The teleost acute-phase inflammatory response and caspase activation by a novel alarmin-like ligand

Abstract

This study tested the hypothesis that NCAMP-1 has alarmin-like properties and activates the caspase-1-binding site in cells of the teleost bone marrow (equivalent). In mammals, alarmins have been studied extensively; however, in teleosts, little is known about their identity and functions. Similar to alarmins, NCAMP-1 has a broad spectrum of bacteriolytic activity. NCAMP-1 is constitutively present in CF serum, and levels were increased following infection with Edwardsiella ictaluri Binding to AK cells was determined with rNCAMP-1 and an anti-His-tag antibody. In vitro treatment of AK (bone marrow equivalent) or spleen cells with rNCAMP-1 increased the IL-1β message three- to fivefold at 3 h, 6 h, and 9 h post-treatment. The association of NCAMP-1 with the activities of alarmin ATP and the acute inflammatory response was demonstrated by NCAMP-1-induced P2XR pore opening and YO-PRO-1 cellular influx. The association of NCAMP-1 binding with inflammasome activation was demonstrated by NCAMP-1 activation of the caspase-1-binding site for tetrapeptide Z-YVAD-FMK. In competition assays, this tetrapeptide competitively inhibited subsequent binding by the pan-caspase substrate tripeptide FAM-VAD-FMK. Lymphocyte-like cells from the spleen were 16%, and epithelial cells were also positive for NCAMP-1. IHC staining and confocal microscopy confirmed the cytosolic existence of NCAMP-1 in lymphoreticular tissue and IL-1β in AK cells. CF T cell lines G14D and 28S.3 expressed NCAMP-1 in the cytosol and in storage granules. These studies strongly suggested that NCAMP-1 is an alarmin-like ligand with similar but distinct activities to those of ATP and HMGB-1.

Nonspecific Cytotoxic Cell Antimicrobial Protein (NCAMP-1): A Novel Alarmin Ligand Identified in Zebrafish

“…3 ). The molecular weight discrepancy between the secreted and recombinant NCAMP-1 forms has been previously noted in catfish [ 38 ]. The very basic nature of the protein is partly responsible for inaccuracies in SDS gel electrophoresis migration.…”

“…The purified and subsequently sequenced protein [nonspecific cytotoxic cell cationic antimicrobial protein (e.g. NCAMP-1)] is a novel phylogenetically conserved H1X-like protein [ 38 , 40 ]. We now show similarities and distinctions between the alarmin functions of NCAMP-1 and ATP, and we suggest that NCAMP-1 shares similarities also with other alarmins such as high mobility group box-1/HMGB-1 [ 7 – 8 ].…”

“…Trypsin activity was inactivated with FCS after harvesting the cells. When indicated as ZFCC, the cells used for analysis were not gated and as shown before contained distinct populations of monocytes, lymphocytes, granulocytes, nonspecific cytotoxic cells and macrophages [ 38 ].…”

“…Fish were euthanized with an overdose of MS222, and 10 minutes after cessation of movement the body cavity was injected with Dietrich’s solution. Total body fixation was accomplished by submersion into Dietrich’s solution [ 38 ]. An entire fish was placed into standard tissue cassettes for routine overnight processing.…”

“…In mammalian cells NCAMP-1 is expressed on the membrane of a human NK cell line (YT-INDY), it binds dGT20 and CpG ODNs [ 36 ]; it is found in granule extracts from RAW 264.7 cells; and NCAMP-1 is expressed by a variety of mouse leukocytes derived from the blood, mesenteric lymph nodes, and spleen [ 37 ]. The alarmin activity of NCAMP-1 has been reported in catfish cells [ 38 ]. Here, NCAMP-1 is compared to those effects induced by the established endogenous alarmin ATP.…”