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Inflammatory responses in influenza A virus infection
Vaccine 19 (2001) S32 – S37 www.elsevier.com/locate/vaccine
Inflammatory responses in influenza A virus infection Ilkka Julkunen *, Krister Mele´n, Maria Nyqvist, Jaana Pirhonen, Timo Sareneva, Sampsa Matikainen Department of Virology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Abstract Influenza A virus causes respiratory tract infections, which are occasionally complicated by secondary bacterial infections. Influenza A virus replicates in epithelial cells and leukocytes resulting in the production of chemokines and cytokines, which favor the extravasation of blood mononuclear cells and the development of antiviral and Th1-type immune response. Influenza A virus-infected respiratory epithelial cells produce limited amounts of chemokines (RANTES, MCP-1, IL-8) and IFN-a/b, whereas monocytes/macrophages readily produce chemokines such as RANTES, MIP-1a, MCP-1, MCP-3, IP-10 and cytokines TNF-a, IL-1b, IL-6, IL-18 and IFN-a/b. The role of influenza A virus-induced inflammatory response in relation to otitis media is being discussed. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Influenza A; Chemokines; Cytokines; Transcription factors
1. Influenza as a disease Influenza viruses are classified in three types (influenza A, B and C) of which influenza A is clinically the most important one. Influenza A virus is highly contagious and it infects upper respiratory tract of humans in all age groups. It causes infections ranging from sporadic cases to large epidemics or pandemics. The infection is characterized by fever and chills often accompanied by cough, sore throat, headache and myalgia [reviewed in [1]]. The patients may also suffer from general symptoms such as loss of appetite, malaise or vomiting. Influenza infection is usually self-limiting and does not require any special treatment apart from antipyretic medication and rest. However, in some individuals other symptoms and complications may develop. Children often suffer from otitis media, conjunctivitis, pharyngitis, sputum production and more severe upper respiratory tract symptoms such as croup during the acute phase of the infection. Also arthralgia, chest pain and lympadenopathy and may occur. The most severe complication is primary viral pneumonia, which develops rapidly within 1 – 2 days * Corresponding author. Tel.: +358-9-47448372; fax: + 358-947448355. E-mail address: [email protected] (I. Julkunen).
and may result in respiratory failure and death. Patients suffering from cardiovascular diseases, chronic bronchitis, obstructive pulmonary disease or asthma have a greater risk of developing influenza-associated secondary bacterial infections [1]. Bacterial pneumonia, which usually starts several days after onset of influenza, is associated with Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae or group A b-hemolytic streptococci. S. pneumoniae, H. influenzae and Moraxella catarrhalis are the pathogens often found in otitis media. Large influenza epidemics are associated with an increase in the overall mortality rate, especially among the elderly [2]. The means to prevent and control influenza include vaccines and antiviral substances [1]. The traditional vaccines, containing inactivated influenza A (H1N1 and H3N2) and influenza B viruses, have shown a protection rate ranging from 50 to 90%. Clinical trials with live, attenuated, cold-adapted influenza virus vaccines have shown a slightly higher protection rate. Prophylactically given antivirals, amantadine and rimantadine have been effective against influenza A virus. Novel sialic acid analogs, zanamavir and oseltamivir have prophylactic and therapeutic effects against all influenza types. Although some development in controlling influenza has taken place during the last few years, the disease is by no means under control. Therefore, re-
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I. Julkunen et al. / Vaccine 19 (2001) S32 – S37
search work on the pathogenesis of influenza infection and analysis of host immune response against the virus are still needed.
