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Host–bacterial interactions in the initiation of inflammation
PAEDIATRIC RESPIRATORY REVIEWS (2001) 2, 245–252 doi:10.1053/prrv.2001.0147, available online at http://www.idealibrary.com on
SERIES: NEW BIOLOGY OF THE AIRWAYS
Host–bacterial interactions in the initiation of inflammation D. Rastogi, A. J. Ratner and A. Prince∗ College of Physicians & Surgeons, Columbia University, Black Building Room 416, 650 West 168th Street, New York, New York 10032, USA KEYWORDS inflammation, P. aeruginosa, cystic fibrosis.
Summary The respiratory epithelium provides both a physical and an immunological barrier to inhaled pathogens. In the normal host, innate defences prevent bacteria from activating inflammation by providing efficient muco-ciliary clearance and antimicrobial activity. Bacteria that persist in the airway lumen, as in cystic fibrosis, activate both the professional immune cells in the respiratory mucosa as well as the more abundant airway epithelial cells. As most of the bacteria become entrapped in airway mucin, shed bacterial products such as pili, flagella, peptidoglycan and lipopolysaccharide from lysed bacteria are likely to be the stimuli most important in activating epithelial signalling. The airway cells respond briskly to bacterial components through several signalling systems which activate epithelial expression of pro-inflammatory cytokines and chemokines. These signals recruit neutrophils to the airways where they eliminate the contaminating bacteria causing C 2001 Harcourt Publishers Ltd inflammation and the ensuing clinical signs of infection. °
INTRODUCTION The normal host defences of the airway epithelium are proficient in preventing infection of the respiratory tract. Despite repeated exposures to a wide variety of potentially pathogenic organisms, the lower respiratory tract usually remains sterile and the host is unaware of the ongoing clearance mechanisms. However, when bacterial contamination of the airways overwhelms the routine muco-ciliary defences and polymorphonuclear leukocytes (PMN’s) are recruited, the resulting inflammation, whilst serving to clear the infecting organisms, also causes the signs and symptoms of respiratory tract infection. An exaggerated host response to bacterial contamination of the airways and the resulting PMN-dominated inflammation is characteristic of cystic fibrosis (CF). In examining the interactions between the CF airway epithelium and specific bacterial pathogens such as Pseudomonas aeruginosa, a great deal has been learned, not only about how the
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Correspondence to: Alice Prince; Tel: +1 212 305-4193; Fax: +1 212 305-2284; E-mail: [email protected]
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CF patient responds to airway infection but also how the normal airway clears contaminating bacteria. This review explores how innate defence mechanisms prevent bacteria from reaching the surface of the airway epithelial cell, what happens when bacteria or bacterial components activate epithelial or immune cells in the lung and which bacterial components are involved in stimulating airway inflammation. In addition, the epithelial signalling pathways which evoke inflammation will be reviewed as well as some of the cytokines and chemokines responsible for recruiting inflammatory cells into the lung.
INNATE DEFENCES The innate defences of the airway mucosa are complex, consisting of several physical, cellular and antimicrobial components (Fig. 1). These defences do not necessarily require the coordination of the ‘professional’ immune system (macrophages, T cells and B cells) which is also involved in bacterial clearance, nor do they require previous experience with a specific antigen or infecting agent. Mechanical defences prevent particulate matter and micro-organisms from entering the lung. They begin at ° C 2001 Harcourt Publishers Ltd
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the nose which functions as a filter. Large particulate matter is trapped in nasal hair or fimbriae and particles >10 µm are caught on the surface of the turbinates and septum. Smaller particles, including bacteria, ranging from 2–10 µm are inhaled and deposited in the lower airways. The airways between the larynx and the respiratory bronchioles are lined with ciliated columnar epithelium covered by fluid 5 to 100 µm thick. The fluid consists of a periciliary layer adjacent to the epithelial surface and a layer of mucus which appears to float on top. The composition of the mucus is determined by the epithelial glycoproteins, mostly sialo- and sulfoglycoproteins, and proteoglycans secreted by Goblet and serous cells. Ciliated cells and the Clara cells of the distal epithelium also contribute to the mucus production. The airway mucins are high-molecular weight glycoproteins which are secreted into the airway as well as being anchored to the epithelial surface by trans-membrane domains. Many bacteria are bound to mucins and swept away by the ‘muco-ciliary escalator’. For example P. aeruginosa, a ubiquitous opportunistic pathogen, is a common contaminant of sinks, showers and puddles. When inadvertently inhaled by the normal host, the organisms are efficiently cleared without eliciting any inflammatory response. Normal innate defences prevent ‘professional’ immune cells from having to process P. aeruginosa antigens. Even laboratory workers exposed to these bacteria in large numbers do not produce high titres of antibody against them. P. aeruginosa flagella, (long appendages involved in motility and chemotaxis) bind to mucin and are physically removed. The expression of specific mucin genes by airway epithelial cells are up-regulated in response to bacterial ligands.1 Thus, contact with the organisms stimulates mucin expression to facilitate mechanical clearance. In CF, due to failure of normal CFTR (cystic fibrosis transmembrane conductance regulator) Cl− channel function, airway mucin is dehydrated, forming a mechanical obstruction which blocks the muco-ciliary clearance.2
Antimicrobial agents in the airway Lysosyme, lactoperoxidase, lactoferrin The airways also contain several types of antimicrobial proteins with a broad range of antimicrobial activity.3 Lysosyme and lactoferrin are particularly abundant and have very potent antimicrobial activity efficiently killing both Gram negative and Gram positive organisms. Lactoperoxidase was recently demonstrated to be expressed in the airways and significantly inhibits the growth of many microbes.4 Surfactant proteins The surfactant proteins A and D, members of the collectin family also participate in bacterial clearance. These have
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been studied by comparing the susceptibility of transgenic mice lacking either surfactant protein to pulmonary infection caused by various pathogens. The Sp-A null mice were particularly susceptible to P. aeruginosa infection.5 Antimicrobial peptides The airway mucosa is also protected by small peptides which have potent antibacterial activity. Several classes of these have been described and their properties established. They lyse Gram negative pathogens by intercalating through the lipopolysaccharide and forming pores in the cytoplasmic membrane. The identification and classification of the related defensins in the airway epithelium was first detailed in the bovine tissue and the human homologues were identified shortly thereafter. Epithelial cells produce the β-defensins (types 1 and 2) both of which have been isolated from bronchoalveolar lavage fluids. Several different peptides have been recently identified including a member of the cathelicidin family LL-37/CAP-18.6 Diminished airway defensin activity has been postulated to contribute to bacterial contamination of the lungs in CF.6 Defensin function in CF In a series of experiments performed by Smith et al., airway epithelial cell secretions in vitro were able to kill a small inoculum of P. aeruginosa added to the secretions in vitro.7 Cell culture supernatants from CF cells were deficient in their ability to kill the organisms and this defect could be reproduced in the normal cell culture supernatant by the addition of high concentrations of NaCl.7 As the defensins are known to be NaCl sensitive in their antibacterial activity, a likely conclusion was that the Na+ in the CF lung airway fluid (expected to be increased as a consequence of CFTR-Cl− channel dysfunction) inhibits defensin activity causing the increased susceptibility to infection in CF. Studies from the Wilson laboratory further suggested that human defensin-1 was, in fact, the NaClsensitive defensin which was associated with this defect in CF.8 These investigators ‘corrected’ the bacterial killing defect by expression of a wild-type CFTR in the epithelial cells. However, this hypothesis has now been challenged. There is no consensus that the NaCl composition of the airway surface fluid in the CF lung in vivo, is ever high enough to inhibit defensin activity to the degrees suggested by the in vitro data. Moreover, the contribution of human defensin to the total mix of anti-microbial agents on the airway surface is difficult to ascertain. There is little evidence that defensins are critical in the clearance of S. aureus, another major CF pathogen. There is little doubt that the antimicrobial peptides, lactoferrin and lysosyme are very important in the normal protection of the mucosa but it is less clear if a relative dysfunction
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of them is a major factor in the pathogenesis of bacterial infection in CF.
