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(Circulation. 2001;103:743.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Tumor Necrosis Factor- (TNF-) Plays a Protective Role in Acute Viral Myocarditis in Mice

A Study Using Mice Lacking TNF-

Hisayasu Wada, MD, PhD; Kuniaki Saito, PhD; Tsugiyasu Kanda, MD, PhD; Isao Kobayashi, MD, PhD; Hidehiko Fujii, MD, PhD; Suwako Fujigaki, BSc; Naoya Maekawa, BSc; Hisato Takatsu, MD, PhD; Hisayoshi Fujiwara, MD, PhD; Kenji Sekikawa, PhD; Mitsuru Seishima, MD, PhD

From the Department of Laboratory Medicine (H.W., K. Saito, H. Fujii, S.F., N.M., M.S.) and Second Department of Internal Medicine (H.T., H. Fujiwara), Gifu University School of Medicine, Gifu; Department of Laboratory Medicine, Gunma University School of Medicine, Gunma (T.K., I.K.); and Department of Immunology, National Institute of Animal Health (K. Sekikawa), Japan.

Correspondence to Hisayasu Wada, MD, PhD, Department of Laboratory Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. E-mail wadah{at}cc.gifu-u.ac.jp

	Abstract

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Background—It has been reported that tumor necrosis factor- (TNF-) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF- on viral myocarditis, however, have not been fully elucidated.

Methods and Results—To investigate the role of TNF- in the progression of viral myocarditis, we used TNF- gene–deficient mice (TNF-^-/-) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-^-/- mice after EMCV infection was significantly lower than that of TNF-^+/+ mice (0% versus 67% on day 14). Injection of recombinant human TNF- (0.2 to 4.0 µg/mouse IV) improved the survival of TNF-^-/- mice in a dose-dependent manner, indicating that TNF- is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-^-/- than in TNF-^+/+ mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-^-/- than in TNF-^+/+ mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-^-/- mice compared with TNF-^+/+ mice.

Conclusions—These observations suggested that TNF- is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF- plays a protective role in the acute stage of viral myocarditis.

Key Words: tumor necrosis factor- • myocarditis • viruses • cell adhesion molecules

	Introduction

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Tumor necrosis factor- (TNF-) is considered to be a proinflammatory cytokine¹ and to play a crucial role in the initiation and continuation of inflammation and immunity. In this regard, this cytokine may be implicated in the pathogenesis of cardiovascular diseases, especially in viral myocarditis. In fact, elevated plasma levels of TNF- have been reported in patients with acute myocarditis.² The enhanced expression of TNF- mRNA was also reported in a mouse model of viral myocarditis.^{3 4} It is still unclear, however, whether the expression of TNF- is beneficial to the host. Administration of TNF- has been reported to aggravate viral myocarditis,⁵ and neutralization of this cytokine with selective antibodies ameliorates viral⁵ and autoimmune myocarditis.⁶ These findings indicated that endogenous TNF- is deleterious to myocarditis. Conversely, it has also been reported that TNF- has antiviral activity,⁷ and mice lacking TNF- show a high degree of susceptibility to infectious agents^{8 9} and impaired clearance of adenoviral vectors.¹⁰ These observations suggest that TNF- has a protective role against viral infection. The pathophysiological effects of endogenous TNF- in viral myocarditis, therefore, are still controversial and remain to be elucidated. To directly examine the role of endogenous TNF- in viral myocarditis, we used mice with a targeted disruption of the gene encoding TNF-¹¹ and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). Our results indicate that TNF- plays an essential role in protection against viral infection in the acute stage of viral myocarditis.

