Abstract
ABSTRACT Adenovirus is a common infectious pathogen in both children and adults. It is a significant cause of morbidity in immunocompetent people living in crowded living conditions and of mortality in immunocompromised hosts. It has more recently become a popular vehicle for gene therapy applications. The host response to wild-type infection and gene therapy vector exposure involves both the innate and adaptive immune systems. The initial innate immune response is associated with the severe acute manifestations of adenovirus infection and also plays a significant role in acute toxicity owing to adenovirus vector exposure. This review discusses the innate immune response primarily during wild-type adenovirus infection because this serves as the basis for understanding the response during both natural infection and exposure to adenovirus vectors.
The host response to adenovirus (Ad) infection involves both the innate and the adaptive or acquired immune system. The innate immune system consists of the local immediate response to infection. This results in the acute inflammation seen during wild-type (WT) infection and, in the case of Ad gene therapy, dose-limiting toxicity. The adaptive immune system plays a role in preventing reinfection with Ad. In gene therapy applications, this system limits both the duration of transgene expression and the effectiveness of repeated exposure to the same vector in an attempt to prolong transgene expression. These issues were recently reviewed.1-4
The purpose of this review is to summarize the current knowledge of how the innate immune system is triggered during WT Ad exposure. A major interest in this topic may be the role of induction of the innate immune system by Ad vectors, which is partly covered in the reviews discussed above. However, most of us are likely never to encounter this response, either professionally or personally. For the most part, this review focuses on the immune response to respiratory infection, but the concepts presented are generalizable to infection of other organ systems. Host defense in the respiratory tract is complicated by the fact that the lung has to provide close access of incoming air to the bloodstream to accomplish gas exchange. This necessitates exposure to pathogens in the environment. Because of its importance in respiration, the immune response to pathogens has to be measured or the host response can be deleterious, as occurs in adult respiratory distress syndrome.
All of us, however, have treated patients with Ad infections, have suffered from these infections, or are even currently infected with WT Ad. Furthermore, to understand how the response to Ad vectors occurs, it will likely be important to understand how the immune system responds to WT infections. Therefore, the focus of this review is the innate immune response to WT Ad. This involves primarily how the innate immune response is activated by Ad. A review of how expressed Ad proteins suppress part of this response was recently conducted.5Some of the information discussed in this review, of course, comes from experiments using gene therapy vectors.
It will become apparent in this broad-based review that there are considerable uncertainties in how or even whether basic components of the innate immune system respond to Ad. This may be partially responsible for the unexpected toxicity in some cases of gene therapy administration6and why there is a lack of effective therapy for acute consequences of WT infection.
AD STRUCTURE AND SEROTYPES
Ads were first cultured and reported as distinct viral agents in 1953.7Ad infections cause a broad spectrum of illness in immunocompromised and immunocompetent hosts. Ads are the cause of a large number of acute febrile respiratory syndromes among military recruits.8They are also a cause of ocular, respiratory, and gastrointestinal infections in the general population. Structurally, Ads are nonenveloped, regular, icosahedral, and 65 to 80 nm in diameter.9The virion has 20 triangular faces and 12 vertices, with 240 hexons and 12 pentons. Each penton consists of a base and a fiber with a terminal knob. Inside the capsid is a single molecule of double-stranded deoxyribonucleic acid (DNA) with a molecular weight of 2.0 × 107to 2.4 × 107(or ˜35 kb). Antigenic determinants that are important in the serologic classification of Ad are inherent to the hexon, penton, and fiber. All human Ad serotypes share a cross-reacting group antigen that is carried on the inner surface of the hexon capsomere.10To date, 51 serotypes of human Ads have been recognized11and grouped into six species (formerly called subgenera) on the basis of their hemagglutinating properties and biophysical and biochemical criteria and, more recently, DNA sequence homology: species A, B (subdivided into subspecies B1 and B2), C, D, E, and F.12Subgroup identification is of more than simple academic interest because the type of infections caused by the virus depends on the subgroup to which the virus belongs.13,14This is also of relevance to the material that follows because the innate immune response may also depend on the Ad subgroup studied.15,16
INNATE IMMUNE SYSTEM
Many components of the innate immune system act to prevent infection on exposure to infectious agents, including viruses. These can be generally classified into mechanical defenses, anti-infective chemical defenses, cellular defenses, and chemokine or cytokine defenses. A detailed description of these components and their role in Ad infection follows; this material is also summarized in Table 1.