IMMUNE CELLS IN THE LUNG Bacteria which elude these innate defences and contaminate the lower respiratory tract stimulate a brisk inflammatory response. The inflammation may be signalled either by the professional immune cells which provide a surveillance function in the lung, such as alveolar macrophages, or may be transmitted by dendritic cells which are stimulated by bacterial gene products. PMN’s are recruited to the site of infection to phagocytose and kill the organisms. Macrophages and other cells of immune origin have different types of responses to specific bacterial gene products than do the airway epithelial cells.
Macrophages Macrophages play a central role in host–bacterial interactions. They are the most prevalent non-parenchymal cells in the airways of normal subjects and are important regulators of airway inflammation. Their origin is either from the bone-marrow-derived blood monocytes or by local multiplication. The former is mainly responsible for the increase in their numbers during inflammation. Alveolar macrophage function is dependent upon mobility, phagocytic activity, expression of receptors and their ability to signal other immune cells through the expression of chemokines and cytokines. Many of their functions are turned off unless they are specifically activated by organisms or cytokines. Macrophages have different affinities for microbial components compared with epithelial cells and are activated by whole organisms or small components of the organisms such as lipopolysaccharide. On activation, alveolar macrophages produce enzymes (acid hydrolases, neutral hydrolases and lysozymes), arachidonic acid derivatives, reactive oxygen metabolites, cytokine and other bioactive peptides to kill the organisms and to recruit additional immune cells. Macrophages also regulate the functions of the dendritic cells, the primary antigen presenting cells of the airway.
PMN’s Neutrophils are the most important cells recruited to the airway after exposure to a pathogen. Their primary function is to recognise, phagocytose and destroy the pathogens. This is accomplished through opsonisation followed by Fc-mediated binding or antigen recognition using complement receptors. The pathogen is ingested and killed in the PMN phagosome through the expression of peptides and reactive oxygen intermediates. Neutrophils release lipid mediators, leukotrienes and reactive oxygen species which are important in bacterial killing and in the inflammatory response. Whilst neutrophils are critically important to phagocytose and kill bacteria, their
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own lysis and release of elastase is immunostimulatory by itself. PMN elastase is a potent stimulus of epithelial interleukin-8 (IL-8) expression by the airway cells, further serving to promote a cycle of continued inflammation. In CF, excessive amounts of neutrophil elastase are thought to contribute to the cleavage of appropriate complement receptors and associated ligands, impeding efficient phagocytosis.9 DNA from lysed PMN’s is a major contributing factor to the increased viscosity of CF airway secretions.
AIRWAY EPITHELIAL CELLS AND THEIR RESPONSE TO BACTERIA The most numerous cells lining the airways are the airway epithelial cells which have evolved complex signalling pathways to enable them to respond to the threat of bacterial infection. They are probably most important in providing a surveillance function for the common opportunistic pathogens, such as S. aureus and P. aeruginosa. They actively respond to adherent and invasive bacteria as well as to isolated bacterial gene products. Their major role is to signal the presence of the pathogen and recruit the influx of PMN’s to remove the organisms. This inflammatory response is controlled primarily by proinflammatory cytokines. These are small polypeptides or glycoproteins between 10 and 30 kDa produced by the respiratory epithelial cell in response to different stimuli. They are not stored in the cells but are actively synthesised for secretion. Cytokines generally act locally by binding to cellular receptors and activating gene expression. An important function of these chemicals is cell to cell communication, which can be autocrine, paracrine or endocrine in mechanism. Pro-inflammatory cytokines help coordinate the local response to bacterial infection. While epithelial cells are capable of expressing numerous cytokines and chemokines, here, the discussion is focused on those shown to be important in CF-associated airway inflammation.
Interleukin-8 (IL-8) IL-8 is a prototype of the family of cytokines which are chemotactic for neutrophils and monocytes. It is synthesised by the circulating monocytes, macrophages, fibroblasts, endothelial cells, and epithelial cells. Endotoxin (LPS), tumour necrosis factor (TNF)α, IL-1, granulocytemacrophage colony-stimulating factor (GM-CSF), lectins, immune complexes and phagocytosis all stimulate IL-8 production.
Recruitment and activation of PMN’s in the airway by airway epithelial cells For PMN’s to migrate to the site of bacterial infection, they must be recruited and activated by local signals. This complex process involves the local expression of
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chemokines such as IL-8 as well as the upregulation of cellular adhesion molecules to signal the circulating PMN’s to a site and direct their migration toward the increased amount of chemokine expression. The activated PMN’s secrete many products that play an important role in microbial killing10 but may also have major toxic effects on the host tissues. Additional cytokines such as granulocytecolony stimulating factor (G-CSF) and GM-CSF act to prolong the half-lives of PMN’s by inhibiting their apoptosis. While this induced inflammatory response results in the local accumulation of PMN’s to phagocytose and eradicate the bacteria, other consequences of inflammation on the host, airway oedema, bronchospasm and consolidation can impair the normal function of air-exchange producing the clinical signs and symptoms of pneumonia. In airway diseases such as CF, the failure to resolve such episodes of local inflammation may eventually result in chronic inflammation and fibrotic changes.
