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The role of the collectin system in pulmonary defence
PAEDIATRIC RESPIRATORY REVIEWS (2001) 2, 70–75 doi: 10.1053/prrv.2000.0104, available online at http://www.idealibrary.com on
SERIES: NEW BIOLOGY OF THE AIRWAYS
The role of the collectin system in pulmonary defence J. Davies1, M.Turner2 and N. Klein2 1
Department of Paediatric Respiratory Medicine, Royal Brompton Hospital and Department of Gene Therapy, Imperial College, London; and 2Immunobiology Unit, Institute of Child Health, London, UK KEYWORDS collectin, mannose-binding lectin, surfactant protein, pulmonary infection, cystic fibrosis
Summary The human collectin system comprises the serum protein, mannosebinding lectin and the hydrophilic surfactant proteins A and D. The three proteins possess structural and functional similarities and are important components of innate immunity. Through a variety of mechanisms, including direct opsonisation and complement activation, they assist in host defence against a wide array of micro-organisms. Investigation of the roles of the surfactant proteins in pulmonary disease has been assisted recently by the development of transgenic knockout mice. Animals deficient in these proteins display susceptibility to certain bacterial and viral pathogens, stimulating research into the role of polymorphisms in these genes in human respiratory disease. The role of MBL in human pulmonary disease is less well established, although accumulating evidence suggests that it is a modifier for lung disease in tuberculosis and cystic fibrosis. © 2001 Harcourt Publishers Ltd
BACKGROUND
collagenous region. In each of the proteins three such chains assemble into a classical collagenous triple helix. The C-terminal region and these three chain subunits are then assembled into higher order oligomers, partially stabilised by disulphide bonds. As shown in Figure 1, the fully assembled MBL and SP-A molecules have a bouquet structure whereas SP-D has an elongated cruciform structure. Each of the proteins recognises carbohydrate arrays on the surface of micro-organisms, with the ability to differentiate self from non-self through three-dimensional structures that fail to bind terminal sugars on mammalian glycoproteins. The collectins exert their host defence functions through direct and indirect mechanisms, with evidence that they can act both as opsonins, to facilitate ingestion and killing of micro-organisms by phagocytes, and as initiators of complement-mediated bacterial lysis. They also influence the inflammatory response in a complex manner, which requires further elucidation. With regard to respiratory infection, more is known about the roles of SP-A and D, although recent evidence suggests that MBL may also be an important component of pulmonary innate immunity.
Despite the fact that normal adults breathe in over 7000 litres of air laden with a wide array of micro-organisms and inorganic particles every day, lower respiratory tract infection is rare in the healthy host. This is largely due to a series of effective defence mechanisms, comprising both the innate and adaptive arms of the immune system. This review will focus on one component of the innate system, the group of proteins named collectins. The collectins (so called because of their collagenous and lectin domains), together with the mucociliary escalator, professional phagocytes and other soluble proteins (lysozyme, lactoferrin, defensins), form the first line of airway defence. This defence is required early in an infective challenge, at a stage before either the humoral or cellmediated adaptive immune systems come into play. In man, the collectins identified include the hydrophilic surfactant proteins SP-A and SP-D and mannose-binding lectin (MBL). MBL, SP-A and SP-D possess structural and functional similarities, based on individual peptide chains, each of which is characterised by a lectin domain and a 1526–0550/01/010070+ 06 $35.00/0
© 2001 Harcourt Publishers Ltd
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
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surfactant volume, type II cell hyperplasia and an abundance of fat-laden macrophages.2
How do SP-A and SP-D enhance clearance of organisms? They have the potential to enhance clearance of organisms through a variety of mechanisms, which will be discussed briefly. For a more detailed discussion see Crouch.3 1.
Figure 1. Structures of the three human collectins. Each protein consists of several three chain subunits in which each chain comprises a collagenous region and carbohydrate recognition domains. MBL exists as a series of oligomers and the tetrameric form is illustrated. Mutations in exon 1 prevent normal function of the protein through lack of oligomer formation.
SURFACTANT PROTEINS-A AND D Pulmonary surfactant is a mixture of lipid and associated proteins, the main function of which is to protect the alveoli from the sheer forces associated with breathing, and to prevent alveolar collapse at low lung volumes.1 Four major surfactant proteins have been identified, falling into two distinct groups: SP-B and C are small hydrophobic proteins which are involved in lipid adsorption at the air/water interface (although recently, in vitro studies have suggested that they may also limit lung inflammation, by stabilising neutrophil membranes, and decreasing chemotaxis, respiratory burst and elastase release). In contrast, SP-A and D are larger, hydrophilic proteins, produced by alveolar type II cells and Clara cells of the distal conducting airways. As the structure and function of these proteins has been elucidated, it seems likely that their major role is in host defence. However, the proteins do appear to play a minor role in lowering surface tension, with SP-A involved in the formation of tubular myelin, a component of the surfactant lipids, and SP-D involved in regulation of surfactant protein homeostasis. The latter mechanism is incompletely understood, but SP-D knockout mice demonstrate a significant increase in
2.
3.
