Inducible Nitric Oxide Synthase Protection Against Coxsackievirus Pancreatitis1 Carlos Zaragoza,* Christopher J. Ocampo,* Marta Saura,* Clare Bao,* Michelle Leppo,* Anne Lafond-Walker,* David R. Thiemann,* Ralph Hruban,† and Charles J. Lowenstein2* Coxsackievirus infection causes myocarditis and pancreatitis in humans. In certain strains of mice, Coxsackievirus causes a severe pancreatitis. We explored the role of NO in the host immune response to viral pancreatitis. Coxsackievirus replicates to higher titers in mice lacking NO synthase 2 (NOS2) than in wild-type mice, with particularly high viral titers and viral RNA levels in the pancreas. Mice lacking NOS have a severe, necrotizing pancreatitis, with elevated pancreatic enzymes in the blood and necrotic acinar cells. Lack of NOS2 leads to a rapid increase in the mortality of infected mice. Thus, NOS2 is a critical component in the immune response to Coxsackievirus infection. The Journal of Immunology, 1999, 163: 5497–5504.
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oxsackievirus is a ssRNA virus that causes a variety of clinical syndromes, including meningitis, myocarditis, diabetes, and pancreatitis (1–5). Coxsackievirus causes a necrotizing pancreatitis in humans (6 –9) and mice (10 –17). Coxsackievirus infection of mice induces an inflammatory infiltrate in the pancreas, acinar cell degeneration, and loss of zymogen granules, followed by fatty tissue replacement of exocrine cells (10 – 12, 15). Electron microscopy of pancreatic acinar cells shows loss of organelles and zymogen granules, and alterations in the rough endoplasmic reticulum (18). The severity of viral pancreatitis in mice depends in part upon the background genetics of the host (19). The immune response to Coxsackievirus infection in the pancreas and other organs can be divided into two components, a rapid, nonspecific innate response, and a delayed but highly specific acquired response. The delayed acquired immune response includes T lymphocytes that not only inhibit viral proliferation but also damage the myocardium (20 –27). This inflammatory response is mediated by a variety of molecules, including macrophage-inflammatory protein-1, CD40, and perforin (27–29). The early, nonspecific anti-Coxsackievirus response includes NK cells (16, 30 –32), macrophages (32–38), and IFN-g (30, 33, 39 – 46). Recent work shows that IFN-g protects pancreatic islet cells from Coxsackievirus infection by activating macrophages (47). NO is an antiviral effector of the rapid, innate immune response to viral infection. Expressed in activated macrophages, the inducible NO synthase (NOS2)3 produces large amounts of NO, a rad-
ical molecule with diverse properties (48, 49). In various cellular and animal models, viral infection induces NOS2 expression, and exogenous NO inhibits the replication of a wide variety of viruses (reviewed in Refs. 49 –53). However, the role of NOS2 in the host response to viral infection is variable: in some animal models of viral infection, chemical inhibition of NOS exacerbates the course of disease, whereas in other models inhibition improves the clinical course (50 –57). This variability in the clinical effect of NOS inhibition upon viral infection may be due to variations in pathogen susceptibility to NO, differences in host immune responses to infection, or nonspecific effects of NOS inhibitors. We hypothesized that the host responds to Coxsackievirus B3 (CVB3) infection by rapidly expressing NOS2, which in turn synthesizes NO, reducing viral replication and damage to the host. Although Coxsackievirus infection induces NOS2 expression in mice, some reports show that NO is beneficial to the host, while others show that NO exacerbates the clinical course of infection (40, 58, 59). We now demonstrate that compared with wild-type controls, mice lacking NOS2 have higher viral titers, develop a more severe pancreatitis, and die more rapidly to Coxsackievirus infection. These results emphasize that NOS2 is a critical component of the innate immune system, a rapidly activated response to infection that delays viral replication long enough for amplification of a highly specific immune response that can eradicate the infection (60).
