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Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
Target cell defense prevents the development of diabetes ...
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Target cell defense prevents the development of diabetes ...

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  • 1. A RTICLES Target cell defense prevents the development of diabetes after viral infection © 2002 Nature Publishing Group http://immunol.nature.com Malin Flodström1, Amy Maday1, Deepika Balakrishna1, Mary Malo Cleary1, Akihiko Yoshimura2 and Nora Sarvetnick1 Published online: 4 March 2002, DOI: 10.1038/ni771 The mechanisms that regulate susceptibility to virus-induced autoimmunity remain undefined. We establish here a fundamental link between the responsiveness of target pancreatic β cells to inter- ferons (IFNs) and prevention of coxsackievirus B4 (CVB4)-induced diabetes. We found that an intact β cell response to IFNs was critical in preventing disease in infected hosts. The antiviral defense, raised by β cells in response to IFNs, resulted in a reduced permissiveness to infection and subse- quent natural killer (NK) cell–dependent death.These results show that β cell defenses are critical for β cell survival during CVB4 infection and suggest an important role for IFNs in preserving NK cell tolerance to β cells during viral infection. Thus, alterations in target cell defenses can critically influence susceptibility to disease. Both genetic and environmental factors are involved in the etiology addition, CVB antigens have been found in residual β cells from of autoimmune disease and viral infections have been implicated as humans who succumbed to a lethal virus infection7,8,10, and nongenetic triggers of autoimmune reactions to self. Type 1 diabetes enteroviruses, including CVBs such as CVB4, have been isolated from is an immune-mediated disease that results from selective loss of the newly diagnosed type 1 diabetes patients10,15,22,23. These reports togeth- insulin-producing pancreatic β cell. Numerous epidemiological and er with the in vitro findings described above, raise the possibility that clinical studies have linked enteroviral infections, particularly infec- β cell permissiveness to CVB4 infection may, in part, regulate suscep- tions with coxsackievirus B4 (CVB4), with a progression to type 1 tibility to CVB4-induced diabetes. Until now, the host factors regulat- diabetes1–3. Based on animal studies, different models for virus- ing the permissiveness of pancreatic β cells to CVB4 infection have induced reactions to self, including CVB4-induced diabetes, have not been fully explored. been proposed. They include molecular mimicry, bystander activa- Interferon-α (IFN-α), IFN-β and IFN-γ are produced early during tion of self-reactive T cells and a direct viral cytolysis of infected tar- viral infections, including infection with picornaviruses (for example, get cells4–6. Although coxsackie viral antigens have been found in the encephalomyocarditis virus24 and CVB4, M. Flodström and N. pancreatic β cells of newly affected type 1 diabetic patients7–9, little Sarvetnick, unpublished data). By inducing an antiviral state in IFN- is known about the antiviral defenses generated by target β cells or responsive target cells, IFNs minimize the permissiveness of target how these defenses can regulate susceptibility to diabetes induced by cells to viral infection and/or replication25–27. We sought to understand viral infection. the role played by IFNs in regulating the cellular permissiveness of In vitro, CVB4 and other members of the CVB family infect human pancreatic β cells to CVB4 infection. We assessed the relevance of and rodent β cells and many such infections result in widespread β cell IFN-induced β cell–specific antiviral defenses in regulating CVB4- death10–15. In contrast, studies with mice have revealed that although induced diabetes. We show that the β cells critically depended on IFNs systemic CVB4 infection can cause almost complete destruction of the to lower their permissiveness to CVB4 infection. We also show that in exocrine pancreas, the pancreatic islet cells, including β cells, are mice with pancreatic β cells that had defective IFN responses, CVB4 selectively spared from CVB4-induced pathology16–19. These observa- caused an acute form of diabetes, which resembled the type 1 diabetes tions show that although there is strong viral tropism for the exocrine that develops in humans after severe enteroviral infection. Finally, we pancreas during systemic infection, the net infectivity of β cells show that permissiveness to CVB4 infection resulted in significant appears to be very low. Accordingly, the majority of systemic infec- β cell damage and an increased susceptibility to natural killer (NK) tions with CVBs are cleared without β cell destruction and develop- cell–dependent death in vivo. Thus, our findings show that the β cell ment of diabetes17,19–21. Nonetheless, several reports of diabetes that itself contains critical circuits that control its survival, which suggests occurred in close association with a CVB infection suggest that infec- that target cell defenses can regulate autoimmune reactions triggered by tion in susceptible individuals may still lead to β cell destruction3,6. In viral infections. 1 Department of Immunology, IMM-23,The Scripps Research Institute, 10550 N.Torrey Pines Road, La Jolla, CA 92037, USA. 2Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Correspondence should be addressed to N. S. (noras@scripps.edu). http://immunol.nature.com • april 2002 • volume 3 no 4 • nature immunology 373
  • 2. A RTICLES Figure 1. STAT1 is phosphorylated in Similar to their non-Tg littermates, some of the islets in SOCS-1–Tg insulin-producing cells after stimula- NOD mice showed a mild peri-insulitis or insulitis at 8 weeks of age tion with IFNs. NIT-1 cells were exposed (Table 1). In addition, the first incidence of spontaneous diabetes in to IFN-α (α) or IFN-γ (γ). STAT1 phospho- rylation (PY-STAT) 10 or 20 min after expo- SOCS-1–Tg NOD mice paralleled that in non-Tg littermates at the age sure was assessed by immunoblotting. Data of 15 weeks (data not shown). are representative of two independent experiments. CVB4 replicates in SOCS-1–expressing islet cells CVBs can infect and replicate in isolated human and rodent © 2002 Nature Publishing Group http://immunol.nature.com Results β cells10,11,13–15,35. We tested the ability of IFNs to inhibit CVB4 replica- Activation of the Jak-STAT signaling pathway tion in primary islet cells (Fig. 3). In initial experiments, pancreatic Upon binding to their respective receptors, type I (IFN-α and