Binding to microbial surfaces (a) In some cases this can lead to enhanced recognition and phagocytosis by macrophages and neutrophils. This has been demonstrated in vitro for a variety of micro-organisms, but there are differences between the two surfactant proteins (Table 1). (b) Binding can directly reduce virulence, as is the case with SP-D and influenza A virus. Binding leads to inactivation of haemagglutinin and neuraminidase, (both essential virulence factors), and reduces viral infectivity. (c) In other cases, the proteins act in an opsonic fashion and are able to bind to specific macrophage receptors, several of which have now been identified. Stimulation of chemotaxis. Both SP-A and SP-D enhance chemotaxis of monocytes, neutrophils and alveolar macrophages. Enhancing leucocyte function, e.g. increasing intracellular killing, through stimulation of reactive oxygen or nitrogen species, effects on cytokine production, regulation of lymphocyte production and apoptosis.
In addition to effects on microbial pathogens, both SP-A and SP-D bind to allergens, including house dust mite and pollen, inhibit binding of IgE to allergens and block allergeninduced histamine release from basophils, suggesting a role in non-infective pulmonary diseases. Furthermore, a recent study has identified SP-D mRNA and protein in human tissues as diverse as brain, heart, gut, kidney and skin, raising the possibility that a much wider role exists for these collectins than has previously been thought. Not all activities of SP-A and SP-D are necessarily of benefit to the host. A recent study reported that SP-A binding to CMV enhanced entry of the virus into pneumocytes4, suggesting in certain cases the proteins may contribute to microbial pathogenesis. Similar hypotheses have been raised regarding SP-A and Mycobacterium tuberculosis and Pneumocystis carinii.
Genetics The genes encoding SP-A and D are located on the long arm of chromosome 10, in close proximity to MBL.5 The SP-A locus consists of two similar but non-identical genes,
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J. DAVIES ET AL.
Table 1 Reported interactions between each of the human collectins and respiratory pathogens.The majority of the data come from in vitro studies. Data from in vivo animal studies are in italics. Some of the interactions are thought to be detrimental to host defence; these are in bold type. AM = alveolar macrophage.
Bacteria P. aeruginosa S. aureus Group B streptococcus S. pneumoniae H. influenzae B. cepacia Klebsiella species Viruses RSV Influenza
HSV Mumps CMV Fungi Aspergillus fumigatus
SP-A
SP-D
MBL
Decreased airway clearance Increased inflammation AM and neutrophil phagocytosis Susceptibility of -/-mice Decreased airway clearance Increased inflammation AM and Neutrophil phagocytosis AM phagocytosis
AM phagocytosis
No significant binding
Neutrophil phagocytosis
Binds and activates complement
AM phagocytosis and killing
Aggregation
SP-A deficient mice more susceptible AM phagocytosis
Disease severity reduced by exogenous SP-D Increases aggregation Neutrophil binding No enhanced phagocytosis
Mycobacterium TB Pneumocystis carinii
Neutrophil phagocytosis
No binding Some binding Binds and activates complement Some binding
Binds to influenza A strains
AM phagocytosis Reduced infectivity Binding and entry into pneumocytes and AMs Binds
Cryptococcus neoformans Other Mycoplasma
Poor binding
Macrophage killing impaired in -/-mice AM binding May facilitate cell entry
SP-A1 and SP-A2, which are closely linked. It is thought that the trimeric subunit of SP-A normally arises from the association of two chains of SP-A1 and one chain of SPA2. The regulation of the SP genes is complex and incompletely understood; SP-A1 and A2 are situated head to head, indicating that they probably share regulatory elements. In vitro studies suggest up regulation by γ-interferon and epidermal growth factor, with inhibition by tumour necrosis factor (TNF)-α, lipopolysaccharide and transforming growth factor (TGF)-β, and a concentration-dependent effect of corticosteroids. Studies from human fetal lung indicate that SP-A1 is upregulated by cAMP and inhibited by glucocorticoids, whereas SP-A2 is constitutively expressed. Sequences upstream of SP-D are similar to those identified for other acute phase proteins, and glucocorticoids have been shown to upregulate expression of this protein. Polymorphisms have been identified in SP-A and also more recently in SP-D, although links between the latter
Neutrophil binding and uptake Aggregation
Binds Binds
Reduced AM phagocytosis Aggregation and AM binding
and disease states have not been described. Certain SP-A polymorphisms have been associated with risk factors for infant respiratory distress syndrome (IRDS). No association between polymorphisms in the surfactant collectins and pulmonary infection have been reported, although in vivo data on their role are available from animal models. Interestingly, mutations in SP-B have been associated with IRDS and with alveolar proteinosis.
Animal models of surfactant protein deficiency SP-A -/Contrary to the initial view that SP-A was closely involved in surfactant composition, newborn SP-A -/-mice demonstrate little disturbance in respiratory function or surfactant lipid metabolism. However, there is evidence of decreased clearance of a variety of pathogens (group B
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
streptococcus, Pseudomonas aeruginosa, Staphylococcus aureus and RSV) and more severe disease in response to these infections, with evidence of disease amelioration following the administration of exogenous SP-A6.
SP-D-/Unlike SP-A -/-animals, the phenotype of SP-D deficient mice is characterised by significant disturbance of surfactant homeostasis, with increased surfactant volume, type II cell hyperplasia and an abundance of fat-laden macrophages.2 Studies addressing the susceptibility of SP-D deficient mice to various pulmonary pathogens are awaited.
Secondary abnormalities in surfactant composition Surfactant proteins can be degraded in vitro by neutrophil elastase, a susceptibility which is further enhanced in the presence of oxidants. It is therefore of no surprise that surfactant composition is altered in chronic pulmonary disease states such as cystic fibrosis. Whether or not primary abnormalities in the surfactant proteins influence progression of such lung disease remains unknown.