	Methods

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Animals
TNF- gene–deficient (TNF-^-/-) mice were produced by gene targeting as described previously.¹¹ Briefly, a TNF-–targeting vector was constructed from a 7-kb DNA fragment isolated from a 129 mouse FIXII genomic library. The HincII-HindIII fragment containing exon 3 and part of exon 4 was replaced with a neomycin-resistant gene cassette. This targeting vector was introduced into TT2 ES cells by electroporation. Resistant clones were screened, and homologous recombination events were confirmed by Southern blotting analysis. Three clones were injected into 8-cell–stage C57BL/6J embryos, which were transferred into the uteri of pseudopregnant ICR females. Chimeric mice derived from 1 clone transmitted the mutation to offspring. TNF-^+/- heterozygotes were backcrossed with C57BL/6J mice, and the F8 generation was used for preparation of TNF-^-/- homozygotes. The H2 haplotype of homozygotes was determined to be H2b. C57BL/6J mice obtained from Japan SLC (Shizuoka, Japan) were used as wild-type controls (TNF-^+/+).

Viral Inoculation
A myocarditic variant of EMCV was generously provided by Dr Seto (Keio University, Tokyo, Japan). The virus stock was stored at -80°C in Hanks’ balanced salt solution with 0.1% BSA until use. Mice were inoculated intraperitoneally with 50 or 500 plaque-forming units (pfu) of EMCV in 0.1 mL of saline. Eight-week-old male mice were used for inoculation and housed in an isolated room. The day of virus inoculation was defined as day 0 in the following studies. The experiments were performed according to the institutional guidelines of Gifu University.

Tumor Necrosis Factor-
Recombinant human TNF- (rhTNF-) produced in Escherichia coli by recombinant DNA technology was provided by Dainippon Pharmaceutical Co Ltd.¹² rhTNF- (0.2 to 4.0 µg) was dissolved in 0.1 mL of saline and injected intravenously.

Measurement for Plasma TNF- Concentration
Plasma TNF- levels were determined with a solid-phase sandwich ELISA (BioSource International). Ninety-six–well microplates were coated with an antibody specific for mouse TNF-. Fifty microliters of each sample was added in duplicate to the microplates, and 50 µL of biotinylated anti–TNF- antibody solution was pipetted into each well. After 1.5-hour incubation at room temperature, the microplates were washed 5 times, and 100 µL of streptavidin–horseradish peroxidase conjugate solution was added to each well. Standard curves were made with mouse TNF- used as a standard. The minimum detectable concentration of TNF- was <3 pg/mL.

RNA Extraction and Reverse Transcription–Polymerase Chain Reaction Analysis
Total RNA was extracted from the heart with Isogen (Nippon Gene) and determined by the absorbance at 260 nm. Reverse transcription–polymerase chain reaction (RT-PCR) was carried out with mRNA Selective PCR Kits (Takara Biochemicals). The following oligonucleotide primer pairs were synthesized: EMCV sense, 5'-GTCGTG-AAGGAAGCAGTTCC-3'; antisense, 5'-CACGTGGCTTTTGGC-CGCAGAGGC-3'; ß-actin sense, 5'-GGACTCCTATGTGGGTGA- CGAGG-3'; antisense, 5'-GGGAGAGCATAGCCCTCGTAGAT-3'.

The PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining. The optimum number of cycles was determined experimentally for each gene product and to verify uniform amplification.

Viral Titer
The viral titers in the hearts of infected mice on day 7 were measured as described previously.¹³ Briefly, the heart was weighed and homogenized in 2 mL of Eagle’s MEM. After centrifugation, the supernatant was added to 96-well microtiter plates containing human amnion (FL) cells in MEM with 10% FCS. The microtiter plates were examined daily for 5 days for the appearance of any cytopathic effect. The viral titers were expressed as the 50% tissue culture infectious doses (TCID₅₀).

Histopathological Study
One half of the heart was fixed in 10% buffered formalin and embedded in paraffin. Transverse sections of the ventricles were stained with hematoxylin and eosin (HE) and observed by microscopy at x200 magnification. The extent of cellular infiltration and myocardial necrosis was graded blindly by 2 experienced pathologists who had no knowledge of the study design and was scored as follows¹³ : 0, no lesions; 1+, lesions involving <25%; 2+, lesions involving 25% to 50%; 3+, lesions involving 50% to 75%; and 4+, lesions involving 75% to 100%.

