Single-base oligodeoxyguanosine-binding proteins on nonspecific cytotoxic cells: identification of a new class of pattern-recognition receptors

The present study was designed to identify a possible new class of pathogen-recognition proteins that bind single-base oligodeoxynucleotide (ODN) ligands. Binding by the teleost natural killer cell equivalent [referred to as nonspecific cytotoxic cells (NCC)] was compared with mammalian cells (mouse RAW264.7 cells and human THP-1 cells). The ODN analysed were composed of 20-mers of guanosine (dG20), adenosine (dA20), thymidine (dT20) or cytosine (dC20). Binding studies first determined the 50% saturation levels for NCC (1.25 microg/ml), RAW264.7 (0.2 microg/ml) and THP-1 (0.8 microg/ml). Binding by dG20 to all the three cell types was saturable. Ligand blots of NCC membrane lysates with biotinylated dG20 revealed two different major molecular weight species (16-18 and 29 kDa) of binding proteins. The 29-kDa protein was identified with the help of Western blot analysis using a polyclonal antibody specific to an NCC antimicrobial protein (ncamp-1). The membrane expression of the 29-kDa ncamp-1 was determined by the binding of surface-biotinylated NCC membrane proteins with digoxigenin dG20 followed by immunoprecipitation using anti-digoxigenin agarose beads. The 29 and 14-18 kDa NCC membrane proteins were cross-reactive using Western blot examination with a polyclonal anti-histone 1 antibody. Function studies revealed that dG20 activated a twofold upregulation of membrane binding by homologous dG20-biotin. dG20 also stimulated NCC-increased membrane expression of NCC receptor protein 1. Additional experiments were performed to determine the DNase sensitivity of the different ODN. dG20 appeared to be more resistant to DNase treatment, compared to dC20, dA20 and dT20. The single-base ODN-binding proteins may represent a new class of pattern-recognition receptors that are involved in innate anti-bacterial resistance mediated by NCC.     

Activation of natural killer-like YT-INDY cells by oligodeoxynucleotides and binding by homologous pattern recognition proteins

The present study was designed to examine the binding and signalling effects of single base and CpG dinucleotide phosphodiester (Po) oligodeoxynucleotides (ODN) on the human natural killer (NK)-like cell line (YT-INDY). Single base Po ODN composed of 20-mers of guanosine (dG20), adenosine (dA20), cytosine (dC20) or thymidine (dT20) as well as 'conventional' Po CpG ODN were examined for their ability to bind and activate YT-INDY cells. Binding by dG20 and CpG ODN to YT-INDY cells was saturable and specific. dG20 binding was competitively inhibited by homologous dG20 and heterologous CpG ODN but not by dC20 and dA20. Two different YT-INDY membrane proteins (18 and 29 kDa) were identified by ligand (Southwestern) blotting with biotinylated dG20 and CpG. The specificity of the ODN-binding protein(s) was further confirmed by ODN depletion experiments using a teleost recombinant protein orthologue [nonspecific cytotoxic cells (NCC) cationic antimicrobial protein-1 (ncamp-1)] known to bind CpG and dG20. Cell proliferation and activation studies showed that dG20 and CpG treatment of YT-INDY cells induced cellular DNA synthesis (i.e. G1 to S-phase conversion). This signalling function was accompanied in dG20-treated cells by proliferation 10 h posttreatment. Both dG20 and CpG ODN binding induced a calcium flux in YT-INDY cells within seconds of treatment. These experiments demonstrated that Po single base dG20 and CpG ODN bind to a (potential) new class of cell-surface proteins that mediate the activation of YT-INDY cells.     

Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: binding, specificity and identification of unique immunostimulatory motifs.

