1 College of Public Health,
2 Microbiology and Immunology, College of Medicine and
3 University Animal Care, University of Arizona, Tucson, Arizona, USA
Received 23 July 2001; in revised form 4 September 2001; accepted 1 October 2001
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ABSTRACT |
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INTRODUCTION |
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Alcohol also acts on the cardiovascular system to promote hypertension, cerebrovascular disease, and stroke. This may occur in part via the promotion of immunosuppression, which facilitates infection and increased pathogenesis. Chronic EtOH consumption in mice causes harmful effects on the host immune response, oxidative balance stress, and nutritional status of the animals (Lee et al., 1999). EtOH reduces the T-cell response to mitogens, suppresses natural killer cell function, reduces granulocyte migration, and lowers macrophage phagocytic activity (Wang and Watson, 1995a
,b
,c; Wang et al., 1997
). Thus, EtOH can act to enhance the host's susceptibility to retroviral infection and to accelerate the onset of murine aquired immunodeficiency syndrome (MAIDS). These factors make it paramount to understand the impact of alcohol consumption on the progression of HIV disease.
There is a high incidence of cardiopathology in AIDS which is not related to HIV, but may be related to other pathogens such as coxsackievirus. Coxsackievirus B3 (CVB3), an enterovirus in the Picornaviridae family, is an aetiological agent of virus-induced myocarditis (Woodruff, 1980; Tracy et al., 1991
). Patients with myocarditis have elevated antibody levels against enterovirus proteins and enterovirus nucleic acids (Bowles et al., 1986
; Easton and Eglin, 1988
; Tracy et al., 1991
). CVB3-induced myocarditis is an immunopathological disease in which the host's immune system, once triggered by the virus, is the major contributor to heart pathology (Woodruff, 1980
). Suggested mechanisms include the development of heart neoantigen (Paque et al., 1979
), the cross-reactivity between virus and heart myosin (Gauntt et al., 1991
, 1993a
,b
), and the development of heart-reactive cytolytic T-cells (Friedman et al., 1998
; Seko et al., 1998
). Clearly, the immune system is required for virus clearance, as severe combined immunodeficient mice develop cardiac necrosis with a high rate of mortality during CVB3 infection (Chow, 1993
).
The present study was designed to determine whether retrovirus-induced murine AIDS, alone or with EtOH consumption, would accentuate the pathological outcome of CVB3 infection, using a coxsackievirus-resistant strain of mouse.
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MATERIALS AND METHODS |
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Infection and treatment of mice
Four-week-old female C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, DE, USA). They were housed in polycarbonate cages with stainless-steel wire lids (four mice per cage) in the Central Animal Facility of the University of Arizona. Animals were cared for as required by the University of Arizona Animal Care and Use Committee. The housing facility was maintained at 2022°C and 3070% relative humidity, with a 12-h light/12-h dark cycle. Mice were maintained for 4 months in the facility on an NIH-31-modified mouse sterilizable diet (mouse diet #7001; Teklad, Madison, WI, USA) and water ad libitum. Mice were then randomly assigned to one of the following groups: uninfected control mice; uninfected mice given EtOH (v/v) in drinking water; mice infected with retrovirus; mice infected with retrovirus given EtOH; mice infected with retrovirus and coxsackievirus; mice infected with retrovirus and coxsackievirus given EtOH; mice infected with coxsackievirus; and mice infected with coxsackievirus given EtOH. The EtOH concentration was increased in increments of 10%, at 1-week intervals, from 10% to a final concentration of 40% (v/v) in drinking water. The mice drinking EtOH were also given 25 g agar gel containing EtOH (40% v/v plus 2% peanut butter) for 16 weeks, five times a week. This dietary approach to EtOH supplementation in mice was based on its development by Bautista in rats (Batey and Patterson, 1991; Bautista, 1997
). Pair-fed control mice received isocaloric water and agar gels in which EtOH was substituted with sucrose. Administration of EtOH and/or water supplemented with sucrose, as well as agar, began 2 weeks after LP-BM5 infection. Fresh drinking water and/or EtOH were provided twice weekly and an agar block every day.
