*3.3. BLT2 Mediates the Production of G-CSF in Neutrophilic Airway Inflammation*

Since previous studies suggested a mediating role of BLT2 in neutrophilic airway inflammation [29–31], we investigated whether BLT2 had any role in the production of G-CSF. Lung inflammation and mucus secretion were markedly suppressed by treatment with the BLT2 antagonist, LY255283 (Figure 3A,B). Under our experimental conditions, the protein level of BLT2 in lung tissue was suppressed by LY255283 (Figure 3C). Clearly, G-CSF, as well as MPO levels increased by LPS/OVA treatment, were alleviated by LY255283 treatment (Figure 3D,E). We also found that the number of total cells and neutrophils in BALF was significantly suppressed by LY255283 (Figure 3F). IF staining also showed that the levels of G-CSF and MPO, as well as that of BLT2, in lung tissue were attenuated by LY255283 treatment (Figure 3F,G). Together, these data suggest that BLT2 is essential for the production of G-CSF, thus contributing to neutrophilic airway inflammation.

**Figure 3.** BLT2 mediates the production of G-CSF in neutrophilic airway inflammation. The BLT2 antagonist LY255283 (10 mg/kg) or DMSO was administered by i.p. injection 1 h before each challenge. The negative controls (NC) were untreated. (**A**,**B**) H&E and PAS staining of mice lung tissues.

Perivascular and peribronchial inflammation in addition to mucus secretion, was examined and scored (400×). Inflammation scores are shown as the mean ± SD (*n* = 5 per group). (**C**) Lung tissue was homogenized, and the protein was isolated to assess the level of BLT2 by Western blotting. (**D**,**E**) G-CSF and MPO levels in BALF were analyzed by ELISA. (**F**) The number of immune cells in BALF was estimated using a CytoSpin and staining with Diff-Quik. The results are shown as the mean ± SD (*n* = 5 per group). (**G**,**H**) IF staining of mice lung tissue for BLT2 (green, Alexa Flour 488), G-CSF (red, Alexa Flour 647), and MPO (magenta, Alexa Flour 594). The nuclei were counterstained with DAPI (blue; 400×). The IF images are representatives of three independent trials with similar results. All experiments were performed in triplicate. \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 versus each control group.
