x31-infected WT and CCR2^−/− mice lack TRAIL expression on exudate macrophages and reveal equal levels of AEC apoptosis and alveolar barrier function. (A) Quantification of BAL exudate mononuclear phagocytes from Pappenheim-stained cytocentrifuge preparations in x31-infected WT (filled bars) or CCR2^−/− (empty bars) mice. (B) Alveolar leakage of uninfected (day 0) or x31-infected (day 7) WT (filled bars) or CCR2^−/− (empty bars) mice. (C, right) FACS analysis of apoptotic AEC type I from mock- or x31-infected WT or CCR2^−/− mice on day 7 pi. (C, left) Quantification of FACS data. Bar graphs show the annexin V⁺ proportion of CD45⁻ T1α⁺ cells. (D) GR1^intF4/80⁺ BAL cells from x31-infected WT or CCR2^−/− mice were gated and analyzed for TRAIL surface expression. *, P < 0.05; ***, P < 0.005. All bar graphs represent the means ± SD from three independent experiments.

Relative mRNA expression of the proapoptotic factors TNF-α, TRAIL, and FasL in peripheral blood (PB) monocytes and alveolar exudate macrophages. (A) BALF exudate alveolar macrophages from PR/8-infected WT mice were high-purity flow sorted on day 8 pi according to their surface expression of F4/80, GR1, and CD11c (F4/80⁺GR1^intCD11c^int [population 3 (P3)], left). PB monocytes from the same (PR/8 infected) animals or from mock-infected mice were flow sorted according to their scatter characteristics (SSC^low) and CD11b and CD115 expression (CD11b⁺CD115⁺ [population 3 (P3)], right). SSC, side scatter; FSC, forward scatter. (B) Relative mRNA expression of TNF-α, TRAIL, and FasL from sorted PB monocytes of mock-infected (PB-Mo mock) or PR/8-infected (PB-Mo PR/8) mice or from exudate macrophages of infected mice (ExMa PR/8). Values are the means ± SD from three independent experiments, including pooled BALF or blood cells, from n = 8 mice each. *, P < 0.05.

AEC apoptosis and lung leakage are reduced in PR/8-infected mice recruiting TRAIL-deficient exudate macrophages to their lungs. (A) Digested lungs of three different groups of PR/8-infected BM chimeric mice (WT recipient mice with transplantation of WT BM cells; WT recipient mice with transplantation of TRAIL^−/− BM cells; and TRAIL^−/− recipient mice with transplantation of WT BM cells) were analyzed for AEC type I apoptosis (left, representative FACS plots; right, quantitative analysis of five independent experiments). Error bars show SD. (B) Alveolar leakage of different BM chimeric mice that were either left untreated or injected with isotype IgG or anti-CCR2 mAb, respectively, was analyzed at day 7 pi. (C) CCR2^−/− mice were adoptively transferred with CCR2^+/+/TRAIL^+/+, CCR2^+/+/TRAIL^−/−, or CCR2^−/−/TRAIL^+/+ MNC, and alveolar leakage was analyzed at day 7 after PR/8 infection. BMT, BM transplantation; iso, isotype IgG; MNC, mononuclear cells; ***, P < 0.005; *, P < 0.05. Bar graphs in B and C represent the means ± SD of three to five mice per group from three independent experiments.

TRAIL is expressed on the cell surface of alveolar exudate macrophages. (A) WT or CCR2^−/− mice were mock or PR/8 infected and BAL cells were stained with GR1-FITC, F4/80–Alexa Fluor 647, and TRAIL PE or isotype PE mAbs, respectively. GR1^intF4/80⁺ cells were gated and analyzed for TRAIL surface expression (left). Quantitative analysis of the proportion of TRAIL⁺ from F4/80⁺ BALF cells gained from PR/8-infected WT (filled bars) or CCR2^−/− mice (empty bars) on days 5, 8, and 11 pi (right). (B) FACS analysis of TRAIL expression on BALF NK cells (SSC^lowNK1.1⁺), neutrophils (GR1^highF4/80⁺), CD8 T cells (SSC^lowCD8⁺), and CD4 T cells (SSC^lowCD4⁺) of PR/8-infected WT (top) or CCR2^−/− (bottom) mice gained at the indicated time points. Values are the means ± SD of two to four mice per group from at least two independent experiments. ***, P < 0.005.