LP-BM5 retrovirus was administered intraperitoneally to mice in 0.1 ml of minimum essential medium with an esotropic titre of 4.5 log10 plaque forming units x 10-3/l, which induces disease with a time course comparable with that previously published (Wang and Watson, 1994a,b
). Infection of female C57BL/6 mice with LP-BM5 murine leukaemia virus leads to the rapid induction of clinical symptoms with virtually no latent phase (Liang et al., 1996
). The mice were infected 2 weeks before initiation of treatment as done previously in numerous studies (Wang and Watson, 1994a
,b
, 1995a
,b
,c
,d
; Wang and Liang, 1997
). The infection period was 20 weeks, while the treatment went on for 18 weeks for all groups. After 16 weeks of EtOH treatment, the appropriate groups of mice were inoculated intraperitoneally with 3 x 105 TCID50 of CVB3/59 in 0.1 ml of Roswell Park Memorial Institute 1640 medium. Mice were killed 12 days after inoculation.
When murine AIDS had developed at 16 weeks after retrovirus infection, all mice in all groups were killed the same week while under ether anaesthesia. Spleens were then removed, and kept at 4°C.
Enzyme-linked immunosorbent assays (ELISA) for cytokines
The production of interleukin (IL)-2, IL-4, IL-6, interferon (IFN)-, and tumour necrosis factor (TNF)-
from mitogen-stimulated splenocytes was determined as described previously (Chouaib et al., 1985
). Briefly, spleens were gently teased with forceps in culture medium (CM; RPMI 1640 containing 10% fetal bovine serum, 2 mmol/l glutamine, 1 x 105 units/l of penicillin and streptomycin) producing a suspension of spleen cells. Red blood cells were lysed by incubation with a lysis buffer (0.16 mol/l ammonium chloride Tris buffer, pH 7.2) at 37°C for 3 min. The cells were then washed twice with culture media (CM). Cell concentrations were determined and adjusted to 1 x 107 cells/ml. Splenocyte viability was >95%, as determined by trypan blue exclusion. Splenocytes [0.1 ml/well (1 x 107 cells/ml)] were cultured in triplicate on 96-well flat-bottomed culture plates (Falcon 3072, Lincoln Park, NJ, USA) with CM. Splenocytes were then stimulated with concanavalin A (Con A, 10 mg/l, 0.1 ml/well; Sigma Chemical Co., St Louis, MO, USA) to determine the production of IL-2 and IL-4 after 24 h and of IFN-
after 72 h in a 37°C, 5% CO2 incubator. Splenocytes were also incubated for 24 h after the addition of lipopolysaccharide (LPS; 10 mg/l; Gibco) to induce IL-6 and TNF-
production. After incubation, supernatants were collected and stored at 70°C until analysis. Cytokines were determined by sandwich ELISA as described previously (Lee et al., 1999
). Rat anti-murine IL-2, IL-4, IL-6 and TNF-
purified antibodies; rat anti-murine IL-2, IL-4, IL-6, IFN-
and TNF-
biotinylated antibodies; and recombinant murine IL-2, IL-4, IL-6, IFN-
and TNF-
were obtained from Endogen (Cambridge, MA, USA).
Mitogenesis of splenocytes
Splenic T- and B-cell proliferation was determined by [3H]thymidine incorporation as described previously (Chouaib et al., 1985). Briefly, splenocytes in 0.1 ml of CM (1 x 107 cells/ml) were cultured in 96-well flat-bottomed cultured plates (Falcon) with Con A and LPS (10 µg/ml). The cells were incubated at 37°C, 5% CO2 for 44 h to allow for Con A-induced T-cell proliferation and LPS-induced B-cell proliferation and then pulsed with [3H]thymidine (0.5 µCi/well; New England Nuclear, Boston, MA, USA). Four hours after the [3H]thymidine pulse, the cells were harvested by a cell sample harvester (Cambridge Technology, Cambridge, MA, USA). Radioactivity was determined by a liquid scintillation counter (Tri-Carb, 2200 CA; Packard, Lagunahills, CA, USA). Data are presented as counts per minute (cpm).
