Biomedical subjects

L Jaso-Friedmann

Publications and source records attributed to L Jaso-Friedmann.

At least 55 records · Page 3Linked to original sources

PubMed1990

A monoclonal antibody-purified soluble target cell antigen inhibits nonspecific cytotoxic cell activity.

We have previously reported the characterization of mAb derived against NC-37 target cells. mAb 18C2 and 1E7 inhibit fish cytotoxicity by binding to target cells and thus preventing the formation of conjugates with fish nonspecific cytotoxic cells (NCC). It was therefore presumed that these inhibitory mAb were specific for the target cell Ag necessary for effector cell recognition. mAb 1D4 and 7C6 bind to NC-37 cells but do not inhibit fish cytotoxic activity. We now report the isolation and purification of the Ag recognized by mAb 18C2 (inhibitor) and 1D4 (noninhibitor) by affinity chromatography of solubilized NC-37 target cell extracts. The 18C2-purified soluble target Ag (STAg) caused inhibition of cytotoxicity when preincubated with fish NCC. This inhibitory activity was reversible and dose-dependent ranging from 20 to 70% inhibition with 25 to 100 micrograms 18C2 purified STAg/10(6) NCC. STAg purified by 1D4 affinity chromatography had no effect on fish cytotoxicity. mAb 18C2 and 1E7 preabsorbed with 18C2 STAg lost their inhibitory activity when tested in the fish NCC cytotoxicity assay. Preabsorption of the same mAb with 1D4 STAg had no effect on their activity.

Acetylglucosamine↗

PubMed1990

Pathways of signal transduction in teleost nonspecific cytotoxic cells.

In the present study evidence is presented that both a putative "receptor" binding monoclonal antibody (mAb) and the calcium ionophore A23187, either singly or together, increase receptor expression and lysis of IM-9 target cells by catfish NCC. NCC activity against IM-9 target cells was increased 55% after 1 h mAb treatment. Receptor expression determined by flow cytometry increased 95% following 18-h treatment. A23187 treatment of NCC produced greater than 200% increases in receptor expression. Combined treatments of NCC with 10(-4) M A23187 and 10(-7) M PMA however augmented receptor expression only 22% above that produced by A23187 alone. MAb and A23187 comodulated cytotoxicity by a 65% increase over ionophore treatment alone. MAb and 10(-10) M PMA comodulation produced only 10.6% increases in cytotoxicity compared to mAb. These data demonstrate that the moiety on NCC recognized by 5C6 may provide an activation signal for increased cytotoxicity and receptor expression. Calcium ionophore, either singly or together with mAb, provided an even stronger activation signal for increased lysis and receptor expression.

Animals↗

PubMed1990

Effects of phorbol esters and calcium ionophore on nonspecific cytotoxic cells.

In the present study mechanisms of signal transduction were investigated by treatment of NCC with phorbol esters and calcium ionophore. NCC were stimulated to produce 3-fold increased cytotoxicity by 10(-4) to 10(-5) M A23187. PMA, PDD, and 4-beta phorbol either suppressed NCC lysis (PMA) or had no enhancing effects (PDD and 4-beta). At a concentration of 10(-4) M increased cytotoxicity occurred after 0.5 to 3 hours treatment. Four-hour preincubation enhanced cytotoxicity at suboptimal A23187 concentrations. A23187 produced increased NCC activity at each E:T ratio tested, however, greatest differences in A23187 concentrations were observed at 20:1 and 40:1. The effects of simultaneous treatment of NCC and/or sequential with PMA and A23187 indicated that different transduction pathways might operate to augment cytotoxicity. PMA, 10(-8) M, combined with 10(-4) M concentrations of A23187 produced a 164.7% increase in cytotoxicity compared to media controls, and a 37.7% increase compared to A23187 controls. These data indicate that teleost cytotoxic cells, unlike mammalian NK, are augmentable by increased levels of extracellular calcium without the presence of any other comodulating signals.

Animals↗

PubMed1990

Detection of function-associated molecules on rat leukemic NK cells: activation by monoclonal antibody or phorbol ester.

In the present study a rat leukemia NK cell line designated CRC- (derived from RNK-16 cells) was shown to spontaneously transform into a noncytolytic (NL) line referred to as CRC-/NL cells. CRC- and CRC-/NL cells were utilized to study pathways of NK activation by phorbol esters, calcium ionophore (A23187), and monoclonal antibody (mAb). 10(-6)-10(-7) M phorbol myristate acetate (PMA) but not phorbol didecanoate or 4-beta-phorbol activated CRC-/NL to lyse YAC-1 targets. Activated CRC-/NL cells produced 20-90% specific cytotoxicity compared to 0-5% for nonactivated cells. 10(-7) M PMA inhibited normal CRC- cytotoxicity. The optimum concentration of PMA for activation was 10(-6)-10(-7) M and 3-6 h treatment time. Augmentation of cytotoxicity by PMA occurred at different E:T ratios. The time required to reverse the PMA activation of CRC-/NL cells was approximately 9-10 h posttreatment. In an effort to attempt to differentiate pathways which initiated activation, CRC-/NL cells were treated with FAM binding mAb, or with combinations of mAb and ionophore, mAb and PMA, or PMA and A23187. mAb singly or in combination with 10(-7) M PMA increased cytotoxicity. However, A23187 either singly or when combined with PMA or mAb did not produce an augmented lysis of YAC-1 target cells. Additional experiments were conducted to determine if PMA activation was associated with FAM binding. This was accomplished by analyzing redirected killing of various FAM mAb-producing myeloma cells in the presence of 10(-7) M PMA. PMA treatment of the CRC-/NL cells caused a significant increase in the lysis of myeloma/mAb-producing cells compared to control cells. Further evidence that FAM binding was associated with cytotoxicity was presented by demonstrating specific inhibition of redirected lysis by homologous mAb. Phenotype analysis of CRC- and CRC-/NL cells demonstrated that OX-7 and OX-1 expression on CRC-/NL cells was increased by 71.8 and 86.8% respectively compared to CRC-. FAM expression (78-83% positives) by CRC- and CRC-/NL cells was not different. These experiments indicated at the functional level that rat NK cells can be activated for increased cytotoxicity by FAM-specific mAb binding and/or by treatment with the diacylglycerol analogue PMA. This implies that protein kinase C mobilization either singly or in concert with inositol-1,4,5-trisphosphate activation following FAM mAb binding may play important roles in NK cell cytotoxicity.

