Role of Clara Cell Secretory Protein in Lung Inflammation

Background: Recurrent airway obstruction (RAO) is a chronic inflammatory lung disease affecting mature horses following repeated exposure to environmental substances, and similar to environmentally-induced asthma of humans. Early during inhalation of noxious substances, non-ciliated lung epithelial cells produce Clara cell secretory protein (CCSP), the most abundantly secreted protein of the airway mucus, to couteract the tissue damage caused by inflammatory cells. Horses are unique by having 3 copies of the CCSP gene distributed over a region of ~520 kilobases. CCSP copies differ from each other at 8 to 10 nucleotides, and are expected to produce different proteins. Since reduced total CCSP is detected in lung tissue of RAO animals, we investigated whether CCSP variants are differentially expressed and have distinct functions. Methods: A long-range polymerase chain reaction (LR-PCR) strategy was used to isolate individual CCSP genomic sequences from each horse analyzed. CCSP tissue and copy-specific expression patterns were evaluated by end-point limiting dilution (EPLD-PCR) and by reverse transcription PCR. A quantitative real-time PCR assay was developed to measure the expression level of each distinct copy. Sequences were determined using an automatic DNA sequencer and analyzed using Geneious ProTM 3.4 software. Results: Three distinct CCSP genomic sequences were identified in every horse analyzed (n > 10). Polymorphisms detected in specific copies from different horses were mostly conservative, indicating preservation of each copy over time. In support of this finding, multiple CCSP copies were also identified in other equids, such as donkeys and Przewalskii’s horses. CCSP tissue-specific expression was evaluated in a total of 29 tissues. Strong expression was detected in lung, uterus, trachea, urethra, Fallopian tube, and mammary gland. Interestingly, only 2 of 3 copies were found expressed in adult lung and uterine tissues. Additionally, RAO affected animals (n = 5) had a significantly different expression ratio of the 2 gene copies compared to controls (n = 10), suggesting diverse roles for different CCSP genes. Conclusions: The precise causes underlying most airway diseases are poorly understood, and most treatments are aimed at reducing the signs of disease rather than at achieving a cure. Findings from these studies may increase our understanding of inflammatory mechanisms in asthma-related diseases and identify better targets for therapy.