Faculty Bibliography

The immunopathological mechanisms as well as the role played by influenza A virus infection of human leukocytes and induction of apoptosis have not been fully elucidated. We confirm here that the percentage of cells that are infected is less than the percent of apoptotic cells. Depletion of monocytes/macrophages and depletion of cells expressing influenza neuraminidase from the cultures after exposure to virus decreased lymphocyte apoptosis. Treatment of virus-exposed leukocyte cultures with anti-neuraminidase antibodies but not with anti-hemagglutinin antibodies, reduced lymphocyte production of active caspase-3 and induction of apoptosis. Different strains of virus induced different levels of apoptosis. Variations in induction of apoptosis correlated with production and expression of viral neuraminidase by infected leukocytes. The data suggest that cell surface expression of neuraminidase plays an important role in the induction of apoptosis in human lymphocytes. The benefit, or cost, to the host of lymphocyte apoptosis warrants continued investigation.

Monocytes-macrophages and lymphocytes are recruited to the respiratory tract in response to influenza virus challenge and are exposed to the virus during the establishment of immune defenses. The susceptibility of human lymphocytes to infection was assessed. The presence of monocytes-macrophages was required to attain infection of both resting and proliferating lymphocytes. Lymphocyte infection occurred in the context of immune cell clusters and was blocked by the addition of anti-intercellular adhesion molecule-1 (ICAM-1) antibody to prevent cell clustering. Both peripheral blood-derived and bronchoalveolar lymphocytes were susceptible to infection. Both CD4⁺ and CD8⁺ T lymphocytes were susceptible to influenza virus infection, and the infected CD4⁺ and CD8⁺ lymphocytes served as infectious foci for other nonpermissive or even virus-permissive cells. These data show that monocytes-macrophages and both CD4⁺ and CD8⁺ lymphocytes can become infected during the course of an immune response to influenza virus challenge. The described leukocyte interactions during infection may play an important role in the development of effective anti-influenza responses.

Both respiratory syncytial virus (RSV) and influenza A virus (IAV) may infect human peripheral blood mononuclear leukocytes (PBMC) during the immune response to viral challenge as the cells are recruited to the respiratory tract. The current studies demonstrated differences in PBMC responses to the two viruses very early after exposure, including reduced fos protein and CD69 expression and IL-2 production by RSV-exposed T lymphocytes. Exposure to RSV resulted in reduced lymphocyte proliferation despite evidence of a virus-specific T lymphocyte frequency equivalent to that for influenza virus. Reduced RSV-induced proliferation was not due to apoptosis, which was itself reduced relative to that of influenza virus-exposed T lymphocytes. The data indicate that differential immune responses to RSV and influenza virus are determined early after exposure of human PBMC and support the concept that the anamnestic immune response that might prevent clinically evident reinfection is attenuated very soon after exposure to RSV. Thus, candidate RSV vaccines should be expected to reduce but not prevent clinical illness upon subsequent infection by RSV. Furthermore, effective therapeutic agents for RSV are likely to be needed, especially for high-risk populations, even after vaccine development.

The current studies were undertaken to determine the susceptibility of human alveolar macrophages (AM) to influenza A virus (IAV) infection in comparison with autologous peripheral blood-derived monocytes-macrophages (PBM). AM and PBM were exposed to IAV in vitro and examined for their ability to bind and internalize IAV, and synthesize viral proteins and RNA. PBM but not AM demonstrated binding and internalization of the virus, synthesizing viral proteins and RNA. Exposure of AM in the presence of a sialidase inhibitor, or in the presence of anti-IAV antibody resulted in viral protein synthesis by the cells. Exposure of AM to FITC-labeled IAV in the presence of anti-FITC antibody also resulted in viral protein synthesis. Thus, human AM are apparently not susceptible to direct infection by a human IAV, but are likely to be infected indirectly in the setting of exposure in the presence of antibody that binds the challenging strain of IAV.

