Evidence of Primary b-Cell Destruction by ... - Diabetes Care

[Pages:7]Pathophysiology/Complications

ORIGINAL ARTICLE

Evidence of Primary -Cell Destruction by T-Cells and -Cell Differentiation From Pancreatic Ductal Cells in Diabetes Associated With Active Autoimmune Chronic Pancreatitis

SHOICHIRO TANAKA, MD1,2 TETSURO KOBAYASHI, MD1,2 KOJI NAKANISHI, MD1,2 MINORU OKUBO, MD1,2 TOSHIO MURASE, MD1,2 MASAJI HASHIMOTO, MD2,3 GORO WATANABE, MD2,3

HIROSHI MATSUSHITA, MD2,4 YUZO ENDO, MD2,5 HIDEO YOSHIZAKI, MD6 TOMOO KOSUGE, MD7 MICHIIE SAKAMOTO, MD8 KAZUO TAKEUCHI, MD2,6

OBJECTIVE -- Diabetes associated with autoimmune chronic pancreatitis (ACP) is a subtype of diabetes that is responsive to corticosteroid treatment of progressive endocrine and exocrine dysfunction. However, little is known about pathological changes of islet and exocrine pancreas in ACP.

RESEARCH DESIGN AND METHODS -- We examined pancreatic specimens obtained on biopsy from four diabetic men with ACP (mean [range]: age 62 years [48 ?78], duration of ACP 3 months [1?5], duration of diabetes 1 month [0 ?3]) morphologically, immunohistochemically, and morphometrically.

RESULTS -- The pancreatic specimens in all cases exhibited inflammatory cell infiltration surrounding ductal cells and extensive fibrosis. Some islets were infiltrated with mononuclear cells with disrupted -cells. The subsets of T-cells infiltrated to the islets were mainly CD8. Islet -cell volume was decreased; the mean percentage area of -cells in the islets in four cases with ACP were 16% (range 13?20) (P 0.0015 vs. type 2 diabetic patients, 48% [27?73], n 8; P 0.0002 vs. nondiabetic control subjects, 58% [39 ?77], n 7). Preserved ductal cells were surrounded predominantly by CD8 or CD4 T-cells. Some cytokeratin 19 ?positive ductal cells contained insulin and glucagon, representing upregulated differentiation of islet cells from ductal cells. Insulin promoter factor-1 (IPF-1) was hyperexpressed in insulin-containing ductal cells.

CONCLUSIONS -- Diabetes associated with ACP is caused by T-cell?mediated mechanisms primarily involving islet -cells as well as pancreatic ductal cells. In ACP, ductal islet precursor cells were associated with IPF-1 hyperexpression, suggesting a critical role of IPF-1 on islet cell differentiation and eventual -cell restoration.

Diabetes Care 24:1661?1667, 2001

From the 1Department of Endocrinology and Metabolism, Toranomon Hospital, Tokyo; the 2Okinaka Memorial Institute for Medical Research, Tokyo; the Departments of 3Surgery, 4Pathology, 5Immunology, and 6Gastroenterology, Toranomon Hospital, Tokyo; and the 7Department of Surgery and the 8Division of Pathology, National Cancer Center, Tokyo, Japan.

Address correspondence and reprint requests to Dr. Tetsuro Kobayashi, Department of Endocrinology and Metabolism, Toranomon Hospital, 2-2-2, Toranomon, Minato-ku, Tokyo 105-8470, Japan. E-mail: tetsuro@po.sphere.ne.jp.

Received for publication 27 February 2001 and accepted in revised form 6 June 2001. Abbreviations: ACP, autoimmune chronic pancreatitis; CAII, carbonic anhydrase II; ELISA, enzymelinked immunosorbent assay; GADAb, autoantibodies against GAD; IA-2Ab, autoantibodies against insulinoma-associated protein 2/islet cell antigen 512; ICA, islet cell antibody; IDX-1, islet duodenum homeobox-1; IPF-1, insulin promoter factor-1; PDX-1, pancreatic and duodenal homeobox factor-1; PFD test, pancreatic function diagnostic test. A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion factors for many substances.

