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Histopathological Study of Intrahepatic Islets Transplanted in the Nonhuman Primate Model Using Edmonton Protocol Immunosuppression

Transplantation and Autoimmunity Branch (B.H., N.P., J.L., D.M.H.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and Pathology Department (S.M.), Naval Medical Research Center, Silver Spring, Maryland 20910

Address all correspondence and requests for reprints to: David M. Harlan, M.D., Chief, Transplantation and Autoimmunity Branch, NIDDK, National Institutes of Health, Building 10, Room 11S210, Bethesda, Maryland 20892. E-mail: DavidMH{at}intra.niddk.nih.gov.

Abstract

While islet cell transplantation is a promising way to restore insulin independence to patients with type I diabetes mellitus, a detailed histological analysis of the transplanted, intraportal islets has not yet been reported. Rhesus macaques underwent total pancreatectomy, then had allogeneic isolated islets infused into their portal vein, followed by daclizumab, tacrolimus, and sirolimus to prevent islet rejection. Islets were evenly distributed among the liver lobes. Liver sections from a primate given allogeneic islets 5 d earlier did not display any islet capillary formation, whereas intrahepatic islets transplanted 30 and 90 d before euthanasia showed an abundant capillary supply. Localized hepatocellular glycogenosis was observed surrounding the islets in a primate with functioning islets 7 months post transplant. Liver sections from a primate that rejected islets transplanted 2 months prior displayed only islet remnants with prominent local lymphohistiocytic inflammation and an occasional capillary. We conclude that islets develop an abundant vascular supply within 30 d following transplant and because capillaries persist even following rejection, that the vascular cells are likely from the recipient. While transplanted islets were not vascularized early post transplant, the primates remained insulin independent. The long-term consequence of islets in the liver, marked by the glycogenosis, remains unknown and warrants further study.

TYPE 1 DIABETES MELLITUS (T1DM) results from the immune mediated destruction of the insulin producing pancreatic ß-cells (ß-cells) located in cell clusters called the islets of Langerhans (1). Though these islets can be replaced through allogeneic transplantation, the approach languished clinically for the past two decades because insulin independence was only rarely achieved, presumably as a consequence of difficulties surrounding islet isolation, implantation, and the subsequent immunosuppression (2, 3). Following recent clinical successes, however, the field has reemerged as a promising way to restore patients with T1DM to insulin independence (2, 4, 5). For the field to further develop, many questions difficult to address from clinical studies must be answered. Toward that end, nonhuman primates are highly relevant due to their phylogenetic relationship to humans (6), and because many of the newer immunomodulatory agents (antibodies and receptor fusion proteins) specific for human epitopes will cross-react with corresponding primate epitopes (6, 7, 8, 9). Thus, we established a nonhuman primate model using a recently reported steroid sparing regimen (10) and noted that a detailed histological analysis of the transplanted, intraportal islets has not been reported (5).

Materials and Methods

General

The procedures described in this study were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals Institute of Laboratory Animals Resources, National Research Council, DHHS, Publication No. (NIH) 86–23 (19850) and were approved by Animal Care and Use Committees of both the NIH and the Armed Forces Radiobiology Research Institute.

Methods employed to induce diabetes, isolate islets, transplant those islets, and follow the animals post transplant have recently been published elsewhere (11, 12). In general, rhesus macaques underwent total pancreatectomy to induce diabetes. Pancreases were procured from donor primates and islet isolation was performed using the automated and Liberase-based method for human islet isolation (12, 13). The portal vein was identified in the operating room, and the islets were infused into the portal vein.

Daclizumab (Zenapax, Roche Molecular Biochemicals, Indianapolis, IN) was administered at 2 mg/kg iv on the day of transplantation and every 2 wk for a total of five doses. Rapamycin (Rapamune, sirolimus, Wyeth-Ayerst, Collegeville, PA) was given twice a day orally to target 24-h trough levels of 10–15 ng/ml. Levels were determined by Mayo Medical Laboratories (Rochester, MN) using liquid chromatography and tandem mass spectroscopy. The average daily dose required was 6 mg/kg. Tacrolimus (Prograf, Fuji Photo Film Co., Ltd., Tokyo, Japan) was given orally twice daily at 2.5–5 mg with dosage adjusted to achieve 24-h trough levels of 4–6 ng/ml (12). Tacrolimus levels were measured by the Clinical Chemistry Laboratory at the NIH. Islet function was assessed by daily tail stick blood glucose measurements and by periodic serum arginine stimulated c-peptide levels. Primates were considered insulin independent if they had normal fasting and postprandial glucose levels (compared with healthy primates) without requirement for exogenous insulin.

