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This thesis examines the role played by the lung collectins, surfactant proteins A and D (SP-A and SP-D) in innate immunity in the lung. The generation of SP-A and SP-D deficient mice has confirmed the important part played by these innate immune molecules in the defence of the lung against infection and in the control of inflammation. SP-A deficient mice are susceptible to infection with a range of microbes. The critical role played by SP-D in controlling inflammation in the lung has been convincingly demonstrated in the SP-D deficient mouse which shows a chronic low grade lung inflammation and the spontaneous development of emphysema. The thesis examines the in vivo activity of recombinant forms of surfactant proteins, in particular a recombinant fragment of surfactant protein D, in the control of lung inflammation using SP-D deficient mice as a model of emphysema. Murine models of allergic inflammation to fungal and common house dust mite allergens were employed to assess the efficacy of recombinant collectins in modulating allergic processes in vivo. The animal models employed and the generation and structural characterisation of recombinant SP-Dare described in the first sections of the thesis. SP-A and SP-D are known to bind to fungal aeroallergens and to allergens of the common house dust mite. Therapeutic testing of the recombinant fragment of surfactant protein D was carried out in mouse models of allergy against allergens of the common house dust mite Dermatophagoides pterynossinus and those of the fungus Aspergillus fumigatus. Exogenously administered collectin reduced serum eosinophilia, IgE and specific IgG 1 levels as well as airway hyperresponsiveness to challenge with both fungal and house dust mite allergens. Further characterisation of the lung phenotype of the SP-D deficient mouse and the use of replacement therapy in correcting the phenotype revealed that SP-D deficient mice have an increased burden of apoptotic and necrotic inflammatory cells in the alveolar space. Treatment of these mice with a recombinant fragment of surfactant protein D led to a marked reduction of the number of apoptotic and necrotic alveolar macrophages in the alveolar space and a partial resolution of the inflammation and excess production of surfactant phospholipids. These results indicate the critical role played by SP-D in regulating inflammation in the lung and the efficacy of a recombinant fragment of SP-D in reducing inflammation in the SP-D deficient lung. Finally the results of a pilot investigation into the levels of SP-Din a population of intubated neonates is presented. The clinical implications of the findings of the thesis are discussed in chapter four, focussing on the therapeutic potential of the recombinant fragment of SP-D to reduce inflammation in neonatal chronic lung disease, cystic fibrosis and adult emphysema.

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via -helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the -helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via -helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the -helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development

Properdin is a serum glycoprotein that upregulates the alternative pathway of complement by stabilizing the C3bBb complex. It also binds sulphated glycoconjugates, such as sulphatide, in vitro. Properdin is composed of cyclic dimers, trimers and tetramers of a 53 kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately sixty amino acids. The sixth TSR of properdin contains an insertion of approximately 30 amino acids which corresponds to the position of an intron in the human properdin gene. In order to identify the regions of properdin important for function, human properdin, and mutant forms each lacking a single TSR, were expressed in Chinese Hamster Ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of the normal and altered forms of properdin were investigated. Wild type recombinant properdin is similar to properdin purified from plasma in size, immunoreactivity, N-terminal sequence, possession of N-linked sugar, oligomerization (as determined by electron microscopy and gel exclusion chromatography), and functional activity in an alternative pathway haemolytic assay, and in C3b and sulphatide binding assays. Properdin "nicked" in TSR5 is unable to bind C3b, while retaining its overall structure and its ability to bind sulphatide. The removal of TSRS prevents C3b and sulphatide binding. Properdin lacking TSR4 is unable to stabilize the C3bBb complex, but is able to bind C3b and sulphatide, and shows the presence of monomers and dimers in the electron microscope. Properdin without TSR3 is able to stabilize the C3bBb complex, to bind CSb and sulphatide, and forms dimers, trimers and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3bBb complex. Monoclonal antibodies which bind to the N-terminal region, TSR1, or TSR2 are able to inhibit properdin binding to CSb. A monoclonal antibody which binds TSR4 is able to inhibit properdin binding to sulphatide, but not to CSb. The results confirm that TSRs are folded as independent units. The N-terminal end and TSR5 of properdin are implicated in CSb binding. The vertices of properdin oligomers may be important for interaction with CSb. TSR4 may also be involved in stabilization of the C3bBb complex. The sulphatide binding site is distinct from the CSb binding site, but TSR5, which contains many basic residues, may be important for both activities.