John H. Leary

The non-specific cytotoxic cell receptor (NCCRP-1): molecular organization and signaling properties

The evolutionary precursor to mammalian natural killer cells in teleost fish is called non-specific cytotoxic cells (NCC). NCC collaborate with other non-specific effector mechanisms to provide innate resistance during acute stress responses. The NCC receptor protein (NCCRP-1) contains 238 amino acid residues and is believed to be a type III membrane protein with three distinct functional domains. The antigen-binding domain has been mapped to amino acids nos. 104-119. The intracellular C-terminus contains a high concentration of potential phosphorylation sites (Y, S, T). Indeed, we have shown that activation of NCC by crosslinking of NCCRP-1 leads to receptor tyrosine and serine phosphorylation. The N-terminus of the molecule is also inside the cells and has as well signature amino acids, proline-rich motifs (PRM), that are indicative of functional relevance. The cytokine/hormone receptor-like PRMs are known docking sites for JAK kinases. We have evidence that following activation, NCCRP-1 comes in contact with JAK kinase and as a result of this interaction, STAT 6 is translocated into the nucleus. These results suggest that NCCRP-1 may play a dual role in the activation of NCC: first, as an antigen recognition molecule necessary for target cell lysis, and second, as an initiator of cytokine release from NCC. Both of these processes are required for a competent innate immune response.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish.

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

Mapping of the Epitope Recognized by Non-Specific Cytotoxic Cells: Determination of the Fine Specificity Using Synthetic Peptides

NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N‐terminal, C‐terminal and internal NKTag (deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20‐mer peptide equivalent to amino acids (aa) nos. 55–74 specifically inhibited NCC lysis of human EBV transformed target cells (IM‐9). Inhibitory effects were nonreversible and concentration dependent; and 30 min pre‐incubation produced optimum inhibition. The inhibitory 20‐mer peptide was truncated into 17, 14, 10, 9 and 6‐mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6‐mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68–74 (ARG‐ASN‐LEU‐THR‐PHE‐ILE‐LEU‐)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14‐mer peptide composed of aa nos 61–74 inhibited lysis of HL‐60, IM‐9, DAUDI, YAC‐1, U937 and NC‐37 target cells. Flanking peptides (aa nos 35–54 and 75–94) were negative. Biotinylated aa nos 61–74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9‐mer obliterated the inhibitory effects. The 17‐mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM‐9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7–9 amino acids in the N‐terminal region of NKTag.

Monoclonal antibody binding to a receptor on nonspecific cytotoxic cells (NCC) increases the expression of proto-oncogene kinases and protein kinase C

Teleost nonspecific cytotoxic cells (NCC) initiate various cell triggering responses following receptor-target cell interactions. A putative receptor protein on NCC may alternatively initiate signalling processes following crosslinkage by homologous anti-receptor mab 5C6. In the present study, we demonstrated that binding to this receptor by mab 5C6 produced increased levels of expression of cytoplasmic src family proto-oncogene kinases lck, fyn and src. The phosphorylated isoforms of each kinase were approximately the same molecular weight (p60). Unlike their mammalian T-cell and natural killer (NK) cell counterparts, NCC p56lck did not autophosphorylate on tyrosine residues. This was determined by a lack of Western blot reactivity of teleost p56lck with anti-phosphotyrosine specific antibodies PT-66 or 4G10. Additional evidence for this lack of tyrosine phosphorylation was shown by experiments treating mab 5C6 activated NCC with sodium orthovanadate. This protein tyrosine phosphatase inhibitor did not affect levels of p56lck autophosphorylation. Mab 5C6 activated NCC were also examined to determine if levels of protein kinase C (PKC) expression were affected during triggering responses. Maximum increased PKC levels occurred 5-10 min following binding. The NCC receptor-activated PKC consisted of a 60,000 M(r) isoform and a 30,000 M(r) homologue equivalent to the mammalian PKC catalytic subunit. Not all kinases examined, however, were affected by mab 5C6 binding. Levels of expression of c-myc and cdc2p34 did not change following NCC activation. This is the first study of NK-like cells in cold-blooded vertebrates regarding the expression of these vital intermediary transducing kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial Amino Acid Sequence of a Novel Protozoan Parasite Antigen that Inhibits Non-Specific Cytotoxic Cell Activity

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC‐37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e. g. NC‐37, YAC‐1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46–50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25–40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM‐9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited NCC lysis of a variety of human and murine transformed targets (e. g. HL‐60, MOLT‐4, DAUDI, NC‐37, U‐937, YAC‐1, EL‐4). Inhibition was specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequencc analysis. Direct N‐terminal analysis yielded a 12 residue sequence. Additional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology to known sequences (Swiss‐Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

ABSTRACT Hepadnaviruses (hepatitis B viruses [HBVs]) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the known host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs, which allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motifs I, II, and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topologies, characterized by three transmembrane domains. Most striking was the fact that BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host codivergence over millions of years and host species jumping. IMPORTANCE Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full-length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegills, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that, although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

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.

Evidence for antigen recognition by nonspecific cytotoxic cells: initiation of 3H-thymidine uptake following stimulation by a protozoan parasite and homologous cognate synthetic peptide.

