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Detection of Lymphokines and Lymphokine Receptors in Pulmonary Sarcoidosis Immunohistologic Evidence That Inflammatory Macrophages Express IL-2 Receptors

WAYNE W. HANCOCK, MD, PhD, LESTER KOBZIK, MD, AMY J. COLBY, BS, CARL J. O'HARA, MD, AMIEL G. COOPER, MD, and JOHN J. GODLESKI, MD

From the Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, New England Deaconess Hospital, Tufts-New England Medical Center Hospital, Boston, Massachusetts

In an investigation of the immunopathogenesis of sarcoidosis, the authors undertook the tissue localization of those lymphokines and lymphokine receptors which are known to play a central role in T-cell and macrophage activation. Using monoclonal antibodies and an ABC immunoperoxidase technique. They determined the distribution of gamma interferon (IFN-g), interleukin-2 (IL2), and corresponding IL-2 receptors (IL-2R), plus T cells and T cell subsets, B cells, and macrophages within thoracic lymph nodes and lung specimens of 9 patients with active pulmonary sarcoidosis. Epithelioid and multinucleate giant cells within sarcoid granulomas of all specimens showed membrane labeling for IL-2R and IFN-g, in addition to IL-2, suggesting that these cells indeed express

functional IL-2 receptors. Infiltrating T cells, largely T4+, were also EL-2R+, and many showed IL-2 and IFN-g labeling. By comparison, macrophages within sections of normal lung or lymph node failed to stain for IL-2, IL-2R, or IFN-g. These immunohistologic studies extend recent in vitro observations by these authors and others that normal human blood monocytes and alveolar macrophages are induced by IFN-g or IL-2 to express functional membrane-bound IL-2 receptors. The in vivo expression of IL-2R by mononuclear phagocytes in pulmonary sarcoidosis is demonstrated, and a new role is suggested for T-cell-derived lymphokines in macrophage activation. (Am J Pathol 1986, 123:1-8)

SARCOIDOSIS is a multisystem disease of unknown origin which in almost 900/o of patients involves formation of noncaseating granulomas within the lungs and intrathoracic lymph nodes. '2 The wide variety of immunologic abnormalities identified in patients with active pulmonary sarcoidosis was reviewed by Daniele et al.3 In peripheral blood there is a generalized lymphopenia, characterized by a marked reduction in helper

Supported by Grant HL 20062 from the National Heart, Lung and Blood Institute. Dr. Hancock is recipient of a Fogarty Award from the National Institutes of Health. Accepted for publication January 31, 1986. Address reprint requests to Wayne Hancock, MD, PhD, Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.

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T cells, a decreased response to mitogens,4 and depressed B-cell function.5 By contrast, the lungs of affected individuals are infiltrated by large numbers of T cells, particularly helper T cells,4'6 many of which show features of activation, including spontaneous division and production of the lymphokines, monocyte chemotactic factor,7 macrophage migration inhibition factor,8 interleukin-2 (IL-2),9 and B-cell-inducing factors,5 in addition to an increased response to mitogens. These findings have resulted in the concept that sarcoidosis is a disease of heightened immunologic activity at specific disease sites such as the lung, leading to lymphocyte infiltration, monocyte recruitment, and granuloma formation. In attempts to further understand the immunopathology of sarcoidosis, a number of investigators have used monoclonal antibodies to study the cellular constituents of sarcoid granulomas within sections of affected lymph nodes'0-13 and skin specimens,14-16 or to analyze the cells obtained by bronchoalveolar lavage of patients with sarcoidosis.6 "20 Almost all of these previous studies have concentrated upon identification of the lymphocyte populations involved using various cellsurface markers. In contrast, the study we now report undertook an examination of the functional characteristics of those cells which comprise or surround sarcoid. In particular, we sought to localize those lymphokines, products of immunocompetent cells which are central to the development of cell-mediated immune reactions, and lymphokine receptors, which have recently been defined using monoclonal antibodies.

