Skip Navigation

Cardiovascular Research 2000 45(3):579-587; doi:10.1016/S0008-6363(99)00267-9
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Huber, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huber, S. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

T cells expressing the {gamma}{delta} T cell receptor induce apoptosis in cardiac myocytes

Sally A. Huber*

Department of Pathology, University of Vermont, 55A South Park Drive, Colchester, VT 05446, USA

* Corresponding author. Tel.: +1-802-656-8944; fax: +1-802-656-8965 shuber{at}salus.uvm.edu

Received 2 June 1999; accepted 9 August 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 4 Results
 5 Discussion
 References
 
Objective: Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the {gamma}{delta} T cell receptor (TcR), and that only mouse strains which accumulate {gamma}{delta} T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that {gamma}{delta} T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the {alpha}β TcR. Methods: Bl.Tg.E{alpha} mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin-fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of {gamma}{delta}+ and {alpha}β+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC-conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by 51Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h 51Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A. Results: Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18–24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the {gamma}{delta} TcR with the remainder expressing the {alpha}β TcR. Both {gamma}{delta}+ and {alpha}β+ T cells lysed myocyte targets. Blocking studies indicate that {gamma}{delta}+ T cells induced predominantly Fas-mediated killing, while {alpha}β+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well. Conclusions: These studies demonstrate that T cells expressing the {gamma}{delta} TcR are more effective mediators of myocyte apoptosis than {alpha}β+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis.

KEYWORDS Apoptosis; Cell culture/isolation; Immunology; Myocarditis; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 4 Results
 5 Discussion
 References
 
Enterovirus infections are accepted as major etiologic factors in clinical myocarditis, and, to a lesser extent, in dilated cardiomyopathy [1–4]. Although most individuals with myocarditis recover, some either die or progress to chronic disease, congestive heart failure and dilated cardiomyopathy [5–7]. Little is known about the factors predisposing individuals to a serious disease outcome. Experimental models have been developed to investigate the pathogenic mechanisms of viral myocarditis. These studies have shown multiple mechanisms of myocyte injury ranging from direct virus-induced injury through lysis of myocytes [8], enteroviral protease cleavage of dystrophin resulting in cytoskeletal disruption [9], and virus replication-mediated suppression of myocyte contractility [10], to virus-induced autoimmunity to heart-specific antigens [11–13]. Cytokines, such as IL-1β and TNF{alpha}, and nitric oxide which are either released by invading inflammatory cells or produced by infected myocardial cells, may additionally disrupt cardiac function [14–16].

Apoptosis is an important part of normal heart development [17,18], but is also implicated in many forms of cardiovascular disease [19–22]. Apoptotic myocytes identified by DNA strand breaks and TdT-TUNEL staining are most often found in patients with chronic myocarditis and dilated cardiomyopathy, but are absent or rare in patients with acute myocarditis [23,24]. Circulating levels of soluble FasL may correlate with severity of congestive heart failure [25]. Studies from this laboratory demonstrate that distinct immunopathogenic mechanisms occur in genetically different mouse strains [26]. Furthermore, only selected types of myocyte injury lead to cardiomyopathy-like disease, characterized by decreased velocity of sarcomere shortening, increased left ventricular atria natriuretic factor (ANF) mRNA expression and a shift to embryonic (β-myosin heavy chain) gene expression [27,28]. These changes were dependent upon the infiltration of the heart with a population of CD8+ T lymphocytes, and correlated to the appearance of apoptotic myocytes. Strains of mice developing severe cardiac inflammation without cardiomyopathy-like alterations uniformly lacked apoptotic myocytes, although apoptosis of inflammatory or interstitial cells could be observed [26]. Two type of CD8+ T cells infiltrate the myocardium during coxsackievirus B3-induced myocarditis. One type expresses {alpha}β T cell receptors (TcR) and represents the classical antigen-specific T cells which are major histocompatibility complex (MHC) antigen restricted and dominate in peripheral lymphoid tissues. The second type expresses {gamma}{delta} TcR [29]. {gamma}{delta}+ T cells are usually a minor component of peripheral lymphoid tissues, but tend to accumulate in inflammatory lesions. Generally, these lymphocytes recognize antigen independently of MHC molecules and are often responsive to heat shock or stress proteins [30]. Among the various lymphocyte subpopulations, {gamma}{delta}+ T cells express the highest levels of FasL [31], making these effectors the logical mediators of Fas-dependent apoptosis. The goal of the present communication is to evaluate the relative abilities of both {alpha}β+ and {gamma}{delta}+ T cells and CD8+ T cells derived from the hearts of myocarditic mice to kill cultured myocytes through either perforin or Fas-dependent mechanisms.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
4 Results
 5 Discussion
 References
 