2. Influenza A virus and its replication Influenza viruses are enveloped, negative-stranded RNA viruses, which belong to the family of Orthomyxo6iridae. Influenza A virus RNA is composed of eight segmented genes, which encode for ten different proteins; envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), matrix protein (M1), nucleoprotein (NP), three polymerases (PB1, PB2 and PA), ion channel protein M2, and nonstructural proteins NS1 and NS2 [3]. Influenza A viruses are classified according to their hemagglutinin (H1 – H15) and neuraminidase (N1–N9) genes. Viruses with HA types H1, H2 and H3 and NA types N1 and N2 are pathogenic in humans. Influenza viruses preferentially replicate in the epithelial cell layers of the upper respiratory tract (Fig. 1), but also macrophages and other leukocytes may be infected [4]. Most cell types carrying the influenza A
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virus receptor, sialic acid containing cell surface glycoprotein, are susceptible to the virus. Cell-bound virus is endocytosed, and low pH in endosomes triggers the fusion of virus and endosome membranes, which leads to liberation and nuclear import of viral nucleocapsids followed by mRNA synthesis by viral RNA polymerases [3]. Viral mRNAs are then transported into the cytoplasm, translated, and newly synthesized NP and polymerases are transported into the nucleus, where they take part in viral RNA replication and subsequent secondary mRNA synthesis. This leads to a second round of viral RNA and protein synthesis, packing and export of viral nucleocapsid from the nucleus followed by budding of the mature virions from the plasma membrane. Productive influenza A virus infection in epithelial cells leads to the destruction of host cell pre-mRNAs, translational inhibition of cellular mRNAs and death of the host cells either by cytolytic or apoptotic mechanisms [3]. However, during the infection cells may respond in many ways to restrict the spread of the virus. Several different transcription factor systems are activated with subsequent production of chemokines and cytokines. Chemokines recruit inflammatory cells to the site of infection and cytokines activate host antiviral defence systems. In addition, antigen presentation by antigen presenting cells and apoptotic pathways are also activated.
3. Cellular responses to influenza A virus infection
3.1. Chemokine production
Fig. 1. Kinetics of influenza A nucleoprotein (NP) and host antiviral MxA gene expression. A549 lung carcinoma cells were infected with influenza virus strain A/Beijing/353/89 (H3N2), cells were harvested at times indicated and total cellular RNA was isolated. In Northern blot analysis both influenza A NP and MxA mRNA expression was found to be enhanced. Immunofluorescence analysis of typical influenza A NP (anti-NP; at 20 h after infection) and MxA protein (anti-MxA; IFN-a-treated cells 1000 IU/ml 24 h) expression pattern in A549 cells.
Chemokines are small secretory molecules that are produced by a variety of cells constitutively or in response to microbial infection. Chemokines bind to their specific cell surface receptors in leukocytes, which is followed by a rapid change in cell shape and behavior enabling them to migrate from the blood stream through the vascular endothelium into the site of inflammation [5]. Chemokine receptors are expressed differently on distinct leukocyte subpopulations and chemokines produced vary depending on the cell type and activating stimulus. Epithelial cells produce RANTES, MCP-1 and IL-8 in response to influenza A virus infection (Fig. 2) [[6,7], Nyqvist et al., unpublished]. Influenza A virus-infected monocytes/macrophages, instead, secrete MIP-1a, MIP-1b, RANTES, MCP-1, MCP-3, MIP-3a and IP-10, whereas the production of IL-8 appears to be limited [[8,9], Matikainen et al. unpublished]. The presence of MIP-1a/b, MCP-1 and IL-8 has been detected in nasopharyngeal secretions of influenza A virus-infected individuals [10,11]. The chemokines produced in influenza A virus infection preferentially favor the recruitment of blood mononuclear cell population to the site of infection [10,11].
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macrophages efficiently produce large quantities of these cytokines [4,15]. Macrophages infected with influenza A virus also produce IL-18, but fail to produce IL-12 [15]. Dendritic cells, however, have been found to produce relatively high levels of both IFN-a/b and IL-12 in response to influenza A virus infection [17]. It appears that tissue macrophages and dendritic cells are the key cell types responsible for the production of antiviral and immunostimulatory cytokines during influenza A virus infection.
3.3. Acti6ation of transcription factors Virus infection activates several transcription factors that are involved in the induction of chemokine and cytokine gene expression. These include nuclear factor kappa B (NF-kB), interferon regulatory factors (IRFs), activating protein (AP)-1, signal transducers and activators of transcription (STATs) and nuclear factor-interleukin 6 (NF-IL-6 or C/EBPb). At least NF-kB, IRF and STAT signaling pathways are activated during influenza A virus infection [[4,18], Matikainen et al., unpublished]. Influenza A virus-induced activation of NF-kB is biphasic. NF-kB activation is first detected at 1 h after infection while the later phase of NF-kB activation correlates with virus replication and viral
Fig. 2. Chemokine production in influenza A virus-infected A549 cells and macrophages. A549 lung carcinoma or primary human macrophages were infected with influenza A/Beijing/353/89 virus, samples were collected at times indicated and RANTES, MCP-1, IL-8 and IP-10 chemokine levels were determined by specific EIA assays. Note the differences in scale.
Respiratory syncytial virus (RSV) infection in epithelial cells has been shown to result in the production of RANTES, MIP-1a, MCP-1 and IL-8 [12,13], which may lead to slightly different inflammatory cell recruitment compared to influenza A virus infection.