Materials and Methods *Division of Cardiology, Department of Medicine, and †Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Received for publication October 26, 1998. Accepted for publication August 17, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Spanish Ministry of Education and Culture (to M.S.), Grants R01 HL53615 and P50 HL52315 from the National Institutes of Health (to C.Z. and C.J.L.), a grant from the Cora and John H. Davis Foundation (to C.J.L.), a grant from the Bernard Bernard Foundation (to C.J.L.), and the Ciccarone Center for Preventive Cardiology of the Johns Hopkins Medical Institutions (to C.J.L.). 2 Address correspondence and reprint requests to Dr. Charles Lowenstein at Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, 950 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. E-mail address:
[email protected] 3
Abbreviations used in this paper: NOS, NO synthase; CVB3, Coxsackievirus B3.
Copyright © 1999 by The American Association of Immunologists
Animals Wild-type 129/Sv mice, wild-type C57BL/6 mice, wild-type hybrid (129, C57BL/6)F2 mice, and (C57BL/6, 129) NOS2 null mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were infected at age 3 wk and then housed in isolated rooms in microisolator cages.
Cell and viral culture CVB3 (Nancy strain; generous gift of Charles J. Gauntt, University of Texas Health Science Center, San Antonio, TX) was grown and titered using HeLa cells. In brief, HeLa cells were cultured in growing medium (MEM; Life Technologies Laboratories, Bethesda, MD) supplemented with 1% L-glutamine (100 mmol), 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS. After infection, HeLa cells were fed with infecting medium (MEM with 1% FCS). Viral stocks were prepared by infecting an 80 –90% confluent monolayer culture of HeLa cells at a multiplicity of infection of 10. Two days after incubation at 37°C, the cells were frozen 0022-1767/99/$02.00
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VIRAL PANCREATITIS IN NOS2 NULL MICE
and thawed three times, and the suspension was centrifuged. Viral supernatants were titered by the plaque assay method and stored at 280°C. The CVB3 stock titer was 2 3 109 PFU/ml. Plaque assays were used to measure the amount of virus in the tissue (from Charles J. Gauntt). Serial dilutions of CVB3 were added to six-well plates of 90% confluent HeLa cells in a volume of 200 ml for 1 h at 37°C with gentle rocking of the plate every 15 min. Equal volumes of 2% agar (Difco, Detroit, MI) and 23 infecting media at 42°C were mixed, and then 2 ml of the mixture was added to each well. Plates were incubated for 2 days at 37°C, the wells were fixed with Carnoy’s solution (25% acetic acid and 75% ethanol), the agar plugs were removed, the cells were stained with Coomassie reagent, and the plaques were counted.
Viral infections Mice that were 3 wk old were infected by i.p. injection of 0.1 ml solution containing 103 PFU/ml of CVB3 in infecting medium. Controls received a 0.1 ml solution with no virus. Animals were killed at different times after infection, according to the institutional guidelines of The Johns Hopkins University. Blood, hearts, livers, kidneys, pancreas, and spleens were collected, and one-half of each specimen was frozen in liquid nitrogen for viral culture, RNA, and protein isolation. The other portion was fixed in 10% Formalin buffer and embedded in plastic matrix for histopathological examination.
Northern analysis of CVB3 and NOS2 RNA Total RNA was harvested from different organs by the guanidinium thiocyanate-phenol-chloroform method (61). RNA was fractionated by gel electrophoresis, transferred to a membrane, and probed with a CVB3 cDNA fragment (generous gift of R. Kandolf, Tubingen, Germany (62)), and an autoradiogram was generated according to standard methods (63).