D iabetes occurs in 50% of patients with chronic pancreatitis (1,2). Although chronic pancreatitis is often related to excessive alcohol consumption, in 25% of patients with chronic pancreatitis, the disorder is not related to alcohol (1,2). Autoimmune-mediated mechanisms often underlie such nonalcoholic impairment of the pancreas, so-called "autoimmune chronic pancreatitis" (ACP) (3?7). ACP is characterized by diffuse swelling and severe fibrosis associated with mononuclear cell infiltration involving the exocrine pancreas (3?7). The disorder can induce severe pancreatic swelling or tumor formation, with malabsorption and often cholestasis, and is sometimes misdiagnosed as pancreatic carcinoma or lymphoma. Patients with ACP are sometimes subjected to pancreatectomy or chemotherapy (6,7). Recently, we have reported that a large proportion of patients with ACP had diabetes and responded well to corticosteroid therapy, and after treatment, those patients had normal or mild glucose intolerance with improved insulin response (8). Current knowledge is limited regarding the changes in both affected endocrine as well as exocrine pancreas. In this study, we report the findings of the affected pancreas of patients with diabetes associated with ACP on a morphological, immunological, and morphometric bases.

RESEARCH DESIGN AND METHODS

Subjects Our cohort study of ACP was performed between 1993 and 1999 and included 2,483 Japanese patients with suspected chronic pancreatitis and symptoms of abdominal and/or back pain, malnutrition, or steatorrhea. ACP was diagnosed in four patients according to the following criteria (3?7): 1) increased pancreatic enzyme

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and/or increased serum -globulin level and/or presence of autoantibodies against pancreatic exocrine antigens, 2) diffuse pancreatic enlargement on ultrasonography or computed tomography, 3) abnormally narrow pancreatogram on endoscopic retrograde cholangiopancreatography , 4) presence of lymphocyte infiltration in pancreatic specimens.

All four patients with ACP were men (cases 1? 4; age 62 12 years [(mean SD) range 48 ?78], BMI 19.5 3.4 kg/m2 [17.4 ?24.5]) who had diabetes (duration 1 2 months [0 ?3]). In all cases, development of diabetes followed the onset of ACP (duration of ACP 3 3 months [1?5]) with prodrome of mild epigastralgia and weight loss (9 2 kg [7?10]) before hospitalization. Insulin was required in three patients (cases 1?3), and an oral hypoglycemic agent (gliclazide) in one patient (case 4) for control of hyperglycemia. None of the patients had a history of heavy alcohol consumption or a family history of pancreatic disease or diabetes. After the diagnosis of ACP, prednisolone was administered at an initial dose of 0.45? 0.91 mg/kg body wt per day and was tapered to 0.07? 0.10 mg/kg body wt per day as a maintenance dose. Digestive enzyme preparations were administered in two of the four patients (cases 1 and 2).

Laboratory examinations Pancreatic function diagnostic test (PFD test) (9) was performed using a kit (Eisai, Tokyo). The value of the PFD test shows the digestive function of p-aminobenzoic acid containing compound by pancreatic chymotrypsin. Islet cell antibody (ICA) was detected by the indirect immunofluorescence method as previously described (10). The cutoff point of our assay was 5 Juvenile Diabetes Foundation units (sensitivity 90%, specificity 92%). Autoantibodies against GAD (GADAb) and insulinoma-associated protein 2/islet cell antigen 512 (IA-2Ab) were assayed as previously reported (11,12). The sensitivity and specificity of the GADAb assay were 80 and 100%, respectively, in the second International GADAb Workshop (11). The IA-2Ab assay was evaluated in the third proficiency IA-2Ab test, organized by the Research Institute for Children, and the results showed 100% sensitivity and 100% specificity (12).

Enzyme-linked immunosorbent assay for autoantibodies against carbonic anhydrase II Human carbonic anhydrase II (CAII) was purchased from Sigma (St. Louis, MO). Serum autoantibodies against CAII were quantified by enzyme-linked immunosorbent assay (ELISA) using a standard method with minor modification (5,13). The absorbance was determined at 405 nm. Inter- and intra-assay coefficients of variation were 9.1 2.4 and 2.0 2.0%, respectively. Mean absorbance (SD) of nondiabetic control subjects was 0.172 0.013 (n 21). In the present study, positive results were defined as absorbance values higher than 0.198, which corresponds to a value that is the mean 2 SD of control values.