3.2. Cytokine production The putative interplay of the cytokine network during influenza A virus infection is shown in Fig. 3. Type I interferons (IFN-a/b) are the key cytokines produced by influenza A virus-infected epithelial cells and monocytes/macrophages [3,14,15]. Experiments using knockout mice that have disrupted IFN-a/b receptor or STAT1 genes have demonstrated the importance of IFN system in antiviral defence against influenza A [16]. It has been shown that influenza A virus-infected lung epithelial cell lines are poor producers of IFN-a/b and proinflammatory cytokines (IL-1, IL-6, TNF-a) [[4], Nyqvist et al., unpublished], whereas monocytes/
Fig. 3. Cytokine interplay during influenza A virus infection. Influenza A virus is capable of infecting both epithelial cells and tissue macrophages. Epithelial cells produce RANTES, MCP-1, IL-8 and IFN-a/b, whereas macrophages produce a wider range of chemokines and cytokines as shown in the figure. Chemokines are involved in recruitment of leukocytes from the circulation to the site of infection. Cytokines IL-1b and TNF-a enhance the expression of cellular adhesion molecules, which contribute to enhanced leukocyte binding to endothelial cells. IFN-a/b, in addition to its antiviral effects, stimulates NK and T cell IFN-g production in synergy with IL-18. IFN-g production is also enhanced by direct cellular interactions between NK or T cells and virus-infected cells. Based on references [[4 – 9,12,14,15,22], Matikainen et al., unpublished].
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protein synthesis [4]. The molecular mechanisms of virus-induced NF-kB activation are unresolved, but Toll-like receptors or protein kinase R (PKR) may be involved. IRF and STAT family members, which are important in IFN and IFN-induced gene expression, are also activated by influenza A virus infection [[4], Matikainen et al., unpublished]. Stimulation of AP-1 and C/EBP has also been demonstrated to take place during influenza A virus infection [19]. Recent evidence suggests that there is considerable variation how different viruses activate these transcriptional systems [Matikainen et al., unpublished]. This results in differential chemokine and cytokine production by different viruses. Influenza A virus infection results in host cell death by cytolytic or apoptotic mechanisms. In most cell types influenza A virus replication is fast and efficient and it involves blocking of host cell mRNA transport into the cytoplasm and destruction of cellular pre-mRNAs by viral endonucleases [3]. In epithelial cells this together with maintenance of viral protein synthesis, packing and production of new viral particles leads to cytolytic death at later times (20 – 40 h) of infection. Apoptosis of leukocytes and epithelial cells has been described to occur during influenza A virus infection [20,21]. Activation of caspase-1 enzyme, which has been associated to apoptosis, was a prerequisite for efficient influenza A virus-induced IL-1b and IL-18 production in human macrophages [22].
4. Cytokine interplay in innate and adaptive immunity IFN-a/b functions as a direct antiviral substance. It upregulates the expression of PKR, oligoadenylate synthetases and Mx, which are known to mediate resistance to viral infections [23]. MxA protein has been shown to directly interfere with influenza A virus replication. MxA gene is induced during influenza A virus infection (Fig. 1) [4,14]. In humans, host’s antiviral mechanisms restrict the replication of influenza A virus at early times of infection, which gives the host more time to activate virus-specific humoral and cell-mediated immune responses, that are needed for the clearance of the virus infection. In addition to having direct antiviral properties, IFN-a/b modifies the host immune response in different ways. First, IFN-a/b upregulates MCP-1, MCP-3 and IP-10 gene expression, which results in further recruitment of monocytes/macrophages and Th1-type cells to the site of infection. Second, IFN-a/b enhances the antigen presentation of macrophages and dendritic cells by upregulating HLA gene expression [23]. Third, IFN-a/b is an important cofactor in the development of Th1-type immune response. IFN-a/b is involved in T cell survival, upregulation of IL-12 receptor expression and enhancement of
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IFN-g gene expression in human NK and T cells, especially in synergy with IL-18 [15,24,25]. The data emphasize an essential role of IFN-a/b in both innate and adaptive immunity against influenza A and other virus infections [26]. Proinflammatory cytokines IL-1b, IL-6 and TNF-a, which are readily produced by influenza A virus-infected leukocytes, do not directly contribute to the antiviral activity of cells [23]. IL-1b and TNF-a are involved in the enhancement of MCP-1 and MCP-3 gene expression and functional maturation of tissue macrophages and dendritic cells. This leads to enhanced inflammatory response and activation of efficient antigen presentation system. IL-18, which is produced by influenza A virus-infected macrophages, has also proinflammatory properties, since it enhances the production of IL-1b, TNF-a and certain chemokines. One important function of IL-18 is to activate NK and T cell IFN-g production, which occurs in synergy with either IL-12 or IFN-a/b. In many viral infections the production of IL-12 seems to be limited and therefore IFN-a/b may be the cytokine that activates the Th1 response in human cells [15,26]. IFN-g, which is produced by cytokine stimulation (IL-12, IL-18, IFN-a/b) or via cellular interactions (T cell receptor stimulation), enhances the overall development of cell-mediated immunity, macrophage activation, antigen presentation, and chemokine gene expression. The intimate interplay between IL-1b, TNF-a, IFN-a/b, IL-18, IFN-g, and chemokines forms a complex positive feed-back system leading to an efficient development of influenza-specific cell-mediated immune response (Fig. 3).
