Expression analysis of lipopolysaccharide-and/or concanavalin A/phorbol myristate acetate-stimulated black rockfish peripheral blood leukocytes using a cDNA microarray to identify up-regulated genes

The responses of peripheral blood leucocytes (PBLs) from the black rockfish Sebastes schlegelii stimulated with lipopolysaccharides (LPS) and/or concanavalin A/phorbol myristate acetate (Con A/PMA) were investigated using a cDNA microarray consisting of 5,088 clones. This analysis showed that 254 unique genes were more than twofold upregulated, and they were selected for sequencing. Among the mitogens, 84 genes were more than twofold upregulated in LPS-stimulated PBLs, and 112 genes were induced in Con A/PMA-stimulated PBLs. Moreover, 58 other genes were more than twofold upregulated in PBLs stimulated with both LPS and Con A/PMA. Other genes were not significantly induced. Overall, these results suggest that certain genes in black rockfish have important roles in physiological and pathological processes. Furthermore, the microarray analysis suggested a promising tool for immune mechanisms in teleost fish.     

Molecular identification and expression analysis of the CC chemokine gene in rock bream (Oplegnathus fasciatus) and the biological activity of the recombinant protein

We identified the CC chemokine cDNA designated as RbCC1 (CC chemokine 1 in rock bream, Oplegnathus fasciatus), which was isolated using expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCC1 cDNA (850 bp) contained an open reading frame (ORF) of 366 bp encoding 122 amino acids. Results from our phylogenetic analysis demonstrated that the RbCC1 was closest relationship to the orange-spotted grouper and Mi-iyu croaker CC chemokines located within the fish CC chemokine group. RbCC1 was significantly expressed in the intestine, spleen, liver, and PBLs (peripheral blood leukocytes). Rock bream PBLs were stimulated with several mitogens, LPS and Con A/PMA which significantly induced the expression of RbCC1 mRNA in the PBLs. The RbCC1 mRNA expression in several tissues under conditions of bacterial and viral challenge was examined. The experimental challenge revealed that the kidney and spleen of fish infected with Streptococcus iniae showed the most significant increases in RbCC1 expression compared to the control. In the case of RSIV infection, the RbCC1 mRNA expression was markedly up-regulated in the liver. In this study, recombinant RbCC1 (approximately 53 kDa) was produced using an Escherichia coli expression system followed by purification. Subsequently, the addition of purified rRbCC1 was examined to investigate the impact on the proliferative and chemotactic activity on kidney leukocytes from rock bream. The results demonstrated that the rRbCC1 induces significant biological activity on kidney leukocyte proliferation and attraction at concentrations in the range of 10–300 μg/mL and suggests that rRbCC1 could be utilized as an immune-stimulant and/or molecular adjuvant to enhance the immune effects of vaccines.     

Cooperation of erythrocytes with leukocytes in immune response of a teleost <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis>

The most abundant cell type in the blood of mammals and fish is erythrocyte. Unlike mammalian erythrocyte, fish erythrocyte is nucleated. The functional differentiation of teleost erythrocyte is insufficient compared with that of mammals. Therefore, fish erythrocyte may have different functions from that of mammals. Functional interaction between erythrocyte and leukocyte was confirmed by the cDNA microarray newly constructed in this study to investigate characterization of rock bream erythrocyte. In this study, different immune related genes of erythrocytes were annotated by microarray analysis. Microarray analysis showed that a total of 338 genes were up-regulated in co-cultured erythrocytes with leukocytes by LPS stimulation when comparing to erythrocytes stimulated LPS. Many genes in erythrocyte of rock bream were up-regulated in presence of leukocytes, suggesting that erythrocytes interact with leukocytes to trigger expression of various genes associated with the immune system. Our results provide valuable information that direct and indirect immunological function of fish erythrocyte.     