2.1 Mice
Genetically modified C57Bl mice (Bl.Tg.E{alpha}) were originally obtained as breeding pairs from Dr. Chella David (Mayo Clinic, Rochester, MN) and were bred and housed at the University of Vermont Animal Care facility. These animals were made by injecting a cloned E{alpha}k gene from A/J mice into male pronucleus of F2 hybrids from C57Bl/6xSJL animals. Transgenic mice were backcrossed through 18 generations to C57Bl/6 mice [32]. Previous studies have demonstrated that Bl.Tg.E{alpha} male mice are highly susceptible to coxsackievirus B3 (CVB3)-induced myocarditis [33]. Males, 4–5 weeks of age, were used for these studies. The investigation conforms with the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.2 Virus
Animals were infected intraperitoneally with 0.5 ml phosphate buffered saline (PBS) containing 5x104 plaque-forming units (PFU) CVB3 (H3 variant) derived from Cos cells transfected with the infectious cDNA of this variant [34].

2.3 Histology and TdT-TUNEL staining
Hearts were removed, fixed in 10% buffered formalin and paraffin embedded. Ten micron sections were placed on slides and either stained with hematoxylin and eosin, or deparaffinized and stained for nicked DNA using the TdT-FragEL DNA Fragmentation Detection kit from Oncogene Research Products (Cambridge, MA) according to the maufacturer's directions.

2.4 Antibodies
The following antibodies were used: monoclonal mouse anti-{alpha}-sarcomeric actin (clone 5C5; Sigma), Cy-Chrome rat anti-mouse CD3 (clone 17A2; Pharmingen; San Diego, CA); rat anti-mouse CD4 (clone GK 1.5; American Type Culture Collection (ATCC), Bethesda, MD); FITC-rat anti-mouse CD4 (clone GK 1.5; Pharmingen); biotin-rat anti-CD8a (clone 53-6.7; Pharmingen); mouse anti-NK1.1 (clone PK136; Pharmingen); PE-hamster anti-FasL (clone MFL3; Pharmingen); FITC-hamster anti-Fas (CD95) (clone Jo2; Pharmingen); purified, FITC, and biotin-hamster anti-{gamma}{delta} (clone GL3; Pharmingen); purified, FITC hamster anti-TcRβ (clone 57-597; Pharmingen); mouse anti-hamster IgG (clone G70-204; Pharmingen); biotin-hamster anti-mouse CD69 (clone H1.2F3; Pharmingen); mouse anti-CVB3 VP1 (clone 8A6); PE sheep anti-mouse IgG (polyclonal; Sigma); rat IgG isotype (clone R35-95; Pharmingen); hamster IgG isotype (polyclonal; Pharmingen); and mouse IgG isotype (clone 107.3; Pharmingen). Streptavidin-PE and Streptavidin-Red670 were obtained from GIBCO BRL (Grand Island, NY).

2.5 Preparation of lymphocytes
Mice were euthanized by injecting 120 mg kg–1 sodium pentobarbitol in PBS intraperitoneally. Hearts are removed, minced finely and digested stepwise three times with 10 ml of 0.4% collagenase Type II (Cooper Biomedical, Freehold, NJ). Cells in the supernatant were washed twice, centrifuged at 1048g for 15 min on Histopaque (Sigma, St. Louis, MO), and the cells at the interface were retrieved, and washed once with RPMI 1640 medium and incubated for 30 min on nylon wool and non-adherent cells were retrieved by washing the nylon wool with 30x volume medium [35]. An aliquot of 2x105 of these lymphocytes was stained with Cy-Chrome-rat anti-mouse CD3, FITC-hamster-anti-mouse {alpha}β TcR, and biotin-hamster-anti-mouse {gamma}{delta} TcR and PE-streptavidin as described below. Purification of cell populations was done using BioMag Magnetic Particles (PerSeptive Biosystems, Framingham, MA). Briefly, lymphocytes were incubated in 1 ml medium containing a combination of anti-NK 1.1, anti-CD4 and anti-{alpha}βTcR monoclonal antibodies (to isolate {gamma}{delta}+ enriched cells) or a combination of anti-NK 1.1, anti-CD4 and anti-{gamma}{delta} TcR (to enrich for CD8+ {alpha}β+ T cells). All antibodies were at a final concentration of 1:50 dilution in medium containing 1% bovine serum albumin (BSA) (Sigma). Cells and antibody were incubated for 30 min at 4°C, centrifuged and resuspended in medium+BSA containing a 1:50 dilution of mouse anti-hamster IgG. After incubation for 20 min on ice, the cells were centrifuged, resuspended in 3 ml medium and 1 ml each of magnetic particles conjugated with goat anti-mouse IgG and goat anti-rat IgG. After incubation for 30 min, cells were placed on a BioMag magnetic support. The supernatant was removed, placed in a new petri dish and put on the magnetic support to remove any residual magnetic particles. Remaining cells were counted by trypan blue exclusion.

