Intranasal LPS administration up-regulates neutrophil recruitment and enhances metastasis to the lung. (A) Representative immunohistochemistry of myeloperoxidase (brown) in PBS-treated control (Ctrl) or LPS-treated mouse lung, liver, and kidney (n = 5 mice per group, four sections per mouse, 7–10 fields per section). (Scale bar: 100 μm.) (B) Representative quantitative RT-PCR depicting the normalized fold expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in LPS-challenged lungs compared with control PBS-challenged lungs. (C, Top) Stereological quantitation of metastasis in lungs of PBS-challenged (Tum-Ctrl) or LPS-challenged (Tum-LPS) mice 6 wk after primary tumor inoculation in the skin. Data are represented as the mean ± SD (n = 3 Ctrl mice and n = 4 LPS mice, five to eight sections per lung). *P = 0.025 (one-tailed Mann–Whitney U test). (C, Bottom) Representative H&E stains of lungs. (Scale bars: 2 mm.) (D, Top) Stereological quantitation of B16-BL6 melanoma lung metastases in Ctrl or LPS-challenged mice 18 d following tail vein injection of 2 × 10⁵ B16-BL6 cells. Data are plotted as the mean ± SEM (n = 10 mice per group). **P = 0.002 (one-tailed t test with Welch’s correction). (D, Bottom) Representative H&E stains. (Scale bars: 2 mm.) (E, Top) Quantitation using bioluminescence imaging (BLI) of LLC pulmonary metastases in Ctrl or LPS-challenged mice 15 d following tail vein injection of 1 × 10⁵ LLC cells. Data are plotted as the mean ± SEM (n = 5 mice per group, similar data in two repeat experiments). *P = 0.048 (one-tailed Mann–Whitney U test). (E, Bottom) Representative BLI images.

Up-regulation of proinflammatory mediators and recruitment of BM-derived cells to lungs after intranasal instillation of LPS. (A) Representative quantitative RT-PCR depicting the normalized fold expression of the proinflammatory mediators IL-1β, IL-6, TNF-α, and Cox-2 in Ly6G⁺ cells sorted from LPS-challenged lungs compared with control PBS-challenged lungs. Control expression levels are set to 1. Numbers signify fold expression relative to controls (n = 3 mice per group). Experiments were repeated three times with comparable results. (B) Representative flow cytometry-based quantitation of recruited BM-derived cells in the lungs of PBS-treated mice (Ctrl) and LPS-challenged mice 3 d after the last PBS/LPS dose. Fold increase is depicted as the percentage of each cell population in LPS-treated lungs compared with the percentage of each cell population in control lungs. The red dotted line represents a fold increase of 1 (n = 3 mice per group). (C) Representative flow cytometry scatter plots of BM-derived cells in the lungs of PBS-treated mice and LPS-treated mice. B220, B cells; CD11b, myeloid cells; CD11c, dendritic cells; CD25, lymphocytes; CD3, T cells; CD45, hematopoietic cells; CD49b, NK cells; cKit, hematopoietic progenitor cells; F4/80, macrophages; Gr1, myeloid cells; Ly6C, monocytes; Ly6G, granulocytes (neutrophils); VEGFR1, hematopoietic progenitor cells. Experiments were repeated three times with similar results.

LPS challenge enhances neutrophil recruitment to the lung in an orthotopic model of melanoma. (A) Immunofluorescence-based quantitation of Ly6G⁺ neutrophil recruitment to lungs in tumor-bearing mice 3 d after the last intranasal administration of PBS (Tum-Ctrl) or LPS (Tum-LPS). Data are represented as the mean ± SEM (n = 3 mice per group, two sections per lung, nine fields per section). ***P < 0.0001 (one-tailed Mann–Whitney U test). (B) Immunofluorescence-based quantitation of CD45⁺ hematopoietic cell recruitment to lungs in non–tumor-bearing mice (Ctrl) and tumor-bearing mice 3 d after the last intranasal administration of PBS or LPS. Data are represented as the mean ± SEM (n = 3 mice per group, two sections per lung, nine fields per section). ***P < 0.0001 (one-tailed t test) for comparisons between Ctrl and Tum-Ctrl, as well as between Ctrl and Tum-LPS.

