Patterns of neutrophil serine protease-dependent cleavage of surfactant protein D in inflammatory lung disease

• Published in: Journal of leukocyte biology Vol. 83; no. 4; pp. 946 - 955
• Main Authors: Cooley, Jessica, McDonald, Barbara, Accurso, Frank J., Crouch, Erika C., Remold‐O'Donnell, Eileen
• Format: Journal Article
• Language: English
• Published: United States Society for Leukocyte Biology 01.04.2008
• Subjects: Adolescent; Adult; Asthma - blood; Bronchoalveolar Lavage Fluid - chemistry; Bronchoalveolar Lavage Fluid - cytology; calcium ion; Child; Child, Preschool; Cough - blood; cystic fibrosis; Cystic Fibrosis - blood; Cystic Fibrosis - enzymology; elastase; Humans; Infant; Leukocyte Elastase - blood; Lung Diseases - blood; Lung Diseases - enzymology; Neuromuscular Diseases - blood; Neutrophils - enzymology; proteinase‐3; Pulmonary Surfactant-Associated Protein D - blood; Reference Values; Serine Endopeptidases - metabolism
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1189/jlb.1007684

The manuscript presents definitive studies of surfactant protein D (SP‐D) in the context of inflammatory lung fluids. The extent of SP‐D depletion in bronchoalveolar lavage fluid (BALF) of children affected with cystic fibrosis (CF) is demonstrated to correlate best with the presence of the active neutrophil serine protease (NSP) elastase. Novel C‐terminal SP‐D fragments of 27 kDa and 11 kDa were identified in patient lavage fluid in addition to the previously described N‐terminal, 35‐kDa fragment by the use of isoelectrofocusing, modified blotting conditions, and region‐specific antibodies. SP‐D cleavage sites were identified. In vitro treatment of recombinant human SP‐D dodecamers with NSPs replicated the fragmentation, but unexpectedly, the pattern of SP‐D fragments generated by NSPs was dependent on calcium concentration. Whereas the 35‐ and 11‐kDa fragments were generated when incubations were performed in low calcium (200 μM CaCl2), incubations in physiological calcium (2 mM) with higher amounts of elastase or proteinase‐3 generated C‐terminal 27, 21, and 14 kDa fragments, representing cleavage within the collagen and neck regions. Studies in which recombinant SP‐D cleavage by individual NSPs was quantitatively evaluated under low and high calcium conditions showed that the most potent NSP for cleaving SP‐D is elastase, followed by proteinase‐3, followed by cathepsin G. These relative potency findings were considered in the context of other studies that showed that active NSPs in CF BALF are in the order: elastase, followed by cathepsin G, followed by proteinase‐3. The findings support a pre‐eminent role for neutrophil elastase as the critical protease responsible for SP‐D depletion in inflammatory lung disease.