2.6 Preparation of myocytes
The procedure for preparation of myocyte cultures has been published previously [36]. Briefly, hearts are removed from neonatal mice within 48 h of birth, minced finely and then subjected to stepwise enzymatic digestion with 0.4% collagenase II and 0.25% pancreatin (Sigma). The cellular debris was allowed to settle and the single cell suspension was retrieved, washed twice and incubated on plastic 25 cm2 tissue culture flasks in medium containing 10% fetal bovine serum (FBS) for three sequential 1-h adsorptions at 37°C to remove endothelial cells and fibroblasts. Non-adherent cells were retrieved washed once with medium and counted for trypan blue exclusion. An aliquot of the cells was stained with monoclonal anti-{alpha}-sarcomeric actin and PE-sheep anti-mouse IgG to evaluate the purity of myocyte preparation (Fig. 1).


Figure 1
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Expression of {alpha}-sarcomeric actin in neonatal myocyte cultures. Myocytes were isolated from neonatal mice within 48 h of birth by stepwise collagenase II digestion. Cells were stained with monoclonal mouse anti-{alpha}-sarcomeric actin and phycoerythrin (PE)-conjugated sheep anti-mouse IgG. Complexity of the cell population was evaluated by forward (FS) vs. side (SS) scatter. The gated population for evaluation is indicated in the boxed area of the first panel.

 
3.1 Flow cytometry
3.1.1 Cell surface staining
105 cells were washed in PBS containing 1% BSA (buffer) and resuspended in 100 µl buffer containing a 1:100 dilution of fluorochrome labeled antibody and a 1:100 dilution of Fc Block, then incubated on ice for 20 min. For biotinylated antibodies, cells were incubated for 20 min with biotin-conjugated antibody and Fc-Block (1:100 dilution of each), centrifuged and resuspended in buffer containing a 1:50 dilution PE-conjugated streptavidin in a two-step labeling procedure. After incubation on ice for 30 min, the cells were washed twice in buffer and fixed in 2% paraformaldehyde for flow analysis.

3.1.2 Intracellular staining
Intracellular antigens were identified by incubating washed cells in 2% paraformaldehyde for 10 min, followed by washing the cells once in buffer alone and once in buffer containing 0.25% saponin (Sigma). Cells were incubated in buffer–saponin containing a final 1:100 dilution each of Fc Block, normal mouse serum and fluorochrome-conjugated antibody for 30 min on ice. The cells were washed once in buffer–saponin and once in buffer alone, then resuspended in 2% paraformaldehyde for flow analysis.

3.1.3 Flow analysis
Cells were evaluated using a Coulter Epics Elite instrument with a single excitation wavelength (488 nm) and band filters for Cy-Chrome/Streptavidin-Red670 (670 nm), PE (575 nm) and FITC (525 nm). Each cell population was classified for cell size (forward scatter) and complexity (side scatter), gated on a population of interest, and evaluated using a minimum of 10 000 cells. Criteria for positive staining were established based on the intensity of the isotope controls. Percent positive cells represents the specifically stained cells minus percent positive cells in the isotype control preparation. Each study was performed at least twice.

3.2 Cytotoxicity assay
Cultures of 3T3 fibroblasts which were stably transfected with plasmid alone or plasmid containing the FasL gene were graciously supplied by Dr. Ralph Budd, Department of Medicine, University of Vermont. These cells were maintained by serial passage in RPMI 1640 medium containing 10% FBS and 0.5 µg ml–1 neomycin. 104 3T3 fibroblasts were plated into wells of 96 well tissue culture plates, incubated overnight, washed once and incubated in medium without neomycin for at least 4 h before addition of target cells. Myocytes were isolated as described above, infected with 100 PFU CVB3/myocyte for 1 h at 37°C in medium containing 10% FBS, washed twice and cultured for varying times as indicated below. Infected and uninfected myocytes were retrieved by trypsinization, washed once and labeled with 100 µCi 51Cr (Na51CrO4; ICN, Costa Mesa, CA) in 1 ml medium at 37°C for 90 min. The cells were washed 3 times and resuspended at 105 cells/200 µl medium with 10% FBS and aliquoted onto 3T3 monolayers. For cytotoxicity assays with T lymphocytes, myocyte targets were labeled as above and 104 targets were co-cultured with 3x105 lymphocytes (30:1 effector–target cell ratio) for 6 h at 37°C in a final volume of 200 µl in 96 well tissue culture plates. In some cultures 100 nM concanamycin A (CMA; Sigma) or 10 µg ml–1 Fas-Fc (kindly supplied by Dr. Jurg Tschopp, Institute of Biochemistry, University of Lausanne Epalinges, Switzerland) was added. Spontaneous release (medium control) cultures contained labeled myocytes only. Maximum release cultures contained myocytes to which 20 µl 6N HCl was added. The cultures were incubated at 37°C for 4 h and 100 µl supernatant was removed and counted using a Packard Gamma Counter (Packard; Downers Grove, IL). Percent cytotoxicity represents: (cpm in supernatant)/(cpm in acid release cultures)x100. Cultures were run in triplicate.

