An immunogold-silver enhancement technique, which combines effective labeling of viable isolated islets with the ultrastructural resolution of cytological details, was applied in electron microscopy to identify major histocompatibility complex (MHC) structures on islet cells. Incubation of freshly isolated islets from CAP (RT1C) and LEW (RT1') rats with OX18, an MHC class I antibody, showed strong positive reactivity in macrophages and/or dendritic-like cells (M0-DCs) and vascular endothelial cells (VEs) and a comparatively weaker reactivity in endocrine a-, p-, and 8-ce"s. With MHC class" antibody OX6 (anti-I-A), M0-DCs were strongly labeled in both rat strains on the surface and on internal structures. Three of five particularly high titered batches of OX6 revealed MHC class" expression on VE and p-ce"s. Four days of in vitro culture in combination with a high concentration of glucose and interferon-'Y induced strong enhancement of MHC class I structures and, to a lesser extent, class " structures on p-ce"s

Müller-Ruchholtz, Wolfgang

Ulrichs, Karin

Von Gaudecker, Brita

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Modulation of Islet Immunogenicity Immunoelectron Microscopic Localization of MHC Structures in Isolated Pancreatic Rat Islets BRITA VON GAUDECKER, KARIN ULRICHS, AND WOLFGANG MULLER-RUCHHOLTZ An immunogold-silver enhancement technique, which combines effective labeling of viable isolated islets with the ultrastructural resolution of cytological details, was applied in electron microscopy to identify major histocompatibility complex (MHC) structures on islet cells. Incubation of freshly isolated islets from CAP (RT1C) and LEW (RT1') rats with OX18, an MHC class I antibody, showed strong positive reactivity in macrophages and/or dendritic-like cells (M0-DCs) and vascular endothelial cells (VEs) and a comparatively weaker reactivity in endocrine a-, p-, and 8-ce"s. With MHC class" antibody OX6 (anti-I-A), M0-DCs were strongly labeled in both rat strains on the surface and on internal structures. Three of five particularly high titered batches of OX6 revealed MHC class" expression on VE and p-ce"s. Four days of in vitro culture in combination with a high concentration of glucose and interferon-'Y induced strong enhancement of MHC class I structures and, to a lesser extent, class " structures on p-ce"s. Diabetes 38 (Suppl. 1 ):150-53, 1989 Whereas major histocompatibility complex (MHC) class I structures are expressed on a wide range of cell types, the expression of MHC class II structures, including pallcreatic islets, appears to be limited to bone marrow-derived cells (1). However, there is increasing evidence that class II struc-tures can also be expressed on non lymphoid cells, including islet cells under certain functional conditions (2,3). Because class II molecules are known to play an essential role in the induction of T-Iymphocyte-dependent immune responses, their detailed analysis appears to be of great importance for determining the immunogenicity of the future islet graft. From the Departments of Anatomy and Immunology. Medical School of the University of Kiel. Kiel. FRG. Address correspondence and reprint requests to Priv. Doz. Dr. Brita von Gaudecker. Department of Anatomy, University of Kiel, Olshausenstrasse 40, 0-2300 Kiel, FRG. 150 Light-microscopic (LM) studies do not allow a precise iden-tification of all islet cell types. Therefore, we applied the immunogold-silver enhancement technique (ISET), which combines effective labeling of viable pancreatic islet cells with the ultrastructural resolution of cytological details, in electron microscopy (EM) to isolated rat islets. In contrast to solid organs, the much smaller islets allowed successful immunolabeling in toto before fixation and embedding. MATERIALS AND METHODS The inbred rat strains CAP (RT1 C) and LEW (RT1') were bred at the Department of Immunology (Univ. of Kiel) and used at the age of 2-3 moo Rat islets were isolated according to the technique of Lacy and Kostianovsky (4) Only clean, hand picked islets were used. OX18 (against a monomorphic determinant of the rat MHC class I molecule; Camon, Wies-baden, FRG) and OX6 (against a monomorphic I-A deter-minant of the rat MHC class II molecule; Camon) were