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)

An antigen receptor (NCCRP-1) on nonspecific cytotoxic cells is a phosphoprotein associated with the JAK-STAT activation pathway.

The antigen receptor (nonspecific cytotoxic cell receptor protein-1/NCCRP-1) on nonspecific cytotoxic cells (NCC) is a 32-kDa predicted Type III membrane protein. The N-terminal cytoplasmic portion of this receptor contains full length and truncated BOX-1 motifs. These motifs are also found on cytokine, erythropoietin and growth hormone receptors and provide docking sites for JAK kinases. In the present study, we investigated a relationship between NCCRP-1 and JAK2 kinase binding. A possible association with further downstream STAT activation was suggested. NCCRP-1 was phosphorylated on C-terminal domain serine residues. To examine the possibility that NCCRP-1 was associated with JAK kinase(s), NCC were purified and lysates were probed by Westen blotting (WB) for the presence of JAK2 kinase. Unlike their mammalian counterparts, NCC JAK2 kinase existed as a 90-95-kDa primary and a 35-40-kDa secondary breakdown product. Both mol wt. forms were significantly smaller than those reported for human JAK kinases. To determine if NCCRP-1 was physically associated with JAK2 kinase, chemical cross-linking experiments were conducted. NCC membrane preparations were treated with the chemical cross-linker DSS, solubilised and immunoprecipitated with anti-NCCRP-1 (e.g., 32 kDa) mab 5C6. WB analysis using anti-JAK2 mab and mab 5C6 demonstrated that the immunoprecipitate contained both the 32-kDa NCCRP-1 and 85-90-kDa JAK2 kinase. To examine further the possibility that STAT proteins may be associated with NCC/NCCRP-1 activation, NCC lysates were probed (WB) with various anti-STAT mabs. The strongest signal was produced by a 100-kDa STAT-6 protein. Lysates were negative for STAT-1, STAT-3 and STAT-5. These data indicate that the N-terminus of NCCRP-1 may initiate cytokine gene transcription by the JAK-STAT signalling pathway.

NCCRP-1: a novel receptor protein sequenced from teleost nonspecific cytotoxic cells.

The recognition structure responsible for binding to conventional antigen on target cells has not previously been described for nonspecific cytotoxic cells (NCC) or for mammalian natural killer (NK) cells. Although several biochemical pathways may be available for initiation of the lytic cycle in NCC, evidence presented indicates that initial contact with a tumor cell or protozoan parasite is facilitated by recognition of a target antigen by a membrane protein of Mr 34,000 on NCC (NCC receptor protein, NCCRP-1). Binding to NCCRP-1 by monoclonal antibody 5C6, by target cell antigen or by cognate synthetic peptide initiates a signalling response leading to increased cytotoxicity. In the present study, three 20-mer microsequences were obtained from tryptic digests of purified NCCRP-1. Degenerate primers were synthesized (based on each peptide sequence) and were used for RT-PCR with mRNA purified from homogeneous NCC populations. An NCCRP-1 specific cDNA sequence was used to synthesize nondegenerate primers. These primers were used in a 5'/3' RACE PCR to obtain the entire NCCRP-1 specific cDNA. A deduced aa sequence consisted of 235 aa with a derived molecular weight of 30,628 Da. NCCRP-1 is proline rich (9%), has two glycosylation sites and 18% of all amino acids are potential phosphorylation sites (serine, threonine, tyrosine). The identity of the protein was confirmed by finding the previously microsequenced peptides in the derived sequence. Homology searches revealed that NCCRP-1 is a novel protein. Northern blot analysis of mRNA content from teleost NCC, B-cells and T-cells revealed only one band in NCC preparations. Functional studies demonstrated a decrease in membrane NCCRP-1 expression and inhibition of NCC cytotoxicity following treatment with NCCRP-1 anti-sense oligonucleotides. Treatment of NCC with sense oligonucleotides had no inhibitory effects on cytotoxicity. An algorithm predicting the membrane conformation of NCCRP-1 suggests one extracellular proline-rich domain, a transmembrane portion of 15 18 aa and a cytoplasmic tail composed of a high frequency of phosphorylation sites. Current studies suggest that NCC and NCCRP-1 may participate in innate resistance functions in teleost fish.

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.

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.

Nonspecific cytotoxic cells and innate immunity: regulation by programmed cell death.