5. Influenza A virus, cytokines and bacterial infections Influenza A virus infection in the epithelial surfaces is sometimes associated with enhanced bacterial colonization and infection [reviewed in [27,28]]. Influenza A virus-associated secondary bacterial pneumonia and otitis media [1] are examples of infections, where both viral and bacterial pathogens may be found. The primary target for influenza A virus is the epithelial cell layer of the whole nasopharyngeal area. Virus-infected cells undergo cytolytic or apoptotic death, which impairs mechanical removal of mucus and bacteria by the cilia of epithelial cells. Simultaneous infection of neutrophils with both influenza A virus and bacteria was shown to potentiate apoptotic cell death [29]. Epithelial cell death may also allow enhanced bacterial adherence to damaged epithelial surfaces by exposing basement membrane structures rich in extracellular matrix proteins collagens, laminin and fibronectin. Influenza A virus-infected epithelial cells have enhanced capacity to adhere bacterial pathogens [27]. Some bacteria, such as S. aureus produce proteolytic enzymes, which may be
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Moilanen is acknowledged. The original work was supported by the Medical Research Council of the Academy of Finland and the Sigrid Juselius Foundation.
References
Fig. 4. A model of influenza A virus-induced inflammatory responses and the development of otitis media. Influenza A virus infects epithelial cell of the nasopharynx (1) and induces chemokine and cytokine production (2). Cytokines upregulate the expression of cellular adhesion molecules and enhance mucosal swelling (3). Chemokines enhance leukocyte extravasation (4), which further enhances inflammatory responses in the site of infection. Virus infection may also induce cell death (5) allowing exposure of epithelial basement membranes. Bacteria may adhere to inflamed epithelium and cause otitis media (6).
involved in the cleavage of influenza A virus hemagglutinin rendering the virus more pathogenic [28]. The production of chemokines and cytokines by virus-infected epithelial cells and leukocytes enhance the inflammatory response in the site of infection. In influenza A virus infection the chemokines produced [8,9] favors the extravasation of blood monocytes, NK, T and B cells to inflamed tissues. These cells in turn are capable of producing proinflammatory cytokines IL-1b, TNF-a, IFN-a/b, IL-18 and IFN-g [15,22], which favor the development of Th1-type immunity that is a prerequisite for specific antiviral immunity and eradication of viral pathogens. A strong Th1-type response may, however, result in tissue damage. Certain cytokines like IL-1b and TNF-a enhance the expression of cellular adhesion molecules [30] and increase cellular interactions, which further favors the extravasation of inflammatory cells. Cellular adhesion molecules may enhance bacterial colonization to the epithelial surfaces of inflamed tissues (Fig. 4). Better knowledge of the inflammatory responses during influenza A virus infection may give us tools to control the appearance of complications and reduce the tissue damage associated with influenza A virus infection.
Acknowledgements We are grateful to Dr Tapani Hovi for critical comments of the manuscript. The expert technical assistance of Valma Ma¨kinen, Marika Ylisela¨, and Katja
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[23] Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem 1998;67:227 – 64. [24] Rogge L, Barberis-Maino L, Biffi M, Passini N, Presky DH, Gubler U, Sinigaglia F. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J Exp Med 1997;185(5):825– 31. [25] Matikainen S, Sareneva T, Ronni T, Lehtonen A, Koskinen PJ, Julkunen I. Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ra, c-myc, and pim-1 genes in human T cells. Blood 1999;93(6):1980–91. [26] Biron C. Initial and innate responses to viral infections-pattern setting in immunity or disease. Curr Opin Microbiol 1999;2(4):374 – 81. [27] Hament JM, Kimpen JL, Fleer A, Wolfs TF. Respiratory viral infection predisposing for bacterial disease: a concise review. FEMS Immunol Med Microbiol 1999;3 – 4:189 – 95. [28] Steinhauer DA. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 1999;258(1):1 – 20. [29] Colamussi ML, White MR, Crouch E, Hartshorn KL. Influenza A virus accelerates neutrophil apoptosis and markedly potentiates apoptotic effects of bacteria. Blood 1999;93(7):2395–403. [30] Meager A. Cytokine regulation of cellular adhesion molecule expression in inflammation. Cytokine Growth Factor Rev 1999;10(1):27 – 39.
