Elevated generation of reactive oxygen/nitrogen species in hantavirus cardiopulmonary syndrome

Abstract

Hantavirus cardiopulmonary syndrome (HCPS) is a life-threatening respiratory disease characterized by profound pulmonary edema and myocardial depression. Most cases of HCPS in North America are caused by Sin Nombre virus (SNV), which is carried asymptomatically by deer mice (Peromyscus maniculatus). The underlying pathophysiology of HCPS is poorly understood. We hypothesized that pathogenic SNV infection results in increased generation of reactive oxygen/nitrogen species (RONS), which contribute to the morbidity and mortality of HCPS. Human disease following infection with SNV or Andes virus was associated with increased nitrotyrosine (NT) adduct formation in the lungs, heart, and plasma and increased expression of inducible nitric oxide synthase (iNOS) in the lungs compared to the results obtained for normal human volunteers. In contrast, NT formation was not increased in the lungs or cardiac tissue from SNV-infected deer mice, even at the time of peak viral antigen expression. In a murine (Mus musculus) model of HCPS (infection of NZB/BLNJ mice with lymphocytic choriomeningitis virus clone 13), HCPS-like disease was associated with elevated expression of iNOS in the lungs and NT formation in plasma, cardiac tissue, and the lungs. In this model, intraperitoneal injection of 1400W, a specific iNOS inhibitor, every 12 h during infection significantly improved survival without affecting intrapulmonary fluid accumulation or viral replication, suggesting that cardiac damage may instead be the cause of mortality. These data indicate that elevated production of RONS is a feature of pathogenic New World hantavirus infection and that pharmacologic blockade of iNOS activity may be of therapeutic benefit in HCPS cases, possibly by ameliorating the myocardial suppressant effects of RONS.

FIG. 1.

Plasma NO_x (a), NT (b), and total protein (c) levels in HCPS patients. Plasma NO₂⁻ levels were determined by using Greiss reagents after conversion of NO₃⁻ to NO₂⁻ with Escherichia coli reductase. Plasma protein-associated NT content was determined by an ELISA with a polyclonal anti-NT antibody and nitrated BSA as a standard. NT values were normalized to plasma protein values measured by a standard BCA assay. A single asterisk indicates a significant difference from results for normal volunteers at a P value of <0.05; triple asterisks indicate a significant difference from results for normal volunteers at a P value of <0.0005. Results are expressed as the mean and SEM.

FIG. 2.

NT adduct formation and iNOS expression in lungs from patients for whom HCPS was fatal. (a) NT immunoreactivity (brown) in lung tissue from an HCPS patient. NT immunoreactivity is particularly prominent in activated alveolar macrophages. (b) Absence of NT immunoreactivity in lung tissue from the same HCPS patient when antibody was preincubated with 10 mM free NT prior to use. (c) Absence of NT immunoreactivity in autopsy-derived normal human lung tissue. (d) Elevated iNOS immunoreactivity (brown) in infiltrating interstitial immunoblasts and alveolar macrophages in lung tissue from an HCPS patient. (e) NT immunoreactivity (brown) in cardiac tissue from an HCPS patient. (f) Absence of NT immunoreactivity in cardiac tissue from the same HCPS patient when antibody was replaced with normal rabbit serum. Paraffin-embedded sections from tissues fixed at autopsy were deparaffinized, rehydrated, and subjected to peroxidase and protein blocking. Tissue NT was detected with a rabbit anti-NT polyclonal IgG antibody. Human iNOS was detected by using clone 3 anti-human/mouse iNOS antibody (after antigen unmasking by boiling in citrate buffer).

FIG. 3.

Plasma NO_x (a), NT (b), and total protein (c) levels in LCMV-13-infected NZB mice. NZB mice were sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. Plasma NO₂⁻ levels were determined by using Greiss reagents after conversion of NO₃⁻ to NO₂⁻ with E. coli reductase. Plasma protein-associated NT content was determined by an ELISA with a polyclonal anti-NT antibody and nitrated BSA as a standard. NT values were normalized to plasma protein values measured by a standard BCA assay. A single asterisk indicates a significant difference from results for mock-infected animals at a P value of <0.05; triple asterisks indicate a significant difference from results for mock-infected animals at a P value of <0.0005. Results are expressed as the mean and SEM.

FIG. 4.

Pulmonary NT in SNV-infected deer mice and LCMV-13-infected NZB mice. (a) Absence of NT immunoreactivity (red) in lung tissue from a deer mouse sacrificed at 21 days after infection with SNV (time of peak viral replication in the lungs). (b) Macrophage infiltration and alveolar flooding (arrows) in lungs of an NZB mouse sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. (c) NT immunoreactivity (red) in lung tissue (particularly alveolar macrophages; arrows) from an NZB mouse sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. (d) Absence of NT immunoreactivity (red) in lung tissue from a mock-infected NZB mouse. (e) NT immunoreactivity (red) in cardiac tissue from an NZB mouse sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. (f) Absence of NT immunoreactivity (red) in cardiac tissue from a mock-infected NZB mouse (staining of erythrocytes is nonspecific; arrow). Paraffin-embedded lung sections were deparaffinized, rehydrated, and subjected to peroxidase and protein blocking. Tissue NT was detected with a rabbit anti-NT polyclonal IgG antibody. The specificity of the NT reaction was confirmed by preincubating the antibody with 10 mM free NT prior to use.

FIG. 5.

Wet lung/dry lung weight ratios in LCMV-13-infected NZB mice. NZB mice were sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. Following exsanguination, the lungs were removed, weighed, and dried in an oven at 55°C for 7 days. After being dried, the lungs were weighed again. The wet lung/dry lung weight ratio was then calculated as an index of intrapulmonary fluid accumulation. Triple asterisks indicate a significant difference from results for mock-infected animals at a P value of <0.0005. Results are expressed as the mean and SEM.

FIG. 6.

Effect of 1400W treatment on LCMV-13-infected NZB mice. (a) Increased survival of LCMV-13-infected 1400W-treated NZB mice. (b) 1400W treatment does not affect splenic virus titers. (c) 1400W treatment reduces splenic weight in LCMV-13-infected NZB mice. (d) 1400W treatment does not reduce wet lung/dry lung weight ratios. (e) Macrophage activation and alveolar flooding in lung tissue from an LCMV-13-infected 1440W-treated NZB mouse. (f) Absence of NT immunoreactivity (red) in lung tissue from an LCMV-13-infected 1400W-treated NZB mouse. Untreated (black bar), saline-treated (grey bar), and 1400w-treated (white bar) NZB mice were sacrificed at 6.25 days after intravenous infection with 4 × 10⁶ PFU of LCMV-13. Virus recovered from spleens was titrated by standard LCMV plaque assays, and values were normalized to the splenic wet weight. Following exsanguination, the lungs were removed, weighed, and dried in an oven at 55°C for 7 days. After being dried, the lungs were weighed again. The wet lung/dry lung weight ratio was then calculated as an index of intrapulmonary fluid accumulation. Tissue NT was detected as described in the legend to Fig. 4. A single asterisk indicates a significant difference from results for mock-infected animals at a P value of <0.05; double asterisks indicate a significant difference from results for mock-infected animals at a P value of <0.005; triple asterisks indicate a significant difference from results for mock-infected animals at a P value of <0.0005. Results are expressed as the mean and SEM.