Ficolin-1 is present in a highly mobilizable subset of human neutrophil granules and associates with the cell surface after stimulation with fMLP

TY - JOUR

T1 - Ficolin-1 is present in a highly mobilizable subset of human neutrophil granules and associates with the cell surface after stimulation with fMLP

AU - Rørvig, Sara

AU - Honoré, Christian Le Fèvre

AU - Larsson, Lars-Inge

AU - Ohlsson, Sophie

AU - Pedersen, Corinna C.

AU - Jacobsen, Lars C.

AU - Cowland, Jack B.

AU - Garred, Peter

AU - Borregaard, Niels

N1 - Keywords: Animals; CHO Cells; Carcinogens; Cricetinae; Cricetulus; Exocytosis; Gene Expression Regulation; Humans; Lectins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Recombinant Proteins; Secretory Vesicles; Tetradecanoylphorbol Acetate

PY - 2009

Y1 - 2009

N2 - Ficolins are soluble molecules that bind carbohydrate present on the surface of microorganisms and function as recognition molecules in the lectin complement pathway. Three ficolins have been identified in humans: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but it is not known which subset of the neutrophils' secretory granules harbors ficolin-1. To determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA, by Western blotting, and by immunohistochemistry. Real-time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, biosynthesis studies of neutrophils precursor cells showed that ficolin-1 was primarily synthesized in myelocytes, metamyelocytes, and band cells. Immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules but also in highly exocytosable gelatinase-poor granules, not described previously. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and the majority becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily exocytosed by stimulation.

AB - Ficolins are soluble molecules that bind carbohydrate present on the surface of microorganisms and function as recognition molecules in the lectin complement pathway. Three ficolins have been identified in humans: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but it is not known which subset of the neutrophils' secretory granules harbors ficolin-1. To determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA, by Western blotting, and by immunohistochemistry. Real-time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, biosynthesis studies of neutrophils precursor cells showed that ficolin-1 was primarily synthesized in myelocytes, metamyelocytes, and band cells. Immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules but also in highly exocytosable gelatinase-poor granules, not described previously. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and the majority becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily exocytosed by stimulation.

U2 - 10.1189/jlb.1008606

DO - 10.1189/jlb.1008606

M3 - Journal article

C2 - 19741154

SN - 0741-5400

VL - 86

SP - 1439

EP - 1449

JO - Journal of Leukocyte Biology

JF - Journal of Leukocyte Biology

IS - 6

ER -