Coxsackievirus B3-Induced Myocarditis
1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65585-x
ISSN1525-2191
AutoresJohn R. Gebhard, Christopher Perry, Stephanie Harkins, Thomas E. Lane, Ignacio Mena, Valérie C. Asensio, Iain L. Campbell, J. Lindsay Whitton,
Tópico(s)interferon and immune responses
ResumoViral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1α expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1α knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus. Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1α expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1α knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus. Epidemiological and other studies suggest that 70% of the general public will be exposed to cardiotropic viruses, and half of them will have an episode of acute viral myocarditis.1O'Connell JB The role of myocarditis in end-stage dilated cardiomyopathy.Tex Heart Inst J. 1987; 14: 268-275PubMed Google Scholar Necropsy studies of victims of violent or accidental deaths show a remarkably high prevalence of myocarditis, approximately 1%,2Gravanis MB Sternby NH Incidence of myocarditis: a 10-year autopsy study from Malmo, Sweden.Arch Pathol Lab Med. 1991; 115: 390-392PubMed Google Scholar although only a subset of these individuals, probably around 10%, have clinical disease, with symptoms such as chest pains, palpitations, or signs of heart failure. Although the majority of symptomatic patients recover well from acute myocarditis, the disease can have serious long-term sequelae; some 10 to 20% of symptomatic patients will develop chronic disease, which may progress over time to dilated cardiomyopathy (DCM),1O'Connell JB The role of myocarditis in end-stage dilated cardiomyopathy.Tex Heart Inst J. 1987; 14: 268-275PubMed Google Scholar, 3Sole MJ Liu P Viral myocarditis: a paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy.J Am Coll Cardiol. 1993; 22: 99A-105AAbstract Full Text PDF PubMed Scopus (144) Google Scholar in which one or both ventricles dilate and decompensate, with resulting cardiac failure. Dilated cardiomyopathy affects 20,000 to 40,000 Americans annually and has a 50% mortality in the 2 years after diagnosis,4Goodwin JF Cardiomyopathies and specific heart muscle diseases: definitions, terminology, classifications and new and old approaches.Postgrad Med J. 1992; 68: S3-S6PubMed Google Scholar and the most effective treatment is cardiac transplantation.5Heck CF Shumway SJ Kaye MP The Registry of the International Society for Heart Transplantation: sixth official report, 1989.J Heart Transplant. 1989; 8: 271-276PubMed Google Scholar Many viruses can cause myocarditis, but enteroviruses in general, and coxsackieviruses in particular, are important human pathogens. Coxsackieviruses are members of the Picornaviridae family and fall into groups A and B, defined by their pathogenicity in newborn mice.6Hyypia T Kallajoki M Maaronen M Stanway G Kandolf R Auvinen P Kalimo H Pathogenetic differences between coxsackie A and B virus infections in newborn mice.Virus Res. 1993; 27: 71-78Crossref PubMed Scopus (30) Google Scholar The proportion of cases of acute myocarditis, and of dilated cardiomyopathy, with suspected coxsackievirus etiology varies among studies, but is usually 25 to 50% and most commonly involves B group viruses (CVB). CVB has been isolated from stool or pharyngeal specimens of many patients with acute myocarditis.7Gelfand HM The occurrence in nature of the coxsackie and ECHO viruses.Prog Med Virol. 1961; 3: 193-244PubMed Google Scholar, 8Woodruff JF Viral myocarditis: a review.Am J Pathol. 1980; 101: 425-484PubMed Google Scholar Isolation of infectious virus particles directly from the human myocardium is less common, because myocardial biopsy remains unusual, but specimens obtained at necropsy after a lethal infection have yielded infectious CVB,7Gelfand HM The occurrence in nature of the coxsackie and ECHO viruses.Prog Med Virol. 1961; 3: 193-244PubMed Google Scholar, 9Sutton GC Harding HB Trueheart RP Clark HP Coxsackie B4 myocarditis in an adult: successful isolation of virus from ventricular myocardium.Clin Aviation Aerospace Med. 1967; 38: 66-69Google Scholar, 10Sutton GC Tobin JR Fox RT Freeark RJ Driscoll JF Study of the pericardium and ventricular myocardium: exploratory mediastinotomy and biopsy in unexplained heart disease.JAMA. 1963; 185: 786-788Crossref PubMed Scopus (6) Google Scholar which causes myocarditis in mice.9Sutton GC Harding HB Trueheart RP Clark HP Coxsackie B4 myocarditis in an adult: successful isolation of virus from ventricular myocardium.Clin Aviation Aerospace Med. 1967; 38: 66-69Google Scholar Although five of the six CVB types can cause myocarditis, the majority of animal studies have focused on CVB3 and have shown unequivocally that this virus can cause cardiac disease. Studies have been carried out in many species, including primates,11Paque RE Gauntt CJ Nealon TJ Assessment of cell-mediated immunity against coxsackievirus B3-induced myocarditis in a primate model (Papio papio).Infect Immun. 