IFN-β) islets were isolated from the two murine strains C57BL/6 and NOD and type II (IFN-γ) IFNs transmit their intracellular signals through the (Fig. 3a,b). The islets were treated with IFN-α (1000 U/ml) or PBS for phosphorylation of Janus kinases (Jaks) and the signal transducers and 24 h and then infected with CVB4. When no IFN-α was added, the activators of transcription (STAT) family of transcription factors28. islets from both strains continuously produced infectious CVB4 (Fig. Because IFN-γ activates the Jak-STAT pathway in insulin-producing 3a,b). On day 6 after infection, light microscopy showed that most of cells29, we tested whether type I IFNs could also initiate a response in the islets had lost their integrity (data not shown). In contrast, viral β cells by measuring the phosphorylation of STAT1 in NIT-1 cells, a titers from IFN-α–treated islets remained two to three logs lower dur- pancreatic β cell line established from nonobese diabetic (NOD) ing the 6-day study period (Fig. 3a,b) and the islets retained their mice30. We found that STAT1 phosphorylation increased in cells round dense structure (data not shown). IFN-γ (1000 U/ml) also inhib- exposed to IFN-α or IFN-γ compared to control cells, which were not ited the generation of infectious CVB4 in pancreatic islets (data not exposed to IFNs (Fig. 1). Therefore, insulin-producing β cells respond shown). To verify the biological relevance of the IFN action on islet to both type I and type II IFNs. cells, islets from mice that lacked intact IFN receptors (IFN-αβγR–/– mice)36 and their wild-type counterparts were treated with IFN-α or Mice with IFN-resistant β cells PBS for 24 h, then infected with CVB4. In wild-type islets, IFN-α To determine whether IFN responsiveness is critical for pancreatic treatment significantly reduced the production of infectious CVB4 β cell survival during a systemic infection with CVB4, we generated (Fig. 3c, P<0.05) and loss of integrity (data not shown). IFN-α treat- transgenic (Tg) NOD mice that expressed the suppressor of cytokine ment did not inhibit CVB4 replication and islet degeneration in IFN- signaling 1 (SOCS-1)31,32 under the control of the human insulin pro- αβγR–/– islets (Fig. 3d and data not shown). Taken together, these data moter33. SOCS-1 is a negative regulator of IFN signaling that acts by show that both type I and II IFNs induced the rapid transition of pan- inhibiting Jak1 and Jak231,32; expression of SOCS-1 blocks IFN- creatic islet cells to an antiviral state, which was critical for a block in γ–induced STAT1 activation in an insulin-secreting cell line34. CVB4 replication in vitro. Transgene expression in both the two SOCS-1–Tg NOD lines (A and To determine whether IFNs have a biological effect on the SOCS- B) that we had established was confirmed by immunohistochemistry 1–expressing cells, pancreatic islets were isolated from SOCS-1–Tg (Fig. 2 and data not shown). Although SOCS-1 was absent from islet NOD mice and their non-Tg littermates. In culture media from cells and exocrine tissue from non-Tg NOD mice (Fig. 2a), as well as infected non-Tg islet cells, IFN-α and IFN-γ inhibited, in a dose- from the exocrine tissue of their SOCS-1–Tg littermates (Fig. 2d), we dependent manner, the generation of infectious CVB4 (Fig. 3e and observed SOCS-1 expression in the islets of Tg mice (Fig. 2d). The data not shown). In contrast, CVB4 replication proceeded with little structure of the pancreatic islets in SOCS-1–Tg NOD mice was nor- or no inhibition in IFN-α–treated (Fig. 3f) or IFN-γ–treated (data not mal and insulin+, glucagon+ (Fig. 2e,f) and somatostatin+ cell distribu- shown) SOCS-1–Tg islet cells. In addition, the infection-induced tion (data not shown) was similar to that in non-Tg mice (Fig. 2b,c). loss of islet integrity in SOCS-1–Tg islets treated with IFN-α or a b c d e f Figure 2. SOCS-1 is expressed in pancreatic islets from SOCS-1–Tg NOD mice, but is absent in islets from non- Tg littermates. Paraffin sections of formalin-fixed pancreata from 8-week-old (a–c) non-Tg and (d–f) Tg mice were stained with antibodies to (a,d) SOCS-1 (b,e) insulin or (c,f) glucagon. Original magnifications were (a,d) ×20 and (b,c,e,f) ×40. 374 nature immunology • volume 3 no 4 • april 2002 • http://immunol.nature.com
  • 3. A RTICLES Figure 3.An intact β cell response to IFNs a b g h is critical for the inhibition of CVB4 repli- cation in vitro. Pancreatic islets were isolated from (a) C57BL/6 (b) NOD (c) 129S6/SvEv (IFN-αβγR+/+) (d) IFN-αβγR–/– (e) non-Tg NOD and (f) SOCS-1–Tg NOD mice that were infect- ed with CVB4 in vitro. Replication of CVB4 was impaired after treatment with IFN-α. (d) This effect was dependent on intact IFN receptors © 2002 Nature Publishing Group http://immunol.nature.com and (f) was prevented by SOCS-1 expression. c d Data are mean±s.e.m. from two to eight inde- pendent experiments. *P<0.05 and **P<0.01 ver- i j sus PBS-treated islets from the same mouse strain. (g–l) CVB4 infection caused ultrastruc- tural changes in pancreatic islet cells. Pancreatic islet cells were taken from (g,i,k) non-Tg or (h,j,l) SOCS-1–Tg NOD mice after 5 days of exposure (g–h) to IFN-α alone (i–j) CVB4 infection for 4 days or (h–l) exposure to IFN-α e f for 24 h followed by infection with CVB4 for 4 days. (k) Islet cells from non-Tg NOD mice were protected from CVB4-induced damage when treated with IFN-α. (l) In contrast, IFN-α k l treatment did not prevent islet cell destruction in SOCS-1–Tg NOD mice. Images are repre- sentative of two independent experiments. Original magnification: ×5200. IFN-γ was as severe as the degeneration in islets infected with CVB4 and ruptured plasma membranes (Fig. 3i,j,l). Others showed the hall- alone. Indeed, replication (Fig. 3f) and islet degeneration (data not marks of apoptosis37, including deformed nucleoli, partial chromatin shown) resembled that in IFN-α–treated islet cells from IFN- condensation and enlarged peri-nuclear spaces (Fig. 2j,l). In addition, αβγR–deficient mice (Fig. 3d and data not shown). Uninfected islet many β cells had undergone some degree of degranulation. In contrast, cells treated with IFN-α or IFN-γ remained intact for the 6-day study no such cellular destruction affected non-Tg islets subjected to IFN-α period, as determined by light microscopy and/or electron micro- treatment and CVB4 infection (Fig. 3k). In fact, the ultrastructure of scopy (data not shown). these islets was nearly indistinguishable from that of uninfected cells Because the light microscopy analysis showed degenerative changes treated with IFN-α alone (Fig. 3g) or PBS (data not shown). IFN-α did in the infected islets, an ultrastructural analysis was done. This analy- not rescue