T lymphocyte subsets in the skin lesions of patients with leprosy

T lymphocyte subsets in the skin lesions of patients with leprosy Robert L. Modlin, M.D., Florence M. Hofman, Ph.D., Clive R. Taylor, M.D., D.Phil., and Thomas H. Rea, M.D. Los Angeles, CA Lymphocyte subsets in the tissues of fourteen patients with leprosy were studied using monoclonal antibodies and a modified immunoperoxidase technic. Two immunohistologic patterns were observed. In tuberculoid leprosy, helper-inducer cells were present among the aggregates of mononuclear phagocytes (epithelioid cells), but the suppressor-cytotoxic cells were predominantly in the lymphocytic mantle surrounding the epithelioid cell aggregates. In reversal reaction and lepromatous tissues, the helper-inducer and the suppressor-cytotoxic cells were both distributed among the mononuclear phagocytes (histiocytes). In tuberculoid specimens the Langerhans cells of the epidermis were increased in number as compared to lepromatous and normal tissues. The technic used appears to be of value in studying some of the cellular components of the immune response in situ. (J AM ACAD DERMATOL8" 182-189, 1983.) hnmunologic mechanisms are probably important in the pathogenesis of many aspects of leprosy. For example, it is generally believed that in tuberculoid leprosy there is high host resistance, characterized by an epithelioid differentiation of the rnononuclear phagocytes, 1 rare bacilli, 1 a positive lepromin skin test, and in vitro peripheral blood lymphocyte blastogenic responses to Mycobacterium teprae. 2 By contrast, in lepromatous leprosy it is thought t h a t host resistance is diminished, resulting in an absence of epithelieid From the Section of Dermatology, Department of Medicine, and the Department of Pathology, University of Southern California Schoot of Medicine, and the Departments of Dermatology and Pathology, Los Angeles County/University of Southern California Medical Center. Supported by a gift from The Military and Hospitaller Order of St. Lazarus of Jerusalem and by a grant from the Heiser Program for Research in Leprosy. Accepted for publication Sept. 22, 1982. Reprint requests to: Dr. Thomas H. Rea, Section of Dermatology, Room 8441, Los Angeles County/University of Southern California Medical Center, t200 N. State St., Los Angeles, CA 90033.

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differentiation of mononuclear phagocytes, I abundant bacilli,1 and an absence of the delayed type of hypersensitivity to M. leprae, z In addition, it has been postulated that the "reactional states" of leprosy may be immunologically mediated, reversal reactions being examples of the delayed type of hypersensitivity responses, with gain in cellmediated immunity, a and erythema nodosum leprosum (ENL) resulting from immune complexes. 4 Attempts have been made to study the lymphocyte populations that form an integral part of the tissue reactions in leprosy. The number of lymphocytes obtainable in suspension from inflammatory infiltrates has been too small to characterize accurately by the usual surface marker technics. For this reason, efforts have been made to adapt some of the surface marker methods for use in tissue sections, as exemplified by the use, by Ridley et al, ~ of complement rosettes (EAC) and spontaneous sheep red cell rosettes (E) for B and T cells, respectively, in tissue sections. Results obtained by these methods are, however, difficult to 0190-9622/83/020182+08500.80/0 © 1983 A m Acad Dermatol

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Fig. 1. Many closely apposed tuberculoid granulomas. The suppressor-cytotoxic cells stain as small black rings located predominantly in the mantle surrounding the granulomas. (Frozen section; OKT8 counterstained with hematoxylin; ×50.) interpret and difficult to reproduce. The availability of monoclonal antibodies that have specificity for lymphocyte surface antigens provides an alternative approach to the analysis, by surface phenotype, of lymphocyte populations in situ in tissue sections. This report documents the use of an immunoperoxidase method employing monoclonal antibodies against T cell surface antigens for the demonstration in leprosy tissues of lymphocytes bearing the T helper-inducer or the T suppressorcytotoxic cell phenotype. The method described has the further advantage of permitting correlation of immunologic findings with the histologic features that distinguish the tuberculoid and lepromatous forms of leprosy. METHOD