Postmortem necropsy examination, collection of tissue specimens, and tissue processing

Complete necropsies were performed on all rhesus monkeys (Macaca mulatta) as soon as possible following an unexpected death (one animal), or when criteria for euthanasia were achieved. Our criteria for euthanasia were: rejection as determined by 4 wk of high glucose levels (above 200 mg/dl) and negative c-peptide, failure to thrive for any reason, weight loss greater than 15%, or our determination that the animal should be euthanized for detailed immunohistological analysis (see Figs. 3 and 4). All major organ systems were sampled including all four liver lobes. The liver lobes were sliced at 0.5- to 1.0-cm intervals to facilitate complete fixative penetration. Tissues were immersion fixed in 10% buffered formalin with ionized zinc (Z-Fix) for 5 d, routinely processed with paraffin infiltration, embedded in paraffin, and then sections were cut on a rotary microtome at 5 mm and placed on positively charged slides. Sections were routinely stained with hematoxylin and eosin for histopathological examination. A specific sampling plan of the liver to detect possible islet cell clusters was devised to thoroughly sample every lobe. This resulted in between 10 and 20 liver slides per monkey, and 1–6 liver sections per slide.

View larger version (141K): [in this window] [in a new window]   Figure 3. Hematoxylin and eosin stain (x200): well-established capillary network adjacent to the portal vein. A notable thin endothelial layer separating the islet from the portal vein lumen can be seen. Arrow, Endothelial layer. Lymphocytic infiltration suggesting subclinical rejection (dashed arrows).

View larger version (127K): [in this window] [in a new window]   Figure 4. Section of liver from a rhesus macaque 7 months after successful portal vein transplantation (hematoxylin and eosin stain, x200 magnification). Within the portal triad, there is an islet cell cluster with adjacent localized hepatocellular glycogenosis that only occurs in hepatic cords that extend from this portal islet to the central vein. We performed the Periodic Acid Schiff (PAS) reaction/procedure to demonstrate glycogen. The PAS positive material is red. cv, Central vein. The lymphocytes probably represent an immune cell response (rejection) resulting from this cluster of transplanted cells.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue blocks were sectioned (4–6 µm), adhered to Superfrost/Plus (A. Daigger & Co., Wheeling, IL) glass microscope slides, dried, and immunostained within 3 d. Slides were cleared, rehydrated in decreasing reagent alcohols and water, flushed with APK wash buffer [Ventana Medical Systems, Inc. (VMSI), Tucson, AZ], and immunostained the same day. Immunohistochemistry was performed on the NEXES (VMSI) automated immunostainer. All endogenous biotin was blocked using the Endogenous Biotin Blocking Kit (VMSI). Slides were incubated with primary polyclonal antibodies (i.e. rabbit negative control immunoglobulin (VMSI), or polyclonal antibodies to insulin (VMSI), somatostatin (VMSI), glucagon (DAKO Corp., Carpinteria, CA), and pancreatic polypeptide (DAKO Corp.). Immunostaining was completed using the Basic DAB Detection Kit (VMSI), which included an endogenous peroxidase inhibitor. Slides were counterstained with Gill’s hematoxylin, washed then blued in 1% acid alcohol and 1% ammonium hydroxide, respectively, dehydrated in increasing reagent alcohols, cleared with xylene, then coverslipped with Cytoseal 60. Photomicrographs were generated using the Flashpoint Imaging Software (Durham, NC) and video capture.