SOCS-1–Tg islet cells from severe degenerative changes sis revealed a considerable number of dead cells in CVB4-infected (Fig. 3l). Indeed, at day 4 p.i. the injury to those islets resembled that islets from both SOCS-1–Tg NOD mice and their non-Tg littermates of Tg islets infected with CVB4 alone (Fig. 3j,l). This islet cell destruc- on day 4 post-infection (p.i.) (Fig. 3i,j). Some cells showed the features tion appeared to correlate with the replication of CVB4 (Fig. 3f). These of virus-mediated cytopathic effects, such as cytoplasmic degeneration experiments showed that ectopic expression of SOCS-1 rendered β cells insensitive to IFN stimulation. a b CVB4 induces diabetes in SOCS-1–Tg NOD mice To determine the consequences of defective β cell responses to IFNs during systemic CVB4 infection, SOCS-1–Tg NOD mice and their non-Tg littermates were infected with CVB4 at 8–9 weeks of age (at least 6 weeks before the spontaneous onset of diabetes in both groups) and were examined. At this age, the nonfasting blood glucose concen- trations in uninfected SOCS-1–Tg NOD mice were 127±4 mg/dl (n=20) and 114±6 mg/dl (n=20) for lines A and B, respectively, where- as in non-Tg littermates it was 117±4 mg/dl (n=20). In agreement with published data17, non-Tg mice developed severe hypoglycemia 3 or 4 days after virus inoculation and 4–6 days p.i. 7/40 (18%) mice died. By 12 days p.i., only 1/33 (3%) surviving mice became hyperglycemic Figure 4. Acute onset of diabetes in SOCS-1–Tg NOD and NOD-SCID (Fig. 4a and data not shown). The rest of the mice remained normo- or mice infected with CVB4. (a) Non-Tg (n=5) and (b) SOCS-1–Tg (n=5) NOD mice were infected with CVB4, although only SOCS-1–Tg mice developed diabetes hypoglycemic (data not shown). In contrast, after infection with after infection. Data are representative of 40 and 21 CVB4-infected non-Tg and Tg CVB4, most of the SOCS-1–Tg NOD mice developed early and severe mice, respectively. hyperglycemia; after initial hypoglycemia during the first 3–4 days http://immunol.nature.com • april 2002 • volume 3 no 4 • nature immunology 375
  • 4. A RTICLES a b c d © 2002 Nature Publishing Group http://immunol.nature.com Figure 5. Insulin-staining β cells are lost in SOCS-I–Tg NOD mice infected with CVB4. Representative immunostaining of pancreatic sections from (a,b) non-Tg and (c,d) SOCS-1– Tg NOD mice infected with CVB4 and killed 8–10 days later. Consecutive serial sections were stained with antibodies to (a,c) insulin or (b,d) glucagon. (a,b) The pancreas from a non-Tg mouse had intact islets with both (a) insulin+ and (b) glucagon+ cells. (c,d) The pancreas of a SOCS-1–Tg mouse had a severely distorted islet with (c) only a few insulin+ cells but (d) near to normal numbers of glucagon+ cells. Sections are representative of pancreata from at least ten infected animals. Original magnification: ×40. p.i., their blood glucose concentrations increased greatly, usually disrupted, and the number of insulin-staining β cells was greatly exceeding 500 mg/dl (27.5 mM) by day 6–8 p.i. (Fig. 4b). Only 1/21 reduced (Fig. 5c). These islets showed severe insulitis (Table 1) or (5%) Tg mice succumbed to the viral infection before day 6 p.i., but were atrophied, containing mainly glucagon+ (Fig. 5d) and somato- the accumulated incidence of diabetes among the survivors was 95% statin+ (data not shown) cells. Hence, the infected mice whose pancre- (19/20) at 12 days p.i. (P<0.01, non-Tg versus SOCS-1–Tg NOD mice atic β cells were incapable of responding to IFNs lost their insulin+, but by χ2-test). not glucagon+ and somatostatin+ islet cells after CVB4 infection. CVB4 infection may precipitate autoimmune-mediated destruction Therefore, as a consequence of the β cell loss, the SOCS-1–Tg NOD of pancreatic β cells in NOD mice38, so we crossed the SOCS-1–Tg mice developed diabetes. NOD mouse onto a C57BL/6 background for one generation. (C57BL/6×NOD)F1 mice do not develop diabetes or insulitis39, and we CVB4 infects SOCS-1–expressing β cells in vivo found that no diabetes occurred in CVB4-infected non-Tg We next determined whether β cell destruction in SOCS-1–Tg NOD (NOD×C57BL/6)F1 mice after CVB4 infection (n=4). In contrast, the mice correlated with a general increase in CVB4 replication in the host SOCS-1–Tg (NOD×C57BL/6)F1 mice rapidly developed hyper- and/or with a specific increase in the pancreatic islets. Inoculation of glycemia, and 4/5 mice were diabetic by day 12 p.i. (data not shown). mice with CVB4 typically results in rapid dissemination of the virus to Hence, the rapid onset of diabetes was not restricted to SOCS-1–Tg vital organs such as the liver, kidneys, spleen, heart and pancreas19,40. mice on a pure NOD background. We measured high titers of replicating virus in several organs on day 3 Histological evaluation of the pancreata showed that, although the p.i., a time when viral titers generally peak in such organs19,40. We found exocrine tissue had undergone massive degeneration in the infected that the titers in pancreata, spleens, kidneys and livers, respectively, SOCS-1–Tg NOD mice and their non-Tg littermates, marked differ- were similar in both groups; data are mean±s.e.m. log10 plaque-forming ences were apparent. In non-Tg mice, most pancreatic islets were units (PFU) per gram of tissue. SOCS-1–Tg NOD mice (n=3): intact, with a normal distribution of insulin+, glucagon+ (Fig. 5a,b) and 12.1±0.3, 9.1±0.3, 7.3±0.2 and 7.4±0.3; non-Tg littermates (n=3): somatostatin+ cells (data not shown). The degree of insulitis and islet 11.6±0.3, 8.7±0.3, 7.1±0.4 and 7.7±0.7. Also on day 4 p.i. (when hyper- destruction (Table 1) was similar to that in uninfected non-Tg NOD glycemia was first observed in infected SOCS-1–Tg NOD mice, see mice (Table 1). However, the islets of SOCS-1–Tg NOD mice were Fig. 4b), the viral loads in pancreata from both groups were similar. Table 1. Histological analysis of pancreatic sections from non-Tg and SOCS-1–Tg NOD mice that were untreated or treated with anti–AGM-1 and infected with CVB4 Insulitis rank Number of mice Number of islets CBV4 infection Anti–AGM-1 A+Ba C D Line A Non-Tg NOD 5 128 – – 77% (99) 19% (24) 4% (5) Non-Tg NOD 3 30 + – 73% (22) 27% (8) 0% (0) SOCS-1–Tg NOD 5 116 – – 81% (94) 11% (13) 8% (9) SOCS-1–Tg NOD 5 90 + – 0% (0) 11% (10) 89% (80) Line B Non-Tg NOD 3 50 + – 90% (45) 8% (4) 2% (1) Non-Tg NOD 2 33 + + 36% (12) 58% (19) 6% (2) SOCS-1–Tg NOD 5 64 + – 0% (0) 3% (2) 97% (62) SOCS-1–Tg NOD 8 98 + + 30% (29) 43% (42) 28% (27) Pancreatic sections (from two or three levels per organ) were stained with H&E and insulitis was ranked A–D (see Methods). aDue to difficulties in separating peri-insulitis that normally occurs in pancreata of NOD mice from mononuclear cells that were infiltrating the exocrine area of the pancreata of infected mice, the two first classes were grouped together. 376 nature immunology • volume 3 no 4 • april 2002 • http://immunol.nature.com