Mechanical Defenses
Mucociliary clearance and coughing are the two mechanical methods of pathogen clearance in the lung. Mucociliary clearance is a process that involves distal to proximal transport of a bilayer of mucus and a periciliary fluid by cilia, collectively referred to as the airway surface fluid. The composition of the bilayer allows infectious particles to be bound in the surface mucus, whereas the cilia are able to function in a lower-density layer. This bilayer increases the efficiency of transport and achieves clearance of infectious particles from the peripheral airways in relatively short periods of time.17Clearance of bacteria from peripheral airways by mucus transport may require up to 6 hours. Bacterial replication is inhibited for 3 to 6 hours by various endogenous antimicrobial factors, including lactoferrin, lysozyme, and defensins.18Viruses, on the other hand, require cellular machinery to reproduce, so mucosal inhibitors act by interfering with the ability of these organisms to infect cells.
There are several inhibitors of viral infectivity present in mucus. The structure of mucus contains multiple sialic acid residues. These residues compete with cellular receptors used by viruses such as influenza virus and therefore decrease virus infectivity. Mucus protease inhibitor prevents the action of airway tryptase clara, which enhances influenza A virus infectivity.19The role of these substances in protection from Ad infection has not been established, but it is likely that sialic acid residues in mucus inhibit viral infectivity in a similar manner because sialic acid is involved in attachment of some subgroup D Ad types to epithelial cells.20-22Also, a recently synthesized sialic acid derivative inhibits the subgroup D Ad 37 to corneal epithelia cells.23With regard to subgroup C Ad vectors, the mucous bilayer is perhaps a limiting factor for optimal Ad gene transfer.24Also, there is charge-dependent repulsion between Ad 5 group C capsid protein and cell surface sialic acid that can be diminished; thus, infectivity is enhanced by neuraminidase.20,25-27Thus, the effect of sialic acid on Ad infection depends on the Ad type and cellular environment, in some cases enhancing and in others inhibiting this process.
The airway epithelium, which lies underneath the mucus bilayer, is also resistant to Ad infections. Two proteins in the adenovirus capsid, the fiber and the penton base, are thought to be involved in the first steps of adenovirus infection. The globular head of the Ad fiber attaches to either the coxsackie and adenovirus receptor (CAR), the major histocompatibility complex (MHC) class I α-2 domain, or the CD46 receptor, depending on the Ad subtype.28-31Some subgroup D Ads use sialic acid for fiber binding to cells as previously described.21,22The penton base of Ad binds to the αv-β3 and αv-β5-integrins or αv-β1-integrins, which is important for virus internalization.32Ads have been shown to be inefficient in targeting proximal human and murine airway epithelium.33One important reason for this inefficiency appears to be the lack of appropriate Ad receptors on ciliated airway epithelial cells.34Resistance to Ad vectors could be partially overcome by increasing the incubation time.35These interactions are further discussed in the section on epithelial cells.
Chemical Defenses
Antimicrobial Peptides
Generally, particles less than 5 μm bypass the mechanical defenses and gain access to the terminal airways. Therefore, additional defense mechanisms are needed to maintain lung sterility. Important classes of antimicrobial compounds are the cationic antimicrobial peptides. These peptides are present intracellularly in cellular phagocytic components of the innate immune system but are also secreted into the airway by epithelial cells. They contain three major classes of molecules: (1) the defensins produced by epithelial cells, neutrophils, monocytes and macrophages, and dendritic cells36; (2) the cathelicidins produced by neutrophils, mast cells, and epithelial cells37-39; and (3) the thrombocidins, which are produced by platelets.40
Defensins
Defensins are single-chain, strongly cationic peptides with molecular weights of 3 to 4.5 kDa.41,42They are divided into two classes, α- and β-defensins, based on their chemical structure. α-Defensins human neutrophil peptides 1 (HNP-1) to HNP-4 are produced by neutrophils, whereas α-defensins human defensin 5 (HD-5) and HD-6 are produced by respiratory tract epithelia, as well as the female reproductive tract and intestinal Paneth cells. β-Defensins human {beta} defensin 1 (HBD-1) to HBD-3 are produced by epithelial cells of many organ systems, whereas HBD-4 is primarily expressed in the testis and gastric antrum.43-49These molecules have a broad spectrum of microbicidal activity against gram-positive and gram-negative bacteria, mycobacteria, fungi, and certain enveloped viruses.41,50,51The antibacterial but not the antiviral activity of defensins is dependent on low salt concentrations.47,49,52This has implications in patients with cystic fibrosis, in whom the salt concentration is altered owing to the genetic defect.