Histology Pancreas was harvested from mice, embedded in plastic, and stained with hematoxylin and eosin, as described previously (58). Immunofluorescence was performed on sections of OCT-embedded pancreas using primary Abs against NOS2 (rabbit anti-murine NOS2 made by us (58)) and against murine macrophages (rat anti-mac 3), and using secondary Abs (goat antirabbit Ig conjugated to FITC, and goat anti-rat conjugated to Cy3, from Jackson ImmunoResearch, West Grove, PA). Slides were scanned into a Macintosh computer and superimposed using Adobe Photoshop. The severity of pancreatitis was graded by a pathologist who did not know the identity of the specimens, using a modification of a previously reported scale (64), evaluating white blood cell infiltration (periductal, 1.0; periductal and occasional parenchymal infiltration, 1.5; periductal and mild parenchymal inflammation, 2.0; periductal and moderate parenchymal infiltration, 2.5; and periductal and severe parenchymal infiltration, 3.0), acinar cell vacuolation (occasional vacuolation, 0.5; prominent vacuolation, 1.0), acinar cell necrosis (occasional necrosis, 0.5; prominent necrosis, 1.0), and intensity of zymogen granule staining (normal staining, 0; mild decrease in staining, 1; absent staining, 2.0).
FIGURE 1. Lack of NOS2 decreases survival in CVB3-infected mice. Wild-type and NOS2 null mice were infected with 103 PFU CVB3. Survival was observed for 15 days following infection. (n 5 45–50 mice for each strain; p , 0.001 for survival between NOS2 null mice and each of the wild-type strains by log rank survival analysis.)
NOS2 null mice. The most profound abnormalities in infected mice that lack NOS2 are the enzymes that measure pancreatic exocrine cell integrity. Viral infection elevates the serum levels of lipase and amylase in mice that lack NOS2 much more than in wild-type mice (Fig. 2). There are no other significant differences between infected wild-type animals and infected NOS2 null animals in other blood chemistries (including sodium, potassium,
Statistics Data were analyzed using ANOVA (Microsoft Excel application run on a Power Macintosh 8500 from Apple Computer). Survival data were analyzed with Kaplan-Meier curves (SAS version 6.12).
Results Mortality is greater in infected NOS2 null mice than in infected wild-type mice To explore the role of NOS2 in the host response to viral infection, we infected wild-type and NOS2 null mice with Coxsackievirus 103 PFU i.p. Mice lacking NOS2 died earlier and in much greater numbers than wild-type mice (Fig. 1). Some variation between wild-type strains exists: the mortality for the C57BL/6 strain is highest, for the 129 is lowest, and for the hybrid is intermediate. However, even the most susceptible wild-type strain, C57BL/6, lives longer (50% mortality at 8 days vs 3 days) and survives in greater numbers (mortality 80% vs 100%, after 7 days) than the NOS2 null mice. NOS2 null mice have abnormal pancreatic enzyme levels To explore the cause of increased death in mice lacking NOS2, we next measured the blood chemistries of infected wild-type and
FIGURE 2. CVB3 induces higher serum levels of pancreatic enzymes in NOS2 null than wild-type mice. Blood was collected from fasting mice 3 days after infection with 103 PFU CVB3, and analyzed for lipase (A) and amylase (B). (n 5 3 6 SD; p , 0.007 for lipase between NOS2 null and controls; p , 0.01 for amylase and lipase between NOS2 null and controls.)
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Table I. Viral titers in NOS2 null micea Organ
Wild Type
NOS2 Null
Blood Pancreas Heart Kidney Liver Spleen
5.0 6 0.1 3 10 1.8 6 0.2 3 106 1.2 6 0.2 3 103 6.2 6 0.2 3 103 74 6 0.9 3 103 28 6 0.1 3 103 6
24 6 0.05 3 106 23 6 0.1 3 106 51 6 0.1 3 103 14 6 0.8 3 103 44 6 0.1 3 103 54 6 0.2 3 103
a
Wild-type (C57BL/6, 129) hybrid mice and NOS2 null mice were infected with CVB3, and the amount of virus in various organs was measured after 3 days (n 5 triplicate measurements of 3 mice 6 SD; p , 0.05 for all comparisons of wild-type to NOS2 null mice).