MBL MBL was first isolated from the serum and liver of rabbits towards the end of the 1970s, and was later identified as an important serum component lacking in children with an opsonisation defect. Since then, much interest has focused on its role in host defence against a variety of infections.
Functions of MBL MBL has the potential to enhance clearance of microorganisms via several mechanisms. These include: acting directly as an opsonin, facilitating ingestion by phagocytes; activation of the lectin complement pathway leading to C3b and iC3b deposition on bacterial surfaces, mediating phagocytosis and killing; or activation of complement leading to the formation of membrane attack complexes and direct bacterial lysis. MBL also has an influence over inflammatory responses, for example, by influencing levels of cytokine production, although this requires further study.
Genetics Within both the structural (exon 1) and promoter regions of human MBL, polymorphisms have been identified which result in mutant forms of the protein incapable of forming high order oligomers.7 Of these, a glycine → aspartic acid mutation in codon 54 is relatively common in the British population with a gene frequency of 0.14, and results in
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expression of a protein incapable of normal polymerisation.8 Individuals homozygous for the mutation have profoundly reduced levels of MBL (≤10 ng/ml), median levels are approximately 350 ng/ml in heterozygotes and over 1600 ng/ml in wild type homozygous individuals. A second point mutation in codon 57 (glycine → glutamic acid) of the MBL gene, is common in Sub-Saharan Africans but rare in Caucasians, and a third mutation results in a cysteine for arginine substitution in codon 52. Polymorphisms have also been identified within the promoter region of MBL, in linkage disequilibrium with the structural mutations, and some result in insignificantly reduced protein levels. The frequency with which these mutations are found in the healthy population supports the concept that MBL is likely to play an adjunctive role in host defence. However, low levels of the protein have been shown to relate to an increased risk of recurrent infections, of meningococcal disease, and to an adverse outcome in HIV infection, although the latter is controversial.
MBL and lung disease Much less is known about the role of MBL in respiratory infection than is the case for SP-A and SP-D, where our understanding has been increased by studies on transgenic mice. There is no published animal model of MBL deficiency; in the mouse the development of such a model is complicated by the presence of two MBL genes, MBL-A and C, with the former encoding the primary functional serum collectin, and the latter encoding an essentially hepatic form of MBL. In vitro studies have, however, indicated that MBL binds to a variety of pulmonary pathogens, including S. aureus, various streptococci, H. influenzae and A. fumigatus9. In a study of the role of MBL in murine influenza infection, there was evidence of anti-viral activity and the protein was detected in respiratory secretions in response to infection. The case for this collectin playing a role in pulmonary defence has been strengthened by human epidemiological studies.
Human disease In a large hospital-based study, the presence of MBL mutations was examined in over 600 children.10 Increased susceptibility to infection was found in children who were either homozygous or heterozygous for mutations in MBL. Of 133 heterozygotes presenting with infection, the site of infection was the respiratory tract in over 30%. MBL has been detected, albeit in low levels, in human nasopharyngeal secretions and middle ear effusions, confirming that the activity of the protein is not restricted to the serum, and that a role for MBL in local airway defence is plausible.
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Tuberculosis It has been suggested that MBL may confer both beneficial and detrimental effects on the infected host. Tuberculosis, leprosy and leishmaniasis are examples of disease in which high levels of MBL may be disadvantageous. Theoretically, by mediating macrophage binding, MBL could enhance infectivity of Mycobacterium tuberculosis (mTB). This is supported by several epidemiological studies. Higher levels of MBL were seen in African adults with TB than in those without, and two further studies in Africa have found an unexpectedly low frequency of mutant MBL alleles in TB patients. In one of these, there seemed to be a beneficial effect on both pulmonary and, more significantly, meningeal infection.11
Cystic fibrosis Recently, a compelling epidemiological link has been made between MBL and respiratory infection in cystic fibrosis (CF). CF results from mutations in cystic fibrosis transmembrane conductance regulator protein (CFTR), a chloride ion channel situated in the apical membrane of epithelial cells. The pulmonary component of the disease is characterised by early and sustained respiratory infection with a narrow range of organisms, various respiratory viruses and S. aureus and H. influenzae in early life.12 From mid-childhood to adolescence, up to 85% of CF patients will become chronically infected with Pseudomonas aeruginosa, which, once acquired, is rarely eradicated. The organism protects itself within a thick layer of alginate, leading to an exaggerated but frustrated inflammatory response, which contributes greatly to the irreversible lung damage characteristic of this disease. B. cepacia is an important, but fortunately less prevalent, organism, which in humans is found almost exclusively within the CF airway. The significance of this pathogen relates to its high levels of transmissibility and antibiotic resistance and to the progression of septicaemia and multi-organ failure in approximately 20–25% of patients. Individuals with CF, even those with the same CFTR mutations and within families, display widely different clinical phenotypes. This has led to the search for genes other than CFTR that may modify disease progression, such as those involved in host defence and the inflammatory response. Garred et al. examined the effect of MBL polymorphisms on the progression of lung disease in a cohort of 149 CF patients.13 Within this group, the frequency of MBL mutations was similar to that observed in the healthy population (approximately 34% heterozygotes and 2–3% homozygotes). Low serum levels were observed as expected and did not differ from those observed in healthy individuals. Lung function, measured by FEV1 and FVC, was significantly lower in the group with MBL deficiency. A trend towards this was visible as early as 8 years of age, although this became significant only at 16 years of
