Liliana Jaso-Friedmann

Nonspecific cytotoxic cells as effectors of immunity in fish

Abstract Nonspecific cytotoxic cells (NCC) may be the teleost fish equivalent of mammalian natural killer (NK) cells. Although significant differences exist between species regarding many characteristics of these cells, both NCC and NK cells share similarities: in the types of target cells sensitive to lysis; in mechanisms of target cell recognition; in the requirements for a competent lytic cycle; and both types of effectors participate in mediating the lysis of infectious microorganisms. A putative antigen binding receptor obtained from catfish NCC has now been characterized using monoclonal antibodies (mabs). This receptor is a vimentin-like protein. Preliminary studies indicate that NCC recognize a 40 kD protein on the membranes of susceptible target cells. Solubilized target cell protein can specifically bind to NCC and inhibit killing. Similar to NK cells, NCC require cell contact with the target cell to deliver the lethal cytotoxic hit. NCC appear to be the more potent cytotoxic cells because fewer are required to kill an individual target cell and less time is required for this action to occur than for NK cells. Unlike NK cells, NCC do not recycle under experimental conditions. Preliminary studies were also reviewed to characterize signal transduction responses. Monoclonal antibody against the vimentin-like protein receptor activates NCC cytotoxicity, initiates the production of significant increased levels of free cytoplasmic calcium, and causes the production of inositol lipid intermediates (specifically phosphotidylinositol 1, 4–5 trisphosphate). NCC may be important effectors of anti-parasite immunity. Although these cells probably do not elicit memory responses, data suggest that they do recognize antigen and can be activated and recruited into peripheral tissue where they mediate cytolytic responses.

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.

Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells.

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with ~50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure–function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.

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.

Mechanisms of cellular cytotoxic innate resistance in tilapia (Oreochromis nilotica).

Mechanisms of innate cytotoxic immunity in tilapia (O. nilotica) were measured by characterization of the activity, distribution and functions of nonspecific cytotoxic cells (NCC). Active cytotoxic cells were obtained from anterior kidney. spleen and peripheral blood whereas nonlytic but anti-NCC monoclonal antibody 5C6 positive cells were obtained from tilapia liver. Thymocytes were not cytotoxic and were mab 5C6+. Unfractionated anterior kidney cells were 6% mab 5C6+ and had very low cytotoxicity of HL-60 target cells. Percoll (45.5%) purified NCC were 44% mab 5C6+ and had 35% HL-60 cytotoxicity (160:1 E:T ratio). Transformed mouse and human target cells were tested for sensitivity to NCC lysis. HL-60, U937, K562, IM-9 and NC-37 human targets were lysed by NCC. YAC-1 targets were insensitive to lysis. The killing of HL-60 targets by tilapia NCC was inhibited by mab 5C6. Experiments to determine optimal conditions for the cytotoxicity assay revealed that tilapia required 15-20h for optimum lysis of targets. Incubation at 37 C produced the highest cytotoxicity. The proliferative competence of Percoll purified anterior kidney cells was determined. A significant increase in in vitro uptake of tritiated thymidine by anterior kidney cells occurred following stimulation by mab 5C6, Con-A, PMA and calcium ionophore A23187. Purified spleen cells also produced significant increased uptake of tritiated thymidine following in vitro activation with PMA and mab 5C6, but not Con-A. These studies indicated that NCC may provide innate cytotoxic immunity similar to that provided by the NCC of catfish.

Function associated molecules on nonspecific cytotoxic cells: Role in calcium signaling, redirected lysis, and modulation of cytotoxicity

Monoclonal antibodies (mAbs) specific for a putative antigen receptor (function associated molecule, FAM) on fish NCC were tested for their ability to initiate signal transduction responses. Anti-FAM hybridoma target cells were significantly lysed by NCC when tested by redirected lysis (RL). Redirected lysis was enhanced by 1-h pretreatment of the NCC with anti-FAM mAb; however, 18-h pretreatment produced significant inhibition. The effects of the calcium ionophore A23187 on RL were also examined: 10(-4) M but not 10(-5) M A23187 significantly increased RL. Purified NCC were treated with mAb and cellular DNA synthesis was determined. Significant increases in NCC incorporation of tritiated thymidine were measured 72 h after treatment with mAb. Experiments were also conducted to determine levels of free cytosolic calcium in NCC following mAb binding. Anti-FAM mAb binding produced a rapid rise in cytosolic calcium (200-500 nM) determined by Fura-2/AM fluorescence. Calcium ionophore A23187, PHA, and Con-A treatment of NCC also produced significantly increased [Ca2+]i. Target cell binding by NCC caused a three-fold increase in [Ca2+]i. These data suggest that triggering of the FAM may initiate a signal(s) that activates cytotoxicity, increases cytosolic free calcium concentrations, and initiates DNA synthesis.

The antigen receptor (NCCRP-1) on catfish and zebrafish nonspecific cytotoxic cells belongs to a new gene family characterized by an F-box-associated domain.

The catfish nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. This report identifies and characterizes a zebrafish orthologue of the catfish NCCRP-1. The zebrafish NCCRP-1 cDNA contains an open reading frame that encodes a predicted protein of 237 amino acids with a MW of 27 kDa and a pI of 5.5. Sequence similarities comparisons show that the NCCRP-1 receptors from these two phylogenetically distant species share a high degree of identity. These results suggested that NCCRP-1 performs a crucial function in innate immunity in teleosts. Further, a zebrafish 17-mer peptide corresponding to the catfish NCCRP-1 antigen-binding domain inhibited (catfish) cytotoxicity toward conventional tumor target cells (HL-60). These data appeared to indicate that the zebrafish NCCRP-1 protein may function as an antigen recognition molecule and, as such, may participate in innate immunity in teleosts. A homology search of the zebrafish NCCRP-1 protein revealed that it shares a significant level of identity with another group of proteins belonging to an F-box subfamily. These proteins share an F-box domain in the N terminus (not present in NCCRP-1) and an extremely conserved C-terminal region that has been termed the F-box-associated domain (FBA). The FBA is currently of unknown function. A new gene family is proposed in this work, based on similarities in the FBA sequences with the catfish and zebrafish NCCRP-1 peptides. This new gene family includes several F-box domain-containing proteins and a predicted C. elegans protein.

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.