Neutrophil GM-CSF receptor dynamics in acute lung injury

Abstract

GM-CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM-CSF receptor (GM-CSFRα) complex, a process essential for signaling. Whereas GM-CSF controls many aspects of neutrophil biology, regulation of GM-CSFRα expression is poorly understood, particularly the role of GM-CSFRα in ligand clearance and whether signaling is sustained despite major down-regulation of GM-CSFRα surface expression. We established a quantitative assay of GM-CSFRα surface expression and used this, together with selective anti-GM-CSFR antibodies, to define GM-CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand-induced GM-CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro-survival effects of GM-CSF required ongoing ligand-receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM-CSFRα expression, which along with mGM-CSFRβ declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM-CSF concentration. Treating mice in an LPS challenge model with CAM-3003, an anti-mGM-CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM-CSF. Consistent with neutrophil consumption of GM-CSF, human neutrophils depleted exogenous GM-CSF, independent of protease activity. These data show that loss of membrane GM-CSFRα following GM-CSF exposure does not preclude sustained GM-CSF/GM-CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM-CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM-CSF levels or GM-CSFRα expression.

Keywords: LPS; alveolar; apoptosis; inflammation; signaling.

Figure 1

GM‐CSFR blockade and quantification in TF‐1 cell line. (A) TF‐1 cells, washed to remove residual human recombinant GM‐CSF from the routine culture conditions, were treated with 0.25 ng/ml GM‐CSF, in the presence of a serial dilution of CAM3001 or isotype control and cultured for 72 hr. CellTiter‐Glo was used to measure ATP as an indirect measure of number of viable cells. Data represent mean ± sem of n = 4 independent experiments. (B) TF‐1 cells that had been maintained in 4 ng/ml human GM‐CSF were washed three times to ensure complete removal of GM‐CSF. The cells were then returned to culture in the presence or absence of 4 ng/mL human GM‐CSF for 18 hr. The cells were stained with CAM3001 followed by PE‐conjugated secondary antibody to assess surface levels of GM‐CSFRα. In the cells that had been cultured in the absence of GM‐CSF the GM‐CSFRα was detectable above background. In the cells that had been maintained in GM‐CSF the GM‐CSFRα was considerably lower. Image shown is a representative experiment of four independent experiments

Figure 2

GM‐CSFRα quantification and kinetics in human neutrophils. (A) Neutrophils were incubated with DAPI and AlexaFluor647‐CAM‐3001 (GM‐CSFRα antibody, left panel) or AlexaFluor647‐NIP228 (Isotype control, right panel) and the presence of GMCSF receptor expression determined by immunofluorescence. Images represent one of three independent experiments. (B) Neutrophils were incubated with AlexaFluor647‐CAM‐3001 and GM‐CSFRα receptor density was measured by flow cytometry using Quantum Simply Cellular beads. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n = 28 donors assessed in 28 independent experiments. (C and D) Human neutrophils were treated with the indicated cytokine (GM‐CSF [1 ng/ml], TNFα [20 ng/ml] and LPS [100 ng/ml]) before GM‐CSFRα number/cell was assessed by flow cytometry (as for B) at the indicated time and compared to baseline. In some experiments (D) samples treated with GM‐CSF (1 ng/ml) were washed (x2) and resuspended in GM‐CSF free media for the remainder of the experiment. Data represent mean ± sem of n = 3 (C) or n = 4 (D) independent experiments. Statistical analysis was performed by 2‐way ANOVA with Bonferroni's post‐test (Significant at ***P < 0.001, **P < 0.01, *P < 0.05)