3.3 Statistics
Statistical evaluation was performed using Student's t-test.


   4 Results
Top
 Abstract
 1 Introduction
 2 Methods
 4 Results
5 Discussion
 References
 
4.1 CVB3 infections upregulate Fas expression on cultured cardiac myocytes
To determine whether CVB3 alters Fas expression in cultured myocyte populations, neonatal myocytes were infected with CVB3 and then cultured for up to 24 h. Cells were stained for surface expression of Fas and intracellular expression of CVB3 VP1 protein (Fig. 2). Controls consisted of uninfected myocytes. Approximately 20% of uninfected myocytes expressed low levels of Fas. This increased to over 60% of infected myocytes by 18 h. By 24 h, the numbers of virus protein positive myocytes decreased, but the numbers of Fas positive myocytes remained elevated.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Fas and CVB3 VP1 expression in myocytes after infection. Neonatal myocytes were isolated as indicated in Section 2, infected with 100 PFU/cell for 1 h and cultured for up to 24 h, stained for Fas expression on the cell surface using FITC-hamster anti-mouse Fas, fixed with 2% paraformaldehyde, permeabilized and stained for intracellular expression of CVB3 VP1 protein using monoclonal mouse anti-CVB3 VP1 and PE-conjugated sheep anti-mouse IgG.

 
4.2 Virus infection of myocytes increases their susceptibility to Fas-dependent apoptosis
To determine whether CVB3 infection affected susceptibility of myocytes to Fas-dependent lysis, uninfected cells and cells infected for either 8, 18 or 24 h were 51Cr-labeled and incubated on either 3T3-mock or 3T3-FasL transfected cell monolayers for 4 h (Fig. 3). Myocytes cultured on 3T3-Mock monolayers showed increased 51Cr release with length of infection indicating some lysis which is most likely due to virus infection. However, myocytes cultured on FasL-transfected 3T3 cells demonstrated significantly increased cytolysis with cells infected 18 and 24 h earlier having greatest susceptibility.


Figure 3
View larger version (7K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 FasL-mediated cytotoxicity of neonatal myocytes. Myocytes were infected with CVB3 for up to 24 h, labeled with 51Cr, and cultured for 4 h on monolayers of either mock or FasL-transfected 3T3 cells. Cultures were done in triplicate. S.E.M. are indicated by bars. *, data is significantly different to Mock-transfected cultures at P≤0.05.

 
4.3 Demonstration of TdT-TUNEL-positive cells in the myocardium of CVB3-infected Bl.Tg.E{alpha} mice
Hearts from three Day-7 virus-infected mice were sectioned and adjacent sections were evaluated by either hematoxylin and eosin staining for inflammation, or by TdT-TUNEL staining for evidence of apoptosis (Fig. 4). TdT-TUNEL-positive cells were identified both within the inflammatory infiltrate, and in areas without evident inflammation [see arrow in Fig. 4(B)]. Higher magnification of a positively stained cell demonstrates a highly swollen myocyte nucleus with apparently clumped nuclear material [Fig. 4(C)].


Figure 4
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 TdT-TUNEL staining of Day 7 CVB3-infected Bl.Tg.E{alpha} myocardium. Hearts were fixed in 10% buffered formalin, paraffin embedded, sectioned and stained with either hematoxylin and eosin (A) or by TdT-TUNEL (B) (magnification, 80x). The cell indicated in (B) is shown at higher magnification (200x) in panel (C).

