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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8595-8600, August 1996 Medical Sciences

Insulin-secreting non-islet cells are resistant to autoimmune destruction (transgenic models//intermediate pituitary/ gene expression) MYRA A. LIPES*, ERIC M. COOPER, ROBERT SKELLY, CHRISTOPHER J. RHODES, EDWARD BOSCHETrI, GORDON C. WEIR, AND ALBERTO M. DAVALLIt Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, MA 02215 Communicated by Roger H. Unger, The University of Texas Southwestern Medical Center, Dallas, IX, April 2, 1996 (received for review December 11, 1995)

ABSTRACT Transgenic nonobese diabetic mice were cre- niques, to POMC-expressing pituitary cells in NOD mice. We ated in which insulin expression was targeted to proopiomela- demonstrate that the transgenic intermediate lobe pituitary cells nocortin-expressing pituitary cells. - efficiently process and secrete mature insulin via a regulated expressing intermediate lobe pituitary cells efficiently secrete secretory pathway and yet, unlike insulin-producing 13 cells, they fully processed, mature insulin via a regulated secretory path- are resistant to immune-mediated destruction. In view of these way, similar to islet .8 cells. However, in contrast to the insulin- immunological and biochemical features, the feasibility of using producing islet (3 cells, the insulin-producing intermediate lobe these cells as an insulin gene delivery system in IDDM was tested. pituitaries are not targeted or destroyed by cells of the immune The methodology used may provide a novel means of identifying system. Transplantation of the transgenic intermediate lobe other B3-cell autoantigens targeted by the autoimmune cascade. tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic METHODS symptoms. The absence of autoimmunity in intermediate lobe Construction of the Transgene. The POMC-Ins transgene pituitary cells engineered to secrete bona fide insulin raises the consisted of the POMC promoter region linked to the structural potential ofthese cell types for (3-cell replacement therapy for the region of the mouse preproinsulin II (Ins) gene (see Fig. 1A). To treatment of insulin-dependent diabetes mellitus. excise the 5' regulatory region of the Ins gene and yet preserve the translation initiation start site at position 1132, a novel Insulin-dependent diabetes mellitus (IDDM) in humans and in HindlIl restriction site was created at position 985 by site-directed nonobese diabetic (NOD) mice is an immune-mediated dis- mutagenesis using the recombination polymerase chain reaction order in which mononuclear cells invade the (PCR) technique (12). A 2.4-kb genomic BamHI Ins fragment of Langerhans (insulitis), resulting in the selective destruction (13) was cloned into pBluescript (pBS, Stratagene). The recom- of the insulin-secreting pancreatic 13 cells, while sparing other binant Ins-pBS vector was linearized in two separate restriction islet cell types (1). Because the clinical applicability of pan- enzyme reactions with BalI (position 846) and PfiMI creatic islet transplantation is severely limited by the scarce (position 1237). These templates were then amplified in two supply of suitable donor islet tissue, poor islet viability, and separate PCR reactions using primer 3, 5'-CAATCAAAAAGCT- aggressive recurrence of autoimmunity in transplanted islet TCAGCAAGCAGGAAGGTAC-3' (corresponding to sense nu- grafts (2), efforts have been made to engineer non-islet cells cleotides 977-1008, mutagenesis sites are underlined and the for insulin gene delivery in IDDM. region of complementarity to primer 3 is in boldface type), and Hepatocytes and the tumor cell line, AtT20, primer 2, 5'-TCG TGT AGA TAA CTA CGA TAC G-3' have been studied. Proinsulin processing to insulin is extremely (corresponding to nucleotides 2050-2071 of pBS). The PfiMI inefficient in hepatocytes (3, 4), which have only a constitutive template was amplified with primer 5'-GCTGAAGCTTTTT- pathway of secretion. In contrast, AtT20 cells also have a 1, regulated secretory pathway, with characteristic secretory gran- GATTGTAGCGGATCACTTAG-3' (corresponding to anti- ules containing the prohormone endopeptidases PC2 (5), PC1/ sense nucleotides 994-962, mutagenesis sites are underlined and PC3 (6), and carboxypeptidase H (7) that