Molecular characterization, expression, and functional analysis of two thioredoxins in the black rockfish (Sebastes schlegelii)

Thioredoxins (TRxs) are a family of small evolutionarily conserved proteins that are essential for the maintenance of cellular homeostasis. Two TRx homologue cDNAs were isolated from a black rockfish concanavalin A (Con A)/phorbol myristate acetate (PMA)-stimulated leucocyte cDNA library and named BrTPx1-1 and BrTPx1-2. As compared with other known TRx peptide sequences, the most conserved regions of both BrTRx1-1 and BrTRx1-2 peptides were found to be the redox-active site Trp-Cys-X-X-Cys (WCXXC). The TRx present in most species is a TRx1-2 protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in the larger 13 kDa BrTRx1-1 protein, a Cys-Pro-Pro-Cys (CPPC) active site was identified. Here, we report the identification of a new member of the TRx protein family from the teleost black rockfish, which defines a new subclass of 13-kDa TRx1-1 proteins. Phylogenetic analysis indicated that both BrTRx1-1 and BrTRx1-2 were grouped with other vertebrate TRx1 peptides. BrTRx1-1 expression was strongly induced in peripheral blood leucocytes (PBLs) 12-24 h following Con A/PMA stimulation, with peak expression at 24 h post-stimulation. BrTRx1-2 was induced in PBLs after stimulation with lipopolysaccharide (LPS), Con A/PMA, or poly I:C at 24 h. The BrTRx1-1 gene was predominantly expressed in the liver and gills, while BrTRx1-2 was expressed in PBLs and gills. After treatment with a high concentration (10 μg/mL) of rBrTRx1-1 or rBrTRx1-2, kidney leucocytes exhibited increased cell proliferation and viability under oxidative stress.     

Molecular cloning and expression analysis of three Mx isoforms of rock bream, Oplegnathus fasciatus

Complementary DNAs (cDNAs) corresponding to three isoforms of rock bream (Oplegnathus fasciatus) Mx (RbMx1, RbMx2 and RbMx3) were cloned using RACE reactions. Analysis of deduced amino acid sequences revealed that the tripartite GTP-binding domain, the dynamine family signature and the leucine zipper repeat were present in all three rock bream Mx isoforms. Cloning of genomic DNA sequence and Southern blot analysis showed that three rock bream Mx isoforms were encoded by different genomic loci, and they were not alternative splicing variants, although some alternative splicing variants were found in RbMx1 and RbMx2. When comparing amino acid sequence identity, RbMx1 shares about 60-70% identities with other fish Mx proteins, whereas both RbMx2 and RbMx3 share slightly high identity of 70-90%. As a result of expression analysis using RT-PCR, RbMx1 was constitutively expressed in the spleen and kidney of rock bream yearling, but RbMx2 and RbMx3 were rarely detected in both organs. When injected with synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C), expression of all rock bream Mx isoforms was up-regulated in spleen and head kidney. RbMx1 was continuously up-regulated throughout experimental period of 72 h but RbMx2 and RbMx3 were down-regulated to almost non-detectable level at 48 h post-injection.     

Molecular characterisation and biological activity of a novel CXC chemokine gene in rock bream (Oplegnathus fasciatus)

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues. In mammals, these cytokines can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in the sequence, and include the CXC(α), CC(β), C(γ), and CX3C(δ) classes. We identified CXC chemokine cDNA, designated RbCXC, isolated using expressed sequence tag analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCXC cDNA (742 bp) contained an open reading frame of 342 bp encoding 114 amino acids. Results from phylogenetic analysis showed that RbCXC was strictly separated into a distinct clade compared to other known CXC chemokine subgroups. RbCXC was significantly expressed in the trunk kidney, liver, spleen, gill, peripheral blood leukocytes (PBLs), and head kidney. Rock bream PBLs were stimulated with several mitogens, including LPS and polyinosinic-polycytidylic acid (poly I:C), which significantly induced the expression of RbCXC mRNA. RbCXC mRNA expression was examined in several tissues under conditions of bacterial and viral challenge. Experimental challenges revealed that all examined tissues from fish infected with Edwardsiella tarda and red sea bream iridovirus showed significant increases in RbCXC expression compared to the control. In the case of Streptococcus iniae infection, RbCXC mRNA expression was markedly upregulated in the kidney, spleen, and liver. In addition, a maltose binding protein fusion recombinant RbCXC (～53 kDa) was produced in an Escherichia coli expression system and purified. Subsequently, the addition of purified recombinant RbCXC (rRbCXC) to kidney leukocytes was examined to investigate the impact of proliferative and chemotactic activity. The rRbCXC induced significant kidney leukocyte proliferation and attraction at concentrations ranging from 10 to 300 μg/mL, suggesting that it can be utilised as an immune stimulant and/or molecular adjuvant to enhance the immunological effects of vaccines.     

Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes

Interferon (IFN)-alpha and -beta are important antiviral mediators. IFN-alpha is widely used for the treatment of chronic hepatitis B. In our previous studies, a subtype of woodchuck IFN-alpha (wIFN-alpha) was characterized and has been shown to be active in suppressing the replication of woodchuck hepatitis virus (WHV) in vitro and vivo. Here, we refined the analysis of the IFN-alpha/beta system of the woodchuck and studied the expression of wIFN-alpha/beta in peripheral blood lymphocytes (PBLs) from na?ve and WHV-infected woodchucks. A number of wIFN-alpha genes were sequenced and could be classified into 10 subtypes and 3 pseudotypes. The biological activity of different subtypes of wIFN-alpha was demonstrated by their ability to protect woodchuck cells against encephalomyocarditis virus infection and to induce MxA expression in woodchuck cells. Additionally, a partial sequence of wIFN-beta was characterized. A subtyping method for wIFN-alpha based on restriction length polymorphism analysis was developed. Further, the expression of wIFN by woodchuck PBLs after stimulation with polyI/C was investigated. The maximal production of wIFN by woodchuck PBLs occurred within the first 48 h after addition poly I/C. The wIFN-alpha subtypes 1, 4, and 5 were found to be produced by poly I/C-stimulated woodchuck PBLs, indicating a selective expression of wIFN-alpha subtypes. PBLs from chronically WHV-infected woodchucks showed a reduced ability to produce wIFN when stimulated with poly I/C. The results suggest that woodchucks with chronic WHV infection have impaired immunological responses to poly I/C.     

Identification and expression analysis of an interferon stimulated gene 15 (ISG15) from black rockfish, Sebastes schlegeli

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by IFNs, viral infection and double-stranded RNA (poly I:C). The ISG15 homolog cDNA was isolated from the black rockfish poly I:C stimulated leukocyte cDNA library. The black rockfish ISG15 homolog was found to consist of 1070bp encoding 160 amino acid residues. Compared with other known ISG15 peptide sequences, the most conserved regions of the black rockfish ISG15 peptide were found to be the tandem ubiquitin-like domains and a C-terminal LRLRGG conjugating motif, characteristic of mammalian and non-mammalian ISG15 proteins. A phylogenetic analysis based on the deduced amino acid sequence revealed a homologous relationship between the ISG15 sequence of black rockfish and that of Atlantic salmon, Atlantic cod, crucian carp and rainbow trout. The expression of the black rockfish ISG15 molecule was induced in the peripheral blood leukocytes (PBLs) from 1 to 12h following poly I:C stimulation, with a peak at 6h post-stimulation. The black rockfish gene was predominantly expressed in the PBLs and the spleen.     

