Fig 2.

Contrasting TNF-α responses in human and pig cells. (A and B) TNF-α mRNA expression (normalized to 18S rRNA expression levels) in human and pig respiratory epithelial cells and macrophages following infection by HPAI H5N1 (A/turkey/Turkey/1/05) and USSR H1N1 (A/USSR/77) viruses at an MOI of 1.0. TNF-α RNA expression levels in human epithelial cells (A) and human macrophages (B) were at least 100-fold higher than those in the corresponding pig cells for both viruses and at both time points. Note that for human or pig, the virus-induced TNF-α mRNA expression level in macrophages was many times (1 to 2 orders) higher than that in epithelial cells. (C and D) Detection of the TNF-α protein (ELISA) in culture supernatants was performed on infected human and pig epithelial cells (C) and macrophages (D). (C) TNF-α protein induction in human epithelial cells by HPAI H5N1 virus was higher than that by USSR H1N1. TNF-α was undetectable in pig epithelial cells infected by the same virus subtypes. (D) Macrophages infected for 48 h with HPAI H5N1 and USSR H1N1 viruses showed strong TNF-α induction in human cells, with the highest induction by HPAI H5N1. In contrast, TNF-α was undetectable or barely detectable in pig macrophages infected by all 3 virus subtypes. TNF-α protein production levels from infected human macrophages were about 3 orders of magnitude higher than those from infected human respiratory epithelial cells. All data shown are the means of data from duplicate wells and are representative of data from 3 experimental replicates. Error bars indicate standard errors of the means. (E) Lack of apparent TNF-α and IFNβ1 responses in lungs of 4-week-old pigs challenged with HPAI H5N1 virus. One pig was used per time point. The absence of TNF-α and IFNβ1 after challenge induction coincided with only transient signs of pyrexia in the first 48 h and intermittent nasal virus shedding within the first 4 days of virus challenge (data not shown), and no virus M gene RNA was detected in the lung at all time points. The relative mRNA expression level was determined by real-time PCR and normalized to the 18S rRNA level.