normally convert the the region of complementarity to primer 1 is in boldface type), prohormone proopiomelanocortin (POMC) to corticotropin and primer 4 (the entire primer 4 was complementary to primer (ACTH) and other . When transfected into AtT20 cells, 2). The PCR products were mixed together and cotransfected into proinsulin is processed to mature insulin, identical in structure to bacteria. The BalI/PfiMI fragment of a plasmid containing the native (i.e., 1-cell derived) insulin (8, 9). A major limitation of Hindlll mutation was then ligated into Ins-pBS that had not using transfected anterior pituitary cells for insulin gene delivery undergone PCR amplification. DNA sequencing of the PCR- is that their major endogenous secretory product is ACrH, and amplified HindIII/PfiMI region did not reveal any cloning arti- thus implantation ofthese cells into diabetic recipients resulted in facts or polymerase errors. a severe Cushings-like hypercortisolemic syndrome (10). In ad- By successive ligations, the HindIlI Ins structural gene and the dition, like many transformed 13-cell lines, AtT20 cells have an POMC promoter [position -703 bp to position +61 bp (14)] were active constitutive pathway, with proinsulin comprising up to 25% subcloned into pBS. This promoter region has previously been of the secreted immunoreactive insulin (11). shown to confer cell-specific expression and correct hormonal Whereas the advantages of various candidate cell lines have regulation in anterior and intermediate lobe cells of transgenic been compared in terms of proinsulin processing and other mice (14, 15). characteristics, their ability to survive the autoimmune attack that destroys 13 cells has not been evaluated. In the present study, Abbreviations: IDDM, insulin-dependent diabetes mellitus; NOD, insulin expression was targeted, using transgenic mouse tech- nonobese diabetic; POMC, proopiomelanocortin; ACTH, cortico- tropin; Ins, preproinsulin II; BG, blood . *To whom reprint requests should be addressed at: Research Division, The publication costs of this article were defrayed in part by page charge Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. payment. This article must therefore be hereby marked "advertisement" in tPresent address: San Raffaele Scientific Institute, Via Olgettina 58, accordance with 18 U.S.C. §1734 solely to indicate this fact. Milan 1-20132, Italy. 8595 Downloaded by guest on September 28, 2021 8596 Medical Sciences: Lipes et al. Proc. Natl. Acad. Sci. USA 93 (1996) Generation of the NOD Transgenic Mice. The POMC-Ins intermediate lobe pituitary tissue was digested in M-199 contain- fusion gene cassette was excised from pBS by digestion with ing 1 mg/ml collagenase and 0.5 mg/ml type IV hyaluronidase at XhoI/EcoRV (see Fig. 1A). The cassette was purified for micro- 37°C. Pituitary cell clusters >10-20 ,M were hand-picked, injection and was microinjected directly into the pronuclei of 1-cell washed, and placed in M-199 with 10% fetal bovine serum and embryos of NOD mice (16). Founders were identified by PCR and Pen/Strep at 37C. After 72 hr, the primary pituitary cell cultures Southern blot analysis of tail DNA. One transgenic NOD line were washed with -free RPMI 1640 medium and (POMC-Insl) was studied in detail and is described here. radiolabeled with 0.4 mCi (1 Ci = 37 GBq) of L-[35S]methionine RNA Blot (Northern) Analysis. Total cellular RNA was iso- for 15 hr in the same medium containing 10 mM L- and lated with RNAzol (Biotecx Laboratories, Houston) and North- 10% fetal bovine serum. After removal ofthe radiolabeled media, ern blot analysis was performed as described previously (16). The the cells were washed with 2.0 ml of modified Krebs-Ringer blots were hybridized sequentially with 32P-labeled insulin and buffer containing 20 mM Hepes, 0.1% bovine serum actin probes. albumin, and 11 mM glucose, and incubated for 30 min in 500 ,ul Immunohistochemistry. For paraffin sections, whole pituitar- of the same medium at 37°C. The medium was removed and the ies were fixed in 10% buffered formalin at 4°C overnight. Sections cells were incubated for 90 min first in the same medium ("basal") (4-,um thick) were stained with hematoxylin and eosin and and then in the same medium containing 1 mM forskolin and 1 immunostained with anti-human ACTH-(1-39) (1:2, mM 