 
4.4 CD8+/{gamma}{delta}+ T cells are more effective in inducing myocyte apoptosis than CD8+/{alpha}β+ T cells
Flow analysis of the total lymphocyte population isolated from the heart indicates that 38±9% of CD3+ cells express the {gamma}{delta} TcR with the remaining 53±12% expressing the {alpha}β TcR. 71% of {gamma}{delta}+ cells are CD69+ indicating a high level of activation in this cell population. Previous studies demonstrated that CD8+ T cells are the major pathogenic effectors in mouse strains prone to cardiomyopathy-like disease [28,37]; but both CD8+/{gamma}{delta}+ and CD8+/{alpha}β+ T cells infiltrate the heart. To determine the relative cytolytic potential of these two populations, 51Cr-labeled 8 and 18 h infected myocytes were cultured with highly enriched CD8+/{gamma}{delta}+ and CD8+/{alpha}β+ T cells derived from the hearts of mice 7 days after viral infection. Fig. 5 characterizes the two lymphocyte populations staining for CD8, {gamma}{delta} TcR and {alpha}βTcR antigens. Fig. 6 gives the cytolytic activity of the lymphocyte populations at effector–target cell ratios of 30:1. CD8+/{alpha}β+ T cells are predominantly cytolytic to 8 h myocytes. These effectors will kill infected targets, but levels of cytotoxicity are lower for this effector cell population than for CD8+/{gamma}{delta}+ cells. The opposite pattern occurs with CD8+/{gamma}{delta}+ cell population, which is predominantly cytolytic to 18 h infected myocytes. Since CD8+/{gamma}{delta}+ and CD8+/{alpha}β+ T cells might kill myocyte targets through either perforin or Fas-dependent mechanisms, cytolytic assays were also performed in the presence of either 10 µg ml–1 Fas-Fc or 100 nM CMA, a specific inhibitor of perforin-mediated lysis [38]. As shown in Fig. 6, CD8+/{gamma}{delta}+cell-mediated cytotoxicity was predominantly inhibited by Fas-Fc but not CMA treatment indicating that these effectors were killing primarily by Fas-dependent mechanisms. In contrast, CD8+/{alpha}β+ cell-mediated cytotoxicity was most susceptible to CMA inhibition, indicating a predominant perforin-dependent mechanism for these effectors. Finally, both cytotoxic lymphocyte populations were stained of FasL expression (Fig. 7). Cells expressing the {gamma}{delta} TcR expressed higher levels of FasL than cells expressing the β TcR.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Characterization of CD8+ cell enriched populations isolated from the hearts of CVB3-infected mice. Cells were stained for CD8 (PE), β TcR (FITC) and {gamma}{delta} TcR (FITC).

 

Figure 6
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 T cell mediated lysis of myocyte targets. Myocytes were infected for either 8 or 18 h with 100 PFU/cell CVB3, 51Cr-labeled, and co-cultured with either {alpha}β+ or {gamma}{delta}+ TcR-enriched lymphocytes at an effector–target cell ratio of 30:1 for 6 h. Additional cultures contained either 100 nM concanamycin A (CMA) or 10 µg ml–1 Fas-Fc to block perforin and FasL-dependent killing, respectively. Results represent mean % cytotoxicity in triplicate cultures minus lysis in medium control cultures (% lysis in 8 h infected myocyte cultures was 10.5% and in 18 h cultures was 16.2%). Bars represent S.E.M. *, data is significantly less than cultures without CMA or Fas-Fc at P≤0.05.

 

Figure 7
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Lymphocytes isolated from the heart were stained with either FITC-anti-β TcR or FITC-anti-{gamma}{delta} TcR and PE-anti-FasL or PE-isotype control antibody (hamster IgG). Cells were gated on the FITC+ populations and evaluated for PE staining as an indication of relative FasL expression.

 

   5 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 4 Results
 5 Discussion
References
 
Although reports by this and other laboratories [13,28,37] associate CD8+ T cells with the more severe forms of coxsackievirus B3-induced myocarditis, how these effectors mediate myocardial injury is less certain. CD8+ T cells could damage cardiac myocytes in several different ways. The most likely mechanisms would be through perforin-induced myocyte necrosis or apoptosis. While once considered distinct processes, apoptosis and necrosis may share similarities. Generally, necrosis is viewed as osmotic lysis of a cell with release of cytosolic contents and stimulation of inflammation. In contrast, apoptosis results in cell shrinkage, fragmentation, DNA degradation and phagocytosis of the ‘apoptotic bodies’ [39–42]. The cytolytic T lymphocyte binds to its target, usually through recognition of MHC class I molecules and associated specific peptides in the MHC molecular groove. This triggers polarized secretion of perforin and granzyme B (serine protease) into the intracellular space between the lymphocyte and its target. Perforin is homologous to the ninth complement component and integrates into the target cell membrane opening pores which both promote osmotic lysis and allow entrance of granzyme B in the target. Granzyme B, by activating effector caspases, can itself cause apoptosis [43]. A second method for inducing apoptosis is through interactions of trimeric FasL on the cytolytic T cell membrane with Fas (CD95) on the target cell. This interaction aggregates the Fas molecules allowing FADD (Fas-associated death domain) to bind, activate caspase 8, and initiate apoptosis.

