Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs

Abstract

Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

FIG. 1.

IHC detection of PI-3 viral antigen in the lungs of lambs inoculated with PI-3 virus or sterile medium 3 (A and B), 6 (C and D), or 17 (E and F) days p.i. (A) PI-3 viral antigen is present within the cytoplasm of bronchiolar epithelial cells. (C) PI-3 viral antigen is present within the cytoplasm of macrophages and bronchiolar epithelial cells. (E) Bronchioles in lungs from lambs 17 days p.i. lack PI-3 viral antigen. (B, D, and F) Control animals lack PI-3 viral antigen. Bar = 100 μm.

FIG. 2.

SBD-1 mRNA levels detected in whole-lung homogenates of PI-3 virus-inoculated and control lambs by fluorogenic real-time RT-PCR. There was a trend toward increased SBD-1 mRNA levels in the PI-3 virus-infected group on days 3 and 6 and a significant increase (*) on day 17 compared to the control animals (P = 0.06). A t test assuming unequal variances was used. The error bars indicate SEM.

FIG. 3.

SP-A mRNA levels detected in whole-lung homogenates of PI-3 virus-inoculated and control lambs by fluorogenic real-time RT-PCR. SP-A mRNA levels were significantly increased (*) in PI-3 virus-inoculated lambs 6 and 17 days p.i. compared to the control animals (P = 0.09 for day 6 p.i. and P = 0.05 for day 17 p.i.). A t test assuming unequal variances was used. The error bars indicate SEM.

FIG. 4.

SP-A protein levels assessed by quantitative-competitive ELISA in whole-lung homogenates of PI-3 virus-inoculated and control lambs. SP-A protein levels were not significantly changed by PI-3 virus inoculation compared to the control animals. A t test assuming unequal variances was used. The error bars indicate SEM.

FIG. 5.

SP-D mRNA levels detected in whole-lung homogenates of PI-3 virus-inoculated and control lambs by fluorogenic real-time RT-PCR. SP-D mRNA levels were significantly increased (*) in PI-3 virus-inoculated lambs 3, 6, and 17 days p.i. compared to the control animals (P = 0.09 for day 3 p.i., P = 0.09 for day 6 p.i., and P = 0.06 for day 17 p.i.). A t test assuming unequal variances was used. The error bars indicate SEM.