Original Ligand for LTßR Is LIGHT: Insight into Evolution of the LT/LTßR System.

The lymphotoxin (LT)/LTß receptor (LTßR) axis is crucial for the regulation of immune responses and development of lymphoid tissues in mammals. Despite the importance of this pathway, the existence and function of LT and LTßR remain obscure for nonmammalian species. In this study, we report a nonmammalian LTßR and its ligand. We demonstrate that TNF-New (TNFN), which has been considered orthologous to mammalian LT, was expressed on the cell surface as a homomer in vitro. This different protein structure indicates that TNFN is not orthologous to mammalian LTα and LTß. Additionally, we found that LTßR was conserved in teleosts, but the soluble form of recombinant fugu LTßR did not bind to membrane TNFN under the circumstance tested. Conversely, the LTßR recombinant bound to another ligand, LIGHT, similar to that of mammals. These findings indicate that teleost LTßR is originally a LIGHT receptor. In the cytoplasmic region of fugu LTßR, recombinant fugu LTßR bound to the adaptor protein TNFR-associated factor (TRAF) 2, but little to TRAF3. This difference suggests that teleost LTßR could potentially activate the classical NF-κB pathway with a novel binding domain, but would have little ability to activate an alternative one. Collectively, our results suggested that LIGHT was the original ligand for LTßR, and that the teleost immune system lacked the LT/LTßR pathway. Acquisition of the LT ligand and TRAF binding domain after lobe-finned fish may have facilitated the sophistication of the immune system and lymphoid tissues.

Teleost Basophils Have IgM-Dependent and Dual Ig-Independent Degranulation Systems.

Recently, mammalian basophils have been highlighted as having roles in allergy and antiparasitic immunity; however, there is little information about the functions and evolutionary origin of basophils, because they are the least abundant leukocyte in most vertebrates. In this study, we characterized the teleost basophils that are abundant in the peripheral blood of fugu (Takifugu rubripes). Fugu basophils have two distinct granules: reddish-purple and dark violet ones. Teleost fish do not have IgG and IgE, but we found that fugu IgM bound on the surface of the basophils, and the cross-linked IgM induced degranulation of both types of granules. This indicates that teleost basophils can be activated in an Ab-dependent manner. Furthermore, papain induced the degranulation of the reddish-purple granules, which contain histamine, and the released granules stimulated the migration of various leukocytes. In contrast, chitin elicited the degranulation of the dark violet granules, which resulted in CD4+ T cell-specific migration. Thus, fugu basophils control immune responses via two distinct Ab-independent mechanisms. In addition, fugu basophils endocytosed soluble Ag and expressed MHC class II and B7-H1/DC. These findings suggested that fugu basophils can interact with T cells as APCs. Thus, the Ab-dependent basophil activation predates the emergence of IgG and IgE, and fish basophils exhibit different dynamics and features of degranulation to distinct stimuli compared with mammalian basophils. Some features of teleost basophils are more similar to those of mammalian mast cells than to those of mammalian basophils.

SJL-1, a C-type lectin, acts as a surface defense molecule in Japanese sea cucumber, Apostichopus japonicus.

The surface defense molecules of aquatic invertebrates against infectious microorganisms have remained largely unexplored. In the present study, hemagglutinins were isolated from an extract of body surface layer of Japanese sea cucumber, Apostichopus japonicus, by affinity chromatography with fixed rabbit erythrocyte membranes. The N-terminal sequence of a 15-kDa agglutinin was almost identical with that of SJL-1, a C-type lectin formerly identified in this species. Because cDNA sequence and tissue distribution of SJL-1 have not been reported, we performed cDNA sequencing, gene expression analysis, and western blotting and immunohistochemical evaluation with anti-recombinant SJL-1 (rSJL-1) antibodies. The hemagglutinin gene was transcribed mainly in the integument, tentacles, and respiratory tree. Western blotting revealed that SJL-I is present in a body surface rinse, indicating that SJL-1 is secreted onto the body surface. SJL-1-positive cells scattered beneath the outermost layer of the integument were detected by immunohistochemistry. Furthermore, rSJL-1 agglutinated Gram-positive and Gram-negative bacteria, and yeast. These results indicate that SJL-1 acts as a surface defense molecule in A. japonicus.

Serum GlcNAc-binding IgM of fugu (Takifugu rubripes) suppresses the growth of fish pathogenic bacteria: a novel function of teleost antibody.

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.

Teleost IL-6 promotes antibody production through STAT3 signaling via IL-6R and gp130.

Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM(+) B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM(+) B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes).

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM+CD8α⁻ cells and IgM+CD8α+ cells. Further characterization of the IgM+CD8α⁻ population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM+CD8α⁻ cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.