We have analyzed the effects of synthetic oligodeoxynucleotides (sODNs) and bacterial DNA (bDNA) on the in vitro activation of NCC. Teleost NCC recognition of DNA appeared to differ from that which occurs in higher vertebrates. NCC contain at least two different receptor specificities for DNA. Both oligodeoxyguanosine 20-mers (dG20) and 5'-TGCTGCTTGTGCTTGTGCTT-3' (4GC-2T) bound specifically to NCC. The existence of different receptor specificities was indicated by reciprocal cold target inhibition experiments. dG20 competed with 4GC-2T binding but sODNs composed of GpC or CpG nests did not compete with recognition by NCC of the dG20. ODN binding by NCC primarily depended on the presence of GpC or CpG nests with a preference for -G- serving as the anchor nucleotide. Secondarily, and similar to models of ODN activation in mammals, palindrome sequences of pu-pu-CpG-py-py activated NCC cytotoxicity. Additional analysis of the requirements for ODN activation indicated that guanosine could not substitute for adenosine as a purine spacer and that CpG motifs containing flanking thymidine (i.e.-GTCpGTT-) augmented the activity of the sODN containing this flanking base. Other evidence for the participation of both G and C in the recognition of specific nucleotides by NCC was that poly-dC20, dA20 or dT20 had no activating properties. Methylation of all cytosine nucleotides within an ODN abrogated activation. A canonical ODN motif of 5'-C/AT/AGCTT-3' can now be suggested for teleosts. Additional studies were done to examine the effects of in vitro treatment of NCC with bDNA. bDNA from three different disease isolates of Streptococcus iniae activated NCC cytotoxicity. Treatment of the bDNA with DNase abrogated the enhancement of cytotoxicity. Also, treatment of NCC with eukaryotic DNA had no effects on cytotoxicity. These studies suggested that NCC recognize bacterial nonmethylated DNA. The consequences of these interactions may be increased innate and acquired anti-bacterial immunity.     

Nonspecific cytotoxic cells of teleosts are armed with multiple granzymes and other components of the granule exocytosis pathway

Granzymes are members of the serine protease family and major components of cytotoxic granules of professional killer cells. Multiple granzymes have been identified from human and rodents with different substrate specificities. Although the significance of granzymes A and B in cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other granzymes may have either equal or greater importance in mediating cell death. Studies on the evolution of these closely related proteases were hindered by the lack of sequence and biochemical information of granzymes from "lower vertebrates." Here we report the generation of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester substrates verified its tryptase activity in full agreement with previous observations by sequence comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these cells. Evidence for additional granzymes with different substrate specificities in NCC was obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity assays. Searches of the catfish EST database further confirmed the presence of teleost granzymes with different substrate specificities. Granzyme activity measurements suggested a predominance of chymase and tryptase activities in NCC. Further proof that the granule exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts.     

Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus)

A tumor necrosis factor (TNF) -like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNF and TNFβ homologs, overall sequence identity/similarity was slightly higher vs. TNF genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNF and β genes, and supported the suggestion that TNF and β genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNF mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNF has been identified, and indicate that catfish TNF is expressed in catfish in a manner similar to that seen in mammals.     

Characterization and expression of an EB1 protein in Ictalurus punctatus neutrophils

We have identified an EB1 gene (CfEB1) and protein in channel catfish neutrophils. The complete cDNA sequence is 1725 bp and the putative protein is composed of 258 amino acids. Western blot analysis of channel catfish neutrophil cell membrane with a monoclonal antibody (14I) yielded a 28 kD protein band with the protein preparation. Aside from neutrophils only a small percentage of other cells tested expressed detectable amounts of EB1 as determined by flow cytometry. Furthermore, EB1 expression increased after phorbol dibutyrate stimulation of neutrophils or incubation of catfish neutrophils with Edwardsiella ictaluri. Nonpermeabilized, fixed catfish neutrophils demonstrated immunofluorescent staining with 14I indicating that EB1 is apparently externally oriented in catfish neutrophils. This is the first report of the external orientation of the EB1 molecule. Because of its increase after stimulation and detection on cell membranes, EB1 may participate in catfish neutrophil cell regulation, signal transduction, or cell membrane changes necessary for phagocytosis.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). III. Biophysical and biochemical properties affecting cytolysis