1981; 31: 470-479PubMed Google Scholar but the most popular animal model is the mouse. The murine model systems show early death, acute myocarditis, and longer-term disease, thus paralleling the human situation. CVB3 infection of mice can result in death at around 4 days postinfection (p.i.), at which time histologically apparent myocarditis is rarely detectable.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar In surviving mice of several strains, a biphasic myocarditis occurs; an acute stage, at which infiltration is first seen 4 to 7 days p.i., resolving by day 14 or so, and a chronic stage, in which low-grade inflammation is maintained. CVB can be isolated from myocardium up to day 9 to 10 p.i., but no infectious virus is detected beyond day 14 (at least in immunocompetent mice). Although there is no doubt that CVB3 can cause myocarditis and myocyte destruction in mice, the precise mechanisms underlying these processes remain uncertain. The role of cytotoxic T lymphocytes (CTLs) in CVB-induced myocarditis was first shown more than 20 years ago13Woodruff JF Woodruff JJ Involvement of T lymphocytes in the pathogenesis of coxsackie virus B3 heart disease.J Immunol. 1974; 113: 1726-1734PubMed Google Scholar and has been confirmed in subsequent studies (see, eg, Refs. 12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar, 14Hashimoto I Tatsumi M Nakagawa M The role of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease.Br J Exp Pathol. 1983; 64: 497-504PubMed Google Scholar, 15Ytterberg SR Mahowald ML Messner RP T cells are required for coxsackievirus B1 induced murine polymyositis.J Rheumatol. 1988; 15: 475-478PubMed Google Scholar, 16Kishimoto C Abelmann WH In vivo significance of T cells in the development of Coxsackievirus B3 myocarditis in mice: immature but antigen-specific T cells aggravate cardiac injury.Circ Res. 1990; 67: 589-598Crossref PubMed Scopus (50) Google Scholar). Acute myocarditis becomes histologically evident 4 to 7 days p.i., and cellular infiltrate comprises CD8+ T cells, natural killer cells, and macrophages.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar, 17Godeny EK Gauntt CJ In situ immune autoradiographic identification of cells in heart tissues of mice with coxsackievirus B3-induced myocarditis.Am J Pathol. 1987; 129: 267-276PubMed Google Scholar, 18Seko Y Shinkai Y Kawasaki A Yagita H Okumura K Takaku F Yazaki Y Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.Circulation. 1991; 84: 788-795Crossref PubMed Scopus (78) Google Scholar, 19Chen SX Mei SW Bao SH Zheng XJ Chang PL Yao JS Yao S Zhang DQ Immunological status and pathology of coxsackie B viral myocarditis and dilated cardiomyopathy.Chin Med J (Engl). 1993; 106: 659-664PubMed Google Scholar This infiltration usually resolves after viral clearance, leading to host recovery (sometimes with residual myocardial fibrosis). We have shown that CTLs play a major role in myocarditis; functional paralysis of this T-cell population (either by monoclonal antibody depletion, or by the use of mice deficient in class I recognition) greatly diminished the acute inflammatory response and prevented subsequent myocardial scarring.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar However CD8+ T-cell depletion is a double-edged sword: in the absence of functional CTLs, viral titers were significantly increased, indicating that CTLs are both protective (diminishing virus titers) and immunopathological (causing extensive myocarditis). The mechanisms by which CTLs exert their biological effects are varied. Major histocompatibility complex class I expression, required for CTL-mediated recognition and lysis, is low to undetectable on normal myocardiocytes in vivo, but can be readily detected in vivo after CVB infection,20Seko Y Tsuchimochi H Nakamura T Okumura K Naito S Imataka K Fujii J Takaku F Yazaki Y Expression of major histocompatibility complex class I antigen in murine ventricular myocytes infected with Coxsackievirus B3.Circ Res. 1990; 67: 360-367Crossref PubMed Scopus (50) Google Scholar perhaps being induced by interferon. CTLs have at least three methods whereby they may affect virus-infected target cells: 1) perforin-mediated lysis, 2) Fas-dependent lysis, and 3) cytokine release. Perforin is critical to the control of some virus infections, given that perforin knockout (PKO) mice are unable to clear infection by lymphocytic choriomeningitis virus.21Kagi D Ledermann B Burki K Seiler P Odermatt B Olsen KJ Podack ER Zinkernagel RM Hengartner H Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.Nature. 