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Surfactant and lung inflammation
  1. K B M Reid1,
  2. H Clark1,
  3. N Palaniyar2
  1. 1MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 ODQ, UK
  2. 2Lung Biology Research Program, Hospital for Sick Children Research Institute, Toronto, Ontario 5G 1X8, Canada
  1. Correspondence to:
    Professor K B M Reid
    MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 ODQ, UK; kenneth.reidbioch.ox.ac.uk

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SP-A and SP-D, either on their own or in combination with existing surfactant therapy, may have a role in the treatment of lung inflammation

The thin alveolar lining consists of a single layer of epithelial cells and an overlay of an oily substance, the pulmonary surfactant, which contains surfactant proteins (SPs) (10% w/w) and lipids (90% w/w). In addition to the well established ability of the surfactant system to reduce alveolar surface tension and thereby prevent collapse of the alveoli on expiration, it is also involved in the very efficient removal of microbes and their debris,1–3 dying epithelial cells, and phagocytes.4 The therapeutic use of exogenous surfactant is well established and has been shown to be effective in the treatment of premature infants with respiratory distress syndrome. The surfactant preparations normally used are natural surfactants of porcine (Curosurf) or bovine (Alveolfact, Survanta) origin, or synthetic protein free preparations. Surfactant preparations derived from natural sources contain the hydrophobic peptides SP-B and SP-C but none—or very low levels—of the much larger hydrophilic surfactant proteins SP-A and SP-D which are lost during the extraction procedure. Although surfactant lipids may reduce lung inflammation, recent studies suggest that these hydrophilic proteins are the major anti-inflammatory components of pulmonary surfactant.

EXISTING SURFACTANT THERAPY

Over the past 10 years it has become recognised that SP-A and SP-D—members of the innate immune collectin family of proteins which are secreted by type II alveolar epithelial cells—play important host defence and immunomodulatory functions in the surfactant system.5,6 SP-A (∼90% w/w of SPs) and SP-D (∼3% w/w of SPs) are the major surfactant proteins and are involved in maintaining an infection-free and inflammation-free lung.7,8 During acute lung infections these innate immune molecules can kill9 and/or opsonise and enhance the phagocytosis of microbes by freshly recruited phagocytes. They also bind to apoptotic polymorphonuclear leucocytes and alveolar macrophages and enhance their clearance by healthy resident macrophages.10,11 It has recently been suggested that surfactant therapy may also be beneficial in cases of adult acute respiratory distress syndrome, since clinical trials in patients with respiratory failure due to pneumonia showed improved oxygenation and no serious side effects following surfactant therapy.12,13 This is consistent with in vitro studies showing that surfactant does not interfere significantly with the phagocytic capacity of polymorphonuclear neutrophils stimulated by bacteria opsonised with specific IgG antibody.13 If surfactant preparations are to be used more widely, it may also be worth considering administering SP-A and/or SP-D to the same groups of patients.

Surfactant proteins SP-A and SP-D

These two human surfactant proteins are extremely well characterised, both having a typical collectin structure composed of polypeptide chains containing a short interchain disulfide bond forming N-terminal domain, a collagen-like region with Gly-X-Y repeats (where X is any amino acid and Y is often hydroxyproline or hydroxylysine), an α-helical hydrophobic neck region, and a C-terminal globular carbohydrate recognition domain (CRD).14–16 Three of these polypeptides form a trimeric subunit which assembles to form higher order structures. The oligomeric assembly of SP-D16,17 resembles that of an “X” (4 subunits) or “asterisk” (>12 subunits) whereas the SP-A, by the association of up to six trimeric subunits, appears as a “bouquet of flowers”.5,17–19 Although the trimeric subunits of the collectins have only limited affinity (mM) for carbohydrate targets on their own, their oligomeric assembly provides a high avidity so that the oligomeric proteins bind to ligands selectively and with high affinity (pM). One reason that SP-A has not been included in surfactant preparations was the concern that humans may raise antibodies to animal surfactant proteins. However, recently it has become possible to generate smaller recombinant fragments of these lung collectins which retain their activity without being immunogenic (in mice).

In the lung, SP-A and SP-D opsonise microbes, allergens, and other foreign bodies to varying degrees and signal their clearance by resident alveolar macrophages and other leucocytes.6 These proteins also bind some of the well known inflammation causing ligands from bacterial cell walls—such as lipopolysaccharide, peptidoglycans and lipoteichoic acid—primarily via hexose sugars.5 Recent studies show that SP-A and SP-D recognise leucine-rich repeat (LLR) containing proteins such as Toll-like receptor 2 (TLR-2)20 and decorin,21 respectively. Binding of SP-A to TLR-2 inhibits the peptidoglycan induced inflammatory signal produced by phagocytes,20 which explains some of the possible mechanisms involved in the anti-inflammatory property of collectins. SP-A may also bind to C1q and dampen the complement activation and, hence, could reduce lung inflammation.22