the primary monoclonal antibodies used. Three of five batches of OX6 showed a particularly high avidity for their determi-nant and were thus called OX6-HT. Antibodies were used in dilutions ranging from 1:40 to 1 :80 in phosphate-buffered saline (PBS). Goat anti-mouse IgG coupled to 5-nm gold particles (dilution 1:5 in PBS; Janssen Life Sciences, Bel-gium) was used as secondary antibody. Two hundred iso-lated, handpicked, viable islets were incubated at 24°C suc-cessively with 200 fLl diluted primary and secondary antibody for 40 min each. Islets were subsequently fixed in a mixture of 2% formaldehyde and 2.5% glutaraldehyde overnight. After treatment with a silver enhancemellt solution from Janssen, islets were osmicated and embedded in aral-dite. Ultrathin sections were evaluated with a Zeiss 902 electron microscope. There were two negative control groups-islets incubated with the nonspecific anti-HLA-DR antibody 1 aB3 and those incubated without the primary an-tibody. Nonspecific binding of IgG was never observed. RESULTS The EM technique resulted in very good tissue preservation. Silver enhancement of the 5-nm gold particles allowed rapid DIABETES, VOL. 38, SUPPL 1, JANUARY 1989 and precise identification of positively reacting cells , partic-ularly at lower magnifications. Ultrathin sections of freshly isolated islets showed a rather loose architecture. Groups of endocrine cells were separated by interstitial spaces con-taining numerous capillaries and some macrophage and /or dendritic-like cells (M0-DCs) . Occasionally, but very rarely , a Iymphocyte or fibrocyte was recognized. The insulin-se-"t . , It - •• ,': . , '. / c : ~ .... '. .. ' ' . ; .. . . .. ~ "'! ... I " • • B VON GAUDECKER K ULRICHS AND W MULLER-RUCHHOLTZ creting l3-cell as predominant cell type comprised - 80% and the glucagon-secreting a-cell - 20% of the total tissue. a-Cells were located mainly in groups or strands along the periphery of the islets . Single somatostatin-secreting o-cells appeared to be sandwiched between a- and l3-cells at the islet periphery. Reaction pattern with MHC class I antibody OX18. Incu-FIG. 1. Isolated LEW islets labeled in toto with immunogold-silver enhancement technique for MHC class I and class 11 expression. a: Freshly isolated islet incubated with OX6-HT; capillaries (VE) label fairly strongly for class 11 molecules. In contrast, l3-cells (8) are labeled more weakly and show signs of internalization (arrows) (x 5100). b: Islet cultured in vitro with OX18 for 4 days in presence of high concentrations of glucose and interferon-y. Class I expression is seen only at islet periphery, possibly because of condensation of endocrine cells during in vitro culture. Capillaries and macrophages and dendritic cells are missing (x 2400). c: FresHly isolated islet incubated with OX6 revealed macrophage with strong class 11 cell surface reaction and some positive Cy10plasmic vacuoles (x 7900). d: Freshly isolated islet incubated with OX18; a-cell (A) and Ii-cell (D) are weakly labeled for class I molecules ; adjacent l3-cell remains unstained. Note different morphologies of secretion granules of a- and Ii-cells (x 7900). e: Freshly isolated islet incubated with high-titer OX6. I3-Cells show dotlike cell surface reactivity for class 11 molecules. Notice specific l3-cell secretion granules with homogeneous round granule core surrounded by a distinct electron-lucent space ( x 8800). DIABETES, VOL. 38, SUPPL. 1, JANUARY 1989 151 IMMUNOELECTRON MICROSCOPY OF ISLETS bation of CAP and LEW islets with the OX18 antibody showed strong positive cell surface reactivity in M~-DCs and vascular endothelial cells (VEs). The endocrine cells were labeled comparatively more weakly for MHC class I molecules (0.-cells> r3-cells = o-cells). a-Cell secretion granules (~300 nm diam) were characterized by a clearly demarcated, spherical, extremely electron-opaque core and a small elec-tron-lucent space separating the core from the limiting mem-brane (Fig. 1d). r3-Cell secretion granules (~300 nm diam) lacked the paracrystalline appearance that is typical for hu-man r3-cells. The homogeneous granule core appeared to be round and separated from the limiting membrane by a distinct electron-lucent space (Fig. 1, a and e) Numerous r3-cells contained labeled antibody within cytoplasmic ves-icles near the cell surface, indicating internalization during incubation with the secondary, gold-coupled antibody (Fig. 1a). o-Cells labeled rather weakly with OX18 (Fig. 1d) and very rarely showed internalization. They were characterized by a uniform population of secretory granules homogene-ously filled with a moderately electron-opaque mass. Their granules tended to be slightly smaller than 0.