Lymphocytic insulitis in a juvenile dog with diabetes ... - Springer Link

Animal Model

Insulitis in a Juvenile Dog with Diabetes Mellitus

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Lymphocytic Insulitis in a Juvenile Dog with Diabetes Mellitus G. Jouvion, DVM, MS,1,* J. Abadie, DVM, PHD, ECVP,1,* J. M. Bach, DVM, PHD,2 F. Roux, DVM, MS,2,3 J. Miclard, DVM,1 J. Y. Deschamps, DVM, PHD,2,3 L. Guigand,1 P. Saï, DVM, PHD,2 and M. Wyers, DVM, ECVP1 Abstract Autoimmune diabetes has never been described in a juvenile dog, whereas serological evidence has established its development in adult dogs. Diabetes mellitus was diagnosed in a 3-mo-old Donge de Bordeaux dog suffering from persistent hyperglycemia and concurrent insulinopenia. Histological analysis of the pancreas revealed inflammatory lesions in 40% of the islets of Langerhans, with infiltration predominantly by T lymphocytes (more than 90%), either at the edge (peri-insulitis: 10%) or in the islets (insulitis: 30%). The remaining 60% of the islets showed a marked atrophy due to massive beta cell loss with no loss of alpha cells. This pattern is quite similar to that observed in humans in which a characteristic insulitis containing high numbers of T lymphocytes is found in 20% of the islets at diabetes diagnosis. By contrast, in rodent models, nearly 70% of the islets of Langerhans show inflammation at diagnosis and macrophages and dendritic cells predominate in the inflammatory lesions. This is the first report of lymphocytic insulitis in a juvenile dog exhibiting diabetes mellitus. Our observations suggest an autoimmune origin for the disease in this dog that is similar to human type 1 diabetes mellitus, for which there is no accurate spontaneous large animal model. Key Words: Autoimmune disease; diabetes mellitus; dog; juvenile onset; lymphocytic insulitis.

*These authors contributed equally to this work. 1

UMR 703 INRA-ENVN, Department of Pathology, National Veterinary School of Nantes, France; 2 IECM Unit, INRA U707, University, National Veterinary School of Nantes, France; and 3Emergency and Critical Care Unit, National Veterinary School of Nantes, France. Address correspondence to Jérôme Abadie, Département de pathologie, Ecole Nationale Vétérinaire de Nantes, Atlanpôle– La Chantrerie, BP 40706, 44307 Nantes Cedex 3, France. E-mail: abadie@vet-nantes.fr Endocrine Pathology, vol. 17, no. 3, 283–290, Fall 2006 © Copyright 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/1559-0097 (Online)/ 06/17:283–290/$30.00

Introduction Diabetes mellitus (DM) is a group of metabolic disorders characterized by chronic hyperglycemia caused by either absolute or relative insulin deficiency [1,2]. Most cases of human diabetes can be classified into insulin-dependent type 1 diabetes (T1D), non-insulin-dependent type 2 diabetes, and diabetes secondary to pancreatic diseases or other endocrinopathies depending on the clinical, genetic, and immunological features [2]. It is not easy to adapt this human diabetes classification to dog diabetes, as there is no evidence for a canine equivalent of human type 2

diabetes and dogs seem mostly to develop an insulin-dependent form of diabetes, which has a latent onset [3]. In humans, T1D most commonly develops in early childhood, as the result of a severe lack of insulin secretion caused in most cases by autoimmune destruction of beta cells. Beta cell injury is known to involve both CD4+ and CD8+ T lymphocytes that recognize autoantigen-derived peptides presented either by MHC class II or class I molecules, respectively [4]. Most of the mononuclear cells that infiltrate the islets of Langerhans in recent onset diabetes are activated T cells [5]. 283

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Unlike human T1D, canine diabetes is mostly found in middle-aged and older dogs, with a peak incidence occurring at 7–9 yr of age. Juvenile onset of DM is very rarely diagnosed in dogs and represents less than 1% of all canine diabetes cases [6]. Furthermore, among the rare cases of canine juvenile DM that have been investigated histologically, insulitis, which is considered the pathological hallmark of beta cell autoimmune aggression, has never been described [3,6–9]. We report the first case of juvenile diabetes mellitus in a 3-mo-old dog exhibiting a lymphocytic insulitis and a severe atrophy of the islets of Langerhans associated with a specific beta cell loss. This case shares certain key pathological features with human juvenile T1D, for which there is not yet an accurate large animal spontaneous model. Material and Methods Clinical and Biochemical Evaluation