Fourteen patients were classified according to the criteria of Ridley? Four were determined to be tuberculoid because they had a few sharply marginated anesthetic plaques clinically, and they had epithelioid cell aggregates surrounded by a mantle of lymphocytes but no bacilli histologically. Two patients were on the borderline in reversal reaction as determined by the abrupt onset of multiple edematous, erythematous plaques clinically and by epithelioid cell aggregates, edema, few lymphocytes, and few bacilli histologically. One

patient had a borderline lepromatous condition as determined by multiple poorly defined plaques clinically and by histiocytes, diffuse lymphocytes, and many bacilli histologically. Seven patients were lepromatous as determined by nodular lesions or widespread diffuse infiltrations of the skin clinically and by foamy histiocytes with few lymphocytes but multiple bacilli and bacillary aggregates (globi) histologically. Five of the lepromatous subjects had ENL as determined by erythematous, tender subcutaneous lesions arising in crops clinically and by a neutrophilic infiltrate superimposed on the lepromatous architecture histologically. Six patients were treated, but the results in these patients did not differ from those in the untreated patients. Tissue from eight biopsies of normal skin and other inflammatory processes were also studied. Tissues were rapidly frozen in liquid nitrogen after being placed in OCT compound (embedding medium for frozen tissue specimens, Lab-Tek Products, Naperville, IL; Miles Laboratories, Inc., Elkhart, IN) and were then stored at - 7 0 ° C until sectioned. Antigens were detected by a modified immunoperoxidase method. ~'7 Briefly, sections were fixed for 10 minutes in cold acetone (4° C), after which the following reagents were added in sequence: normal horse serum, a primary mouse antihuman monoclonal antibody, biotinylated horse antimouse serum, biotin-avidin complex with horseradish peroxidase linked to the avidin, the chromogenic substrate, diaminobenzidine, and cupric sulfate for darkening of the reaction product.

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Fig. 2. One large and two small tuberculoid granulomas. The cells staining positively for the suppressor-cytotoxic phenotype are seen as small dark rings. These positive cells are predominantly at the periphery of the granulomas, but a few are located centrally. (Frozen section; OKT8 counterstained with hematoxylin; x 150.)

Fig. 3. The same aggregate of three tuberculoid granulomas, as seen in Fig. 2. The cells bearing the helper-inducer phenotype, small dark rings, are distributed throughout the granuloma and show no predilection for the peripheral mantle. (Frozen section; Leu 3 with no counterstain; x 150.) After each stage, sections were washed in changes of phosphate-buffered saline solution. Control sections were stained with an antibody of irrelevant specificity or with the omission of primary antibody. The primary monoclonal antibodies used were directed against a pan T cell marker (Leu l, Becton Dickinson Con-

sumer Products, Rochelle Park, NY), a T helperinducer marker (Leu 3), a T suppressor-cytotoxic marker (Leu 2 or OKT8, Ortho Pharmaceutical Corp., Raritan, NJ), a thymocyte marker (OKT6), the Ia or HLA-Dr group-specific antigen (H4, Dr. Ron Billing, UCLA, CA), a natural killer marker (HNK 1, Drs. Abo

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z

Fig. 4. Multiple small lepromatous granulomas. The cells staining for the suppressorcytotoxic phenotype are distributed throughout the granuloma and show no predilection for the mantle, in contrast to the tuberculoid granulomas shown in Fig. 1. Staining for the helper-inducer phenotype in these granulomas yielded a similar diffuse pattern. (Frozen section; Leu 2 with no couaterstain; × 150.) and Balch), and a macrophage and monocyte marker (OKM1, Ortho). RESULTS Tuberculoid lesions Histologically the four tuberculoid tissues showed the anticipated organization of circumscribed epithelioid cell granulomas surrounded by a mantle of lymphocytes. 1 Following immunoperoxidase staining, lymphocytes bearing the pan T cell antigen were found to be present throughout the mantle and also, less densely but regularly, within the epithelioid cell aggregates. Lymphocytes expressing the T suppressor-cytotoxic phenotype were predominantly in the mantle (Figs. 1 and 2) and were found only rarely within granulomas. In contrast, lymphocytes staining for the T helper-inducer antigen were found in large numbers within the epithelioid cell aggregates and showed no predilection for the mantle (Fig. 3). There were twice the number of helper ceils as compared with suppressor cells. HNKl,positive cells were relatively few in number (less than 5% of lymphocytes), were present in small aggregates, and appeared to be confined to the mantle. Approximately half of the epithelioid cells stained with the OKM 1 antibody, and these were arranged

iii Fig. 5. Single tuberculoid granuloma with OKT6-posirive cells present in the surrounding mantle. (Frozen section; OKT6 counterstained with hematoxylin; × 150.) in clusters. Occasional lymphocytes observed within the epidermis stained for helper or suppressor antigens in approximately equal numbers. Lepromatous lesions In the seven lepromatous specimens, cells ex-

pressing the pan T, helper, or suppressor phenotypes were randomly distributed among the histiocytes, without any semblance of mantle formation (Fig. 4). The five cases with ENL lesions showed

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Fig. 6. The epidermis in a tuberculoid specimen. Langerhans cells staining for OKT6 antigen are numerous (approximately 10% to 15% of epidermal cells), and increased numbers of cell bodies and dendrites are seen as compared to normal specimens and lepromatous epidermis. The evident apposition of dendrites and adjacent Langerhans cells in tuberculoid epidermis was rarely observed in normal or lepromatous specimens. (Frozen section; OKT6 with no countersta!n; × 300.)

