Respiratory syncytial virus and neutrophil activation

Abstract

Respiratory syncytial virus infects almost all children by 2 years of age. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and they are activated in the presence of infection. However it is not clear whether RSV can directly signal to activate neutrophil cytotoxic function. To investigate this we have used a preparation of RSV washed using a new centrifugal diafiltration method to rapidly remove inflammatory molecules produced by the epithelial cells used to propagate the RSV stock. Human neutrophils were isolated from peripheral blood and activated with either the unwashed crude RSV preparations or the purified intact RSV. Neutrophils were also challenged with purified RSV G-glycoprotein. The effect of challenging human neutrophils with these preparations of intact RSV, or the RSV G-glycoprotein, was assessed by measuring the cell surface expression of CD11b and CD18b, the phagocytic oxidative burst, and intracellular release of calcium pools. Neutrophils challenged with the washed RSV exhibited significantly lower activation of surface marker expression (P < 0.001) and oxidative burst (P < 0.001) than those challenged with unwashed virus or with virus free supernatant. There was no increase in intracellular calcium release on exposure to the washed RSV. Purified G glycoprotein did not stimulate neutrophils, whilst the use of a blocking antibody to the F protein did not prevent unwashed RSV from activating cytotoxic responses. These results suggest that neutrophils have no innate signalling system that recognizes RSV but they are activated at sites of RSV infection as a result of the cytokines and inflammatory molecules released by virally infected cells.

Fig. 1

10% SDS PAGE mini gel stained in silver and showing removal of proteins following centrifugal dia filtration of the RSV sample. The samples were separated by SDS-PAGE under denaturing dissociating conditions at the dilution equivalent to the original sample and show the following. Lane A: shows the sample of unwashed RSV; Lane B: shows the sample of filtrate from the RSV preparation and the line marks the presence of the casein in the filtrate; Lane C: shows the washed RSV sample; Lane D: shows a lane with no sample added but demonstrates the non specific silver staining with the ion front.

Fig. 2

A bar graph showing the reduction of IL8 in the RSV for samples (n = 3 + SEM) before washing (UR), in the filtrate sample (F), and in the washed sample (WR). The concentrations were determined by regression using a standard curve of IL8 (from 62·5 pg/ml; where r = 0·998).

Fig. 3

Graph showing the mean (± SE) values of fluorescence as measured by flow cytometry for the expression of CD11b (PE) and CD18 (FITC) on the surface of purified neutrophils. The cells were either left nonstimulated (C), or were stimulated with 1 × 10⁴ pfu/ml washed (WR), or 1 × 10⁴ pfu/ml of unwashed (UR) RSV for 30 min at 37°C. In addition, the change in CD expression was compared to the positive control 1 µm FMLP (fMLP), and the viral specific effects were compared to virus free filtrate (F).

Fig. 4

Graph showing the mean (± SE) values of fluorescence for measurement of free radical production using DC-FDA as measured by flow cytometry over a three minute period. The cells were either left nonstimulated (C) or stimulated with the virus sample filtrate (F); either at 1 × 10⁴ pfu/ml of unwashed (UR) RSV, or 1 × 10⁴ pfu/ml of washed (WR) RSV. The X axis shows the increase in units of FITC fluorescence.

Fig. 5

Graph showing the peak mean values (± SE) values of Indo-1 fluorescence in purified neutrophils with different stimulants. The samples were compared to the control (C) of no stimulants, positive control of 1 µm fMLP (fMLP) and 100 n m Ionomycin (I). Both washed (WR) and unwashed (UR) RSV preparations were used at 1 × 10⁴ pfu/ml.