Histopathology
Upon the death of mice, hearts were removed, rinsed in saline and transversely cut in halves. One half of each heart was immediately placed in Histochoice tissue fixative (Amresco, Solon, OH, USA) and stored at 0°C. Fixed heart tissues were sectioned (6 µm) on a Zeiss HM 505 N cryostat (Carl Zeiss, Inc., Thornoweed, NY, USA) and stained with haematoxylin and eosin. The extent of inflammatory lesions within the myocardium was graded by a pathologist without knowledge of the other experimental variables. The grading was performed in a semi-quantitative manner according to the relative degree (from heart to heart) of mononuclear cell infiltration and the extent of necrosis (Fig. 1). Mild damage is considered as <10% of heart tissue affected, moderate = 1025% and severe as >25% of heart tissue affected.
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RESULTS |
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Mitogenesis of splenocytes
The proliferation of splenocytes stimulated by LPS and Con A was significantly (P < 0.05) decreased by LP-BM5 retrovirus (Fig. 2A and B). EtOH supplementation for 49 days increased T- and B-cell mitogenesis in uninfected mice, but decreased T-cell and increased B-cell mitogenesis in coxsackievirus infected mice (P < 0.05). EtOH-treated retrovirus-infected mice showed a significant decrease in both T- and B-cell mitogenesis, when compared to EtOH-treated uninfected mice (P < 0.05). Infection with coxsackievirus increased the proliferation of T-cells in the non-retrovirus-infected group; on the other hand, B-cells mitogenesis in the same group was decreased, when compared to the ethanol-treated CVB infected group. Both non-treated and EtOH-treated retrovirus-infected and coxsackievirus-superinfected groups showed a significant decrease in their T- and B-cell mitogenesis, when compared to uninfected and/or CVB infected groups treated or not treated with EtOH (P < 0.05).
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Cardiac histopathology of immunodeficient mice infected with coxsackievirus and treated with EtOH
Coxsackievirus B3 (CVB3)-infected immunodeficient mice treated with EtOH showed significant myocarditis, whereas CVB3-infected immunocompetent mice treated with EtOH showed no heart lesions. Mice co-infected with CVB3 and retrovirus developed significant myocarditis in 75% of the animals. Supplementation with EtOH to mice co-infected with CVB3 and retrovirus, increased the percentage of animals with myocarditis to 85% as well as increasing lesion severity (Fig. 3). Mice infected only with retrovirus and supplemented with EtOH had histopathological evaluations equal to those of the controls.
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DISCUSSION |
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Alcohol (or any variable that could result in a shift in Th1/Th2 profiles) could serve as a co-factor in the onset and progression of AIDS (Shearer and Clerici, 1993). EtOH can alter immune function either directly through its action on immunocompetent cells (Jerrels and Sibley, 1995; Lopez et al., 1997
; Chen et al., 1998
; Ahluwalia et al., 2000
; Hosseini et al., 2000
) or indirectly through modulation of different neuroendocrine hormones (Doll, 1998
) and neurotransmitters (De Witte, 1996
) that regulate the immune system.
Coxsackieviruses cause a wide spectrum of clinical diseases, including aseptic meningitis, colds, myalgia, myocarditis, pharyngitis, conjunctivitis, handfoot-and-mouth disease, and possibly some cases of juvenile diabetes mellitus. Ten million Americans become infected by group B coxsackieviruses every year. At least 5% of these people experience heart infection, with an unknown proportion developing myocarditis. About 1065% of biopsies are coxsackievirus-positive for patients with clinical diagnoses of myocarditis or unexplained heart failure (Gauntt and Pallansch, 1996). These percentages might be much higher, as screening for coxsackievirus infections is not usually carried out.
