The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Abstract

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca 2+ -independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.

Ciona intestinalis interleukin 17-like genes expression is upregulated by LPS challenge

“…Previous findings on inflammatory genes expression, including CiTNFα and galectins (Vizzini et al, 2012), revealed a prompt (4-8 h) upregulation of immune-related genes transcription by pharynx hemocytes, and the present paper suggest that CiIL17 genes, such as vertebrate ortologs, are involved in the inflammatory response (Parrinello et al, 2007Vizzini et al, 2012). In situ hybridization assay showed that CiIL17 genes are expressed by inflammatory hemocytes (granulocytes and URGs) in the pharynx vessels, and, according to previous findings (Bonura et al, 2009(Bonura et al, , 2010Cammarata and Parrinello, 2009;Cammarata et al, 2008;Parrinello et al, 2008Parrinello et al, , 2010Vizzini et al, 2008Vizzini et al, , 2012, the LPS inoculation enhanced the population density of hemocytes expressing immune-related genes including CiIL-17s. Finally these findings suggest that CiIL-17 genes may be key genes in cellular defence reaction.…”

Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis

“…Indeed, a future research direction might be the possibility that this cell gathered in clusters could represent epithelia host stationary cells derived from the lymph nodules (Ermak, 1976). According to previous findings Vizzini et al, 2008Vizzini et al, , 2012Cammarata et al, 2008;Bonura et al, 2009Bonura et al, , 2010, the LPS inoculation enhanced the population density of hemocytes expressing immune-related genes including CiTGF-b. In invertebrates, TGF1 affects immunocyte and human monocyte migration in dose-correlated fashion and induces hemocytes to migrate in a chemotactic manner (Ottaviani et al, 1997a).…”

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

“…It is known that during a pathogenic infection, the expression of galectin genes is often upregulated and their production increase [2,4]. The LPS locally inoculated through the ascidian body wall directly permeates the underlying pharynx tissue and stimulates a prompt local inflammatory reaction including upregulation of collagen-IX-like, C3a [43], TNFalfa-like [23,24] and MBL-like genes [25], together with enhanced phenoloxidase activity [22,44] and an overproduction of galectin-like molecules with opsonic properties found in the hemolymph [27]. Here we show that LPS promptly upregulates CiLgals-a and -b genes being CiLgals-a more represented as shown by a higher expression rate.…”