The other half of the heart was embedded in OCT compound (Miles Laboratory), snap-frozen in liquid nitrogen, and cut into sections 5 µm thick. Sections were air-dried and fixed in cold acetone for 10 minutes. The primary antibodies used were hamster anti-mouse intercellular adhesion molecule-1 (ICAM-1, clone 3E2, PharMingen, diluted 1:20) and rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1, clone 429, PharMingen, diluted 1:10). Secondary antibodies were biotinylated goat anti-hamster IgG (EY Laboratories, diluted 1:300) and rabbit anti-rat IgG (Dako, diluted 1:300). The streptavidin-peroxidase complex (Dako) and 3,3'-diaminobenzidine were used for visualization. The sections were counterstained with methyl green (Sigma). The levels of immunoreactivities of ICAM-1 and VCAM-1 were examined by 2 experienced pathologists in a blinded fashion.

Measurement of Plasma Creatine Kinase, Lactate Dehydrogenase, Alanine Aminotransferase, and Blood Urea Nitrogen
Activities of creatine kinase (CK), LDH, and ALT and concentrations of BUN in the plasma were measured with commercially available kits and an automatic analyzer Hitachi 736.

Statistical Analysis
Results are expressed as mean±SEM. Survival rates of mice were analyzed by the Kaplan-Meier method. Comparisons between 2 groups were performed by Student’s t test, and those among 3 groups were performed by 1-way ANOVA. Histopathological scores were examined by the Mann-Whitney test. StatView 4.5 and Super ANOVA software were used for statistical analyses on a Power Macintosh G3 (Apple Computer). Results were considered statistically significant at a value of P<0.05.

	Results

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Survival After Inoculation of EMCV
Figure 1 shows the survival rates of TNF-^+/+ and TNF-^-/- mice. The survival rates of TNF-^+/+ mice on day 14 after inoculation with 50 (Figure 1A) or 500 (Figure 1B) pfu of EMCV were 95% and 67%, respectively. All TNF-^-/- mice died within 14 days after inoculation of either dose of EMCV.

View larger version (17K): [in this window] [in a new window]   Figure 1. Survival rates of TNF-+/+ and TNF--/- mice 14 days after inoculation with 50 (A) or 500 (B) pfu of EMCV. Survival rate of TNF-+/+ mice was significantly higher than that of TNF--/- mice. *P<0.01 vs TNF--/- mice.

RT-PCR Analysis of Viral Genomic RNA and Viral Titer in the Heart
The cardiac viral genomic RNA levels on day 7 after inoculation with 50 or 500 pfu of EMCV in TNF-^-/- mice quantified by RT-PCR analysis were significantly increased compared with those in TNF-^+/+ mice (Figure 2B). The cardiac viral titer in TNF-^-/- mice was also significantly higher than that in TNF-^+/+ mice (Figure 2C). These results suggested that TNF-^-/- mice have impaired viral elimination.

View larger version (49K): [in this window] [in a new window]   Figure 2. RT-PCR analysis of viral genomic RNA and viral titer in cardiac tissue on day 7. A, Representative results of agarose gel electrophoresis of RT-PCR products of EMCV genomic RNA and ß-actin mRNA. B, Summarized data of cardiac EMCV genomic RNA levels normalized to ß-actin mRNA. EMCV genomic RNA level was significantly higher in TNF--/- than in TNF-+/+ mice. C, Viral titer in TNF--/- mice was significantly higher than that in TNF-+/+ mice. Viral titers are expressed as TCID50. Each group consisted of 9 to 10 mice. Results are expressed as mean±SEM. *P<0.01.

Changes in Plasma TNF- Levels in EMCV-Treated Mice
As shown in Figure 3, plasma TNF- levels of TNF-^+/+ mice were significantly elevated 6 hours after inoculation with 500 pfu of EMCV and reached the maximal value 24 hours after inoculation. Plasma TNF- levels on day 7 were still higher than before inoculation. Plasma TNF- could not be detected in TNF-^-/- mice.