J. DAVIES ET AL.
age. Although P. aeruginosa has been identified as one of the major contributory factors in progression of lung damage in CF, MBL-deficient patients were not found to have higher rates of pseudomonal infection. Rather, it appeared that, following pseudomonal colonisation, MBLdeficient patients experienced a marked decline in lung function, which was not apparent in the MBL-sufficient group and which appeared to adversely influence survival. MBL is known to affect inflammation in various ways and it seems likely therefore that the host response is in some way responsible for this clinical deterioration. This story is confused by results from another smaller study comparing patients deficient in MBL with controls closely matched for age and CF genotype (all homozygous ∆F508).14 In contrast to the earlier study, these authors only found a significant reduction in lung function in patients with severe MBL deficiency (homozygotes or compound heterozygotes). They also reported increased P. aeruginosa colonisation in these patients, although greater numbers would be required to confirm statistical significance. B. cepacia was not mentioned in this report, although in the first study the frequency of MBL mutations was significantly higher than would have been expected amongst 10 cepacia-colonised patients. These findings led us to examine a possible mechanism for these observations. Using strains obtained from the sputum of CF patients, we were able to demonstrate that MBL does not bind significantly to the surface of either mucoid or non-mucoid P. aeruginosa. However, high levels of binding were seen to B. cepacia.15 This binding led to the activation of complement, which in vivo would be expected to result in bacterial killing. Taken together, these results suggest that MBL is not likely to play a major role in the acquisition of P. aeruginosa. Once colonisation has taken place, however, there may be an adverse inflammatory response to the chronic presence of this organism in the airway of MBL-deficient patients, which accounts for the faster decline in pulmonary function. However, as MBL does bind significantly to B. cepacia, MBL-deficient patients may be at greater risk of acquiring this pathogen, a hypothesis which would be supported by the Garred study. We are currently in the process of examining the MBL status of cepacia-colonised adults with CF and preliminary data suggest that this will confirm the earlier study.
SUMMARY Both the pulmonary (SP-A and D) and serum (MBL) collectins, appear to be involved in the innate defence of the airway. They have broadly similar functions, aimed both directly at the micro-organism (opsonisation, complement activation and killing), and indirectly through modulation of the host inflammatory response, which are incompletely understood. Polymorphisms exist in the human genes encoding these proteins, and certain links
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
have been identified between deficiency states and predisposition to pulmonary infection. A further understanding of the effects of such polymorphisms, through both animal models and human studies, may lead to an increased understanding of disease pathogenesis, the identification of subgroups at risk and possibly to new therapeutic avenues. RESEARCH DIRECTIONS Laboratory and animal studies Work is continuing to examine the exact roles of the collectins both in killing of pathogens and in the host inflammatory response. Studies on SP-A and D knockout mice will seek to identify the roles of these proteins in a variety of lung diseases both infective and inflammatory. The development of a similar mouse model for MBL deficiency would greatly assist research in this area. Human disease Further epidemiological and laboratory studies are required to elucidate the role of the collectins in human lung diseases. As well as allowing identification of subgroups of patients at particular risk of certain infections or severe disease, results of such studies may lead to novel therapeutic approaches.
REFERENCES 1. Floros J, Kala P. Surfactant proteins: molecular genetics of neonatal pulmonary diseases. Annu Rev Physiol 1998; 60: 365–384.
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2. Botas C et al. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc Natl Acad Sci USA 1998; 95: 11869–11874. 3. Crouch EC. Collectins and pulmonary host defense. Am J Respir Cell Mol Biol 1998; 19: 177–201. 4. Weyer C, Sabat R, Wissel H, Kruger DH, Stevens PA, Prosch S. Surfactant protein A binding to cytomegalovirus proteins enhances virus entry into rat lung cells. Am J Respir Cell Mol Biol 2000; 23: 71–78. 5. Floros J, Hoover RR. Genetics of the hydrophilic surfactant proteins A and D. Biochim Biophys Acta 1998; 1408: 312–322. 6. LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J, Korfhagen T. Surfactant protein-A enhances respiratory syncytial virus clearance in vivo. J Clin Invest 1999; 103: 1015–1021. 7. Madsen HO et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol 1995; 155: 3013–3320. 8. Turner MW. Mannose-binding lectin (MBL) in health and disease. Immunobiology 1998; 199: 327–393. 9. Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000; 68: 688–693. 10. Summerfield JA, Sumiya M, Levin M, Turner MW. Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series. BMJ 1997; 314: 1229–1232. 11. Hoal-Van Helden EG et al. Mannose-binding protein B allele confers protection against tuberculous meningitis. Pediatr Res 1999; 45: 459–464. 12. Govan JR, Nelson JW. Microbiology of cystic fibrosis lung infections: themes and issues. J Royal Soc Med 1993; 86(Suppl 20): 11–18. 13. Garred P et al. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J Clin Invest 1999; 104: 431–437. 14. Gabolde M, Giulloud-Bataille M, Feingold J, Besmond C. Association of variant alleles of mannose binding lectin with severity of pulmonary disease in cystic fibrosis: cohort study. BMJ 1999; 319: 1166–1167. 15. Davies J, Neth O, Alton E, Klein N, Turner M. Differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis. Lancet 2000; 355: 1885–1886.
