Cardiac Disease in Transgenic Mice Expressing Human Immunodeficiency Virus-1 Nef in Cells of the Immune System
2002; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64184-3
ISSN1525-2191
AutoresDenis G. Kay, Ping Yue, Zaher Hanna, Serge Jothy, Étienne Tremblay, Paul Jolicoeur,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe previously reported that a severe acquired immune deficiency syndrome-like disease develops in transgenic (Tg) mice expressing the human immunodeficiency virus-1 in its natural target cells: immature and mature CD4+ T cells and cells of the macrophage/dendritic lineage. Here, we show that these mice also develop cardiac disease, characterized most prominently by a focal myocytolysis, occasionally by myocarditis and by deposition of endogenous immunoglobulin on cardiomyocytes. Microfil perfusion demonstrated widespread coronary arteriospasm and echocardiographic analysis revealed depressed cardiac function in Tg mice. A higher (but still modest) level of cardiomyocyte apoptosis was detected in Tg as compared to non-Tg hearts. Tg expression was detected in some of the infiltrating mononuclear cells, but not in cardiomyocytes or in cells of the heart vessels, suggesting a human immunodeficiency virus-1-induced disease process mediated by cells of the immune system. The similarity of the heart disease observed in these Tg mice to that observed in acquired immune deficiency syndrome patients suggests a common pathogenesis. We previously reported that a severe acquired immune deficiency syndrome-like disease develops in transgenic (Tg) mice expressing the human immunodeficiency virus-1 in its natural target cells: immature and mature CD4+ T cells and cells of the macrophage/dendritic lineage. Here, we show that these mice also develop cardiac disease, characterized most prominently by a focal myocytolysis, occasionally by myocarditis and by deposition of endogenous immunoglobulin on cardiomyocytes. Microfil perfusion demonstrated widespread coronary arteriospasm and echocardiographic analysis revealed depressed cardiac function in Tg mice. A higher (but still modest) level of cardiomyocyte apoptosis was detected in Tg as compared to non-Tg hearts. Tg expression was detected in some of the infiltrating mononuclear cells, but not in cardiomyocytes or in cells of the heart vessels, suggesting a human immunodeficiency virus-1-induced disease process mediated by cells of the immune system. The similarity of the heart disease observed in these Tg mice to that observed in acquired immune deficiency syndrome patients suggests a common pathogenesis. 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We recently developed a novel murine Tg model of AIDS (CD4C/HIV) in which wild-type or mutant HIV-1 genomes are expressed under the control of regulatory sequences (CD4C) comprising the murine CD4 gene enhancer and the promoter elements of the human CD4 gene.31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar, 32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Consequently, these CD4C/HIV Tg mice express HIV-1 gene products in the natural target cell populations of the virus, ie, in immature CD4+CD8+ T cells, in mature CD4+ T cells, and in cells of the macrophage/dendritic lineage, including peritoneal and alveolar macrophages, Kupffer cells, and dendritic cells. They also exhibit most of the phenotypes associated with this syndrome in human patients: weight loss/failure to thrive, wasting, early death, thymic atrophy, lymphadenopathy, preferential and progressive loss of CD4+ T cells, down-regulation of CD4 cell-surface expression, increase in CD8+ T cell and of B cell number, T cell activation, immunodeficiency, lymphocytic interstitial pneumonitis, interstitial nephritis.31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar, 32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Most recently, we have documented B cell activation, elevated levels of autoantibody production, and an impairment of germinal center formation in these mice.33Poudrier J Weng X Kay DG Paré G Calvo EL Hanna Z Kosco-Vilbois MH Jolicoeur P The AIDS disease of CD4C/HIV transgenic mice shows impaired germinal centers and autoantibodies and develops in the absence of IFN-γ and IL-6.Immunity. 