Inflammatory responses in influenza A virus infection Ilkka Julkunen *, Krister Mele´n, Maria Nyqvist, Jaana Pirhonen, Timo Sareneva, Sampsa Matikainen Department of Virology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Abstract Influenza A virus causes respiratory tract infections, which are occasionally complicated by secondary bacterial infections. Influenza A virus replicates in epithelial cells and leukocytes resulting in the production of chemokines and cytokines, which favor the extravasation of blood mononuclear cells and the development of antiviral and Th1-type immune response. Influenza A virus-infected respiratory epithelial cells produce limited amounts of chemokines (RANTES, MCP-1, IL-8) and IFN-a/b, whereas monocytes/macrophages readily produce chemokines such as RANTES, MIP-1a, MCP-1, MCP-3, IP-10 and cytokines TNF-a, IL-1b, IL-6, IL-18 and IFN-a/b. The role of influenza A virus-induced inflammatory response in relation to otitis media is being discussed. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Influenza A; Chemokines; Cytokines; Transcription factors
1. Influenza as a disease Influenza viruses are classified in three types (influenza A, B and C) of which influenza A is clinically the most important one. Influenza A virus is highly contagious and it infects upper respiratory tract of humans in all age groups. It causes infections ranging from sporadic cases to large epidemics or pandemics. The infection is characterized by fever and chills often accompanied by cough, sore throat, headache and myalgia [reviewed in [1]]. The patients may also suffer from general symptoms such as loss of appetite, malaise or vomiting. Influenza infection is usually self-limiting and does not require any special treatment apart from antipyretic medication and rest. However, in some individuals other symptoms and complications may develop. Children often suffer from otitis media, conjunctivitis, pharyngitis, sputum production and more severe upper respiratory tract symptoms such as croup during the acute phase of the infection. Also arthralgia, chest pain and lympadenopathy and may occur. The most severe complication is primary viral pneumonia, which develops rapidly within 1 – 2 days * Corresponding author. Tel.: +358-9-47448372; fax: + 358-947448355. E-mail address: [email protected] (I. Julkunen).
and may result in respiratory failure and death. Patients suffering from cardiovascular diseases, chronic bronchitis, obstructive pulmonary disease or asthma have a greater risk of developing influenza-associated secondary bacterial infections [1]. Bacterial pneumonia, which usually starts several days after onset of influenza, is associated with Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae or group A b-hemolytic streptococci. S. pneumoniae, H. influenzae and Moraxella catarrhalis are the pathogens often found in otitis media. Large influenza epidemics are associated with an increase in the overall mortality rate, especially among the elderly [2]. The means to prevent and control influenza include vaccines and antiviral substances [1]. The traditional vaccines, containing inactivated influenza A (H1N1 and H3N2) and influenza B viruses, have shown a protection rate ranging from 50 to 90%. Clinical trials with live, attenuated, cold-adapted influenza virus vaccines have shown a slightly higher protection rate. Prophylactically given antivirals, amantadine and rimantadine have been effective against influenza A virus. Novel sialic acid analogs, zanamavir and oseltamivir have prophylactic and therapeutic effects against all influenza types. Although some development in controlling influenza has taken place during the last few years, the disease is by no means under control. Therefore, re-
0264-410X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 2 7 5 - 9
I. Julkunen et al. / Vaccine 19 (2001) S32 – S37
search work on the pathogenesis of influenza infection and analysis of host immune response against the virus are still needed.