LPS does not increase extravasation or seeding of LLC cells tail vein injected 3 d after the last LPS dose. (A, Left) Flow cytometry-based quantitation of the number of Cell Tracker dye-positive cells in lungs 2 h after cell injection. Data are represented as the mean ± SEM (n = 3 mice per group). P = 0.629 (two-tailed Mann–Whitney U test). (A, Right) Representative flow cytometry scatter plots of Cell Tracker dye-positive LLC cells in lungs. (B, Left) Flow cytometry-based quantitation of the number of Cell Tracker dye-positive cells in lungs 48 h after cell injection. Data are represented as the mean ± SEM (n = 3 mice per group). P = 0.2 (two-tailed Mann–Whitney U test. (B, Right) Representative flow cytometry scatter plots of Cell Tracker dye-positive LLC cells in lungs.

Depletion of Ly6G⁺ cells suppresses metastasis. (A, Top) Quantification of pulmonary metastases following LLC tail vein injection using BLI in mice administered IgG isotype Ab or anti-Ly6G neutralizing Ab (n = 5 per group). (A, Bottom) Representative BLI images. (B) Flow cytometry-based quantification of Ly6G⁺ cells in lungs of isotype-treated and anti–Ly6G-treated mice (n = 3 per group). ***P = 0.00016.

Neutrophil recruitment in LPS-inflamed lungs is associated with Tsp-1 degradation. (A) Western blot analysis of Tsp-1 levels in the lungs of WT mice challenged with PBS (Ctrl, n = 3 mice) or LPS (n = 4 mice). β-Actin was used as a loading control. The experiment was reproduced three times with comparable results. (B) Quantitative RT-PCR analysis of Tsp-1 in flow cytometry-sorted Ly6G⁺ cells from the lungs of PBS-challenged (Ctrl) or LPS-challenged mice. Samples were normalized based on equal Ly6G⁺ cell numbers, and normalized to GAPDH mRNA. Data are plotted as the mean ± SEM (n = 3 mice per group, in duplicates). The PBS group was used as a control for relative expression (2^−ΔΔCt). (C) Flow cytometry analysis of CD63 median fluorescence intensity (MFI) in the CD11b⁺ Ly6G⁺ population in lungs of control and LPS-challenged mice. Data are plotted as the mean ± SEM (n = 4 mice per group). *P = 0.029 (one-tailed Mann–Whitney U test). The experiment was reproduced twice with comparable results. (D) NE activity (Left) and CG activity (Right) in lungs of PBS-treated (Ctrl) and LPS-treated mice. Activity is presented as relative fluorescence units per minute (RFU/min) and plotted as the mean ± SEM (n = 4 mice per group). *P = 0.018 and *P = 0.029 for NE activity and CG activity, respectively (one-tailed Mann–Whitney U test).

Conditional expression of IL-1β in lungs promotes neutrophil recruitment, Tsp-1 degradation, and enhanced metastatic outgrowth. (A) Flow cytometry-based quantification of Ly6G⁺ cells in lungs of CCSP–tetracycline-controlled transactivator (rtTA); tetO–IL-1β or age-matched control littermates (WT or CCSP-rtTA single-transgenic) administered doxycycline for 10 d (n = 5 controls and n = 7 CCSP-rtTA; tetO–IL-1β). **P = 0.0064 (one-tailed t test). Data are represented as the mean ± SEM. (B) Representative quantitative RT-PCR depicting the normalized expression of the proinflammatory mediators IL-1β, TNF-α, and Cox-2 in lungs of CCSP-rtTA; tetO–IL-1β mice and controls administered doxycycline for 10 d (n = 5 controls and n = 7 CCSP-rtTA; tetO-IL-1β). **P = 0.005 and *P = 0.025 for IL-1β and TNF-α, respectively (one-tailed t test). *P = 0.014 for Cox-2 (one-tailed Mann–Whitney U test). Data are represented as the mean ± SEM. (C) NE activity (Left) and CG activity (Right) in lungs of CCSP-rtTA; tetO–IL-1β mice and controls administered doxycycline for 10 d (n = 5 controls and n = 7 CCSP-rtTA; tetO–IL-1β). Activity is represented as RFU/min. *P = 0.038 and *P = 0.013 for NE and CG activity, respectively (one-tailed t test). Data are represented as the mean ± SEM. (D) Western blot analysis of Tsp-1 in pooled lungs of bitransgenic mice or littermates administered doxycycline for 10 d (n = 5 controls and n = 7 CCSP-rtTA; tetO–IL-1β). β-Actin is used as a loading control. (E, Left) Stereological measurements of lung metastases 28 d after tail vein injection of 1 × 10⁶ mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) (FVB/N strain)–derived breast cancer single-cell suspensions into syngeneic CCSP-rtTA; tetO–IL-1β or age-matched control littermates (FVB/N strain) that were administered doxycycline for 8 d. Data are plotted as the mean ± SEM (n = 6 controls and n = 10 CCSP-rtTA; tetO–IL-1β). **P = 0.005 (one-tailed Mann–Whitney U test). (E, Right) Representative H&E stains. (Scale bars: 2 mm.)

