Figure 1.
Virulence of influenza viruses for C57BL/6 mice.
Mice were infected with 105 PFU of BJx109, HKx31 or PR8. (A) Weight loss. Mice were weighed daily and results expressed as mean percent weight change of each group (± SEM), compared to the weight immediately prior to infection. (B) Titres of virus in the lungs at day 5 post-infection. Lungs were removed and titres of infectious virus were determined in clarified homogenates by standard plaque assay. Data represent mean virus titres ± 1 SEM. The detection limit for the assay is indicated by the dotted line. Data shown are for groups of 5 mice and are representative of 2 or more independent experiments. * = virus titres from PR8-infected mice were significantly higher than those from HKx31-infected mice (p<0.001, one-way ANOVA).
Figure 2.
Neutrophil response in the airways after infection of mice with BJx109, HKx31 or PR8.
Groups of 5 mice were infected with 105 PFU of each virus via the intranasal route and at the times indicated, mice were killed and lavage performed. Numbers of (A) total BAL and nasal tissue cells, and (B) total BAL and nasal tissue neutrophils from virus-infected mice. Cell numbers in the BAL fluids from naïve mice are included for comparison. Neutrophils were identified by flow cytometry as CD45+ Gr-1high cells. A minimum of 50,000 living cells (PI−) were collected and analyzed from each mouse. * = BJx109 is significantly reduced compared to HKx31 and PR8; # = HKx31 is significantly reduced compared to PR8; x = HKx31 is significantly reduced compared to BJx109 and PR8 (p<0.05, one-way ANOVA). (C) Levels of KC, MIP-2 and MIP-1α in BAL supernatants at various times after influenza virus infection. Naïve (N) animals were included for comparison. Chemokine levels were determined by ELISA and results are expressed in pg/mL. The detection limit for each ELISA is indicated by the dotted line. ND = not done as all PR8-infected mice were euthanized at day 5 post-infection.
Figure 3.
Neutrophils are not susceptible to influenza virus infection.
Mature murine neutrophils were purified from the bone marrow as described in Materials and Methods. Aliquots of 106 (i) LA-4 epithelial cells, or (ii) neutrophils were incubated with 107 PFU of BJx109, HKx31 or PR8. Control cells were incubated with BJx109 that had been UV-inactivated to destroy virus infectivity (UV BJx109). After 1 hr, cells were washed and incubated an additional 8 hrs. (A) Cells were fixed and levels of intracellular viral NP protein determined by flow cytometry. Histograms shown represent cells incubated with virus (grey) or uninfected control cells (white). A gate was set to include 5% of cells in the uninfected control and the percentage of cells staining positive for intracellular NP are shown relative to this in each panel. (B) RNA was extracted from cells and levels of (i) vRNA and (ii) mRNA encoding NP, PA and M genes determined via quantitative RT-PCR. Cells incubated in media alone (mock) were included as a control. Data is expressed in pg/ml and represent the mean ± SD of 3 independent experiments.
Figure 4.
Effect of mAb 1A8 and mAb RB6-8C5 treatment of mice prior to and during infection with BJx109, HKx31 or PR8.
Groups of 5 B6 mice were treated with purified (A) anti-Ly6G (1A8) or (B) anti-Gr-1 (RB6-8C5) antibodies 24 hrs prior to virus infection and every second day thereafter. Control mice received rat IgG (IgG). Data show weight loss for mice infected with 105 PFU of (i) BJx109, (ii) HKx31 or (iii) PR8. Mice were weighed daily and results expressed as the mean percent weight change of each group (± SEM), compared to original body weight. Animals that had lost ≥25% of their original body weight and/or presented with evidence of pneumonia were euthanized. Data shown are from one experiment and are representative of two independent experiments. The dashed line indicates days on which the weight loss induced by treatment with (A) mAb 1A8 or (B) mAb RB6-8C5 was statistically different to IgG-treated controls (p<0.05, Student's t-test).
Figure 5.
Virus replication in neutropenic and control mice following infection with BJx109, HKx31 and PR8.
Groups of 5 B6 mice received i.n. and i.p. treatments with purified anti-Ly6G (1A8, striped bars) 24 hrs prior to virus infection and every second day thereafter. For comparison, virus-infected mice were also treated with anti-Gr-1 (RB6-8C5, grey bars) antibodies and control mice with rat IgG (IgG, black bars). Mice were infected with 105 PFU of BJx109, HKx31 or PR8 and at day 5 post-infection mice were killed, lungs removed and titres of infectious virus were determined in clarified homogenates by standard plaque assay. Data represent mean viral titres titres ± 1 SD. The detection limit for the assay is indicated by the dotted line. Viral titres from treated mice (RB6-8C5 or 1A8) that were significantly higher than those from control (IgG-treated) animals are indicated by * = p<0.05, ** = p<0.01 (Student's t-test).
Figure 6.
Neutrophils play a beneficial role during severe influenza infections of mice.
(A) Groups of 5 B6 mice were treated with purified anti-Ly6G (1A8) or rat IgG (IgG) antibodies 24 hrs prior to virus infection and every second day thereafter. Data show weight loss for mice infected with 102 PFU of PR8. (B) Groups of 5 B6 or B6.RAG-2γc−/−mice were infected with 102 PFU of HKx31 and at day 5 and 9 post-infection, mice were killed and BAL performed. Numbers of BAL neutrophils from virus-infected mice are shown. Neutrophils were identified via differential counts and Diff Quick staining, as described in Material and Methods. * = neutrophil numbers from B6.RAG-2γc−/− mice were significantly higher compared to numbers from B6 mice (p<0.01, one-way ANOVA). (C) Immunocompetent B6 mice (upper panels) or B6.RAG-2γc−/−mice (lower panels) were treated with purified anti-Ly6G antibodies (1A8) (i) at days −1, +1, +3, +5, +7, +9 and +11 (Continuous treatment, left panels), or (ii) at days +5, +7, +9 and +11 (Late treatment, right panels) relative to infection with 102 PFU of HKx31 at day 0. Control mice received a similar treatment regime of rat IgG (IgG). Arrows indicate the days mice were treated with mAb 1A8 or control rat IgG. Mice were weighed daily and results expressed as the mean percent weight change of each group (± SEM), compared to original body weight. Animals that had lost ≥25% of their original body weight and/or presented with evidence of pneumonia were euthanized. Data shown are from one experiment and are representative of two independent experiments. Dashed lines indicate days on which the weight loss induced by treatment with (A/C) mAb 1A8 was statistically different to IgG-treated controls (p<0.05, Student's t-test).