Although programmed cell death (PCD) and the cellular pathology of apoptosis have been extensively studied in mammals and invertebrates, little is known regarding these important regulatory processes in cold blooded vertebrates, especially teleost fish. In the present review, select immunoregulatory properties of PCD/apoptosis in nonspecific cytotoxic cells (NCC) from catfish and tilapia were identified. The techniques used to define the characteristics of PCD in NCC were DNA ploidy, Annexin-V binding and cellular morphology. Using these procedures, we determined that the biochemical/genetic changes that NCC undergo during PCD are similar to those described in mammalian cells. We hypothesize that one immediate response of NCC to acute stress in teleost fish is the release of apoptosis regulatory factors (ARF) or stress activated serum factors (SASF) into the peripheral blood. These cytokine-like factors activate NCC by protecting them from initiation of: "activation induced cell death" (AICD); from "receptor induced apoptosis"; and from initiation of dexamethasone induced DNA hypoploidy. We predict that the mechanism of these actions is enhanced NCC recycling capacity and initiation of migration of NCC into sites of inflammation. In this review, studies were also summarized regarding the expression and release of "death and survival proteins" by NCC. Although the survey was not exhaustive, we showed that tilapia NCC that were activated in vitro with SASF contained increased levels of two adaptor proteins (i.e. CAS, FADD) and soluble FasL. At present the relevance of expression of the adaptor proteins by NCC is not known, however, additional evidence for the role of FasL in NCC innate immune responses was presented. Interestingly, NCC contained constitutive cytosolic FasL, and activation with tumor cells caused a significant decrease in the cytoplasmic levels of this "death protein". This indicated that FasL in NCC may function as a secretory cytokine-like molecule. Unlike mammalian NK cells and T-cells, activated NCC do not express membrane FasL. A level of phosphatase regulation of NCC apoptosis was indicated by demonstrating a reduced camptothecin induce DNA hypoploidy by pretreatment of NCC with the tyrosine phosphatase inhibitor sodium orthovanadate. This review emphasized the important regulatory functions of PCD/apoptosis for NCC in innate immune responses.

Identification of a target cell antigen recognized by nonspecific cytotoxic cells using an anti-idiotype monoclonal antibody.

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian natural killer (NK) cells. In the present study an anti-idiotypic monoclonal antibody (mAb 7D12) was generated against idiotopes on an mAb (mAb 6D3.2) that recognizes a putative receptor on NCC. The idiotypic specificity of mAb 7D12 was determined in competition assays by incubating biotinylated mAb 7D12 with mAb 6D3.2 hybridoma cells following preincubation with combinations of biotinylated 7D12 with either nonbiotinylated homologous or heterologous mAb. The ligand recognized by mAb 7D12 (determined by flow cytometry) was found on cells from the anterior kidney, spleen, thymus, PBL, liver, and brain. NCC lysis of IM-9 targets was inhibited 76% following preincubation of the target cells with different concentrations of mAb 7D12. The involvement of the ligand recognized by mAb 7D12 in the NCC lytic cycle was determined by showing that this mAb produced 50% inhibition of NCC conjugate formation with NC-37 target cells. Biochemical analysis using SDS-PAGE and Western blotting revealed that mAb 7D12 recognized 54 and 65 M(r) proteins in IM-9 target cell lysates. These studies demonstrated that an idiotope on a NCC specific anti-receptor mAb was an "internal image" of a target cell ligand. The anti-id mAb generated against this image (idiotope) inhibited NCC cytotoxicity and thus was equivalent to an NCC receptor that binds to a target cell ligand involved in NCC recognition.

Prevalence of Arkansas-type infectious bronchitis virus in Delmarva peninsula chickens.

The prevalence of Arkansas (Ark)-type infectious bronchitis virus (IBV) in Delmarva peninsula broiler-type chickens was determined. The immunity of 5-to-11-week-old commercial broilers was evaluated by intraocular inoculation with Ark-type DPI strain (Ark DPI) challenge virus and collection of tracheal swabbings 5 days later. Serum Ark-type antibody titers were obtained using the virus-neutralization test. Eighty-five flocks were tested from January to August 1981. Nearly 60% of the flocks had substantial (greater than or equal to 70%) local immunity of the upper respiratory tract. Twenty-two percent had intermediate (50-69%) and 19% of the flocks had low (less than or equal to 40%) levels of local immunity. Serum antibody titers generally agreed with challenge results. In addition, high Ark-type IBV neutralizing-antibody titers were found in 16 Delmarva broiler breeder flocks. Seven current IBV field isolates were characterized for antigenic similarity to Ark DPI. Four isolates contained Ark antigen(s) based on significant neutralization in virus-neutralization tests and on substantial immunity to challenge afforded by Ark DPI virus immunization. Three isolates did not appear to contain Ark antigen(s). Immunization of chickens with Ark DPI virus afforded substantial protection against Connecticut- and homologous-type virus challenge, partial immunity (63%) against JMK, and no protection against the Massachusetts 41 strain of IBV.

