Gender differences in the immune system activities of sea urchin Paracentrotus lividus.

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.

Enhanced expression of a cloned and sequenced Ciona intestinalis TNFalpha-like (CiTNF alpha) gene during the LPS-induced inflammatory response.

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.

Inducible lectins with galectin properties and human IL1alpha epitopes opsonize yeast during the inflammatory response of the ascidian Ciona intestinalis.

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1alpha (IL1alpha) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1alpha antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1alpha epitopes, probably because of steric configuration and the oligomerization process.

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 Ca(2+)-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.