Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome

Abstract

Rationale: Acute respiratory distress syndrome (ARDS) affects over 200000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined.

Methods: Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls.

Main results: Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis.

Conclusions: We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then 'deprime' and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.

Keywords: ARDS; Neutrophil Biology.

Figure 1

Representative images obtained from the posterior head of γ camera 40 min after injection of autologous human neutrophils. Autologous ^99mtechnetium (Tc)-labelled neutrophils were primed with granulocyte macrophage colony-stimulating factor (GM-CSF) (100 ng/mL, 30 min, n=8), platelet-activating factor (PAF) (1 μM, 5 min, n=5; or 30 min, n=6), or control (phosphate-buffered saline (PBS), 5 min, n=8), washed twice with autologous plasma (150 g, 5 min) and resuspended in plasma, before being reinjected into the left antecubital fossa of healthy spontaneously breathing volunteers, lying supine in a dual-headed γ camera. Images were acquired at 1/s for 2 min, followed by 1/20 s for 38 min, from the time of injection. (A–C) Shows representative images from the posterior head of the γ camera for unprimed (A), deprimed (PAF for 30 min; B) and GM-CSF primed (C) autologous neutrophils 40 min post injection into the left antecubital fossa. ¹¹¹In, ¹¹¹indium tropolonate; RBC, red blood cell; ROI, region of interest.

Figure 2

Effect of neutrophil priming on pulmonary transit kinetics. Autologous ^99mtechnetium-labelled neutrophils were primed with granulocyte macrophage colony-stimulating factor (100 ng/mL, 30 min, n=8), platelet-activating factor (1 μM, 5 min, n=5; or 30 min, n=6) or control (phosphate-buffered saline, 5 min, n=8), washed twice with autologous plasma (150 g, 5 min) and resuspended in plasma, before being reinjected into the left antecubital fossa of healthy spontaneously breathing volunteers, lying supine in a dual-headed γ camera. Images were acquired at 1/s for 2 min, followed by 1/20 s for 38 min, from the time of injection. Regions of interest were drawn around the lungs and the average count per pixel recorded. The data were corrected for radioisotope decay and plotted against time. A γ variate was fitted to the control data to simulate a first-pass transit curve for unprimed neutrophils (A), from which a mean transit time of 14.18 s (14.06–14.61 s) was derived. (C) Shows the median and IQR of data obtained from all 27 independent experiments. *Represents p<0.05 compared with control. To validate our findings regarding the transit kinetics of unprimed neutrophils, autologous ^99mtechnetium-labelled erythrocytes and ¹¹¹indium-labelled neutrophils were mixed with lithium chloride and injected as a single bolus into the right internal jugular veins of patients with healthy lungs (normal spirometry and thoracic CT) under surgical anaesthesia. Starting immediately prior to injection, continuous blood sampling was undertaken from the left radial artery (collected in 3.6 s fractions) using a peristaltic pump and fraction collector. Blood ^99mtechnetium and ¹¹¹indium activity was measured, with appropriate corrections for background, crosstalk and radionuclide decay, and expressed as a fraction of the administered activities. Each first-pass curve was fitted with a γ variate function to calculate the area under the first-pass curve and the difference in lung mean transit times of erythrocytes and neutrophils. (B) Shows the median and IQR of six independent patient studies.

Figure 3

Measurement of neutrophil priming gradients across the lungs. Paired samples of whole blood were obtained from the radial artery and internal jugular veins of critically ill patients with systemic sepsis (n=6), acute respiratory distress syndrome (ARDS) (n=8), or perioperative controls (n=5). Absolute neutrophil count was measured using a Coulter DXH. Neutrophil shape change and cell surface expressions of CD11b and CD62L were measured using no lysis whole blood flow cytometry. Gradients were expressed as the ratio of the arterial value over the venous value. (A) Shows a scatter plot (with mean and IQR) for neutrophil count, (B) shape change (expressed as mean forward scatter), (C) shows CD11b cell surface expression, and (D) shows CD62L cell surface expression. Analysed using Kruskal–Wallis test with Dunn's post hoc comparisons. *Represents p<0.05.

Figure 4

Correlation between A and V gradient of neutrophil CD62L cell surface expression and oxygenation status in patients with acute respiratory distress syndrome (ARDS). Paired samples of whole blood were obtained from the radial artery and internal jugular veins of patients with ARDS (n=8 patients). Neutrophil cell surface expression of CD62L was measured using no lysis whole blood flow cytometry. Gradients were expressed as the ratio of the arterial value over the venous value. The P/F ratio (pO₂ in arterial blood/fraction of inspired oxygen) was recorded at the time each sample was taken. Figure 4 shows the Spearman correlation between A and V gradient of neutrophil CD62L cell surface expression and P/F ratio (R=0.7857, p<0.05).