The Role of Fc Receptors in the Innate Immune System of Flounders Purported to Be Homologs of FcγRII and FcγRIII.

FcγR is a significant opsonin receptor located on the surface of immune cells, playing a crucial role in Ab-dependent cell-mediated immunity. Our previous work revealed opposite expression trends of FcγRII and FcγRIII in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda. This observation suggests that FcγRII and FcγRIII might serve distinct functions in Ig-opsonized immune responses. In this study, we prepared rFcγRIII as well as its corresponding Abs to investigate the potential roles of FcγRII and FcγRIII in the Ab-dependent immune response of IgM+ B cells. Our findings indicate that, unlike FcγRII, FcγRIII does not participate in Ab-dependent cellular phagocytosis. Instead, it is involved in cytokine production and bacterial killing in mIgM+ B lymphocytes. Additionally, we identified platelet-derived ADAM17 as a key factor in regulating FcγRIII shedding and cytokine release in mIgM+ B lymphocytes. These results elucidate the functions of FcγRII and FcγRIII in the innate immunology of mIgM+ B lymphocytes and contribute to an improved understanding of the regulatory roles of FcγRs in the phagocytosis of teleost B lymphocytes.

Cluster of differentiation antigens: essential roles in the identification of teleost fish T lymphocytes.

Cluster of differentiation (CD) antigens are cell surface molecules expressed on leukocytes and other cells associated with the immune system. Antibodies that react with CD antigens are known to be one of the most essential tools for identifying leukocyte subpopulations. T lymphocytes, as an important population of leukocytes, play essential roles in the adaptive immune system. Many of the CD antigens expressed on T lymphocytes are used as surface markers for T lymphocyte classification, including CD3, CD4 and CD8 molecules. In this review, we summarize the recent advances in the identification of CD molecules on T lymphocytes in teleosts, with emphasis on the functions of CD markers in the classification of T lymphocyte subsets. We notice that genes encoding CD3, co-receptors CD4 and CD8 have been cloned in several fish species and antibodies have been developed to study protein expression in morphological and functional contexts. T lymphocytes can be divided into CD4+ and CD8+ cells discriminated by the expression of CD4 and CD8 molecules in teleost, which are functionally similar to mammalian helper T cells (Th) and cytotoxic T cells (Tc), respectively. Further studies are still needed on the particular characteristics of teleost T cell repertoires and adaptive responses, and results will facilitate the health management and development of vaccines for fish.

Kinetics of T lymphocyte subsets and B lymphocytes in response to immunostimulants in flounder (Paralichthys olivaceus): implications for CD4+ T lymphocyte differentiation.

CD4+ T lymphocytes play crucial roles in the adaptive immune system. CD4, as the most effective marker to delineate the T-helper subsets, was identified in many fish species. Two CD4 homologs, CD4-1 and CD4-2, have been reported in flounder (Paralichthys olivaceus). In this study, monoclonal antibodies (mAbs) against CD4-1 and CD4-2 of flounder were produced, CD4+ T lymphocytes were isolated and identified, and the variations in CD4+ and CD8+ T lymphocytes and IgM+ B lymphocytes after Poly I:C, PMA or ß-glucan stimulation were investigated. Then, the expression of transcription factors and cytokines in sorted CD4+ T lymphocytes was analyzed. The results showed that the mAbs were specific to flounder CD4-1+ and CD4-2+ T cells. CD4-1+ and CD4-2+ cells responded to all three stimulants, while CD8+ T lymphocytes only give a strong response to Poly I:C, and the percentages of IgM+ B lymphocytes showed a tendency to increase. After stimulation, the expression of transcription factors and cytokines of Th1, Th2 and Th17 cells varied in CD4+ T cells. These results will provide crucial foundations for the differentiation and function of teleost CD4+ T lymphocytes.

Immune responses of flounder Paralichthys olivaceus vaccinated by immersion of formalin-inactivated Edwardsiella tarda following hyperosmotic treatment.

The aim of the present study was to evaluate the effects of hyperosmotic immersion (HI) vaccination and determine the optimum hyperosmotic salinity for flounder Paralichthys olivaceus by investigating its immune responses following vaccination. Flounder were immersed in 1 of 3 hyperosmotic solutions at 50, 60 and 70‰ salinity, then transferred into 30‰ salinity normal seawater containing formalin-inactivated Edwardsiella tarda for vaccination (3 HI groups), or were immersed in normal seawater as direct immersion (DI group). The results showed that the percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood leukocytes and spleen leukocytes induced by HI were significantly higher than that with DI (p < 0.05), and the 50‰ salinity group showed the strongest response among the HI groups, which reached peaks at Week 4. ELISA assay showed that the specific serum antibodies gradually increased after vaccination and reached peak at Day 32, and the fish treated with HI showed stronger antibody responses; among the HI groups, a significantly higher specific antibody level was detected in the 50‰ salinity group at Day 32 (p < 0.05). Similarly, the fish treated with HI showed higher specific mucosal antibody levels compared to the DI group, and the mucosal antibody showed a faster response, with peak time arriving 1 wk earlier than for the serum antibody. The relative percent survival (RPS) of flounder treated with HI at 50, 60 and 70‰ salinities were 79, 71 and 57% respectively, while this was 43% in the DI group. These results demonstrated that HI, especially the 50‰ salinity, could efficiently enhance the immune response of flounder and show higher RPS. This has significant value for immunological prevention of edwardsiellosis in flounder.

