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Published online before print January 22, 2008

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(Journal of Leukocyte Biology. 2008;83:946-955.)
© 2008 by Society for Leukocyte Biology

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^*,,1

^* 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

¹ Correspondence: Immune Disease Institute, 800 Huntington Avenue, Boston, MA 02115, USA. E-mail: remold{at}cbr.med.harvard.edu

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