The British Columbia Research Institute for Children's and Women's Health, Department of Pathology and Laboratory Medicine, University of British Columbia, #318, BCRICWH, 950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4
Correspondence
Janet Chantler
chantler{at}interchange.ubc.ca
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ABSTRACT |
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INTRODUCTION |
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The prevailing view has long been that type 1 diabetes is an autoimmune disease with genetic susceptibility linked to HLA DR3, DR4 and DQ alleles (Gale, 2001
; Lipton et al., 1992
; Nerup et al., 1976
). In part due to lack of concordance in identical twins, an environmental trigger is also thought to be involved, which has been suggested to be a virus infection or in certain instances drugs or even diet (Couper, 2001
; Yoon, 1995
). A number of viruses have been linked to the onset of type 1 diabetes including rubella and mumps viruses (Yoon & Ray, 1985
), but the strongest association is with enteroviruses and, in particular, members of the group B coxsackieviruses (CVBs) (Hyoty et al., 1998
; Szopa et al., 1993
; Ramsingh et al., 1997a
). Evidence that CVBs have a role includes epidemiological data (Hyoty et al., 1998
; Hovi, 1998
) and individual case studies that have described patients who developed diabetes shortly after a documented infection with one of the CVBs (Toniolo et al., 1988
). In particular, reports of patients who developed fatal diabetic ketoacidosis during a documented CVB infection and in whom the virus was detected in islets of Langerhans post-mortem (Iwasaki et al., 1985
; Jenson et al., 1980
) provide strong evidence for the ability of coxsackieviruses to damage the pancreas and cause diabetes. In addition, beta cells have been shown to be susceptible to coxsackievirus infection in vitro (Szopa et al., 1993
; Roivainen et al., 2000
) and various mouse strains have been reported to develop hyperglycaemia following injection with certain CVB isolates (Yoon et al., 1978
; Szopa et al., 1993
; See & Tilles, 1995
).
Epidemiological evidence suggesting a role for CVBs in the pathogenesis of diabetes includes the detection of anti-CVB IgM, indicating recent infection, in up to 50 % of patients at time of onset of symptoms, compared with approximately 5 % of age-matched controls (Frisk et al., 1992). In addition, elevated T-cell responses to CVB antigen (Jones & Crosby, 1996
) have been found and shown to display different specificities in patients who develop type 1 diabetes relative to patients who do not, potentially affecting virus clearance (Varela-Calvino et al., 2002
). The most direct evidence, however, is the detection of CVB-specific RNA in serum by RT-PCR in 4060 % of recently diagnosed patients with insulin-dependent diabetes (Clements et al., 1995
; Andreoletti et al., 1997
). Therefore, there is substantial evidence for the presence of coxsackieviruses but what is currently unclear is the underlying mechanism of pathogenesis. In the rare fatal cases, fulminant infection of the pancreas and consequent destruction of both exocrine and endocrine tissue occurs, but if coxsackieviruses have a wider role in type 1 diabetes, how does virus infection induce a slow depletion of beta cells consistent with the normal onset of this disease, where there is a prolonged subclinical period of months or years during which some islet function is preserved? Moreover, what is the role of autoimmunity, in the form of both autoreactive T cells and islet cell antibodies, which are a hallmark of the majority of cases of type 1 diabetes?
To address the question of how CVBs cause beta-cell depletion, we have undertaken a detailed study of the pathological events occurring in the pancreas following CVB infection of SJL mice, a mouse strain that has previously been shown to be susceptible to virus-induced diabetes (Yoon et al., 1978). A comparison of the pancreatic damage caused by the diabetogenic E2 strain of CVB4 with that induced by the non-diabetogenic JVB strain was carried out with the purpose of defining the pathogenic properties of each strain that lead to the differences in disease outcome. The results suggest that the fundamental cause of beta-cell depletion in the E2-infected animals is lack of islet neogenesis, rather than virus-induced beta-cell death, although some islets are infected and this undoubtedly accelerates the process of beta-cell loss.