Perforin-positive lymphocytes are observed in the myocardium during myocarditis [44,45], and studies by Gebhard et al. [46] indicate that perforin-dependent mechanisms are primarily responsible for the inflammation and fibrosis in this disease. However, apoptosis of myocytes was only observed in perforin knockout animals, indicating that perforin-independent responses are responsible for this type of injury. Both our studies [26] and those of others [47] indicate that apoptosis correlates to myocyte dysfunction and that apoptosis can be observed independent of inflammation and over extended areas of the myocardium [26]. Thus, the total damage mediated through apoptosis can be significantly greater than necrosis of myocytes intimately associated with an inflammatory infiltrate. Despite these studies, other investigators have found only rare apoptotic myocytes in CVB3-infected mice [48]. Pro-apoptotic factors (Bax, Fas and FasL) showed no correlation to histology, but the expression of the anti-apoptotic Bcl-2 generally increased as histopathology increased, especially in CD1 mice. Our previously published study [26] also failed to demonstrate apoptotic myocytes in either CVB3-infected DBA/2 or MRL+/+ mice despite severe cardiac inflammation. We concluded in that study that the presence of apoptotic myocytes depended upon the pathogenic mechanism of myocardial injury with only a very specific (CD8+ cell-mediated) immunopathogenic response causing this effect. Myocarditis in both DBA/2 and MRL+/+ mice, while severe, was CD8+ T cell-independent. Whether myocarditis in C3H and CD1 mice as reported by Colson et al. [48] requires CD8+ T cells is unclear.

This report shows that CD8+ T cells expressing the {gamma}{delta} TcR are more effective mediators of Fas-dependent apoptosis and less effective mediators of necrosis of cardiac myocytes than CD8+ T cells expressing the {alpha}βTcR. The difference in killing patterns observed between {alpha}β+ and {gamma}{delta}+ T cells may reflect the types of antigens most likely recognized by these two effectors. {alpha}β+ T cells recognize processed antigenic epitopes in the context of MHC molecules. {gamma}{delta}+ cells are usually less antigen specific, or rather react to antigens that are often more broadly distributed. Thus, one of the major antigens recognized by {gamma}{delta}+ cells is heat shock protein [30] which can be upregulated by many forms of stress, including infections [49,50], free radical or cytokine mediated injury [51], and hyperthermia (possibly fever?) [52]. Indeed, Kanda et al. [52] report that heat stress substantially aggravates viral myocardial disease in mice. Finally, soluble factors released during inflammation likely upregulate Fas expression on both infected and uninfected myocytes and at considerable distances from the inflammatory lesions. This means there probably are far more targets for FasL-mediated killing than for antigen-specific, perforin-induced lysis. Therefore, we hypothesize that {gamma}{delta}+ T cells are a dominant factor in the pathology of viral myocarditis and cardiomyopathy.

Time for primary review 29 days.


   Acknowledgements
 
The author wishes to thank Ms. Debbie Perrotte for help in preparing this manuscript. The work was supported by grants from the American Heart Association (AHA9750081N) and the National Institutes of Health, Heart, Lung and Blood (HL58583).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 4 Results
 5 Discussion
 References
 

