Tumor-derived granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor prolong the survival of neutrophils infiltrating bronchoalveolar subtype pulmonary adenocarcinoma

We evaluated the role of the tumor environment in the regulation of apoptos is of tumor-infiltrating neutrophils, the number of which correlates negati vely with outcome, in patients with adenocarcinoma of the bronchioloalveola r (BAC) subtype. We examined three different parameters of apoptosis, namel y morphological aspect, annexin-V expression, and DNA fragmentation. Bronch oalveolar lavage fluid (BALF) supernatants from patients with BAC significa ntly inhibited the 24-hour spontaneous apoptosis of normal peripheral blood neutrophils in vitro compared to BALF supernatants from control patients ( 64 +/- 4% versus 90 +/- 2% measured by annexin-V flow cytometry, P = 0.04). The alveolar neutrophil count correlated positively with the granulocyte c olony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulat ing factor (GM-CSF) concentrations in the patient's BALF. Furthermore, neut ralizing antibodies (Abs) against GM-CSF and G-CSF significantly inhibited BALF anti-apoptotic activity (15 to 40% and 34 to 63% inhibition, respectiv ely), whereas neutralizing Abs against interleukin (IL)-8, IL-6, IL-1 beta and tumor necrosis factor-alpha had no significant effect. In an attempt to identify the cell origin of anti-apoptotic cytokines, we tested in vitro t he effect of BAC cells (A549 cell line and primary culture derived from a p atient's BAC tumor) on the apoptosis of peripheral blood neutrophils. Cell- free supernatants from tumor cells did not inhibit neutrophil apoptosis. In contrast, cell-free supernatants from tumor cells previously exposed to co nditioned media from peripheral blood mononuclear cells and alveolar macrop hages significantly inhibited spontaneous neutrophil apoptosis. This inhibi tion was partially lifted when conditioned media from mononuclear cells wer e previously treated with Abs against IL-1 beta and tumor necrosis factor-a lpha. As in vivo, neutralizing Abs against GM-CSF significantly inhibited t he anti-apoptotic activity of cell culture supernatants, and combination wi th Abs against G-CSF had an additive effect. In vivo, GM-CSF and G-CSF were strongly expressed by tumor cells and moderately or not expressed by the n ormal epithelium, as assessed by immunohistochemical studies. These finding s demonstrate that the tumor environment generates local conditions that pr olong alveolar neutrophil survival through the production of soluble factor s, thereby contributing to the persistence of the neutrophil alveolitis obs erved in BAC.