In our study, the mice co-infected with retrovirus and CVB3 and treated with EtOH exhibited more severe cardiopathology, when compared to the same group without EtOH treatment (Fig. 2). This correlates with an accentuation of suppression of Th1-cells and cytokine production due to EtOH treatment in retrovirus-immunosuppressed mice. LP-BM5 infection in mice, as well as HIV infection in humans, suppresses the Th1-cell response (Liang et al., 1996
), which should increase coxsackievirus-induced myocarditis. We observed that the decreased production of IL-2 by splenocytes from mice with murine AIDS was further reduced by EtOH consumption, as with coxsackievirus infection. INF-
production was also significantly inhibited in the group of mice with AIDS treated with EtOH when compared to the healthy animals. INF-
production was considerably decreased in the murine AIDS group superinfected with coxsackievirus. This pronounced decrease correlates with the degree of cardiopathology observed in this group. In turn, Th2 cytokine secretion, specifically of IL-4 and IL-6, was increased in retrovirus-infected mice treated with EtOH, as well as in controls fed sucrose water.
Our data suggest that EtOH shifts the cytokine balance in favour of a Th2 response by enhancing Th2 function and/or by suppressing Th1 function (Tables 1 and 2). EtOH may thereby promote the conditions that favour the opportunistic viral infections that cause cardiac pathology.
In mice infected with the murine retrovirus, there was an increase in the secretion of Th2 cytokines facilitating suppression of the cellular immune response (Wang et al., 1997). This type of response promotes an antibody-mediated immunity. The cardiac pathology observed may have occurred via several mechanisms: (1) due to infection with CVB3 which induces synthesis of antibodies to the pathogen, some of the antibodies may cross-react with cardiac myosin, inducing myocarditis (Fujinami et al., 1983
; Gaunt et al., 1991, 1993a,b; Barnet and Fujinami, 1992
; Cunningham, 1993
; Gauntt and Pallansch, 1996
); (2) myocyte damage by the coxsackievirus with the concomitant release of myosin by the myocyte cells (Rose et al., 1992
). Myosin can very easily be taken up by dendritic cells activating autoreactive T-cells. As the mice develop AIDS, and especially during chronic EtOH treatment, there was a marked increase in the Th2 cytokines: IL-4 and IL-6. This results in subsequent B-cell activation and production of antibodies and anti-myosin autoantibodies that induce myocarditis (Rose et al., 1992
). If this occurred in the murine AIDS model, anti-myosin antibodies should cause some of the cardiac pathology observed during coxsackievirus infection.
Coxsackievirus heart lesions in mice closely resemble those seen in humans infected with the same virus (Baboonian et al., 1997). The primary lesions consist of necrotic foci surrounded by mononuclear cells that are found throughout the ventricles and, to a lesser extent, in the atria. In the chronic stage, necrotic foci are replaced with connective tissue and calcification, and the number of mononuclear cells diminishes.
Several studies have established a strong relationship between alcohol and AIDS (Fong et al., 1994; Fein et al., 1995
; Meyerhoff et al., 1995
; Pol et al., 1996
; Lee et al., 1999
). In a study done by the Multicenter AIDS Cohort, 209 bisexual and homosexual men who were heavy alcohol drinkers or drug users, became HIV-seropositive 6 months after initiation of the study (Penkover et al., 1991
) compared to a control group of bisexual and homosexual men who did not become infected within the same time period. Consumption of EtOH by experimental animals and by humans negatively affects the immune system (Hosseini et al., 2000
). One notable change in the immune system is a loss of lymphoid cells from primary and secondary lymphoid organs (Jerrells et al., 1990
; Jerrells and Pruett, 1994
). Specifically, EtOH will deplete lymphocytes from gut-associated lymphoid tissue (Jerrels et al., 1986), thymus-derived lymphocytes (T-cells) and bone marrow-derived lymphocytes (B-cells) from mesenteric lymph nodes (Sibley et al., 1995
). Finally, chronic alcoholism in humans can cause lymphopenia (Lui, 1980
) with a specific decrease in CD8+ cells (Kronfol et al., 1993
).
In conclusion, alcohol consumption induces a decrease in the cellular immune response. In retrovirus-infected mice, this cellular response is further handicapped due to the low Th1 response, making the host with AIDS highly susceptible to secondary infections. One such infection caused by coxsackievirus can create severe tissue damage resulting in the development of cardiopathology.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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