2-isobutyl-1-methylxanthine ("stimulated"). At the end of Biomeda, Foster City, CA) and guinea pig anti-insulin (1:200, each incubation period, the medium was removed and placed at Incstar, Stillwater, MN), with goat anti-guinea pig (1:200, Linco -20°C pending analysis. Research Immunoassay, St. Charles, MO) as a secondary anti- Pancreatic islets (n = 100), isolated from Sprague-Dawley rats, body, followed by rabbit PAP (1:200, Dako). For plastic sections, were radiolabeled for 15 hr in methionine-free RPMI 1640 pituitary and islet grafts were fixed overnight in Bouins' fixative, medium containing 11 mM glucose, 2 mM leucine, 10% fetal then washed and stored in 10% buffered formalin until they were bovine sera, and 0.25 mCi [35S]methionine. After this incubation embedded in plastic (Araldite; Ernest F. Fullam, Latham, NY). period, the medium was removed and the islets were placed in 1 Sections (1-,um thick) were affixed to glass by heat; the plastic ml modified Krebs-Ringer bicarbonate buffer containing 2 mM resin was removed by sodium methoxide. Immunoperoxidase methionine, 2.8 mM glucose, 20 mM Hepes, and 0.1% BSA. staining was performed with 1:200 guinea pig anti-human insulin After a 90-min preincubation period at 37°C, the medium was antisera; a 1:3000 cocktail of antibodies to , somatosta- removed, replaced with 1 ml fresh medium, and further incubated tin, and (gift of Michael Appel, University for 60 min. At the end of this incubation period, this medium of Massachusetts) or rabbit anti-human ACTH-(1-39) antisera ("basal") was collected and placed at -20°C. The same isletswere (Biomeda). Primary antibodies were incubated at 4°C for 12 hr then incubated for a further 60 min with the same medium (ACTH) or for 48 hr (insulin and cocktail). containing 16.7 mM glucose. This medium ("stimulated") was For frozen sections, pituitaries were fixed in 4% paraformal- then removed and placed at -20°C. dehyde in PBS at 4°C overnight. Tissues were cryoprotected in Pituitary and islet cell lysates and the media samples were then increasing concentrations of (10%, 15%, and 20%) in immunoprecipitated for (pro)insulin and analyzed by alkaline- PBS at 4°C and embedded in TISSUE-TEK O.C.T. (Miles, urea PAGE as previously described (18). Elkhart, IN). Pituitaries were sectioned at -20°C onto S-P Brand RESULTS Superfrost Plus glass slides (Baxter Diagnostics, Deerfield, IL). Expression of Insulin in the Pituitaries of Transgenic NOD Sections were coincubated with guinea pig anti-human insulin Mice. Northern blot analysis revealed an abundant 550 bp insulin (Incstar, 1:800) and rabbit anti-porcine ACTH (1:150) antisera transcript in the pituitary, identical in size to the endogenous overnight at 4°C. Primary antibodies were detected with goat pancreatic insulin transcript. In contrast, RNA from transgenic anti-guinea pig IgG-Cy3 (Sigma, 1:200) and goat anti-rabbit , brain, , spleen, lymph nodes, testes, , IgG-FITC (Sigma, 1:50). , and salivary gland did not show any detectable signals (Fig. Pituitary and Islet Isolation and Transplantation. Islets were 1B). Immunocytochemistry of the pituitaries from the transgenic obtained from young (<6 weeks) female homozygous POMC- animals (Fig. 2A) showed that a small percentage of cells in the Insl transgenic NOD mice by collagenase infusion of the pan- anterior lobe (A) and the great majority of cells in the interme- creas through the common bile duct as described (17). Aliquots diate lobe (I) stained positive for insulin (Top), similar in distri- of 100 islets (>75 and <250 ,um in diameter) were hand-picked bution to ACTH immunostaining (Middle). The posterior pitu- under a stereomicroscope. Intermediate lobe pituitaries from itary (P) was devoid of specific insulin immunostaining and transgenic female NOD mice were diced into fragments <250 showed background signals similar to nontransgenic control ,um. Grafts consisted either of 100 islets placed under the capsule pituitaries (Bottom). Colocalization of insulin and ACTH (or of one kidney and the diced transgenic intermediate lobes placed POMC) immunoreactivity to the same pituitary cells was dem- under the contralateral kidney capsule or of 100 islets mixed with onstrated by double immunolabeling the same frozen section the transgenic intermediate lobe fragments placed into a single (Fig. 