CCR2 deficiency is associated with increased survival, reduced body weight loss, attenuated alveolar leakage, and slightly reduced viral clearance during the time course of PR/8 infection. (A) Body weight and survival of PR/8-infected sex- and age-matched WT (♦, n = 23) or CCR2^−/− (▪, n = 23) were determined until days 14 and 21 pi, respectively in four independent experiments. Error bars show SD. (B) Alveolar leakage was analyzed in PR/8-infected WT (filled bars) or CCR2^−/− (empty bars) mice. The ratio of BALF and blood FITC fluorescence is expressed in arbitrary units (AU). (C) Lung virus titers of WT (♦) and CCR2^−/− (▪) mice in the time course of PR/8 infection. Virus titers were determined from lung homogenate supernatants and values are given as log₁₀ foci-forming units (FFU)/lung. Data in B and C are means ± SD of three to five mice per group from at least three independent experiments. *, P < 0.05; ***, P < 0.005.

Alveolar barrier dysfunction is associated with CCR2 expression on circulating leukocytes during PR/8 infection. (A) Three different groups of PR/8-infected BM chimeric mice (WT recipient mice with transplantation of 100% WT BM cells; WT recipient mice with transplantation of mixed 50% WT/ 50% CCR2^−/− BM cells; and WT recipient mice with transplantation of 100% CCR2^−/− BM cells) were subjected to BAL at day 8 pi. Exudate macrophage numbers were calculated from BALF leukocyte numbers and differential counts of Pappenheim-stained cytocentrifuge preparations. (B) FACS analysis of BALF cells from PR/8-infected chimeric mice of each transplantation group was performed as described in Fig. 2 D, and representative dot plots from three independent experiments are shown. Note that population 3 (P3; exudate macrophages), population 4 (P4; resident alveolar macrophages), and population 5 (P5; alveolar DCs) are subgates of population 2 [P2]. (C) Alveolar leakage from PR/8-infected chimeric mice at day 7 pi. All bar graphs represent the means ± SD of four to eight mice per group from three independent experiments. ***, P < 0.005.

AEC apoptosis is reduced in PR/8-infected CCR2^−/− mice compared with WT mice. (A) WT or CCR2^−/− mice were mock or PR/8 infected and TUNEL assay was performed on cryosections from lavaged lungs at day 7 pi. Nuclei of apoptotic cells appear in brown. Arrows, apoptotic intraalveolar leukocytes; arrowheads, apoptotic AEC. Bars, 100 μm. (B) Flow cytometric quantification of apoptotic AEC. Lungs from mock- or PR/8-infected WT or CCR2^−/− mice were digested on day 7 pi and analyzed for annexin V binding. Representative dot plots show expression of the AEC type I marker T1α and annexin V staining of viable (propidium iodide negative) CD45⁻ cells. Apoptotic cells are mainly AEC type I (left). Quantification of FACS data as obtained in B from PR/8-infected mice (right). The bar graph represents the annexin V⁺ proportion of CD45⁻ T1α⁺ cells. (C) Quantification of FACS analysis of the NP⁺ proportion of CD45⁻ T1α⁺ cells from lung homogenates in the time course after PR/8 infection. Values are means ± SD of three to four mice per group from three independent experiments. *, P < 0.05; ***, P < 0.005. NP, IV nucleoprotein; filled bars, WT; empty bars, CCR2^−/−.