SERIES: NEW BIOLOGY OF THE AIRWAYS
The role of the collectin system in pulmonary defence J. Davies1, M.Turner2 and N. Klein2 1
Department of Paediatric Respiratory Medicine, Royal Brompton Hospital and Department of Gene Therapy, Imperial College, London; and 2Immunobiology Unit, Institute of Child Health, London, UK KEYWORDS collectin, mannose-binding lectin, surfactant protein, pulmonary infection, cystic fibrosis
Summary The human collectin system comprises the serum protein, mannosebinding lectin and the hydrophilic surfactant proteins A and D. The three proteins possess structural and functional similarities and are important components of innate immunity. Through a variety of mechanisms, including direct opsonisation and complement activation, they assist in host defence against a wide array of micro-organisms. Investigation of the roles of the surfactant proteins in pulmonary disease has been assisted recently by the development of transgenic knockout mice. Animals deficient in these proteins display susceptibility to certain bacterial and viral pathogens, stimulating research into the role of polymorphisms in these genes in human respiratory disease. The role of MBL in human pulmonary disease is less well established, although accumulating evidence suggests that it is a modifier for lung disease in tuberculosis and cystic fibrosis. © 2001 Harcourt Publishers Ltd
BACKGROUND
collagenous region. In each of the proteins three such chains assemble into a classical collagenous triple helix. The C-terminal region and these three chain subunits are then assembled into higher order oligomers, partially stabilised by disulphide bonds. As shown in Figure 1, the fully assembled MBL and SP-A molecules have a bouquet structure whereas SP-D has an elongated cruciform structure. Each of the proteins recognises carbohydrate arrays on the surface of micro-organisms, with the ability to differentiate self from non-self through three-dimensional structures that fail to bind terminal sugars on mammalian glycoproteins. The collectins exert their host defence functions through direct and indirect mechanisms, with evidence that they can act both as opsonins, to facilitate ingestion and killing of micro-organisms by phagocytes, and as initiators of complement-mediated bacterial lysis. They also influence the inflammatory response in a complex manner, which requires further elucidation. With regard to respiratory infection, more is known about the roles of SP-A and D, although recent evidence suggests that MBL may also be an important component of pulmonary innate immunity.
Despite the fact that normal adults breathe in over 7000 litres of air laden with a wide array of micro-organisms and inorganic particles every day, lower respiratory tract infection is rare in the healthy host. This is largely due to a series of effective defence mechanisms, comprising both the innate and adaptive arms of the immune system. This review will focus on one component of the innate system, the group of proteins named collectins. The collectins (so called because of their collagenous and lectin domains), together with the mucociliary escalator, professional phagocytes and other soluble proteins (lysozyme, lactoferrin, defensins), form the first line of airway defence. This defence is required early in an infective challenge, at a stage before either the humoral or cellmediated adaptive immune systems come into play. In man, the collectins identified include the hydrophilic surfactant proteins SP-A and SP-D and mannose-binding lectin (MBL). MBL, SP-A and SP-D possess structural and functional similarities, based on individual peptide chains, each of which is characterised by a lectin domain and a 1526–0550/01/010070+ 06 $35.00/0
© 2001 Harcourt Publishers Ltd
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
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surfactant volume, type II cell hyperplasia and an abundance of fat-laden macrophages.2
How do SP-A and SP-D enhance clearance of organisms? They have the potential to enhance clearance of organisms through a variety of mechanisms, which will be discussed briefly. For a more detailed discussion see Crouch.3 1.
Figure 1. Structures of the three human collectins. Each protein consists of several three chain subunits in which each chain comprises a collagenous region and carbohydrate recognition domains. MBL exists as a series of oligomers and the tetrameric form is illustrated. Mutations in exon 1 prevent normal function of the protein through lack of oligomer formation.
SURFACTANT PROTEINS-A AND D Pulmonary surfactant is a mixture of lipid and associated proteins, the main function of which is to protect the alveoli from the sheer forces associated with breathing, and to prevent alveolar collapse at low lung volumes.1 Four major surfactant proteins have been identified, falling into two distinct groups: SP-B and C are small hydrophobic proteins which are involved in lipid adsorption at the air/water interface (although recently, in vitro studies have suggested that they may also limit lung inflammation, by stabilising neutrophil membranes, and decreasing chemotaxis, respiratory burst and elastase release). In contrast, SP-A and D are larger, hydrophilic proteins, produced by alveolar type II cells and Clara cells of the distal conducting airways. As the structure and function of these proteins has been elucidated, it seems likely that their major role is in host defence. However, the proteins do appear to play a minor role in lowering surface tension, with SP-A involved in the formation of tubular myelin, a component of the surfactant lipids, and SP-D involved in regulation of surfactant protein homeostasis. The latter mechanism is incompletely understood, but SP-D knockout mice demonstrate a significant increase in
2.
3.