2001; 15: 173-185Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar In a mutational analysis of the HIV-1 genome, we determined that the expression of a single HIV-1 gene, nef, is both necessary and sufficient to cause this AIDS-like disease in Tg mice.32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Indeed, Tg mice harboring three distinct mutant HIV transgenes, CD4C/HIVMutA (expressing Rev, Env, and Nef), CD4C/HIVMutB (expressing all HIV-1 gene products except Env), and CD4C/HIVMutG (expressing only Nef) all develop an indistinguishable AIDS-like disease, whereas Tg mice harboring a mutant genome lacking Nef expression (CD4C/HIVMutH) failed to develop the AIDS-like disease.32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Nef is a structurally complex, multifunctional accessory protein encoded by primate lentiviruses [HIV-1, HIV-2, and simian immunodeficiency virus (SIV)].34Cullen BR The role of Nef in the replication cycle of the human and simian immunodeficiency viruses.Virology. 1994; 205: 1-6Crossref PubMed Scopus (85) Google Scholar, 35Harris M From negative factor to a critical role in virus pathogenesis: the changing fortunes of Nef.J Gen Virol. 1996; 77: 2379-2392Crossref PubMed Scopus (70) Google Scholar, 36Peter F HIV nef: the mother of all evil?.Immunity. 1998; 9: 433-437Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar It plays a critical role in virus virulence. Individuals harboring HIV-1 with defects in the nef gene remain healthy after more than a decade of infection.37Kirchhoff F Greenough TC Brettler DB Sullivan JL Desrosiers RC Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection.N Engl J Med. 1995; 332: 228-232Crossref PubMed Scopus (894) Google Scholar, 38Deacon NJ Tsykin A Solomon A Smith K Ludford-Menting M Hooker DJ McPhee DA Greenway AL Ellett A Chatfield C Lawson VA Crowe S Maerz A Sonza S Learmont J Sullivan JS Cunningham A Dwyer D Dowton D Mills J Genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients.Science. 1995; 270: 988-991Crossref PubMed Scopus (1032) Google Scholar Similarly, Rhesus macaques infected with nef deleted SIV, fail to develop simian AIDS.39Kestler HW Ringler DJ Mori K Panicali DL Sehgal PK Daniel MD Desrosiers RC Importance of the nef gene for maintenance of high virus loads and for development of AIDS.Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1438) Google Scholar Therefore, it seems that Nef expression in CD4+ cells of the immune system of CD4C/HIV Tg mice mimics its action in human AIDS. On further investigation, we recently observed a novel feature of the AIDS-like disease in these Tg mice, namely the development of cardiac disease. We report here our study on this cardiac disease observed in Nef-expressing CD4C/HIVMutA and CD4C/HIVMutG Tg mice. We provide pathological and functional evidence of both focal as well as global cardiac disease including cardiac vasculature abnormalities. We conclude that the cardiac disease is similar to that documented in human AIDS, like other phenotypes of this severe murine AIDS-like disease and that it is mediated directly or indirectly by Nef-expressing cells of the immune system or factors produced by these cells. The generation and characterization of the CD4C/HIVWT, CD4C/HIVMutA, CD4C/HIVMutB, and CD4C/HIVMutG Tg mice have been described previously.31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar, 32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Tg and non-Tg littermates were housed together in the same cage, and were maintained on a standard 12-hour light/dark cycle. Mice were fed ad libitum on a diet of Purina Laboratory Chow (a 50%/50% mixture of Chow no. 5001/5015). For routine histological analysis, mice were killed by CO2 inhalation, and organs to be evaluated were dissected and fixed by overnight immersion in 3.7% formaldehyde buffered in phosphate-buffered saline (PBS). For all analyses of heart tissues in situ, the heart was divided by transverse sectioning into three equally sized pieces. The middle one third of the heart was used for analysis. For terminal dUTP nick-end labeling (TUNEL) assay or immunohistochemistry (IHC), tissues were fixed by intracardiac perfusion. Mice anesthetized with Avertin were exsanguinated by PBS perfusion, followed by fixation with 4% paraformaldehyde or periodate-polylysine-paraformaldehyde fixative.40McLean IW Nakane PK Periodate-lysine-paraformaldehyde fixative. a new fixation