Differential epithelial expression of the putative innate immune molecule SPLUNC1 in cystic fibrosis

Abstract

Introduction: 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.

Conclusion: 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.

Figure 1

SPLUNC1 is increased in the small airways of CF lung. Immunohistochemical localisation of SPLUNC1 was performed as described in the materials and methods sections. Sections show submucosal gland staining in "normal" human airways (A) and a lack of staining in the epithelium of smaller airways (B, C, an enlarged inset of B). Expression of SPLUNC1 is increased in cases of CF (D-H) where staining was predominantly found in the epithelial cells of the smaller airways and also within the inflammatory cell containing mass within the plugged lumens. SPLUNC1 did not co-localise with MUC5AC (I). There was no staining within the peripheral lung tissue of cases of CF (J). Cases of emphysema (K) and bacterial pneumonia (L) also did not exhibit SPLUNC1 staining.

Figure 2

SPLUNC1 is not expressed in neutrophils, monocytes or macrophages. Expression of SPLUNC1 was investigated by the use of RT-PCR with exon spanning primer pairs as described in the materials and methods section. Total RNA from B and T cells, neutrophils, monocytes, mononuclear cells and macrophages (either mock treated or infected with Neisseria meningitidis mc58) as well as positive control tissues (nasal epithelium and SPLUNC1-CHO cells), were used as template. The negative controls were a negative reverse transcription reaction performed in the absence of RT enzyme and a PCR reaction performed without Taq polymerase. Primers to the myeloid enriched anti apoptotic gene Bcl2A1 were used as a positive control for all samples.

Figure 3

SPLUNC1 is not expressed in inflammatory cells in CF or pneumonia. Serial sections of lung tissue were stained as described in the materials and methods section. SPLUNC1 (A) is not found to be expressed in CD68 expressing cells (Macrophages, B) nor in neutrophil elastase expressing cells (Neutrophils, C) in CF. Arrows in A point to the SPLUNC1 negative lumen as well as the positively staining epithelial cell layer. In sections from a case of bacterial pneumonia neither macrophages (E) nor neutrophils (F) appear to stain for SPLUNC1 (D), which is negative in this field.

Figure 4

Expression of SPLUNC1 in TBE cells grown at the ALI is not induced by pro-inflammatory mediators but requires continued RA driven differentiation. A. TBE cells isolated from three different donors were grown at the ALI as described in the materials and methods section. Cells were treated for increasing lengths of time with 25 ng/ml of IL-1β prior to isolation of RNA. Expression of SPLUNC1 was investigated by the use of RT-PCR with exon spanning primer pairs as described in the materials and methods section. Primers to Elafin and WFDC2 were used as controls. B. The effect of withdrawal of RA from the ALI growth medium was tested in TBE cells from the same three donors. ALI cells were established using standard grow conditions in medium containing 50 nM all trans retinoic acid (RA). After 14 days in culture RA was removed from the medium of one group of cells and culture was continued for an additional 18 days. RNA was harvested at 7 and 14 days during ALI growth and 20, 26 and 32 days following RA removal for 6, 12 and 18 days respectively prior to the isolation of RNA. Expression of SPLUNC1 was investigated by the use of RT-PCR with exon spanning primer pairs as described in the materials and methods section with primers to GAPDH serving as a positive control.