A 3-mo-old male Donge de Bordeaux dog was presented to the Emergency and Critical Care Unit at the National Veterinary School of Nantes. The dog was assessed by a conventional physical examination, and blood and urine samples were taken for biochemical analysis. Glycemia was assessed by reflectometry (Vettest chemistry analyzer, Idexx Laboratories Inc., Westbrook, ME, USA), fructosaminemia by spectrophotometry (FRUC, 11930010216, Roche, Basel, Switzerland), and insulinemia by radioimmuno assay (INSIK-5, P2796, Diasorin, Saluggia, Italy) at the Emergency and Critical Care Unit and at the Laboratoire des Dosages Hormonaux, Veterinary School of Nantes, France. Normal values for glycemia are 0.6 to 1.2 g/L, for fructosaminaemia they are less than 350 µmol/L, and for insulinaemia they are

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10–40 µIU/mL. Glycosuria was evaluated using a urine dipstick test (Boehringer Ingelheim, Ingelheim, Germany). Histology and Immunohistochemistry

Tissue samples from organs, including each part of the pancreas (head, tail, and body), were fixed in 10% buffered formalin and embedded in paraffin. Fivemicrometer sections were cut and stained with hematoxylin, eosin, and saffron (HES). Immunolabeling for glucagon (clone K79bB10, dilution 1:2000, Sigma, St. Louis, MO, USA), CD3 (M0452, dilution 1:200, Dako, Glostrup, Denmark), CD79 (M7051, dilution 1:50, Dako), MCA 874G (clone MAC 387, dilution 1:500, Serotec, Raleigh, NC, USA), and proliferation marker Ki-67 (clone MlB-1, dilution 1:50, Dako) was carried out with the relevant antibodies followed by an anti-mouse biotinylated secondary antibody (E433, dilution 1:300, Dako), and reactions were revealed with streptavidin–peroxidase complex (P397, dilution 1:300, Dako). The immunohistochemical reaction was developed with 3,3'-diaminobenzidine (DAB) and slides were counterstained with Mayer’s hematoxylin. Immunolabeling for insulin (A0564, dilution 1:50, Dako) was carried out using the same protocol, but with an alkaline phosphatase detection (D306, dilution 1:25, Dako) with fast red as chromogen. Lesions of the islets of Langerhans were categorized as peri-insulitis, insulitis, and atrophy without significant inflammation, and were counted from 50 randomly selected fields in each part of the pancreas (head, tail, and body) at a ×200 magnification (LUCIA imaging software, Laboratory Imaging Inc., Prague, Czech Republic). The mean surface area of the islets and of the insulin- and glucagon-secreting cells were measured by morphometric analysis


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Fig. 1. (left) Pancreas, general view. Multifocal inflammatory lesion, centered on the islets of Langerhans, and occurring over the entire organ except for the exocrine part of the pancreas. HES staining. Bar = 200 µm. Fig. 2. (right) Pancreas, islet of Langerhans. Islets show a massive lymphocytic infiltration associated with decreased and lost endocrine cells. HES staining. Bar = 40 µm.

in 30 randomly selected fields in every pancreas part and compared with those from two age-matched control dogs. Reliability was assessed by 15 repeated measurements over several days (coefficient of variation < 1.5%). Statistical Analysis

All reported data are expressed as mean ± standard deviation. The statistical significance of comparative analysis between the diabetic and control dogs was analyzed using an unpaired Student’s t test (Stat View, Abacus Concept, Berkeley, CA, USA). A value of p < 0.05 was considered as significant. Results The 3-mo-old male Donge de Bordeaux dog (10.5 kg) was presented with a 2-wk history of depression, anorexia, polydipsia, and polyuria, and with progressive weakness. Persistent hyperglycemia (demonstrated by three successive dosages, range 3.3–4.5 g/L) and hyperfructosaminemia

(534 µmol/l) associated with a marked glucosuria were detected. The dog had reduced insulinemia (14 µIU/mL). After consultation with the owner, euthanasia and a complete post-mortem examination were carried out. At necropsy, we observed no macroscopic modification of the pancreas. Its size and development were normal for the age and size of the dog. Only a diffuse, moderate hepatic glycogen and fatty storage were present, consistent with a prolonged hyperglycemic status. Histological analysis of the pancreas revealed a multifocal lesion centered on the islets of Langerhans, present in all parts of the organ but not in the exocrine part of the pancreas (Fig. 1). In 40% of the islets, the lesion consisted in inflammatory changes. Among these 40%, 10% displayed a mild inflammatory primarily lymphocytic infiltration located at the edge of the islets (peri-insulitis) with no endocrine cell destruction; and 30% showed a massive lymphocytic infiltration within the islets (insulitis), associated with reduced and lost endocrine cells (Fig. 2). The remain-

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Fig. 3. (left) Pancreas, islet of Langerhans. Marked atrophy of the endocrine tissue. HES staining. Bar = 40 µm. Fig. 4. (right) Pancreas, islet of Langerhans. Immunolabeling of insulitis lesion revealed a high percentage (> 90%) of T lymphocytes. CD3 immunolabeling (peroxidase, DAB chromogen). Bar = 40 µm.