Binding to microbial surfaces (a) In some cases this can lead to enhanced recognition and phagocytosis by macrophages and neutrophils. This has been demonstrated in vitro for a variety of micro-organisms, but there are differences between the two surfactant proteins (Table 1). (b) Binding can directly reduce virulence, as is the case with SP-D and influenza A virus. Binding leads to inactivation of haemagglutinin and neuraminidase, (both essential virulence factors), and reduces viral infectivity. (c) In other cases, the proteins act in an opsonic fashion and are able to bind to specific macrophage receptors, several of which have now been identified. Stimulation of chemotaxis. Both SP-A and SP-D enhance chemotaxis of monocytes, neutrophils and alveolar macrophages. Enhancing leucocyte function, e.g. increasing intracellular killing, through stimulation of reactive oxygen or nitrogen species, effects on cytokine production, regulation of lymphocyte production and apoptosis.
In addition to effects on microbial pathogens, both SP-A and SP-D bind to allergens, including house dust mite and pollen, inhibit binding of IgE to allergens and block allergeninduced histamine release from basophils, suggesting a role in non-infective pulmonary diseases. Furthermore, a recent study has identified SP-D mRNA and protein in human tissues as diverse as brain, heart, gut, kidney and skin, raising the possibility that a much wider role exists for these collectins than has previously been thought. Not all activities of SP-A and SP-D are necessarily of benefit to the host. A recent study reported that SP-A binding to CMV enhanced entry of the virus into pneumocytes4, suggesting in certain cases the proteins may contribute to microbial pathogenesis. Similar hypotheses have been raised regarding SP-A and Mycobacterium tuberculosis and Pneumocystis carinii.
Genetics The genes encoding SP-A and D are located on the long arm of chromosome 10, in close proximity to MBL.5 The SP-A locus consists of two similar but non-identical genes,
72
J. DAVIES ET AL.
Table 1 Reported interactions between each of the human collectins and respiratory pathogens.The majority of the data come from in vitro studies. Data from in vivo animal studies are in italics. Some of the interactions are thought to be detrimental to host defence; these are in bold type. AM = alveolar macrophage.
Bacteria P. aeruginosa S. aureus Group B streptococcus S. pneumoniae H. influenzae B. cepacia Klebsiella species Viruses RSV Influenza
HSV Mumps CMV Fungi Aspergillus fumigatus
SP-A
SP-D
MBL
Decreased airway clearance Increased inflammation AM and neutrophil phagocytosis Susceptibility of -/-mice Decreased airway clearance Increased inflammation AM and Neutrophil phagocytosis AM phagocytosis
AM phagocytosis
No significant binding
Neutrophil phagocytosis
Binds and activates complement
AM phagocytosis and killing
Aggregation
SP-A deficient mice more susceptible AM phagocytosis
Disease severity reduced by exogenous SP-D Increases aggregation Neutrophil binding No enhanced phagocytosis
Mycobacterium TB Pneumocystis carinii
Neutrophil phagocytosis
No binding Some binding Binds and activates complement Some binding
Binds to influenza A strains
AM phagocytosis Reduced infectivity Binding and entry into pneumocytes and AMs Binds
Cryptococcus neoformans Other Mycoplasma
Poor binding
Macrophage killing impaired in -/-mice AM binding May facilitate cell entry
SP-A1 and SP-A2, which are closely linked. It is thought that the trimeric subunit of SP-A normally arises from the association of two chains of SP-A1 and one chain of SPA2. The regulation of the SP genes is complex and incompletely understood; SP-A1 and A2 are situated head to head, indicating that they probably share regulatory elements. In vitro studies suggest up regulation by γ-interferon and epidermal growth factor, with inhibition by tumour necrosis factor (TNF)-α, lipopolysaccharide and transforming growth factor (TGF)-β, and a concentration-dependent effect of corticosteroids. Studies from human fetal lung indicate that SP-A1 is upregulated by cAMP and inhibited by glucocorticoids, whereas SP-A2 is constitutively expressed. Sequences upstream of SP-D are similar to those identified for other acute phase proteins, and glucocorticoids have been shown to upregulate expression of this protein. Polymorphisms have been identified in SP-A and also more recently in SP-D, although links between the latter
Neutrophil binding and uptake Aggregation
Binds Binds
Reduced AM phagocytosis Aggregation and AM binding
and disease states have not been described. Certain SP-A polymorphisms have been associated with risk factors for infant respiratory distress syndrome (IRDS). No association between polymorphisms in the surfactant collectins and pulmonary infection have been reported, although in vivo data on their role are available from animal models. Interestingly, mutations in SP-B have been associated with IRDS and with alveolar proteinosis.
Animal models of surfactant protein deficiency SP-A -/Contrary to the initial view that SP-A was closely involved in surfactant composition, newborn SP-A -/-mice demonstrate little disturbance in respiratory function or surfactant lipid metabolism. However, there is evidence of decreased clearance of a variety of pathogens (group B
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
streptococcus, Pseudomonas aeruginosa, Staphylococcus aureus and RSV) and more severe disease in response to these infections, with evidence of disease amelioration following the administration of exogenous SP-A6.
SP-D-/Unlike SP-A -/-animals, the phenotype of SP-D deficient mice is characterised by significant disturbance of surfactant homeostasis, with increased surfactant volume, type II cell hyperplasia and an abundance of fat-laden macrophages.2 Studies addressing the susceptibility of SP-D deficient mice to various pulmonary pathogens are awaited.
Secondary abnormalities in surfactant composition Surfactant proteins can be degraded in vitro by neutrophil elastase, a susceptibility which is further enhanced in the presence of oxidants. It is therefore of no surprise that surfactant composition is altered in chronic pulmonary disease states such as cystic fibrosis. Whether or not primary abnormalities in the surfactant proteins influence progression of such lung disease remains unknown.
