Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells
© Chan et al. 2005
Received: 16 June 2005
Accepted: 11 November 2005
Published: 11 November 2005
Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.
We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.
We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.
The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.
Influenza pandemics arise from genetic reassortment between avian and human influenza viruses or alternatively by the direct adaptation of a avian influenza viruses to efficient human-to-human transmission . Avian influenza A subtype H5N1 transmitted from poultry to humans in Hong Kong in 1997 (H5N1/97) causing fatal human respiratory disease [2, 3]. The subsequent re-emergence of human H5N1 disease in southern China , Vietnam , Thailand and Cambodia  has raised the specter of a new influenza pandemic. While human-to-human transmission of the H5N1 subtype influenza virus appears to be inefficient so far, the disease has exceptional severity in those affected with reported mortality rates ranging from 33% in Hong Kong in 1997 to 55% in Thailand and Vietnam in 2004. The reasons for this unusual severity of human disease have remained unclear.
While dissemination outside the respiratory tract was not demonstrated in human H5N1 disease in 1997 and 2003 [4, 7], there is some evidence that more recent H5N1 viruses may occasionally disseminate to multiple organs contributing to unusual disease manifestations such as meningo-encephalitis . However, most patients with H5N1 disease had a primary viral pneumonia complicated by the syndromes of acute respiratory distress and multiple organ dysfunction [4–7, 9] with lymphopenia and haemophagocytosis being notable findings. The syndromes of acute respiratory distress and multiple organ dysfunction as well as haemophagocytosis have previously been associated with cytokine dysregulation [10, 11].
Influenza virus infection of blood-monocyte-derived murine and human [12, 13] macrophages and porcine alveolar macrophages  have been shown to result in induction of pro-inflammatory cytokines. Furthermore, we have previously demonstrated that, when compared to human H1N1 and H3N2 influenza viruses, infection of H5N1/97-like viruses lead to the hyper-induction of proinflammatory cytokines in human primary macrophage cultures in vitro . We also reported that patients with H5N1 disease have unusually high serum concentrations of chemokines IP-10 (interferon-gamma-inducible protein-10) and MIG (monokine induced by interferon γ) . We have therefore hypothesized that this differential hyper-induction of cytokines and chemokines may contribute to the unusual severity of human H5N1 disease [4, 12].
While macrophages are a key sentinel cell of the immune system and are permissive to influenza virus replication, the primary target cell for the virus are respiratory epithelial cells . In primates experimentally infected with H5N1/97 virus, the type I and II pneumocytes and alveolar macrophages were found to contain viral antigen . Virus infection of alveolar pneumocytes was also demonstrated in the lung of a patient with fatal H5N1 disease . Human alveolar epithelial cells are vital for the maintenance of lung function and the pulmonary air-blood barrier. In addition, human respiratory epithelial cells respond to viral infections by mounting a cytokine response that contributes both to the innate and adaptive host defenses . Furthermore, type II pneumocytes express class II major histocompatibility complex (MHC) molecules in vivo . Expression of class II MHC is usually limited to specialized cells of the immune system whose role is to present foreign antigen to helper T cells [20, 21]. The expression of these molecules on alveolar epithelial cells is likely to be of relevance to the adaptive immune response. Therefore it is important to study cytokine responses induced by infection of epithelial cells with influenza viruses including H5N1 viruses.
Human influenza A viruses have been previously reported to induce interleukin 6 (IL-6), interleukin 8 (IL-8) and RANTES (regulated on activation, normal T cell expressed and secreted) in vitro from the transformed bronchial epithelial cell line (NCI-H292) . However, the physiological relevance of findings from transformed cell lines is uncertain and primary alveolar epithelial cell cultures would be a more relevant model . Here, we have compared the cytokine profiles induced by H5N1/97 and H1N1 viruses in human primary type II pneumocytes and bronchial epithelial cells in vitro to test the hypothesis that H5N1/97 and H5N1/04 viruses differentially hyper-induce pro-inflammatory cytokines in respiratory epithelial cells.
