Figure 1.

LLC cells induce neutrophil chemotaxis via secreting ELR+CXC chemokine and neutrophils had a cytotoxic effect on these cells in vitro. (A) The production of CXCL1, CXCL2 and CXCL5 by NSCLC and mesothelioma lines was measured by ELISA. (B) Neutrophils were purified from bone marrow of C57BL/6 mice and were analyzed by flow cytometry for purity by CD11b+Ly6G+ staining. (C) Purified neutrophils from C57BL/6 or Cxcr2−/− mice were assayed by transwell migration for chemotaxis toward LLC cells. Negative control was neutrophil migration toward a non-neutrophil secretion lung cancer cell line. Cells were counted from both the top and bottom of transwell plate after 16 h. (D) Neutrophils isolated from bone marrow of naive (C BM Neut) or tumor-bearing (T BM Neut) mice were cocultured with luciferase-labeled LLC cells at 20:1 and 40:1 neutrophil to tumor cell ratio. Following overnight incubation, luciferase activity was measured using the Clarity (Bio-Tek) microplate luminescence reader as a measure of cytotoxicity. (E) As above neutrophils isolated from bone marrow of naive (C BM Neut) or AE17 tumor bearing (AE17 BM Neut) mice were assayed for cytotoxicity against LLC cells, at a 20:1 neutrophil:tumor cell ratio. (F) Addition of 10ng/mL recombinant TNF-α or IL-1β induced enhanced neutrophil cytotoxic effect, measured as in D. *p <0.05 **p <0.01; ***p <0.001. Values are mean ± SEM, n = 3/experiment, representative of three experiments).