Sheep lung segmental delivery strategy demonstrates adenovirus priming of local lung responses to bacterial LPS and the role of elafin as a response modulator.

Viral lung infections increase susceptibility to subsequent bacterial infection. We questioned whether local lung administration of recombinant adenoviral vectors in the sheep would alter the susceptibility of the lung to subsequent challenge with bacterial lipopolysaccharide (LPS). We further questioned whether local lung expression of elafin, a locally produced alarm anti-LPS/anti-bacterial molecule, would modulate the challenge response. We established that adenoviral vector treatment primed the lung for an enhanced response to bacterial LPS. Whereas this local effect appeared to be independent of the transgene used (Ad-o-elafin or Ad-GFP), Ad-o-elafin treated sheep demonstrated a more profound lymphopenia in response to local lung administration of LPS. The local influence of elafin in modulating the response to LPS was restricted to maintaining neutrophil myeloperoxidase activity, and levels of alveolar macrophage and neutrophil phagocytosis at higher levels post-LPS. Adenoviral vector-bacterial synergism exists in the ovine lung and elafin expression modulates such synergism both locally and systemically.

SLPI and elafin: one glove, many fingers.

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.

Characterization of the ovine ortholog of secretory leukoprotease inhibitor.

There is great interest in the use of the sheep as a model for the investigation of inflammation in the lung. The serine antiproteases secretory leukoprotease inhibitor (SLPI) and elafin are important "alarm antiproteases" in the lung and have potentially important roles in the innate immune response. SLPI was first characterized in man and subsequently in murine, porcine, and rat tissues. Here we present the first data concerning the gene and cDNA sequence encoding for the ovine ortholog of SLPI, a protein of 132 amino acids with 66% sequence identity at the amino acid level with human SLPI. A 24-amino-acid signal sequence signifies that, like the other mammalian orthologs, ovine SLPI is a secreted protein. Tissue distribution of expression is demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and shows features similar to SLPI expression in other mammals, specifically at mucosal surfaces such as the upper respiratory and intestinal tracts, and also the skin, liver, and kidney. This distribution lends credence to SLPI having important roles in innate immunity. We have also cloned the ovine SLPI cDNA into an expression vector and expressed the ovine SLPI protein in vitro. This has enabled us to demonstrate that ovine SLPI is correctly processed (Western blot analysis and SELDI-TOF mass spectrometry analysis) and has biological antihuman neutrophil elastase activity. In summary, the ovine ortholog of SLPI shows similarities to other members of the SLPI family and has all the features of a modulator of innate immunity.

Trappin ovine molecule (TOM), the ovine ortholog of elafin, is an acute phase reactant in the lung.

As large animal models continue to play an important role in translating lung-directed therapeutic strategies from laboratory animals to humans, there is an increasing interest in the analysis of endogenous regulators of inflammation at both a genomic and a therapeutic level. To this end, we have sought to characterize the ovine ortholog of elafin, an important regulator of inflammation in humans. We have isolated both the elafin cDNA and gene, which have a similar structure to other species' orthologs. Interestingly, we have isolated two alleles for ovine elafin, which contain a very high number of transglutamination repeats, thought to be important in binding elafin to the interstitium. The mainly mucosal mRNA distribution for ovine elafin suggests that ovine elafin may, like its human ortholog, have functions in innate immunity. This is supported by analysis of elafin and the related protein secretory leukocyte protease inhibitor (SLPI) in ovine bronchoalveolar fluid in response to locally administered lipopolysaccharide and confirmation of them acting as "alarm" antiproteases. We have also cloned the ovine elafin cDNA into an adenoviral vector and have demonstrated correct processing of the secreted protein as well as biological activity. Overexpression of ovine elafin in a lung-derived epithelial cell line has a protective effect against the enzymes human neutrophil and porcine pancreatic elastase. The identification of the ovine elafin gene and its translated protein are important in developing practical strategies aimed at regulating inflammation in the large mammalian lung.