Published ahead of print on May 25, 2006, doi:10.1164/rccm.200601-005OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200601-005OC
HIV-1 Nef Is Associated with Complex Pulmonary Vascular Lesions in SHIV-nef–infected MacaquesDivision of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, and Pulmonary Hypertension Center, University of Colorado at Denver and Health Sciences Center, Denver, Colorado; New England National Primate Research Center, Harvard Medical School, Southborough, Massachusetts; California National Primate Research Center, University of California at Davis, Davis, California; Allegheny General Hospital, Pittsburgh, Pennsylvania; and Johns Hopkins University School of Medicine, Baltimore, Maryland Correspondence and requests for reprints should be addressed to John C. Marecki, Ph.D., Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado at Denver and Health Sciences Center, 4200 East Ninth Avenue, Box C272, Denver, CO 80262. E-mail: John.Marecki{at}UCHSC.edu
Rationale: HIV-infected patients with pulmonary arterial hypertension have histologic manifestations that are indistinguishable from those found in patients with idiopathic pulmonary arterial hypertension. In addition, the role of pleiotropic viral proteins in the development of plexiform lesions in HIV-related pulmonary hypertension (HRPH) has not been explored. Simian immunodeficiency virus (SIV) infection of macaques has been found to closely recapitulate many of the characteristic features of HIV infection, and thus hallmarks of pulmonary arterial hypertension should also be found in this nonhuman primate model of HIV. Objectives: To determine whether pulmonary arterial lesions were present in archived SIV-infected macaque lung tissues from Johns Hopkins University and two National Primate Research Centers. Methods: Archived macaque and human lung sections were examined via immunohistochemistry for evidence of complex vascular lesions. Results: Complex plexiform-like lesions characterized by lumenal obliteration, intimal disruption, medial hypertrophy, thrombosis, and recanalized lumena were found exclusively in animals infected with SHIV-nef (a chimeric viral construct containing the HIV nef gene in an SIV backbone), but not in animals infected with SIV. The mass of cells in the lesions were factor VIII positive, and contained cells positive for muscle-specific and smooth muscle actins. Lung mononuclear cells were positive for HIV Nef, suggesting viral replication. Endothelial cells in both the SHIV-nef macaques and patients with HRPH, but not in patients with idiopathic pulmonary arterial hypertension, were also Nef positive. Conclusions: The discovery of complex vascular lesions in SHIV-nef– but not SIV-infected animals, and the presence of Nef in the vascular cells of patients with HRPH, suggest that Nef plays a key role in the development of severe pulmonary arterial disease.
Key Words: idiopathic pulmonary arterial hypertension • HIV-1 • Nef • pulmonary hypertension • SHIV-nef The first case of HIV infection associated with severe pulmonary hypertension appeared in 1987 (1) and confirmation by several follow-up studies (2–5) indicated that HIV infection may be causally related to pulmonary hypertension, with histologic lesions that are indistinguishable from those present in idiopathic pulmonary arterial hypertension (IPAH) (6, 7). Although HIV-related pulmonary hypertension (HRPH) is still a rare disease, the incidence is many times higher than that of IPAH in the general population (approximately 1 in 4,000 vs. 2 in 1 million in the United States [N. Gallie, personal communication] or 1 in 200 in the Swiss or French populations [2, 8, 9]). Importantly, there is no evidence of viral particles in the complex pulmonary vascular lesions (3, 10) or bone morphogenetic protein receptor II mutations (11) in HRPH. The mechanisms underlying HRPH may rely on indirect action of HIV proteins and/or the associated immune dysregulation present in patients with HIV infection. Among proteins of HIV that could participate in the pathogenesis of HRPH, the Tat protein causes increased endothelial cell permeability and injury via production of IL-6, shown to be present in lungs of patients with severe pulmonary hypertension (12). Nef, a 27-kD, N-myristoylated protein first misidentified as a negative regulatory factor for viral replication, is critical for the maintenance of high viral loads during the course of HIV infection in vivo (13, 14), and is an adaptor protein with multiple domains important for the interaction with the cellular signaling machinery. Its proline-rich (PxxP)3 domain interacts with Src homology 3–containing signaling proteins (15, 16), while other Nef domains interact with the endocytotic cellular machinery responsible for the down-regulation of CD4 (17–19). Nonhuman primate models of HIV-1 infection