BACTERIAL INDUCTION OF HOST DEFENCES IN CF Bacteria which evade the activity of antimicrobial peptides and mucociliary clearance may persist in the airway lumen and replicate in the absence of effective host defences. These organisms can then remain sequestered within the airway secretions and adapt to this milieu, as occurs in CF, or invade the mucosal barrier in susceptible hosts to cause sepsis, a more common complication in immunocompromised hosts. The lung, unlike other mucosal surfaces such as the gastrointestinal epithelium or the urogenital tract, is normally sterile. Thus, even the presence of superficial organisms is sufficient to activate an epithelial response. In the CF lung the vast majority of organisms are entrapped in mucin and surrounded by a florid PMN response. They form a biofilm but few, if any, bacteria are seen directly apposed to the epithelial cells. Histopathological studies of CF lung tissue do not demonstrate any intraepithelial bacteria, nor is sepsis seen, despite the huge intralumenal burden of bacteria, except as a terminal event. Bacterial invasion of the airway mucosal cells by the common CF pathogens is highly unusual. In a fatal case of B. cepacia infection, organisms were shown to be invasive.11 The presence of common pathogens such as P. aeruginosa within airway epithelial cells seems limited to experimental models of infection or to cells in culture. Initial reports that CFTR might function as a receptor for internalisation of P. aeruginosa have not been corroborated by data from patients. Studies using transgenic mice did not confirm any relationship between the level of CFTR expression in the airway epithelium and the ingestion or invasion of the epithelial cell by P. aeruginosa.12 Superficial interactions between the common CF pathogens, S. aureus, H. influenzae and P. aeruginosa and
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the respiratory epithelium are sufficient to cause inflammation as documented in many histopathological studies of the CF lung.13 Even very early in the disease process, before bacteria are consistently isolated from pulmonary secretions, broncho-alveolar lavage studies consistently show a marked pro-inflammatory response. While there may be endogenous factors that contribute to this inflammation, clearly the presence of bacteria in the airway lumen is a sufficient stimulus to activate epithelial cells. As most of the organisms are entrapped in mucin, it seems likely that shed bacterial components as well as organisms that become adherent to the epithelial cells themselves, are capable of stimulating pro-inflammatory responses. Epithelial production of IL-8 is particularly important in recruiting and stimulating PMN’s in the lung and is widely used as a clinical marker of inflammation. Several laboratories have examined which bacterial components activate epithelial IL-8 expression to recruit PMN’s into the airways. These include both adherent intact organisms as well as isolated bacterial gene products which may be present in the airway lumen, even in the absence of viable bacteria.14
BACTERIAL ACTIVATION OF HOST INFLAMMATORY RESPONSES Adherent organisms stimulate epithelial IL-8 expression Many pulmonary pathogens bind to the GalNAc β1-4Gal moiety which is available on cells with asialylated glycolipids.15 These include the common respiratory pathogens S. aureus, H. influenzae and P. aeruginosa. While such asialylated receptors are not normally available on the airway surfaces to any great degree, they are significantly increased in areas of cell damage and regeneration.16 Cells with CFTR mutations have increased amounts of asialylated glycoconjugates and hence, more adherent organisms.17,18 There is a direct correlation between adherent P. aeruginosa and the amount of IL-8 expression in epithelial cells. Cells with CFTR mutations, or cell lines specifically constructed to lack normal CFTR expression were found to have increased numbers of adherent P. aeruginosa, which correlated with the increased numbers of asialylated glycolipid receptors. For P. aeruginosa, epithelial binding is mediated by pili, small polar appendages which bind directly to the GalNAcβ1-4Gal moiety of asialylated glycolipids, as well as by non-pilin adhesins. The significance of this interaction is that pilin mediated adherence is a potent stimulus of epithelial IL-8 expression.19 Therefore, increased bacterial binding results in increased IL-8 production. This was clearly demonstrated in experiments using cftr -/- transgenic mice which were also found to have increased proinflammatory responses to P. aeruginosa infection.20
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Bacterial gene products that stimulate IL-8 expression Pili P. aeruginosa pili were studied to establish that they mediate epithelial adherence and inflammation and are essential in the pathogenesis of airway infection. P. aeruginosa and other mucosal pathogens such as V. cholera and Neisseriae, express type IV pili. These are relatively well conserved 15–16 kDa appendages which mediate the transfer of DNA among the bacteria and also serve to ligate glycolipid receptors on eukaryotic cells. An 8 amino acid adhesin domain has been mapped and includes a di-sulfide bridge, which is essential for binding. The antigenically dominant region of pili is not the same as the binding domain. In addition, pili are glycosylated, a modification that further complicates their antigenicity. Piliated P. aeruginosa, but not pil mutants can colonise neonatal mice and cause pulmonary inflammatory responses. The expression of these type IV pili is required for P. aeruginosa to cause invasive infection. The secretion of many P. aeruginosa toxins which act within eukaryotic cells requires pilin-mediated attachment. Purified pili in vitro are potent stimuli for epithelial IL-8 expression. Sheared pili from the surface of organisms are sufficient to stimulate epithelial IL-8 expression by themselves, even in the absence of intact bacteria.14 While pili are antigenic, there is relatively little cross-reactivity among native P. aeruginosa pili. Thus, antibody raised to one pilin type does not necessarily provide cross protection to prevent infection from another strain of P. aeruginosa with a different pilin type. While pili have been proposed as a potential vaccine antigen, their antigenic diversity has precluded the development of a clinically useful vaccine.
Flagella P. aeruginosa also express polar flagella, 45–53 kDa appendages which provide motility and chemotaxis toward preferred carbon sources. Flagella are important in the pathogenesis of lung disease,14 they are highly immunogenic and have far fewer antigenic types than pilin. Flagella are an important ligand for non-opsonic phagocytosis and also bind mucin components, facilitating mucociliary clearance. In a mouse infection model, the introduction of flagella into the lungs of a neonatal mouse causes intense inflammation, virtually indistinguishable from that induced by wild-type bacteria. In vitro, flagella have been shown to activate epithelial Ca2+ fluxes, nuclear factor (NF)κB, and IL-8 expression. Flagella are shed into the culture media in vitro, and are likely to be present in the airway. Thus, organisms with either attached flagella or secreted flagella can activate airway epithelial cells through ligation of asialylated glycolipid signalling systems. Flagella also activate
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the expression of epithelial mucin and are under consideration as a potential vaccine candidate to try to induce antibodies that would block the initial colonisation stage of P. aeruginosa infection. LPS P. aeruginosa lipopolysaccharide is a potent stimulus of macrophages and T cells. While LPS was thought to signal through CD14, a cell surface receptor of macrophages, this is not sufficient to mediate pro-inflammatory signalling by itself. As described below, CD14 along with the toll-like receptors signal LPS. Though CD-14 is not found on the surface of epithelial cells, soluble CD-14 and lipopolysaccharide binding protein are constituents of normal alveolar fluids. Their concentration increases more than 10-fold in the BAL fluid of patients of acute lung injury. In the airways, P. aeruginosa LPS can stimulate NFκB activation and mucin gene expression but does not stimulate IL-8 production by airway epithelial cells. Moreover, its effects on the airway cells require prolonged (24– 48 hours) exposure, unlike the brief (few minutes or less) exposure of flagella or pili that is sufficient to induce the Ca2+ fluxes associated with NFκB activation and IL-8 transcription. LPS is abundantly shed from actively growing bacteria and could be important in stimulating host responses. LPS has been shown to stimulate mucin gene expression through an NFκB-dependent pathway.1 More importantly, LPS is an effective activator of macrophages, activating pro-inflammatory signalling cascades through recognition of the toll-like receptors. G-CSF and GM-CSF expression in the airway In addition to IL-8 the cytokines G-CSF and GM-CSF are expressed by pulmonary epithelial cells and are important in both activating and maintaining PMN’s at the site of infection. Increased amounts of GM-CSF are produced by cells with CFTR mutations, similar to the findings with IL-8.21 GM-CSF has many effects on the immune functions of the lung. It is important in the normal lipid metabolism of alveolar macrophages and surfactant homeostasis.22 GM-CSF-deficient mice have significantly increased susceptibiltiy to infection with Group B Streptococci.23 Activation of the signalling pathways which lead to epithelial IL-8 expression, through NF-κB, also stimulate these NFκB-dependent cytokines. Thus, the epithelial response to pathogenic bacteria includes the expression of many signalling components to activate immune defences of the lung. Signalling through toll-like receptors in the lung Lipopolysaccharide and bacterial peptidoglycan also stimulate professional immune cells in the lung through