Nonspecific cytotoxic cells (NCC) from the catfish (Ictalurus punctatus) may comprise a population of cells that are responsible for cellular immunity in the fish. NCC kill a wide variety of transformed target cells, and previous studies have indicated that NCC share properties with mammalian natural killer cells. In the present study, many biophysical and biochemical properties of NCC were defined. NCC were nylon wool nonadherent and adherent. NCC activity was also enriched in plastic nonadherent cells. NCC were nonphagocytic (for carbonyl iron), and they did not bind to Sephadex G-10. Characterization of NCC by density gradient centrifugation indicated that they comprise a relatively homogenous population of cytolytic cells that band at 45.5% Percoll. Moderate to high doses (500-2500 R) of X-irradiation produced a stimulatory effect on NCC lysis of labeled target cells. Additional studies indicated that a soluble suppressor protein in catfish serum (CFS) regulated NCC activity. This S. aureus protein A binding component isolated from CFS suppressed NCC activity. Analysis by SDS-PAGE indicated that the soluble regulatory protein had properties similar to immunoglobulin. These data indicate that NCC share some biophysical properties with mammalian natural killer cells. In addition, NCC appear to be under partial cell regulation by a radiation sensitive suppressor cell and also by a soluble regulator serum immunoglobulin component.     

The two channel catfish intelectin genes exhibit highly differential patterns of tissue expression and regulation after infection with Edwardsiella ictaluri

Intelectins (IntL) are Ca(2+)-dependent secretory glycoproteins that play a role in the innate immune response. The mammalian IntL is also known as lactoferrin receptor (LfR) that is involved in iron metabolism. The objective of this study was to characterize the intelectin genes in both channel catfish and blue catfish, to determine their genomic organization and copy numbers, to determine their patterns of tissue expression, and to establish if they are involved in defense responses of catfish after bacterial infection. Two types of IntL genes have been identified from catfish, and IntL2 was completely sequenced. The genomic structure and organization of IntL2 were similar to those of the mammalian species and of zebrafish and grass carp, but orthologies cannot be established with mammalian IntL genes. The IntL genes are highly conserved through evolution. Sequence analysis also indicated the presence of the fibrinogen-related domain in the catfish IntL genes, suggesting their structural conservations. Phylogenetic analysis suggested the presence of at least two prototypes of IntL genes in teleosts, but only one in mammals. The catfish IntL genes exhibited drastically different patterns of expression as compared to those of the mammalian species, or even with the grass carp gene. The catfish IntL1 gene is widely expressed in various tissues, whereas the channel catfish IntL2 gene was mainly expressed in the liver. While the catfish IntL1 is constitutively expressed, the catfish IntL2 was drastically induced by intraperitoneal injection of Edwardsiella ictaluri and/or iron dextran. Such induction was most dramatic when the fish were treated with both the bacteria and iron dextran. While IntL1 was expressed in all leukocyte cell lines, no expression of IntL2 was detected in any of the leukocyte cell lines, suggesting that the up-regulated channel catfish IntL2 expression after bacterial infection may be a consequence of the initial immune response, and/or a downstream immune response rather than a part of the primary immune responses.     

Expression and characterization of human group C rotavirus virus-like particles in insect cells

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.     

Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of ⁵¹Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee × Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutirain to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.     

Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes

Neurocysticercosis (NCC) caused by infection with the larvae of Taenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using an Escherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.     

Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus

Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder, Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids. The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is composed of three exons and two introns. The Japanese flounder tumor necrosis factor (TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame natural embryo (HINAE) cells upon stimulation with synthesized CpG oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid differentiation factor 88 (MYD88) were increased in some organs including blood, gill, kidney and spleen after Edwardsiella tarda challenge. Immunohistochemical analysis revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9-expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but many were found in these organs after E. tarda challenge and were coincident with lesions that had been colonized by the bacteria.     

S100 protein is a useful and specific marker for hair cells of the lateral line system in postembryonic zebrafish

The neuromast of the lateral line system of zebrafish has become an ideal model for the study of both developmental genetics and the vertebrate auditory system. Interestingly, the hair cells of this system have been found to selectively display immunoreactivity for S100 protein in some teleosts. In order to provide a selective marker for the sensory cells of the lateral line system, we have analyzed immunohistochemically the expression of S100 protein in zebrafish from the larval to the adult stage. In larval and adult animals S100 protein immunoreactivity was detected restricted to the hair cells of both superficial and canal neuromasts. Apparently the expression of S100 protein by hair cells was independent of the age, but it was expressed heterogeneously in the hair cells of canal neuromasts. The results of this work provide a feasible method to easily identify sensory cells in the neuromasts, and may be of interest in studies regarding development, differentiation or turnover of hair cells.     