2B). Of note, the ACTH antibody used in these studies was site under the kidney capsule. Recipients consisted of diabetic raised against the entire ACTH molecule (i.e., 1-39) and would [blood glucose (BG) levels > 350 mg/dl for at least 1 week] thus be expected to recognize its cleavage products, ca-MSH female NOD mice. Two weeks after transplantation, the grafts [ACT'H-(1-13)], and corticotropin-like intermediate lobe pep- were visualized under a stereomicroscope, excised, and fixed in tide [CLIP, ACTH-(18-39)] that are present in the intermediate Bouins' solution for histological processing. lobe. For the therapeutic transplantations, diabetic female NOD To further characterize transgene expression, insulin content of mouse recipients (n = 6) were transplanted under the kidney the anterior and intermediate lobes and the of 6-week- capsule with four diced intermediate lobe transgenic pituitaries. old transgenic NOD mice were measured by acid ethanol extrac- The control group consisted of diabetic female NOD mice (n = tion followed by radioimmunoassay. These studies revealed that 3) transplanted with four diced intermediate lobes from non- the great majority of the pituitary-derived insulin was made in the transgenic NOD mice. After transplantation, mice were followed intermediate lobe with immunoreactive insulin content averaging with serial BG analyses using a One Touch II meter (Lifescan, 0.56 ± 0.05 ,ug per intermediate lobe (n = 5), compared with only Mountain View, CA) and body weights. Five weeks after trans- 0.02 jig of immunoreactive insulin per anterior lobe (n = 5). In plantation, mice were killed and their grafts were fixed in Bouins' contrast, pancreatic insulin content in young NOD mice averaged solution for histological examination. 23.7 ± 0.9 jig per gland (n = 5). To ascertain whether the Metabolic Labeling of Freshly Isolated Pancreatic Islets and transgenic insulin was secreted into the circulation, a 48-hr fast Primary Cultures of Transgenic Pituitary Cells. Transgenic was performed. Because pituitary cells lack key elements of the Downloaded by guest on September 28, 2021 Medical Sciences: Lipes et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8597

A Preproinsulin 11 Coding Region studies showed that, similar to proinsulin processing in normal

985 ATG islets, little insulin was detectable in the media in the basal state, 1132 1899 2404 Promoter C but secretion was greatly enhanced in response to forskolin and POMC p B C A 3'UTI I IBMX (Fig. 3). The predominant secretory product in the media I F W !1 E 2 ~~~MU A~1 E2 E3 || ||of transgenic intermediate lobe pituitary cells was mature insulin. Xho 1 Sal l *61 Bam HI Eco RV -703 Thus, similar to islet 13 cells, intermediate lobe pituitary cells Hind Hind III III efficiently process proinsulin to mature insulin via the regulated pathway of protein secretion. PRIMER 3: 5'...CAAA AAGCTT CAG 3 PPI -2 ...CAAA AACCAT CAG Insulin-Producing Intermediate Lobe Pituitary Cells Are Not PRIMER 1: 3'...GTTT TTCGAA GTC...5 Susceptible to Autoimmune Attack Because insulin has been implicated as an autoantigen in IDDM (20, 21) it was of interest to examine whether the expression of insulin in the pituitary, which is not normally involved by autoimmunity in NOD mice, B EX would engender the development of ectopic lymphocytic infil- trates (i.e., hypophysitis). However, serial examination of the - - ' E v ' v! 4' -- = CL ; I. c - , E I! > w M pituitaries from these transgenic NOD mice (and from other POMC-Ins transgenic NOD mouse lines that we generated) did not reveal the presence of ectopic lymphocytic infiltration, even

28s after the onset of diabetes. To exclude the possibility that either the anatomical location of the pituitary gland or the sustained production of insulin in the transgenic NOD mice might be impairing immune responsiveness to the ectopically expressed