View larger version (16K): [in this window] [in a new window]   Figure 3. Changes in plasma TNF- concentrations measured by ELISA in response to inoculation with 500 pfu of EMCV. Plasma TNF- concentrations in TNF-+/+ mice were significantly elevated at 6 hours after inoculation and reached maximal value at 24 hours after inoculation. Each group consisted of 5 mice. *P<0.01 vs Before.

Survival of EMCV-Treated TNF-^-/- Mice Supplemented With rhTNF-
Because a significant increase in plasma TNF- level was observed 6 hours after inoculation of EMCV in TNF-^+/+ mice, rhTNF- (0.2 to 4.0 µg/mouse IV) was injected through the tail vein into TNF-^-/- mice 6 hours after inoculation with 500 pfu of EMCV. As shown in Figure 4, intravenous injection of rhTNF- improved the survival of TNF-^-/- mice in a dose-dependent manner. Survival of TNF-^-/- mice receiving rhTNF- at a dose of 2.0 µg was equivalent to that of TNF-^+/+ mice. These findings indicated that TNF- is required for protection against acute viral myocarditis.

View larger version (19K): [in this window] [in a new window]   Figure 4. Effects of intravenous administration of rhTNF- on survival of TNF--/- mice infected with 500 pfu of EMCV. Intravenous injection of rhTNF- (0.2 to 4.0 µg/mouse) improved survival of TNF--/- mice in a dose-dependent manner. Each group consisted of 10 mice. *P<0.01 vs Control (TNF--/- mice without rhTNF- injection).

Immunohistochemical Findings
As shown in Figure 5, TNF-^+/+ mice showed apparent cellular infiltration of the heart on day 7 after inoculation with 500 pfu of EMCV. Interestingly, the cellular infiltration in TNF-^-/- mice was suppressed compared with that in TNF-^+/+ mice. Histopathological scores of cellular infiltration and myocardial necrosis were significantly decreased in TNF-^-/- mice (Table).

View larger version (164K): [in this window] [in a new window]   Figure 5. Histopathological findings in murine heart sections stained with hematoxylin-eosin. A, Uninfected TNF-+/+ mice. B, Uninfected TNF--/- mice. No cellular infiltration was seen in hearts of uninfected mice. C, Infected TNF-+/+ mice (day 7). D, Infected TNF--/- mice (day 7). Marked cellular infiltration was observed in infected hearts of TNF-+/+ mice but not in those of TNF--/- mice.

View this table: [in this window] [in a new window]   Table 1. Myocardial Histopathological Scores on Day 7

Figure 6 shows the results of immunostaining for ICAM-1 and VCAM-1 in the infected heart on day 7. The levels of ICAM-1 and VCAM-1 immunoreactivity in TNF-^-/- mice were diminished compared with those in TNF-^+/+ mice.

View larger version (158K): [in this window] [in a new window]   Figure 6. Immunohistochemical findings in hearts of infected mice (day 7). A, ICAM-1 in TNF-+/+ mice. B, ICAM-1 in TNF--/- mice. C, VCAM-1 in TNF-+/+ mice. D, VCAM-1 in TNF--/- mice. Levels of ICAM-1 and VCAM-1 immunoreactivity were decreased in TNF--/- mice compared with TNF-+/+ mice.

Plasma CK, LDH, ALT, and BUN
Plasma CK and LDH activities of TNF-^-/- mice were significantly higher than those of TNF-^+/+ mice (Figure 7), suggesting that severe myocardial damage occurred in TNF-^-/- mice despite the slight changes in histopathological findings compared with TNF-^+/+ mice. There were no significant differences in plasma ALT or BUN levels, however, between TNF-^-/- and TNF-^+/+ mice (Figure 7).

View larger version (28K): [in this window] [in a new window]   Figure 7. Plasma CK, LDH, ALT, and BUN in uninfected and infected mice on day 7 after inoculation with 500 pfu of EMCV. Plasma CK (A) and LDH (B) activity of TNF--/- mice was significantly higher than that of TNF-+/+ mice on day 7, indicating that severe myocardial damage occurred in TNF--/- mice. There were no significant differences in plasma ALT (C) or BUN (D) levels between TNF--/- and TNF-+/+ mice. Each group consisted of 8 to 11 mice. *P<0.01.