for immunoelectron microscopy.J Histochem Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3200) Google Scholar In detail, after anesthesia induction, a midline incision was made through the anterior abdominal wall ∼2 cm below the xyphoid process. The incision was carried superiorly to the xyphoid process and then laterally to the midaxillary lines, exposing the diaphragm. The diaphragm was punctured just below the xyphoid process and then rapidly cut away from the thoracic cage. Bilateral midaxillary incisions were made through the ribs beginning at the subcostal margin and terminating in the axilla. The rib flap was raised and a 26-gauge needle introduced into the left ventricle close to the apex of the heart. Simultaneously, the free wall of the right atrium was pierced with a sharp scissors. Perfusate was pumped at mean arterial pressure (∼100 mm Hg) using a peristaltic pump. Exsanguination (by PBS) was considered to be complete once the liver had blanched and the effluent PBS was clear. Fixative was then pumped for 5 minutes, at which point the limbs of the mice were rigidly fixed in place. Mice were postfixed by overnight immersion in fixative. After dissection, the organs were embedded in paraffin using a Shandon tissue infiltrator (Hypercenter XP, Pittsburgh, PA), sectioned at 5 μm, and stained with hematoxylin and eosin, as described previously31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar or processed for TUNEL or IHC. Tg and control non-Tg tissues were assessed independently by two investigators (SJ and DGK). Myocarditis was scored following the Dallas criteria.41Aretz HT Billingham ME Edwards WD Factor SM Fallon JT Fenoglio JJJ Olsen EGJ Schoen FJ Myocarditis: a histopathologic definition and classification.Am J Cardiovascular Pathol. 1986; 1: 3-14Google Scholar In situ hybridization was performed on paraffin-embedded tissues, using 35S- UTP-labeled anti-sense and control sense RNA probes as described previously.31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar, 32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Tissues from non-Tg control animals hybridized with anti-sense probes as well as Tg animal tissues hybridized with sense probes failed to exhibit any specific hybridization signal. The detection of DNA fragmentation in cells undergoing apoptosis was made using the TUNEL assay in situ with a digoxigenin-derivitized UTP (Boehringer Mannheim, Montreal, Canada). The technique was essentially that recommended by the manufacturer. Only animals in which the tissue processing resulted in rapid and complete fixation of hearts and other positive control tissues (lymph nodes, intestine) were considered. Indeed, we have noted TUNEL-positive artifacts in control mice that were overanesthetized (poor respiratory effort, cyanotic appearance) before perfusion fixation, or in whom the perfusion resulted in an incomplete fixation. These artifacts were most notable in the central nervous system where a significant proportion of hippocampal and cortical neurons were TUNEL-positive, and in the heart where large numbers of myocyte nuclei were TUNEL-positive. However, TUNEL-positive cells were rarely observed in these control tissues (eg, zero to two positive cells per transsection of heart) when the perfusion fixation was sufficiently rapid and complete. Consequently, hypoxic or poorly fixed mice were excluded from TUNEL analysis. Heart tissue analyzed was included in paraffin blocks together with several other tissues including small intestine and mesenteric lymph nodes. Because overfixation can result in a false-negative TUNEL signal, only hearts from blocks in which the internal positive controls (apical intestinal epithelial cells and clusters of lymph node follicular cells) were TUNEL-positive, were considered for analysis. From one to three sections were analyzed per heart. Endogenous Ig were visualized in Tg hearts by immunohistochemistry using anti-mouse Ig or IgM (Sigma) antibodies, essentially as previously described.31Hanna Z Kay DG Cool M Jothy S Rebai N Jolicoeur P Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.J Virol. 