G-CSF and GM-CSF Modify Neutrophil Functions at Concentrations found in Cystic Fibrosis

Abstract

The role of colony stimulating factors (CSFs) in cystic fibrosis (CF) circulating neutrophils has not been thoroughly evaluated, considering that the neutrophil burden of lung inflammation in these subjects is very high. The aim of this study was to assess granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) levels in CF patients in various clinical conditions and how these cytokines impact on activation and priming of neutrophils. G-CSF and GM-CSF levels were measured in sputum and serum samples of stable CF patients (n = 21) and in CF patients with acute exacerbation before and after a course of antibiotic therapy (n = 19). CSFs were tested on non CF neutrophils to investigate their effects on reactive oxygen species (ROS) production, degranulation (CD66b, elastase, lactoferrin, MMP-9), and chemotaxis. At very low concentrations found in CF patients (0.005-0.1 ng/ml), both cytokines inhibited ROS production, while higher concentrations (1-5 ng/ml) exerted a stimulatory effect. While either CSF induced elastase and MMP-9 secretion, lactoferrin levels were increased only by G-CSF. Chemotaxis was inhibited by GM-CSF, but was increased by G-CSF. However, when present together at low concentrations, CSFs increased basal and fMLP-stimulated ROS production and chemotaxis. These results suggest the CSF levels that circulating neutrophils face before extravasating into the lungs of CF patients may enhance their function contributing to the airway damage.

Figure 1

Effect of G-CSF and GM-CSF on oxidative burst. Neutrophils were treated with single (A) or both CSFs (C) for 50 min, or treated with single (B) or both (D) CSFs and further incubated with 1 μM fMLP for 5 min. ROS production is given as percent of untreated cells (A,C), or cells treated with fMLP (B,D). Concentrations of CSFs are in ng/ml. TNF-α (5 ng/ml) was used as positive control. Results are shown as mean ± SEM of six experiments. Kruskall-Wallis (with Dunn’s test as post hoc test) and Mann-Whitney U-test: *P < 0.05; **P < 0.01; ***P < 0.0001 as compared with untreated controls (A,C) or fMLP-treated samples (B,D).

Figure 2

Effect of GM-CSF and G-CSF on elastase secretion. Neutrophils were incubated with either GM-CSF (A) or G-CSF (B) at the concentration indicated for 50 min in the absence (white columns) or presence (grey columns) of 1 μM fMLP. fMLP was used as positive control. Elastase levels were measured in the supernatants. Results are shown as mean ± SEM of five experiments. ANOVA (with Tukey’s post hoc test) and Student t-test. In both panels, **P < 0.01: +fMLP controls vs. -fMLP controls; *P < 0.05 and **P < 0.01 as compared with untreated and –fMLP controls or untreated and +fMLP controls.

Figure 3

Effect of GM-CSF and G-CSF on CD66b expression and lactoferrin secretion. Neutrophils were incubated with either GM-CSF (A,C) or G-CSF (B,D) at the concentration indicated for 50 min in the absence (white columns) or presence (grey colums) of 1 μM fMLP. fMLP was used as positive control. CD66b levels (A,B) were analyzed by cytofluorimetry and are expressed as MFI. Lactoferrin levels (C,D) were measured in the supernatants. Results are shown as mean ± SEM of five experiments. ANOVA (with Tukey’s post hoc test) and Student t-test. In all four panels, **P < 0.01: + fMLP controls vs. -fMLP controls; *P < 0.05, **P < 0.01, and ***P < 0.0001 as compared with untreated and –fMLP controls or untreated and + fMLP controls.

Figure 4

Effect of GM-CSF and G-CSF on MMP-9 secretion. Neutrophils were incubated with either GM-CSF (A) or G-CSF (B) at the concentration indicated for 50 min in the absence (white columns) or presence (grey columns) of 1 μM fMLP. fMLP was used as positive control. MMP-9 levels were measured in the supernatants. Results are shown as mean ± SEM of five experiments. ANOVA (with Tukey’s post hoc test) and Student t-test. In both panels, **P < 0.01: +fMLP controls vs. −fMLP controls; *P < 0.05, **P < 0.01, and ***P < 0.0001 as compared with untreated and −fMLP controls or untreated and +fMLP controls.

Figure 5

Effect of GM-CSF and G-CSF on neutrophil chemotaxis. (A) Neutrophils were allowed to migrate for 2.5 h in the absence or presence of either GM-CSF or G-CSF at the indicated concentrations. fMLP (1 μM) was used a positive control. (B) Neutrophils were pre-treated with either CSF for 50 min and allowed to migrate along a fMLP gradient for 2.5 h. (C) Neutrophils were either allowed to migrate for 2.5 h in the absence or presence of both CSFs contemporarily presented in the lower chamber at low concentrations (0.005 or 0.1 ng/ml) and with fMLP as positive control, or first pre-treated with both CSFs contemporarily for 50 min at the same concentrations and then allowed to migrate along a fMLP gradient for 2.5 h. Data are shown as percentage of migrated neutrophils as compared with total neutrophils added in the apical compartment. Results are shown as mean ± SEM of six experiments. ANOVA (with Tukey’s post hoc test) and Student t-test. In all panels, ***P < 0.0001: +fMLP controls vs. -fMLP controls; *P < 0.05, **P < 0.01, and ***P < 0.0001 as compared with untreated and –fMLP controls or untreated and +fMLP controls.