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METHODS |
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Animal care and virus inoculation.
Five-week-old SJL/J male mice (n=6) were obtained from Jackson Laboratories and housed in a Level 3 containment facility at the BCRICWH. They were acclimatized for 1 week before inoculation intraperitoneally (i.p.) with 105 p.f.u. of either CVB4 (JVB) or CVB4 (E2) diluted in PBS to a final volume of 0·2 ml per mouse. Control mice were injected with an equal volume of PBS. Mice were sacrificed on selected days between day 1 and day 95 post-infection (p.i.) and tissues (pancreas, heart, liver and spleen) as well as heart blood were collected. One half of each tissue was snap-frozen in dry ice and stored at -70 °C for virus titration, while the other half was fixed in 4 % formaldehyde in sodium phosphate buffer, pH 7·4, for histopathological examination and immunohistochemistry. For mice that were supplemented with exocrine enzymes, each pellet was fortified with 150 µl of enzyme suspension containing 30 mg lipase, 50 mg amylase, 15 mg cellulase, 50 mg papain and 30 mg bromelain. Food intake was monitored daily by weighing the pellets provided and those remaining after 24 h.
Serum glucose measurements.
Heart blood was obtained at sacrifice from all animals. In addition, another set of age-matched mice was inoculated as part of a longitudinal experiment to monitor blood glucose levels in individual animals over time (between days 1 and 95 p.i.). Blood was collected from the tail vein or the saphenous vein in a hind leg and serum glucose was measured by the glucose Trinder assay.
Insulin ELISA.
The whole pancreas of selected mice was removed and homogenized in 1 ml of cold (-20 °C) acid-alcohol homogenizing solution. The homogenate was centrifuged at 12 000 r.p.m. to remove tissue debris and the concentration of insulin measured using a sandwich ELISA, with streptavidinHRP as secondary antibody and TMB as substrate. Absorbance was read at 450 nm using a microtitre plate reader (SpectraMax 190).
Histopathological examination.
Tissues were fixed in 4 % PF at 4 °C overnight and stored in 70 % ethanol until processing. They were embedded in paraffin, sliced into 3 µm sections with a Leica microtome and mounted on slides for immunohistochemical staining, in situ hybridization or histological staining with Masson's trichrome. Regions of fibrosis and connective tissue deposition stained blue against a pink-purple background tissue stain. Necrotic areas in the pancreatic acinar tissue stained pale pink.
Immunohistochemistry.
The sections of paraffin-embedded tissue were dipped in xylene and rehydrated in graded alcohols (100 %, 70 %, 50 % ethanol). Endogenous peroxidases were quenched with 3 % H2O2 and non-specific proteins were blocked with 2 % BSA in PBS. Primary antibodies for insulin (Dako), glucagon (Dako), pIAPP (provided by B. Verchere, Vancouver) or viral antigen (Chemicon) were diluted in blocking buffer and 25 µl was added per section. The slides were incubated at 37 °C for 1 h in a humidified chamber, washed with PBS and then for 30 min with 100 µl HRP-conjugated secondary antibody diluted 1 : 500 in blocking buffer. After a further PBS wash, the DAB substrate was added for 10 min at room temperature. Sections were counterstained with haematoxylin (blue nuclear stain) and then mounted under coverslips.
Immunofluorescent staining for insulin and Glut-2.
Tissue sections were treated with xylene and rehydrated in ethanol as described above. The slides were then blocked with 2 % normal goat serum, 0·2 % Tween 20 (polyoxyethylenesobitan monolaurate), 2 % BSA and 7 % glycerol for 30 min. After washing in PBS, the slides were incubated in rabbit anti-insulin (Dako) or anti-Glut 2 antibody (Chemicon) for 1 h, washed again and incubated with a secondary antibody conjugated to fluorescein isothiocyanate or Alexa-Fluor red 594 for insulin or Glut-2, respectively. After mounting, the slides were viewed under a Zeiss epifluorescent microscope with a digital image capturing system.
