Use of autologous 99mTechnetium-labelled neutrophils to quantify lung neutrophil clearance in COPD

Abstract

Rationale: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung.

Aim: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT).

Methods: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with 99mTechnetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis.

Results: There was increased accumulation of 99mTc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge.

Conclusions: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.

Trial registration: ClinicalTrials.gov NCT02551614.

Keywords: copd pathology; imaging/ct mri etc; innate immunity; neutrophil biology.

Conflict of interest statement

Competing interests: MB, AC, KP, SM, JM, ML, GB, EJ, MdG, FW and EMH are employees of GSK. NT, NF, RS, DG, CS, AW, JB, AMP and ERC have no competing interests to declare.

© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.

Figures

Figure 1

Schematic diagram of the study design. Eighteen healthy volunteers randomised 2:1 to receive LPS or saline and 20 patients with stable COPD (stage 2/3) completed the protocol. Ten patients with COPD were scanned 7–10 days later to determine reproducibility. 99mTc, 99mTechnetium; HVs, healthy volunteers; LPS, lipopolysaccharide; SPECT, single-photon emission CT.

Figure 2

SPECT/CT and Patlak-Rutland analysis. Panel A shows a two-dimensional representation (coronal, sagittal and transverse views, respectively) of a reconstructed 45 min SPECT/CT scan in (i) a saline-challenged HV, (ii) an LPS-challenged HV and (iii) a patient with COPD. Peak areas of radioactivity are seen as yellow/white, with lower levels in blue. The large airspaces, with negligible radioactivity, are black and can be seen in the emphysematous lung (iii). Panel B shows the composite Patlak-Rutland graphical plot (±SD) in saline- challenged HVs (n=6), LPS-challenged HVs (n=12) and patients with COPD (n=20) (V1). For visual purposes the right and left lungs have been displayed separately. The plot gradient represents blood clearance of 99mTc-neutrophils to the lungs in mL/min/mL lung volume. The y-axis intercept corresponds to the 99mTc-neutrophil distribution volume. 99mTc, 99mTechnetium; HV, healthy volunteer; LPS, lipopolysaccharide; SPECT, single-photon emission CT.

Figure 3

Neutrophil clearance data. Panel A shows 99mTc-neutrophil clearance (slope/intercept) in each individual patient, with mean and SD for all saline-challenged HVs (n=6), LPS-challenged volunteers (n=12) and patients with COPD (V1) (n=20). Green triangles represent patients with a diagnosis of chronic bronchitis. Non-parametric tests were used to compare groups (only significant p values are presented). Panel B displays the correlation in neutrophil clearance between the two studies in patients with COPD with repeat scans. No correlation was observed between neutrophil clearance and baseline neutrophil counts in COPD (panel C) or the absolute increase in circulating neutrophil counts post-LPS (panel D). The absolute neutrophil count prior to LPS challenge was 3.9±1.2 10×9/L. Panel E shows clearance values in relation to (1) peak circulating IL-6 levels in saline HVs (open circles) and LPS HVs (closed circles) and (2) baseline IL-6 levels in patients with COPD (green circles). HVs and patients with COPD were analysed independently with results for each group displayed. Correlations for all data sets were determined using a Pearson correlation coefficient and are displayed on each data set along with the corresponding p value. 99mTc, 99mTechnetium; HV, healthy volunteer; IL-6, interleukin 6; LPS, lipopolysaccharide.