Conditional expression of IL-1β in lungs promotes neutrophil recruitment, Tsp-1 degradation, and enhanced metastatic outgrowth. (A) Representative scatter plots depicting flow cytometry analysis of Ly6G⁺ cells in lungs of CCSP-rtTA; tetO–IL-1β or age-matched control littermates (WT or CCSP-rtTA single-transgenic) administered doxycycline for 10 d. (B) Quantitation of number of surface metastases in lungs 28 d after tail vein injection of 1 × 10⁶ mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) (FVB/N strain)–derived breast cancer single-cell suspensions into syngeneic CCSP-rtTA; tetO–IL-1β or age-matched control littermates (FVB/N strain) that were administered doxycycline for 8 d. Data are plotted as the mean ± SEM (n = 7 controls and n = 10 CCSP-rtTA; tetO–IL-1β). *P = 0.018 (one-tailed t test with Welch’s correction).

Neutrophil-derived Ser proteases mediate proteolysis of Tsp-1. (A and B) In vitro degradation assays of recombinant Tsp-1 with recombinant NE and CG proteases, alone or in combination with the specific inhibitors of NE or CG, and sivelestat. Recombinant (r) protein incubations were followed by Western blot analysis for Tsp-1 levels. Experiments were repeated three times with similar results. (C) Representative flow cytometry analysis of degranulation marker CD63 in CD11b⁺ Ly6G⁺ cells cultured in vitro with 0.01% DMSO (Ctrl, solid gray histogram), 20 nM TPA (TPA, empty blue histogram), or 20 nM TPA + 0.05 μg/μL sivelestat (Siv, solid red histogram). Experiments were repeated three times with similar results. (D) Representative protease activity assays for NE (Left) and CG (Right) in CM of Ly6G⁺ cells cultured in vitro with 20 nM TPA or 20 nM TPA + 0.05 μg/μL Siv. Activities are represented as RFUs as a function of time. Experiments were repeated twice with similar results. (E) Western blot analysis of Tsp-1 in CM of Ly6G⁺ cells cultured in the presence of TPA or TPA + Siv at t = 0, 30, 60, and 120 min. Ponceau S staining shows equal protein loading.

Neutrophil Ser proteases NE and CG work in concert to degrade Tsp-1 and enhance metastasis. (A) Representative flow cytometry analysis of the degranulation marker CD63, in CD11b⁺ Ly6G⁺ cells isolated from WT mouse lungs (solid purple histogram) and NE^−/− CG^−/− mouse lungs (empty green histogram) and cultured in vitro with 20 nM TPA. Experiments were repeated three times with similar results. (B) NE activity (Left) and CG activity (Right) in CM of Ly6G⁺ cells isolated from WT and NE^−/− CG^−/− mouse BM and cultured with 20 nM TPA for 30 min. Data are plotted as the mean ± SEM (n = 3 per group). ***P < 0.001 for NE activity and for CG activity [analysis of covariance (ANCOVA), comparing activity curves]. (C) Representative Western blot analysis of Tsp-1 in CM from Ly6G⁺ cells isolated from WT and NE^−/− CG^−/− mouse BM and cultured with 20 nM TPA for t = 0, 2, or 4 h. Ponceau S (Ponc) staining is used as a loading control. Experiments were repeated twice with similar results. (D) Western blot analysis of Tsp-1 protein in lungs of WT and NE^−/− CG^−/− mice treated with LPS. Protein samples are pooled from four mice per group. β-Actin serves as a loading control. (E) Flow cytometry analysis of the MFI of the degranulation marker CD63 in CD11b⁺ Ly6G⁺ cells in lungs of WT and NE^−/− CG^−/− mice treated with LPS. Data are plotted as the mean ± SEM (n = 4 lungs per group). ns, not significant (two-tailed Mann–Whitney U test). (F) Quantification of metastatic burden via BLI in WT BMT and NE^−/− CG^−/− BMT mice treated with LPS and tail vein injected with LLC tumor cells. Signals were normalized to the readings obtained at day 4 for each individual animal. Data at each time point are plotted as the mean ± SEM (n = 8 mice per group). *P = 0.038 at day 21 (one-tailed t test with Welch’s correction). (G) Western blot analysis of Tsp-1 in lung samples from WT BMT and NE^−/− CG^−/− BMT mice treated with LPS. Samples are pooled from three mice per group. β-Actin serves as a loading control.