Figure 3

Sustained GM‐CSF signaling required for GM‐CSF‐mediated neutrophil survival. (A–D) Freshly isolated neutrophils were treated with the indicated concentration of GM‐CSF and the percentage of apoptotic cells analyzed after 20 hr culture by flow cytometry. (B) Neutrophils were pretreated with increasing concentrations of CAM‐3001 for 20 min prior to GM‐CSF treatment. (C) Cells were maintained in media containing the indicated concentration of GM‐CSF or the culture media were replaced after 1.5 or 6 hr of treatment with GM‐CSF‐free media for the remainder of the incubation time. Media change alone did not impact apoptosis of control samples without GM‐CSF incubation (data not shown). (D) Human neutrophils were treated with GM‐CSF (1 ng/ml) and this was followed by addition of CAM‐3001 (1 μM) at the indicated time after GM‐CSF treatment began. The bars indicate the response to GM‐CSF and the effect of pretreatment with CAM‐3001 (20 min) prior to treatment with GM‐CSF. Data represents mean ± sem of n = 8 (A), n = 3 (B), n = 5 (C) or n = 3 (D) independent experiments

Figure 4

GM‐CSFRα blockade inhibits inflammation in response to inhaled LPS in a mouse model of acute lung injury. Mice were treated with inhaled PBS or LPS (nebulized 1 mg/mL for 10 min) and 24 hr later BALF total cell counts (A), BALF neutrophils (B) and lung homogenate IL‐1β (C) were assessed 24 hr after LPS challenge. Additionally, mice were treated intranasally with PBS, isotype control, or CAM‐3003 (at doses indicated), or budesonide (3 mg/kg, p.o.) 3 hr prior to LPS exposure as indicated. n = 8 mice per group, except control groups without LPS (n = 4 mice per group). Statistical analysis was performed by 2‐way ANOVA with Bonferroni's post‐test (Significant at ***P < 0.001, **P < 0.01, *P < 0.05 compared to either PBS group, isotype control or LPS group as indicated by bars)

Figure 5

GM‐CSFRα blockade causes a sustained increase in LPS‐induced alveolar GM‐CSF concentration. Mice were treated with PBS or LPS (10 μg, intranasally [i.n.]) for the indicated time before the percentage of neutrophils (as a percentage of CD45+ cells) in the blood, bone‐marrow and BALF was determined by (A) flow cytometry and (B) the concentration of GM‐CSF in the BALF measured by ELISA. (C) CAM‐3003 (anti‐mouse GM‐CSFRα mAb) or isotype control (400 μg, i.n.) was administered either 3 hr prior or 6 hr post‐LPS administration (10 μg, i.n.) and the concentration of GM‐CSF after 24 hr was determined by ELISA. Data show mean ± sem for each mouse group (A, n = 6; B, n = 6; C n = 5). Statistical analysis was performed by 2‐way ANOVA with Bonferroni's post‐test (Significant at ***P < 0.001, **P < 0.01, *P < 0.05, compared to PBS group (A and B) or LPS group (C)). Data in A are representative of 2 independent experiments

Figure 6

Dynamic changes in BALF neutrophil GM‐CSFRα and common β chain expression during LPS‐induced acute lung injury. Mice were treated with PBS or LPS (10 μg, intranasally [i.n.]) for the indicated time. The expression of GM‐CSFRα (A) or GM‐CSFRβ (B) was then determined, using flow cytometry, on neutrophils isolated from BALF, whole blood and bone marrow. Data are expressed as geometric mean fluorescent intensity. Data show single points as well as mean ± sem for each mouse group (n = 4–6). Statistical analysis was performed by 2‐way ANOVA with Bonferroni's post‐test (Significant at ***P < 0.001, **P < 0.01, *P < 0.05)

Figure 7

Human neutrophils deplete exogenously added GM‐CSF from media independent of ligand degradation. Human neutrophils were treated with 30 pg/mL GM‐CSF for the indicated time before the concentration of GM‐CSF was determined from cell supernatant by ELISA. Where indicated, neutrophils were pretreated with protease inhibitors (10 μM Sivelestat and 2 mM EDTA) prior to treatment with GM‐CSF. Data are expressed as mean ± sem (Data shown are for n = 3 donors in a single experiment; another donor showed the same effect in a further independent experiment)