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Published online before print January 22, 2008

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1007684

Received for publication October 10, 2007.
Revised December 11, 2007.
Accepted for publication December 20, 2007.

Article

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

Jessica Cooley ^*, Barbara McDonald , Frank J. Accurso , Erika C. Crouch , and Eileen Remold-O’Donnell ^*@

*Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA; Departments of Pathology and Immunology, Washington University School of Medicine, Saint Louis, Missouri, USA; and Department of Pediatrics, University of Colorado School of Medicine and The Children’s Hospital, Denver, Colorado, USA

^@ To whom correspondence should be addressed. E-mail: remold{at}cbr.med.harvard.edu.

	Abstract

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.

Key Words: cystic fibrosis • calcium ion • elastase • proteinase-3