Interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection

Abstract

Interleukin-1alpha (IL-1alpha) and IL-1beta are proinflammatory cytokines, which induce a plethora of genes and activities by binding to the type 1 IL-1 receptor (IL-1R1). We have investigated the role of IL-1 during pulmonary antiviral immune responses in IL-1R1(-/-) mice infected with influenza virus. IL-1R1(-/-) mice showed markedly reduced inflammatory pathology in the lung, primarily due to impaired neutrophil recruitment. Activation of CD4(+) T cells in secondary lymphoid organs and subsequent migration to the lung were impaired in the absence of IL-1R1. In contrast, activation of virus-specific cytotoxic T lymphocytes and killing of virus-infected cells in the lung were intact. Influenza virus-specific immunoglobulin G (IgG) and IgA antibody responses were intact, while the IgM response was markedly reduced in both serum and mucosal sites in IL-1R1(-/-) mice. We found significantly increased mortality in the absence of IL-1R1; however, lung viral titers were only moderately increased. Our results demonstrate that IL-1alpha/beta mediate acute pulmonary inflammatory pathology while enhancing survival during influenza virus infection. IL-1alpha/beta appear not to influence killing of virus-infected cells but to enhance IgM antibody responses and recruitment of CD4(+) T cells to the site of infection.

FIG. 1.

Delayed viral clearance and enhanced mortality in IL-1R1^−/− mice after infection with influenza virus. (A) C57BL/6 wild-type and IL-1R1^−/− mice were infected intranasally with influenza virus PR8 (100 PFU). Lungs were removed at the indicated days and kept frozen until determination of viral titers. Values show log₁₀ PFU/ml for individual mice (n = 4/group/day). Averages are indicated with horizontal lines. *, significant differences (Wilcoxon test). Groups of mice (n = 11/group) were infected intranasally with the influenza virus 50% lethal dose (2 × 10³ PFU), and (B) survival and (C) weight were monitored during the course of infection. **, significant differences (P = 0.0023 by the Mantel-Haenszel test).

FIG. 2.

Reduced pulmonary inflammation in IL-1R1^−/− mice. C57BL/6 wild-type (WT) mice (top row) and IL-1R1^−/− mice (bottom row) were infected with influenza virus PR8 (100 PFU), and lungs were taken for histology on day 3. C57BL/6 mice showed areas of severe pathology, with massive peribronchial lymphocytic inflammation and the lumen filled with exudates of neutrophils and mucus. Affected areas of the lungs from IL-1R1^−/− mice showed mild pathology with few intraepithelial neutrophils and peribronchial lymphocytes (hematoxylin-eosin staining was used; magnifications [left to right], ×5, ×50, and ×200).

FIG. 3.

Impaired recruitment of neutrophils and CD4⁺ T cells to the lung in the absence of IL-1R1. Groups of mice were infected intranasally with 100 PFU influenza virus PR8. Lung-infiltrating cells were isolated by BAL. (A) Total BAL cell number (T) was measured using a Coulter Counter (Instrumenten Gesellschaft IG, Basel, Switzerland), and eosinophils (E), neutrophils (N), monocytes/macrophages (M), and lymphocytes (L) were determined by differential counts according to morphological criteria as described in Materials and Methods. Numbers represent average values for four mice ± standard deviations. (B) On days 7 and 10, BAL cells were incubated with anti-CD32/CD16 MAb to block unspecific binding and subsequently stained with allophycocyanin-labeled anti-CD4 and PE-labeled anti-CD8 MAbs. Cells were acquired using a FACSCalibur (Becton Dickinson) and data analyzed with Cell Quest software. CD4⁺ and CD8⁺ T-cell numbers were calculated from percentages of BAL total cell numbers excluding dead cells. Values show averages for four mice per group. *, significant differences (Student test). (C) On days 7 (upper panels) and 10 (lower panels), aliquots of BAL cells were stained with PE-conjugated class I tetramers loaded with influenza virus peptide NP68 and allophycocyanin-conjugated anti-CD8 MAb before analysis by flow cytometry. Values show percentages of virus-specific CD8⁺ T cells gated on lymphocytes of an individual mouse representative of the group. Average percentages from groups of mice (n = 4) are shown in parentheses.

FIG. 4.

IL-1R1^−/− mice exhibit normal cytotoxic T-cell effector responses. Mice were infected with 100 PFU influenza virus PR8 and analyzed on day 10 postinfection. (A) To determine primary CTL responses at the site of virus infection, pools of BAL cells from groups of mice (n = 4/group) were serially diluted threefold in microtiter plates starting with 1 × 10⁶ cells before addition of ⁵¹Cr-labeled NP68 peptide-pulsed EL-4 target cells (1 × 10⁴ per well) and incubated for 5 h before measurement of ⁵¹Cr release. (B) Mediastinal lymph node cells were restimulated in vitro with specific peptide for 5 days. Effector cells were mixed with EL-4 target cells at the ratios indicated. Values show percent lysis. Nonspecific lysis of target cells not pulsed with peptide was below 2% (not shown).

FIG. 5.

Strongly reduced priming of spleen CD4⁺ T cells from influenza virus-infected IL-1R1^−/− mice. To determine proliferation of virus-specific Th cells, groups of mice were infected (n = 4/group) with 100 PFU influenza virus, and 7 days later CD4⁺ T cells were purified from the spleens of individual mice via positive selection by MACS (purity, >95). Isolated cells were stimulated with irradiated splenocytes from uninfected C57BL/6 mice together with graded doses of UV light-inactivated influenza virus for 72 h. [³H]thymidine was added for the final 12 h of the culture. Numbers represent the average [³H]thymidine incorporation (cpm) of wild-type and knockout cells.

FIG. 6.

Reduced IgM and IgA antibody response in influenza virus-infected IL-1R1^−/− mice. Groups of mice were infected with 100 PFU influenza virus. To examine antibody responses, mice were bled and BAL fluid was isolated on days 7 (A) and 10 (B and C). Virus-specific antibody levels of the indicated isotypes were measured by ELISA. Values represent the BAL fluid (A and B) and serum (A and C) dilutions at half-maximum absorption (optical density at 405 nm). Lines indicate averages. *, significant differences (Student test).