1994; 369: 31-37Crossref PubMed Scopus (1533) Google Scholar, 22Walsh CM Matloubian M Liu CC Ueda R Kurahara CG Christensen JL Huang MT Young JD Ahmed R Clark WR Immune function in mice lacking the perforin gene.Proc Natl Acad Sci USA. 1994; 91: 10854-10858Crossref PubMed Scopus (445) Google Scholar In this article, we evaluate the role of perforin both in the etiology of CVB3 myocarditis and in CVB3 infection and clearance. Perforin-mediated lysis of CVB-infected cells is a plausible foundation for myocarditis and for control of myocardial virus titers, both of which we found to be CTL dependent.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar Perforin-containing cells enter the myocardium soon after CVB infection,18Seko Y Shinkai Y Kawasaki A Yagita H Okumura K Takaku F Yazaki Y Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.Circulation. 1991; 84: 788-795Crossref PubMed Scopus (78) Google Scholar and circumstantial evidence indicates that perforin may be required for CVB-induced myocarditis;23Seko Y Shinkai Y Kawasaki A Yagita H Okumura K Yazaki Y Evidence of perforin-mediated cardiac myocyte injury in acute murine myocarditis caused by Coxsackie virus B3.J Pathol. 1993; 170: 53-58Crossref PubMed Scopus (42) Google Scholar in addition, splenocytes from CVB-infected mice can lyse CVB-infected myocytes in vitro.24Huber SA Job LP Cellular immune mechanisms in Coxsackievirus group B, type 3 induced myocarditis in BALB/C mice.Adv Exp Med Biol. 1983; 161: 491-508Crossref PubMed Scopus (37) Google Scholar In this communication, we show that the absence of perforin is, broadly, beneficial to CVB-infected mice. PKO mice survive a CVB3 challenge that is lethal to 90% of perforin +/+ littermates, and although myocarditis occurs in the absence of perforin, it is much less extensive than in perforin+ mice, resolves more rapidly, and does not result in severe myocardial fibrosis. Despite the diminished myocardial infiltration in PKO mice, viral titers, myocardial distribution, and growth kinetics are indistinguishable in PKO and perforin+ mice, and virus is undetectable by 21 days p.i. in both mouse phenotypes. Therefore, in CVB myocarditis, the antiviral and immunopathological functions of CTLs can be uncoupled. We also evaluate apoptosis and chemokine expression in PKO and perforin+ mice. In both mouse genotypes, chemokine expression approximately parallels myocarditis. Expression of MIP-1α, a chemokine whose disruption renders mice resistant to myocarditis,25Cook DN Beck MA Coffman TM Kirby SL Sheridan JF Pragnell IB Smithies O Requirement of MIP-1α for an inflammatory response to viral infection.Science. 1995; 269: 1583-1585Crossref PubMed Scopus (564) Google Scholar is barely detectable in PKO mice. These findings have important therapeutic implications; the development of a specific inhibitor of perforin or of the chemokine pathways might permit us to prevent immunopathology without diminishing the antiviral efficacy of the immune response. The cardiopathogenic H3 strain of CVB3 (Nancy) was utilized in all experiments. The titer of the virus was determined by plaque assay in HeLa cells according to previously published procedures. PKO mice were generated by Drs. C. M. Walsh and W. R. Clark (University of California, Los Angeles, Los Angeles, CA), using AB-1 embryonal stem cells, which were introduced into the C57 background and bred on this background.22Walsh CM Matloubian M Liu CC Ueda R Kurahara CG Christensen JL Huang MT Young JD Ahmed R Clark WR Immune function in mice lacking the perforin gene.Proc Natl Acad Sci USA. 1994; 91: 10854-10858Crossref PubMed Scopus (445) Google Scholar Heterozygous PKO mice were sent to Scripps Research Institute, where they were bred to generate homozygous knockout mice, as well as heterozygous and homozygous positive littermates (as controls). We determined the perforin gene status by polymerase chain reaction on tail DNA, for every mouse bred. All experiments utilized adult (at least 8 weeks old) male PKO mice and their transgenic wild-type (homozygous or heterozygous) littermates. Mice were infected (0.2 ml intraperitoneally) with CVB3 diluted in saline. For evaluation of susceptibility to early death, mice were given 100 plaque-forming units (pfu) CVB3 on day 0 and observed for 21 days. For evaluation of myocarditis, mice were given a sublethal dose (35 pfu) to ensure survival, and at the indicated time points, the organs were removed, dissected in equal parts, washed, and either snap frozen in liquid nitrogen or fixed in 10% zinc normal buffered formalin for