[Show abstract][Hide abstract] ABSTRACT: Cells from the coelomic cavity of adult zebrafish (zf) were used to study the alarmin-like activities of nonspecific cytotoxic cell antimicrobial protein-1 (NCAMP-1). Immunohistochemistry studies using polyclonal anti-NCAMP-1 identified constitutive NCAMP-1 in epithelial cells of the zf anterior kidney, in liver parenchyma and in the lamina propria of the intestine. NCAMP-1 was also located in the cytosol of mononuclear cells in these tissues. Cytosolic NCAMP-1 was detected in a diverse population of coelomic cells (CC) using confocal microscopy and polyclonal anti-NCAMP-1 staining. Large mononuclear and heterophil-like CC had intracellular NCAMP-1. These studies indicated that NCAMP-1 is constitutively found in epithelial cells and in ZFCC. To establish a relationship between NCAMP-1 and the alarmin functions of ATP, a stimulation-secretion model was initiated using zf coelomic cells (ZFCC). ZFCCs treated with the alarmin ATP secreted NCAMP-1 into culture supernatants. Treatment of ZFCC with either ATP or NCAMP-1 activated purinergic receptor induced pore formation detected by the ZFCC uptake of the dye YO-PRO-1. ATP induced YO-PRO-1 uptake was inhibited by antagonists oxidized-ATP, KN62, or CBB. These antagonists did not compete with NCAMP-1 induced YO-PRO-1 uptake. Binding of ZFCC by both ATP and NCAMP-1 produced an influx of Ca2+. Combined treatment of ZFCC with ATP and NCAMP-1 increased target cell cytotoxicity. Individually NCAMP-1 or ATP treatment did not produce target cell damage. Similar to ATP, NCAMP-1 activates cellular pore formation, calcium influx and cytotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.