  1. Huber S.A., Gauntt C.J., Sakkinen P. Enteroviruses and myocarditis: Viral pathogenesis through replication, cytokine induction and immunopathogenicity. Advances in Virus Research (1998) 51:35–80.[Medline]
  2. Huber S. Animal models of human disease—Autoimmunity in myocarditis: Relevance of animal models. Clin Immunol Immunopathol (1997) 83:93–102.[CrossRef][ISI][Medline]
  3. Bowles N., Richardson P., Olsen E., Archard L. Detection of coxsackie B virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet (1986) I:1120–1122.
  4. Kandolf R., Hofschneider P. Viral heart diseases. Springer Semin Immunopathol (1989) 11:1–13.[ISI][Medline]
  5. Archard L., Bowles N., Cunningham L., et al. Molecular probes for detection of persisting enterovirus infection of human heart and their prognostic value. Eur Heart J (1991) 12:56–59.[ISI][Medline]
  6. Olsen E. Prognosis and treatment of cardiomyopathies and myocarditis. Sekiguchi M., ed. (1994) Tokyo: University of Tokyo Press. 121–127.
  7. Hiramitsu S., Morimoto S., Uemura A., et al. Long term outcome of 28 patients with viral or idiopathic myocarditis. (1994) Tokyo: University of Tokyo Press.
  8. McManus B., Chow L., Wilson J., et al. Direct myocardial injury by enterovirus: A central role in the evolution of murine myocarditis. Clin Immunol Immunopathol (1993) 68:159–169.[CrossRef][ISI][Medline]
  9. Badorff C., Lee G., Lamphear B., et al. Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nature Med (1999) 5:320–326.[CrossRef][ISI][Medline]
  10. Wessely R., Klingel K., Santana L., et al. Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation–contraction coupling and dilated cardiomyopathy. J Clin Invest (1998) 102:1444–1453.[ISI][Medline]
  11. Neumann D., Rose N., Ansari A., Herkowitz A. Induction of multiple heart autoantibodies in mice with coxsackievirus B3- and cardiac myosin-induced autoimmune myocarditis. J Immunol (1994) 152:343–350.[Abstract]
  12. Neu N., Beisel K., Traystman M., Rose N., Craig S. Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to coxsackievirus B3-induced myocarditis. J Immunol (1987) 138:2488–2492.[Abstract]
  13. Huber S., Lodge P. Coxsackievirus B3 myocarditis in Balb/c mice: Evidence for autoimmunity to myocyte antigens. Am J Path (1984) 116:21.[Abstract]
  14. Lowenstein C., Hill S., Lafond-Walker A., et al. Nitric oxide inhibits viral replication in murine myocarditis. J Clin Invest (1996) 97:1837–1843.[ISI][Medline]
  15. Iwasaki A., Matsumori A., Yamada T., et al. Pimobendan inhibits the production of proinflammatory cytokines and gene expression of inducible nitric oxide synthetase in a murine model of viral myocarditis. J Am Coll Cardiol (1999) 33:1400–1407.[Abstract/Free Full Text]
  16. Matsumori A. The use of cytokine inhibitors. A new therapeutic insight into heart failure. Int J Cardiol (1997) 62(Suppl. 1):S3–S12.
  17. Kasjtura J., Mansukhani M., Cheng W., et al. Programmed cell death and the expression of the protooncogene Bcl-2 in myocytes during postnatal maturation of the heart. Exp Cell Res (1995) 219:110–121.[CrossRef][ISI][Medline]
  18. James T. Normal and abnormal consequences of apoptosis in the human heart: From postnatal morphogenesis to paroxysmal arrhythmias. Circ (1994) 90:556–573.[Abstract/Free Full Text]
  19. James T., Nichols M., Sapire D., DiParre P., Lopez S. Complete heart block and fatal right ventricular failure in an infant. Circ (1996) 93:1588–1600.[Free Full Text]
  20. Kajstura J., Cheng W., Reiss K., et al. Apoptotic and necrotic myocyte deaths are independent contributing variables of infarct size in rats. Lab Invest (1996) 74:86–107.[ISI][Medline]
  21. Mallat Z., Tedgui A., Fontaliran F., et al. Evidence of apoptosis in arrhythmogenic right ventricular dysplasia. N Engl J Med (1996) 335:1190–1196.[Abstract/Free Full Text]
  22. Narula J., Haider N., Virmani R., et al. Apoptosis in myocytes in end-stage heart failure. N Engl J Med (1996) 335:1182–1189.[Abstract/Free Full Text]
  23. Kawano H., Okada R., Kawano Y., Sueyoshi N., Shirai T. Apoptosis in acute and chronic myocarditis. Jpn Heart J (1994) 35:745–750.[Medline]
  24. Seki Y., Kai H., Muraishi A., Adachi K., Imaizumi T. Myocardial DNA strand breaks are detected in biopsy tissues from patients with dilated cardiomyopathy. Clin Cardiol (1998) 21:591–596.[ISI][Medline]
  25. Toyozaki T., Hiroe M., Tanaka M., et al. Levels of soluble Fas ligand in myocarditis. Am J Cardiol (1998) 82:246–248.[CrossRef][ISI][Medline]
  26. Huber S. Coxsackievirus-induced myocarditis is dependent on distinct immunopathogenic responses in different strains of mice. Lab Invest (1997) 76:691–701.[ISI][Medline]
  27. Hamrell B., Huber S., Leslie K. Reduced unloaded sarcomere shortening velocity and a shift to a slower myosin isoform in acute murine coxsackievirus myocarditis. Circ Res (1994) 75:462–472.[Abstract/Free Full Text]