- or r3-cell gran-ules. Reaction pattern with MHC class 11 antibodies OX6 and OX6-HT. Incubation of CAP and LEW islets with OX6 and OX6-HT confirmed and extended our earlier findings in im-munofluorescence LM (3). The commercially available OX6 antibody, directed against the I-A determinant of the MHC class II molecule, labeled only M~-DCs and occasionally a Iymphocyte in both rat strains (Fig. 1 c). Most of these cells had the ultrastructural appearance of macrophages with phagocytic-inclusion bodies of different sizes and density. The macrophages showed a vivid internalization of positively stained membrane fractions, more clearly identifiable in preparations without silver enhancement. At higher magni-fication it became obvious that the small gold particles were located at the cell surface and also in cytoplasmic vacuoles of varying sizes. This internalization was probably due to the remaining viability of the cells during the incubation period with the gold-coupled secondary antibody. Occasionally, but very rarely, this type of cell resembled nonphagocytic DCs, which lack phagosomes. However, OX6-HTs (i.e., those batches with higher avidity for their determinant) additionally reacted strongly with islet capillaries (Fig. 1 a). Moreover, ISET with this highly reactive antibody clearly revealed a dotlike, weak, and cell surface-restricted MHC class II expression on r3-cells as well (Fig. 1 e). a-Cells and o-cells TABLE 1 were negative. Although the general reaction pattern ap-peared to be similar in both rat strains, the intensity of reac-tivity differed by allotype (CAP> LEW; data not shown). Ultrastructure and MHC expression of islets after in vitro culture with high concentration of glucose and inter-feron-y (IFN-y). It is widely believed that in vitro culture reduces islet immunogenicity and in particular the number of bone marrow-derived cells (5). To evaluate this phenom-enon ultrastructurally, EM studies with cultured CAP and LEW islets were performed with the OX18, OX6, and OX6-HT antibodies. Islets were stimulated during the 4 days of culture with 350 mg/dl glucose and 1.200 U/mIIFN-')'. Most of the cultured islets were composed of viable 0.-, r3-, and o-cells still containing their secretion granules. Twenty-four hours after in vitro culture, the VE apparently degenerated and had completely disappeared after 4 days. M~-DCs were also missing in viable islets. With OX18, more r3-cells than a-cells of both rat strains showed increased labeling of all membrane fragments that face the islet periphery (Fig. 1 b). Cells within the islet center remained unstained, obviously because of tissue condensation, and thus reduced antibody diffusion. In cultured islets, none of the cell types stained with OX6; however, OX6-HT reacted with the peripherally located membrane fragments of the r3-cells more strongly than in uncultured controls (data not shown). DISCUSSION The excellent contrast of the ISET contributes to the detailed ultrastructural analysis of observations made by indirect im-munofluorescence/immunoperoxidase LM (1). Earlier at-tempts with isolated islets and immunoperoxidase EM, lim-ited by nonspecific peroxidase staining, had led to less precise results (6). Although LM with viable total islets proved to be superior to frozen-tissue-section LM insofar as the total number of MHC class II-positive cells could be counted rapidly (3,7), the exact binding site of the antibody and the ultrastructure of the positively reacting cell type (e.g., en-docrine cell) remained unclear despite double staining with cell-type-specific antibodies. The ISET proves to be of great help in overcoming this methodological limitation, which has also been observed by others (2). However, there is one slight disadvantage when incubating a tissue in