Results

Histopathological studies

Liver sections were available for study from five primates:

1) An insulin independent primate that died 5 d after transplantation due to aspiration pneumonia (Fig. 1),

View larger version (87K): [in this window] [in a new window]   Figure 1. An islet cell cluster in a portal vein shows evidence for localized cellular degeneration and individual cell necrosis. A low-grade lymphocytic portal hepatitis surrounding the islet cell cluster is seen (red arrows). Note the hepatic artery and bile duct beside the islet. Cytosolic hyalinization and pycnotic nuclei (dashed arrows) suggest degenerative changes. Magnification, x200.

View larger version (113K): [in this window] [in a new window]   Figure 2. Immunohistochemistry for insulin (a), pancreatic polypeptide (b), glucagon (c), and somatostatin (d). There are multiple fine capillaries (arrows) extending from the wall of the vein into the islet cluster (e). Magnification, x400. An islet cell cluster (f) shows a mitotic figure (arrowhead), Magnification, x1000.

4) An insulin independent primate 3 months following transplantation (Fig. 3), and

5) A primate euthanized 7 months after receiving allogeneic islets and that periodically required small insulin doses beginning 6 months post transplant (Fig. 4).

Islets were found to be evenly distributed among lobes studied: An average of 7.3 islets per slide in the right lateral lobe, 7.1 islets per slide in the left lateral lobe, and in the large central lobe we found 5.8 islets per slide in the left medial fragment, 8.4 islets per slide in the gallbladder proximal fragment, and 1.5 per slide islets in the right medial fragment. We also found 3 islets per slide in the caudate process and 4.3 islets per slide in the papillary process. These observations demonstrate that islets, at least in this animal, are fairly evenly distributed throughout the portal vascular bed.

Several observations are worthy of particular note. While islets in the native pancreas are known to be highly vascular, we did not see any evidence of neovascularization in the animal that died 5 d after receiving the islet transplant (Fig. 1). In addition, cellular degeneration is evident (cytosolic hyalinization, pyknotic nucleii) possibly the result of the animal’s unexpected death some hours earlier, and/or from islet damage from the recent isolation and transplant procedure.

Intrahepatic islets from the animal transplanted with islets 30 d prior, stained for insulin, pancreatic polypeptide, glucagon, and somatostatin (Fig. 2, a–d). Liver sections from that animal displayed an abundant capillary supply (Fig. 2e) and occasional mitotic figures (Fig. 2f). We are unable to state with certainty the exact cell types undergoing mitosis within the islet.

The intrahepatic islets of the primate transplanted 3 months prior also had a well-established capillary network (Fig. 3). The islets in this animal were often adjacent to the portal vein but were remarkable for the thin endothelial layer separating the islet from the portal vein lumen.

Liver sections from the primate that rejected the transplanted islets 2 months after grafting (presumably secondary to inadequate immunosuppressive drug levels) contained only sparse surviving endocrine cells within the lymphohistiocytic periportal inflammatory lesion, but nevertheless still contained a prominent capillary network. This observation suggests that the vessels were from the recipient (figure not shown).

Islet vascularity could not be assessed in the primate euthanized 7 months after islet transplant because the islets were small and sparse, despite the animal’s persistent insulin independence. We did note prominent areas of localized hepatocellular glycogenosis around the islets (Fig. 4). We subsequently carefully screened all cases for hepatocellular glycogenosis with hematoxylin and eosion and planned to confirm suspect cases with Periodic Acid Schiff staining. Only the primate with intrahepatic islets for 7 months displayed alterations in hepatocellular morphology suggesting glycogen deposits.

Discussion

We have established a clinically relevant nonhuman primate (rhesus macaque) model of islet transplantation in an effort to address many questions associated with this developing technique (12). We report here the histological characteristics of pancreatic islets transplanted into the portal vein of five rhesus monkeys because, and for obvious reasons, detailed histological analysis of clinical samples is not possible.

