173 research outputs found
Cytotoxic activity of Ciona intestinalis (Tunicata) hemocytes: Properties of the in vitro reaction against erythrocyte targets
Hemocytes (effectors) of Ciona intestinalis showed a natural cytotoxic capacity (HCA) when assayed in vitro against erythrocytes (targets). Cytotoxic cells lysed, to a variable extent, rabbit (RE), human (A, B, O), guinea pig, and sheep (SE) erythrocytes. Hemocyte cytotoxic activity (HCA) assayed against SE is a calcium-dependent reaction, occurs rapidly (15-30 min), at 25-37°C over a wide range of pH (5.4-8.0). Assays were carried out using: 1) the medium in which hemocytes were maintained, 2) the soluble portion of hemocyte lysates, and 3) debris prepared from hemocyte lysates. Results suggest that HCA is a cell-mediated process that requires effector-target cell contacts. Anti-SE (calcium-dependent) and anti-RE (calcium-independent) agglutinins were also found in the reaction medium, probably released by hemocytes as a consequence of the in vitro experiments. The occurrence of HCA was independent of any allogeneic reaction between mixed hemocytes. Various levels of cytotoxic activity reveal hemocyte specificity. © 1993
Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus coelomocytes
The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured
spherulocytes involved in immune defences. In the present paper, we show a Ca2+-dependent cytotoxic activity for the unfractionated
coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations
as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high
cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released by
amoebocytes. With regard to this, we show that an enhanced cytotoxic activity was found by adding the supernatant from sonicated amoebocytes
or hemocyte culture medium into spherulocyte preparations
Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis
Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona
intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins.
In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried
out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory
responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis,
Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms
(Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgalsa
N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding,
whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable
as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on
CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD
were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling
process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgalsa
and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and
expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and
immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting
analysis indicated that CiLgals can form oligomer
Ciona intestinalis peroxinectin is a novel component of the peroxidase– cyclooxygenase gene superfamily upregulated by LPS
Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reac-
tion. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase–cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the out-group ofmammalianMPO, EPO and TPO clades. The CiPxtmolecular structuremodel resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid glan
Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFα-producing cells
In situ hybridisation and mmunohistochemistry
analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFα), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development.
Granulocytes with large granules and compartment/morula cells are CiTNFα-producing cells in both inflamed pharynx
and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the
involvement of CiTNFα in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory haemocytes.
Specific antibodies against a CiTNFα peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS
inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFα-positive tissue response. Larval
histological sections and whole-mount preparations have revealed that CiTNFα is expressed by trunk mesenchyme,preoral lobe and tunic cells, indicating CiTNFα-expressing cell immigration events and an ontogenetic role
Inducible lectins with galectin properties and human IL1 alpha epitopes opsonize yeast during the inflammatory response of the ascidian Ciona intestinalis
Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca2+-dependent cytotoxic activity
toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte
populations were separated (B1-B6 bands) through a Percoll discontinuous density gradient, the
hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS)
were assayed. In addition the separated hemocytes were cultured and the cell free medium (CFM)
assayed after 3h culture. Results support that unilocular refractile hemocytes (URGs), enriched in
B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysisns can
be released into a culture medium. The B5 activity was blocked by D-Galactose, α-Lactose,
Lactulose, LacNAc, thiodigalactoside (TDG), L-Fucose, D-Mannose, D-Glucose, sphingomyelin
(SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS
chemico-physical properties (alkaline medium, high termostability, Ca2+-dependence, trypsin
treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2
could be responsible for changes and large alterations of the target cell membrane. An apoptotic
activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted
HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells
respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme
activity
The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis
Phenoloxidase (PO) activity was examined in the
tunic tissue of Ciona intestinalis following lipopolysaccharide(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.
(LPS) intratunic injection. Tunic homogenate supernatant
(THS), assayed with the Dopa-MBTH reaction,
displayed Ca2+-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 enzym
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