In situ hybridization.
The tissue sections were baked overnight at 60 °C, deparaffinized using xylene and rehydrated in graded alcohols. The tissues were permeabilized using 0·2 M HCl, 2x SSC, 20 mM Tris/HCl (pH 7·4) containing 2 mM calcium chloride and 1 µg proteinase K ml-1 and then quenched in 0·25 % acetic anhydride containing 0·1 M triethanolamine. The slides were then dehydrated using graded alcohols. The hybridization solution (25 µl) containing 100 ng ml-1 of the sense or antisense probes labelled with digoxigenin (Boehringer Mannheim) was added to each section, glass coverslips were overlaid and the slides were placed in a sealed humidified dish at 42 °C overnight. Post-hybridization washes were performed overnight using 50 % formamide, 10 mM Tris/HCl (pH 7·4), 1 mM EDTA and 600 mM NaCl in a 56 °C rocking water-bath, followed by several washes in 2x SSC. The slides were equilibrated in buffer 1 containing 0·15 M NaCl and 0·1 M Tris/HCl (pH 7·5) and blocked with 2 % lamb serum. Development was carried out according to the Boehringer Mannheim instructions for digoxigenin-labelled probes. The slides were counterstained with eosin and were examined with a light microscope for a positive reaction indicated by a blue-black colour.
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RESULTS |
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In order to quantify the levels of insulin in JVB- and E2-infected animals relative to controls, the total insulin content of pancreata from a group of mice (n=5) at selected timepoints up to day 55 was measured by ELISA assay (Fig. 6B). On day 3, the E2-infected mice contained reduced levels of insulin (approximately 50 % of controls) and by day 10 this had further diminished to around 10 %, remaining at around this level up to day 55. The JVB-infected pancreata also contained diminished levels of insulin on day 3 (70 % of controls) and this was further reduced to approximately 50 % of normal levels on day 10, remaining at 5070 % up to day 55. In subsequent experiments we found that total insulin in JVB-infected mice never returned to control levels but stabilized at between 60 and 80 % that of the controls up to day 95, while following E2 infection the insulin levels remained at around 1015 % of the control group.
To determine whether the beta cells in E2-infected pancreata retained other functional properties, we also immunostained for islet amyloid polypeptide (pIAPP), a beta-cell-specific protein, which is normally secreted together with insulin. As shown in Fig. 6(A, b, e, h and k), pIAPP immunostaining was strongly positive up to day 95 indicating that the remaining beta cells still retain some secretory function. TUNEL staining, a measure of cells undergoing apoptosis, also showed that the majority of cells within the E2-infected islets were viable on day 35, while we have found increasing numbers of TUNEL-positive cells up to day 95 (not shown).
Does JVB or E2 infection affect glucose homeostasis?
Following the acute stage of infection, both E2- and JVB-infected mice became active and resumed normal feeding. The E2 mice, however, started to lose weight and were significantly smaller by day 35 p.i. We measured non-fasting serum glucose levels in saphenous vein blood and found that E2-infected mice were extremely hypoglycaemic, despite the lack of insulin synthesis (Fig. 7A). To determine whether this was due to the lack of pancreatic exocrine function limiting digestion, mice were supplemented with pancreatic enzymes (including trypsin, chymotrypsin, lipase and amylase) from the time of infection. Blood glucose levels were measured, following heart bleed, at time of sacrifice (shown as a scatter plot in Fig. 7B
). Even some of the unsupplemented E2-infected mice were found to be hyperglycaemic when blood was drawn directly from the heart instead of from the saphenous vein. In addition, the enzyme-supplemented mice became significantly hyperglycaemic after day 10 and up to day 56, the final timepoint of the experiment. The surviving islets stained more strongly for insulin in the presence of supplementation, suggesting that the reduced insulin synthesis in E2-infected animals was at least in part due to a regulatory response to the low glucose levels. However, even after enzyme supplementation, total pancreatic insulin remained low and insufficient to maintain glucose homeostasis, so that hyperglycaemia resulted.