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the ligation of TLR’s (toll-like receptors).24 These are transmembrane receptors that activate a downstream signalling pathway resulting in the activation of NFκB and expression of pro-inflammatory cytokines and chemokines. TLR’s share a common structure with extracellular repeats of leucine-rich domains, which participate in ligand recognition. The cytoplasmic portion is homologous to the intracellular signalling domain of the type 1 IL-1 receptor. The extracellular leucine-rich repeat domains recognise bacterial ligands and the intracellular domains trigger the activation of transcription factor NFκB leading to activation of pro-inflammatory genes. Several adaptor proteins are also important in this signalling cascade and include CD14, found in macrophages and other immune cells. Similarly, while airway epithelial cells express both TLR2 and TLR4 their contribution to epithelial-derived inflammatory responses remains to be established. TLR-2 is a signal transducer for peptidoglycan and lipotechoic acid, stimulatory components of Gram positive bacteria and functions as the pattern recognition factor for other diverse bacterial products such as membrane lipoproteins and lipopeptides from B. burgdorferi, M. avium, and T. pallidum.25
EPITHELIAL SIGNALLING PATHWAYS Activation of NFκB Interaction of bacterial ligands with their receptors on epithelial cells elicits a specific response. In a screening study using A549 lung epithelial cells the selective activation of certain proinflammatory genes was observed in response to adherent, piliated P. aeruginosa, as opposed to non-adherent mutants.26 Many of these pro-inflammatory responses, such as IL-8 expression, are dependent upon
Figure 1 Innate defences of the respiratory epithelium.30
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gene transcription. The genes involved in chemokine and cytokine expression are regulated by NFκB, a transcription factor that is mobilised to the cell nucleus in response to bacterial stimulation. NFκB exists in the cytoplasm as a phosphorylated complex with an inhibitor IκB. Upon stimulation IκB’s are dephosphorylated and targeted to the proteosome for degradation. NFκB components then translocate to the nucleus where they can bind appropriate sequences and initiate transcription. The activation of airway epithelial cells by P. aeruginosa pili or adherent bacteria was shown to involve the translocation of NFκB and transcription of IL-8. Since the activation of NFκB leads to the expression of many other proinflammatory responses as well, this process is important in the general response to infecting organisms.
Ca2+ -dependent epithelial activation Many cellular signalling pathways are mediated by changes in the intracellular concentration of calcium, [Ca2+ ]i , which may be transient and rapidly regulated. Several mucosal pathogens activate host responses by changing the local [Ca2+ ] including the pulmonary pathogens that recognise asialylated glycolipid receptors.27 Ligation of the asialoGM1 receptor by either P. aeruginosa or S. aureus was shown to stimulate the release of [Ca2+ ] followed by the activation of p38 and ERK (extracellular response kinase) 1/2 mitogen-activated kinases, NF-κB activation and IL-8 expression. This pathway could be stimulated by other organisms that ligate the asialoGM1 receptor as presented on the apical surfaces of airway epithelial cells. For example, S. aureus strains that recognise the asialoGM1 receptor, but not mutants which bind elsewhere, activated this Ca2+ -dependent signalling cascade (Fig. 2).
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and the antimicrobial activities of the airway secretions, enables organisms to persist in the airway lumen. Both adherent bacteria and shed gene products are potent stimuli for epithelial IL-8 expression, activating epithelial signalling pathways and resulting in NFκB translocation and IL-8 expression. This serves to recruit PMN’s from the circulation and mobilise them to the airway to respond to the perceived infection. The analysis of the exaggerated response of cells with abnormal CFTR function to bacteria has been especially useful, not only in explaining the airway pathology characteristic of CF lung disease, but in defining the normal responses to inhaled bacteria.
REFERENCES Figure 2 Activation of signalling pathways in airway epithelial cells (modified from Prince A. Bacterial induction of cytokine secretion in pathogenesis of airway inflammation. In: Virulence Mechanisms of Bacterial Pathogens, 3rd edition, Ed. Brogden KA, Roth JA, Stanton TB, Bolin CA, Minion FC, Wannemuchler MJ 2000, ASM Press, Washington, D.C.).
BACTERIAL INVASION OF THE AIRWAY EPITHELIUM— CONSEQUENCES OF THE TYPE III SECRETION SYSTEMS In animal models of infection as well as in immunocompromised hosts, airway infection can lead to invasion, bacteraemia, sepsis and mortality. With regard to P. aeruginosa, invasion from the respiratory tract appears to be limited chiefly to strains that bind via pili28 and are able actively to secrete specific virulence factors into the mucosal cells through type III secretion systems.29 These toxins, such as exoenzyme S and T, interact with specific epithelial cell components and interfere with the normal function of the actin cytoskeleton. Thus, they are able to interfere with the normal production of tight junctions and enable organisms to invade paracellularly. Invasive bacteria gain access to the blood stream where, in the absence of opsonic antibody, they cause high-grade bacteraemia and sepsis. These invasive organisms must coordinate their expression of pili to facilitate close apposition of bacteria and the epithelial cells, as well as an intact type III secretion system. These virulence factors also trigger epithelial apoptosis, further enabling the organism to invade beyond the mucosal barrier and cause disseminated infection.29
SUMMARY Bacteria are capable of interacting with airway epithelial cells by way of a number of discrete ligands which stimulate pro-inflammatory responses. Failure of the normal innate clearance mechanisms, mucociliary clearance
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type II epithelial cell hyperplasia in transgenic mice. Am J Physiol 1997; 273: L715–725. LeVine AM, Reed JA, Kurak KE, Cianciolo E, Whitsett JA. GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection. J Clin Invest 1999; 103: 563–569. Kaisho T, Akira S. Critical roles of Toll-like receptors in host defense. Crit Rev Immunol 2000; 20: 393–405. Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, Carroll JD, Espevik T, Ingalls RR, Radolf JD, Golenbock DT. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 1999; 274: 33419–33425. Ichikawa JK, Norris A, Bangera MG, Geiss GK, van ’t Wout AB, Bumgarner RE, Lory S. Interaction of Pseudomonas aeruginosa with epithelial cells: identification of differentially regulated genes by expression microarray analysis of human cDNAs. Proc Natl Acad Sci USA 2000; 97: 9659–9664. Ratner AJ, Bryan R, Weber A, Nguyen S, Barnes D, Pitt A, Gelber S, Cheung A, Prince A. Cystic fibrosis pathogens activate Ca2+ -dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells. J Biol Chem 2001; 276: 19267–19275. Lee A, Chow D, Haus B, Tseng W, Evans D, Fleiszig S, Chandy G, Machen T. Airway epithelial tight junctions and binding and cytotoxicity of Pseudomonas aeruginosa. Am J Physiol 1999; 277: L204–217. Rajan S, Cacalano G, Bryan R, Ratner AJ, Sontich CU, van Heerckeren A, Davis P, Prince A. Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am J Respir Cell Mol Biol 2000; 23: 304–312. Ratner AJ, Prince A. Lactoperoxidase. New recognition of an “old” enzyme in airway defenses. Am J Respir Cell Mol Biol 2000; 22: 642–644.
