*3.2. G-CSF Is Critical for Neutrophilic Airway Inflammation*

Recent studies showed that G-CSF had a critical role in the recruitment of neutrophils into airways in severe asthma [10]. To investigate the contributory role of G-CSF in our neutrophilic asthma model, a neutralizing monoclonal antibody against G-CSF was i.p. injected 1 h before each challenge. Histopathological analysis by H&E and PAS staining showed that the lung inflammation and mucus secretion induced by LPS/OVA were reduced by anti-G-CSF treatment (Figure 2A,B). We also observed that the increased levels of G-CSF and MPO activity were significantly suppressed by anti-G-CSF treatment (Figure 2C,D). Similarly, increases in the number of total cells and neutrophils infiltrating the airways induced by LPS/OVA administration were reduced to basal levels after anti-G-CSF treatment (Figure 2E). IF staining showed that G-CSF neutralization decreased G-CSF and MPO levels in lung tissue (Figure 2F,G). Together, these results suggest a critical contributory role of G-CSF in neutrophilic airway inflammation.

**Figure 1.** Elevated levels of G-CSF in the steroid-resistant neutrophilic lung airway inflammation model. For the experiment, mice were administered dexamethasone (Dexa; 1 mg/kg) or DMSO by i.p. injection 1 h before each challenge (*n* = 4 per group). The negative controls (NC) were not treated. (**A**) Scheme of the LPS/OVA-induced steroid-resistant neutrophilic airway inflammation model. (**B**,**C**) 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* = 4 per group). (**D**,**E**) MPO and G-CSF 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* = 4 per group). (**G**,**H**) Immunofluorescence (IF) staining of mice lung tissues for MPO (magenta, Alexa Flour 594) and G-CSF (red, Alexa Flour 647). 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* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, ns: not significant versus each control group.

**Figure 2.** G-CSF is critical for neutrophilic airway inflammation. For the analysis of the effect of anti-G-CSF, mice were administered anti-G-CSF (5 mg/kg) or control IgG1 (5 mg/kg) by i.p. injection 1 h before each challenge (*n* = 3–5 per group). 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* = 3–5 per group). (**C**,**D**) G-CSF and MPO levels in BALF were analyzed by ELISA. (**E**) 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* = 3–5 per group). (**F**,**G**) IF staining of mouse lung tissues for 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* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, ns: not significant versus each control group.