Materials and Methods Patients

Tissue samples from 9 patients with the clinical diagnosis of sarcoidosis were studied. A confirmatory pathologic diagnosis was made on the basis of lung or thoracic lymph node biopsy showing characteristic noncaseating, epithelioid granulomas, and an absence of mycobacterial, fungal, or parasitic infection, or history of exposure to materials known to cause granulomatous lung diseases.

AJP

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April 1986

Tissue and Cell Preparation

Tissues were obtained as part of the diagnostic evaluation of each patient. Lymph nodes (n = 7), one open lung biopsy, and one transbronchial biopsy were quickfrozen with dry ice and isopentane, and stored at -80 C. Specimens were sectioned on a cryostat (46 i) and fixed in either acetone or freshly prepared 2%7o paraformaldehyde in 0.1 M phosphate-buffer, pH 7.2, for 5 minutes at 4 C. Control samples of normal lung (n = 4) and lymph node (n = 4) were obtained from fresh surgical specimens and were handled as described for sarcoid tissues. Cytospin smears of normal peripheral blood mononuclear cells, isolated by centrifugation over Ficoll-Hypaque (n = 3), were prepared as previously described.21 Monoclonal Antibodies A panel of monoclonal antibodies22-29 were selected on the basis of reactivity with a lymphokine or lymphokine receptor or usefulness as a relevant leukocyte cell marker. TWo anti-interleukin-2 (IL-2) monoclonal antibodies,22 DMS-1 (IgGI) and DMS-3 (IgG3), were obtained from Dr. Kendall Smith, Dartmouth Medical School, Hanover, New Hampshire; both antibodies were used as undiluted tissue culture supernatant. Anti-gamma interferon (IFN-g) monoclonal antibodies were purchased from Interferon Sciences, New Brunswick, New Jersey, and Chemicon, Los Angeles, California; the former anti-IFN-g antibody (IgGI) was used at 1:20 dilution, and the latter antibody (IgG2a) was diluted 1:50. Three antibodies to the human IL-2R were used: anti-Tac (IgG2a)23 was obtained from Dr. Thomas Waldman, NIH, Bethesda, Maryland, and diluted 1:5000; anti-IL-2R, (IgG2a)24 was purchased from Coulter, Hialeah, Florida, and diluted 1:50, and 2A3 antibody (IgGl)21 was obtained from Becton-Dickinson, Mountain View, California, and diluted 1:20. A monoclonal antibody to Dr (Ia-like) antigens was purchased from Becton-Dickinson; this IgG2a antibody, termed L243,26 was diluted 1:100. The pan-T-cell marker OKT3 (IgGl),27 the helper/inducer T-subset marker OKT4 (IgG2a),27 and the cytotoxic-suppressor

Figure 1-Immunoperoxidase staining of cryostat sections of a thoracic lymph node from a patient with pulmonary sarcoidosis. (ABC immunoperoxidase technique; hematoxylin counterstain) a-lsotype control monoclonal antibody (IgG2a) failed to bind to sections containing sarcoid granulomas (G). b-A monoclonal antibody to human IFN-g (IgG2a) bound to the cellular membranes of epithelioid cells within granulomas (G), in addition (x250) c-A monoclonal antibody to the human IL-2R (anti-Tac, IgG2a) lato adjacent isolated inflammatory macrophages (M) and lymphocytes (L). (x250) dbeled the cellular membranes of epithelioid macrophages within granulomas (G). In addition, IL-2R lymphocytes (arrows) were also stained. (x400) Higher magnification of the field from c, showing anti-Tac binding to large cells with pale, eccentric, indented nuclei within granulomas; these are transe-A monoclonal antibody to human IL-2 (DMS1, IgGl) bound formed macrophages, which have undergone maturation into epithelioid cells. (x800) Inset-A higher magnification to epithelioid and multinucleate giant cells (arrow) within granulomas, as well as to the nucleoli of all cells examined. illustrate membrane labeling of epitheloid cells and dense staining of a lymphocyte (arrow) within the sarcoid granuloma. (x400; Inset, x800)

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