Interleukin-6(1) (IL-6(1)) and tumor necrosis factor alpha(3) (TNFalpha(3)) are high-cytokine-producing genotypes that are known to increase the susceptibility to infectious diseases, but their influence on cytokine production induced by respiratory viruses is unknown. We exposed human monocyte-derived macrophages from IL-6(1), TNFalpha(3), and normal genotype donors to different respiratory viruses. Respiratory syncytial virus (RSV) stimulation was associated with higher IL-6 concentrations in IL-6(1) donors than in normal donors (P = 0.015); 2 of 7 (29%) polymorphic donors were poor responders compared with 6 of 7 (86%) normal donors (P = 0.002). Adenovirus, influenza virus, and RSV stimulations were associated with higher TNFalpha concentrations in TNFalpha(3) donors than in normal donors (P = 0.03, <0.01, <0.01). A similar trend was seen with rhinovirus stimulation, but this was not significant. These results show that IL-6(1) and TNFalpha(3) gene polymorphisms lead to enhanced production of the respective cytokines when exposed to specific respiratory viruses. This, in turn, may influence the susceptibility to, severity of, and recovery from respiratory virus infections, or influence the immune response to and reactogenicity of viral vaccines.

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in infants and the elderly. Like many other pH-independent enveloped viruses, RSV is thought to enter at the cell surface, independently of common endocytic pathways. We have used a targeted small interfering RNA (siRNA) library to identify key cellular genes involved in cytoskeletal dynamics and endosome trafficking that are important for RSV infection. Surprisingly, RSV infection was potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis, including clathrin light chain. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. We conclude that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH. These findings raise the possibility that other pH-independent viruses may share a similar dependence on endocytosis for infection and provide a new potential avenue for treatment of infection.

Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.

Immune function was observed for 144 weeks in 643 human immunodeficiency virus (HIV)-infected subjects who (1) had nadir CD4+ cell counts of <50 cells/mm3, followed by a sustained increase to > or =100 cells/mm3 after the initiation of HAART, and (2) were enrolled in a randomized trial of continued azithromycin prophylaxis versus withdrawal for prevention of Mycobacterium avium complex disease. The median CD4+ cell count was 226 cells/mm3 at entry and 358 cells/mm3 at week 144. Anergy (80.2% of patients) and lack of lymphoproliferative response to tetanus toxoid (TT; 73%) after immunization and impaired antibody responses after receipt of hepatitis A (54%) and TT (86%) vaccines were considered to be evidence of impaired immune reconstitution. Receipt of azithromycin did not have an effect on CD4+ cell count but was associated with higher rates of delayed-type hypersensitivity responses to TT (25% of subjects who received azithromycin vs. 15% of those who did not; P=.009) and mumps skin test antigen (29% vs. 17%; P=.001). Although the subjects had only partial responses to immune function testing, the rate of opportunistic infections was very low, and none of the tests was predictive of risk

This study examined the effects of nitrogen dioxide (NO(2)) exposure on airway inflammation, blood cells, and antiviral respiratory defense. Twenty-one healthy volunteers were exposed on separate occasions to air and 0.6 and 1.5 ppm NO(2) for 3 h with intermittent moderate exercise. Phlebotomy and bronchoscopy were performed 3.5 h after each exposure, and recovered cells were challenged with respiratory viruses in vitro. Blood studies revealed a 4.1% NO(2) dose-related decrease in hematocrit (P = 0.003). Circulating total lymphocytes (P = 0.024) and T lymphocytes (P = 0.049) decreased with NO(2) exposure. Exposure to NO(2) increased the blood lymphocyte CD4(+)-to-CD8(+) ratio from 1.74 +/- 0.11 to 1.85 +/- 0.12 in males but decreased it from 1.88 +/- 0.19 to 1.78 +/- 0.19 in females (P < 0.001 for gender difference). Polymorphonuclear leukocytes in bronchial lavage increased with NO(2) exposure (P = 0.003). Bronchial epithelial cells obtained after exposure to 1.5 ppm NO(2) released 40% more lactate dehydrogenase after challenge with respiratory syncytial virus than with air exposure (P = 0.024). In healthy subjects, exposures to NO(2) at levels found indoors cause mild airway inflammation, effects on blood cells, and increased susceptibility of airway epithelial cells to injury from respiratory viruses.