Materials and methods
An influenza virus isolated from a patient with fatal influenza A H5N1 disease in Hong Kong in 1997, A/Hong Kong/483/97 (H5N1/97), viruses from patients with H5N1 disease in Vietnam in 2004, A/Vietnam/1194/04 and A/Vietnam/3046/04 (both abbreviated as H5N1/04) and a human H1N1 virus A/Hong Kong/54/98 (H1N1) were studied. Viruses were initially isolated in Madin-Darby canine kidney (MDCK) cells. They were cloned by limiting dilution, and seed virus stocks were prepared in MDCK cells. Virus infectivity was assessed by titration of tissue culture infection dose 50% (TCID50) in MDCK cells. The H5N1 influenza viruses used in this study were handled in a BL3 biocontainment facility.
Primary human bronchial epithelial cells (NHBE) were obtained from Cambrex Bio Science (Walkersville, Inc., Maryland, USA). NHBE cells were grown according to the suppliers instructions in serum-free and hormone supplemented bronchial epithelial growth media (BEGM) which included supplements of 13 g/l bovine pituitary extract, 0.5 g/l hydrocortisone, 0.5 mg/l human recombinant epidermal growth factor, 0.5 g/l epinephrine, 10 g/l transferrin, 5 g/l insulin, 0.1 mg/l retinoic acid, 6.5 mg/l 3,3',5-triiodo-L-thryonine, 50 g/l gentamicin, and 50 mg/l amphotericin B (Cambrex Bio Science, Walkersville, Inc., Maryland, USA). Medium was changed daily starting from the day after seeding. Cells reached confluency in approximately 9 to 10 days, and nearly confluent cells were subcultured using trypsin/EDTA (Cambrex) at a ratio of 1:5. Experiments were carried out on the same batch of cells at passage 3 to 4. The cells were incubated in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions.
Primary human alveolar epithelial cells (type II pneumocytes) were isolated from human non-tumor lung tissue obtained from 13 patients (mean age 65 yr [range, 46–77 yr], 10 males and 3 females) undergoing lung resection in Grantham Hospital, Hong Kong. The research protocol was approved by the ethics committee of the University of Hong Kong and Hospital Authority Hong Kong West Cluster. Human type II pneumocytes were isolated using a modification of the methods previously described [19, 23]. Briefly, after removing visible bronchi, the lung tissue was chopped into pieces of >0.5 mm thickness using a tissue chopper, washed with balanced salt solution (BSS, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 10 mM HEPES, 5.5 mM glucose, pH 7.4) for 30 min at 37°C three times to partially remove macrophages and blood cells. The tissue was digested using a combination of trypsin (0.5%, GIBCO BRL, Gaithersburg, MD, USA) and elastase (2 units/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) twice for 15 min at 37°C in a shaking water-bath. The partially digested tissue was minced in the presence of 40% fetal bovine serum (FBS) in DMEM/F12 medium and DNase I (350 units/ml) (GIBCO BRL, Gaithersburg, MD, USA), and cell clumps dispersed by repeatedly pipetting the cell suspension for 10 minutes. After filtration through gauze and a 40 μm cell strainer to ensure a single cell suspension, the cells were incubated with a 1:1 mixture of DMEM/F12 medium and small airway growth medium (SAGM, Cambrex Bio Science Walkersville, Inc., Maryland, USA) containing 5% FBS and 350 units/ml DNase I, on tissue-culture treated plastic Petri dishes in a humidified incubator (5% CO2, 37°C) for 2 hours in order to let macrophage attach on the plastic surface. The non-adherent cells were layered on a discontinuous Percoll density gradient (densities 1.089 and 1.040 g/ml) and centrifuged at 25 × g for 20 min. The cell layer at the interface of the two gradients was collected and washed four times with BSS to remove the Percoll. To remove remaining alveolar macrophages, the cell suspension was incubated with magnetic beads coated with anti-CD-14 antibodies at room temperature for 20 min under constant mixing. After the removal of the beads using a magnet and assessment of cell viability by trypan-blue exclusion, the purified type II pneumocyte suspension was suspended in SAGM supplemented with 1% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin, and plated at a cell density of 300,000 cells/cm2. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions, and growth medium was changed daily starting from 60 hours after plating the cells.