Alveolar macrophage recruitment in PR/8-infected mice is CCR2 dependent. (A and B) PR/8-infected WT (filled bars) or CCR2^−/− (empty bars) mice were subjected to BAL, followed by quantification of total leukocyte BALF numbers (A) and BAL leukocyte subpopulations (B) from Pappenheim-stained cytocentrifuge preparations. (C) Quantification of exudate mononuclear phagocytes calculated from differential counts of BAL leukocytes from infected WT mice treated with isotype IgG or anti-CCR2 mAb (day 8 pi, left). Alveolar leakage of isotype- or anti-CCR2–treated PR/8-infected mice (day 7 pi, right). (D) FACS analysis of alveolar mononuclear phagocyte subpopulations in BALF from infected WT versus CCR2^−/− mice or from WT mice treated with isotype IgG or anti-CCR2 mAb was performed by three-hierarchy gating on day 8 pi. CD45⁺ cells (population 1 [P1]) were gated according to their GR1 and F4/80 expression. GR1^intF4/80⁺ cells represent alveolar mononuclear phagocytes (population 2 [P2]). Subgate analysis of population 2 revealed a CD11c^int (population 3 [P3], exudate macrophages) and a CD11c^high (population 4 [P4], resident AM) subpopulation as well as a CD11c^highMHCII^high subpopulation (population 5 [P5]) representing alveolar DCs. (E) Flow cytometric quantification of CD11c^highMHCII^high DCs and lymphocyte subpopulations from BAL leukocytes of PR/8-infected WT (filled bars) versus CCR2^−/− (empty bars) mice using the gating characteristics described in D for DCs. For analysis of lymphocyte subpopulations, CD45⁺ cells were subgated on NK cells (SSC^lowNK1.1⁺), CD4 T cells (SSC^lowCD4⁺), and CD8 T cells (SSC^lowCD8⁺). Data in E are given as total cells in BAL calculated from the respective percentage of CD45⁺ cells. All bar graphs represent the means ± SD from five experiments. *, P < 0.05; ***, P < 0.005. AM, alveolar macrophages; ex, exudate; mn, mononuclear; iso, isotype IgG.

Anti-TRAIL treatment attenuates AEC apoptosis as well as alveolar leakage and enhances survival upon PR/8 infection. (A) PR/8-infected WT mice were treated with IgG isotype or anti-TRAIL mAb on days 3 and 5 pi and TUNEL assay was performed on lung cryosections at day 7 pi (arrows, apoptotic intraalveolar leukocytes; arrowheads, apoptotic AECs). Bars, 100 μm. (B) FACS analysis (representative dot plots, left) and quantification (four to six mice per group; right) of apoptotic AEC I of PR/8-infected, IgG isotype–treated, or anti-TRAIL–treated mice (day 7 pi). Error bars show SD. (C) Alveolar leakage of PR/8-infected WT mice at day 7 pi that were treated with IgG isotype or anti-TRAIL mAb at days 3 and 5 pi. (D) Survival of PR/8-infected WT mice treated with isotype IgG (▴, n = 10) or anti-TRAIL mAb (•, n = 12) at days 3, 5, 7, and 9 pi in three independent experiments. (E) TRAIL receptor DR5 is expressed on cultured AEC and up-regulated upon PR/8 infection dose dependently in vitro. Bar graphs depict DR5 mRNA expression as fold induction of mock-infected AEC. (F) DR5 is expressed on AEC and up-regulated upon PR/8 infection in vivo. Left, FACS analysis of DR5 and NP expression of gated AEC type I (CD45⁻ T1α⁺) from digested lungs of uninfected (day 0, top) or PR/8-infected (day 5 pi) WT mice. Right, histograms depict DR5 expression of NP-negative (medium gray histograms) and NP-positive CD45⁻ T1α⁺ AEC I (dark gray histograms) from noninfected (day 0) and PR/8-infected WT mice in the time course of infection. Isotype PE–stained cells are shown in light gray histograms. An NP-positive population is missing at days 0 and 14 pi. Bar graphs in C and E represent the means ± SD of three to five independent experiments. *, P < 0.05; ***, P < 0.005; iso, isotype IgG; NP, IV nucleoprotein.