chloride, bicarbonate, urea nitrogen, creatinine, glucose, g-glutamyl transpeptidase, bilirubin, alkaline phosphatase, albumen, total protein, globulin, phosphate, magnesium, calcium, cholesterol, triglyceride, and osmolarity). NOS2 null mice have more CVB3 in the pancreas than wild-type mice Because pancreatic enzymes are elevated in the blood of NOS2 null mice, we examined the amount of CVB3 in the pancreas, as well as in other organs. Wild-type and NOS2 null mice were infected with CVB3 as above, and the pancreas, blood, heart, liver, spleen, and kidney were harvested after 3 days. Both infected wildtype and infected NOS2 null mice have large amounts of virus in blood and in pancreas (Table I). However, more CVB3 is found in the blood and organs of NOS2 null mice than in wild-type mice (Table I). In particular, there is 10-fold more virus in the pancreas of NOS2 null mice than in the pancreas of all strains of wild-type mice (Fig. 3). Furthermore, there is more viral RNA in the pancreas of NOS2 null mice than in wild-type mice by Northern analysis (Fig. 4). Although CVB3 viral particles are present in reduced numbers in the wild-type pancreas, the Northern analysis does not detect viral RNA in the wild-type pancreas after infection; the reason for this is unclear. Because the titer of CVB3 in the pancreas of NOS2 null mice is elevated, because the serum lipase and amylase are abnormally high, and because others have shown that Coxsackievirus causes pancreatitis in mice (10 –16), we therefore focused our attention on the pancreas.
FIGURE 4. CVB3 viral RNA levels in pancreas of wild-type and NOS2 null mice. Mice were infected with 103 PFU CVB3. Pancreas was harvested from hybrid wild-type mice noninfected (lane 1) or infected for 3 days (lane 2); and also harvested from NOS2 null mice noninfected (lane 3) or infected for 3 days (lane 4). CVB3 expression was analyzed by Northern analysis of total pancreatic RNA. (This experiment was repeated twice with n 5 3 mice per group with similar results.)
by expressing NOS2. To test this hypothesis, we measured NOS2 mRNA steady state levels in the pancreas of wild-type mice (Fig. 5). Northern analysis shows that NOS2 mRNA is absent in noninfected wild-type mice, but is present 3 days after infection of wild-type mice (Fig. 5A). RT-PCR shows that NOS2 mRNA is present 3 days after infection, and then decreases 5 days after infection in normal mice (Fig. 5B). Thus, NOS2 is a component of the host response to CVB3 infection. To identify the cells expressing NOS2, we performed immunofluorescence on the pancreas of infected wild-type mice, using Abs to the macrophage marker mac-3 and Abs to NOS2. Single cells in the pancreas express both mac-3 and NOS2 (Fig. 6). In addition, some cells in the pancreas stain for NOS2, but not for macrophage markers; the identity of these cells is unknown (Fig. 6D). Thus, macrophages and other cells express NOS2 in the pancreas 3 days after infection with CVB3.
Macrophages express NOS2 in the pancreas in response to viral infection If the absence of NOS2 permits an increase in viral replication in the pancreas, then the host must normally respond to viral infection
FIGURE 3. Mice lacking NOS2 have higher amounts of CVB3 in the pancreas than wild-type mice. Wild-type and NOS2 null mice of various strains were infected with 103 PFU CVB3, and the amount of CVB3 was measured in the pancreas at 3 and 5 days after infection. (n 5 triplicate measurements of 3 animals 6 SD; p , 0.01 for differences between wildtype and NOS2 null mice.)