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells.

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.

Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder (Paralichthys olivaceus).

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72 kDa (heavy chain) and 26 kDa (light chain), and a total molecular weight of 798 kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

Response of mucosal and systemic sIgM-positive cells in turbot (Scophthalmus maximus L.) immunization with Edwardsiella tarda.

Indirect fluorescence analyze technology (IFAT) and fluorescence-activated cell sorter (FACS) analysis were used to detect surface IgM positive cells (sIgM+ cells) in lymphoid tissues of unvaccinated turbots and vaccinated ones with inactivated Edwardsiella tarda. The results were as follows: (1) The percentage of sIgM+ cells respectively was 0+/-0.00% of skin, 1.58+/-0.40% of gills, 17.05+/-0.39% of peripheral blood (PBL), 21.06+/-1.79% of kidney in unvaccinated healthy turbots (average length: 7.12+/-0.5 cm; average height: 3.8+/-0.2 cm). (2) After direct immersion (d.i.), the response of sIgM+ cells was produced at first in the gills and thereafter in the kidney and PBL, and a significant increase of the number of sIgM+ cells was found in gills. (3) After immunization via intraperitoneal injection (i.p.), the response of sIgM+ cells was produced simultaneously in the gills, kidney and PBL after i.p., and a significant increase of the number of sIgM+ cells was found in PBL and kidney instead of in gills. These results strongly supported the different immunological role of gills, PBL and kidney upon the different administration routes, and the presence of two compartmental models for immune response in turbot.

Purification, virulence and characterization of an extracellular peptidase produced by Aeromonas salmonicida ssp. masoucida isolated from cultured sea cucumber Apostichopus japonicus.

Strain H1, Aeromonas salmonicida ssp. masoucida isolated from an ulcer on the sea cucumber Apostichopus japonicus proved to be virulent toward this holothurian at 12 degrees C. Extracellular products (ECP) of strain H1 were prepared, and an extracellular peptidase of 42 kDa was purified from the ECP using Sephadex G-100 gel filtration chromatography and diethylaminoethyl Sepharose ion exchange chromatography. Stepwise virulence tests on sea cucumbers indicated that the 42 kDa peptidase was the virulence factor of strain H1, and its LD50 for these holothurians was 1.12 microg g(-1). A peptidase activity test using azocasein as a substrate showed that optimum pH and temperature for the peptidase are 8.0 and 40 degrees C, respectively; the peptidase was completely inactivated above 70 degrees C. The peptidase was inhibited strongly by 5 mM phenylmethanesulphonyl fluoride, indicating that the enzyme is a serine peptidase.

Whitespotted puffer Arothron hispidus, a new host for lymphocystis in Qingdao Aquarium of China.

In April 2004 white nodular lesions were found on the fins of whitespotted puffer Arothron hispidus (Linnaeus). Diagnostic studies were carried out to confirm the disease using light and electron microscopy, histochemical methods and PCR. The results revealed that the nodules were composed of giant cells up to 400 microm in diameter. These cells were surrounded by a periodic acid-Schiff (PAS)-positive hyaline capsule containing dot-shaped, Feulgen-positive inclusion bodies in the cytoplasm and an irregular nucleus. Numerous virus particles 200 nm in diameter and with hexagonal profiles were observed in the cytoplasm. These features were consistent with those of lymphocystis disease. Additionally, based on the gene sequences of major capsid protein (MCP) of lymphocystis disease virus (LCDV) from Japanese flounder Paralichthys olivaceus, 2 pair primers were designed; after a nested PCR was performed for detection of LCDV in A. hispidus, a positive amplified product was obtained showing the presence of LCDV. Therefore, the white nodules were the lymphocystis lesions caused by LCDV infection and A. hispidus was demonstrated to be a new host for LCDV.

Elimination of shrimp endogenous peroxidase background in immunodot blot assays to detect white spot syndrome virus (WSSV).

False positive results were obtained in immunodot blot assays to detect white spot syndrome virus when horseradish peroxidase-conjugated sheep anti-mouse immunoglobin (Ig) serum was used as a secondary antibody with 3-3'-diaminobenzine tetrahydrochloride dihydrate as the detection substrate. The cause was considered to be a reaction of shrimp endogenous peroxidase (POD) with the substrate. In experiments designed to inhibit POD activity, 9 different reagents were used at different concentrations and for different treatment times. EDTA, sodium azide, HEPES-Na, NaHSO3, H2O2 and phenylthiourea (PTU) were able to inhibit POD activity by 44, 60, 64, 67, 79, and 90%, respectively. Phenylmethylsulfonyl fluoride did not inhibit POD, and neither periodic acid nor H2O2 in methanol were appropriate due to the formation of flocculant precipitates when added to shrimp extracts. It was concluded that of the treatments tested, 10 mM PTU for 2 h yielded optimal inhibition and that such pretreatment of samples eliminates false positive results in immunodot blot assays.

Endoenzymes associated with haemocyte types in the scallop (Chlamys farreri).

Haemocyte types of the scallop (Chlamys farreri) were identified by Giemsa stain and flow cytometry (FCM). Additionally, the activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analysed by immunocytochemical and biochemical methods. The results indicate that there were two types of haemocytes in the scallop, hyalinocytes and granulocytes, and that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.