Nonspecific cytotoxic cell antimicrobial protein (NCAMP-1): a novel alarmin ligand identified in zebrafish.

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

Western blot analysis of NCAMP-1 release from ATP treated zebrafish CC.

<p>ZFCC were untreated (NA) or treated for 30 min with 5 mM ATP (+A). Supernatants were harvested and analyzed by Western blot using rabbit polyclonal anti-NCAMP-1 antibody. (+) is the positive control of R-NCAMP-1-Histag binding. (-) is the negative control (e.g. no primary antibody).</p

NCAMP-1 or ATP do not cause cellular damage.

<p>(A) ZFCC were incubated with 1ug NCAMP-1 for 30min at room temperature and analyzed with YO-PRO-1 and PI. (B) 20mM and 30mM ATP were added in presence of YO-PRO-1 and PI and immediately analyzed (0min). Representative of 3 independent experiments.</p

Constitutive expression of NCAMP-1 in zebrafish tissues.

<p>Serial thin sections of zf trunk kidney and intestine were examined by H&E staining; rabbit polyclonal anti-NCAMP-1 serum and normal rabbit isotype control.</p

Cytotoxic activity of zebrafish effectors is increased following pretreatment of NCAMP-1 or ATP.

<p>HL-60 target cells were labeled with 5mM CFSE for 15 minutes at 37°C and washed. Targets were added to zebrafish effectors, pretreated for 30 minutes with either 5mM ATP, 1ug NCAMP-1, or no treatment, at an effector to target cell ratio of 8:1. Flow analysis was conducted at times 0, 1, 2, and 4 hours of co-incubation. Data points represent the mean ± standard error of 3 independent experiments. At times of 60 and 120 minutes, significant differences were observed between NCAMP-1 and media (** P≤ 0.01); NCAMP-1 and ATP *P≤ 0.05 as calculated by two-way ANOVA according to Bonferroni method.</p

NCAMP-1 and ATP bind to CC and induce an influx of Ca²⁺ ions.

<p>ZFCC were loaded with 4 uM Fluo-4 for 20 minutes at 37°C, washed with media, then analyzed for mean fluorescence intensity by flow cytometry. 50 seconds into analysis the calcium ionophore (positive control) or 1ug NCAMP-1 (A) or different concentrations of ATP (B) were added and analysis was continued. In C, cells were treated with the indicated ATP antagonist before addition of 30mM ATP and analyzed. Arrows indicate the addition of the agonist. Representative of 3 independent experiments.</p

YO-PRO-1 Uptake by ZFCC in response to NCAMP-1 treatment in the absence or presence of ATP.

<p>* P = ≤ 0.05 Different from NCAMP-1 only; Duncan’s (new) Multiple Range Test.</p><p>YO-PRO-1 Uptake by ZFCC in response to NCAMP-1 treatment in the absence or presence of ATP.</p

ATP induced YO-PRO-1 uptake by ZFCC is abrogated with P2X₇R inhibitors.

<p>30mM ATP was added to ZFCC with 5mM YO-PRO-1 and analyzed for percent fluorescence immediately or incubated with oxidized-ATP (oATP) (A), KN62 (B), or CBB (C) for 15 minutes prior to addition of ATP. Results are shown as the mean of 3 independent experiments ± S.E. * P≤ 0.01 significantly different from media controls as calculated by two-way ANOVA according to Bonferroni method.</p