Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis

Abstract

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.

Fig. 1

Time courses of opsonized zymosan particle phagocytosis. iC3b opsonized zymosan A particles (1 mg/ml) and neutrophils from the sources indicated were incubated on glass slides at 37 °C for 10, 30 and 60 min before washing with Krebs buffer to remove nonadhered material. The cells were then fixed and stained and the number of zymosan particles per cell counted in 100 neutrophils per slide. The time courses of zymosan uptake by neutrophils isolated from either the blood or lung fluid of patients with CF (21 patients) or the non-CF group (17 subjects) are shown. Neutrophils from non-CF (NCF) lung and circulation are compared in (a); neutrophils from CF lung and circulation are compared in (b); neutrophils from NCF and CF circulation are compared in (c) and neutrophils from NCF and CF lung are compared in (d). The mean and standard error are shown for each time point and * indicate time points at which there is a significant difference (P < 0·05).

Fig. 2

Uptake of C3bi-opsonized zymosan particles by pulmonary neutrophils. (a) A montage of images is shown which indicates how each individual neutrophil within a population was classified as containing a specific number of zymosan particles. In each example, the number of particles per cell is indicated in the bottom left hand corner. Histograms show distributional data representative from 10 separate experiments of particles per neutrophil isolated from non-CF lung (b) and CF lung (c). In each case at least 100 neutrophils were counted. The white columns shows cells that have not taken up zymosan and the final column shows the percentage of neutrophils in which it was not possible to accurately count the number of zymosan particles within the cell but was at least 9 particles/cell.

Fig. 3

The histograms show (a) the mean of median fluorescence FACS values (arbitrary units) of CD18 surface expression on CF neutrophils (n = 5, striped columns) and non-CF neutrophils (n = 5, stippled columns) (P = 0·04). (b) CD18 expression on neutrophils from CF lung (b) and non-CF lung (c) was visualize confocally in two planes xy (i.e. standard view) and xz (i.e. perpendicular to the standard view). In the perpendicular view, a white line has been to indicate where the position of the coverslip, but is slightly below its true position so as not to obscure the level of CD18 expression at the cell-coverslip contact surface. These examples were typical of at least 5 other examples and also reflected the distribution of CD11b stained separately.

Fig. 4

Ca²⁺ signalling by CR3 on CF neutrophils. Relative changes in cytosolic free Ca²⁺ concentration during particle uptake are shown for neutrophils from (a) CF lung and (b) non-CF lung and (c) for successive particle uptake by a CF pulmonary neutrophil. In (a) and (b) the upper series show the delivery of a single zymosan a particle using a micropipette, the second series iof images show the cytosolic free Ca²⁺ pseudo-coloured image for the same view as in the upper series of images, where blues are low Ca²⁺ and greens are high Ca²⁺. The Ca²⁺ data is quantified in each case in the graph below the image series and the time at which the images time indicated by the lines. The data in (c) shows that an individual CF pulmonary neutrophil was able to elicit a number of phagocytic events and that each was accompanied by a typical Ca²⁺ signal. These data are representative of 7 separate experiments.

Fig. 5

CR3 function on CF pulmonary neutrophils. The time course shows the rate of uptake by CF pulmonary neutrophils of zymosan particles either not opsonized (▪) and C3bi-opsonized zymosan particles (•) (n = 8). Opsonized zymosan were taken up at a faster rate than the unopsonized particles over the first 30 min, but had approximately the same maximum capacity. * indicates that data at that time point was significantly different (P < 0·05).