	Discussion

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In the present study, EMCV was used to induce myocarditis in mice. EMCV is a member of the Picornaviridae family, which includes the Enterovirus genus. EMCV can cause acute myocarditis in various animal species, including mice. This is an established mouse model for viral myocarditis leading to dilated cardiomyopathy and congestive heart failure.¹⁴ On entry of EMCV into the host cell, the single-positive-strand viral RNA is released from the capsid, and viral proteins are synthesized by the host cell translational machinery. One of the viral proteins is an RNA-dependent RNA polymerase that allows replication of viral genomic RNA. Replicated positive-strand viral RNAs are encapsidated and released by host cell lysis.¹⁵ EMCV infection is thus usually cytopathic.

TNF- plays a crucial role in the progression of inflammatory responses in the heart as well as other vital organs. In fact, it has been reported that plasma TNF- levels are elevated in patients with various cardiac diseases, including acute myocarditis,² and that the cardiac expression of TNF- mRNA is enhanced in mice with viral myocarditis.^{3 4} The production of TNF- is generally considered to be harmful to the cardiovascular system, because systemic administration of TNF- results in myocardial depression¹⁶ and cardiomyopathy.¹⁷ Cardiac-specific overexpression of TNF- has been reported to cause severe myocarditis in mice.^{18 19} Moreover, the neutralization of TNF- with selective antibodies ameliorates viral⁵ and autoimmune⁶ myocarditis and prevents myocardial dysfunction induced by lipopolysaccharide.²⁰ In marked contrast, TNF- has also been proposed to have beneficial actions. TNF- production might enhance contractility, mediate compensatory hypertrophy, or be involved in cardiac adaptation to stress.^{21 22} Furthermore, TNF- has been reported to have antiviral activity.⁷ Mice lacking TNF- show a high degree of susceptibility to infectious agents^{8 9} and impaired clearance of adenoviral vectors,¹⁰ suggesting that TNF- has a protective role against viral infection. Thus, the precise role of TNF- in the pathogenesis of viral myocarditis is still unclear. To directly address this issue, we induced acute viral myocarditis in TNF-^-/- mice¹¹ by infection with EMCV.

In the present study, EMCV caused severe, lethal myocarditis in TNF-^-/- mice. The survival rate of TNF-^-/- mice after EMCV infection was significantly lower than that of TNF-^+/+ mice (Figure 1). Intravenous administration of rhTNF- (0.2 to 4.0 µg/mouse) improved the survival of TNF-^-/- mice in a dose-dependent manner (Figure 4). Moreover, the myocardial viral titer and the level of viral genomic RNA in TNF-^-/- mice were significantly higher than those in TNF-^+/+ mice (Figure 2). These observations indicated that lack of TNF- resulted in failure to eliminate infecting viruses, showing that TNF- is essential for protection from viral myocarditis in the acute stage. A recent study using knockout mice demonstrated that the effectors most important for elimination of adenovirus vectors from infected hepatocytes are TNF- > Fas > perforin.¹⁰ The antiviral effect of TNF- has already been demonstrated both in vitro and in vivo.^{7 23} Administration of TNF-, however, is unable to cause elimination of the adenovirus vectors in severe combined immunodeficient mice,¹⁰ suggesting that TNF- does not act directly on infectious agents. TNF- is thought to exert its antiviral effects via activation of the immune system. Chisari²⁴ reported that hepatitis B virus (HBV)–specific cytotoxic T lymphocytes can abolish HBV gene expression and replication noncytopathically by secreting TNF- and interferon-. TNF- delivers noncytopathic antiviral signals to the hepatocytes to degrade the cytoplasmic transcript and the nucleocapsid particles of HBV.²⁵ TNF-, therefore, is thought to be an important immune mediator in host defense.