MBL MBL was first isolated from the serum and liver of rabbits towards the end of the 1970s, and was later identified as an important serum component lacking in children with an opsonisation defect. Since then, much interest has focused on its role in host defence against a variety of infections.
Functions of MBL MBL has the potential to enhance clearance of microorganisms via several mechanisms. These include: acting directly as an opsonin, facilitating ingestion by phagocytes; activation of the lectin complement pathway leading to C3b and iC3b deposition on bacterial surfaces, mediating phagocytosis and killing; or activation of complement leading to the formation of membrane attack complexes and direct bacterial lysis. MBL also has an influence over inflammatory responses, for example, by influencing levels of cytokine production, although this requires further study.
Genetics Within both the structural (exon 1) and promoter regions of human MBL, polymorphisms have been identified which result in mutant forms of the protein incapable of forming high order oligomers.7 Of these, a glycine → aspartic acid mutation in codon 54 is relatively common in the British population with a gene frequency of 0.14, and results in
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expression of a protein incapable of normal polymerisation.8 Individuals homozygous for the mutation have profoundly reduced levels of MBL (≤10 ng/ml), median levels are approximately 350 ng/ml in heterozygotes and over 1600 ng/ml in wild type homozygous individuals. A second point mutation in codon 57 (glycine → glutamic acid) of the MBL gene, is common in Sub-Saharan Africans but rare in Caucasians, and a third mutation results in a cysteine for arginine substitution in codon 52. Polymorphisms have also been identified within the promoter region of MBL, in linkage disequilibrium with the structural mutations, and some result in insignificantly reduced protein levels. The frequency with which these mutations are found in the healthy population supports the concept that MBL is likely to play an adjunctive role in host defence. However, low levels of the protein have been shown to relate to an increased risk of recurrent infections, of meningococcal disease, and to an adverse outcome in HIV infection, although the latter is controversial.
MBL and lung disease Much less is known about the role of MBL in respiratory infection than is the case for SP-A and SP-D, where our understanding has been increased by studies on transgenic mice. There is no published animal model of MBL deficiency; in the mouse the development of such a model is complicated by the presence of two MBL genes, MBL-A and C, with the former encoding the primary functional serum collectin, and the latter encoding an essentially hepatic form of MBL. In vitro studies have, however, indicated that MBL binds to a variety of pulmonary pathogens, including S. aureus, various streptococci, H. influenzae and A. fumigatus9. In a study of the role of MBL in murine influenza infection, there was evidence of anti-viral activity and the protein was detected in respiratory secretions in response to infection. The case for this collectin playing a role in pulmonary defence has been strengthened by human epidemiological studies.
Human disease In a large hospital-based study, the presence of MBL mutations was examined in over 600 children.10 Increased susceptibility to infection was found in children who were either homozygous or heterozygous for mutations in MBL. Of 133 heterozygotes presenting with infection, the site of infection was the respiratory tract in over 30%. MBL has been detected, albeit in low levels, in human nasopharyngeal secretions and middle ear effusions, confirming that the activity of the protein is not restricted to the serum, and that a role for MBL in local airway defence is plausible.
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Tuberculosis It has been suggested that MBL may confer both beneficial and detrimental effects on the infected host. Tuberculosis, leprosy and leishmaniasis are examples of disease in which high levels of MBL may be disadvantageous. Theoretically, by mediating macrophage binding, MBL could enhance infectivity of Mycobacterium tuberculosis (mTB). This is supported by several epidemiological studies. Higher levels of MBL were seen in African adults with TB than in those without, and two further studies in Africa have found an unexpectedly low frequency of mutant MBL alleles in TB patients. In one of these, there seemed to be a beneficial effect on both pulmonary and, more significantly, meningeal infection.11
Cystic fibrosis Recently, a compelling epidemiological link has been made between MBL and respiratory infection in cystic fibrosis (CF). CF results from mutations in cystic fibrosis transmembrane conductance regulator protein (CFTR), a chloride ion channel situated in the apical membrane of epithelial cells. The pulmonary component of the disease is characterised by early and sustained respiratory infection with a narrow range of organisms, various respiratory viruses and S. aureus and H. influenzae in early life.12 From mid-childhood to adolescence, up to 85% of CF patients will become chronically infected with Pseudomonas aeruginosa, which, once acquired, is rarely eradicated. The organism protects itself within a thick layer of alginate, leading to an exaggerated but frustrated inflammatory response, which contributes greatly to the irreversible lung damage characteristic of this disease. B. cepacia is an important, but fortunately less prevalent, organism, which in humans is found almost exclusively within the CF airway. The significance of this pathogen relates to its high levels of transmissibility and antibiotic resistance and to the progression of septicaemia and multi-organ failure in approximately 20–25% of patients. Individuals with CF, even those with the same CFTR mutations and within families, display widely different clinical phenotypes. This has led to the search for genes other than CFTR that may modify disease progression, such as those involved in host defence and the inflammatory response. Garred et al. examined the effect of MBL polymorphisms on the progression of lung disease in a cohort of 149 CF patients.13 Within this group, the frequency of MBL mutations was similar to that observed in the healthy population (approximately 34% heterozygotes and 2–3% homozygotes). Low serum levels were observed as expected and did not differ from those observed in healthy individuals. Lung function, measured by FEV1 and FVC, was significantly lower in the group with MBL deficiency. A trend towards this was visible as early as 8 years of age, although this became significant only at 16 years of