Fig. 5. Pancreas, islet of Langerhans; Ki-67 immunolabeling (peroxidase, DAB chromogen). Bar = 40 µm. (A) Control dog. Immunolabeling revealed a very low percentage of proliferating cells in normal islets. (B) Immunolabeling revealed a high percentage of proliferating cells in the islet as well as in the peri-islet area of the pancreas.

ing 60% of the islets displayed a marked atrophy with few, if any, infiltrating inflammatory cells (Fig. 3). In all cases, we detected shrunken apoptotic cells among both endocrine and inflammatory cells, but most often when there was prominent lymphocytic infiltration. We observed no significant lesion in the exocrine tissue, except a moderately extended lymphocytic infil-

tration around the inter- and intralobular ducts and around the blood vessels, with a small increase in interstitial fibrous tissue. Immunohistochemistry revealed a high percentage (> 90%) of CD3-positive cells (T lymphocytes) (Fig. 4), and a low proportion of CD79 (B lymphocytes and plasma cells) and MCA 874G-positive cells (macrophages) (< 10%).


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Fig. 6. Pancreas, islet of Langerhans; Insulin immunolabeling (alkaline phosphatase, fast red chromogen). Bar = 40 µm. (A) Control dog. The total surface area of the insulin-secreting cells represents 2.4% of the pancreas surface area. (B) Diabetic dog. The total surface area of the insulin-secreting cells and the cytoplasmic granulation content of beta cells are considerably reduced.

Fig. 7. Pancreas, islet of Langerhans; Glucagon immunolabeling (peroxidase, DAB chromogen). Bar = 40 µm. (A) Control dog. The total surface area of glucagon-secreting cells represents 0.5% of the pancreas surface area. (B) Diabetic dog. The atrophic islets are exclusively composed of glucagon-immunopositive alpha cells with no beta cells remaining.

We observed a high proliferative activity in the diabetic pancreatic tissue when compared to the control pancreas, as assessed by Ki-67 labeling. Ki-67+ cells were mainly emigrating lymphocytes, mostly found in and around the inflammatory foci (Fig. 5). There was evident atrophy of the endocrine tissue, with the surface of the islets of Langerhans being more than three times smaller in the diabetic dog (2948 ± 1007 µm2)

than those in control dogs (11,929 ± 6752 µm2 and 9607 ± 3321 µm2 for controls) (p < 0.0001). The total surface area of insulin-secreting cells was 2.4% and 2.1% of the pancreas surface area in control animals, but was only 0.1% of the area in the diabetic dog (p < 0.0001). In the diabetic pancreas, the number of beta cells was considerably decreased and they were either isolated or found in very small groups. They had a much lower insulin-

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immunolabeling intensity than those in the control dogs, which was consistent with a decreased cytoplasmic endocrine granulation content (Fig. 6). By contrast, we observed no statistically significant difference in surface area of the glucagon-secreting cells between the diabetic dog and the controls (0.6% of the analyzed pancreas surface area vs 0.5% and 0.5% in the control pancreas). The atrophic islets with almost no lymphocytic infiltration (60% of the diabetic dog pancreatic islets) were exclusively composed of glucagon-immunopositive alpha cells with no remaining beta cells (Fig. 7). Discussion Human juvenile-onset T1D is considered a polygenic disease that is modulated by environmental factors and caused by T cell–mediated destruction of insulin-producing beta cells in the pancreas [2,10]. The infiltration of mononuclear cells in the islets of Langerhans [11] and the detection of serum autoantibodies (islet cell antibodies, and specific antibodies against glutamic acid decarboxylase, insulin, and other islet autoantigens, such as IA-2) associated with DM onset strongly suggest an autoimmune process in the development and progression of the disease [12–16]. This is the first case of lymphocytic insulitis similar to that observed in diabetic juvenile humans reported in a young dog. This insulitis was associated with atrophy of the islets of Langerhans corresponding to the specific loss of endocrine beta cells. Our findings were consistent with a selective destruction of insulin-secreting cells by autoreactive CD3+ T lymphocytes and these are the first evidence that dogs may sometimes present a diabetic pathogenesis similar to T1D in humans. Autoimmunity has previously been suggested to play a role in canine diabetes due