Catfish nonspecific cytotoxic cells bind to and lyse certain protozoan parasites and tumor cells. Target cell binding is facilitated by recognition of (minimally) one antigenic determinant. Binding to this determinant initiates multiple signalling pathways in NCC including protooncogene kinase phosphorylation, regulation of phosphatase activity and increased membrane receptor expression. In the present study, highly purified NCC were activated in vitro with the protozoan parasite Tetrahymena pyriformis, with a multiple antigenic peptide (MAP) composed of the cognate antigenic determinant of this parasite (i.e. natural killer target antigen/NKTag) and NCC were activated with a monoclonal antibody specific for the NCC receptor which binds NKTag. NCC were purified by Percoll density gradients and negative selection by panning (2x) over anti-sIg specific mab 9E1. In 5 day proliferation experiments, treatment of NCC with immobilized Tetrahymena initiated a significant increase in uptake of tritiated thymidine. This appeared to be a primary response in that NCC from in vivo parasite primed catfish did not have secondary-like proliferation responses. Stimulation of NCC with immobilized synthetic peptides composed of the cognate antigenic determinant of this parasite (i.e. MAP) also caused significant increased uptake of tritiated thymidine. An indication that NCC recognize a specific antigenic determinant was that sMAP (i.e. peptides composed of the same amino acids as MAP but in a scrambled sequence) failed to increase incorporation. Similar to the MAP results, mab 5C6 binding to NCC also caused increased thymidine uptake. To determine if an IL-2 cosignal was required to achieve optimum activation responses by NCC, different concentrations of human recombinant IL-2 (rHuIL-2) were tested individually or as costimulants. Co-treatment of NCC with rHuIL-2 and any of the three stimuli (parasite, MAP, mab 5C6) did not produce increased proliferation of NCC. These studies demonstrated that NCC specifically recognize an antigenic determinant on protozoan parasites and binding to this antigen produces an activation signal that may have important consequences for elicitation of innate immunity.

Molecular characterization and expression of a granzyme of an ectothermic vertebrate with chymase-like activity expressed in the cytotoxic cells of Nile tilapia (Oreochromis niloticus)

We have identified the gene coding for a novel serine protease with close similarities to mammalian granzymes from nonspecific cytotoxic cells of a teleost fish Oreochromis niloticus. The genomic organization of tilapia granzyme-1 (TLGR-1) has the signature five-exon–four-intron structure shared by all granzymes and similar hematopoietic Ser proteases. Molecular modeling studies suggested a granzyme-like structure for this protein with four disulfide linkages and two additional Cys residues. The expression of this gene is found to be restricted to cytotoxic cell populations with a low level of constitutive expression when compared to similar granzymes in other teleost species. High levels of transcriptional activation of TLGR-1 with different stimuli suggested that this gene is highly induced during immune reactions. Triplet residues around the active site Ser of TLGR, which determines the primary substrate specificity of granzymes, differ significantly from that of other granzymes. Recombinant TLGR-1 was expressed in the mature and proenzyme forms using pPICZ-alpha vector in the Pichia pastoris expression system. Recombinant TLGR-1 was used to determine the primary substrate specificity of this protease using various synthetic thiobenzyl ester substrates. In vitro enzyme kinetics assays suggested a preference for residues with bulky side chains at the P1 site, indicating a chymase-like activity for this protease. These results indicate the presence of novel granzymes in cytotoxic cells from ectothermic vertebrates.

Detection of function-associated molecules on rat NK cells and their role in target cell lysis

Anti-effector cell mAb 5C6.10.4 (5C6) inhibits cytotoxic activity of fish nonspecific cytotoxic cells (NCC). We now show that 5C6 also inhibits mammalian NK cell activity using fresh and cultured (CRC) leukemic rat NK cells. The inhibitory activity of 5C6 was caused by blocking of conjugate formation between NK cells and YAC-1 targets. Binding studies done by flow cytometry (FCM) showed that mAb 5C6 specifically bound to 8% of unfractionated rat spleen cells. Enrichment by nylon-wool fractionation produced 27.2% specific binding, along with a 3.4-fold enrichment in cytotoxic activity. Tissue distribution studies revealed that the highest number of cells recognized by mAb 5C6 were found in NWNA spleen cells (28.7%), followed by liver (18.9%) and peripheral blood (13.9). Two-color FCM showed that although all 3.2.3 mAb-positive cells were also stained with mAb 5C6, a small percentage of 3.2.3. negative noncytotoxic NWNA spleen T cells were 5C6 positive. Redirected lysis experiments demonstrated that anti-effector mAb-producing myeloma cells could be killed by CRC and NWNA spleen cells. In addition, mAb 5C6 produced specific inhibition of redirected lysis of each myeloma target. Experiments were also conducted to determine the signaling capability of the FAM complex. Binding of the anti-FAM mAbs to NWNA rat spleen cells caused a rapid increase in cytosolic free calcium of approximately 472 nM. Western blot analysis of CRC cell lysates showed that the molecules recognized by anti-FAM mAbs have molecular weights of 38 and 42 kDa. These studies indicate that the anti-effector mAbs recognize a functionally relevant molecule on rat NK cells that is involved in the first steps of cytolysis, i.e., antigen recognition, and which also triggers the activation of signal-transducing events in these cells.