toto: the comparatively large gold-coupled secondary antibody may not reach its target and thus fail to stain if interstitial spaces decrease, e.g., during in vitro culture (Fig. 1b). Major histocompatibility complex reaction pattern of freshly isolated and cultured rat islet cells identified by immunogold-silver enhancement electron microscopy Labeling pattern by cell type Islet condition Antibody MW-DC VE Freshly isolated OX18 +++ +++ ++ + + OX6 ++++ OX6-HT ++++ +++ + Cultured (glucose + inferferon) OX18 Not identified Degenerated ++ +++ OX6 Not identified Degenerated OX6-HT Not identified Degenerated ++ Labeling pattern: + + + +, very strong: + + +, strong, + +, weak; +, very weak; -, negative. MW-DC, macrophages and dendritic-like cells; VE, vascular endothelial cells; OX6-Hl high-titered batch of OX6. 152 DIABETES, VOL. 38, SUPPL. 1, JANUARY 1989 The loss of M~-DCs and VE in cultured islets confirms comparable data of other authors on rat and mouse islets (1,6) Thus, in vitro culture can be taken as a valuable means of reducing the islet immunogenicity provided by these two cell types. However, in vitro culture with IFN-,)" a substance known to stimulate MHC molecule expression, increases class I (Fig, 1 b) and to a lesser extent class II molecule expression on l3-cells (Table 1; EM data have been con-firmed by immunofluorescence LM in unpublished obser-vations). The latter observation, which is being further in-vestigated and quantified with both techniques, LM and EM (ISET), is in some concordance with recent reports from other laboratories (9-11). According to those reports, IFN-')' in-duces rather than increases class II expression. Both modes of MHC expression could be part of a mechanism controlling the induction of autoimmunity and/or allograft rejection and thus should be further elucidated. ACKNOWLEDGMENTS We thank M. Bartels, L. Hoffmann, and M. v. Kolszynski for skillful technical assistance. This work was supported by Deutsche Forschungsge-meinschaft Grants SFB 111 /CN11 and SFB 111 /B 12-B. DIABETES, VOL. 38, SUPPL 1, JANUARY 1989 B VON GAUDECKER, K ULRICHS AND W MULLER-RUCHHOLTZ REFERENCES 1 Faustman D, Hauptfeld v, Davie JM, Lacy PE Murine pancreatic [3-cells express H-2K and H-2D but not la antigens. J Exp Med 1511563-68, 1980 2 Halloran PF, Wadgymar A, Autenried P The regulation of expression of major histocompatibility complex products. Transplantation 41413-20, 1986 3 Ulrichs K, Muller-Ruchholtz W: MHC class 11 antigen expression on the various cells of normal and activated isolated pancreatic islets Diag Immuno/347-55, 1985 4. Lacy PE, Kostianovsky M Method for the isolation of intact islets of Lan-gerhans from the rat pancreas. Diabetes 1635-39, 1967 5 Lafferty KJ, Prowse SJ, Simeonovic CJ, Warren HS Immunobiology of tissue transplantation: a return to the passenger leukocyte concept. Annu Rev Immuno/l:143-73, 1983 6. Rabinovitch A, AleJandro R, Noel J, Brunschwig JP, Ryan US Tissue culture reduces la antigen-bearing cells in rat islets and prolongs islet allograft survival. Diabetes 31 (Suppl. 4)48-54, 1982 7 Gebel HM, Yasunami Y, Diekgraefe B, Davie JM, Lacy PE la-bearing cells within isolated canine islets. Transplantation 36346-48, 1983 8. Parr EL, Bowen KM, Lafferty KJ Cellular changes in cultured rnouse thyroid glands and islets of Langerhans. Transplantation 30 135-41, 1980 9. Wright JR Jr, Lacy PE, Unanue ER, Muszynski C, Hauptfeld V Interferon-mediated induction of la antigen expression on isolated murine whole islets and dispersed islet cells. Diabetes 351174-77, 1986 10. PUJol-Borrell R, Todd I, Doshi M, Bottazzo GF, Sutton R, Gray D, Adolf GR, Feldmann M HLA class 11 induction in human islet cells by interferon-'I plus tumor necrosis factor or Iymphotoxin. NaturF; (Lond) 326304-306, 1987 11 Walker R, Cooke A, Bone AJ, Dean BM, van der Meide P, Ba'lrd JD Induction of class 11 MHC antigens in vitro on pancreatic B cells from BBI E rats. Diabetologia 29 749-51, 1986 153 

Immunoelectron microscopic localization of MHC structures in isolated pancreatic rat islets

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Immunoelectron microscopic localization of MHC structures in isolated pancreatic rat islets

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