Phosphorylation-induced activation of tilapia nonspecific cytotoxic cells by serum cytokines.

Cytokines as soluble mediators of immunity are important in understanding immunological mechanisms against infectious organisms and during stress conditions. In the present study, the role of protein tyrosine phosphorylation is assessed in the activation of nonspecific cytotoxic cells (NCC) from tilapia Oreochromis niloticus by cytokine-like serum factors. NCC are the teleost equivalent of mammalian natural killer (NK) cells. In teleost fish, NCC are important mediators of innate immunity against bacterial and parasite insult and tumor growth. We have previously shown that exposure of tilapia (a tropical fish) to cold water temperatures (3 to 5 min at 5 to 10 degrees C) produces physiological stress responses characterized by immediate phenotypic and immunological changes. The serum obtained from stressed tilapia contains a 'stress activating serum factor' (SASF) which passively increases in vitro naive NCC cytotoxicity 2- to 4-fold over control levels. In an effort to identify the mechanisms of activation of cytotoxicity by SASF, the phosphorylation status of tyrosine residues in proteins from treated NCC was determined. NCC were incubated with heat-inactivated or untreated stress serum and Western blots of the cell lysates were probed with anti-phosphotyrosine monoclonal antibodies (mabs). The levels of tyrosine phosphorylation in several proteins of the SASF-activated NCC were higher than in control cells. Increased tyrosine phosphorylation was also induced by incubation of NCC in the presence of the tyrosine phosphatase inhibitor Na orthovanadate (vanadate). In every case, an increase in phosphorylation status shown by Western blotting was correlated with increases in cytotoxic activity of NCC against HL-60 target cells. The enzyme inhibitor Herbimycin A (HA) has been previously used to inhibit the activity of the src-family of tyrosine kinases. In the present study, a 4 h pretreatment of NCC with HA (2 microM), followed by treatment with SASF blocked the activation of cytotoxicity produced by SASF. These results suggested that activation of NCC by cytokine-like factors is mediated through activation of the src family of protein tyrosine kinases. Activation was associated with increased phosphorylation and higher cytotoxic effector functions.

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.

Freeze-drying today and tomorrow.

The freeze-drying process and equipment have been improved over the years; the cycle times have shortened and the dried products have improved as a result. This talk will deal with these improvements and how we have progressed from the early systems to where we are today. Such areas of discussion will include: vacuum pumping systems, how they are sized and designed to meet the needs for general and special applications; heat transfer systems, and their use in maintaining the drying profile; condensing surface design, and what is best for certain types of dryers; controls and instrumentation, and how these have played a big part in the drying process and have made it possible to get repeatability; refrigeration systems, and the part they play in the performance of freeze-drying; and lastly the effect of internal stoppering, bottomless trays, and other items such as these have had on the present state of the art. It goes without saying that there have been many changes and there will continue to be changes and we shall endeavor to look into the future--as to what might well bo some of these changes. Included in the talk will be a number of slides and illustrations to point out the various items as they are discussed.

Role of nonspecific cytotoxic cells in the induction of programmed cell death of pathogenic protozoans: participation of the Fas ligand-Fas receptor system.

Numerous different species of parasites and pathogenic microorganisms produce programmed cell death (PCD) and apoptosis in eukaryotic targets. How ever, only a few studies have demonstrated that effector cells, cytokines, growth factors, or soluble apoptosis-inducing factors are capable of initiating apoptosis in protozoan parasites. Certain Tetrahymena spp. in teleosts are opportunistic pathogens. In the present study these pathogenic protozoans were developed as a model system to describe the potential role of the Fas ligand (FasL)-Fas receptor (FasR) system as a means of innate immunity in teleosts. Nonspecific cytotoxic cells (NCC) constitutively express soluble FasL (sFasL). Binding of the antigen receptor (i.e., NCCRP-1) on NCC to target cells caused the release of sFasL into the milieu. The presence of functional sFasL in these supernatants was determined by Western blot analysis and by demonstrating the lysis of FasR(+) HL-60 but not IM-9 (FasR(-)) targets. Soluble FasL containing supernatants generated by tumor cell-activated NCC also produced a reduction in 2 N DNA (i.e., DNA hypoploidy) of T. furgasoni. The induction of DNA hypoploidy by NCC supernatants could be neutralized by adsorption of the supernatants with anti-FasL antibody (but not with an isotype control). Experiments were next done to determine the expression of FasR on Tetrahymena and study the effects of anti-FasR monoclonal crosslinkage and treatment with soluble human recombinant FasL (huFasL) on initiation of PCD in Tetrahymena. Cell cycle analysis revealed that both crosslinkage and soluble huFasL binding to Tetrahymena produced DNA hypoploidy. The reduction in diploid DNA was confirmed by observing oligonucleosome fragmentation (DNA laddering) following anti-FasR treatment. Additional evidence for FasR expression on Tetrahymena was obtained using fluorescence microscopy and flow cytometry. Both methods showed that all Tetrahymena examined (three species consisting of four isolates) expressed membrane FasR. These studies demonstrated the potential of the FasL-FasR system in teleosts for initiation of antiparasite innate immunity. Effector NCC may initiate PCD of Tetrahymena that express a FasR-like protein. Induction of apoptosis may be a major mechanism of homeostatic control of protozoan parasite infestations/infections.