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to the presence of serum islet cell cytoplasmic antibodies in dogs with DM [17]. However, whether these autoantibodies can be used as autoimmune markers of the disease is doubtful, as they have also been observed in dogs following exogenous insulin treatment [18] and in dogs with endocrinopathies other than DM (e.g., hyperadrenocorticism, hypoadrenocorticism, or hypothyroidism) [17], and they are rarely detected in untreated diabetic dogs [19]. Moreover, previous histological analyses in diabetic dogs have never revealed the existence of an insulitis, even when serum-associated cytotoxicity against beta cells was observed [20]. Indeed, the lesions observed in canine insulin-dependent diabetes have been (i) congenital islet hypoplasia with no evidence of an autoimmune origin in the familial form of DM (reported in Keeshond, Samoyed dogs, and golden retrievers) [21,22]; (ii) pancreatic islet destruction by chronic relapsing exocrine pancreatitis [7,11,23,24]; and (iii) islet atrophy with endocrine cell hydropic degeneration and beta cell loss [6,7,25]. In these last cases, it has been suggested that lymphocytic infiltration may occur early in the disease process and is no longer present at diagnosis. However, Atkins et al. [6,7,26] did not find lymphocytic insulitis even in young diabetic dogs. Thus, all the previously reported cases of juvenile-onset DM in dogs show distinct pathological features to their human counterpart. The mononuclear infiltrate observed in our diabetic dog and the proportion of infiltrated islets are similar to those described in human T1D, which strongly supports the possibility of using the dog as an animal model for human T1D. Indeed, this infiltrate essentially involves CD3+ T cells and rare macrophages as is observed in recent-onset T1D in humans [5]. By contrast, in rodent T1D animal models,


macrophages and dendritic cells are highly present during the first step of pancreatic islet infiltration [27,28] and persist for a long time in the insulitis lesions [29]. Furthermore, only 30% of analyzed islets in our dog showed a characteristic insulitis, which is close to the 20% of infiltrated islets observed in humans at diabetes diagnosis [30]. This may explain why insulitis is sometimes not detected in human T1D pancreas biopsies [31]. By contrast, in the NOD mouse (non-obese diabetic mouse) model, almost 70% of the islets of Langerhans display characteristic and severe insulitis at diagnosis [32]. We are currently looking for environmental, dietary, or toxic agents that could have contributed to the islet cell damage and may have initiated the autoimmune process. We are also carrying out a pedigree analysis and a familial screening of the dog to investigate the possibility of obtaining related affected dogs for the further study of the genetic and immunological characteristics of this disease. This extensive analysis may also allow a T1D-like spontaneous diabetic dog family to be established that could provide a pertinent animal model for studying longterm preclinical preventive or therapeutic trials that are impossible in rodent models such as the NOD mouse and the BB rat (BioBreeding rat) [33]. Spontaneous canine models, being larger and having a longer life span, may prove very useful as intermediates between rodents and humans for developing novel drug-based and genetic therapeutic approaches.

3.

4.

5.

6.

7.

8.

9. 10. 11.

12.

13.

14.

References 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 29(Suppl 1):S43–48, 2006. 2. Maitra A, Abbas AK. The endocrine pancreas. In: Kumar V, Abbas AK, Fausto N, eds.

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289 Robbins and Cotran pathologic basis of disease. Philadelphia, PA: Elsevier, 2005; 1189– 1207. Catchpole B, Ristic JM, Fleeman LM, Davison LJ. Canine diabetes mellitus: can old dogs teach us new tricks? Diabetologia 48:1948–1956, 2005. Miller BJ, Appel MC, O’Neil JJ, Wicker LS. Both the Lyt-2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J Immunol 140:52– 58, 1988. Bottazzo GF, Dean BM, McNally JM, MacKay EH, Swift PG, Gamble DR. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 313:353–360, 1985. Atkins CE, Hill JR, Johnson RK. Diabetes mellitus in the juvenile dog: a report of four cases. J Am Vet Med Assoc 175:362–368, 1979. Atkins CE, LeCompte PM, Chin HP, Hill JR, Ownby CL, Brownfield MS. Morphologic and immunocytochemical study of young dogs with diabetes mellitus associated with pancreatic islet hypoplasia. Am J Vet Res 49:1577–1581, 1988. Elie M, Hoenig M. Canine immune-mediated diabetes mellitus: a case report. J Am Anim Hosp Assoc 31:295–299, 1995. Gale EA. Do dogs develop autoimmune diabetes? Diabetologia 48:1945–1947, 2005. Gale EA. The discovery of type 1 diabetes. Diabetes 50:217–226, 2001. Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 14:619– 633, 1965. Bottazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 2:1279–1283, 1974. Lendrum R, Walker G, Cudworth AG, et al. Islet-cell antibodies in diabetes mellitus. Lancet 2:1273–1276, 1976. Palmer JP, Asplin CM, Clemons P, et al. Insulin antibodies in insulin-dependent diabetics before insulin treatment. Science 222:1337– 1339, 1983. Verge CF, Stenger D, Bonifacio E, et al. Combined use of autoantibodies (IA-2 autoantibody, GAD autoantibody, insulin autoantibody, cytoplasmic islet cell antibod-