FIGURE 5. CVB3 induces NOS2 in wild-type mice, but not NOS2 null mice. A, CVB3 infection increases NOS2 steady state mRNA levels in the pancreas of wild-type mice by Northern analysis. Pancreatic RNA was harvested from one wild-type mouse before infection (lane 1) and from three individual wild-type mice 3 days after infection with CVB3, fractionated, and analyzed with a radioactive NOS2 probe. B, CVB3 infection increases NOS2 steady state mRNA levels in the pancreas of wild-type mice by RT-PCR. RT-PCR was performed on RNA harvested from pancreas of three individual wild-type mice 3 days (lanes 1–3) and 5 days (lanes 4 – 6) after infection with CVB3.
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FIGURE 6. CVB3 induces NOS2 protein expression in macrophages in wild-type mice. Double-labeled immunofluorescence was performed on sections of pancreas from wild-type mice 3 days after infection. Sections were stained both with Ab to NOS2 (green color in A and D), and Ab to a macrophage marker mac-3 (red color in B and E). Photomicrographs at 3240 taken of the same slide by immunofluorescence using different filters in A and B were then merged to show colocalization of NOS2 and mac-3 (yellow color in C). Some cells that express NOS2 also express macrophage markers (A–C), while other cells that express NOS2 are not macrophages (D–E). (Each photomicrograph is representative of five different slides.)
CVB3 induces an acute pancreatitis in NOS2 null mice much more severe and diffuse than in wild-type mice Because infected mice lacking NOS die rapidly with large amounts of CVB3 in the pancreas, we next examined the pancreas in more detail. Wild-type and NOS2 null mice were infected with CVB3 103 PFU i.p., the pancreas was harvested 3 days after infection or 3 days after mock infection, and pancreas sections were stained with hematoxylin and eosin. Lack of NOS2 is associated with an earlier and more severe pancreatitis (Fig. 7H). This acute pancreatitis is characterized by a diffuse, interstitial mononuclear cell infiltrate associated with acinar cell necrosis (manifested by coagulation necrosis and loss of acinar staining). In contrast, the wildtype strains C57BL/6 (Fig. 7B) and C57BL/6, 129 (Fig. 7F) are not affected, and the wild-type 129 mice have only a mild pancreatitis
VIRAL PANCREATITIS IN NOS2 NULL MICE
FIGURE 7. CVB3 induces a pancreatitis more severe in NOS2 null than wild-type mice. Wild-type mice and NOS2 null mice were infected with 103 PFU CVB3; after 3 days, pancreas was harvested, sectioned, stained with hematoxylin and eosin, and photographed at 3100. Wild-type C57BL/6 mice noninfected (A) and infected (B), wild-type 129 noninfected (C) and infected (D), and wild-type (C57BL/6, 129) hybrid nonnfected (E) and infected (F) are compared with NOS2 null noninfected (G) and NOS2 null infected (H). (Samples are representative of duplicate slides, n 5 3 mice.)
(Fig. 7D). Although the wild-type strain 129 has a mild pancreatitis, it is focal in nature, some segments being affected, and others being spared (not shown). However, the pancreatitis in the NOS2 null mice is diffuse: all segments are affected. (Note that an islet has been included in each photomicrograph for standardization; and note that islet cells are not affected in any animals.) A quantitative assessment of the pancreatitis in wild-type and NOS2 null animals confirms that CVB3 causes a more severe pancreatitis in NOS2 null mice than in wild-type mice (Table II). Finally, assessment of pancreatic function by analyzing serum levels of pancreatic enzymes shows that amylase and lipase are greatly elevated in mice lacking NOS2, compared with all wildtype strains, indicating severe pancreatic damage (Fig. 2). Thus,
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Table II. Severity of acute viral pancreatitis in NOS2 null micea Strain
Severity (6SD)
C57BL/6 129 C57BL/6, 129 C57BL/6, 129 KO
0.0 6 0.0 3.0 6 1.7 0.0 6 0.0 6.3 6 0.3
a Wild-type and NOS2 null mice were injected with CVB3, and after 3 days their pancreas were harvested, sectioned, and stained by hematoxylin and eosin. The severity of pancreatitis was analyzed in a blinded fashion. (n 5 3, p , 0.03 for all groups compared to NOS2 null mice).