The Leu 1, Leu 2, and Leu 3 positively staining cells were diffusely distributed in the granulomas of the two specimens of reversal reaction, but no mantle was present. There were approximately equal numbers of helper and suppressor cells. Occasional cells expressing the helper or suppressor phenotype were observed in the epidermis. Rare HNK 1-positive ceils were present.

larly well developed in lepromatous tissues. A few OKT6-positive cells, type unknown, were found in tuberculoid granulomas (Fig. 5) but were rare in lepromatous tissues and absent in normal tissues. The OKT6-positive cells, when present in the tuberculoid lesion, showed a predilection for the periphery of the granuloma, occasionally encircling the granuloma. In the epidermis of tuberculoid specimens there was heavy staining of Langerhans cells for Ia and OKT6 antigens 8m (Fig. 6) (intraepidermal, dendritic cells staining positively for Ia and OKT6 antigens were considered to be Langerhans cells). The Ia-staining product uniformly filled the intercellular spaces below the stratum granulosum. In contrast, the OKT6 antibody appeared to stain only cellular material, i.e., the cell bodies and dendritic extensions of Langerhans cells. Compared with normal and lepromatous tissues (Fig. 7), the numbers of OKT6-positive cell bodies and associated dendrites were increased in tuberculoid lesions.

Ia and OKT6 staining

DISCUSSION

Virtually every cell in all of the granulomas bore the Ia antigen. In addition, the fibroblasts stained with Ia antibody, a phenomenon particu-

There has been much speculation that the two characteristic histologic forms of leprosy, tuberculoid and lepromatous, in some way reflect basic

a 2:1 predominance of helper cells, whereas in the two lesions without ENL the helper:suppressor ratio was 1 : I or smaller. Lymphocytes were not observed in the epidermis in cases of lepromatous leprosy. The borderline lepromatous tissue differed from simple lepromatous leprosy in that more T cells were present, together with an appreciable number (approximately 10% of lymphocytes) of cells showing reactivity with the antinatural killer cell antibody. A l l T cell subsets were intimately admixed with the mononuclear phagocytes. Reversal reaction

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Fig. 7. The epidermis in a lepromatous specimen. Langerhans cell bodies and dendrites are evident. The number of Langerhans cells staining for OKT6 antigen (approximately 5 % of epidermal cells) is similar to the number in normal skin but is iess than that seen in the tuberculoid specimens. (Frozen section; OKT6 with no counterstain; × 300.) differences in the immune response mounted by the host against the leprosy bacillus. 1 Certainly organisms abound in the Iepromatous form, presumably associated with diminished immune responsiveness, whereas bacilli may be difficult to find in the tuberculoid form, consistent with an active and effective immune response. In our study, immunohistologic technics employing antibodies against particular T lymphocyte subsets, identified by phenotype, provide striking new evidence that the immune response indeed is fundamentally different in tuberculoid and lepromatous leprosy. Immunostaining clearly revealed that the distribution of cells expressing T cell, helper cell, or suppressor cell phenotypes was remarkably different in the two major histologic forms of leprosy, although the overall number of lymphocytes present was similar. In tuberculoid leprosy, cells of the helper-inducer phenotype were present within the granulomas, distributed among the aggregated epithelioid cells, whereas cells of the suppressor-cytotoxic phenotype were predominantly restricted to the mantle region. Thus the intimate admixture of helper T cells and epithelioid histiocytes may reflect the active cooperation of these diverse cell types in promoting an effective immune response. The presence of cells with the suppressor-cyto-

toxic phenotype at the periphery of these granulomas also is intriguing, and it is tempting to speculate that the occurrence of these cells in such numbers may in some way contribute to the confinement of bacilli within circumscribed granulomas. The lepromatous and reversal reaction tissues did not show this architectural separation of T lymphocyte subsets but instead revealed cells of the helper-inducer phenotype and cells o f the suppressor-cytotoxic phenotype admixgd with the rnononuclear phagocytes. It is possible that this relatively haphazard arrangement of helper and suppressor cells might reflect the lack of an organized immune response, thus permitting florid bacillary proliferation. Similarly in reversal reactions, one consequence of the lack of a mantle of cells expressing the suppressor-cytotoxic phenotype might be the associated extensive tissue destruction characteristic of this condition. Recent studies of peripheral blood mononuclear cells in patients with leprosy have produced conflicting results concerning the distribution of M. leprae-inducib[e lymphocytes with suppressor activity. Thus Nath and Sirrgh ~° found more suppressor activity in tuberculoid than in lepromatous patients, but Mehra et al. 11 found suppressive activity primarily in borderline and lepromatous sub-