SERIES: NEW BIOLOGY OF THE AIRWAYS
Host–bacterial interactions in the initiation of inflammation D. Rastogi, A. J. Ratner and A. Prince∗ College of Physicians & Surgeons, Columbia University, Black Building Room 416, 650 West 168th Street, New York, New York 10032, USA KEYWORDS inflammation, P. aeruginosa, cystic fibrosis.
Summary The respiratory epithelium provides both a physical and an immunological barrier to inhaled pathogens. In the normal host, innate defences prevent bacteria from activating inflammation by providing efficient muco-ciliary clearance and antimicrobial activity. Bacteria that persist in the airway lumen, as in cystic fibrosis, activate both the professional immune cells in the respiratory mucosa as well as the more abundant airway epithelial cells. As most of the bacteria become entrapped in airway mucin, shed bacterial products such as pili, flagella, peptidoglycan and lipopolysaccharide from lysed bacteria are likely to be the stimuli most important in activating epithelial signalling. The airway cells respond briskly to bacterial components through several signalling systems which activate epithelial expression of pro-inflammatory cytokines and chemokines. These signals recruit neutrophils to the airways where they eliminate the contaminating bacteria causing C 2001 Harcourt Publishers Ltd inflammation and the ensuing clinical signs of infection. °
INTRODUCTION The normal host defences of the airway epithelium are proficient in preventing infection of the respiratory tract. Despite repeated exposures to a wide variety of potentially pathogenic organisms, the lower respiratory tract usually remains sterile and the host is unaware of the ongoing clearance mechanisms. However, when bacterial contamination of the airways overwhelms the routine muco-ciliary defences and polymorphonuclear leukocytes (PMN’s) are recruited, the resulting inflammation, whilst serving to clear the infecting organisms, also causes the signs and symptoms of respiratory tract infection. An exaggerated host response to bacterial contamination of the airways and the resulting PMN-dominated inflammation is characteristic of cystic fibrosis (CF). In examining the interactions between the CF airway epithelium and specific bacterial pathogens such as Pseudomonas aeruginosa, a great deal has been learned, not only about how the
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Correspondence to: Alice Prince; Tel: +1 212 305-4193; Fax: +1 212 305-2284; E-mail: [email protected]
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CF patient responds to airway infection but also how the normal airway clears contaminating bacteria. This review explores how innate defence mechanisms prevent bacteria from reaching the surface of the airway epithelial cell, what happens when bacteria or bacterial components activate epithelial or immune cells in the lung and which bacterial components are involved in stimulating airway inflammation. In addition, the epithelial signalling pathways which evoke inflammation will be reviewed as well as some of the cytokines and chemokines responsible for recruiting inflammatory cells into the lung.
INNATE DEFENCES The innate defences of the airway mucosa are complex, consisting of several physical, cellular and antimicrobial components (Fig. 1). These defences do not necessarily require the coordination of the ‘professional’ immune system (macrophages, T cells and B cells) which is also involved in bacterial clearance, nor do they require previous experience with a specific antigen or infecting agent. Mechanical defences prevent particulate matter and micro-organisms from entering the lung. They begin at ° C 2001 Harcourt Publishers Ltd
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the nose which functions as a filter. Large particulate matter is trapped in nasal hair or fimbriae and particles >10 µm are caught on the surface of the turbinates and septum. Smaller particles, including bacteria, ranging from 2–10 µm are inhaled and deposited in the lower airways. The airways between the larynx and the respiratory bronchioles are lined with ciliated columnar epithelium covered by fluid 5 to 100 µm thick. The fluid consists of a periciliary layer adjacent to the epithelial surface and a layer of mucus which appears to float on top. The composition of the mucus is determined by the epithelial glycoproteins, mostly sialo- and sulfoglycoproteins, and proteoglycans secreted by Goblet and serous cells. Ciliated cells and the Clara cells of the distal epithelium also contribute to the mucus production. The airway mucins are high-molecular weight glycoproteins which are secreted into the airway as well as being anchored to the epithelial surface by trans-membrane domains. Many bacteria are bound to mucins and swept away by the ‘muco-ciliary escalator’. For example P. aeruginosa, a ubiquitous opportunistic pathogen, is a common contaminant of sinks, showers and puddles. When inadvertently inhaled by the normal host, the organisms are efficiently cleared without eliciting any inflammatory response. Normal innate defences prevent ‘professional’ immune cells from having to process P. aeruginosa antigens. Even laboratory workers exposed to these bacteria in large numbers do not produce high titres of antibody against them. P. aeruginosa flagella, (long appendages involved in motility and chemotaxis) bind to mucin and are physically removed. The expression of specific mucin genes by airway epithelial cells are up-regulated in response to bacterial ligands.1 Thus, contact with the organisms stimulates mucin expression to facilitate mechanical clearance. In CF, due to failure of normal CFTR (cystic fibrosis transmembrane conductance regulator) Cl− channel function, airway mucin is dehydrated, forming a mechanical obstruction which blocks the muco-ciliary clearance.2
Antimicrobial agents in the airway Lysosyme, lactoperoxidase, lactoferrin The airways also contain several types of antimicrobial proteins with a broad range of antimicrobial activity.3 Lysosyme and lactoferrin are particularly abundant and have very potent antimicrobial activity efficiently killing both Gram negative and Gram positive organisms. Lactoperoxidase was recently demonstrated to be expressed in the airways and significantly inhibits the growth of many microbes.4 Surfactant proteins The surfactant proteins A and D, members of the collectin family also participate in bacterial clearance. These have
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been studied by comparing the susceptibility of transgenic mice lacking either surfactant protein to pulmonary infection caused by various pathogens. The Sp-A null mice were particularly susceptible to P. aeruginosa infection.5 Antimicrobial peptides The airway mucosa is also protected by small peptides which have potent antibacterial activity. Several classes of these have been described and their properties established. They lyse Gram negative pathogens by intercalating through the lipopolysaccharide and forming pores in the cytoplasmic membrane. The identification and classification of the related defensins in the airway epithelium was first detailed in the bovine tissue and the human homologues were identified shortly thereafter. Epithelial cells produce the β-defensins (types 1 and 2) both of which have been isolated from bronchoalveolar lavage fluids. Several different peptides have been recently identified including a member of the cathelicidin family LL-37/CAP-18.6 Diminished airway defensin activity has been postulated to contribute to bacterial contamination of the lungs in CF.6 Defensin function in CF In a series of experiments performed by Smith et al., airway epithelial cell secretions in vitro were able to kill a small inoculum of P. aeruginosa added to the secretions in vitro.7 Cell culture supernatants from CF cells were deficient in their ability to kill the organisms and this defect could be reproduced in the normal cell culture supernatant by the addition of high concentrations of NaCl.7 As the defensins are known to be NaCl sensitive in their antibacterial activity, a likely conclusion was that the Na+ in the CF lung airway fluid (expected to be increased as a consequence of CFTR-Cl− channel dysfunction) inhibits defensin activity causing the increased susceptibility to infection in CF. Studies from the Wilson laboratory further suggested that human defensin-1 was, in fact, the NaClsensitive defensin which was associated with this defect in CF.8 These investigators ‘corrected’ the bacterial killing defect by expression of a wild-type CFTR in the epithelial cells. However, this hypothesis has now been challenged. There is no consensus that the NaCl composition of the airway surface fluid in the CF lung in vivo, is ever high enough to inhibit defensin activity to the degrees suggested by the in vitro data. Moreover, the contribution of human defensin to the total mix of anti-microbial agents on the airway surface is difficult to ascertain. There is little evidence that defensins are critical in the clearance of S. aureus, another major CF pathogen. There is little doubt that the antimicrobial peptides, lactoferrin and lysosyme are very important in the normal protection of the mucosa but it is less clear if a relative dysfunction
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of them is a major factor in the pathogenesis of bacterial infection in CF.