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DISCUSSION |
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Glucose dysregulation was therefore induced by CVB4 but whether this manifested as high or low blood glucose levels depended on the availability of pancreatic digestive enzymes and in turn dietary glucose. When E2-infected animals were supplemented with enzymes normally supplied by the exocrine pancreas, they became reproducibly hyperglycaemic, while the unsupplemented mice were hypoglycaemic as dietary insufficiency results in glucose starvation of the tissues. Under these circumstances, the liver becomes the chief source of glucose through a combination of glycogenolysis and gluconeogenesis (Nordlie et al., 1999), but this process does not compensate for the lack of nutritional intake and the blood glucose levels remain low.
Many small islet clusters were seen in JVB-infected pancreata, indicative of islet neogenesis. The majority of these appeared to originate from tubular or ductal tissue that stained for the glucose transporter Glut-2, a marker for both beta cells and precursors (Fig. 3). However, the total pancreatic insulin content following infection with the non-diabetogenic JVB strain did not return to normal levels, at least for the 95 days of observation, suggesting that these animals may also be susceptible to further insult to the pancreas, either by a second virus infection, drugs or even diet. This may explain the conundrum of why the epidemiological evidence incriminates a number of CVB strains in inducing type 1 diabetes, while in experimental systems, few coxsackievirus isolates have been shown to infect islets in animal models. If the key to whether glucose dysregulation occurs lies in the degree to which the exocrine tissue is destroyed and islet neogenesis prevented, then a number of coxsackievirus strains (and other enteroviruses) could play a role (Hyoty et al., 1998
). Moreover, successive infections with different viruses may cause accumulative damage, which could eventually lead to insulin insufficiency, an idea first promoted by A. L. Notkins and collaborators over 20 years ago (Toniolo et al., 1980
).
In E2-infected pancreata in SJL mice, the role of the immune system appeared to involve clearing out the remnants of infection and enhancing tissue recovery. Widespread invasion of islets by lymphoreticular cells after infectious virus was no longer detectable, which would be suggestive of an autoimmune reaction, was not observed. On day 3 p.i., NK cells, involved in innate defence against viruses (Biron & Brossay, 2001), were the major cell type identified in both E2- and JVB-infected pancreata, as has been reported by others (Vella & Festenstein, 1992
). By day 5, the infiltrate comprised largely CD45+ cells, but to our surprise the numbers of infiltrating cells were much higher in JVB-infected pancreata. These lymphocytic cells were dispersed throughout the acinar tissue and around islets, and their presence in juxtaposition to regenerating acinar tissue and islet neogenesis suggests that they may play a role in tissue recovery resulting in the pancreas in JVB-infected mice appearing normal by day 10 p.i. Immune-mediated killing of infected cells undoubtedly occurred but the overall result of lymphocytic infiltration was the restriction of further virus damage followed by tissue repair. In contrast, in the E2-infected animals far fewer infiltrating cells were seen, despite higher levels of virus replication and tissue damage. Perhaps the greater virulence of E2 leads to cytopathology before the infected cells can synthesize cytokines such as interferons to limit virus replication and chemokines to attract lymphocytes into the tissue. The particular importance of interferon-
in protecting islets from CVB4-induced damage was recently shown by N. Sarvetnick and colleagues (Flodstrom et al., 2002
). The rapidity with which E2 infects and spreads throughout the exocrine pancreas may prevent the tissue from mounting an effective antiviral response in time.