The expression and role of miR-301a in human umbilical cord-derived mesenchymal stromal cells

Background aimsToll-like receptors (TLRs) are expressed in human umbilical cord-derived mesenchymal stromal cells (UC-MSCs), and activation of TLRs plays an important role in proliferation, differentiation and immunoregulatory activity of UC-MSCs. We investigated whether TLRs regulated the expression of microRNAs (miRNAs) in UC-MSCs and the role of miRNAs.Methods and ResultsWith miRNA microarray analysis, we demonstrated that the expression of many miRNAs varied when UC-MSCs were stimulated with the ligand of toll-like receptor 4 (TLR4), lipopolysaccharide (LPS). The expression of some miRNAs was verified by polymerase chain reaction. It was found that microRNA-301a (miR-301a) was up-regulated by the ligands of TLR3 and TLR4, LPS and polyinosinic acid:polycytidylic acid poly(I:C). However, the inhibitors of nuclear factor κB NF-κB and interferon regulatory factor 3 IRF3 signal attenuated the effect of LPS and poly(I:C) on miR-301a expression. Over-expression or lower expression of miR-301a affected the cytokine secretion of UC-MSCs.ConclusionsThe expression of miR-301a in UC-MSCs was regulated by TLRs, and miR-301a affected the cytokine secretion of UC-MSCs.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Human T-cell hybridomas producing lymphokines. II. Enhancement of lymphotoxin secretion from human T-cell hybridomas by phorbol myristate acetate

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF/MAF and LT-producing cloned hybrid line E10-20 secreted rather decreased amounts of MIF/MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.     

Erythromycin differentially inhibits lipopolysaccharide- or poly(I:C)-induced but not peptidoglycan-induced activation of human monocyte-derived dendritic cells

Erythromycin (EM) has attracted attention because of its anti-inflammatory effect. Because dendritic cells (DCs) are the most potent APCs involved in numerous pathologic processes including innate immunity, we examined effects of EM on the activation of human DCs by pathogen-derived stimuli. Monocyte-derived DCs were pretreated with EM and subsequently stimulated with peptidoglycan, polyriboinosinic:polyribocytidylic acid (poly(I:C)), or LPS. The activation of DCs was assessed by surface molecule expression and cytokine production. To reveal the signaling pathways affected by EM, TLR expression, NF-kappaB, IFN regulatory factor-3, and AP-1 activation were examined. EM inhibited costimulatory molecule expression and cytokine production that was induced by poly(I:C) and LPS but not by peptidoglycan. EM pretreatment down- and up-regulated mRNA levels of TLR3 and TLR2, respectively, but did not affect that of TLR4. EM suppressed IFN regulatory factor-3 activation and IFN-beta production but not AP-1 activation induced by poly(I:C) and LPS. The inhibitory effect of EM on NF-kappaB activation was observed only in poly(I:C)-stimulated DCs. EM selectively suppressed activation of DCs induced by LPS and poly(I:C) in different ways, suggesting that the immuno-modulating effects of EM depend on the nature of pathogens. These results might explain why EM prevents the virus-induced exacerbation in the chronic inflammatory respiratory diseases and give us the clue to design new drugs to treat these diseases.     

NBD-cholesterol incorporation by rat macrophages and lymphocytes: a process dependent on the activation state of the cells

The time-course of incorporation of NBD-cholesterol by macrophages (Ma) and lymphocytes (LY) obtained from untreated and thioglycollate-injected (thio) rats was investigated. NBD-cholesterol incorporation was also examined in Ma obtained from untreated rats and stimulated in vitro by lipopolysaccharide (LPS) and phorbol-myristate acetate (PMA). The same measurement was performed in LY from untreated rats stimulated by addition of LPS and concanavalin A (Con A) into the culture medium. Thio-treated Ma showed high fluorescence intensity after 1 h of incubation with NBD-cholesterol. Ma submitted concomitant to LPS and NBD-cholesterol showed low fluorescence intensity, as well as Ma stimulated with PMA. Ma from untreated and LPS pre-treated rats showed a similar time-course of incorporation. LY from thio-treated rats showed lower incorporation of NBD-cholesterol in comparison to LY from untreated rats. Incorporation was reduced when LPS was added concomitantly with NBD-cholesterol. On the other hand, LY pre-treated with LPS for 48 h showed a very high incorporation of NBD-cholesterol. Con A treatment did not cause a significant effect on NBD-cholesterol incorporation. The findings presented herein led us to conclude that the uptake of NBD-cholesterol by Ma and LY is markedly affected by the activation state of the cells.