  28. Huber S, Hamrell B, Knowlton K. Lessons from animal models of viral myocarditis. In: Schultheiss H-P, Schwimmbeck P (Eds.), Role of Immune Mechanism in Heart Disease. Springer-Verlag, Berlin, 1996:35–43.
  29. Huber S., Mortensen A., Moulton G. Modulation of cytokine expression by CD4+ T cells during coxsackievirus B3 infections of Balb/c mice initiated by cells expressing the y8+ T cell receptor. J Virol (1996) 70:3039–3045.[Abstract]
  30. Born W., Happ M., Dallas A., et al. Recognition of heat shock proteins and gamma delta cell function. Immunol Today (1990) 11:40–43.[CrossRef][ISI][Medline]
  31. Suda T., Okazaki Y., Naito T., et al. Expression of the Fas ligand in cells of the T lineage. J Immunol (1995) 154:3806–3813.[Abstract]
  32. LeMeur M., Gerlinger P., Benoit C., Mathis D. Correcting an immune-response deficiency by creating Ea gene transgenic mice. Nature (1985) 316:38–42.[CrossRef][Medline]
  33. Huber S., Stone J., Wagner D. Jr., et al. Gamma delta-positive T cells regulate MHC class II (IA and IE)-dependent susceptibility to coxsackievirus B3-induced autoimmune myocarditis. J Virol (1999) 73:5630–5636.[Abstract/Free Full Text]
  34. Knowlton K., Jeon E., Berkley N., Wessely R., Huber S. A mutation in the puff region of VP2 attenuates the myocarditic penotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3. J Virol (1996) 70:7811–7818.[Abstract]
  35. Huber S., Job L., Woodruff J. Sex-related differences in the pattern of coxsackievirus B3-induced immune spleen cell cytotoxicity against virus-infected myofibers. Infect Immunology (1981) 32:68–73.
  36. Huber S., Job L., Woodruff J. Lysis of infected myofibers by coxsackievirus B3 immune T lymphocytes. Am J Pathol (1979) 98:681–694.[ISI]
  37. Henke A., Huber S., Stelzner A., Whitton J. The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis. J Virol (1995) 69:6720–6728.[Abstract]
  38. Kataoka T., Shinohara N., Takayama H., et al. A powerful tool for characterization and estimation of contribution of perforin and Fas based lytic pathways in cell mediated cytotoxicity. J Immunol (1996) 156:3678.[Abstract]
  39. Tschopp J., Nabholz M. Perforin-mediated target cell lysis by cytolytic T lymphocytes. Ann Rev Immunol (1996) 8:279–302.[Medline]
  40. Nagata S., Golstein P. The Fas death factor. Science (1995) 267:1449–1456.[Abstract/Free Full Text]
  41. Zakeri Z., Bursch W., Tenniswood M., Lockshin R. Cell death: Programmed, apoptosis, necrosis, or other. Cell death and differentiation. Cell Death Diff. (1995) 2:87–94.[ISI][Medline]
  42. Raff M. Cell suicide for beginners. Nature (1998) 396:119–122.[CrossRef][Medline]
  43. Talanian R., Yange X., Turbov J., et al. Granule-mediated killing: Pathways for granzyme B-initiated apoptosis. J Exp Med (1997) 186:1323–1331.[Abstract/Free Full Text]
  44. Seko T., Shinkai Y., Kawasaki A. Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3. Circulation (1991) 84:788–795.[Abstract/Free Full Text]
  45. Young L., Joag S., Zheng L., Lee Y., Young J. Perforin-mediated myocardial damage in acute myocarditis. Lancet (1990) 336:1019–1021.[CrossRef][ISI][Medline]
  46. Gebhard J., Perry C., Harkins S., et al. Coxsackievirus B3-induced myocarditis: Perforin exacerbates disease, but plays no detectable role in virus clearance. Am J Pathol (1998) 153:417–428.[Abstract/Free Full Text]
  47. Felzen B., Shilkrut M., Less H., et al. Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytolytic T lymphocytes from perforin-deficient mice: A major role for inositol 1,4,5-triphosphate. Circ Res (1998) 82:438–450.[Abstract/Free Full Text]
  48. Colston J., Chandrasekar B., Freeman G. Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis. Cardiovasc Res (1998) 38:158–168.[Abstract/Free Full Text]
  49. Huber S. Heat-shock protein induction in adriamycin and picornavirus-infected cardiocytes. Lab Invest (1992) 67:218–224.[ISI][Medline]
  50. Sciammas R., Johnson R.M., Sperling A.I., et al. Unique antigen recognition by a herpes virus-specific gamma delta cell. J Immunol (1994) 152:5392–5397.[Abstract]
  51. Heng M.K., Heng M.C. Heat-shock protein 65 and activated gamma/delta T cells in injured arteries. Lancet (1994) 344:921–923.[CrossRef][ISI][Medline]
  52. Kanda T., Nakano M., Yokoyama T., et al. Heat stress aggravates viral myocarditis in mice. Life Sci (1999) 64:93–101.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
B.-C. Yu, C.-K. Chang, H.-Y. Ou, K.-C. Cheng, and J.-T. Cheng
Decrease of peroxisome proliferator-activated receptor delta expression in cardiomyopathy of streptozotocin-induced diabetic rats
Cardiovasc Res, July 9, 2008; (2008) cvn172v2.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Huber, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huber, S. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?