2. Influenza A virus and its replication Influenza viruses are enveloped, negative-stranded RNA viruses, which belong to the family of Orthomyxo6iridae. Influenza A virus RNA is composed of eight segmented genes, which encode for ten different proteins; envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), matrix protein (M1), nucleoprotein (NP), three polymerases (PB1, PB2 and PA), ion channel protein M2, and nonstructural proteins NS1 and NS2 [3]. Influenza A viruses are classified according to their hemagglutinin (H1 – H15) and neuraminidase (N1–N9) genes. Viruses with HA types H1, H2 and H3 and NA types N1 and N2 are pathogenic in humans. Influenza viruses preferentially replicate in the epithelial cell layers of the upper respiratory tract (Fig. 1), but also macrophages and other leukocytes may be infected [4]. Most cell types carrying the influenza A
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virus receptor, sialic acid containing cell surface glycoprotein, are susceptible to the virus. Cell-bound virus is endocytosed, and low pH in endosomes triggers the fusion of virus and endosome membranes, which leads to liberation and nuclear import of viral nucleocapsids followed by mRNA synthesis by viral RNA polymerases [3]. Viral mRNAs are then transported into the cytoplasm, translated, and newly synthesized NP and polymerases are transported into the nucleus, where they take part in viral RNA replication and subsequent secondary mRNA synthesis. This leads to a second round of viral RNA and protein synthesis, packing and export of viral nucleocapsid from the nucleus followed by budding of the mature virions from the plasma membrane. Productive influenza A virus infection in epithelial cells leads to the destruction of host cell pre-mRNAs, translational inhibition of cellular mRNAs and death of the host cells either by cytolytic or apoptotic mechanisms [3]. However, during the infection cells may respond in many ways to restrict the spread of the virus. Several different transcription factor systems are activated with subsequent production of chemokines and cytokines. Chemokines recruit inflammatory cells to the site of infection and cytokines activate host antiviral defence systems. In addition, antigen presentation by antigen presenting cells and apoptotic pathways are also activated.
3. Cellular responses to influenza A virus infection
3.1. Chemokine production
Fig. 1. Kinetics of influenza A nucleoprotein (NP) and host antiviral MxA gene expression. A549 lung carcinoma cells were infected with influenza virus strain A/Beijing/353/89 (H3N2), cells were harvested at times indicated and total cellular RNA was isolated. In Northern blot analysis both influenza A NP and MxA mRNA expression was found to be enhanced. Immunofluorescence analysis of typical influenza A NP (anti-NP; at 20 h after infection) and MxA protein (anti-MxA; IFN-a-treated cells 1000 IU/ml 24 h) expression pattern in A549 cells.
Chemokines are small secretory molecules that are produced by a variety of cells constitutively or in response to microbial infection. Chemokines bind to their specific cell surface receptors in leukocytes, which is followed by a rapid change in cell shape and behavior enabling them to migrate from the blood stream through the vascular endothelium into the site of inflammation [5]. Chemokine receptors are expressed differently on distinct leukocyte subpopulations and chemokines produced vary depending on the cell type and activating stimulus. Epithelial cells produce RANTES, MCP-1 and IL-8 in response to influenza A virus infection (Fig. 2) [[6,7], Nyqvist et al., unpublished]. Influenza A virus-infected monocytes/macrophages, instead, secrete MIP-1a, MIP-1b, RANTES, MCP-1, MCP-3, MIP-3a and IP-10, whereas the production of IL-8 appears to be limited [[8,9], Matikainen et al. unpublished]. The presence of MIP-1a/b, MCP-1 and IL-8 has been detected in nasopharyngeal secretions of influenza A virus-infected individuals [10,11]. The chemokines produced in influenza A virus infection preferentially favor the recruitment of blood mononuclear cell population to the site of infection [10,11].
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macrophages efficiently produce large quantities of these cytokines [4,15]. Macrophages infected with influenza A virus also produce IL-18, but fail to produce IL-12 [15]. Dendritic cells, however, have been found to produce relatively high levels of both IFN-a/b and IL-12 in response to influenza A virus infection [17]. It appears that tissue macrophages and dendritic cells are the key cell types responsible for the production of antiviral and immunostimulatory cytokines during influenza A virus infection.
3.3. Acti6ation of transcription factors Virus infection activates several transcription factors that are involved in the induction of chemokine and cytokine gene expression. These include nuclear factor kappa B (NF-kB), interferon regulatory factors (IRFs), activating protein (AP)-1, signal transducers and activators of transcription (STATs) and nuclear factor-interleukin 6 (NF-IL-6 or C/EBPb). At least NF-kB, IRF and STAT signaling pathways are activated during influenza A virus infection [[4,18], Matikainen et al., unpublished]. Influenza A virus-induced activation of NF-kB is biphasic. NF-kB activation is first detected at 1 h after infection while the later phase of NF-kB activation correlates with virus replication and viral
Fig. 2. Chemokine production in influenza A virus-infected A549 cells and macrophages. A549 lung carcinoma or primary human macrophages were infected with influenza A/Beijing/353/89 virus, samples were collected at times indicated and RANTES, MCP-1, IL-8 and IP-10 chemokine levels were determined by specific EIA assays. Note the differences in scale.