with either SIV or genetically manipulated pathogenic viruses containing HIV-1 genes recapitulate many of the end-organ pathologies and immune deficiencies seen in humans (20). Only a few investigations using this model for noninfectious lung complications in SIV infection have been done. However, one report by Chalifoux and coworkers described a pulmonary arteriopathy in the lungs of 19 of 85 macaques infected with SIV at the New England National Primate Research Center (NENPRC, Southborough, MA) (21). As HIV modeling in monkeys has significantly evolved with the use of recombinant, chimeric SIV vectors containing potentially pathogenetic HIV genes (SIV/HIV, or SHIV constructs), or SIV with tropism to organs such as brain or lung, we therefore investigated whether SIV-infected macaque lungs displayed the characteristic complex vascular lesions, including the characteristic plexiform lesions, present in HRPH. We further extended the scope of our study to address whether the HIV-1 Nef protein might be detected in the lungs of patients with HRPH and in macaques infected with a chimeric viral construct containing the HIV nef gene in an SIV backbone (SHIV-nef), but not in normal or uninfected IPAH lungs. Our findings indicate that HIV-1 Nef may be involved in the development of vascular lesions found in HRPH. Some of the results of these studies have been previously reported in the form of an abstract (22).
Animal Selection From the NENPRC, we obtained eight animals: two infected with a chimeric SHIV-nef virion (23, 24) and six infected with SIVmac251 or SIVE660 containing the native SIV nef allele. From the California National Primate Research Center (CNPRC; University of California at Davis, Davis, CA) we obtained five macaque lungs: one infected with SIVmac239 and four infected with SHIV-nef chimeric viruses (25). From the Retrovirus Laboratory, Department of Comparative Medicine, Johns Hopkins University (JHU; Baltimore, MD) we obtained 12 lungs from macaques infected with a mixture of lymphocytotropic and monocytotropic SIV strains (26). Table 1 describes the different animals and their known characteristics. No hemodynamic parameters were available from any of the macaque cohorts. All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved before implementation by the Institutional Animal Care and Use Committees at the University of California at Davis, JHU, and Harvard University.
Human Tissues Archived, paraffin-embedded lung tissue sections were obtained from two patients with IPAH, two patients with HRPH, and one patient with no clinical or histologic evidence of IPAH. These tissues were used for immunohistochemistry of the HIV-1 Nef protein. Patient characteristics have already been described by Cool and coworkers (6, 27).
Tissue and Histologic Examination
Immunohistochemistry
Detection of HIV-1 Nef.
Statistics
Pulmonary Pathologies in SIV- and SHIV-nef–infected Macaques We examined a total of 24 different monkey lungs from two separate primate centers based in New England (NENPRC), California (CNPRC), and Johns Hopkins University (JHU), with a total of 18 monkeys infected with SIV and six infected with SHIV-nef (Table 1). The SIV strains contained the simian nef allele whereas the SHIV-nef strains were chimeric viruses with HIV nef from various HIV isolates cloned in place of the native SIV nef sequence. Figure E1 of the online supplement shows a diagrammatic depiction of the genomic structures of parental and chimeric SIV strains. From the NENPRC, we obtained eight animals: two infected with a chimeric SHIV-nef virion that contained the SIVmac251 backbone and the HIV nef gene, and six infected with SIVmac251 or SIVE660 containing the native SIV nef allele. We found that the two SHIV-nef animals (designated NENPRC7 and NENPRC8 in Table 1) showed pulmonary vessels with medial smooth muscle cell hypertrophy and lumenal obstruction with endothelial cells (Figures 1B and 1C). The lesions were histologically similar to the plexiform lesions seen in patients with HRPH. One animal infected with SIVmac251 (Figure 1A and Table 1; designated as NENPRC1), showed significant perivascular cellular infiltration, but little disruption of the intimal or medial layers of the pulmonary vasculature. Furthermore, none of the animals infected with parental SIV strains (SIVmac251 or SIVE660, NENPRC1–NENPRC6 in Table 1) had any evidence of endothelial layer disruption or smooth muscle cell proliferation. Several of the prominent histopathologic features in these lungs included pulmonary edema, organizing thromboemboli, nonnecrotizing perivascular lymphocytic infiltrates, neutrophilic infiltration, and hyperplasia of bronchial-associated lymphoid tissue (Table 1). In four animals, alveolar proteinosis from Pneumocystis carinii was also evident, suggesting severe immunodeficiency with opportunistic infections.