The characterisation of the natural ligands and regulatory proteins in the lungs involved—together with SP-A and SP-D—in the identification and clearance of apoptotic cells is presently a very active area of research.23–26 The likely critical involvement of SP-D in the modulation of lung homeostasis is indicated by the examination of the lungs of gene targeted mice deficient in SP-D, which show a low grade macrophage mediated lung inflammation and ultimately exhibit structural alterations such as hypertrophy and hyperplasia of type II cells, a diminished number of alveoli, increased alveolar size and decreased alveolar surface area, consistent with the definition of pulmonary emphysema.27 This is accompanied by an intra-alveolar accumulation of alveolar macrophages, many of which are enlarged and foamy. Surfactant homeostasis is disturbed, indicated by the presence of giant lamellar bodies in some type II cells and the development of alveolar lipoproteinosis.7,8 In addition to the increased intra-alveolar surfactant pool, stereological analysis has revealed the existence of an increased intracellular surfactant pool due to an increase in the number and size of lamellar bodies per type II cell and the number of type II cells per lung.7

ANIMAL MODEL OF SP-D DEFICIENCY

SP-D deficiency brings about a chronic inflammatory state characterised by increased levels of reactive oxygen species as well as a raised expression of matrix metalloproteinases, which could be responsible for the pulmonary remodelling process resulting in alveolar destruction consistent with these structural findings.10,28 Moreover, a considerable number of apoptotic and necrotic alveolar macrophages was observed in the bronchoalveolar lavage (BAL) fluid, providing evidence that a delayed clearance of dead cells may be involved in the chronic inflammatory state of SP-D deficiency. Previous reports had identified a role for SP-A and SP-D in the phagocytosis of apoptotic and necrotic cells in vitro,29 though SP-D appeared to be more important for this function than other collectins in vivo.10,30 This essential role of SP-D has been emphasised by experiments involving the intranasal administration of a truncated 60 kDa fragment of human SP-D (rfhSP-D) to SP-D knock-out mice, which resulted in a significant reduction in the number of apoptotic and necrotic alveolar macrophages in BAL fluid and a decrease in inflammatory mediators and intra-alveolar lipid accumulation.10,30 Application of rfhSP-D to mice suffering from allergic asthma also showed a dampening of the allergic response due to allergen inhalation with a modification of cytokine levels and a decrease in airway hyperresponsiveness on allergen challenge.31,32 Allergen challenge upregulates collectin (particularly SP-D) expression in the lungs, both in allergic mice33 and patients.34,35 The allergic inflammation may directly be modulated by collectins altering cytokine production and macrophage functions.36

INTERACTION OF SP-A AND SP-D WITH DNA

A recent study has shown that SP-A and SP-D can bind to DNA,37 which may be of physiological significance in terms of control of inflammation since apoptotic cell death results in the fragmentation of DNA and its subsequent display on the surface as blebs.4 Inefficient removal of apoptotic cells leads to disintegration of their contents and the formation of necrotic cells.4 These leaky cells eventually release their intracellular components and many of these components elicit tissue inflammation generation. Furthermore, certain pulmonary pathogens such as Pseudomonas aeruginosa actively secrete DNA onto the extracellular matrix to form active biofilm and subsequently to establish chronic infection.3 Patients with cystic fibrosis suffer from (and may die of) chronic microbial infection, lung inflammation, and accumulation of apoptotic and necrotic neutrophils and their cell contents such as DNA. These patients show a deficiency in levels of SP-A and SP-D,38 and the concentrations of these collections are inversely related to inflammation in early cystic fibrosis.39 Removal of dying cells and their components (including DNA) is therefore essential for maintaining inflammation-free tissues and preventing autoimmune diseases.4 Although the multiple pathways and proteins involved in the apoptotic process have been studied in great detail, clearance of these cell debris and autoantigens is poorly understood. Free DNA and RNA—as well as the DNA present on apoptotic cells—constitute a novel class of ligands for the collectins SP-A, SP-D and MBL,37,40 and it has been found that collectins, particularly SP-D, effectively enhance the uptake of DNA and DNA containing beads by macrophages in vitro, and the absence of SP-D in vivo leads to increased anti-DNA autoantibody generation.

CONCLUSIONS

Thus there is increasing support for the view that recombinant forms of SP-A or SP-D, either on their own or with existing surfactant therapy, may be worth considering as therapeutic agents in the treatment of lung inflammation associated with injury, allergy, or infection. However, the intact collectin proteins are very large (around 600 kDa), there are two functional SP-A genes, and several allelic variants of both proteins (which may show significantly different structural and functional properties). The preparation of functionally active recombinant forms of the whole SP-A and SP-D proteins is therefore complicated and their efficient delivery to the lungs may be difficult and expensive. A recombinant fragment (although still 60 kDa in size) of SP-D, which has been shown to retain many of the anti-inflammatory properties of the whole molecule, may offer a first step in the use of the protective properties of these proteins of innate immunity in the treatment of lung inflammation.

SP-A and SP-D, either on their own or in combination with existing surfactant therapy, may have a role in the treatment of lung inflammation

REFERENCES

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