IMMUNE CELLS IN THE LUNG Bacteria which elude these innate defences and contaminate the lower respiratory tract stimulate a brisk inflammatory response. The inflammation may be signalled either by the professional immune cells which provide a surveillance function in the lung, such as alveolar macrophages, or may be transmitted by dendritic cells which are stimulated by bacterial gene products. PMN’s are recruited to the site of infection to phagocytose and kill the organisms. Macrophages and other cells of immune origin have different types of responses to specific bacterial gene products than do the airway epithelial cells.
Macrophages Macrophages play a central role in host–bacterial interactions. They are the most prevalent non-parenchymal cells in the airways of normal subjects and are important regulators of airway inflammation. Their origin is either from the bone-marrow-derived blood monocytes or by local multiplication. The former is mainly responsible for the increase in their numbers during inflammation. Alveolar macrophage function is dependent upon mobility, phagocytic activity, expression of receptors and their ability to signal other immune cells through the expression of chemokines and cytokines. Many of their functions are turned off unless they are specifically activated by organisms or cytokines. Macrophages have different affinities for microbial components compared with epithelial cells and are activated by whole organisms or small components of the organisms such as lipopolysaccharide. On activation, alveolar macrophages produce enzymes (acid hydrolases, neutral hydrolases and lysozymes), arachidonic acid derivatives, reactive oxygen metabolites, cytokine and other bioactive peptides to kill the organisms and to recruit additional immune cells. Macrophages also regulate the functions of the dendritic cells, the primary antigen presenting cells of the airway.
PMN’s Neutrophils are the most important cells recruited to the airway after exposure to a pathogen. Their primary function is to recognise, phagocytose and destroy the pathogens. This is accomplished through opsonisation followed by Fc-mediated binding or antigen recognition using complement receptors. The pathogen is ingested and killed in the PMN phagosome through the expression of peptides and reactive oxygen intermediates. Neutrophils release lipid mediators, leukotrienes and reactive oxygen species which are important in bacterial killing and in the inflammatory response. Whilst neutrophils are critically important to phagocytose and kill bacteria, their
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own lysis and release of elastase is immunostimulatory by itself. PMN elastase is a potent stimulus of epithelial interleukin-8 (IL-8) expression by the airway cells, further serving to promote a cycle of continued inflammation. In CF, excessive amounts of neutrophil elastase are thought to contribute to the cleavage of appropriate complement receptors and associated ligands, impeding efficient phagocytosis.9 DNA from lysed PMN’s is a major contributing factor to the increased viscosity of CF airway secretions.
AIRWAY EPITHELIAL CELLS AND THEIR RESPONSE TO BACTERIA The most numerous cells lining the airways are the airway epithelial cells which have evolved complex signalling pathways to enable them to respond to the threat of bacterial infection. They are probably most important in providing a surveillance function for the common opportunistic pathogens, such as S. aureus and P. aeruginosa. They actively respond to adherent and invasive bacteria as well as to isolated bacterial gene products. Their major role is to signal the presence of the pathogen and recruit the influx of PMN’s to remove the organisms. This inflammatory response is controlled primarily by proinflammatory cytokines. These are small polypeptides or glycoproteins between 10 and 30 kDa produced by the respiratory epithelial cell in response to different stimuli. They are not stored in the cells but are actively synthesised for secretion. Cytokines generally act locally by binding to cellular receptors and activating gene expression. An important function of these chemicals is cell to cell communication, which can be autocrine, paracrine or endocrine in mechanism. Pro-inflammatory cytokines help coordinate the local response to bacterial infection. While epithelial cells are capable of expressing numerous cytokines and chemokines, here, the discussion is focused on those shown to be important in CF-associated airway inflammation.
Interleukin-8 (IL-8) IL-8 is a prototype of the family of cytokines which are chemotactic for neutrophils and monocytes. It is synthesised by the circulating monocytes, macrophages, fibroblasts, endothelial cells, and epithelial cells. Endotoxin (LPS), tumour necrosis factor (TNF)α, IL-1, granulocytemacrophage colony-stimulating factor (GM-CSF), lectins, immune complexes and phagocytosis all stimulate IL-8 production.
Recruitment and activation of PMN’s in the airway by airway epithelial cells For PMN’s to migrate to the site of bacterial infection, they must be recruited and activated by local signals. This complex process involves the local expression of
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chemokines such as IL-8 as well as the upregulation of cellular adhesion molecules to signal the circulating PMN’s to a site and direct their migration toward the increased amount of chemokine expression. The activated PMN’s secrete many products that play an important role in microbial killing10 but may also have major toxic effects on the host tissues. Additional cytokines such as granulocytecolony stimulating factor (G-CSF) and GM-CSF act to prolong the half-lives of PMN’s by inhibiting their apoptosis. While this induced inflammatory response results in the local accumulation of PMN’s to phagocytose and eradicate the bacteria, other consequences of inflammation on the host, airway oedema, bronchospasm and consolidation can impair the normal function of air-exchange producing the clinical signs and symptoms of pneumonia. In airway diseases such as CF, the failure to resolve such episodes of local inflammation may eventually result in chronic inflammation and fibrotic changes.