NOS2 plays a critical role in defending the pancreas from viral infection. The severity of viral pancreatitis increases as the viral inoculum increases Although NOS2 expression can protect an infected host from the cytopathic effects of viruses that are susceptible to NO, NOS2 expression can be harmful to the host if the virus is impervious to NO (65). To examine the contribution of NOS2 to host damage, we infected wild-type and NOS2 null mice with increasing amounts of CVB3. The damage to the pancreas, as assessed by serum lipase and amylase levels, increases as the inoculum of virus increases in the NOS2 null mice (Fig. 8). This dose-dependent effect of virus suggests that damage to the pancreas is mediated by direct viral injury. In contrast, the pancreatitis in the wild-type infected mice is mild, even in the presence of a high dose of virus. This would suggest that NO production is not damaging the pancreas of the normal host. Lack of myocarditis and encephalitis in infected mice Coxsackievirus B can infect other target organs in addition to the pancreas, including brain and heart. Although virus is detected in the heart of infected mice (Table I), no inflammatory response was seen histologically. Histological analysis of brain harvested from both wild-type and NOS2 null mice 3 days after infection, and of heart harvested from wild-type and NOS2 null mice 3, 5, 7, 10, and 15 days after infection shows an absence of inflammation or cytopathic effect (data not shown).
Discussion Our data show the consequences of a lack of NOS2 upon a systemic viral infection. Macrophages normally express NOS2 in response to many types of viral infections; NOS2 limits Coxsackievirus replication in all tissues. But in mice that lack NOS2, Coxsackievirus replicates to higher titers, and infection is associated with much more severe pancreatitis in this particular strain of mouse. Mice lacking NOS2 die much sooner and in greater numbers than wild-type mice. Thus, NOS2 is part of a rapid immune response that helps the host survive an acute Coxsackievirus infection. These results confirm and extend the initial reports by Karupiah et al. (67) and Croen (66) that NOS inhibitors permit viral replication to increase in cells and mouse footpads. Other investigators have subsequently shown that inhibition of NOS permits viral replication to increase in a variety of in vitro models (54, 55, 68). However, the effect of inhibition of NOS in infected animals is variable: some investigators report an exacerbation of the clinical course of viral disease (40, 54, 55), others an improvement (56, 57, 59), still others no change (69, 70). Mice deficient in NOS2 permit a more specific assessment of the role of NOS2 in viral infection (51, 71, 72). For example, others have recently reported that lack
FIGURE 8. Greater inoculum of CVB3 causes more severe pancreatitis. Wild-type and NOS2 null mice were infected with increasing amounts of CVB3, and serum lipase (A) and amylase (B) were measured 3 days after infection. (n 5 triplicate measurements of 3 animals 6 SD; p , 0.01 for differences between wild-type and NOS2 null mice at 103 PFU.)