  • 5. A RTICLES a b c d © 2002 Nature Publishing Group http://immunol.nature.com Figure 6.The tropism of CVB4 for islet cells is altered when IFN signaling is perturbed. Pancreatic sections were analyzed with VP-1 (an antibody that detects a CVB4 coat protein). No VP-1 staining was evident (a) in the pancreas of an uninfected SOCS-1–Tg NOD mouse or in (b) the islets of a CVB4-infected non-Tg NOD mouse killed on day 3 p.i. Note the presence of CVB4 in exocrine pancreata from both (b) non-Tg and (c,d) Tg mice killed on day 3 and 4 p.i., respectively; VP-1 staining appeared exclusively in the islets of Tg mice. Original magnifications were (a,c) ×40 and (b,d) ×20. SOCS-1–Tg NOD mice: 11.8±0.3 log10 PFU per gram of tissue (n=4); infected and killed at day 3 or 4 p.i. and examined (Fig. 6). Pancreata non-Tg mice: 11.5±0.3 log10 PFU per gram of tissue (n=4). Because no were removed and CVB4 was detected by immunohistochemistry with generalized increase in the replication of CVB4 occurred in infected an antibody specific for VP-1 (a capsid protein conserved within the SOCS-1–Tg NOD mice, pancreatic viral load apparently did not con- members of the enterovirus family41). Although CVB4 was present in tribute to their marked β cell loss after infection. the exocrine part of the pancreata of non-Tg mice on days 3 and 4 p.i. The pancreatic islets typically represent only 1–2% of the total pan- (Fig. 6b and data not shown), the virus was virtually absent from their creas mass; therefore, the amount of virus in homogenates of the islets (day 3 p.i., n=2, Fig. 6b; day 4 p.i., n=5, data not shown). Virus whole pancreas may not accurately reflect an enhanced amount of was also detected in the exocrine tissue of infected SOCS-1–Tg NOD CVB4 replication specific to the islet cells. Consequently, the ability mice; however, in marked contrast to non-Tg littermates, CVB4 was of SOCS-1 expression to enhance CVB4 replication specifically in detectable in the islets of infected SOCS-1–Tg NOD mice (Fig. 6c,d). islet cells was examined. Because islets are difficult to isolate from Indeed, at 3 days p.i., virus was detected to varying degrees in the infected mice, immunohistochemistry was chosen for this analysis. To islets of all the SOCS-1–Tg NOD mice studied (n=3, Fig. 6c). On day this end, SOCS-1–Tg NOD mice and their non-Tg littermates were 4 p.i., islet structures and/or endocrine cells were visible in only 4/6 a b c d e f g h Figure 7. CVB4 and insulin colocalize in the pancreatic islet cells of infected SOCS-1–Tg NOD mice. (a–d) Little if any colocalization of CVB4 and insulin staining was seen within islet cells from an infected non-Tg NOD mouse killed on day 3 p.i. (e–h) In infected SOCS-1–Tg NOD, however, pancreatic sections showed the localization of CVB4 within insulin+ β cells. Note the presence of CVB4 in pancreatic exocrine tissue from both mice. (a,e) The FITC channel (green) shows cells stained for CVB4 with an antibody to VP-1. (b,f): Rhodamine staining (red) shows insulin+ cells. (c,g) A composite image of both channels. (d,h) Phase contrast images of the respec- tive tissue sections. http://immunol.nature.com • april 2002 • volume 3 no 4 • nature immunology 377
  • 6. A RTICLES a b c d © 2002 Nature Publishing Group http://immunol.nature.com e Figure 8. Adaptive immunity is not necessary for the swift induction of diabetes in CVB4-infected SOCS-1–Tg NOD mice, but depletion of NK cells prevents diabetes development. (a) NOD-SCID mice did not develop diabetes after CVB4 infection (n=4). (b) However, an acute form of diabetes did occur in infected SOCS- 1–Tg NOD-SCID mice (n=5). Data are representative of 11 Tg and five non-Tg mice. (c) Diabetes was not apparent in non-Tg NOD mice that were depleted of NK cells using anti–asialo-GM1. (d) NK cell depletion with anti–asialo-GM1 prevented diabetes development in CVB4-infected SOCS-1–Tg NOD mice (n=6). Data are representative of three non- Tg and 11 Tg SOCS-1–Tg NOD mice; mice were treated with anti–asialo-GM1 both before and after CVB4 infection. (e) Most of the SOCS-1–Tg (NOD×C57BL6)F1 mice (5/9) developed diabetes after infection with CVB4.Treatment with NK cell–depleting anti-NK1.1 blocked diabetes development. infected SOCS-1–Tg NOD mice (Fig. 6d and data not shown), and diabetes development in SOCS-1–Tg mice; these mice all became CVB4 was present in the endocrine cells of all four mice with remain- severely hyperglycemic within 6–7 days p.i. (n=3, data not shown). ing islet structures. In the two other pancreata, the islet cell structures However, in antibody-treated non-Tg littermates, blood glucose con- were so distorted that individual endocrine cells could not be dis- centrations remained low (below 125 mg/dl, n=2). Histological analy- cerned, which precluded analysis. Double immunofluorescence analy- sis of pancreata from mice killed on day 8 p.i. showed that in the sis (Fig. 7) showed that CVNB and insulin colocalized in several cells SOCS-1–Tg NOD mice the pancreatic islets were severely distorted from the islets of SOCS-1–Tg NOD mice (Fig. 7g, and data not and almost devoid of insulin+ cells (data not shown). These experiments shown). Little, if any, colocalization of CVB4 and insulin was found ruled out a prominent role for CD8+ T cells in the destruction of SOCS- in the islets of infected non-Tg mice (Fig. 7c). Taken together, these 1–Tg β cells during systemic CVB4 infection. findings showed that the apparent tropism of CVB4 was altered in the To expand our studies on the role of adaptive immune responses in Tg mice, with virus being present in the pancreatic islets. disease development, the SOCS-1–Tg mice were bred with nonobese- In additional studies, IFN-αβγR–/– mice and their wild-type controls diabetic–severe-combined immunodeficient (NOD-SCID) mice, which were infected with CVB4. All IFN-αβγR–/– mice (n=11) died within lack mature T and B lymphocytes44. SOCS-1–Tg NOD-SCID and their 4 days of infection, whereas most of the receptor-sufficient control non-Tg NOD-SCID littermates both lost a substantial number of pan- mice (10/12) survived the whole 28-day study period. Immunohisto- creatic acinar cells after infection (data not shown). No diabetes (Fig. chemical analysis of pancreata from infected mice killed at days 2, 3 8a) or histological signs of β cell loss were observed in CVB4-infect- or 5 p.i. did not reveal CVB4-staining cells in the pancreatic islets of ed non-Tg littermates (n=5). However, most SOCS-1–Tg NOD-SCID