BACTERIAL INDUCTION OF HOST DEFENCES IN CF Bacteria which evade the activity of antimicrobial peptides and mucociliary clearance may persist in the airway lumen and replicate in the absence of effective host defences. These organisms can then remain sequestered within the airway secretions and adapt to this milieu, as occurs in CF, or invade the mucosal barrier in susceptible hosts to cause sepsis, a more common complication in immunocompromised hosts. The lung, unlike other mucosal surfaces such as the gastrointestinal epithelium or the urogenital tract, is normally sterile. Thus, even the presence of superficial organisms is sufficient to activate an epithelial response. In the CF lung the vast majority of organisms are entrapped in mucin and surrounded by a florid PMN response. They form a biofilm but few, if any, bacteria are seen directly apposed to the epithelial cells. Histopathological studies of CF lung tissue do not demonstrate any intraepithelial bacteria, nor is sepsis seen, despite the huge intralumenal burden of bacteria, except as a terminal event. Bacterial invasion of the airway mucosal cells by the common CF pathogens is highly unusual. In a fatal case of B. cepacia infection, organisms were shown to be invasive.11 The presence of common pathogens such as P. aeruginosa within airway epithelial cells seems limited to experimental models of infection or to cells in culture. Initial reports that CFTR might function as a receptor for internalisation of P. aeruginosa have not been corroborated by data from patients. Studies using transgenic mice did not confirm any relationship between the level of CFTR expression in the airway epithelium and the ingestion or invasion of the epithelial cell by P. aeruginosa.12 Superficial interactions between the common CF pathogens, S. aureus, H. influenzae and P. aeruginosa and
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the respiratory epithelium are sufficient to cause inflammation as documented in many histopathological studies of the CF lung.13 Even very early in the disease process, before bacteria are consistently isolated from pulmonary secretions, broncho-alveolar lavage studies consistently show a marked pro-inflammatory response. While there may be endogenous factors that contribute to this inflammation, clearly the presence of bacteria in the airway lumen is a sufficient stimulus to activate epithelial cells. As most of the organisms are entrapped in mucin, it seems likely that shed bacterial components as well as organisms that become adherent to the epithelial cells themselves, are capable of stimulating pro-inflammatory responses. Epithelial production of IL-8 is particularly important in recruiting and stimulating PMN’s in the lung and is widely used as a clinical marker of inflammation. Several laboratories have examined which bacterial components activate epithelial IL-8 expression to recruit PMN’s into the airways. These include both adherent intact organisms as well as isolated bacterial gene products which may be present in the airway lumen, even in the absence of viable bacteria.14
BACTERIAL ACTIVATION OF HOST INFLAMMATORY RESPONSES Adherent organisms stimulate epithelial IL-8 expression Many pulmonary pathogens bind to the GalNAc β1-4Gal moiety which is available on cells with asialylated glycolipids.15 These include the common respiratory pathogens S. aureus, H. influenzae and P. aeruginosa. While such asialylated receptors are not normally available on the airway surfaces to any great degree, they are significantly increased in areas of cell damage and regeneration.16 Cells with CFTR mutations have increased amounts of asialylated glycoconjugates and hence, more adherent organisms.17,18 There is a direct correlation between adherent P. aeruginosa and the amount of IL-8 expression in epithelial cells. Cells with CFTR mutations, or cell lines specifically constructed to lack normal CFTR expression were found to have increased numbers of adherent P. aeruginosa, which correlated with the increased numbers of asialylated glycolipid receptors. For P. aeruginosa, epithelial binding is mediated by pili, small polar appendages which bind directly to the GalNAcβ1-4Gal moiety of asialylated glycolipids, as well as by non-pilin adhesins. The significance of this interaction is that pilin mediated adherence is a potent stimulus of epithelial IL-8 expression.19 Therefore, increased bacterial binding results in increased IL-8 production. This was clearly demonstrated in experiments using cftr -/- transgenic mice which were also found to have increased proinflammatory responses to P. aeruginosa infection.20
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Bacterial gene products that stimulate IL-8 expression Pili P. aeruginosa pili were studied to establish that they mediate epithelial adherence and inflammation and are essential in the pathogenesis of airway infection. P. aeruginosa and other mucosal pathogens such as V. cholera and Neisseriae, express type IV pili. These are relatively well conserved 15–16 kDa appendages which mediate the transfer of DNA among the bacteria and also serve to ligate glycolipid receptors on eukaryotic cells. An 8 amino acid adhesin domain has been mapped and includes a di-sulfide bridge, which is essential for binding. The antigenically dominant region of pili is not the same as the binding domain. In addition, pili are glycosylated, a modification that further complicates their antigenicity. Piliated P. aeruginosa, but not pil mutants can colonise neonatal mice and cause pulmonary inflammatory responses. The expression of these type IV pili is required for P. aeruginosa to cause invasive infection. The secretion of many P. aeruginosa toxins which act within eukaryotic cells requires pilin-mediated attachment. Purified pili in vitro are potent stimuli for epithelial IL-8 expression. Sheared pili from the surface of organisms are sufficient to stimulate epithelial IL-8 expression by themselves, even in the absence of intact bacteria.14 While pili are antigenic, there is relatively little cross-reactivity among native P. aeruginosa pili. Thus, antibody raised to one pilin type does not necessarily provide cross protection to prevent infection from another strain of P. aeruginosa with a different pilin type. While pili have been proposed as a potential vaccine antigen, their antigenic diversity has precluded the development of a clinically useful vaccine.
Flagella P. aeruginosa also express polar flagella, 45–53 kDa appendages which provide motility and chemotaxis toward preferred carbon sources. Flagella are important in the pathogenesis of lung disease,14 they are highly immunogenic and have far fewer antigenic types than pilin. Flagella are an important ligand for non-opsonic phagocytosis and also bind mucin components, facilitating mucociliary clearance. In a mouse infection model, the introduction of flagella into the lungs of a neonatal mouse causes intense inflammation, virtually indistinguishable from that induced by wild-type bacteria. In vitro, flagella have been shown to activate epithelial Ca2+ fluxes, nuclear factor (NF)κB, and IL-8 expression. Flagella are shed into the culture media in vitro, and are likely to be present in the airway. Thus, organisms with either attached flagella or secreted flagella can activate airway epithelial cells through ligation of asialylated glycolipid signalling systems. Flagella also activate
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the expression of epithelial mucin and are under consideration as a potential vaccine candidate to try to induce antibodies that would block the initial colonisation stage of P. aeruginosa infection. LPS P. aeruginosa lipopolysaccharide is a potent stimulus of macrophages and T cells. While LPS was thought to signal through CD14, a cell surface receptor of macrophages, this is not sufficient to mediate pro-inflammatory signalling by itself. As described below, CD14 along with the toll-like receptors signal LPS. Though CD-14 is not found on the surface of epithelial cells, soluble CD-14 and lipopolysaccharide binding protein are constituents of normal alveolar fluids. Their concentration increases more than 10-fold in the BAL fluid of patients of acute lung injury. In the airways, P. aeruginosa LPS can stimulate NFκB activation and mucin gene expression but does not stimulate IL-8 production by airway epithelial cells. Moreover, its effects on the airway cells require prolonged (24– 48 hours) exposure, unlike the brief (few minutes or less) exposure of flagella or pili that is sufficient to induce the Ca2+ fluxes associated with NFκB activation and IL-8 transcription. LPS is abundantly shed from actively growing bacteria and could be important in stimulating host responses. LPS has been shown to stimulate mucin gene expression through an NFκB-dependent pathway.1 More importantly, LPS is an effective activator of macrophages, activating pro-inflammatory signalling cascades through recognition of the toll-like receptors. G-CSF and GM-CSF expression in the airway In addition to IL-8 the cytokines G-CSF and GM-CSF are expressed by pulmonary epithelial cells and are important in both activating and maintaining PMN’s at the site of infection. Increased amounts of GM-CSF are produced by cells with CFTR mutations, similar to the findings with IL-8.21 GM-CSF has many effects on the immune functions of the lung. It is important in the normal lipid metabolism of alveolar macrophages and surfactant homeostasis.22 GM-CSF-deficient mice have significantly increased susceptibiltiy to infection with Group B Streptococci.23 Activation of the signalling pathways which lead to epithelial IL-8 expression, through NF-κB, also stimulate these NFκB-dependent cytokines. Thus, the epithelial response to pathogenic bacteria includes the expression of many signalling components to activate immune defences of the lung. Signalling through toll-like receptors in the lung Lipopolysaccharide and bacterial peptidoglycan also stimulate professional immune cells in the lung through
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the ligation of TLR’s (toll-like receptors).24 These are transmembrane receptors that activate a downstream signalling pathway resulting in the activation of NFκB and expression of pro-inflammatory cytokines and chemokines. TLR’s share a common structure with extracellular repeats of leucine-rich domains, which participate in ligand recognition. The cytoplasmic portion is homologous to the intracellular signalling domain of the type 1 IL-1 receptor. The extracellular leucine-rich repeat domains recognise bacterial ligands and the intracellular domains trigger the activation of transcription factor NFκB leading to activation of pro-inflammatory genes. Several adaptor proteins are also important in this signalling cascade and include CD14, found in macrophages and other immune cells. Similarly, while airway epithelial cells express both TLR2 and TLR4 their contribution to epithelial-derived inflammatory responses remains to be established. TLR-2 is a signal transducer for peptidoglycan and lipotechoic acid, stimulatory components of Gram positive bacteria and functions as the pattern recognition factor for other diverse bacterial products such as membrane lipoproteins and lipopeptides from B. burgdorferi, M. avium, and T. pallidum.25
EPITHELIAL SIGNALLING PATHWAYS Activation of NFκB Interaction of bacterial ligands with their receptors on epithelial cells elicits a specific response. In a screening study using A549 lung epithelial cells the selective activation of certain proinflammatory genes was observed in response to adherent, piliated P. aeruginosa, as opposed to non-adherent mutants.26 Many of these pro-inflammatory responses, such as IL-8 expression, are dependent upon
Figure 1 Innate defences of the respiratory epithelium.30
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gene transcription. The genes involved in chemokine and cytokine expression are regulated by NFκB, a transcription factor that is mobilised to the cell nucleus in response to bacterial stimulation. NFκB exists in the cytoplasm as a phosphorylated complex with an inhibitor IκB. Upon stimulation IκB’s are dephosphorylated and targeted to the proteosome for degradation. NFκB components then translocate to the nucleus where they can bind appropriate sequences and initiate transcription. The activation of airway epithelial cells by P. aeruginosa pili or adherent bacteria was shown to involve the translocation of NFκB and transcription of IL-8. Since the activation of NFκB leads to the expression of many other proinflammatory responses as well, this process is important in the general response to infecting organisms.