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Endocrine Pathology

16.

17.

18.

19.

20.

21.

22.

23.

24.

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ies) in type 1 diabetes: Combinatorial Islet Autoantibody Workshop. Diabetes 47:1857– 1866, 1998. Yu L, Robles DT, Abiru N, et al. Early expression of antiinsulin autoantibodies of humans and the NOD mouse: evidence for early determination of subsequent diabetes. Proc Natl Acad Sci USA 97:1701–1706, 2000. Haines DM, Penhale WJ Autoantibodies to pancreatic islet cells in canine diabetes mellitus. Vet Immunol Immunopathol 8:149–156, 1985. Haines DM. A re-examination of islet cell cytoplasmic antibodies in diabetic dogs. Vet Immunol Immunopathol 11:225–233, 1986. Davison LJ, Ristic JM, Herrtage ME, Ramsey IK, Catchpole B. Anti-insulin antibodies in dogs with naturally occurring diabetes mellitus. Vet Immunol Immunopathol 91:53–60, 2003. Sai P, Debray-Sachs M, Jondet A, Gepts W, Assan R. Anti-beta-cell immunity in insulinopenic diabetic dogs. Diabetes 33:135–140, 1984. Kimmel SE, Ward CR, Henthorn PS, Hess RS. Familial insulin-dependent diabetes mellitus in Samoyed dogs. J Am Anim Hosp Assoc 38:235–238, 2002. Kramer JW, Nottingham S, Robinette J, Lenz G, Sylvester S, Dessouky MI. Inherited, early onset, insulin-requiring diabetes mellitus of Keeshond dogs. Diabetes 29:558–565, 1980. Alejandro R, Feldman EC, Shienvold FL, Mintz DH. Advances in canine diabetes mellitus research: etiopathology and results of islet transplantation. J Am Vet Med Assoc 193:1050–1055, 1988. Minkus G, Breuer W, Arun S, et al. Ductuloendocrine cell proliferation in the pancreas of

25.

26.

27.

28.

29.

30.

31.

32.

33.

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two young dogs with diabetes mellitus. Vet Pathol 34:164–167, 1997. Gepts W, Toussaint D. Spontaneous diabetes in dogs and cats. A pathological study. Diabetologia 3:249–265, 1967. Atkins CE, Chin HP. Insulin kinetics in juvenile canine diabetics after glucose loading. Am J Vet Res 44:596–600, 1983. Rosmalen JG, Martin T, Dobbs C, et al. Subsets of macrophages and dendritic cells in nonobese diabetic mouse pancreatic inflammatory infiltrates: correlation with the development of diabetes. Lab Invest 80:23–30, 2000. Voorbij HA, Jeucken PH, Kabel PJ, De Haan M, Drexhage HA. Dendritic cells and scavenger macrophages in pancreatic islets of prediabetic BB rats. Diabetes 38:1623–1629, 1989. Reddy S, Liu W, Elliott RB. Distribution of pancreatic macrophages preceding and during early insulitis in young NOD mice. Pancreas 8:602–608, 1993. Foulis AK, Stewart JA. The pancreas in recentonset type 1 (insulin-dependent) diabetes mellitus: insulin content of islets, insulitis and associated changes in the exocrine acinar tissue. Diabetologia 26:456–461, 1984. Hanafusa T, Miyazaki A, Miyagawa J, et al. Examination of islets in the pancreas biopsy specimens from newly diagnosed type 1 (insulin-dependent) diabetic patients. Diabetologia 33:105–111, 1990. Mueller R, Krahl T, Sarvetnick N. Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 184:1093– 1099, 1996. Sai P, Gouin E. [Spontaneous animal models for insulin-dependent diabetes (type 1 diabetes)]. Vet Res 28:223–229, 1997.