No measurements of pulmonary artery pressure or cardiac output were taken in the NENPRC early study. On the other hand, examination of echocardiographic data from monkeys infected with the parental SIV strains showed no evidence of right ventricular changes in these monkeys (i.e., no echocardiographic evidence of right ventricular hypertrophy in approximately 30 SIV-infected animals examined [R.P.S., unpublished data]), despite evidence of widespread myocarditis and left ventricular changes. We then compared the findings in the studies from the NENPRC with those in five additional monkey lungs obtained from the CNPRC (animals designated as CNPRC1–CNPRC5 in Table 1). Animal CNPRC1 had been infected with SIVmac239 and animal CNPRC2 had been infected with a chimeric SHIV-nef containing the HIV-1 nefSF33 allele in the SIVmac239 backbone. CNPRC1 had no involvement of the pulmonary vasculature but showed neutrophilic and lymphocytic infiltration of the perivascular space (Figure 1D). One SHIV-nef–infected monkey (CNPRC2) showed mild vascular remodeling (see Figure E2). Another animal infected with SHIV-nefSF33 (tissues unavailable) died of immunodeficiency, but its SHIV-nef virus was isolated, recovered, and used to infect three additional macaques (CNPRC3, CNPRC4, and CNPRC5 in Table 1). The nef allele, which had acquired four amino acid mutations during passage in vivo, was designated as Nef SF33A (A, for adapted) (25). Two animals (CNPRC3 and CNPRC4; Figures 1E and 1F, respectively) showed abnormal vascular lesions with medial hypertrophy and a disrupted, cuboidal endothelial cell layer. Pentachrome staining corroborated both medial and intimal hypertrophy of the pulmonary arteries (see Figure E3). CNPRC5 died of severe wasting and opportunistic infections 3 mo postinfection, with lung samples showing extensive inflammation, but no vascular remodeling (see Figure E2). As with animals in the NENPRC cohort, no evidence of cardiac abnormalities was found in young animals infected with parental SIVmac251 at the CNPRC (A.F.T., unpublished data).
To further confirm that SIV alone is not associated with complex pulmonary vascular lesions, we examined an additional 12 monkeys infected with an immunosuppressive swarm SIV- Combining the results from the three geographically separate primate centers, four of the six SHIV-nef–infected animals were found to have complex vascular lesions, and one additional animal had evidence of less prominent pulmonary vascular remodeling. Thus, five of the six SHIV-nef–infected animals (83%) with remodeling were chronically infected (more than 12 mo), whereas none of the animals infected with the parental SIV strains containing the SIV nef allele (n = 18) developed complex pulmonary vascular lesions. Taken together, there is a statistically significant association between SHIV-nef infection in macaques and the presence of pulmonary vascular lesions (Fisher's exact test, p < 0.0001).