Respiratory syncytial virus (RSV) infection in epithelial cells has been shown to result in the production of RANTES, MIP-1a, MCP-1 and IL-8 [12,13], which may lead to slightly different inflammatory cell recruitment compared to influenza A virus infection.
3.2. Cytokine production The putative interplay of the cytokine network during influenza A virus infection is shown in Fig. 3. Type I interferons (IFN-a/b) are the key cytokines produced by influenza A virus-infected epithelial cells and monocytes/macrophages [3,14,15]. Experiments using knockout mice that have disrupted IFN-a/b receptor or STAT1 genes have demonstrated the importance of IFN system in antiviral defence against influenza A [16]. It has been shown that influenza A virus-infected lung epithelial cell lines are poor producers of IFN-a/b and proinflammatory cytokines (IL-1, IL-6, TNF-a) [[4], Nyqvist et al., unpublished], whereas monocytes/
Fig. 3. Cytokine interplay during influenza A virus infection. Influenza A virus is capable of infecting both epithelial cells and tissue macrophages. Epithelial cells produce RANTES, MCP-1, IL-8 and IFN-a/b, whereas macrophages produce a wider range of chemokines and cytokines as shown in the figure. Chemokines are involved in recruitment of leukocytes from the circulation to the site of infection. Cytokines IL-1b and TNF-a enhance the expression of cellular adhesion molecules, which contribute to enhanced leukocyte binding to endothelial cells. IFN-a/b, in addition to its antiviral effects, stimulates NK and T cell IFN-g production in synergy with IL-18. IFN-g production is also enhanced by direct cellular interactions between NK or T cells and virus-infected cells. Based on references [[4 – 9,12,14,15,22], Matikainen et al., unpublished].
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protein synthesis [4]. The molecular mechanisms of virus-induced NF-kB activation are unresolved, but Toll-like receptors or protein kinase R (PKR) may be involved. IRF and STAT family members, which are important in IFN and IFN-induced gene expression, are also activated by influenza A virus infection [[4], Matikainen et al., unpublished]. Stimulation of AP-1 and C/EBP has also been demonstrated to take place during influenza A virus infection [19]. Recent evidence suggests that there is considerable variation how different viruses activate these transcriptional systems [Matikainen et al., unpublished]. This results in differential chemokine and cytokine production by different viruses. Influenza A virus infection results in host cell death by cytolytic or apoptotic mechanisms. In most cell types influenza A virus replication is fast and efficient and it involves blocking of host cell mRNA transport into the cytoplasm and destruction of cellular pre-mRNAs by viral endonucleases [3]. In epithelial cells this together with maintenance of viral protein synthesis, packing and production of new viral particles leads to cytolytic death at later times (20 – 40 h) of infection. Apoptosis of leukocytes and epithelial cells has been described to occur during influenza A virus infection [20,21]. Activation of caspase-1 enzyme, which has been associated to apoptosis, was a prerequisite for efficient influenza A virus-induced IL-1b and IL-18 production in human macrophages [22].