of NOS2 is associated with an increase in mortality from ectromelia virus, although the precise physiological cause of the increased mortality is unknown (73). Thus, NOS2 null mice are a useful tool to demonstrate which viruses are susceptible to NO, and which are resistant. Antiviral mechanism of NOS2 We have recently identified one mechanism by which NO inhibits viral replication. NO inactivates the cysteine protease 3Cpro of Coxsackievirus that is critical to the viral life cycle (74). This cysteine protease processes the Coxsackievirus polyprotein into structural and nonstructural viral polypeptides. NO nitrosylates the active site cysteine residue of this cysteine protease, rendering it inactive, and resulting in an accumulation of unprocessed viral polyproteins. However, several aspects of the mechanism by which NOS2 inhibits viral replication are unknown. First, it is unclear whether the antiviral effects of NO are due only to direct effect upon the virus, or whether NO in addition influences the host immune response as well (75). Second, it is unclear whether NO is the effector molecule that inhibits viral replication, or whether a nitrosothiol or other NO derivative is responsible (76, 77). Finally, some viral targets of NO are still unknown. A variety of viruses are inhibited by NO, suggesting that there are many viral targets of NO
5502 (reviewed in Refs. 76, 78, and 79). Furthermore, some types of viruses are resistant to NO. NO can inhibit viral transcription, translation, and DNA synthesis, but the identity of the susceptible viral enzymes is unknown. Others have shown that NO affects protein synthesis and DNA synthesis of vaccinia virus (67, 80); and NO can affect latency of EBV by down-regulating the transcription factor Zta (81). NO can also affect the DNA of microbes, and perhaps can also directly modify RNA (reviewed in Ref. 52). We have shown that NO inhibits protein synthesis, and NO inhibition of the CVB3 cysteine protease may account for this reduction in viral protein expression (79). However, NO also inhibits replication of the CVB3 RNA genome in vitro (79). Our previous in vitro data match this in vivo report, which shows a dramatic reduction in viral RNA only in mice expressing NOS2 (Fig. 4). Thus, NO may inhibit CVB3 replication in mice by reducing replication of the viral RNA genome or by inhibiting other steps in the viral life cycle. Severe pancreatitis in NOS2 null mice Normally, mice express NOS2 in the pancreas in response to CVB3 infection: Northern analysis shows NOS2 mRNA in the pancreas of infected mice, and immunofluorescence shows infiltrating macrophages (as well as other cells) expressing NOS2 in the pancreas of infected mice (Fig. 5). In the presence of NOS2, CVB3 causes a pancreatitis that is mild. But in the absence of NOS2, viral infection causes much more severe organ damage. In this model, CVB3 infection damages the pancreas in NOS2 null mice, as assessed by histology and pancreatic enzyme release (Figs. 2 and 7). Furthermore, this severe pancreatitis is diffuse, whereas the mild pancreatitis in wild-type animals is segmental. How does lack of NOS2 lead to structural and functional damage of the pancreas? One possible explanation is that the virus directly injures the pancreas, as has been reported by others (17, 18): without NOS2 inhibition of viral replication, the greater amount of virus causes more organ damage. However, the amount of virus in the pancreas is only 10-fold more in NOS2 null than in wild-type animals, and a 10-fold increase in viral amount may not be sufficient to account for such a dramatic increase in pancreatitis. Perhaps NOS2 not only reduces viral titers, but also plays another role in protecting the host. For example, many viruses including CVB3 can induce apoptosis (29, 82– 84), and NO can block apoptosis by inhibition of caspase-3 (85– 87). If CVB3 activates proapoptotic molecules, and if NO inhibits apoptosis, then a decrease in NO may result in an increase in apoptosis. (TUNEL assay for DNA fragmentation of infected pancreas reveals widespread staining, but because this staining is in the cytoplasm as well as the nucleus, the positivity probably represents necrosis rather than apoptosis. Data not shown.) Perhaps NO also limits viral damage by inhibiting Coxsackievirus proteases (74), or inhibits endogenous pancreatic proteases. Another possibility is that NO could limit viral damage by decreasing host cell metabolism (88), thereby reducing the amount of inflammatory mediators released. Thus, NO may play more than one role in the immune response to viral infection, not only directly affecting viral enzymes, but also protecting host cells from damage or death. NOS2 decreases mortality from CVB3 infection Infected mice lacking NOS2 die much more rapidly than all three strains of infected wild-type mice. The cause of this accelerated death is unclear. However, several lines of evidence suggest that acute pancreatitis is a factor in the increased mortality. Histology shows a severe inflammation and necrosis in the pancreas (Fig. 7), and no other organ shows as much inflammation (data not shown). Blood chemistries show that lipase and amylase are 6-fold higher