Although Ad is nonenveloped, defensins have activity against this virus. The human α-defensin HNP-1 is present in bronchoalveolar lavage fluid and inhibits Ad 5 vector infection in vitro by 95%.53The α-defensin HD-5 and the β-defensin HBD-1 also reduce Ad 5 vector infectivity in vitro by three- to fivefold.54Some of the defensins, especially HBD-2, are up-regulated by various cytokines, including interleukin (IL)-1β.47,55Likewise, production of cytokines such as IL-6 and IL-8 can be induced by defensins.56This provides for amplification of the antiviral response during infection. It is not known if defensins inhibit the infectivity of WT Ad. It is likely that this occurs, based on its activity inhibiting other viruses, in which preincubation of the virus with defensins inhibits infectivity or viral replication,52,57and also based on its effects on Ad vectors.
Cathelecidins
Cathelecidins are antimicrobial peptides found exclusively in mammals. In vitro and in vivo studies indicate that they are effector molecules of mammalian innate immunity. Although many different cathelecidins have been identified in mammals, only one has been identified in humans. The first human cathelicidin was cloned from complementary DNA isolated from human bone marrow as FALL-39.58It is identical to the independently identified antimicrobial domain of human cationic antimicrobial protein (hCAP)-18 produced by neutrophils.59This peptide, hCAP-18, is cleaved to form a carboxy-terminal fragment with antimicrobial activity called LL-37.60The carboxy terminus of this peptide fragment binds and inactivates lipopolysaccharide.61LL-37 (hCAP-18) is present in neutrophil granules59and is produced by bone marrow and testis,58epithelial cells of the skin,62and respiratory39and squamous epithelia of the human mouth, tongue, esophagus, cervix, and vagina.63
Cathelicidins have activity against bacteria, fungi, and viruses. LL-37 inhibits the growth of Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus, and this activity is enhanced by lysozyme and lactoferrin.39Although the rabbit cathelecidin cationic antimicrobial protein 18 (CAP-18) has activity against Candida species, human CAP-18 (LL-37) does not have this activity.64Data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo.65The lack of plasmacytoid dendritic cell recruitment, together with the missing up-regulation of cathelicidin LL-37 in atopic dermatitis lesions, is considered relevant for an atopic dermatitis patient's susceptibility to eczema vaccinatum.66LL-37 has some activity against herpes simplex viruses 1 and 2 in vitro, but there are no reports for testing against Ad.67These peptides may have an indirect activity against Ad as they are up-regulated by IL-663and interferon (IFN)-γ,68and cathelicidins are chemotactic for neutrophils, monocytes, and T cells.68,69However, LL-37/hCAP-18 was not up-regulated in human colon epithelial cells stimulated with cytokines that play a role in intestinal inflammatory responses, including tumor necrosis factor (TNF)-α, IL-1α, IFN-γ, and IL-6.70Thus, LL-37/hCAP-18 appears to be differentially regulated among different cell types. Cathelicidins are likely partially responsible for the inflammatory infiltrate seen during Ad infection because there is evidence that cathelicidins are up-regulated by cytokines induced by Ad and because cathelicidins are chemotactic.
Thrombicidins
Thrombicidins are antimicrobial peptides released from platelets stimulated with thrombin or found in platelet extracts. They are also released when platelets are exposed to infectious agents. Several of these peptides are either structurally similar to or identical to known chemokines.71Studies of peptides have demonstrated in vitro and in some cases in vivo activity against bacterial species, such as Staphylococcus and Streptococcus, and fungal species, such as Candida and Cryptococcus.40,71-73However, there are as yet no studies of the direct antiviral activities of thrombocidins.