Pancreatic specimens Pancreatic biopsy was performed before initiation of corticosteroid therapy in each patient with ACP. In all four patients with ACP, biopsy specimens were obtained from the quarter part of the pancreatic tail farthest from the end because this area is regarded as fairly representative of the entire pancreas (14). Two pancreatic specimens were obtained from each of the four patients with ACP. There were no apparent sequelae after the pancreatic biopsies. Pancreatic specimens of seven control subjects without diabetes and chronic pancreatitis (four men, three women; age 66 5 years [(mean SD) range 59 ? 75]) were obtained during surgery for gastric carcinoma with warm ischemic time within 15 min. Pancreatic specimens from eight patients with type 2 diabetes (one biopsy and seven autopsies; five men, three women; age 67 8 years [51? 81], duration of diabetes 8 5 years [0 ?15]) were also studied. Written informed consent was obtained from all subjects, and the protocol of this study was approved by the ethics committee of Toranomon Hospital.

Immunohistochemistry Specimens were fixed in 10% formaldehyde or were snap-frozen in OCT compound (Tissue-Tek, Miles, IN). -, -, and -cells were stained immunohistochemically with specific anti-insulin, anti-glucagon, and anti-somatostatin sera (Dako, Santa Barbara, CA) by peroxidaseantiperoxidase methods previously described (14). Specific anti-serum against insulin promoter factor-1 (IPF-1, also

known as pancreatic and duodenal homeobox factor-1 [PDX-1] and islet duodenum homeobox-1 [IDX-1]) was provided by Dr. Kajimoto (Osaka University) (15,16). The specific antibodies for cytokeratin 19 (clone BA17; Dako) were used to stain pancreatic ductal cells (14,17). Indirect immunofluorescence method was used for staining HLA class II molecules (clone 9-49 [I3]; Immunotech, Westbrook, ME), CD8 T-cells (clone SFCI21Thy2D3 [T8]; Immunotech), CD4 T-cells (clone SFCI12T4D11 [T4]; Immunotech), B-cells (clone 89B [B4]; Immunotech), macrophages (clone 94 [Mo1]; Immunotech), and NK cells (clone 3G8; Immunotech) in unfixed pancreatic sections (17). Presence of lymphocyte infiltration to the islet was examined on all islets of each pancreatic section of ACP cases 1? 4 (number of islets: case 1, n 48; case 2, n 10; case 3, n 8; case 4, n 21).

Morphometric analysis Morphometry was computed as follows (14): seven photographs of randomly chosen islets taken at magnification 100 or 200 were used for determination of the area of endocrine cell. The area of islets and each type of endocrine cell, including -, -, and -cells, was automatically calculated by tracing their outlines with a digitizer-assisted computer program (MEAS 1; Graphtec, Tokyo). The percentage of the areas of each type of endocrine cell, including -, -, and -cells, in relation to the islet was obtained by dividing each area by the area of the corresponding islet.

Ultrastructural analysis by electron microscopy Pancreatic specimens from case 1 were fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde dissolved in 0.1 mol/l phosphate buffer (pH 7.4), postfixed for 1 h at 4?C in 1% osmium tetroxide, diluted with the phosphate buffer, dehydrated with graded alcohols, and embedded in an epoxy resin mixture (Araldite M; Ciba-Geigy, Basel, Switzerland).

Statistical analysis Fisher's exact test was performed to examine the presence of lymphocyte infiltration to the islets. Data comparison in morphometric analysis was performed

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3,525 mg/dl, respectively [844 ?2,314]), whereas the remaining two patients had normal levels of serum -globulin and IgG. One patient (case 3) had an elevated level of pancreatic enzymes (elastase 1 655 ng/dl [22?221], lipase 389 units/l [25?170]). In two patients (cases 1 and 2), results of PFD test showed a decreased level of pancreatic digestive function (35 and 59%, respectively [68]).

Figure 1--Pancreatic parenchyma extensively destroyed and replaced by massive fibrosis with

mononuclear cell infiltration in case 2. A: Severe fibrous change of the pancreas (hematoxylineosin stain, 100). CD8 T-cells (B, arrows) infiltrated around pancreatic ductal cells that were stained for cytokeratin 19 (C). B and C are serial sections (200).

using bilateral unpaired Student's t test. Values were expressed as means SD.

