Regulation of systemic and local neutrophil responses by G-CSF during pulmonary Pseudomonas aeruginosa infection

Abstract

Granulocyte colony-stimulating factor (G-CSF) regulates the production, maturation, and function of neutrophils. Its expression is often induced during infection, resulting in high concentrations of G-CSF in inflammatory exudates and in the blood, suggesting that it may regulate both local and systemic neutrophil responses. Herein, we characterize the neutrophil response in G-CSFR(-/-) mice following intratracheal injection with Pseudomonas aeruginosa-laden agarose beads, modeling the pulmonary infection observed in many patients with cystic fibrosis. G-CSFR(-/-) mice are markedly susceptible to bronchopulmonary P aeruginosa infection, exhibiting decreased survival and bacterial clearance as well as extensive damage to lung tissue. The systemic neutrophil response was mediated primarily by enhanced neutrophil release from the bone marrow rather than increased neutrophil production and was attenuated in G-CSFR(-/-) mice. Despite normal to increased local production of inflammatory chemokines, neutrophil accumulation into the infected lung of G-CSFR(-/-) mice was markedly reduced. Moreover, the percentage of apoptotic neutrophils in the lung was elevated, suggesting that G-CSF signals may play an important role in regulating neutrophil survival at the inflammatory site. Collectively, these data provide new evidence that G-CSF signals play important but specific roles in the regulation of the systemic and local neutrophil response following infection.

Figure 1

G-CSFR^−/− mice display increased susceptibility to bronchopulmonary infection with P aeruginosa. (A) Wild-type (n = 22) and G-CSFR^−/− mice (n = 23) were challenged with a single intratracheal injection of P aeruginosa–laden beads. Kaplan-Meier estimates demonstrate significantly decreased survival in G-CSFR^−/− mice compared with wild-type mice (log rank, P = .006). Bacterial load of P aeruginosa in the lung was estimated by measuring the number of bacterial CFUs in the BALF (B) at the indicated times. The number of animals at each time point ranges from n = 4 at 24 hours to n = 21 at 48 hours. Bacterial load was also measured in whole lung homogenates (C) at 48 hours (n = 4). (D) Representative Giemsa-stained sections of lung tissue 72 hours after infection with Pseudomonas-laden beads are shown. Original magnification, × 40 (left panel) or × 200 (right panel). (E) Lungs harvested at 72 hours were sectioned and assigned a histologic score as described in “Materials and methods” (n = 4). Data represent the mean ± SEM; *P ≤ .05.

Figure 2

The systemic neutrophil response following P aeruginosa infection is impaired in G-CSFR^−/− mice. (A) The serum concentration of G-CSF in wild-type mice at the indicated times following P aeruginosa infection was measured by ELISA. (B) The absolute neutrophil count in the blood was measured in wild-type and G-CSFR^−/− mice (n = 8-26, each time point). (C) The number of mature neutrophils per femur was measured in wild-type and G-CSFR^−/− mice (n = 4-17, each time point). (D) The percentage of the total body pool of mature neutrophils present in the blood was estimated as described in “Materials and methods” (n = 4-17, each time point). All data represent the mean ± SEM. ‡P < .05 compared with untreated mice of the same genotype. *P < .05 compared with G-CSFR^−/− mice.

Figure 3

Local production of key inflammatory chemokines and cytokines in the lung is normal to increased in G-CSFR^−/− mice following P aeruginosa infection. The concentration of (A) G-CSF, (B) TNF-α, (C) IL-6, (D) MIP-2, and (E) KC in BALF was measured by ELISA (n = 4-7 for each time point). *P ≤ .05 compared with wild-type mice. (F) Blood monocytes isolated from wild-type mice were stimulated with LPS (10 μg/mL) for 24 hours in the presence (■) or absence (□) of 100 ng/mL G-CSF (n = 6). TNF-α in the supernatant of these cultures was measured by ELISA. Graph shown is representative of 3 independent experiments. *P ≤ .05. All data represent the mean ± SEM.

Figure 4

Neutrophil accumulation into the BALF of G-CSFR^−/− mice is reduced following P aeruginosa infection. (A) Kinetics of neutrophil accumulation into the BALF of wild-type and G-CSFR^−/− mice following P aeruginosa infection (n = 3-13, each time point). (B) To correct for the effect of circulating neutrophil number on neutrophil recruitment to the lungs, the number of neutrophils in the BALF of each mouse was divided by the total number of neutrophils present in the blood. All data represent the mean ± SEM. ‡P < .05 compared with untreated mice of the same genotype. *P < .05 compared with G-CSFR^−/− mice.

Figure 5

Accumulation of neutrophils in pulmonary vessels. Forty-eight hours after P aeruginosa inoculation, wild-type and G-CSFR^−/− animals were injected with PE-Gr-1 antibody. Five minutes later, neutrophils in blood (A) and lung homogenate (B) were harvested as described in “Materials and methods.” Shown are representative fluorescence-activated cell sorted (FACS) plots of wild-type (left) and G-CSFR^−/− mice (right) stained ex vivo with FITC-7/4 (antineutrophil) antibody. (C) Quantitation of the percentage of 7/4⁺Gr-1⁺ cells in lung homogenate of wild-type (□) and G-CSF^−/− (■) animals (n = 5). Data represent the mean ± SEM.

Figure 6

A higher percentage of apoptotic neutrophils is present in the BALF of G-CSFR^−/− mice following P aeruginosa infection. (A) Neutrophils isolated from the bone marrow of wild-type mice were cultured for 48 hours under conditions that induced apoptosis (“Materials and methods”). Cells were harvested at the indicated times and stained for Gr-1, annexin V, and 7AAD. Whereas the majority of neutrophils is viable (annexin V⁻, 7AAD⁻) at time 0 (left panel), an increasing percentage of early apoptotic (annexin V⁺, 7AAD⁻) and then late apoptotic (annexin V⁺, 7AAD⁺) cells was seen with prolonged culture (middle panel, right panel). Histograms are gated for Gr-1⁺ (granulocytic) cells. (B) Representative dot plots of neutrophils recovered from the BALF of wild-type (left panel) and G-CSFR^−/− (right panel) mice. Data are gated for Gr-1⁺ (granulocytic) cells.