wild-type mice (n=2 mice for each timepoint, data not shown). In IFN- mice (8/11, 73%) infected with CVB4 underwent rapid and extensive αβγR–/– mice that survived 2 or 3 days p.i., 5–10% of the total number islet damage and developed severe hyperglycemia (Fig. 8b). Thus, the of endocrine cells within each islet stained positively for CVB4 (n=3 adaptive immune response played little, if any, role in CVB4-induced or 4 mice per timepoint, data not shown), which showed that islet cell diabetes in SOCS-1–Tg NOD mice. CVB4 tropism was altered in IFN-αβγR–/– mice. Together, these results suggested that intact β cell responses to IFNs were critical in NK cells contribute to CVB4-induced diabetes preventing CVB4 from infecting β cells in vivo. We next assessed the role played by NK cells in causing β cell destruction and diabetes in CVB4-infected SOCS-1–Tg NOD mice. Diabetes evolves without adaptive immunity NK cells contribute to the host’s first line of defense against infect- Because virus-infected cells are often attacked by activated immune ing viruses by producing cytokines and directly destroying virus- cells (in particular CD8+ T cells42), we next determined whether the infected cells45,46. Because our previous experiments showed that unresponsiveness to IFN caused infected SOCS-1–Tg β cells to SOCS-1 expression rendered β cells permissive to early CVB4 become the targets of immune-mediated destruction during CVB4 infection, we examined NK cells as mediators of β cell destruction. infection. In addition, the NOD mouse has a pool of autoreactive To determine whether uninfected SOCS-1–Tg β cell were destroyed T lymphocytes that are responsive to pancreatic β cell antigens43. nonspecifically by activated NK cells, SOCS-1–Tg NOD mice and Hence, antigen leakage from CVB4-infected SOCS-1–Tg β cells could their non-Tg littermates were exposed to double-stranded RNA in potentially have activated quiescent autoreactive T cells in SOCS-1–Tg the form of poly(I)·poly(C)47. Despite mimicking a viral infection NOD mice. To assess the participation of CD8+ T cells, SOCS-1–Tg and activating NK cells, SOCS-1–Tg NOD mice (n=4, data not NOD mice and their non-Tg littermates were depleted of CD8+ T cells shown) and non-Tg mice (n=3, data not shown) treated with before infection with CVB4. We found that depletion did not inhibit poly(I)·poly(C) remained normoglycemic for the 28-days study 378 nature immunology • volume 3 no 4 • april 2002 • http://immunol.nature.com
  • 7. A RTICLES which infection-induced reactions to autologous cells can be regulated. a b Our data show that target cell responses will critically determine the out- come of a viral infection: antiviral defenses expressed by the pancreatic β cell are necessary in preventing diabetes after infection with CVB4. Type I IFNs secreted during early viral infection act in a paracrine manner to lower the permissiveness of neighboring and distant cells to viral infection. During certain viral infections, early innate production of IFN-γ may also contribute to the transition to an antiviral state25–27. © 2002 Nature Publishing Group http://immunol.nature.com Our data suggest that IFNs provide the stimuli necessary to activate antiviral defenses in pancreatic β cells. Treatment of pancreatic islets with IFNs before and during infection in vitro blocked CVB4 replica- tion and cell death, which shows that IFNs prevented islet cells from Figure 9. Depletion of NK cells prevents the loss of insulin-staining β cells undergoing viral cytolysis and/or infection-induced apoptosis. CVB4 after CVB4 infection of SOCS-1–Tg NOD mice. Representative immuno- was detected in the islet cells of mice with β cells that were either staining of pancreatic sections from the pancreas of an infected SOCS-1–Tg NOD expressing SOCS-1 or lacking IFN receptors. This shows that the tro- mouse treated with anti–asialo-GM1 to deplete NK cells: the structure of the pan- creatic islet is close to normal and contains numerous (a) insulin+ and (b) glucagon+ pism of CVB4 for islet cells was altered when the engagement of IFN cells. Sections are representative of at least eight infected animals. Original magnifi- receptors or IFN signaling was perturbed in β cells. In SOCS-1–Tg cation: ×40. mice, infection resulted in β cell loss and diabetes, which showed that an intact β cell response to IFN is required to prevent diabetes after a period. This suggested that activated NK cells per se had no pro- systemic CVB4 infection. nounced effect on β cell survival. The SOCS-1–Tg model suggests that IFNs are critical in preventing Although generalized activation of NK cells did not result in islet direct virus-mediated killing of β cells. This deduction is based on a cell destruction, the targeting of CVB4-infected β cells by NK cells correlation between no suppression of CVB4 replication in IFN-treat- was not ruled out. To address this, SOCS-1–Tg NOD mice and their ed SOCS-1–Tg islet cells in vitro and fast induction of diabetes in non-Tg littermates were treated with anti–asialo-GM1 or PBS before CVB4-infected SOCS-1–Tg mice, and the observation that the pres- and during infection with CVB4. Anti–asialo-GM1 treatment effec- ence of CVB4 in the pancreatic islet cells preceded the disappearance tively depletes NK cells48. Although one non-Tg mouse treated with of β cells in infected SOCS-1–Tg mice. From additional studies with anti–asialo-GM1 succumbed to the viral infection on day 4 p.i., none NOD-SCID and (NOD×C57BL6)F1 mice, we deduced that it was of the surviving mice (n=2) developed diabetes (Fig. 8c) and no dia- unlikely that β cell–specific responses to IFNs were required to pre- betes occurred in non-Tg mice treated with PBS (n=5) (data not vent either an attack by the host’s adaptive antiviral immune response shown). Of the antibody-treated SOCS-1–Tg NOD mice, 2/11 died or an attack from autoreactive T cells that could have been activated within 5 days of infection, and only one of the surviving mice devel- through bystander mechanisms17,38. Depletion of NK cells afforded oped hyperglycemia when examined to 10 days p.i. (Fig. 8d). In addi- protection from CVB4-induced diabetes and led to a marked decrease tion, 5/5 mock-treated SOCS-1–Tg NOD mice developed a severe in β cell destruction after infection in SOCS-1–Tg mice. NK cells play hyperglycemia within 5–7 days p.i. (data not shown). Histological a role in the host’s defense against coxsackievirus infection55,56; how- evaluation of pancreata from infected mice showed milder insulitis ever, a direct attack on pancreatic β cells had not previously been (Table 1) and less islet destruction in SOCS-1–Tg NOD mice treated reported. We showed here that systemic