Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

doi:10.1016/S0140-6736(02)11772-7

The Lancet

Volume 360, Issue 9348, 7 December 2002, Pages 1831-1837

https://doi.org/10.1016/S0140-6736(02)11772-7 Get rights and content

Summary

Background

In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role.

Methods

We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor α (TNF α) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-α response.

Findings

The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF α and interferon beta. The concentration of TNF-α protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF α induced by the virus.

Interpretation

The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF α. This characteristic may contribute to the unusual severity of human H5N1 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.

Section snippets

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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Mechanisms of DiseaseInduction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

Summary

Background

Methods

Findings

Interpretation

Introduction

Section snippets

Viruses and cells

Results

Discussion

GLOSSARY

Lancet

Lancet

Chest

J Immunol Methods

Virology

Virology

Lancet

Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness

Science

Molecular basis for high virulence of Hong Kong H5N1 influenza in mammals

Science

Pathogenesis of avian influenza A (H5N1) viruses in ferrets

J Virol

Pathology of fatal human infection associated with avian influenza A H5N1 virus

J Med Virol

Pathogenesis of influenza A (H5N1) virus infection in a primate model

J Virol

Hemophagocytic syndromes and infection

Emerg Infect Dis

Emergence of influenza A viruses

Philos Trans R Soc Lond B Biol Sci

Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans

J Virol

Mechanisms of Disease
Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?