulas infiltrantes. Scanalizaron las biopsias de 21 casos de lepra tubercu-loide o lepromatosa sin tratamiento. Se encontrO quelos granulomas contenian linfocitos y macrOfagos. LosnUmeros de linfocitos fucron mayores en los granu-lomas tuberculoides que en los lepromatosos. Un altoporcentaje de los linfocitos de los granulomas tuber-culoides expresaron antigenos OKT11 y antigenos Ia,indicativos del estado activado de los linfocitos T. LaproporciOn de celulas Leu 3a+ fue mayor que la decelulas OKT8 + en estos granulomas. En los granu-lomas lepromatosos solo se observaron unos cuantoslinfocitos OKT II+ y OKT8 +. La relaciOn de celulasLeu3a+ : OKT8+ (2.79 + 0.61) fue mayor en losgranulomas tuberculoides que en los lepromatosos. Lamayoria de los macrOfagos de ambos tipos de granu-lomas expresaron antigenos Ia.

RÉSUMÉ

On a prepare des suspensions de cellules isolees re-ceuillies dans des granulomes de lepre, en vue de menerune etude in vitro sur les caracteristiques des cellulesd'infiltration. Au total, on a analyse les biopsies re-coltees chez 21 malades atteints de lepre tuberculdideou lepromateuse, non traites. On a observe que lesgranulomes contenaient des lymphocytes et des mac-rophages. Le nombre de lymphocytes etait plus elevêdans les suspensions de granulomes tuberculoides quedans les granulomes lepromateux. Une proportion ele-vee de lymphocytes obtenus a partir de granulomestuberculoides ont exprimê des antigenes OKT1 1 et dutype Ia. Ceci indiquc la presence dc cellules T activees.Dans ces granulomes, la proportion de cellules Leu3a+etait plus &levee que cello de cellules OKT8+. Dansles granulomes lepromateux, par contre, un petit nombreseulement de lymphocytes positifs ont exprime les anti-genes OKT11 ou OKT8. Le rapport des cellulesLeu3a+/OKT8+ etait plus Cleve dans les granulomestuberculoides que dans les granulomes lêpromateux(2.79 ± 0.61). La plupart des macrophages provenantde l'une ou l'autre espece de granulomes exprimaientdes antigenes du type Ia.

Acknowledgments. This work was supported byLEPRA. The technical assistance of Shri V. M. Sobti,Mrs. Pritpal Kaur, Shri P. N. Sharma and Mrs. Sewak,and the secretarial assistance of Shri J. S. Rawat isgratefully acknowledged.

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