histological analysis. Quantitation of virus from heart tissue was performed by homogenization of the tissue and centrifugal clarification of the supernatant, followed by virus plaque assay in HeLa cells as described. For histological analysis, heart tissue was fixed in 10% neutral buffered formalin, sectioned into 5-μm slices, and stained either with hematoxylin and eosin (H&E) or with Masson's trichrome as specified in the text. Inflammation was scored on a scale of 0 to 4 based on the number of lesions present in a section. A score of 1 represents 1 to 10 lesions; 2, 11 to 20; 3, 21 to 40; and 4, >40. The RNase protection assay was performed as previously described.26Asensio VC Campbell IL Chemokine gene expression in the brains of mice with lymphocytic choriomeningitis.J Virol. 1997; 71: 7832-7840Crossref PubMed Google Scholar Probes used were C10 (a murine member of the C-C chemokine family); MIP-1α and MIP-1β (macrophage inflammatory proteins 1α and 1β); MIP-2 (macrophage inflammatory protein 2); MCP-1 and MCP-3 (monocyte chemoattractant proteins 1 and 3); crg-2 (cytokine-regulated gene-2); and RANTES (regulated upon activation normal T cell expressed and secreted). A previously published protocol27Lane TE Paoletti AD Buchmeier MJ Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.J Virol. 1997; 71: 2202-2210PubMed Google Scholar was followed on paraffin sections (5 μm) of heart tissue previously fixed in 10% normal buffered formalin. After preparation of the slides and prehybridization at 42°C, 1 × 106 cpm of a radiolabeled antisense RNA probe was applied and allowed to hybridize at 42°C overnight. After washing, slides were dipped in photographic emulsion and were held at 4°C for 3 to 14 days, when they were developed and fixed. To visualize the cells, the developed slides were stained in H&E for 2 to 3 minutes. The Apoptag kit (Oncor, Gaithersburg, MD) was used according to the manufacturer's instructions. Three groups of mice (10 mice per group) were infected with 100 pfu CVB3 and were observed for 21 days. As shown in Figure 1, 90% of homozygous positive littermates (wild type, +/+) died, whereas 100% of PKO mice survived. Thus, CTL-mediated lysis appears to contribute to a lethal outcome of CVB infection. This is consistent with our findings of diminished susceptibility in β2mKO mice, which lack CD8+ T cells, and of protection against lethal outcome after CD8 depletion of CD4KO mice.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar There may be a gene dosage effect, because 50% of heterozygous mice survived, compared with only 10% of homozygous positive mice. No animals died after day 10 p.i. Thus, perforin appears important to the immunopathological function of CD8+ T cells during lethal acute infection. Results for +/+ and +/− mice were similar, and henceforth both genotypes will be referred to as "perforin+" mice. Figure 2 shows the development of myocarditis lesions over time in PKO and perforin+ mice. At each time point, in each group, a minimum of four mice was used (thus, perforin+ groups include at least eight mice, four +/+ and four +/−). At 4 days p.i., no infiltration was seen in PKO mice, and only very occasional small foci were detected in perforin+mice. By 7 days p.i., mild myocarditis could be seen in both perforin+ and PKO mice. Myocarditis was much more marked by day 10 in perforin+ mice, but declined in PKO littermates. By day 14, myocarditis had almost resolved in PKO mice (four out of four mice), but remained severe in perforin+ animals. Note that this scoring system reflects only the number of individual lesions and not the total area of myocardium involved. In a particularly extensive myocarditis, the lesion score may even be reduced as adjacent lesions "fuse" to produce a single large lesion. Consequently, lesion scores must be complemented by representative histology (Figure 3). The lesions in PKO mice were very much smaller, and more circumscribed, than those in perforin+ animals; the appearance in the PKO mice was similar to that seen after CD8 depletion of CD4KO mice and to that seen in β2mKO mice.