s insulin, we transgenic and control islets _ X transplanted pituitaries ctin 1 under the kidney capsules of "naive" (nontransgenic) overtly diabetic NOD recipients. As expected, when control islet grafts were removed from the kidney capsule of the recipients 2 weeks after transplantation, the grafts showed severe infiltration with O-ci -ct- _ complete loss of the insulin-staining 13 cells, with only non-X3 cells remaining (Fig. 4A). In striking contrast, the transgenic pituitary FIG. 1. (A) Construction of the rat proopiomelanocortin grafts, placed under the capsule of the contralateral kidney, were (POMC)/mouse preproinsulin II (POMC-Ins) fusion transgene. P, devoid of lymphocytic infiltration (Fig. 4B) and showed abundant prepeptide coding region; B, B-chain coding region; C, C- staining for insulin and ACTH. Thus, the ectopic expression of coding region; A, A-chain coding region. Exons 1-3 (E1-3) are as insulin in the pituitary did not elicit the development of patho- indicated. (B) Northern blot analysis of a transgenic NOD mouse logic lesions. demonstrating pituitary-specific expression of insulin that is similar in size to the endogenous (pancreatic) transcript. Twenty micrograms of One possible explanation for these findings was that in the total cellular RNA was used for each of the various tissues, except for absence of antecedent cell injury or of a "triggering event" similar the pituitary and hypothalamus from which the amount of RNA to that which leads to insulitis, the transgenic pituitaries might not extracted per animal was too low for spectrophotometry, and thus the contain sufficient numbers of antigen-presenting cells to activate entire amount of RNA isolated per animal was loaded. The relatively the relevant lymphocytes to effect destruction of the insulin- low insulin signal in the pancreas may reflect partial degradation of the expressing pituitary cells. To address this concern and to examine pancreatic RNA and that the islet cell mass makes up only about 1% the relative ability of the intermediate lobe pituitary cells to of the total pancreas. The Northern blot was sequentially hybridized withstand inflammatory damage, islet and transgenic pituitary to 32P-labeled insulin and ,B-actin probes (16). cells were mixed together and engrafted into a single site under the kidney capsules of diabetic NOD mice. This resulted in the glucose sensing apparatus including the glucose transporter development of severe lymphocytic infiltration over the entire GLUT-2, insulin secreted from the pituitary would not be ex- graft, including areas containing pituitary tissue (Fig. 4C, arrow- pected to respond to ambient glucose levels (19). Indeed, with a head). Although there was complete destruction of insulin- 60% drop in BG in control NOD mice during the fast [from 112 ± producing 1-cells in the islets, the insulin-positive cells of the 6.0 to 46 ± 1.6 mg/dl (n = 12)], serum insulin levels fell below pituitary remained intact. Indeed, the only insulin-positive cells the detection limits of the radioimmunoassay. In contrast, despite that remained in the grafts colocalized with ACTH staining and similar drops in BG levels in heterozygous and homozygous were thus pituitary derived. These studies indicate that, even transgenic mice (from 102 ± 7.6 to 41 ± 2.2 mg/dl and 107 ± 7.4 when placed in direct contact with islet-specific pathogenic to 43 ± 1.0 mg/dl, respectively), serum insulin levels after the lymphocytes [presumptively, many of which are insulin-specific 48-hr fast were markedly elevated, averaging 43.3 ± 4.2 (n = 8) (22)] the insulin-expressing transgenic pituitaries were not sus- and 94.6 ± 8.6 microunit per ml (n = 8), respectively. These in ceptible to immune-mediated destruction. vivo data demonstrate that the ectopically produced insulin was Transplantation of the Transgenic Pituitaries into Diabetic secreted into the circulation. NOD Mice Restores Normoglycemia. The ability ofthe transgenic Proinsulin Is Efficiently Processed to Insulin via a Regulated intermediate lobe pituitary cells to efficiently process and secrete Pathway of Secretion in Transgenic Intermediate Lobe Pituitary mature insulin, along with their resistance to autoimmune attack Cells. To examine the molecular forms of insulin synthesized and and injury, suggested that these cells could be used as a vehicle secreted by the transgenic intermediate lobe pituitaries and to for insulin replacement in IDDM. Indeed, we found that trans- determine whether, characteristic of regulated secretory cells, plantation of four intermediate lobe pituitaries under the kidney secretion in the intermediate lobe pituitaries would be coupled to capsule of spontaneously diabetic NOD mice resulted in a extracellular stimuli, primary intermediate