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age. Although P. aeruginosa has been identified as one of the major contributory factors in progression of lung damage in CF, MBL-deficient patients were not found to have higher rates of pseudomonal infection. Rather, it appeared that, following pseudomonal colonisation, MBLdeficient patients experienced a marked decline in lung function, which was not apparent in the MBL-sufficient group and which appeared to adversely influence survival. MBL is known to affect inflammation in various ways and it seems likely therefore that the host response is in some way responsible for this clinical deterioration. This story is confused by results from another smaller study comparing patients deficient in MBL with controls closely matched for age and CF genotype (all homozygous ∆F508).14 In contrast to the earlier study, these authors only found a significant reduction in lung function in patients with severe MBL deficiency (homozygotes or compound heterozygotes). They also reported increased P. aeruginosa colonisation in these patients, although greater numbers would be required to confirm statistical significance. B. cepacia was not mentioned in this report, although in the first study the frequency of MBL mutations was significantly higher than would have been expected amongst 10 cepacia-colonised patients. These findings led us to examine a possible mechanism for these observations. Using strains obtained from the sputum of CF patients, we were able to demonstrate that MBL does not bind significantly to the surface of either mucoid or non-mucoid P. aeruginosa. However, high levels of binding were seen to B. cepacia.15 This binding led to the activation of complement, which in vivo would be expected to result in bacterial killing. Taken together, these results suggest that MBL is not likely to play a major role in the acquisition of P. aeruginosa. Once colonisation has taken place, however, there may be an adverse inflammatory response to the chronic presence of this organism in the airway of MBL-deficient patients, which accounts for the faster decline in pulmonary function. However, as MBL does bind significantly to B. cepacia, MBL-deficient patients may be at greater risk of acquiring this pathogen, a hypothesis which would be supported by the Garred study. We are currently in the process of examining the MBL status of cepacia-colonised adults with CF and preliminary data suggest that this will confirm the earlier study.
SUMMARY Both the pulmonary (SP-A and D) and serum (MBL) collectins, appear to be involved in the innate defence of the airway. They have broadly similar functions, aimed both directly at the micro-organism (opsonisation, complement activation and killing), and indirectly through modulation of the host inflammatory response, which are incompletely understood. Polymorphisms exist in the human genes encoding these proteins, and certain links
THE COLLECTIN SYSTEM AND PULMONARY DEFENCE
have been identified between deficiency states and predisposition to pulmonary infection. A further understanding of the effects of such polymorphisms, through both animal models and human studies, may lead to an increased understanding of disease pathogenesis, the identification of subgroups at risk and possibly to new therapeutic avenues. RESEARCH DIRECTIONS Laboratory and animal studies Work is continuing to examine the exact roles of the collectins both in killing of pathogens and in the host inflammatory response. Studies on SP-A and D knockout mice will seek to identify the roles of these proteins in a variety of lung diseases both infective and inflammatory. The development of a similar mouse model for MBL deficiency would greatly assist research in this area. Human disease Further epidemiological and laboratory studies are required to elucidate the role of the collectins in human lung diseases. As well as allowing identification of subgroups of patients at particular risk of certain infections or severe disease, results of such studies may lead to novel therapeutic approaches.
REFERENCES 1. Floros J, Kala P. Surfactant proteins: molecular genetics of neonatal pulmonary diseases. Annu Rev Physiol 1998; 60: 365–384.
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2. Botas C et al. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc Natl Acad Sci USA 1998; 95: 11869–11874. 3. Crouch EC. Collectins and pulmonary host defense. Am J Respir Cell Mol Biol 1998; 19: 177–201. 4. Weyer C, Sabat R, Wissel H, Kruger DH, Stevens PA, Prosch S. Surfactant protein A binding to cytomegalovirus proteins enhances virus entry into rat lung cells. Am J Respir Cell Mol Biol 2000; 23: 71–78. 5. Floros J, Hoover RR. Genetics of the hydrophilic surfactant proteins A and D. Biochim Biophys Acta 1998; 1408: 312–322. 6. LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J, Korfhagen T. Surfactant protein-A enhances respiratory syncytial virus clearance in vivo. J Clin Invest 1999; 103: 1015–1021. 7. Madsen HO et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol 1995; 155: 3013–3320. 8. Turner MW. Mannose-binding lectin (MBL) in health and disease. Immunobiology 1998; 199: 327–393. 9. Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000; 68: 688–693. 10. Summerfield JA, Sumiya M, Levin M, Turner MW. Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series. BMJ 1997; 314: 1229–1232. 11. Hoal-Van Helden EG et al. Mannose-binding protein B allele confers protection against tuberculous meningitis. Pediatr Res 1999; 45: 459–464. 12. Govan JR, Nelson JW. Microbiology of cystic fibrosis lung infections: themes and issues. J Royal Soc Med 1993; 86(Suppl 20): 11–18. 13. Garred P et al. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J Clin Invest 1999; 104: 431–437. 14. Gabolde M, Giulloud-Bataille M, Feingold J, Besmond C. Association of variant alleles of mannose binding lectin with severity of pulmonary disease in cystic fibrosis: cohort study. BMJ 1999; 319: 1166–1167. 15. Davies J, Neth O, Alton E, Klein N, Turner M. Differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis. Lancet 2000; 355: 1885–1886.