Mechanisms of DiseaseInduction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?
Introduction
The H5N1 “avian flu” in Hong Kong in 1997 was the first documented instance of human respiratory disease and death associated with a purely avian influenza virus1, 2 and was thought to be an incipient pandemic situation. The human disease was unusually severe, with an overall case-fatality rate of 33%. Except for one patient who was substantially immunocompromised, underlying disorders did not explain the severity of the course of the disease.3
The basis for the unusual clinical presentation and severity of illness in patients with H5N1 disease remains unknown. Although the major determinants of pathogenicity of influenza viruses in avian species are well defined, those in mammals are unclear. Experimental H5N1/97 infection in mice, ferrets, and macaques has given conflicting results. In experimentally infected BALB/c mice, some human H5N1 viruses (eg, 483/97) led to disseminated disease but others (eg, 486/97) did not; these differences were attributed to mutations in the polymerase basic protein 2 (PB2) and haemagglutinin (ha) genes.4 However, differences in virus dissemination between these viruses were not demonstrable in experimentally infected ferrets.5 No evidence of productive virus replication outside the respiratory tract was seen in limited autopsy studies of patients dying from H5N1 disease6 or after experimental infection of macaques in which pathological features similar to those of human H5N1 disease were reproduced.7
Patients with H5N1 disease had a primary viral pneumonia complicated by syndromes of acute respiratory distress and multiple organ dysfunction. Lymphopenia and haemophagocytosis were notable findings.3, 6 Haemophagocytosis and the syndromes of acute respiratory distress and multiple organ dysfunction are associated with cytokine dysregulation.8, 9 We suggest that the clinical features of severe human H5N1 disease are compatible with virus-induced cytokine dysregulation. To test our hypothesis, we used human primary monocyte-derived macrophages as an in-vitro model. The cytokine gene and protein expressions induced by H5N1/97 influenza viruses were compared with those of contemporary human H1N1 or H3N2 viruses and those of avian origin.
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Viruses and cells
Viruses, their abbreviations, origins, and passage history are shown in table 1. An influenza A (H5N1/97) isolate from a patient with fatal H5N1 disease (483/97) and one from a patient with mild disease (486/97) were compared with contemporary human H1N1 (54/98) and H3N2 (1174/99) viruses.
Avian viruses genetically related or unrelated to H5N1/97 were included to help identify the genes causing the biological effects associated with H5N1/97 viruses. These viruses are believed to be natural
Results
The human and avian viruses replicated to similar titres (about 104 log10 tissue-culture infectious dose [TCID]50 per mL) after infection of macrophages at a multiplicity of infection of two, apart from the human H1N1 virus, which replicated to slightly higher titre (105 log10 TCID50 per mL). 8 h after infection, similar proportions (90–100%) of macrophages had evidence of viral antigen (nucleoprotein) expression as assessed by immuno-fluorescence.
Several cytokine genes were upregulated when
Discussion
Previous studies have shown that human influenza viruses infect macrophages inducing the release of TNF-α, interleukins 6 and 1β, interferons alpha and beta, and chemokines such as RANTES, MIP-1α and MIP-1β, MCP-1, MCP-3, and interleukin 10.18, 19 The cytokine mRNA profile and the kinetics of TNF-α production from macrophages in response to infection with human H1N1 virus in our experiments (Figure 1, Figure 2, Figure 3) were similar to those documented previously. However, we found that
GLOSSARY
- haemagglutinin (ha) and neuraminidase (na)
- The two surface glycoproteins of the influenza A virus. Influenza A viruses are separated into subtypes on the basis of these glycoproteins. There are 15 HA (H1–15) and nine NA (N1–9) subtypes recognised at present. A virus is designated on the basis of HA and NA, for example H3N2.
- housekeeping genes
- Genes that are constitutively expressed in virtually all cells, since they are fundamental to all cellular functions. The expression of such genes can serve
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