RESULTS

Clinical features of patients with diabetes associated with ACP All four patients with ACP had apparent diabetes (fasting blood glucose 8.9 2.4 mmol/l [range 6.8 ?11.4], 2-h blood glucose during 75-g oral glucose tolerance test 22.8 2.5 mmol/l [19.4 ?25.2],

HbA1c 6.6 1.2% [5.7? 8.2; normal range 4.3?5.8]). All of these patients had diffuse pancreatic enlargement and narrowing of the main pancreatic duct; results of testing for autoantibodies against CAII, a putative autoantigen in ACP (5), were positive, whereas results of testing for GADAb, IA-2Ab, and ICA were negative. Two of four patients (cases 1 and 2) had increased levels of serum -globulin (2.0 and 2.8 g/dl, respectively [normal range 0.8 ?1.6]) and IgG (2,666 and

Histologic and immunohistologic

findings in the pancreas with ACP

Pancreatic tissue specimens of all these patients showed fibrous change in the acinar and ductal cells and inflammatory cell infiltration surrounding spared ductal cells of the exocrine pancreas (Fig. 1). Pancreatic islets containing -cells were also surrounded by fibrous tissue in all patients (Fig. 2). Intra- and peri-insular mononuclear cell infiltration was observed in 15% (7/48), 20% (2/10), 25% (2/8), and 19% (4/21) of islets in cases 1, 2, 3, and 4, respectively. In eight patients with type 2 diabetes and seven control subjects, intra- and peri-insular mononuclear cell infiltration was not observed (P 0.0020 and P 0.0030, respectively, vs. patients with ACP). The subset of T-cells infiltrated to the islets was mainly CD8. In pancreata of patients with ACP, the percentage area of -cells in the islets was 16 3% (range 13?20, n 4). Remarkably, this value was reduced when compared with patients with type 2 diabetes (48 14% [27?73], n 8, P 0.0015 vs. patients with ACP) and nondiabetic control subjects (58 13% [39 ?77], n 7, P 0.0002 vs. patients with ACP). Percentage areas of -cells and -cells in islets of pancreata in patients with ACP (9 1% [7?10] and 7 1% [6 ? 8], respectively) did not differ when compared with patients with type 2 diabetes (17 8% [8 ?30] and 6 2% [4 ?9], respectively) and nondiabetic control subjects (10 5% [3?18] and 6 3% [4 ?12], respectively). Cytokeratin 19 ?positive ductal cells contained insulin as well as glucagon in all cases (Fig. 3A and B). Some of these endocrine cells were surrounded by fibrous tissues involving migratory mononuclear cells. Immunohistochemical staining on infiltrating mononuclear cells around pancreatic ductal cells showed predominantly CD8 or CD4 T-cells, with smaller numbers of -cells, macrophages, and

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with type 2 diabetes and nondiabetic control subjects, IPF-1 expression was clearly stained in some cells, but the intensity was weaker than pancreatic tissue of patients with ACP, especially in the nucleus.

Electron microscopy Some mononuclear cells were adjacent to -cells and -cells. These -cells showed electron-dense cytoplasm with vacuolated and degranulated insulin vesicles, swollen endoplasmic reticulum, and Golgi apparatus, whereas the morphological structure of -cells did not change (Fig. 4). Some islet precursor cells with endocrine granules in spared ductal cells were vacuolated and degranulated and had electron-dense cytoplasm.

Figure 2--Immunohistochemical staining of islet -cells (A) and CD8 T-cells (B) in serial sections of pancreatic tissue in case 1. Pancreatic -cells were involved in fibrous exocrine tissue (A, 200). The number of -cells (stained brown) was remarkably reduced when compared with nondiabetic control subjects. CD8 T-cells (arrows) infiltrated into or around distorted islets (B, 200).

NK cells in all cases (Fig. 1B and C). Inappropriate HLA class II molecule expression was observed in most of the ductal cells surrounded by these migrating cells in all patients, whereas the HLA class II molecule expression was not observed in spared islet cells.

Immunostaining of IPF-1 In all patients with ACP, IPF-1 was densely stained in the nucleus and less densely stained in the cytoplasm in some insulin-containing ductal cells and some islet -cells (Fig. 3C and D). In the islets and ductal cells of pancreata of patients

CONCLUSIONS -- ACP is characterized by clinical symptoms, including mild abdominal pain and weight loss, the presence of autoantibodies against pancreatic exocrine tissues, radiographic or ultrasonographic findings of pancreatic swelling, and narrowing of the main pancreatic duct (3? 8). The prevalence of ACP is 2% in cases with chronic pancreatitis, as diagnosed by endoscopic retrograde cholangiopancreatography (5). Noteworthy is the finding that most patients with ACP have diabetes (8).