It has long been known that islets are highly vascularized. Pancreatic islets comprise only approximately 2% of the pancreas cell mass and yet consume up to 20% of the arterial blood flow (2, 14, 15, 16, 17). Yet islets lose this vascular supply during the isolation process (18). The data reported herein demonstrate that islets, easily identified in the majority of liver sections studied and always lodged near the portal triad region, were prominently vascularized by d 30 post transplant (Fig. 2), whereas liver sections studied 5 d after islet transplant did not reveal evidence of vascularization (Fig. 1). These findings closely correlate with similar reports evaluating angiogenesis in the rodent model of islet transplantation (19), mostly in grafts placed under the renal capsule (20) or the dorsal skinfold chamber (18, 21, 22, 23, 24). Although we demonstrated no obvious functional correlate associated with the vascularization, it stands to reason that islet vascularization is important for normal function and viability. If so, then these data suggest a vulnerable period following transplant when patients should perhaps be treated with exogenous insulin so as to avoid functionally challenging what may be a metastable state for the islets. Our data suggest a recipient source for the vasculature as a rich capillary network persisted even during the later stages of rejection when very few islet cells remained. If the endothelial cells were of a donor source, one would have imagined that they would be lost as well during a rejection. These findings support the observation by Vajkoczy et al. (25), who showed that transplanted rat islets were revascularized by endothelium of hamster (host) origin.

Most of the islets shown had mild to moderate lymphocyte infiltration suggesting subclinical rejection. Yet, the primates maintained an insulin independent state, underscoring the lack of reliable rejection markers in patients following islet transplantation.

Many have questioned whether the portal vein is the best site to infuse the allogeneic islets. Reasons for this concern include that the portal system is difficult to access, that serious morbidity or even mortality can ensue should the portal vein bleed or clot as a result of the islet transplant, and that the portal vein carries blood with oxygen tension slightly less than that of arterial blood, contains higher concentrations of substances from the gut that may prove toxic to the islets, and that the immunosuppressive agents (known to be toxic to islet cell function) are absorbed from the gut and thus their toxic effects might be magnified for islets bathed in portal blood. Many of these concerns should be allayed by the observation demonstrated in Fig. 3 that suggests that by 3 months post transplant, islets have become essentially extraportal. That is, the islets are surrounded by an endothelial layer separating them from the portal vein blood and instead are vascularized by a rich capillary network.

We were interested in the effect that high local insulin levels secreted by the transplanted islets might have on the surrounding hepatocytes. Liver sections from the primate with functioning islets for 7 months support the notion that a local effect does occur in that localized hepatocellular glycogenosis was observed surrounding islet clusters. The clinical implications of this finding are not clear and dictate long-term follow-up for patients with islets transplanted into their portal circulations, and with particular focus on hepatic structure and function. Indeed, we are intrigued by the fact that we observed these changes only in this one animal. One possibility is that it is a rare event in animals with as yet unidentified factors contributing to the lesion. Another possibility is that the glycogen deposition slowly develops over time and that its onset occurs only after several months. A third possibility is that the islets in this animal were, of the animals we examined, the smallest and the most sparse. This latter observation may suggest that the remaining islets in this animals liver were functioning at higher capacity than islets in primates with a larger islet mass. Perhaps the presumed hyperfunction contributed to the glycogen deposition observed.

The data presented are limited by the study’s primary design. That is, the project was not designed for detailed histological analysis and therefore the clinical course, and not preestablished time points, dictated the availability of liver sections. Nevertheless, we conclude that islets transplanted into the portal vein lodge like a thrombus in the distal portal branches, and that within several weeks, the islets develop an abundant vascular supply. A recipient source for the vasculature is suggested as the capillaries persist even during the later stages of rejection when very few islet cells remain. After the initial capillary network formation, endothelium grows around the islets rendering them essentially extraluminal relative to the portal vein. Localized hepatocellular glycogenosis surrounding islet clusters indicate a possible local effect of the transplanted islets on the surrounding hepatocytes.

We can formulate no firm conclusions from these observations, but our data suggest a need to support islets with exogenous insulin for several weeks until they are vascularized, and that recipients of islet allografts should be followed closely with studies to evaluate liver function and morphology.

Acknowledgments

Footnotes

Abbreviations: CL, Central lobe; T1DM, type I diabetes mellitus; VMSI, Ventana Medical Systems, Inc.

Received May 3, 2002.

Accepted August 18, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirshberg, B. Articles by Harlan, D. M. Search for Related Content PubMed PubMed Citation Articles by Hirshberg, B. Articles by Harlan, D. M.