1998; 72: 121-132Crossref PubMed Google Scholar, 32Hanna Z Kay DG Rebai N Guimond A Jothy S Jolicoeur P Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Induction of experimental autoimmune myocarditis was performed essentially as described.42Coligah JE Animal models for autoimmune and inflammatory disease: autoimmune myocarditis. Current Protocols in Immunology, chapter 15.14. John Wiley & Sons, New York2001Google Scholar Briefly 2-month-old BALB/c mice were challenged twice, at 1week intervals, with 100 μg of cardiac myosin (M-0531, Sigma) emulsified in Freund's complete adjuvant. Control mice received PBS emulsified in adjuvant. Two weeks after the second injection, mice were anesthetized and perfusion-fixed with 4% paraformaldehyde, and hearts were dissected and processed into paraffin. Microfil (Flow Tech, Carver, MA) perfusion (1 ml) was performed after thoracotomy under avertin anesthesia, via the apex of the left ventricle while the heart was still beating, as described.43Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar For hypoxic stress, oxygen tension was automatically controlled using a digital O2 sensor (model VA201T, Vulcain Inc., Quebec, Canada) in airtight chambers. Oxygen tension was progressively lowered to 16%, 12% for 2 minutes each, and then to 8% for 4 minutes before Microfil perfusion. Hearts were fixed by immersion in 3.7% formaldehyde buffered with PBS, sliced into 1-mm-thick transverse sections, dehydrated through ethanol series, and cleared in methyl salicylate, as described.43Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar Digital images of cleared Microfil-infiltrated heart tissues were captured using a Leica stereo dissecting microscope (model MZ12) mated to a Sony color charge-coupled device camera (model DCX-950; Sony). Nonoverlapping images were obtained of the entire cross-sectional area of transverse heart sections of left and right ventricle, at two distinct planes of focus. Two heart sections were analyzed per heart. An average of 30 images was analyzed per heart. Images were captured and processed (see below) blindly from seven Tg and seven non-Tg animals. Image processing was accomplished with Photoshop (Adobe Systems Inc., San Jose, CA, version 5.1) using a combination of automatic thresholding of the Microfil-infiltrated vessels from the tissue background and manual deletion of tissue artifacts from incomplete clearing of the myocardium. Processed images were then quantitated using the threshold, followed by measurement of the total thresholded area functions of the Northern Eclipse image analysis software (Empire Imaging Inc., version 6). IHC to detect the active form of caspase-3 (ab397; D. Nicholson, Merck Frost, Montreal, Canada), dystrobrevin (Novocastra Vector Laboratories (Canada) Inc.), α, ε (J. Sanes, Washington University, St. Louis, MO) and γ sarcoglycan (Novocastra) was performed on fresh frozen 10-μm heart sections. Sections were prepared from heart tissue removed from freshly killed mice. The middle third of the heart, cut transversally, was enrobed in OCT (Sakura Chemicals, Montreal, Canada) and plunged into isopentane prechilled in liquid nitrogen to the freezing point (viscous liquid). Incubations with primary antisera were overnight at 4°C with 1/1000 dilution (α-caspase-3) and 2 hours at room temperature using 1/500 dilution for all anti-sarcoglycan antibodies. After incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase, immunoreaction was detected using diaminobenzidine as chromagen. Tissues were counterstained with hematoxylin. Collagen staining of paraffin-embedded heart tissue was performed as described44Ammarguellat F Larouche I Schiffrin EL Myocardial fibrosis in DOCA-salt hypertensive rats effect of endothelin ETA receptor antagonism.Circulation. 2001; 103: 319-324Crossref PubMed Scopus (130) Google Scholar using a 0.1% solution of Sirius Red (Direct Red 80, Aldrich, Toronto, Canada) in Bouin's solution. Quantitation of Sirius Red staining was performed on transections of hearts of nine Tg and seven non-Tg mice. High-density digital images acquired using a Zeiss Cool Pix digital camera were processed using the thresholding function of Photoshop (version 6, Adobe). Collagen deposition was expressed as a percentage of the total tissue area occupied by Sirius Red-stained tissue. Left ventricular dimensions, wall thickness, and cardiac function were evaluated by echocardiography and compared between CD4C/HIVMutA Tg mice and age- and sex-matched non-Tg cont
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