Inhibition of airway Na+ transport by respiratory syncytial virus

Abstract

In previous studies, we have shown that two major respiratory pathogens, influenza virus and parainfluenza virus, produce acute alterations in ion transport upon contacting the apical membrane of the respiratory epithelium. In the present study, we examine the effects on ion transport by the mouse tracheal epithelium of a third major respiratory pathogen, respiratory syncytial virus (RSV). RSV infections are associated with fluid accumulation in the respiratory tract and cause illnesses that range in severity from rhinitis, sinusitis, otitis media, and bronchitis to bronchiolitis and pneumonia. We find that within minutes of RSV contacting the apical membrane; it inhibits amiloride-sensitive Na+ transport by the epithelium. This effect is mediated by protein kinase C and is reproduced by recombinant viral F (fusion) protein. Since this inhibition is not accompanied by any alteration in the epithelial responses to carbachol or to forskolin plus 3-isobutyl-1-methylxanthine (IBMX), it is not due to a nonspecific toxic action of the virus. The inhibition also appears to require Toll-like receptor 4 and the presence of asialogangliosides in the apical membrane. Since the concentration range over which this inhibition is observed (10(2) to 10(5) PFU/ml) is comparable to the viral concentrations observed in clinical and experimental RSV infections, it seems likely that direct inhibition by the virus of epithelial Na+ transport may contribute to the fluid accumulation that is observed in RSV infections.

FIG. 1.

Effect of RSV on amiloride (10 μmol/liter)-sensitive Na⁺ absorption in the mouse trachea. Original Ussing chamber recordings of V_te before and during apical exposure for 1 h to 2 × 10⁴ PFU/ml RSV (A) or control buffer (B). Dashed lines in panels A and B indicate the effects of amiloride. Panel C summarizes the measurements of the amiloride-sensitive Na⁺ current (I_scEq-Amil) obtained under control conditions and after incubation with either control buffer solution or RSV. The data are presented as means ± SEM (with the number of experiments in parentheses). The asterisk indicates a statistically significant difference compared to the control.

FIG. 2.

Concentration-response curve for the inhibitory effect of application for 1 h to the apical membrane of RSV on the amiloride-sensitive short-circuit current (I_scEq-Amil) in the mouse trachea. In this figure, the I_scEq-Amil measured 1 h after exposure to RSV has been normalized to the I_scEq-Amil measured prior to exposure to give the relative I_scEq-Amil. The data are presented as means ± SEM (with the number of experiments in parentheses).

FIG. 3.

Effect of RSV on carbachol (CCH) (100 μmol/liter)-induced ion transport in the mouse trachea. Original Ussing chamber recordings of V_te and the CCH responses prior to and following apical exposure for 1 h to RSV (1 × 10⁴ PFU/ml) (A) or to control buffer (C). Panel B summarizes the effects of 1 h of exposure to RSV on the CCH-induced change in the short-circuit current (I_scEq-CCH). Dashed lines in panels A and C indicate the effects of CCH. Panel D summarizes the effects of 1 h of exposure to control buffer on the CCH-induced change in I_scEq-CCH. The data are presented as means ± SEM (with the number of experiments in parentheses).

FIG. 4.

Effect of RSV on IBMX (100 μmol/liter) and forskolin (2 μmol/liter)-induced ion transport in the mouse trachea. Original Ussing chamber recordings of V_te and the responses to IBMX plus forskolin (I/F) prior to and following apical exposure for 1 h to RSV (2 × 10⁴ PFU/ml) (A) or control buffer (C). Panel B summarizes the effects of 1 h of exposure to RSV on the IBMX-plus-forskolin-induced change in the short-circuit current (I_scEq-I/F). Dashed lines in panels A and C indicate the effects of IBMX and forskolin. Panel D summarizes the effects of 1 h of exposure to control buffer on the IBMX-plus-forskolin-induced change in I_scEq-I/F. The data are presented as means ± SEM (with the number of experiments in parentheses).

FIG. 5.

(A) Summary of the effects on the amiloride-sensitive short-circuit current (I_scEq-Amil) across the mouse tracheal epithelium of apical application for 1 h of the RSV F protein (2.5 μg/ml), the RSV F protein (2.5 μg/ml) in the presence of the protein kinase C inhibitor BIM-I (100 nmol/liter), and intact RSV (10⁵ PFU/ml) in the presence of BIM-I (100 nmol/liter). (B) Summary of the effects on I_scEq-Amil across cultured mouse collecting duct (M1) cell monolayers of application for 1 h of RSV (2 × 10⁵ PFU/ml) and RSV (2 × 10⁵ PFU/ml) in the presence of BIM1 (100 nmol/liter). Also shown are the effects on the inhibition of I_scEq by RSV (2 × 10⁵ PFU/ml) of preincubation of the monolayers in PPMP (40 μmol/liter) for 24 h or preincubation in neuraminidase (0.5 U/ml) for 30 min. (C) Summary of the effects on I_scEq-Amil across the mouse tracheal epithelium of 1 h of apical exposure to RSV (2 × 10⁵ PFU/ml) or the F protein (2.5 μg/ml) following pretreatment of the apical surface of the epithelium with neuraminidase (Neura) (0.5 U/ml) for 30 min or when apyrase (Apyr) (2 U/ml) or suramin (Sur) (100 μmol/liter) have been included in the apical bathing solution. The data are presented as means ± SEM (with the number of experiments in parentheses). The asterisks indicate statistically significant differences compared to the corresponding controls. con, control.

FIG. 6.

Summaries of the effects of RSV (A) or control (con) (B) incubation on short-circuit current responses (ΔI_scEq) to amiloride (Amil), carbachol (CCH), and forskolin plus IBMX (I/F). Dashed lines in panel C indicate the effects of amiloride. The effects of lipopolysaccharide (LPS) (10 μM) on transepithelial voltage and amiloride-sensitive transport (C) and amiloride-sensitive I_scEq (D) are shown. The data are presented as means ± SEM (with the number of experiments in parentheses). The asterisks indicate statistically significant differences compared to the corresponding controls.