Characterization of Pulmonary Vascular Lesions in SHIV-nef–infected Animals
Presence of Nef in the Lungs of HIV-1–infected Patients and SHIV-nef–infected Macaques The presence of plexiform lesions in the pulmonary vasculature of SHIV-nef but not in the SIV-infected animals suggested a role for HIV-1 Nef in the pathogenesis of HRPH. As the presence of Nef in the lungs of patients with HRPH has not been reported previously, we examined paraffin-embedded lung sections from a normal human lung, two patients with IPAH, two patients with HRPH, and two SHIV-nef–infected macaques (CNPRC3 and CNPRC4) for the presence of the HIV-1 Nef protein. Figure 4 (first column) shows some representative lung sections stained with a monoclonal HIV-1 Nef antibody, whereas a separate section and region of the same subject in the remaining panels show dual-immunofluorescence stains for factor VIII and Nef. We detected no Nef staining in an uninfected control (Figure 4A; and see Figure E5A) or in patients with IPAH (Figure 4E; and see Figure E5B), illustrating the specificity of the antibody and the absence of any molecular mimicry (29). On the other hand, the lungs from patients with HRPH showed distinct positive staining in alveolar mononuclear cells (most likely lymphocytes and macrophages) as well as in endothelial cells lining the vessels (see Figures E5C and E6). The occluding cells and some lumenal cells found in the complex lesion in the patient with HRPH also stained positive for Nef (Figure 4I), and Nef-positive cells were observed clustering at sites adjacent to the vessel walls. In addition, Nef-positive cells were observed in lymphoid-associated tissues (see Figure E6), suggesting a potential source for the viral protein.
Figures 4 and 5 show immunofluorescence staining for factor VIII and Nef in human and macaque lungs, respectively. The pattern of staining in the merged images clearly indicates that Nef colocalizes with endothelial cells. This pattern in the SHIV-nef macaque lungs recapitulated that found in the human lungs, with staining in several endothelial cells within the vasculature as well as in perivascular sites (Figure 5I). We also found factor VIII-negative, Nef-positive medial and parenchymal cells. Interestingly, some adventitial cells in HRPH stained positive for both factor VIII and Nef (see Figure E6). As expected, lymphoid tissue showed staining for Nef, but no factor VIII positivity (Figure 5). These findings provide additional evidence that HIV-1 Nef may be a critical player in the development of complex pulmonary vascular lesions.
The main findings of our study are that complex vascular structures resembling plexiform lesions of IPAH are found in the lungs of macaques infected with a chimeric virus with the SIV backbone and the HIV nef gene. None of the monkeys infected with the parental SIV nef showed pulmonary vascular lesions, suggesting that there are functional differences between HIV and SIV nef. These differences may result in a protein that can act either alone or in conjunction with other viral or cellular factors in promoting alterations of pulmonary arteries characteristic of severe pulmonary hypertension. These findings were somewhat unanticipated, because HIV nef can complement nef deletions in SIV (24, 25), suggesting that at least in terms of immunodeficiency alleles from both lentiviruses are functionally equivalent. We found that Nef was present in endothelial and in factor VIII–negative cells, including the infected mononuclear cells. To our knowledge, this is the first time that any group has identified plexiform lesions in macaque lungs infected with a lentivirus. Although all the animals in cohorts from the New England and California National Primate Research Centers have abnormal endothelial cell morphology, the characteristics of the vasculopathies are different. Vascular lesions in the NENPRC SHIV-nef1486D95 cohort more closely resemble the plexiform lesions seen in HRPH patients with medial smooth muscle cell hypertrophy and multiple lumenal obstructions containing factor VIII-positive endothelial cells. Interestingly, lesions in the SHIV-nefSF33A animals from the CNPRC cohort were dominated by medial hypertrophy and a disrupted, cuboidal endothelial cell layer. The different pathologies in the two cohorts may be related to the stage of plexiform lesion formation in SHIV-nef animals, the rate of progression of lesion development, or the specific nef isolates in the chimeras. Without prospective studies using both of the constructs in cohorts infected in parallel, we are unable to provide direct evidence for the underlying mechanistic reasons for the differences. Nevertheless, the appearance of a disrupted endothelial cell layer