activation of NK cells does with anti-asialo-GM1 (Fig. 9a,b) than in mice treated with vehicle not result in β cell destruction and diabetes in SOCS-1–Tg mice. alone (Table 1 and data not shown). Together, these findings suggest that an enhanced permissiveness of Because prototypical NK cell markers, such as asialo-GM1, are SOCS-1–expressing β cells to CVB4 replication is paralleled by their expressed on NK cells as well as on some cells of the T cell lin- increased sensitivity to NK cell–mediated destruction. Whether a eage49–52, it was possible that anti–asialo-GM1 treatment depleted not direct killing pathway or other mechanisms contribute to the NK only NK cells but also virus-specific T cells52. The cell surface mark- cell–dependent destruction of SOCS-1–expressing β cells, these stud- er NK1.1 is not expressed on NK cells or on NK T cells in the NOD ies suggest that the IFN response not only lowers β cell permissiveness mouse53, but it is present on these cell types in C57BL/6 mice and in to CVB4 infection, but also contributes to the escape from NK (C57BL/6×DBA/2)F1 mice54. The percentage of NK1.1+ TCRαβ– cell–dependent killing. These data raise the possibility that β cell splenocytes in non-Tg and SOCS-1–Tg (NOD×C57BL/6)F1 mice are responses elicited by IFNs preserve self-tolerance of NK cells to pan- similar to those in C57BL/6 mice, and (NOD×C57BL/6)F1 hybrids can creatic β cells during a CVB4 infection. be successfully depleted of NK cells with anti-NK1.1 (M. Flodström The human coxsackievirus adenovirus receptor (CAR) protein is a and N. Sarvetnick, unpublished data). We found that although infec- receptor for several CVB serotypes55–57, although the existence of other tion with CVB4 resulted in a rapid development of diabetes in the receptors has also been proposed58,59. CAR plays a central role in the majority of SOCS-1–Tg (NOD×C57BL/6 mice)F1 (5/9), none of the productive infection of human pancreatic islet cells60. The murine anti-NK1.1–treated SOCS-1–Tg (NOD×C57BL/6)F1 mice (0/6) devel- homolog of CAR (mCAR) can determine CVB tropism in murine oped diabetes at day 28 p.i. (Fig. 8e). Together, these data show that cells61,62, but whether mCAR and/or other receptor(s) contribute to NK cells contribute to β cell destruction in SOCS-1–Tg NOD mice CVB4 attachment and entry to murine pancreatic islet cells is present- after CVB4 infection. ly unknown. It has been suggested that differing amount of mCAR expression might account for the high tropism of CVB3 for pancreatic Discussion acinar cells and a low tropism for pancreatic β cells18. Indeed, viral tro- It has been proposed that viral infections trigger or precipitate autoim- pism is initially determined by the tissue-specific expression of viral mune reactions to self1,2,4–6. We have defined here an additional way in receptors. Nonetheless, for many RNA and some DNA viruses, the http://immunol.nature.com • april 2002 • volume 3 no 4 • nature immunology 379
  • 8. A RTICLES ability of the receptor-expressing cell to mount an antiviral defense in low C peptide concentrations and normal glycosylated hemoglobin response to type I IFNs is the major determinant for successful virus A(1c) (HbA1c) concentrations. Although the etiology remains unknown, replication63. We found that pancreatic islet cells, which are normally it was suggested that a viral infection could have caused the rapid highly permissive to an in vitro infection with CVB410,11,13,35, did not destruction of the pancreatic β cells in these patients73,74. Hence, an support abundant viral replication after treatment with IFNs. In addi- increased understanding of the molecular mechanisms behind IFN- tion, by infecting SOCS-1–Tg or IFN-αβγR–/– mice, we showed that the induced antiviral defenses in pancreatic β cells could facilitate the IFN action was likely to be crucial also in regulating β cell permissive- development of antiviral therapy that may provide effective prophylax- ness to CVB4 infection during infection in vivo. Our data provide a is for humans with acute-onset of type 1 diabetes. Finally, CVB infec- © 2002 Nature Publishing Group http://immunol.nature.com possible explanation for the paradoxical finding that although most sys- tions may result in β cell damage, an event that might be necessary in temic CVB infections pass without causing diabetes in the infected order for an autoimmune-mediated β cell destruction to progress3,17,38,75. host16–19, in vitro infection with CVBs often results in β cell Several clinical studies have emphasized the role of enteroviral infec- death10,11,13–15,35. Indeed, although IFNs released during systemic CVB4 tions in accelerating the progression of diabetes in humans76–79. infection ensured the efficient transition of β cells into an antiviral Defective β cell antiviral defenses could therefore augment cellular state, a lack of adequate IFN stimulation could reasonably account for damage and the release of otherwise sequestered β cell antigens, pro- the marked effects on β cell function and survival during infection in viding a rich pool of epitopes to prime self-reactive lymphocytes and vitro that we observed here and others have also reported10,11,13–15,35. initiating autoimmunity4,80. Thus, enhanced antiviral defenses may also However, whether IFNs mediate inhibition of viral entry and/or viral benefit individuals with a genetic predisposition to develop autoim- replication remains to be determined. mune type 1 diabetes. IFN-α is expressed in the pancreata of diabetic patients64–66. In summary, our data show that by responding to IFNs, the β cell not Published data showing that human pancreatic β cells infected with only restrains CVB4 infection and replication, but also escapes antivi- CVB in vitro produce IFN-α60 support the view that these individuals ral activities raised by the host’s innate immune system. The pancreat- might have carried a persistent viral infection. Animal studies have pro- ic β cell was once called the “innocent bystander” and was believed to vided evidence to suggest that type I IFNs (IFN-α or IFN-β) could be have little involvement in its own demise during type 1 diabetes81. involved in the pathogenesis of type 1 diabetes by exerting negative However, our results suggest that during CVB4 infection, the host is effects on the β cell67–72. In addition, IFN-α induces the expression of critically dependent on the early antiviral defenses raised by the pan- human retroviral superantigens that activate Vβ7+ T cells, which pro- creatic β cell. Conversely, if β cell antiviral defense fails, the host will vides a possible link between viral infections and the activation of immediately succumb to diabetes irrespective of