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jects. Our immunohistologie data indicate that cells with the suppressor phenotype are important in all types of leprosy, and their distinctive anatomic locations correlate with the kind of immunologic response. These results showing positive staining of Langerhans cells with OKT6 and anti-Ia antibodies are in accord with previous reports, s.9 The epidermis of tuberculoid lesions, particularly, showed a large number of Langerhans cells as compared with tissues from lepromatous patients and from normal subjects. The staining patterns of these two antibodies showed definite differences in antigen distribution; Ia antigen appeared to be shed extensively into intercellular spaces, whereas OKT6 antigen was strictly confined to the cells. In view of the finding that early tuberculoid leprosy may be primarily intraepidermal, TM it is possible that the increased numbers of Langerhans cells, associated with the presence of lymphocytes within the epidermis, may reflect the initial host response to intraepidermal bacilli. The hypothesis that Langerhans cells are able to function as macrophages and antigen-handling cells is consistent with this view.8 The observation that the majority of the epithelioid cells found in tuberculoid tissues show positive reactivity with the OKM1 (antimonocyte-macrophage antibody) further reinforces the view that these cells are derived from the mononuclear phagocytic series. The apparent lack of staining of a portion of epithelioid cells may be related to differential expression of antigen at different stages of development, perhaps analogous to the differing patterns of complement and immunoglobulin receptor expression on the maturing epithelioid cells in tuberculoid leprosy, as reported by Ridley. 12 Of considerable interest are the large numbers of lymphocytes demonstrated by immunoperoxidase-monoclonal antibody staining as compared to the numbers of lymphocytes apparent in routine paraffin sections. We think it likely that against a background of numerous mononuclear phagocytes, nonaggregated lymphocytes may be hard to see; alternatively, within granulomas, lymphocytic nuclei may not have their typical morphology.

The functional consequences of these differing immunohistologic patterns are at present still speculative. However, it is of considerable interest that two patterns have been identified, conforming to two major histologic patterns and two radically different immune states. Immunohistologic technics are particularly suited to the study of ceil phenotypes in situ in leprosy tissues. As other cell surface antigens are recognized and as other segments of the granulomatous spectrum of tissue responses in leprosy become available for study, it might be expected that new data will accumulate, enhancing our understanding of the tissue responses in leprosy. Clearly immunohistologic methods now available will not prevent speculation, nor should they, but they may lead to the development of firmer ground on which to base new hypotheses, as well as provide a means of testing them by direct observation of leprosy tissues. We thank Dr. Daniel E. Gormley for providing access to one of the patients studied. We also thank Dr. E. George Thorne, Division of Dermatology, Ortho Pharmaceutical Corp., Raritan, NJ, for providing some of the antibodies used in this study. REFERENCES 1. Ridley DS: Histological classification and the immunological spectrum of leprosy. Bull WHO 51:451-465, 1974. 2. Myrvang B, Godal T, Ridley DS, Froland SS, Song YK: Immune responses to Mycobacterium leprae and other myeobacterial antigens throughout the clinical and histopathological spectrum of leprosy. Clin Exp Immunol 14:541-553, 1973. 3. Barnetson RStC, Bjune G, Pearson JMH, Kronvall G: Cell mediated and humoral immunity in "reversal reactions." Int J Lepr 44:267-274, 1976. 4. Wemambu SNC, Turk JL, Waters MFR, Rees RJW: Erythema nodosum leprosum: A clinical manifestation of the Arthus phenomenon. Lancet 2:933-935, 1969. 5. Ridley MJ, Ridley DS, Turk JL: Surface markers on lymphocytes and cells of the mononuclear phagocytic series in skin sections in leprosy. J Pathol 125:91-98, 1978. 6. Taylor CR: Immunoperoxidase techniques: Practical and theoretical aspects. Arch Pathol Lab Med 102:113-121, 1978. 7. Hsu S-M, Raine L, Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29: 577-580, 1981. 8, Stingl G, Katz SI, Clement L, Green I, Shevach EM: Immunological functions of h-bearing epidermal Langerhans cells. J Immunol 121:2005-2012, 1978.

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9, Fithian E, Kung S, Goldstein G, Rubenfeld M, Fenoglio C, Edelson R: Reactivity of Langerhans cells with hybridoma antibody. Proc Natl Acad Sci USA 78:25412544, 1981. 10. Nath I, Singh R: The suppressive effect ofM. leprae on the in vitro proliferative responses of lymphocytes from patients with leprosy. Clin Exp Immunol 41:406-414, 1980.

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11. Mehra V, Mason LH, Rothman W, Reinherz E, Schlossman SF, Bloom BR: Delineation of a human T cell subset responsible for lepromin-induced suppression in leprosy patients. J Immunol 125:1183-1188, 1980. 12. Ridley DS: The pathogenesis of the early lesions in leprosy. J Pathol 111:191-206, 1972.