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google ScholarFigure 3Myocarditis in perforin+ and PKO mice: perforin is required for extensive infiltration. Representative H&E-stained sections of hearts from CVB-infected perforin+ mice (left) and PKO mice (right) at days 4, 10, and 14 p.i. Original magnification, ×50. The final row shows enlargements (original magnification, ×25) of myocardial lesions at day 10 p.i.View Large Image Figure ViewerDownload Hi-res image Download (PPT) No infiltration was detectable in either genotype by day 21 p.i., but by >30 days p.i., marked myocardial fibrosis was present in perforin+ mice, but not in PKO animals (Figure 4). Thus, perforin exacerbates myocarditis, and its ablation prevents not only acute disease, but also the subsequent scarring. CTLs are critical to the control of many virus infections, but, in combating picornavirus infections, antibodies appear to play a vital role. Nevertheless, we have shown that CD8+ T cells play some role in limiting CVB replication, because depletion of CD8+ T cells leads to increased CVB load in the heart.12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar To determine whether this antiviral effect was mediated by perforin, a time course of CVB3 replication in the heart was carried out, comparing PKO mice with perforin+littermates. As shown in Figure 5, there was no statistically significant difference between the mouse strains. Regardless of the perforin status, the growth curve peaked on day 4 p.i., falling rapidly thereafter, and no virus was detectable by 21 days p.i. (not shown). Furthermore, the absolute titers at each time point were not significantly different. Thus, perforin is not required for clearance of CVB3, and it appears to play little if any role in the limitation of viral replication in the heart. Although we show above that myocardial virus titers are similar regardless of the perforin status of the host, it remained possible that the infection would be more widely distributed in the myocardia of mice lacking perforin. Therefore, at the peak of virus titer, 4 days p.i., myocardial sections were evaluated by in situhybridization with antisense probes for CVB3. For this positive-strand virus, the antisense probe detects the abundant viral genome and the almost identical single mRNA. Although the virus titer is very high (∼108 pfu/g), virus was detectable only in well separated and tightly circumscribed foci in both PKO and perforin+hearts (shown by dark-field microscopy in Figure 6); the lesion numbers were similar in all mice, regardless of perforin status. Bright-field examination of the infected foci revealed no inflammation, consistent with our finding that myocarditis is absent on day 4 p.i., as shown in Figure 3. Apoptosis, or programmed cell death, is an important facet of the host defense against virus infection and also plays a critical part in several aspects of host development, including regulation of the immune response. Apoptosis can be induced by several mechanisms. We looked for apoptosis in +/+ and +/− littermates and found signal in the infiltrate, but not in noninflamed muscle (Figure 7); this is consistent with our previous observation in the CD4KO mouse strain (also perforin+).12Henke A Huber SA Stelzner A Whitton JL The role of CD8+ T lymphocytes in coxsackie virus B3-induced myocarditis.J Virol. 1995; 69: 6720-6728PubMed Google Scholar The picture differed in PKO mice; little or no signal could be detected in the small inflammatory lesions, whereas many myocyte nuclei were positive. Therefore, in perforin+ mice, there was a marked inflammation, with apoptosis restricted to these areas whereas in PKO mice the myocarditis was reduced, but apoptosis was detected in myocyte nuclei. CVB-induced myocarditis does not occur in MIP-1α transgenic knockout mice.25Cook DN Beck MA Coffman TM Kirby SL Sheridan JF Pragnell IB Smithies O Requirement of MIP-1α for an inflammatory response to viral infection.Science. 1995; 269: 1583-1585Crossref PubMed Scopus (564) Google Scholar This chemokine is secreted by macrophages, lymphocytes, and neutrophils and is chemoattractant for T cells. We considered it possible that perforin-mediated lysis of infected cells might increase neutrophil and macrophage infiltration, which would, through release of MIP-1α and other chemokines, increase the influx of CTLs. If so, the limited myocardial infiltration in PKO mice might result, in part, from lower chemokine levels in the infected myocardium, which in turn resulted from the reduced cytolysis. Normal and PKO mice were infected with a sublethal dose of CVB3 and chemokine mRNA expression in the hearts was examined at the indicated time points, using an RNase protection assay. As demonstrated in Figure 8, many of the proinflammatory chemokines were up-regulated during the course of infection. Both the onset and decay of chemokine expression seemed advanced in PKO mice; crg-2 and MCP-1 were detectable at day 2 p.i. and peaked at day 4 p.i. In contrast, chemokine expression in perforin+ mice was undetectable on day 2, and barely detectable on day 4 p.i.; in these mice, expression peaked around day 7, when myocarditis developed. In both PKO and perforin+ mice, signal reached background levels by day 14. When considering specific chemokines, MIP1-α was barely
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