Pulmonary Surfactants
Although the primary role of pulmonary surfactants is to facilitate lung expansion by reducing surface tension, they also play a role in lung defense against infections. Two surfactant proteins, SP-A and SP-D, have antimicrobial activity. In this property, they are classified as collectins or collagenous C-type lectins. These proteins consist of oligomers of trimeric subunits. The subunits contain C-type lectin carbohydrate recognition domains.74These domains bind to a number of different organisms and decrease virulence both by inhibiting infectivity and enhancing phagocytosis by cellular components of the innate immune system. Both surfactant proteins bind to components of P. aeruginosa, Klebsiella pneumoniae, and E. coli, whereas SP-A binds to Haemophilus influenzae, group B streptococci, Streptococcus pneumoniae, and Staphylococcus aureus.74In addition, both proteins bind to unencapsulated forms of Cryptococcus neoformans,75 Aspergillus fumigatus,76and Pneumocystis carinii.77,78
The lung collectins show specific interactions with various respiratory viruses. Purified SP-A and SP-A inhibit the infectivity and hemagglutination activity of influenza A virus in vitro.79,80Collectins also inhibit the infectivity of respiratory syncytial virus (RSV)81and rotavirus.82SP-A also promotes the phagocytosis of herpes simplex virus by rat alveolar macrophages.83
Animal studies using SP-A-deficient or SP-B-deficient mice have verified an in vivo effect on clearance of several of these microbes. Clearance of bacterial pathogens, including group B streptococci, Haemophilus influenzae, P. aeruginosa, and other viral pathogens, such as RSV and influenza A virus, was deficient in SP-A-deficient mice.84SP-D enhanced the phagocytosis and pulmonary clearance of RSV in mice.85SP-D-deficient mice showed decreased viral clearance of certain strains of influenza A virus and increased production of inflammatory cytokines in response to viral challenge. Viral clearance of strains of influenza A virus and the cytokine response were both normalized by the coadministration of recombinant SP-D.86Of particular interest to this review, clearance of Ad DNA from the lung and uptake of fluorescent-labeled Ad vector by alveolar macrophages were decreased in SP-A-deficient mice. Supplementing with SP-A enhanced viral clearance and inhibited lung inflammation during pulmonary adenoviral infection, confirming the importance of SP-A in antiviral host defense.87
Cellular Defenses
Macrophages
Cellular defenses of the innate immune system include both resident cells and those recruited from the bloodstream. Alveolar macrophages are the most prominent resident cells that engulf and kill infectious agents. They also provide a link to the adaptive immune system because they also function as antigen-presenting cells, although less effectively than other cells.88-90Macrophages have a pivotal role in immune surveillance of the respiratory tract, initiation of anti-infective inflammation, and regulation of potentially harmful inflammatory responses. Alveolar macrophages make up 85% of cells recovered in bronchoalveolar lavage.91Alveolar macrophages play an important role in the elimination of Ad vectors from the lung. Alveolar macrophages recovered from the mouse lung 30 minutes following intratracheal administration of an Ad vector showed large amounts of vector genome, whereas much less was evident in alveolar macrophages recovered after 24 hours.92In a murine model, Ad labeled with a fluorescent dye was very rapidly (˜1 minute) localized within the alveolar macrophages.93Although best studied in epithelial cells,94internalization of Ad by mononuclear phagocytes is believed to occur by integrin receptor-mediated endocytosis.95, 96Granulocyte-macrophage colony-stimulating factor regulates endocytosis through a transcription factor, PU.1, which is also involved in production of macrophages by stimulating the differentiation of macrophage progenitors.97-100
Alveolar macrophages were also identified as a cell source of initial cytokine signaling by Ad. Thirty minutes after infection, alveolar macrophages expressed messenger ribonucleic acid (mRNA) for TNF-α and IL-6 but not airway epithelial or vascular endothelial cells. Blockage of virus uptake at specific subcellular sites in the alveolar macrophages completely blocked TNF-α expression.93Other products released by macrophages include IL-12 (which activates natural killer [NK] cells), leukotriene B4, toxic oxygen species, IL-1 (T-cell stimulatory and proinflammatory cytokine), and antibacterial products, such as lactoferrin, lysozyme, and defensins.36, 93TNF, IL-1, and IL-6 are considered primary activators of the immune system. Each of these cytokines effects responses that mediate the host response to infectious agents.
Dendritic Cells
Cells of similar lineage, the dendritic cells, also function in the respiratory tract and are more like tissue macrophages present in other organ systems. As such, they are less efficient at phagocytosis but more efficient at antigen presentation than alveolar macrophages. They are indispensable to the initiation of the adaptive immune response. Tobacco smoke decreases the number of dendritic cells in lung tissue, and chronic tobacco exposure impairs the immune response against Ad.101Antigen presentation by murine dendritic cells is nuclear factor kappa B (NF-κB) dependent, and NF-κB inhibition blocks dendritic cell Ad antigen presentation and has a marked immunosuppressive effect in vivo.102Preferential activation of dendritic cells and macrophages may account for Ad inflammatory responses in vivo. In a murine model, Ad-activated dendritic cells spontaneously produced high levels of IL-6 and IL-12, but Ad-activated splenic macrophages spontaneously secreted only IL-6. Elimination of tissue macrophages and splenic dendritic cells in vivo considerably reduced the early release of IL-6, IL-12, and TNF-α and significantly blocked the specific cellular immune response to Ad.103Thus, dendritic cells and macrophages may play different roles in this process in terms of their ability to produce distinct patterns of inflammatory cytokines.