Characterization of human type II pneumocytes
Staining for alkaline phosphatase
Human type II pneumocytes were identified by staining for alkaline phosphatase. Freshly isolated cells were spun down on glass slides, air-dried, and stained for 20 min at room temperature. The stain was prepared by dissolving 10 mg naphthol AS bi-phosphate (Sigma) in 40 μl DMSO and was diluted in 10 ml of 0.125 M 2-amino-2-methyl propanol buffer (pH 8.9, Sigma) containing 10 mg fast red (Sigma). The slide was washed and counterstained in 1% methylene green (Sigma) for 30 seconds and was mounted in aqueous medium .
Transmission electron microscopy
Cells were fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Washington, PA, USA), washed three times in phosphate buffered saline and serially dehydrated in acetone. The tissue was post-fixed in 1% osmium tetroxide and embedded in an Araldite resin (Polysciences, Inc., Washington, PS, USA). Semi-thin sections (1 μm) were cut using an ultra-microtome (Reichert Ultracut S, Leica Aktiengesellscharft, Wien, Australia) with a diamond knife and were stained with toluidine blue for light microscopic examination. Ultra-thin sections (80 nm) mounted on copper grids were electron contrasted with uranyl acetate (1.5 hours, 30°C, Electron Microscopy Sciences) and lead citrate (40 minutes, 20°C, Electron Microscopy Sciences, Washington, PA, USA), and were examined with a transmission electron microscope (EM 208S, FEI Company, Hillsboro, Oregon, USA).
The expression of cell surface antigen was measured by staining purified type II pneumocytes with optimal dilution of rabbit anti-human surfactant protein-C (SP-C) (Upstate, Lake Placid, NY, USA) monoclonal antibodies (24°C, 30 minutes) followed by a fluorescein isothiocyanate (FITC-conjugated goat anti-mouse IgG antibody; Sigma, F-0257, 24°C, 30 minutes). Each cell preparation was also stained with antibody specific for monocyte/macrophage surface antigen (CD14 conjugated with FITC, MCA2185F; Serotec. Oxford, UK). The cells were examined by the flow cytometry (FACSSCalibur; Becton Dickinson), and the FITC-stained cells were detected by measuring green light emitted at 530 nm (FL1 channel). The percentage of cells expressing the epithelial and macrophage makers were determined.
Influenza virus infection of type II pneumocytes and bronchial epithelial cells
Human type II pneumocytes and bronchial epithelial cells (seeded at 1 × 106 cells per well in 24-well tissue-culture plates) were infected at a multiplicity of infection (MOI) of two unless otherwise indicated. After 60 min of virus adsorption, the virus inoculum was removed, and the cells were washed with warm culture medium (SAGM for type II pneumocytes and BEBM for bronchial epithelial cells) and incubated in medium supplemented with 0.6 mg/L penicillin, 60 mg/L streptomycin, and 2 mg/L N-p-tosyl-L-phenylalanine chloromethyl ketone-treated-trypsin (Sigma, St Louis, MO, USA). Aliquots of culture supernatant were collected and frozen at -80°C for subsequent virus titration and cytokine analysis. The supernatants were titrated on MDCK cells and the viral titre was quantitated as log10TCID50/ml. RNA was extracted from cells for analysis of cytokine gene expression. Ten hours after infection, replicate cell monolayers were fixed and analyzed by immuno-fluorescent staining specific for influenza virus nucleoprotein (DAKO Imagen, Dako Diagnostics Ltd, Ely, UK) to determine the proportion of cells infected.
Quantification of cytokine mRNA by real-time quantitative RT-PCR
DNase-treated total RNA was isolated by means of RNeasy Mini kit (Qiagen, Hilden, Germany). The cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse transcriptase (Life Technologies, Rockville, MD, USA) and quantified by real-time PCR analysis with a LightCycler (Roche, Mannheim, Germany). The mRNA for IP-10, interferon beta, IL-6, RANTES and tumor necrosis factor (TNF) alpha were quantitated using real-time RT-PCR. The oligonucleotide primers and methods used for real-time quantification of cytokines, viral matrix gene and the housekeeping gene product γ-actin mRNA have been described previously [12, 24].
Quantification of cytokine proteins by ELISA
The concentrations of IP-10, RANTES, interleukin 6 and interferon beta proteins in the primary human bronchial and alveolar epithelial cell supernatants were measured by a specific ELISA assay (R&D Systems, Minneapolis, MN, USA). Samples of culture supernatant were irradiated with ultraviolet light (CL-100 Ultra Violet Cross linker) for 15 min to inactivate any infectious virus before the ELISA assays were done. Previous experiments had confirmed that the dose of ultraviolet light used did not affect cytokine concentration as measured by ELISA (data not shown).