any other defense potentially autoreactive T cells72. In contrast to these studies, our data mechanisms that are awakened. These observations also imply that show that an early response to IFNs is essential for β cell survival in individual variations in the target β cell–specific antiviral defense can response to CVB4 infection. Additional support for a critical role influence susceptibility to virus-induced type 1 diabetes. This scenario played by IFN-α in preventing CVB-induced pathologies comes from can most likely be extended to other organ-specific autoimmune dis- data showing that human islets infected with CVBs in vitro rapidly suc- eases where genes expressed within the target tissue itself could influ- cumb to the viral infection when IFN-α in the culture media is neutral- ence inflammation and thereby regulate disease susceptibility. ized60. These findings suggest that IFN-α can act as a “double-edged sword” in the pathogenesis of virus-induced autoimmune disease for Methods two reasons. First, IFN-α ensures that pancreatic β cells and other cells, Animal husbandry. NOD/Shi, NOD-SCID and C57BL/6 were from the rodent-breeding as suggested by the rapid death that occurs in CVB4-infected IFN- colony at The Scripps Research Institute (TSRI). Breeding pairs of IFN-αβγR–/– αβγR–/– mice, enter an antiviral state that is critical for their survival (129S6/SvEv)36 mice were a gift of S. Virgin (Washington University School of Medicine, St. Louis, MO) and wild-type 129S6/SvEv mice were from Taconic Laboratories during early infection. Second, a prolonged increase in systemic or (Germantown, NY). Mice were bred and maintained at TSRI, where they were kept in a spe- local amounts of type I IFN contributes to β cell demise by either act- cific pathogen–free environment. The overall incidence of diabetes in the NOD/Shi colony ing directly on the β cell or through the activation of self-reactive was 60–70% for females and 25–30% for males. All live animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and the Animal Research T cells. Nonetheless, the theory that IFNs play a nonredundant role in Committee (ARC) and were conducted in accordance with institutional guidelines for ani- preventing β cell death after CVB4 infection could certainly extend to mal care and use. infections with other pancreatrophic viruses. Hence, as research seeks to develop methods for preventing type 1 diabetes, these data—which Generation of SOCS-1–Tg mice. SOCS-1–Tg mice were generated by placing a 673–base pair DNA fragment encompassing SOCS-1 cDNA82–84 under the transcriptional control of show that a block of IFN actions can be devastating to pancreatic the human insulin promoter33. The transgene construct (3.3-kb) was microinjected into one β cells and lead to diabetes in hosts infected with CVB4—will be a key of the pronuclei of fertilized eggs derived from NOD/shi donors at the Transgenic and consideration. Embryonic Stem Cell Facility at TSRI. Transgenic progeny were detected as described33. Of the 60 mice born, five founders had integrated copies of the transgene encoding SOCS-1. Although diabetes is an unusual outcome of acute CVB infections, Of these, three mice transmitted the transgene to their progeny, and two of these lines (lines one exception is patients diagnosed with type 1 diabetes when suffer- A and B) were kept for the initial studies. Tg mice from both lines behaved similarly when ing from severe, sometimes fatal, enteroviral infections7,10,15,23. Our infected with CVB4, that is, they quickly developed diabetes after infection with CVB4. Experiments to evaluate the mechanisms behind CVB4-induced diabetes were done with model for CVB4-induced type 1 diabetes resembles this clinical pic- mice from line A. ture. Thus, some cases of diabetes, which occur in close association with a viral infection, could be the result of a failing β cell defense Virus strain and propagation of viral stocks. CVB4 Edwards strain 2 (E2) was from C. Gauntt (University of Texas, San Antonio, Texas). A stock of CVB4 was prepared and against the infecting virus. In addition, the rapid onset of diabetes par- the titer was determined as described17. alleled by pancreatitis in CVB4-infected SOCS-1–Tg mice shares some of the clinical features of a new subtype of nonautoimmune type 1 dia- Pancreatic islet isolation and culture. Pancreatic islets were isolated and cultured as betes73. Diabetes in this group of patients had an acute onset and was described85. Islets were isolated from 5–6-week-old NOD mice in order to retrieve islets that did not have significant numbers of mononuclear infiltrates or significant β cell destruction. characterized by high serum pancreatic enzyme concentrations, the All other mice used were aged 8–12 weeks. The islet preparations were cultured for at least absence of glutamic acid decarboxylase 65 (GAD65) autoantibodies, 6 days before experiments began and, as judged by light microscopy and electron 380 nature immunology • volume 3 no 4 • april 2002 • http://immunol.nature.com
  • 9. A RTICLES microscopy (data not shown), mononuclear cells surrounding the islets were released dur- murine phospho–STAT-1 (Upstate Biotechnology, Lake Placid, NY). Signal detection was ing this preculture period. done as described85. In vivo and in vitro viral infection and in vivo poly(I)·poly(C) treatment. Mice were Flow cytometric analysis and cell depletion studies. Single-cell suspensions of spleen or infected with one i.p. injection of CVB4 (50 or 100 PFU) at age 8–9 weeks. Age-matched lymph nodes were prepared as described17. The following antibodies were generated and animals that were mock-infected with vehicle alone served as controls. In some experi- conjugated to FITC or phycoerythrin (PE): anti-CD16/32 (2.4G2), anti-CD8 (53-6.7) and ments, the mice were given one i.p. injection of 100 µg of poly(I)·poly(C) (Sigma, St. Louis, anti-CD4 (RM4-5). PE-anti–pan NK cell (DX5), allophycocyanin–anti-CD3 (145-2C11) MO). A 14 h poly(I)·poly(C) treatment increased NK cell activities in splenic cell popula- and fluorochrome-labeled isotype-matched controls were from BD PharMingen (San tions by >240% (n=2) compared to controls (determined by lysis of YAC-1 target cells in a Diego, CA). Samples acquired on a FACScan or a FACSCalibur yielded data for analysis standard 51Cr-release assay). For in vitro islet infections, a described method60 was used with by CELLQuest software (both from Becton Dickinson, San Jose, CA). To deplete CD8+ © 2002 Nature Publishing Group http://immunol.nature.com some modifications. Briefly, the pancreatic islets (20 per condition) were washed once in T cells we used monoclonal rat immunoglobulin G2b, which was