Neutrophils
Neutrophils are also important in the response to infectious agents. Following exposure to Ad, the macrophages release various cytokines, including IL-8, which play an important role in orchestrating further neutrophilic inflammatory responses. IL-8 is a major neutrophil chemotactic factor in the lung.104Intravenously administered Ad significantly increased leukocyte rolling and adhesion in the liver within minutes of transduction. P-selectin, α4-integrin, and E-selectin are important in this process because blockade of these receptors inhibited leukocyte rolling and subsequent adhesion. Depletion of circulating neutrophils eliminated leukocyte rolling and adhesion.105Macrophage inflammatory protein 2 (MIP-2) antibody and neutrophil depletion diminished hepatic injury as determined by both reduced serum aspartate transamine and alanine transaminase levels and histology.106This suggests that early tissue injury is largely due to chemokine production and neutrophil recruitment. One strategy to prevent Ad-mediated inflammation and tissue injury would therefore be by interfering with chemokine or neutrophil function.
Epithelial Cells
The airway epithelial cells not only function as a passive barrier to infectious particles, they also actively participate in the innate immune response to foreign antigens. This is particularly important in Ad infections because the epithelium is the major site for Ad replication. The immune response of epithelia to infection and antigen exposure consists of an increase in the release of antimicrobial peptides into the lumen of the airways and the release of chemokines and cytokines into the submucosa that initiate an inflammatory reaction. This results in the recruitment of phagocytes that help clear the invaders and of dendritic cells and lymphocytes, which facilitate an adaptive immune response. The Toll-like receptors (TLRs) are a family of pattern recognition molecules present on various cells in the body that mediate direct cellular responses to microbial exposure. Activation of TLR on epithelial cells has now been shown to be involved in the regulation of expression of a variety of genes, including those encoding cytokines, chemokines, and antimicrobial peptides.107It is not known whether activation of TLR is important in the epithelial response to Ad. However, TLR activation is important in cytokine induction by several other viruses in other cell types and in chemokine and defensin induction in epithelial cells by rhinovirus.108-112
There are other mechanisms whereby components of the innate immune system, in this case, the lung epithelia, respond to Ad infection. Ad vector infection of A549 respiratory epithelial cells induces a significant expression of intercellular adhesion molecule 1 (ICAM-1) and increased CD18-dependent adhesion of activated neutrophils.113
This may be partially responsible for the initial inflammation seen in animal models of Ad infection, although direct stimulation of cytokines both from lung epithelia and alveolar macrophages also plays a role. Intratracheal instillation of Ad 3p and 7h in mice caused a robust inflammatory response in the lung. This initially consisted of neutrophilic and monocytic alveolar infiltration. There is also mild peribronchial and perivascular inflammation. At early time periods after exposure, neutrophils were the predominant cell type recovered by alveolar lavage. This correlated with increased levels of neutrophilic chemokines such as MIP-2 and KC (mouse IL-8 homologues) in lung tissue. Elevation of other proinflammatory cytokines, such as IL-1β, TNF-β, IFN-γ, and IL-12, was also noted.114
It is likely that direct infection or stimulation of distal epithelia is responsible for many of these changes because the peribronchial inflammation was minimal and because proximal bronchial human airway epithelial cells are resistant to infection by Ad, at least as determined for Ad 5 vectors.34Infection of epithelia by Ad involves two steps. First, the globular head of the Ad fiber attaches to either the CAR, the MHC class I α-2 domain, or the CD46 receptor, depending on the Ad subtype.28-31Then, the penton base of Ad binds to the αv-β3-and αv-β5-integrins or αv-β1-integrins, which is important for virus internalization.32The resistance of the bronchial epithelia to Ad infection appears to be due to a lack of the appropriate receptors on the apical membrane. CAR and MHC class I are polarized to the less accessible basolateral membrane,115and αv-β3- and αv-β5-integrins are also minimally expressed on the apical plasma membrane of proximal airway epithelial cells.116
It is important to note that these restrictions are likely not as important in the distal human lung, specifically the alveolar epithelium. Human alveolar epithelial cell lines are easily infected by both type 5 and type 7 WT virus.15Also, human lung tissue slices support WT Ad7 virus replication, infection of the alveolar epithelia, and an innate immune response, as evidenced by increased IL-8 release.117
Cytokines and Adenoviruses