The quantitative cytokine and chemokine mRNA and protein expression profile were compared using one-way ANOVA, followed by Bonferroni multiple-comparison test. Differences were considered significant at p < 0.05.
In vitro infection of human type II pneumocytes
Induction of pro-inflammatory cytokines and chemokines in type II pneumocytes
mRNA profile of cytokine and chemokine gene expression of primary culture of human type II pneumocytes 3 h and 6 h after infection with A/Hong Kong/483/97 (H5N1/97), A/Vietnam/1194/04, A/Vietnam/3046/04 (both H5N1/04) and A/Hong Kong 54/98 (H1N1) influenza viruses denoted as fold increase compared to mock infected cells.
Ratio of expression over mock-infected cells
3 hours post infection
6 hours post infection
483/97 (H5N1/97) (-■-)c
1194/04 (H5N1/04) (-◆-)c
3046/04 (H5N1/04) (-×-)c
54/98 (H1N1) (-▲-)c
483/97 (H5N1/97) (-■-)c
1194/04 (H5N1/04) (-◆-)c
3046/04 (H5N1/04) (-×-)c
54/98 (H1N1) (-▲-)c
Broadly, there were two patterns of kinetics of cytokine gene transcription. Cytokines up-regulated from 3 hr post-infection onwards included IP-10, interferon beta and IL-6 whereas RANTES mRNA was only up-regulated at 6 hr post-infection (Table 1). The observations remained valid whether the cytokine mRNA expression data were analyzed with or without normalization for γ-actin mRNA concentrations.
Infection and cytokine induction profile of primary human bronchial epithelial cells
Secretion of cytokine proteins from bronchial and alveolar epithelial cells
We found that the replication efficiency of the H5N1 and H1N1 viruses was similar in both primary human alveolar (Figure 4) and bronchial epithelial cells (Figure 6). Both influenza virus subtypes induced an IP-10, interferon beta, RANTES, and IL-6 responses. The cytokine induction was dependent on viral replication since UV-inactivated virus did not induce any effect. Interestingly, we found that H5N1/97 and 1194/04 (H5N1/04) viruses were more potent inducers of IP-10, interferon-beta, RANTES and IL-6 mRNA and protein than the human H1N1 virus (Figure 5, 7, 8 to 10). Thus, the observed differences of mRNA are reflected in levels of cytokine and chemokine proteins secreted (Figure 8 to 10). The results with 3046/04 (H5N1/04) were generally similar to 1194/04 (H5N1/04) with the exception that the levels of RANTES protein in type II pneumocytes was not significantly elevated when compared with H1N1 virus infected cells (Figure 10) although the mRNA levels were (Figure 5). Our inability to detect any interferon-beta proteins in our experiments in spite of marked induction of mRNA is probably related to the limited sensitivity of the interferon beta ELISA. A more sensitive bioassay for interferon-beta may be required for this purpose. The type II pneumocytes used in these experiments were derived from a total of 13 donors and each set of experimental data is based on the results of at least three separate experiments from three donors therefore excluding a donor specific artifact. The bronchial epithelial cells were purchased from a commercial source and comes from one donor. However, since the results from these cells are broadly in line with those from the type II pneumocytes, again, we think that donor specific artifacts are unlikely to explain the results we have obtained. Finally, these results are also comparable to our previous observations from primary human monocyte derived macrophages  with the exception that in contrast to macrophages, no TNF alpha and IL-1 beta was induced in respiratory epithelial cells by any of the viruses tested.
This differential hyper-induction of cytokines was not explained by differences in the replication kinetics between the two virus subtypes. H5N1 viruses isolated from patients with H5N1 disease in Hong Kong in 1997, Vietnam in 2004 and human influenza viruses of the H1N1 subtype all replicate with similar efficiency. Increase in the MOI of the H1N1 virus did not result in an increase of cytokine responses to levels comparable to that of the H5N1 viruses. The cellular mechanisms underlying this differential cytokine hyper-induction by H5N1 viruses are presently poorly understood. Studies on the transformed bronchial epithelial cell line A549 previously demonstrated that toll-like receptor 3 (TLR-3) is involved in the influenza virus A initiated cytokine responses . It remains to be determined whether H5N1 viruses also act via TLR-3 signaling in primary human epithelial cells.