The pathogenesis of E2 that we observed in 6-week-old SJL mice closely mimics the disease seen in the patient from whom this CVB4 variant was isolated (Yoon et al., 1979). A more fundamental question is whether our findings support the hypothesis that CVB infections are more widely involved in the onset of type 1 diabetes, a disease long held to be due to autoimmune attack on beta cells by autoreactive T cells. Our results did not indicate that autoimmunity was a major component of the islet damage seen in E2-infected animals. However, recent studies have defined a group of diabetic patients who lack autoreactive antibodies or T cells indicating that autoimmunity is not a prerequisite of the disease. This idiopathic type 1B diabetes comprises between 5 and 20 % of cases of insulin-dependent diabetes in different ethnic populations (Gavin, 2002
). It is associated with younger children and abrupt onset with rapid loss of beta-cell capacity (Urakami et al., 2002
). There is no evidence of autoimmunity, while a history of recent virus-like illness is common. In one study where pancreatic biopsy specimens were examined, T-cell infiltrates into the exocrine tissue were seen but no insulitis and high serum pancreatic enzyme concentrations suggested that damage to exocrine tissue had occurred (Imagawa et al., 2000
). Others have also found high pancreatic enzyme activities in more than 25 % of patients at the time of diagnosis (Semakula et al., 1996
). Our findings with E2 fit well with this type of disease onset.
The fact that the pancreata of 6-week-old mice infected with the non-diabetogenic JVB strain did not regain normal insulin levels raises the possibility that any virus that causes exocrine damage may reduce the ability of the pancreas to reconstitute islet tissue and avert glucose dysregulation. It is possible that the pancreas has a finite capacity to regenerate and that successive insults (different virus infections, perhaps in conjunction with immune-mediated pathology, diet or alcohol) eventually result in an inability to replace damaged tissue. Diabetes would then ensue when insufficient islet neogenesis occurred to maintain glucose homeostasis. In this regard, coxsackievirus-induced diabetes may share similarities with HIV infections where AIDS results only when the capacity to replace CD4 cells is exhausted. Similarly, insulin insufficiency may only become apparent when the beta-cell precursors are no longer available to replenish cells lost due to successive virus infections or indeed to a single fulminant infection in the case of a virulent strain such as E2. This hypothesis invokes a role for all pancreatropic viruses and not just rare variants with a particular tropism for beta cells, which fits much better with the epidemiological data reported (Roivainen et al., 1998; Hyoty et al., 1998
).
While our study switches the focus of coxsackievirus-induced diabetes from the islet and beta-cell destruction to the exocrine tissue and islet neogenesis, other factors most likely play a role. CVB infections in humans are known to be associated with production of autoantibodies and autoreactive T cells that could accelerate islet destruction (Atkinson et al., 1994; Peakman et al., 2001
), and molecular mimicry between CVB antigens VP1 and 2C and two of the major autoantigens identified in type 1 diabetes, IA-2 and GAD65, may play a role in this (Harkonen et al., 2002
; Lonnrot et al., 1996
). In individuals with a genetic predisposition to autoimmune reactions, direct killing of beta cells by virus, while limited, may be sufficient to stimulate pre-existing autoreactive T cells that may participate in islet destruction (Horwitz et al., 2001
). Related to this, bystander activation of immune cells drawn to the islets to eliminate virus could result in induction of cytokines such as TNF
, IL1 and interferon-
, which are known to be capable of triggering beta-cell death, and this could also accelerate the onset of insulin insufficiency (Horwitz et al., 1998
). The extent to which direct damage to the pancreas or islets, by virus or other environmental triggers, plays a role as opposed to immune-mediated damage by cytokines or self-reactive T cells probably varies between patients of differing genetic backgrounds. Thus, while we did not find evidence of insulitis in uninfected islets in the SJL mouse strain, autoimmune reactions may have a role in mice with different genetic backgrounds and have a role in CVB-induced disease in humans.
In conclusion, the results reported lead us to propose a novel mechanism for coxsackievirus-induced diabetes, which focuses on the prevention of islet replacement by neogenesis rather than beta-cell destruction by either virus itself or virus-induced autoimmune mechanisms. The remarkable regeneration of pancreas that occurs in JVB-infected mice lends optimism to the development of treatment strategies for type 1 diabetes based on limitation of acute virus-induced damage and treatment with growth factors and/or stem cells to encourage regeneration of both acinar and islet tissue.
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ACKNOWLEDGEMENTS |
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Received 7 February 2003;
accepted 9 August 2003.