lobe pituitary cultures significant gain in body weight (Fig. 5A) and in the complete were established from the transgenic animals. Intermediate lobe remission from diabetic symptoms. This was associated with the pituitary cells were labeled for 15 hr with [35S]methionine, progressive return to near normoglycemia (Fig. SB), with mean washed, and the insulin forms secreted in response to the BG levels decreasing from 484 ± 21 mg/dl pretransplantation to secretagogues forskolin and 3-isobutyl-1-methylxanthine 150 ± 43 mg/dl after transplantation (n = 6). In parallel with this (IBMX) during subsequent chase periods were identified by drop in BG, random insulin levels increased from 4 ± 0.2 immunoprecipitation followed by alkaline urea-PAGE. These microunits per ml pretransplantation to 42 ± 9 microunits per ml Downloaded by guest on September 28, 2021 8598 Medical Sciences: Lipes et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A

FIG. 2. (A) Serial pituitary sections from a POMC-Ins transgenic NOD mouse demonstrating immunostaining of insulin (Ins, Top) compared with ACTH (1-39) immunostaining (ACTH, Middle) in the anterior and intermediate pituitary lobes. Nontransgenic pituitary showed no specific insulin staining (Ins C, Bottom). Anterior (A), intermediate (I), and posterior (P) pituitary lobes are as indicated. (B) Colocalization of insulin (Ins, red, Upper) and ACFH (green, Lower) immunoreactivity in individual anterior pituitary cells of transgenic NOD mice by double immunofluorescence staining of the same section. Cryostat sections of pituitary tissue from transgenic NOD mice were incubated with a mixture of antibodies to ACTH and insulin, followed by a mixture of secondary antibodies labeled with Cy3 (insulin) and FITC (ACTH). (X290 on a Leitz BH-2 fluorescent microscope.) posttransplantation, in a similar range to random insulin levels of creatic islets of these animals were severely atrophied and devoid nondiabetic control mice [39 ± 9 microunits per ml (n = 6)]. At of insulin-positive cells, confirming that the enhanced insulin the end of the transplantation period, immunohistochemistry of levels posttransplantation were due to the transgenic tissue the grafts of the recipients showed abundant insulin staining with implants (data not shown). These results demonstrate that the no evidence of lymphocytic infiltration. As expected, the pan- intermediate pituitary-derived insulin is biologically active and are consistent with our biochemical studies which showed that the Pituitary Islets great majority of insulin secreted by the transgenic pituitaries is fully processed, mature insulin. As expected, diabetic NOD mice Proins_ :: receiving nontransgenic (control) intermediate lobe pituitaries Proins had no reduction in serum BG levels, and had increasingly severe diabetic symptoms which resulted in their demise within 3 weeks Ins_ 1 after transplantation. - Ins DISCUSSION Cells basal stim basal stim This study describes the generation of transgenic NOD mice in Media Media which insulin expression was targeted to POMC-expressing cells of the pituitary. We demonstrate that, similar to islet X3 FIG. 3. Comparison of storage and secretion of insulin-related cells, the POMC-expressing intermediate lobe pituitary cells peptides in transgenic NOD intermediate lobe pituitary cells ("pitu- from transgenic mice efficiently process proinsulin to mature, itary") and in normal islets ("islets"). Primary cultures of intermediate biologically active insulin via a regulated pathway of protein lobe pituitary cells isolated from transgenic NOD mice and freshly secretion. Unlike the insulin- isolated rat islets were radiolabeled overnight with [35S]methionine, pancreatic 13 cells, however, washed, and chased first with unlabeled medium ("basal") then with expressing pituitary cells are not immunologically attacked in medium containing forskolin and IBMX (pituitary) or glucose (islets) diabetic NOD mice. Indeed, transplantation of the transgenic ("stim"). Cell extracts ("cells") and media were analyzed by immu- intermediate lobe pituitary tissues into spontaneously diabetic noprecipitation with an anti-insulin antiserum as described. NOD mice restored normoglycemia and reversed diabetic Downloaded by guest on September 28, 2021 Medical Sciences: Lipes et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8599