Cytokine and chemokine responses in vivo result from autocrine and paracrine interactions involving many cell types. Chemokines such as IP-10 and MCP-1 are macrophage chemo-attractants and mediate the inflammatory response by further recruitment of circulating leukocytes into the inflamed tissue. We have previously demonstrated that IP-10 and MCP-1 are up-regulated in primary human macrophage by SARS-CoV . The strong induction of chemokines in the lung micro-environment might explain the prominent macrophage infiltrate observed in the lungs of patients with fatal H5N1  as well as SARS .
RANTES attracts monocytes, eosinophils, basophils and T cells, and selectively CD4+ T cells. Its production from the bronchial epithelial cells contributes to the infiltration of the inflammatory cells in airway viral infection . IL-6 is a multifunctional cytokine that can regulate immune and inflammatory responses involved in the activation, growth and differentiation of T-cells  and can contribute to T cell mediated inflammatory reactions. In fact, autopsy examination showed an increased CD3+ T cells in the interstitium of the lung from patients with H5N1 diseases . In addition, IL-6 has been shown to be released by macrophages and epithelial cells during lung injury  and the effects of IL-6 are synergistic with those of IL-1 and TNF-alpha . We have previously demonstrated that other proinflammatory cytokines such as IL-1, TNF-alpha and IL-6 are hyper-induced in H5N1 infected macrophages . Therefore, the differential up-regulation of IL-6 expression in human respiratory epithelial cells and the cytokines induced in macrophages by H5N1 viruses may contribute synergistically to the pathogenesis of human H5N1 disease.
The H5N1 viruses have continued to reassort, acquiring different internal genes from other influenza viruses of avian origin [33, 34]. The H5N1/04 viruses, A/Vietnam/1194/04 and A/Vietnam/3046/04 represent the Z genotype viruses that emerged as the dominant virus genotype affecting poultry in south-east Asia [27, 35]. Thus there appears to be an association between the property of hyper-inducing cytokines and high virulence. Additionally, in pig epithelial cells, H5N1/97 viruses were found to resist the antiviral effects of interferon  and this may also be relevant in pathogenesis. It is notable that patients with avian influenza (H5N1) disease appeared to have higher levels of IP-10 in their sera than those with infections with the human influenza viruses  providing in vivo data that parallels our present findings in vitro. Studies on recombinant viruses bearing the HA and NA of the 1918 "Spanish flu" pandemic virus showed that these viruses have enhanced virulence for mice and induce higher levels of macrophage-derived chemokines in vivo in mice . However, such observations of hyper-induction of cytokines in vivo may simply reflect more extensive replication of the respective virus. The studies in vitro with H5N1 viruses exclude such potential confounding factors and it would be relevant to study the cytokine profiles of the 1918 recombinant viruses in in vitro models similar to those described here.
H5N1 subtype influenza A viruses associated with human disease are more potent than human H1N1 virus at inducing proinflammatory cytokines and chemokines, including IP-10, interferon beta, IL-6 and RANTES, from human primary alveolar and bronchial epithelial cells infected in vitro. Previous findings showed that H5N1/97 viruses also hyper-induce cytokines from macrophages and that patients with H5N1 disease have high levels of IP-10 and other chemokines in the serum. These findings may be relevant to the pathogenesis of H5N1 disease. The recent re-emergence of H5N1 disease in humans is a cause for renewed pandemic concern and highlights the need for a better understanding of the pathogenesis of human H5N1 disease. Such understanding will lead to new strategies for managing human H5N1 disease and enhance our preparedness to confront pandemic influenza, whether from H5N1 or other influenza A subtypes.
This research was supported by a research grants to MCW Chan from the Research Fund for the Control of Infectious Diseases (RFCID 03040712) and the Small Project Funding, CRGC, The University of Hong Kong and research grants to JSM Peiris from the Research Fund for the Control of Infectious Diseases (RFCID 01030172), the Research Grants Councils of Hong Kong (HKU 7459/03M) and The University of Hong Kong Research Achievement Award, 2005.
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