Differential epithelial expression of the putative innate immune molecule SPLUNC1 in cystic fibrosis.
BACKGROUND
Short PLUNC1 (SPLUNC1) is the founding member of a family of proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. It is one of the most highly expressed genes in the upper airways and the protein has been detected in sputum and nasal secretions. The biology of the PLUNC family is poorly understood but in keeping with the putative function of the protein as an immune defence protein, a number of RNA and protein studies have indicated that SPLUNC1 is increased in inflammatory/infectious conditions such as Cystic Fibrosis (CF), COPD and allergic rhinitis.


METHODS
We used immunohistochemistry to localise SPLUNC1 in lung tissue from patients with CF and a range of other lung diseases. We used a range of additional markers for distinct cell types to try to establish the exact site of secretion of SPLUNC1. We have complemented these studies with a molecular analysis of SPLUNC1 gene expression in primary human lung cell cultures and isolated inflammatory cell populations.


RESULTS
In CF, expression of SPLUNC1 is significantly elevated in diseased airways and positive staining was noted in some of the inflammatory infiltrates. The epithelium of small airways of CF lung exhibit significantly increased SPLUNC1 staining compared to similar sized airways in non-CF lungs where staining is absent. Strong staining was also seen in mucous plugs in the airways, these included many inflammatory cells. No alveolar epithelial staining was noted in CF tissue. Airway epithelial staining did not co-localise with MUC5AC suggesting that the protein was not produced by goblet cells. Using serial sections stained with neutrophil elastase and CD68 we could not demonstrate co-localisation of SPLUNC1 with either neutrophils or macrophages/monocytes, indicating that these cells were not a source of SPLUNC1 in the airways of CF lungs. No change in staining pattern was noted in the small airways or lung parenchyma of other lung diseases studied including, COPD, emphysema or pneumonia where significant NE and CD68 staining was noted. Cultures of primary tracheobronchial epithelial cells were analysed by RT-PCR and showed that pro-inflammatory mediators did not induce expression of SPLUNC1. We have also shown that SPLUNC1 gene expression was not seen in isolated human mononuclear cells, macrophages or neutrophils.


CONCLUSIONS
These studies show that SPLUNC1 is specifically and significantly increased in the small airways of lungs from patients with CF. They further suggest that it is the airway epithelium that is responsible for the increased levels of SPLUNC1 in CF and not inflammatory cells; this could be a defensive response to the infectious component of the disease.
Bingle
Lynne
L
Department of Oral Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK. l.bingle@sheffield.ac.uk
Barnes
Frances A
FA
Cross
Simon S
SS
Rassl
Doris
D
Wallace
William A
WA
Campos
Michael A
MA
Bingle
Colin D
CD
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003550:Cystic Fibrosis / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate; D018836:Inflammation Mediators / Q000378:metabolism; D008168:Lung / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D009093:Mucus / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
C485599:BPIFA1 protein, human; D006023:Glycoproteins; D018836:Inflammation Mediators; D010750:Phosphoproteins; D012333:RNA, Messenger