Ads induce different clinical manifestations, ranging from inapparent infection or benign upper respiratory tract disease to necrotizing bronchiolitis or even disseminated fatal disease in immunocompromised patients. Ads are cytopathic in permissive cell cultures, but viral particles are scarce in affected tissue in the whole organism, suggesting that additional mechanisms might be involved in the pathogenesis of tissue damage. Activation of the immune system and the generation of numerous chemokines and cytokines clearly play a role in activation of inflammation and therefore could play a major role in the pathogenesis of tissue damage. Although there is a distinction between chemotactic cytokines (chemokines) and nonchemotactic cytokines by their ability to induce cell migration, their functions are interrelated. For example, chemokines not only attract inflammatory cells, they also activate them and may cause them to proliferate. Also, although nonchemotactic cytokines may also have activating and proliferative effects, they also indirectly stimulate chemotaxis by stimulating the release of chemotactic cytokines. Both chemotactic and nonchemotactic cytokines have been shown to play an important role in pulmonary host defense. These include TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12, IFN-γ, and granulocyte colony-stimulating factor.118There is evidence that the cytokine response by itself is important in the consequences of infection with Ad. Increased concentrations of IL-6, IL-8, and TNF-α were associated with hypoperfusion, febrile peaks, tonic-clonic seizures, and septic shock and were significantly associated with the severity of Ad infection in children.119The activation of the innate immune responses also occurs in the absence of virus gene transcription.15,120,121The alveolar macrophages are proposed to be the source of initial cytokine signaling after acute Ad respiratory tract infections. In a murine model, 30 minutes after infection, alveolar macrophages but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-α and IL-6.93The signal for initiation of TNF-α expression during Ad exposure to RAW264.7, a macrophage cell line, requires virion internalization and likely occurs during or subsequent to endosome acidification because blockage of these specific steps of passage of the virus completely blocked TNF-α expression.93There is some uncertainty as to whether endosome acidification is required because the agent to prevent this process, chloroquine, inhibits signaling pathways known to be important for cytokine production.122Other cells of the immune system also clearly participate in the initial innate immune response because chemokines, particularly IL-8, are induced in airway epithelial cells by Ad in culture and in human tissue.15,16,117The innate immune response of alveolar epithelial cells does not appear to require triggering by the macrophage because IL-8 is induced in pure epithelial cell cultures in the absence of macrophages and IL-8 is induced in a human slice model in which macrophages are not prominent in the preparation.15,16,117
Interleukin-1
IL-1 plays a role in the early inflammatory response seen after Ad infection. In a mouse model, intranasal inoculation of WT 5 Ad produced pneumonia even though the virus did not replicate. Assays showed the appearance of IL-1, TNF-α, and IL-6 in mouse lungs concomitant with the development of an early-phase infiltration with lymphocytes and monocytes/macrophages.123IL-1 is also the major mediator of a very early inflammatory response to Ad vector in the brain.124IL-1β may also play a role in progressive fibrosis and tissue remodeling in mice through the induction of profibrotic cytokines platelet-derived growth factor and transforming growth factor β1.125A key event in virus-induced inflammation is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as ICAM-1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. Fluids from Ad type 37-infected respiratory and ocular epithelial cells activated ICAM-1 and, to a lesser extent, VCAM-1 expression on cultured human umbilical vein cells.126Blocking studies with anticytokine antibodies implicated IL-1α as the epithelial cell-derived factor that activated ICAM-1 expression.126Ad-infected A549 respiratory cells treated with an IL-1 receptor antagonist (IRAP) expressed 75% less IL-8 than did untreated cells, whereas interleukin-1 receptor antagonist protein (IRAP) pretreatment of TNF-α-stimulated cells did not affect IL-8 production.127Thus, IL-8 production by Ad vector-infected cells is partly through IL-1 activation that can be down-regulated by IRAP. It is not known whether IL-1 is important in IL-8 induction by WT Ad.
Interleukin-6