Conversely, the observation that intravenous administration of rhTNF- 6 hours after inoculation with EMCV improved the survival of TNF-^-/- mice (Figure 4) indicates that TNF- is necessary only in the early stage of infection to protect against viral myocarditis. The plasma TNF- level on day 7 after inoculation in TNF-^+/+ mice, however, was still higher than the basal level (Figure 3), suggesting that TNF- production is excessively prolonged. The persistent expression of TNF- in the chronic stage of viral myocarditis in mice has been reported,^{3 4} and TNF- expression was more pronounced in susceptible C3H.HeJ mice, in which marked myocardial depression was observed.⁴ These findings suggest that persistent expression of TNF- in the chronic stage of viral myocarditis may be harmful to the cardiovascular system, whereas TNF- expression in the acute stage of viral myocarditis is beneficial for elimination of infectious viruses. The persistent expression of TNF- may cause chronic infiltration of leukocytes,^{18 19} resulting in myocardial damage. Interleukin-2 has been reported to have a similar action²⁶ in that interleukin-2 suppressed viral myocarditis in the acute stage but exacerbated the condition in the chronic stage. As in the cases of norepinephrine and angiotensin II, sustained production of TNF- might be a maladaptive response to chronic heart diseases.^{27 28}

Histopathological analysis revealed that myocardial cellular infiltration in TNF-^-/- mice was markedly suppressed compared with TNF-^+/+ mice (Figure 5). Similar findings have been reported, in that TNF-^-/- mice showed significant reduction in the number of infiltrating lymphocytes in the liver infected with adenoviral vectors and impaired clearance of adenoviral vectors from the liver.¹⁰ TNF- is a potent inducer of the expression of adhesion molecules,^{29 30} which are necessary to recruit immune cells to sites of inflammation. Immunohistochemical analysis (Figure 6) showed that the levels of adhesion molecule immunoreactivity (ICAM-1, VCAM-1) in the myocardium were reduced in TNF-^-/- mice compared with TNF-^+/+ mice, suggesting that TNF- could recruit leukocytes to inflammatory sites through the upregulation of adhesion molecules. The lack of leukocyte recruitment may be a major factor responsible for impaired clearance of infectious agents in TNF-^-/- mice.¹⁰ In contrast, the leukocyte recruitment in autoimmune myocarditis is thought to be harmful, because there is no infectious agent to be eliminated, and leukocyte infiltration merely causes myocardial damage in autoimmune myocarditis. For this reason, TNF- receptor p55–deficient mice do not develop autoimmune myocarditis,³¹ and anti–TNF- antibodies ameliorate autoimmune myocarditis.⁶

The minimal histopathological changes raised suspicions that myocarditis may not have been the actual cause of death in TNF-^-/- mice. No detectable inflammation was observed, however, in extracardiac organs, such as the brain, spinal cord, liver, pancreas, and kidney, in TNF-^-/- mice (data not shown). Furthermore, the myocardial viral titer (Figure 2) and the plasma CK and LDH levels (Figure 7) of TNF-^-/- mice were markedly increased. EMCV is thought to be a cytopathic virus,¹⁵ and persistent picornavirus infection has been reported to induce cytopathic effects on myocytes.^{32 33} Thus, we considered myocarditis to be the cause of death in TNF-^-/- mice infected with EMCV. Because EMCV is thought to bind to VCAM-1 as the first step in viral entry,³⁴ the mechanism by which EMCV injures myocytes with suppressed VCAM-1 expression in TNF-^-/- mice is unknown, and the exact cause of death in TNF-^-/- mice remains to be elucidated. On the basis of our data, however, it is obvious that the presence of TNF-, particularly at an early stage of infection, is critical to protection from lethal EMCV infection.

In conclusion, TNF- plays a protective role in acute viral myocarditis, possibly through leukocyte recruitment.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid for General Scientific Research from the Japanese Ministry of Education, Science, and Culture (10670641). We thank S. Maeda, M. Takemura, N. Furuta, A. Morise, C. Togashi, and A. Saito for excellent technical assistance.

Received June 9, 2000; revision received August 15, 2000; accepted August 15, 2000.

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