4. Cytokine interplay in innate and adaptive immunity IFN-a/b functions as a direct antiviral substance. It upregulates the expression of PKR, oligoadenylate synthetases and Mx, which are known to mediate resistance to viral infections [23]. MxA protein has been shown to directly interfere with influenza A virus replication. MxA gene is induced during influenza A virus infection (Fig. 1) [4,14]. In humans, host’s antiviral mechanisms restrict the replication of influenza A virus at early times of infection, which gives the host more time to activate virus-specific humoral and cell-mediated immune responses, that are needed for the clearance of the virus infection. In addition to having direct antiviral properties, IFN-a/b modifies the host immune response in different ways. First, IFN-a/b upregulates MCP-1, MCP-3 and IP-10 gene expression, which results in further recruitment of monocytes/macrophages and Th1-type cells to the site of infection. Second, IFN-a/b enhances the antigen presentation of macrophages and dendritic cells by upregulating HLA gene expression [23]. Third, IFN-a/b is an important cofactor in the development of Th1-type immune response. IFN-a/b is involved in T cell survival, upregulation of IL-12 receptor expression and enhancement of
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IFN-g gene expression in human NK and T cells, especially in synergy with IL-18 [15,24,25]. The data emphasize an essential role of IFN-a/b in both innate and adaptive immunity against influenza A and other virus infections [26]. Proinflammatory cytokines IL-1b, IL-6 and TNF-a, which are readily produced by influenza A virus-infected leukocytes, do not directly contribute to the antiviral activity of cells [23]. IL-1b and TNF-a are involved in the enhancement of MCP-1 and MCP-3 gene expression and functional maturation of tissue macrophages and dendritic cells. This leads to enhanced inflammatory response and activation of efficient antigen presentation system. IL-18, which is produced by influenza A virus-infected macrophages, has also proinflammatory properties, since it enhances the production of IL-1b, TNF-a and certain chemokines. One important function of IL-18 is to activate NK and T cell IFN-g production, which occurs in synergy with either IL-12 or IFN-a/b. In many viral infections the production of IL-12 seems to be limited and therefore IFN-a/b may be the cytokine that activates the Th1 response in human cells [15,26]. IFN-g, which is produced by cytokine stimulation (IL-12, IL-18, IFN-a/b) or via cellular interactions (T cell receptor stimulation), enhances the overall development of cell-mediated immunity, macrophage activation, antigen presentation, and chemokine gene expression. The intimate interplay between IL-1b, TNF-a, IFN-a/b, IL-18, IFN-g, and chemokines forms a complex positive feed-back system leading to an efficient development of influenza-specific cell-mediated immune response (Fig. 3).
5. Influenza A virus, cytokines and bacterial infections Influenza A virus infection in the epithelial surfaces is sometimes associated with enhanced bacterial colonization and infection [reviewed in [27,28]]. Influenza A virus-associated secondary bacterial pneumonia and otitis media [1] are examples of infections, where both viral and bacterial pathogens may be found. The primary target for influenza A virus is the epithelial cell layer of the whole nasopharyngeal area. Virus-infected cells undergo cytolytic or apoptotic death, which impairs mechanical removal of mucus and bacteria by the cilia of epithelial cells. Simultaneous infection of neutrophils with both influenza A virus and bacteria was shown to potentiate apoptotic cell death [29]. Epithelial cell death may also allow enhanced bacterial adherence to damaged epithelial surfaces by exposing basement membrane structures rich in extracellular matrix proteins collagens, laminin and fibronectin. Influenza A virus-infected epithelial cells have enhanced capacity to adhere bacterial pathogens [27]. Some bacteria, such as S. aureus produce proteolytic enzymes, which may be
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Moilanen is acknowledged. The original work was supported by the Medical Research Council of the Academy of Finland and the Sigrid Juselius Foundation.
References
Fig. 4. A model of influenza A virus-induced inflammatory responses and the development of otitis media. Influenza A virus infects epithelial cell of the nasopharynx (1) and induces chemokine and cytokine production (2). Cytokines upregulate the expression of cellular adhesion molecules and enhance mucosal swelling (3). Chemokines enhance leukocyte extravasation (4), which further enhances inflammatory responses in the site of infection. Virus infection may also induce cell death (5) allowing exposure of epithelial basement membranes. Bacteria may adhere to inflamed epithelium and cause otitis media (6).
involved in the cleavage of influenza A virus hemagglutinin rendering the virus more pathogenic [28]. The production of chemokines and cytokines by virus-infected epithelial cells and leukocytes enhance the inflammatory response in the site of infection. In influenza A virus infection the chemokines produced [8,9] favors the extravasation of blood monocytes, NK, T and B cells to inflamed tissues. These cells in turn are capable of producing proinflammatory cytokines IL-1b, TNF-a, IFN-a/b, IL-18 and IFN-g [15,22], which favor the development of Th1-type immunity that is a prerequisite for specific antiviral immunity and eradication of viral pathogens. A strong Th1-type response may, however, result in tissue damage. Certain cytokines like IL-1b and TNF-a enhance the expression of cellular adhesion molecules [30] and increase cellular interactions, which further favors the extravasation of inflammatory cells. Cellular adhesion molecules may enhance bacterial colonization to the epithelial surfaces of inflamed tissues (Fig. 4). Better knowledge of the inflammatory responses during influenza A virus infection may give us tools to control the appearance of complications and reduce the tissue damage associated with influenza A virus infection.
Acknowledgements We are grateful to Dr Tapani Hovi for critical comments of the manuscript. The expert technical assistance of Valma Ma¨kinen, Marika Ylisela¨, and Katja
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