Nonspecific cytotoxic cells in fish: antigenic cross-reactivity of a function associated molecule with the intermediate filament vimentin.

Nonspecific cytotoxic cells (NCC) in fish may lyse tumor cells and protozoan parasites in a similar fashion as their mammalian NK counterparts. Previous studies have shown that recognition and binding by NCC may be mediated by a receptor. In the present study the putative receptor or function-associated molecule (FAM) on NCC was shown to contain antigenic cross-reactivity with the mammalian intermediate filament vimentin. This was accomplished by comparing the specificity of anti-FAM monoclonal antibody 5C6 with commercially available anti-vimentin mabs V9 and 13.2 and with a polyclonal anti-vimentin antiserum. Tissue distribution studies indicated that the anti-vimentin antibodies bound to significantly fewer (< 50%) anterior kidney, spleen, kidney, and brain cells than mab 5C6. These mabs, however, produced inhibition of NCC lysis of IM-9 target cells. Conditions of mab incubation (with NCC) which were previously shown to modulate NCC cytotoxicity by 5C6 also produced large increases in NCC lysis of IM-9 targets in the presence of mabs V9 and 13.2. The determinant(s) recognized by these antibodies is present on the cell membrane. Microscopic examination of nonpermeabilized or acetone-treated and antibody-stained cells demonstrated that acetone permeabilization produced a 28-32% increase in staining (due to intracellular binding) compared to the staining of viable cells. Biochemical analysis of the cross-reactive antigens was done using analytical 2D SDS-PAGE and Western blot experiments. Mab 5C6 detected a 32- to 35-kDa protein (pI 6.3-6.5) in NCC extracts. Anti-vimentin mab 13.2 recognized a 57-kDa vimentin protein which had a relatively broad pI range (pH 6.3-6.7). Mab 13.2 also cross-reacted (in the same Western blot) with the NCC receptor protein (pI = pH 6.3-6.5). These data indicate that the FAM on NCC/NK cells contain vimentin-like antigenic determinants which may function in target cell binding.

Role of function-associated molecules in target cell lysis: analysis of rat adherent lymphokine-activated killer cells.

Monoclonal antibodies (MAbs) against fish nonspecific cytotoxic cells (NCC) inhibit the lysis of target cells by NCC and by human and rat natural killer (NK) cells. We now show that the anti-function-associated molecule (anti-FAM) MAb 5C6 also inhibits the cytolytic activity of rat (F344) nonadherent lymphokine-activated killer (LAK) cells and adherent LAK (ALAK) cells. Rat nylon-wool-nonadherent (NWNA) cells were cultured for 48 h in recombinant human interleukin-2 (IL-2). Nonadherent cells were then removed and the plastic-adherent (ALAK) cells were recultured for 9 days. Cells were daily analyzed by cytometry (using MAb 5C6 and the rat NK cell MAb 3.2.3) and cytotoxicity. Approximately 40% of the initially cultured NWNA cells, 50-60% of the LAK cells and 87-98% of the ALAK cells were positive for MAb 5C6. Tissue culture supernatants containing MAb 5C6 caused a dose-dependent inhibition of cytotoxicity against P815 and YAC-1 targets. Approximately 50% inhibition was obtained at MAb concentrations of 1.25-2.5 micrograms/ml. Maximum inhibition of ALAK cytotoxicity was 86% on day 4 of culture. Two-dimensional SDS-PAGE and Western blot analysis of acetone-precipitated cell membrane preparations identified a 55-kD molecule (pI 6.1-6.3) on NWNA and ALAK cells and an additional slightly more acidic 45-kD protein on ALAK cells. These data indicate that rat ALAK cells may initiate lysis of target cells via receptor binding.