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N'P~~~~~~J

2. 2.~~~~~~~~~ ~ ~ ~ ~ il

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f FIG. 4 (A) Representative serial sections of '4| ll | l l s l s l-l -l 1 -_ E _> : :tgrafts that were placed under the kidney capsule of diabetic female NOD mice and ACH retrieved 2 weeks later. Consecutive sections ca_ ; 1 l 1% _ ...... ; . N(1-gmlymphocyticthick)infiltrationof the isletbygraftstainingshowedwith severehema- toxylin and eosin (H & E), (Top) and complete loss of insulin-staining cells (Ins, Middle), with only non-(P islet cells remaining as detected by staining with a cocktail of antibodies to - 0000;;tNglucagon, , and pancreatic polypeptide (Non-Ins, Boom). (B) Represen- - ~~~~~~~~~~~~~tativeserial sections of transgenic intermediate pituitary grafts that were placed under the kidney capsule of the contralateral kidney of the same recipient as in A. No lymphocytic L 2 infiltrates were present in the pituitary grafts by H & E staining (Top); the grafts were intact and - . ~~~~expressed abundant insulin (Ins, Middle) and - bus ~~~ACTH immunoreactivity (Bottom). (C) Rep- -~~~ - -~~~~~~ - - - - ~~~~~~~~~resentative serial sections from a graft contain- - ~~~~~~inga mixture of islet and transgenic interme- diate lobe pituitary cells transplanted under the kidney capsule of a diabetic female NOD mouse for 2 weeks. These grafts demonstrated severe and diffuse lymphocytic infiltration by H :~-~ & E staining (Upper Left, lymphocytes indi- cated by the arrow) with complete destruction - of the insulin-producing (3cells; the residual islets were identified with an antibody cocktail to glu- cagon, somatostatin, and pancreatic polypeptide - ~ ~ ~ ~~~I(Non-Ins, Lower Left). All remaining insulin

(Ins, Upper Right) were ~~~~~~~~~~~~~~~~~~~~~~~stainingderived andcells with pituitary-immuno- u - c co-localizedData shownACTHare Rew.-hm ACTH ~~~~~~~~~~stainingtative- of(Lowerat l-eatRight).five- sepanrate- eperi_ments.represen- symptoms. The absence of autoimmune infiltration in inter- Intermediate lobe pituitary cells have several advantages com- mediate lobe pituitary cells engineered to secrete insulin pared with the previously described ACTH-producing anterior provides encouraging in vivo evidence of the potential of these pituitary cells for targeted insulin gene delivery (10, 19). First, due cell types for gene replacement therapy in IDDM. to tissue specific differences in prohormone processing, ACTH is Downloaded by guest on September 28, 2021 8600 Medical Sciences: Lipes et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A 4 immune responses (28) and/or susceptibility to destruction. In addition, if the primary autoantigen(s) is to be recognized in the context ofMHC antigens, there may be tissue-specific differences in the nature and amount of antigenic peptides processed and .2~~~~~~~~~~~~~~~J presented by these non-islet tissues to cells ofthe immune system. Regardless of the exact mechanism(s) underlying the ability of

-10 -- the insulin-producing intermediate lobe pituitaries to elude im- mune system recognition and attack, this feature is clearly advantageous for transplantation purposes. A limitation of this current gene delivery system is that insulin secretion in the 7 14 21 28 35 intermediate lobe pituitary cells was not glucose regulated. It has Days after transpbantaffon -80 recently been shown, however, that transfection of AtT20 pitu- -6 itary cells with the (-cell glucose transporter GLUT-2 could confer glucose-stimulated insulin synthesis and secretion (19). Thus, while further molecular manipulations will be probably be necessary for these non-islet cell types to be useful as gene replacement therapy in IDDM, these studies demonstrate in vivo the potential of this approach. O--0 We thank Y. Wang and C. Cahill for excellent technical assistance, S. Bonner-Weir, L. Villa-Komaroff, D. H. Jones, and J. Drouin for advice and reagents, the National Institute of Diabetes and Digestive and Kidney Diseases National and Pituitary Program for 0 7 14 21 28 35 pituitary antisera, and C. R. Kahn and H. A. Wolpert for critical Days after transplanffon reading of the manuscript. M.A.L. is a recipient of a Career Devel- opment Award from the American Diabetes Association