Ca2+ -dependent epithelial activation Many cellular signalling pathways are mediated by changes in the intracellular concentration of calcium, [Ca2+ ]i , which may be transient and rapidly regulated. Several mucosal pathogens activate host responses by changing the local [Ca2+ ] including the pulmonary pathogens that recognise asialylated glycolipid receptors.27 Ligation of the asialoGM1 receptor by either P. aeruginosa or S. aureus was shown to stimulate the release of [Ca2+ ] followed by the activation of p38 and ERK (extracellular response kinase) 1/2 mitogen-activated kinases, NF-κB activation and IL-8 expression. This pathway could be stimulated by other organisms that ligate the asialoGM1 receptor as presented on the apical surfaces of airway epithelial cells. For example, S. aureus strains that recognise the asialoGM1 receptor, but not mutants which bind elsewhere, activated this Ca2+ -dependent signalling cascade (Fig. 2).
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and the antimicrobial activities of the airway secretions, enables organisms to persist in the airway lumen. Both adherent bacteria and shed gene products are potent stimuli for epithelial IL-8 expression, activating epithelial signalling pathways and resulting in NFκB translocation and IL-8 expression. This serves to recruit PMN’s from the circulation and mobilise them to the airway to respond to the perceived infection. The analysis of the exaggerated response of cells with abnormal CFTR function to bacteria has been especially useful, not only in explaining the airway pathology characteristic of CF lung disease, but in defining the normal responses to inhaled bacteria.
REFERENCES Figure 2 Activation of signalling pathways in airway epithelial cells (modified from Prince A. Bacterial induction of cytokine secretion in pathogenesis of airway inflammation. In: Virulence Mechanisms of Bacterial Pathogens, 3rd edition, Ed. Brogden KA, Roth JA, Stanton TB, Bolin CA, Minion FC, Wannemuchler MJ 2000, ASM Press, Washington, D.C.).
BACTERIAL INVASION OF THE AIRWAY EPITHELIUM— CONSEQUENCES OF THE TYPE III SECRETION SYSTEMS In animal models of infection as well as in immunocompromised hosts, airway infection can lead to invasion, bacteraemia, sepsis and mortality. With regard to P. aeruginosa, invasion from the respiratory tract appears to be limited chiefly to strains that bind via pili28 and are able actively to secrete specific virulence factors into the mucosal cells through type III secretion systems.29 These toxins, such as exoenzyme S and T, interact with specific epithelial cell components and interfere with the normal function of the actin cytoskeleton. Thus, they are able to interfere with the normal production of tight junctions and enable organisms to invade paracellularly. Invasive bacteria gain access to the blood stream where, in the absence of opsonic antibody, they cause high-grade bacteraemia and sepsis. These invasive organisms must coordinate their expression of pili to facilitate close apposition of bacteria and the epithelial cells, as well as an intact type III secretion system. These virulence factors also trigger epithelial apoptosis, further enabling the organism to invade beyond the mucosal barrier and cause disseminated infection.29
SUMMARY Bacteria are capable of interacting with airway epithelial cells by way of a number of discrete ligands which stimulate pro-inflammatory responses. Failure of the normal innate clearance mechanisms, mucociliary clearance
1. Li JD, Dohrman AF, Gallup M, Miyata S, Gum JR, Kim YS, Nadel JA, Prince A, Basbaum CB. Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease. Proc Natl Acad Sci USA 1997; 94: 967–972. 2. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95: 1005–1015. 3. Travis SM, Conway BA, Zabner J, Smith JJ, Anderson NN, Singh PK, Greenberg EP, Welsh MJ. Activity of abundant antimicrobials of the human airway. Am J Respir Cell Mol Biol 1999; 20: 872–879. 4. Gerson C, Sabater J, Scuri M, Torbati A, Coffey R, Abraham JW, Lauredo I, Forteza R, Wanner A, Salathe M, Abraham WM, Conner GE. The lactoperoxidase system functions in bacterial clearance of airways. Am J Respir Cell Mol Biol 2000; 22: 665–671. 5. LeVine AM, Kurak KE, Bruno MD, Stark JM, Whitsett JA, Korfhagen TR. Surfactant protein-A-deficient mice are susceptible to Pseudomonas aeruginosa infection. Am J Respir Cell Mol Biol 1998; 19: 700–708. 6. Bals R, Wang X, Zasloff M, Wilson JM. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA 1998; 95: 9541–9546. 7. Smith JJ, Travis SM, Greenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 1996; 85: 229–236. 8. Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. Human beta-defensin-1 is a salt sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997; 88: 553–560. 9. Tosi MF, Zakem H, Berger M. Neutrophil elastase cleaves C3bi on opsonized pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J Clin Invest 1990; 86: 300–308. 10. Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, Shapiro SD. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 1998; 4: 615–618. 11. Burns JL, Jonas M, Chi EY, Clark DK, Berger A, Griffith A. Invasion of respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia. Infect Immun 1996; 64: 4054–4059. 12. Chroneos ZC, Wert SE, Livingston JL, Hassett DJ, Whitsett JA. Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo. J Immunol 2000; 165: 3941–3950. 13. Burns JL, Gibson RL, McNamara S, Yim D, Emerson J, Rosenfeld M, Hiatt P, McCoy K, Castile R, Smith AL, Ramsey BW. Longitudinal
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