VIRAL PANCREATITIS IN NOS2 NULL MICE in infected NOS2 null mice compared with noninfected mice. Total serum amylase and serum lipase are screening tests for acute pancreatitis, and serum amylase levels greater than 3 times normal are highly specific for acute pancreatitis (89). The remaining blood values are normal or not significantly different, including electrolytes and tests of renal function (data not shown). However, it is impossible to exclude other causes of death. Nonetheless, the amount of virus in the pancreas is 1000-fold more than in other organs, so it is probable that pancreatic damage plays a major role in the accelerated mortality of infected mice lacking NOS2. In this study, infected mice did not develop myocarditis, even though virus was present in the heart (Table I). One possible reason for the lack of organ pathology other than in the pancreas is the genetic strain of the mice used in this particular study. The host susceptibility to myocarditis depends upon many factors, including the host genetics (90). We and others have detected myocarditis in different strains of mice infected with the same virus (58, 91). Perhaps the particular strains of mice used in this study are resistant to viral myocarditis (92). Huber and colleagues report that the CVB3 causes minimal cardiac lesions in C57BL/6 mice despite high viral titers (93). Variability in strains of virus can also account for the lack of myocarditis (91). Some strains of CVB3 can cause severe pancreatitis and severe myocarditis, while other strains cause severe pancreatitis and only a mild myocarditis (17, 94). (Our experiments use CVB3m Nancy strain from Dr. Gauntt (91), which causes myocarditis in other strains of mice, such as MF1 (95).) Therefore, genetic variation either in mice or in CVB3 can influence the severity of Coxsackievirus myocarditis. A recent report showed that ectopic expression of IFN-g in islet cells protected mice from hypoglycemia due to severe pancreatic inflammation following Coxsackievirus infection (47). Interestingly, although IFN increased the number of activated macrophages and the amount of NO produced, the NO inhibitor aminoguanidine did not reduce the antiviral effect of ectopic IFN. Perhaps activated macrophages possess multiple pathways to eliminate virus, which are active even in the absence of NOS2. Or perhaps aminoguanidine inhibited other NOS isoforms that may have indirectly affected viral replication. Effect of NOS2 on the host Karupiah et al. have shown that NOS2 expression can be harmful to the infected host, especially if the virus is resistant to NO. Specifically, the natural history of influenza A viral infection is the same in wild-type and NOS2 null hosts; but the pneumonitis is much less severe in NOS2 null hosts. In this particular infection, NOS2 expression in the lungs of mice does not affect influenza A replication, but damages the lungs. Two lines of evidence suggest that CVB3 damages the pancreas, and NO does not harm the pancreas. First, infected wild-type mice that are expressing NOS2 have less pancreatitis than infected NOS2 null mice (Figs. 6 and 7). Second, an increase in viral inoculum causes an increase in pancreatitis (Fig. 8). However, an increase in viral titers could also induce an increase in host immune responses. Nonetheless, our data suggest that NOS2 plays a beneficial role in the host response to CVB3 infection. Speculation on the role of NOS2 in the immune response to viral infection Mice that lack NOS2 are useful to study the precise role of NOS2 in the host response to different types of viral infection, and the results of our work and of others lead to the following speculation. An initial viral inoculum is followed by an early viremia and infection of susceptible organs. Cytokines released from damaged tissue attract and stimulate macrophages that synthesize NOS2.
The Journal of Immunology (Other cells synthesize NOS2 as well.) NO or a derivative of NO diffuses into infected cells, blocking viral replication, and perhaps protecting host cells by reducing cell metabolism or apoptosis. This NOS2 response to viral infection occurs within 1 to 3 days of infection, as a critical component of the rapid innate immune system. Mice that express NOS2 inhibit Coxsackievirus replication, allowing time for the slower, more specific, acquired immune response to develop within 1 wk of infection and to eradicate the virus. However, the early Coxsackievirus load is so high in mice that lack the NOS2 component of the innate immune system, that excessive levels of virus kill the host before the acquired immune system becomes fully activated. Our data thus suggest that NOS2 is a critical component of the innate immune response to Coxsackievirus infection.
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