and a New FIG. 5. Effect of transplanting transgenic insulin-producing inter- Investigator Award from the Harcourt Charitable General Cinema mediate lobe tissues into spontaneously diabetic NOD mice. (A) Foundation. A.M.D. was a recipient of a postdoctoral fellowship from Percent weight change and (B) BG levels in diabetic NOD mice after the Juvenile Diabetes Research Foundation International. This work transplantation with transgenic intermediate lobe pituitaries (n = 6; was supported by grants from the National Institutes of Health, the *) or with nontransgenic control intermediate lobe pituitaries (n = 3; Greenwall Foundation, and the Markey Charitable Trust. A) calculated from day 0. Each point was the mean percent weight 1. Eisenbarth, G. S. (1983) N. Engl. J. Med. 308, 322-327. change, or the mean BG level, SE. 2. Lacy, P. E. (1993) Diabetes 1, 76-92. 3. Valera, A., Fillat, C., Costa, C., Sabater, J., Visa, J., Paujol, A. & Bosch, F. further processed in intermediate lobe cells to a-MSH [ACTH- (1994) FASEB J. 8, 440-447. (1-13)] and CLIP [ACTH-(18-39); refs. 22 and 23], neither of 4. Kolodka, T. M., Finegold, M., Moss, L. & Woo, S. L. C. (1995) Proc. Natl. which are known to have adverse metabolic effects. Second, Acad Sci. USA 92 3293-3297. A. despite its relatively small size (<25%o of the mass of the anterior 5. Smeekens, S. P., Avruch, S., LaMendola, J., Chan, S. J. & Steiner, D. F. (1991) Proc. Natl. Acad. Sci. USA 88, 340-344. pituitary), the insulin content of the intermediate lobe was high, 6. Seidah, N. G., Marcinkiewicz, M. & Benjannet, S. (1991) Mol. Endocrinol. '31-fold greater than the anterior lobe, paralleling expression of 5, 111-122. endogenous POMC (Fig. 2 A and B). Third, the intermediate 7. Davidson, H. W. & Hutton, J. C. (1987) Biochem. J. 245, 575-582. pituitary grafts had markedly better viability than anterior pitu- 8. Moore, H.-P., Walker, M. D., Lee, F. & Kelly, R. B. (1983) Cell35, 531-538. 9. Ferber, S. Gross, D. J., Villa-Komaroff, L, Vollenweider, F., Meyer, K, itary lobe grafts: when transplanted long-term (>100 days) under Loeken, M., Kahn, C. R. & Halban, P. A. (1991) Mol. Endocrinol. 5,319-326. the kidney capsule the anterior pituitary cells, with the exception 10. BeltrandelRio, H., Schnedl, W. J., Ferber, S. & Newgard, C. B. (1994) of the lactotrophs, became atrophied, whereas the intermediate Pancreatic Islet Transplantation, eds. Lanza, R. P. & Chick, W. L. (Landes, pituitary grafts remained viable and continued to produce abun- Austin, TX), Vol. 1, pp. 169-178. dant amounts of , similar to previous 11. Gross, D. J., Halban, P. A., Kahn, C. R., Weir, G. C. & Villa-Komaroff, L. studies (24). These (1989) Proc. Natl. Acad. Sci. USA 86, 4107-4111. findings, reminiscent of changes after pituitary stalk transection, 12. Jones, D. H., Sakamoto, K., Vorce, R. L. & Howard, B. H. (1990) Nature are most likely due to the differential regulation of these two cell (London) 344, 793-794. types: whereas secretion from most anterior lobe cells is depen- 13. Wentworth, B. M., Schaefer, I. M., Villa-Komaroff, L. & Chirgwin, J. M. dent upon stimulation by trophic hypothalamic hormones, secre- (1986) J. Mol. Evol. 23, 305-312. 14. Tremblay, Y., Tretjakoff, I., Peterson, A., Antakly, T., Zhang, C. X. & tion from intermediate lobe pituitary cells is predominantly under Drouin, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8890-8894. tonic inhibitory control by (25) and y-aminobutyric 15. Hammer, G. D., Fairchild-Hundtress, V. & Low, M. J. (1990) Mol. Endo- acid (26). crinol. 4, 1689-1697. Insulin has been implicated as an autoantigen in IDDM (20, 16. Lipes, M. A., Rosenzweig, A., Tan, K.-N., Tanigawa, G., Ladd, D., Seid- 21); however, pathologlc lesions did not spontaneously man, J. G. & Eisenbarth, G. S. (1993) Science 259, 1165-1169. develop in 17. Gotoh, M., Maki, T., Kiyoizumi, T., Satoim, S. & Monaco, A. P. (1985) the insulin-secreting pituitaries of the transgenic NOD mice. Transplantation 40, 1715-1720. Likewise, in contrast to transplanted islets, transplantation of the 18. Alarcon, C., Lincoln, B. & Rhodes, C. J. (1993)1J Biol. Chem. 268,4276-4280. transgenic pituitaries under the kidney capsules of diabetic (non- 19. Hughes, S. D., Johnson, J. H., Quaade, C. & Newgard, C. 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