In the present study, we clearly documented that pancreatic islets as well as exocrine pancreatic cells were involved in the inflammatory process of ACP, even though the number of examined pancreatic specimens was low. The extent of destruction of -cells and pancreatic exocrine cells was remarkable in the active stage of ACP. Morphometric and ultrastructural analyses demonstrated predominant and specific loss of -cell volume. These histological findings were consistent with clinical findings that a high proportion of cases with ACP had insulin-deficient (not insulin-resistant) diabetes (8).

The mechanisms that contribute to -cell deficiency in this type of diabetes are speculative. Some islets were infiltrated with CD8 dominant T-cells (Fig. 2A and B). The -cells examined ultrastructurally were adjacent to lymphocytes with morphological features of CD8 lymphocyte (18,19) and showed characteristic changes of cell death (Fig. 4). These results suggest that CD8 T-cells may play an important role in the selective destruction of islet -cells in ACP.

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Figure 3--Immunostaining of insulin and IPF-1 in pancreatic ductal cells of a patient with ACP. Insulin-containing cells (A, brown) were seen in some ductal cells that also were stained for cytokeratin 19 (B, brown). A and B are serial sections of pancreatic specimens in case 2 (400). Ductal

cells were stained with antiserum against IPF-1 (C) and insulin (D) in serial sections of pancreatic specimens in case 3. IPF-1 was heavily expressed in the nucleus and less heavily in the cytoplasm in ductal cells (C). These ductal cells contained insulin (D). C and D, 1,000.

The possibility cannot be excluded that the -cell damage is epiphenomenon of pancreatic exocrine cell damage in ACP. Ductal cells were surrounded predominantly with CD8 or CD4 T-cells and rarely with macrophages and NK cells (Fig. 1B and C). Pancreatic ductal cells surrounded by these cells were also expressed HLA class II molecules. These findings indicate that T-cells and macrophages may release cytokines and upregulate the aberrant expression of HLA class II molecules of pancreatic ductal cells. Residual -cells may be suppressed for insulin secretion by cytokines secreted by these T-cells and macrophages migrating to neighboring exocrine pancreatic tissues (20,21). In the patients with dia-

betes and ACP, insulin response to oral glucose improved remarkably after initiation of corticosteroid therapy, with resultant improved glycemic control (8). Considering the evidence present in this study and previous results (8), administration of corticosteroids also may suppress infiltration of mononuclear cells that contribute to -cell death in ACP. Corticosteroids may also suppress release of cytokines from lymphocytes and macrophages and inhibit expression of HLA class II molecules on ductal cells, blocking progression of the inflammatory cascade (22).

Some of pancreatic exocrine cells positive for cytokeratin 19, which is a marker of pancreatic ductal cells, contained insu-

lin (Fig. 3A and B). These cells have the potential to differentiate toward and replicate so as to replenish islet -cells (23? 26). Presence of ductal cells that contain insulin surrounded by fibrous tissues with infiltrating cells suggest that islet precursor cells among ductal cells could be suppressed by cytokines from these mononuclear cells and could be reversed by corticosteroid treatment.

Interestingly, in our patients with ACP, pancreatic ductal cells containing insulin expressed IPF-1/PDX-1/IDX-1, a transcription factor required for the differentiation of islet as well as for insulin gene transcription (Fig. 3C and D). This striking observation suggests that IPF-1/ PDX-1/IDX-1 plays an important role in

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Figure 4--Lymphocytes (L) were adjacent to -cell (B) and -cell (A) in case 1. The -cell in distorted and fragmented islet exhibited electron-dense cytoplasm with vacuolated and degranulated insulin granules and swollen endoplasmic reticulum, whereas morphological structure of -cell did not change. Bar, 2 m.

differentiation of endocrine cells from ductal cells exposed to immunological insults in ACP. The responsible factor for expression for IPF-1/PDX-1/IDX-1 remains unclear. In transgenic adult mice whose pancreata produced interferon-, interleukin-6, or tumor necrosis factor-, ductal cells expressed insulin (27?29) and IPF-1/PDX-1/IDX-1 (30), suggesting that cytokines play a crucial role in the expression of IPF-1/PDX-1/IDX-1 in ductal cells in patients with ACP.

Acknowledgments -- This study was supported, in part, by a grant from the Ministry of Education, Science, Sports and Culture, Japan.

We thank Dr. Y. Kajimoto (Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine) for his generous gift of antibodies against IPF-1, Dr. A. Takeshita (Department of Endocrinology and Metabolism, Toranomon Hospital) for helping the establishment of ELISA for autoantibodies against CAII, F. Takano for excellent secretarial work, and T. Hughes for editorial assistance.

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