specific for mouse CD8 Hank’s balanced salt solution (HBSS) followed by infection with CVB4 in 2 ml of HBSS (YTS169). Each of three i.p. injections given to mice every other day for 6 days contained containing 2×105 PFU CVB4/ml (2×104 PFU/islet). After 1.5 h of incubation at 37 °C, islets 1.0 mg of antibody. Mice were infected with CVB4 8 days after the first antibody injection. were washed three times in HBSS and placed in Millicell culture plate inserts (Millipore Thereafter, the presence of CD8+ T cells in single-cell suspensions from the spleens and Corp., Bedford, MA) that contained fresh media (1 ml). The plates were incubated at 37 °C peripheral lymph nodes (axillar, inguinal and pancreatic) of treated mice were assessed by and media was changed every second day for up to day 6 p.i. In some experiments, the islets flow cytometry and showed consistent 90–95% depletion of CD8+ cells in all compartments were treated with IFN-α (100 or 1000 U/ml, Calbiochem, La Jolla, CA), IFN-γ (1000 U/ml. tested. To deplete NK cells, SOCS-1–Tg and non-Tg NOD littermates mice were given one Pharmingen, San Diego, CA) or vehicle alone for 24 h before infection. Fresh IFN was intravenous (i.v.) 50 µl injection of anti–asialo-GM1 (Wako Chemicals, Richmond, VA)— added at each media change. Culture supernatants were retrieved for the determination of which was diluted in PBS to a total volume of 200 µl—3 days before CVB4 infection, then viral titers (see below). The HBSS from the last wash p.i. and media from mock-infected i.p. 20 µl injections of anti–asialo-GM1 1 day before and then 1 and 4 days after CVB4 islets served as controls. Initial experiments showed that the amount of virus that was trans- infection. Control mice were given vehicle alone, then were infected with CVB4. To pre- ferred together with islets in the media from the last wash was below the detection limit of vent the animals from developing serum sickness (anti–asialo-GM1 is of rabbit origin) the our plaque assay (see below). SOCS-1–Tg NOD mice were given four doses of anti–asialo-GM1 and the experiments were terminated on days 10–12. Mice on the (NOD×C57BL/6)F1 background received one Virus recovery from infected mice and infected pancreatic islets and determination of i.p. 200 µg injection of anti-NK1.1 (clone PK136) or vehicle 3, 6 or 9 days before infection viral titers. Titers of infectious virus in the separate organs of infected mice or in culture and 2 and 7 days after infection. The depletion of NK cells was verified with flow cyto- media from infected pancreatic islets (the latter retrieved every 48 h p.i.) were quantified in metric analysis of splenocytes and intrahepatic lymphocytes or a standard YAC-1 killing HeLa cells with a standard plaque assay technique19. Viral titers were quantified as PFU per assay. Antibody-treated mice showed <1% and <2% CD3–DX5+ cells in their spleens gram of tissue or per islet, and the results were presented as relative PFU per gram of tissue (anti–asialo-GM1 and anti-NK1.1) and liver (anti-NK1.1), respectively. Compared to vehi- or islet. The lower detection limit of this assay was 50 PFU per gram of tissue (1.7 cle-treated control mice, the YAC-1 killing activities of splenocytes from anti–asialo- log10PFU/g) or 50 PFU per ml of islet culture media (2.5 PFU/islet or 0.4 log10PFU/islet). GM1–treated mice were reduced by 85–95% at an effector:target cell ratio of 50:1. All mice were challenged with 100 µg of poly(I)·poly(C), which was given intraperitoneally 14 h Ultrastructural analysis of cell death. Infected and control islets cells were evaluated by before killing activities were measured. electron microscopy. For this ultrastructural analysis, islets were fixed in glutaraldehyde (2.5% glutaraldehyde, 0.1 M Na cacodylate (pH 7.3) and 1 mM CaCl2) and processed for Statistical analysis. Data are expressed as mean±s.e.m. values. When experiments were epon-araldite resin embedding by standard procedures. Ultrathin sections were stained with done in duplicate, the average of the two values was considered as one independent obser- uranyl acetate followed by staining with Reynold’s lead citrate; they were examined at the vation. Statistical analysis was done with Student’s unpaired t-tests (single comparisons) or Core Electron Microscope Facility, TSRI. ANOVA tests (multiple comparisons). Accumulated incidence of diabetes was determined with χ2-tests. Blood glucose assessment and diabetes monitoring. Diabetes was assessed by measuring venous blood glucose concentrations in nonfasting mice with Glucometer Elite strips Acknowledgments (Bayer, Pittsburgh, Pennsylvania). Animals were considered diabetic after at least two con- We thank L. Mocknic, A. Ilic and L.Tucker for excellent technical assistance; N. Hill, secutive blood glucose measurements of >250 mg/dl (13.8 mM). The date of diabetes onset M. Horwitz, C. King, M. Kritzik, S. Pakala, F. Shi and other members of the Sarvetnick labo- was taken as the first date these measurements were made. Moribund or infected mice pre- ratory for discussions and suggestions; P. Minick for editing the manuscript; and B. Smith senting nonfasting blood glucose readings above 250 mg/dl for more than 2–3 consecutive and M.Wood (Core Electron Microscope Facility,The Scripps Research Institute) for days were killed and the pancreata removed for histological analysis. assistance with electron and confocal microscopy. Supported by National Institutes of Health grants (ROI: AI42231), the National Multiple Sclerosis Society (M. F.) and the Histology, immunohistochemistry and immunofluorescence. Paraffin sections of forma- Juvenile Diabetes Research Foundation (M. F.). lin-fixed organs were prepared, cut into 5-µm thick sections and stained with hematoxylin and eosin (H&E) or with primary antibodies to insulin, glucagon (Dako, Carpinteria, CA), Competing interests statement SOCS-1 (J192, Immuno-Biological Laboratories, Tokyo, Japan) or VP-1 (Dako)19. Slides The authors declare that they have no competing financial interests. were counterstained in Mayer’s hematoxylin (for insulin, glucagon and VP-1 staining) or methyl green (for SOCS-1). For double immunofluorescence, primary antibodies were Received 19 December 2001; accepted 12 February 2002. detected with fluorescein isothiocyanate (FITC)- or Texas red–conjugated secondary anti- bodies (Vector, Burlingame, CA). The Slowfade Light kit (Molecular Probes, Inc. Eugene, 1. Hyoty, H., Hiltunen, M. & Lonnrot, M. Enterovirus infections and insulin dependent diabetes mellitus OR) was used to ensure minimal fluorescence fading. Sections were analyzed by immuno- – evidence for causality. Clin. 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