IL-6 is another cytokine that has been associated with the immune response following Ad infection. IL-6 is produced by vascular endothelial cells, mononuclear phagocytes, fibroblasts, and activated T lymphocytes in response to a variety of stimuli and is referred to as the global response marker.128Nijsten and colleagues reported that IL-6 increases earlier in illness than other acute-phase proteins, such as C-reactive protein and α1-antitrypsin, and is related to the generation of fever.129IL-6 may act as a hepatocyte-stimulating factor and induces various acute-phase proteins in the liver cells.130When classified according to the clinical findings and outcome, IL-6 was detected in the groups with severe and fatal Ad infections in children and not in the group classified as moderate.119Increased concentrations of IL-6 were associated with hypoperfusion, febrile peaks, tonic-clonic seizures, and septic shock in these children. High serum values are associated with the severity of Ad infection. In a study comparing 106 children with Ad, influenza, or RSV infections, the IL-6 concentration was > 50 pg/mL in 78.6% of patients with Ad infections compared with 15.8% in patients with influenza and none in patients with RSV infections. IL-6 was again associated with the severity of Ad infection and correlated with serum concentrations of C-reactive protein in the group with Ad infection.131IL-6 presumably plays a dual effect during viral infections; it may stimulate immune defenses against infected cells and may participate in tissue damage.132
Interleukin-8
The importance of IL-8 in acute Ad infection is suggested by the consistent finding of prominent neutrophilic peribronchial and alveolar infiltration early in the disease process in a number of animal models.133-136Inflammation also occurred in conditions in which the virus did not replicate.123This is important because IL-8 is the major neutrophil chemotactic factor in the lung.104There are data suggesting that IL-8 has a role in the pathogenesis of naturally acquired acute Ad infection in humans. In children with Ad pneumonia, serum IL-8 levels correlate with clinical outcome.119The highest levels are seen in patients with a fatal outcome and approach values seen in patients with sepsis. Both TNF-α and IL-1 produced by alveolar macrophages are potent inducers of IL-8 production by several cell types, including alveolar macrophages, type II epithelial cells, and lung fibroblasts.104Ad strains and vectors vary in their ability to induce IL-8 and cause lung disease. WT 5 virus does not induce IL-8 release in vitro or pneumonia in immunocompetent humans. Both E1A-deficient Ad 5 vectors and WT 7 virus induce IL-8 release and pneumonitis.6,8,15,137-140Ad gene expression is likely not necessary for IL-8 induction because (1) induction occurs prior to Ad protein expression in Ad 7-infected cells and (2) Ad protein expression is minimal in gene therapy vectors. It is therefore likely that induction occurs through interaction of surface proteins with cellular receptors, which are known for Ad 5 but not entirely known for Ad 7. This could result in signaling pathway induction, resulting in IL-8 gene expression. We and others have demonstrated p42/44 mitogen-activated protein kinase activation with exposure of cells to both virus types. We have confirmed that activation of this kinase is essential for IL-8 induction by Ad 7.16The role of other signaling pathways in IL-8 induction by Ad is not known. Also unexamined is the mechanism whereby modification of WT 5 virus for gene therapy results in a vector that is capable of inducing IL-8. A similarly modified Ad 7 replication-deficient vector has been constructed but has not been tested for IL-8 or signal pathway induction.
Recent work by Matsuse and colleagues and Elliott and colleagues suggested that latent Ad may act as a coactivator of the IL-8 gene, predisposing some patients to chronic inflammation and development of chronic obstructive pulmonary disease (COPD). In their studies, increased amounts of Ad DNA, specifically the Ad E1A proteins, were detected in the lungs of patients with COPD compared with age-matched asymptomatic patients with similar smoking exposures.141,142Additional studies showed that quantitative regional expression of the Ad E1A protein in lung epithelial tissue also correlated with the number of inflammatory cells and the amount of lung destruction as determined by computed tomography.143In vitro studies, using lung epithelial cell lines, demonstrated that stable transfection with Ad E1A proteins enhanced lipopolysaccharide-induced expression of IL-8 through NF-κB.144,145Thus, the presumed mechanism of Ad enhancement of COPD is amplification of the cytokine response, specifically IL-8, to external stimuli, such as lipopolysaccharide or cigarette smoke.
Interleukin-12
IL-12 is a heterodimeric protein consisting of two subunits (p35 and p40) and plays an important role in promoting T helper 1-type immune responses. It serves as the major signal for IFN-γ expression from T and NK cells.146It therefore plays a role in linking the innate and adaptive immune systems.
IL-12 is induced during both WT Ad infection and Ad vector exposure. In a mouse model, after infection with mouse WT Ad, message for the p40 component of IL-12 was transiently increased shortly after infection.147IL-12 was expressed mainly by macrophages. Ad vector also stimulates IL-12. Ad vector infection of mouse dendritic cells stimulated IL-12 release or IL-12-dependent dendritic cell activation of NK and T cells,148-150and an increased IL-12 response to Ad vector infection in BALB/c mice correlated with increased Ad vector clearance.151However, knockout of the IL-12 gene did not significantly affect Ad removal from the host.152Therefore, although IL-12 may be important in innate immune activation and the type of subsequent adaptive immune response to Ad infection, IL-12 does not by itself appear to modulate clearance of Ad.