Pathways of signaling in nonspecific cytotoxic cells: effects of protein kinase and phosphatase inhibitors and evidence for membrane tyrosine phosphorylation.

The present study was done to examine the role of protein kinases (PK) and phosphatases in nonspecific cytotoxic cell (NCC) activation processes. Treatment of NCC with 30 or 60 microM H-7 prior to adding IM-9 target cells significantly inhibited cytotoxicity. A similar concentration and time-dependent inhibition response was observed following treatment with H-8. The calcium and cyclic nucleotide antagonist HA1004, however, produced a significant increase in cytotoxicity at 30 and 60 microM concentrations. Treatment with genistein produced almost 100% inhibition of NCC lysis of IM-9 targets at a 142 micrograms/ml concentration. Studies were also carried out to determine the effects of these cytotoxicity modulators on stimulus-induced responses. In all cases where the costimulus consisted of the calcium ionophore A23187, cytotoxicity was markedly increased. In experiments using subsaturating concentrations of mab 5C6 as the costimulus, genistein completely reversed (inhibited) the modulating effects of this mab. Additional evidence for protein phosphorylation was obtained by immunoprecipitation and Western blot analysis (using antiphosphotyrosine mab 4G10) of membranes from mab 5C6-stimulated NCC. The role of protein phosphorylation on cytotoxic activity was further analyzed by the use of phosphatase inhibitors. Preincubation of NCC with either sodium orthovanadate or sodium fluoride significantly increased cytotoxicity. Lithium chloride had little or no effect on the killing of IM-9 target cells. These data indicate that multiple second messenger pathways participate in NCC lytic responses, the most crucial of which appears to facilitate phosphorylation of tyrosine residues on membrane proteins.

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 microsequence 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.

Receptor associated phosphorylation following monoclonal antibody or synthetic peptide binding to nonspecific cytotoxic cells.

We have previously shown that crosslinkage of a receptor protein on catfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initiates signalling responses. Receptor linked signalling was associated with the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosphoserine and anti-phosphotyrosine mabs. These proteins are components of a macromolecular protein complex (>200 kDa) determined by reducing and nonreducing SDS-PAGE. The calcium ionophore A23187 treatment produced the same pattern of phosphoprotein expression as peptide or mab. Maximum phosphoserine expression occurred at 15'-30' post-mab binding. We now show that synthetic peptide or mab treatment initiated the same serine and tyrosine phosphorylation profiles. The PKC specific inhibitor MDL 29,152 produced 50% inhibition of NCC lysis of IM-9 target cells, and completely inhibited serine phosphorylation of peptide activated cells but had no effect on tyrosine phosphorylation of the phosphointermediates. Genistein pretreatment of NCC inhibited cytotoxicity and tyrosine phosphorylation. Sequential immunprecipitation of the phosphointermediate demonstrated that the phosphorylated serine and tyrosine residues were on the same 50-60 kDa protein. These data indicate that both proximal and distal signalling events required for NCC activation may be associated with ATPase phosphorylation.

Nonspecific cytotoxic cell receptor protein-1: a novel (predicted) type III membrane receptor on the teleost equivalent of natural killer cells recognizes conventional antigen.

The phylogenetic model for "conventional" antigen recognition by NK cells may be a protein (NCCRP-1) recently identified from catfish nonspecific cytotoxic cells (NCC). NCCRP-1 may be a Type III membrane protein. The antigen binding domain was identified by competition experiments using synthetic peptides. Within this domain, a 38-mer peptide (aa 104-140) inhibited NCC killing of IM-9, HL-60, NC-37, U937, and MOLT-4 target cells. Biotinylated 38-mer also bound to IM-9 target cells. A mab which inhibited conjugate formation between NCC and target cells also bound to the 38-mer. Nonbiotinylated 38-mer inhibited mab 5C6 binding to immobilized homologous biotinylated peptide in cold competition ELISA experiments. Peptide 104-140 was truncated into two peptides. Amino acid 104-119 bound to (68%) and inhibited lyis of IM-9 target cells, whereas aa 120-140 had no activity. A predicted structure-function algorithm suggested an N-terminal domain containing BOX-1 motifs for cytokine activation; a C-terminal domain containing abundant phosphorylation sites (i.e., Y, S, and T amino acids); and an extracellular antigen binding domain.