Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis

Abstract

The pathogenesis of severe acute respiratory syndrome (SARS) remains unclear. Macrophages are key sentinel cells in the respiratory system, and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus (SARS-CoV) and other respiratory viruses. Primary human monocyte-derived macrophages were infected with SARS-CoV in vitro. Virus replication was monitored by measuring the levels of positive- and negative-strand RNA, by immunofluorescence detection of the SARS-CoV nucleoprotein, and by titration of the infectious virus. The gene expression profiles of macrophages infected with SARS-CoV, human coronavirus 229E, and influenza A (H1N1) virus were compared by using microarrays and real-time quantitative reverse transcriptase PCR. Secreted cytokines were measured with an enzyme-linked immunosorbent assay. SARS-CoV initiated viral gene transcription and protein synthesis in macrophages, but replication was abortive and no infectious virus was produced. In contrast to the case with human coronavirus 229E and influenza A virus, there was little or no induction of beta interferon (IFN-beta) in SARS-CoV-infected macrophages. Furthermore, SARS-CoV induced the expression of chemokines such as CXCL10/IFN-gamma-inducible protein 10 and CCL2/monocyte chemotactic protein 1. The poor induction of IFN-beta, a key component of innate immunity, and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS.

Figures

FIG. 1.

Nonproductive replication of SARS-CoV in human macrophages. Differentiated primary human monocyte-derived macrophages (A) and FRhK-4 cells (B) were seeded in 24-well plates (2 × 105 cells per well) on glass coverslips. Cells were infected at an MOI of 1 to 2, and RNAs were extracted at 3, 6, and 15 h postinfection. The levels of positive (solid lines) and negative (dotted lines) RNA strands of the SARS-CoV Orf1b and nucleoprotein genes were determined by real-time quantitative RT-PCR. The data show means of duplicate cultures from the same donor and are representative of three independent experiments with similar results.

FIG. 2.

Human macrophages were mock treated (A) or infected with SARS-CoV (B and C) and fixed with methanol for 15 min at 15 h postinfection. A mouse monoclonal antibody (4D11) specific for the SARS-CoV nucleoprotein (K. H. Chan, unpublished results) was tested on SARS-CoV-infected and uninfected macrophages (A and C). A mouse monoclonal antibody against influenza A virus hemagglutinin was used as a control (B). All three cell smears were stained with a secondary fluorescein isothiocyanate-conjugated anti-mouse antibody (Zymed Laboratories, San Francisco, Calif.), and Evans blue was used as a counterstain.

FIG. 3.

No induction of IFN-β gene expression in SARS-CoV-infected macrophages. Levels of IFN-β mRNA were determined by real-time quantitative RT-PCR. (A) Macrophages were infected with SARS-CoV (•), HCoV-229E (♦), and influenza A (H1N1) virus (▪) at an MOI of 1 to 2, and RNAs were extracted at 3, 6, and 15 h postinfection. SARS-CoV-infected macrophages did not induce IFN-β at any of the three time points, in contrast with infections with influenza A (H1N1) virus and HCoV-299E.

FIG. 4.

Levels of CXCL10/IP-10 and CCL2/MCP-1 were elevated in SARS-CoV-infected macrophages. Macrophages were infected with SARS-CoV at an MOI of 1 to 2. (A) RNAs were extracted at 3, 6, and 15 h postinfection, and the levels of mRNA for CXCL10/IP-10 and CCL2/MCP-1 were determined by real-time quantitative RT-PCR. (B) Aliquots of the culture supernatant were taken at 6, 15, and 24 h postinfection, and the levels of secreted CXCL10/IP-10 and CCL2/MCP-1 were determined by specific ELISAs. The data shown are means (± standard deviations) of duplicate cultures from the same donor and are representative of three independent experiments with similar results. SARS-CoV infection (•) of macrophages induced higher levels of gene expression and secretion of CXCL10/IP-10 and CCL2/MCP-1 than did mock infection (▪).