Double KO of NE and CG in BM does not affect hematopoietic cell composition, Ly6G⁺ cell circulation in blood, recruitment to lungs, or degranulation after LPS challenge. (A) Flow cytometry-based quantitation of various hematopoietic cell subsets in blood of WT BMT and NE^−/− CG^−/− BMT mice (P = 0.1 for CD45⁺ cells, P = 0.4 for CD11b⁺ cells, P = 0.4 for Ly6G⁺ cells, P = 0.475 for CD3⁺ cells, P = 0.628 for B220⁺ cells). Statistical analysis used is the two-tailed Mann–Whitney U test. (B) Quantitation of CD11b⁺ Ly6G⁺ cells in blood of PBS-treated WT BMT and LPS-treated WT BMT or NE^−/− CG^−/− BMT mice (n = 4 mice per group). *P = 0.044 between WT BMT and WT BMT + LPS (two-tailed t test); *P = 0.016 between WT BMT and NE^−/− CG^−/− BMT + LPS (two-tailed t test). ns, not significant. (C) Flow cytometry-based quantitation of CD45⁺ hematopoietic cells and Ly6G⁺ neutrophils recruited to lungs of WT BMT and NE^−/− CG^−/− BMT mice treated with LPS (n = 4 mice per group). Differences are not statistically significant, as determined by the two-tailed Mann–Whitney U test. Lungs were collected 2 d after LPS treatment. (D) Flow cytometry-based quantitation of CD63 median fluorescence intensity (MFI) of Ly6G⁺ cells in lungs of WT BMT and NE^−/− CG^−/− BMT mice treated with LPS (n = 4 per group). P = 0.305 (two-tailed unpaired t test). (E) Quantitation of BLI showing luciferase-labeled LLC cell outgrowth in lungs of WT BMT and NE^−/− BMT mice (n = 8 mice per group). P = 0.4796 (one-tailed Mann–Whitney U test). (F) Quantitation of BLI showing luciferase-labeled LLC cell outgrowth in lungs of WT BMT and CG^−/− BMT mice (n = 8 mice per group).

Proposed model of inflammation-enhanced metastasis. Under normal physiological conditions (steady state), lungs exhibit sparse neutrophil counts and abundant intact and functional Tsp-1. Inflammation in the lungs causes enhanced influx of BM-derived neutrophils, which are the first responders at the infection site. Activated neutrophils produce potent inflammatory mediators, including IL-1β, TNF-α, IL-6, and Cox-2, and they degranulate their azurophilic granules, releasing the Ser proteases NE and CG. NE and CG degrade Tsp-1, rendering the lung microenvironment conducive to increased metastatic outgrowth. Whether CG and NE also have a non–Tsp-1–mediated role in metastasis is not known and is depicted by a “?”.

Proteases in vivo mediate complete degradation of Tsp-1. (A) Full blot of PBS-challenged and LPS-challenged mouse lungs probed for Tsp-1. Full-length Tsp-1 runs as a double band between 110 kDa and 170 kDa. (B) Tsp-1 blot from Tsp-1^−/− mice is used to determine nonspecific bands, which appear as a result of using a mouse-derived Tsp-1 Ab on mouse lungs and having to use an anti-mouse secondary Ab for detection. Of note, the Tsp-1 Ab clone we use is the Ab-11 clone, which has the ability to recognize the N-terminal heparin-binding domain, the calcium-binding domain, and the collagen type V-binding domain of Tsp-1.

MMTV-PyMT lungs show recruitment of neutrophils and Tsp-1 degradation. (A) Representative flow cytometry-based quantitation of recruited BM-derived cells in the lungs of 10-wk-old FVB/N mice (Ctrl) and 10-wk-old MMTV-PyMT mice. Fold increase is depicted as the percentage of each cell population in LPS-treated lungs compared with the percentage of each cell population in control lungs. The red dotted line represents a fold increase of 1 (n = 3 mice per group). (B) Western blot analysis of Tsp-1 protein in the lungs of FVB/N mice, 10-wk-old MMTV-PyMT mice, and Tsp-1^−/− mice. β-Actin is used as a loading control.