Identification of a cDNA encoding channel catfish interferon

Despite considerable advances in our understanding of teleost immunity, relatively few cytokine genes, including those for interferon (IFN), have been identified at the molecular level. In contrast, numerous studies have shown that following virus infection or exposure to double-stranded RNA, fish or fish cells produce a soluble factor that is functionally similar to mammalian IFN. A putative catfish (CF) IFN cDNA was identified by BLASTX screening of a catfish EST library generated from a mixed lymphocyte culture enriched for NK-like cells. Consistent with its designation as a putative cytokine cDNA, the 3' non-translated region contained multiple copies of an RNA instability motif. Analysis of the deduced amino acid sequence of CF IFN showed low levels of identity/similarity to a panel of mammalian and avian IFN proteins, and markedly higher similarity to a recently identified zebrafish IFN. To determine if the identified cDNA encoded CF IFN, expression was monitored following infection of channel catfish ovary (CCO) cells with UV-inactivated catfish reovirus or exposure to double-stranded RNA, treatments which induce IFN or IFN-like activity in catfish and other species. In both cases, upregulation of putative CF IFN mRNA was detected. Moreover, upregulation of CF IFN mRNA was accompanied by the appearance of an antiviral factor in the culture medium. To confirm these results, recombinant CF IFN was synthesized in COS-7 cells and shown to have antiviral activity in CCO cells. Collectively, these results argue strongly that the identified catfish cDNA is an IFN homolog.     

Cellular and molecular mechanisms in the development of a cleft lip and/or cleft palate; insights from zebrafish (Danio rerio)

Human cleft lip and/or palate (CLP) are immediately recognizable congenital abnormalities of the face. Lip and palate develop from facial primordia through the coordinated activities of ectodermal epithelium and neural crest cells (NCCs) derived from ectomesenchyme tissue. Subtle changes in the regulatory mechanisms of NCC or ectodermal epithelial cells can result in CLP. Genetic and environmental contributions or a combination of both play a significant role in the progression of CLP. Model organisms provide us with a wealth of information in understanding the pathophysiology and genetic etiology of this complex disease. Small teleost, zebrafish (Danio rerio) is one of the popular model in craniofacial developmental biology. The short generation time and large number of optically transparent, easily manipulated embryos increase the value of zebrafish to identify novel candidate genes and gene regulatory networks underlying craniofacial development. In addition, it is widely used to identify the mechanisms of environmental teratogens and in therapeutic drug screening. Here, we discuss the value of zebrafish as a model to understand epithelial and NCC induced ectomesenchymal cell activities during early palate morphogenesis and robustness of the zebrafish in modern research on identifying the genetic and environmental etiological factors of CLP.     

Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.     

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.     

Zebrafish acvr2a and acvr2b exhibit distinct roles in craniofacial development.

To examine the roles of activin type II receptor signaling in craniofacial development, full-length zebrafish acvr2a and acvr2b clones were isolated. Although ubiquitously expressed as maternal mRNAs and in early embryogenesis, by 24 hr postfertilization (hpf), acvr2a and acvr2b exhibit restricted expression in neural, hindbrain, and neural crest cells (NCCs). A morpholino-based targeted protein depletion approach was used to reveal discrete functions for each acvr2 gene product. The acvr2a morphants exhibited defects in the development of most cranial NCC-derived cartilage, bone, and pharyngeal tooth structures, whereas acvr2b morphant defects were largely restricted to posterior arch structures and included the absence and/or aberrant migration of posterior NCC streams, defects in NCC-derived posterior arch cartilages, and dysmorphic pharyngeal tooth development. These studies revealed previously uncharacterized roles for acvr2a and acvr2b in hindbrain and NCC patterning, in NCC derived pharyngeal arch cartilage and joint formation, and in tooth development.