containing the HIV-1 Nef protein suggests a role for Nef in the development of the complex severe pulmonary vascular lesions. Weesner and Kaplan (30) found elevated pulmonary artery pressures in a subset of SIV-infected stumptailed macaques (Macaca arctoides) and only moderate medial thickening of the pulmonary arteries. Chalifoux and coworkers (21) described an arteriopathy characterized by intimal and medial thickening in 19 of 85 macaques infected with SIV at the NENPRC. The medial thickening was seen only in large and medium-sized pulmonary arteries, and was reported to be the result of extracellular matrix deposition. Thirty-seven percent of the macaques with arteriopathy contained lumena with thrombi in various stages of organization and recanalization. In fact, after exhaustive examination, the authors did not find evidence of plexogenic lesions of small pulmonary arterioles and mention that the endothelial layer was always intact in these small arterioles. Infection of C57BL/6 mice with the LP-BM5 retroviral mixture, a murine model of immunodeficiency, leads to a diffuse interstitial pneumonitis similar to AIDS-associated interstitial pneumonitis, characterized by lymphocytic and macrophage infiltration in a setting of immunodeficiency (31). Although these mice develop right ventricular hypertrophy and show an increase in the percentage of muscularized pulmonary arteries with increased perivascular collagen deposition, they are remarkable for not developing plexiform lesions or intimal fibrosis of the pulmonary arteries (32). Transgenic mice expressing the entire HIV-1–coding sequence under the control of the CD4 promoter develop a severe AIDS-like disease and a lymphoid interstitial pneumonitis as well (33). These studies highlight the relevance of our identification of HIV-1 Nef as a potential contributor to advanced pulmonary vascular remodeling and plexiform lesions in HIV-associated pulmonary hypertension. The present study, in combination with the observation that lung tissue samples from many patients with pulmonary hypertension were positive for human herpesvirus type 8 (27), suggests a central role for viral gene products and their influence on endothelial cell biology.
How could Nef exert its effects on endothelial cells? HIV does not appear to infect endothelial cells directly, but extracellular Nef has been found in HIV-infected patients at a level of approximately 10 ng/ml (34). We also found Nef-positive cells in medial layers as well in the parenchyma (Figures 4K and 4L, arrows; and see Figure E6), and the cell morphology suggests that these may be smooth muscle and epithelial cells, respectively. As with endothelial cells, productive infection of these non-CD4+ cells has not been described. Acheampong and coworkers (35) demonstrated that either exogenous or intracellular Nef induced apoptosis of human brain microvascular endothelial cells. In lymphocytes, Nef dose- and time-dependently induced apoptosis; however, infected cells were protected from death through down-regulation of class I major histocompatibility complex (36). Because Nef has been shown to enter lymphocytes via the chemokine receptor CXCR4 (37, 38) which is expressed on endothelial cells in the lung vasculature, active infection is not a prerequisite for HIV-induced pulmonary vascular injury. Furthermore, HIV-infected alveolar macrophages and lymphocytes may act as Nef reservoirs and increase angiogenic factors such as platelet-derived growth factor-
In summary, we describe the associations between HIV-1 Nef and complex pulmonary vascular lesions in SHIV-nef–infected monkeys, which recapitulate the alterations seen in HRPH and IPAH. Our findings should lead to future investigations of the monkey model to elucidate the pathogenesis of these vascular lesions, and molecular examination of how the Nef protein might alter pulmonary vascular cells.
The authors thank M. Christine Zink, Joseph L. Mankowski, and Janice E. Clements of the Johns Hopkins University School of Medicine for providing archived macaque lung tissues, and Dr. Douglas Everett for assistance with the statistical analyses.
Supported by NHLBI R01 HL59785 (supplement to S.C.F.), RR00169 California National Primate Research Center (CNPRC), RR00168 New England National Primate Research Center (NENPRC), and NHLBI RO1HL66554 (R.M.T.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200601-005OC on May 25, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the manuscript. Received in original form January 3, 2006; accepted in final form May 12, 2006
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ABSTRACT
METHODS
-smooth muscle actin (
B670 and a macrophage-tropic molecular clone SIV/17E-Fr mixture (JHU1–JHU12; 