Tumor Necrosis Factor α
TNF-α, initially named for its ability to trigger the necrosis and involution of certain tumors,153is now widely recognized as a mediator of the host response to infection. TNF-α is rapidly produced following either antigen-specific or -nonspecific stimulation of the alveolar macrophages and has been designated as an early alarm response cytokine.118TNF-α and IL-1 do not appear to affect polymorphonuclear (PMN) chemotaxis directly, but both of these cytokines are potent inducers for the production of certain C-X-C chemokines, such as IL-8, by alveolar macrophages, type II epithelial cells, and other cells of the airways.85,104These C-X-C chemokines are the major chemoattractants for PMN recruitment into the lung during pulmonary infection and inflammation.104,154-157A study evaluating the expression of TNF-α and IL-1β after Ad infection of human monocytes (which are nonpermissive for Ad virus infection) and macrophages showed that the production of both cytokines was enhanced in monocytes.158However, in macrophages, a slight enhancement of TNF-α was seen, and no IL-1β was detected. The results suggest that cellular genes might be activated in nonpermissive cells in which no viral gene products could be detected. TNF-α signaling through both the p55 and p75 TNF receptors plays an important role in the magnitude of the humoral immune response. The absence of TNF-α or the p55 receptor significantly attenuates the antibody response to Ad.159
Replication-competent Ads with various E1 modifications designed to restrict their replication to tumor cells are being evaluated as oncolytic agents in clinical trials. In mouse models, such oncolytic Ads showed greater dose-dependent hepatotoxicity than E1-deleted Ad vectors following intravenous administration. Hepatotoxicity correlated with expression of WT E1A in the liver with increases in viral DNA levels. This was correlated with rapid induction of TNF-α to high levels and with rapid elevation of serum alanine transaminase. Hepatotoxicity was significantly reduced for an Ad with deletions in the region E1A (dl01/07) or a virus lacking E1A. The results suggest a mechanism for hepatotoxicity involving virus-induced production of local TNF-α release and E1A-mediated sensitization of hepatocyte killing.160Ad hepatotoxicity does not, however, absolutely require Ad gene expression. High-dose hepatic delivery of an E1A-deficient virus expressing an ornithine transcarbamylase gene resulted in severe hepatic and systemic toxicity and death in a human subject.6Subsequent studies showed that Ad vectors in which the viral gene expression was completely suppressed caused similar toxicity in animal models.121Thus, both Ad capsid and Ad proteins are capable of causing cell damage during Ad vector exposure.
Interferons
IFNs are a family of cytokine mediators that influence the quality of cellular immune responses and amplify antigen presentation to specific T cells. IFN was first discovered by Isaacs and Lindenmann in 1957 by the induction of IFN in chick cells by influenza virus.161In 1967, it was observed that chick embryo fibroblasts inoculated with human Ad produced IFN.162There are three major classes of IFNs: IFN-α, IFN-β (type I IFN), and IFN-γ (type II IFN). Whereas type I IFNs are secreted by virus-infected cells, IFN-γ is produced by T and NK cells and plays an important role in cell-mediated immunity against a broad spectrum of intracellular pathogens. IFN-γ activates several leukocyte functions, including stimulation of respiratory burst, antigen presentation, and priming TNF-α release by macrophages. Ad type 7 at a multiplicity of infection of 1 induced IFN in human leukocyte cultures.163Exposure of cells to IFN induces an antiviral state in which the replication of a wide variety of both DNA and ribonucleic acid viruses is inhibited.164During this antiviral state, a set of cellular genes, termed IFN-stimulated genes, are transcriptionally induced. Ad infection of HeLa cell cultures induced the transcription of these genes, without IFN synthesis or the synthesis of new adenoviral proteins.165However, the E1A gene products specifically suppressed this transcription. The dual effect of Ad on the expression of the IFN-stimulated genes may represent an example of action and evolutionary reaction between virus and host.
SUMMARY
Protection against Ad infection by the innate immune system involves a complex system of mechanical and biologic factors. The biologic factors include soluble inhibitors of viral infectivity, some of which have additional functions, such as surfactant. Other factors, such as cytokines and chemokines, serve to amplify the immune response through recruitment or activation of inflammatory cells. Cellular targets of these biologic response modifiers also assist with elimination of infectious virus and bridge the gap between innate and adaptive immunity.
As much of the damage owing to WT Ad infection appears to be due to dysregulation of this response, modulating this response may prove beneficial in suppressing the immediate damage that occurs. This would not be advantageous if the approach enhanced overall viral replication and thus increased the infectious burden on the host. This highlights a divergence in the concerns of investigators in the field of WT therapeutics from the gene therapist, who does not have to be greatly concerned with replication of the vector.
There is another important difference in the approaches that these two groups of investigators must take. The gene therapist, at least one whose object is to express a foreign or functional native gene product, has to allow the initial stages of the Ad infectious cycle to proceed. Blocking internalization of WT virus may limit the immune response and toxicity, as well as the severity of infection, and be an attractive approach to those who are looking for Ad WT infection therapeutics. The same intervention when performed during gene therapy with Ad vectors may limit acute toxicity but would also have the undesirable effect of limiting transduction. Thus, the gene therapist will likely always be viewing ways to avoid the immune response altogether, whereas the investigator involved in WT Ad therapeutics will perhaps be interested only in modifying it slightly or perhaps even enhancing it. Nevertheless, a deeper understanding of the nature of the innate immune response to both Ad and Ad vectors will likely benefit both fields as they search for novel methods to defend the host against this pathogen or to use a modified pathogen to restore normal function of the host.