Animals↗

PubMed1989

Identification of a human natural killer cell target cell antigen with monoclonal antibodies.

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.

Adolescent↗

PubMed1988

Characterization by monoclonal antibodies of a target cell antigen complex recognized by nonspecific cytotoxic cells.

Fish nonspecific cytotoxic cells (NCC)3 recognize and lyse a large variety of human and mouse transformed cells. In an effort to determine the Ag recognized by NCC on these targets, mAb were raised against NC-37 target cells. Four anti-NC-37 mAb were chosen for further characterization based on their effects on NCC lysis of target cells. Purified mAb 18C2 and 1E7 (IgM isotype) inhibited NCC killing of the following targets: U937, MOLT-4, K562, HL-60, DAUDI, NC-37, P815, and YAC-1. The dose-dependent inhibitory activity occurred at the target cell level and ranged from 50 to 70% at a concentration of 50 micrograms/well when compared to noninhibitory mAb 7C6 and 1D4 (IgG isotype). Similarly, mAb 18C2 protected the fish parasite Tetrahymena pyriformis from lysis by NCC when compared to mAb 7C6. Adsorption experiments demonstrated that the inhibitory effect on NC-37 lysis by NCC could be removed in a titratable fashion by incubation of mAb 1E7 with any one of the other target cell lines, but it could not be removed by incubation with effector cells. The inhibitory activity of mAb 1E7 and 18C2 was shown to be caused by the inhibition of conjugate formation between effector and NC-37 target cells. The relative membrane concentration of the antigenic determinants recognized by these mAb on the target cells was studied by flow cytometry using FITC-labeled mAb. These experiments showed that all four mAb bound to the surface of the cells tested. Biochemical analysis with Western blots and immunoprecipitation showed that mAb 18C2 and 1E7 recognize two Ag in NC-37 lysates: a larger protein of around 80 kDa and a smaller one of 42 kDa.

Animals↗

PubMed1988

Identification of a putative antigen receptor on fish nonspecific cytotoxic cells with monoclonal antibodies.

In the present study mAb were derived against flow cytometry (FCM) purified fish (Ictalurus punctatus) nonspecific cytotoxic cells (NCC). mAb 5C6.10.4 and 6D3.2.10 produced 60 to 65% inhibition of lysis of NC-37 target cells (a human B-lymphoblastoid cell line) by unfractionated NCC. mAb 2B2.4.9 and 6D3.4.4 were noninhibitors of cytotoxicity. All mAb were the same isotype (IgM) and were cloned by limiting dilution (2x). Inhibitory activity was specific for the effector cells because the mAb had no effect on NCC cytotoxicity when only the target cells were treated. Inhibition could be produced by preincubation of the mAb with NCC or by no preincubation, and inhibition was not reversible. Killing by FCM-sorted NCC of NC-37 target cells was inhibited almost 100% by mAb 5C6.10.4. Inhibitor mAb also significantly reduced NCC killing of MOLT-4, K562, P815, U937, Daudi, YAC-1, and HL-60 cells. Experiments also were conducted to determine at which stage of the lytic cycle the mAb acted. Both inhibitor mAb significantly inhibited conjugate formation between effector and NC-37 target cells. The technique of FCM was combined with competitive binding experiments to determine that the Ag recognized by both inhibitor and noninhibitor mAb was found on the membranes of the same cells. These results were confirmed by demonstrating (by using FCM) that FITC-labeled inhibitor and biotinylated noninhibitor mAb bound to the same cells. FCM also was next used to determine mAb binding to various effector cell populations. Inhibitor and noninhibitor mAb bound to approximately 25% (5C6.10.4) and 39% (6D3.4.4) of fish anterior kidney cells; to 42% (5C6.10.4) and 54% (6D3.4.4) of fish spleen cells; and to 2.5% (5C6.10.4 and 6D3.4.4) of fish peripheral blood. mAb were used to purify the target cell binding structure found on NCC. Con A-Sepharose purified mAb were used as the fixed ligand for Affi-Gel-10 affinity chromatography experiments. FCM-purified NCC were solubilized and the receptor was purified by using this technique. Analysis of the NCC-purified receptor by 12% SDS-PAGE indicated that the mAb purified structure may be composed of a dimeric molecule consisting of 41 kDa and 38 kDa proteins. The same dimer was purified by using either inhibitory (6D3.2.10) or noninhibitory (6D3.4.4) mAb. Similar results were obtained with immunoprecipitation experiments by using mAb 5C6.10.4. These studies demonstrate that the Ag-binding receptor structure on fish NCC may be comprised of a dimeric complex.

Animals↗

PubMed1984

Characterization of the mechanism of action of Escherichia coli heat-stable enterotoxin.

The mechanism of activation of intestinal guanylate cyclase by Escherichia coli heat-stable enterotoxin (STa) has been studied by using isolated rat intestinal epithelial cells and purified brush border membrane (BBM) preparations. Inhibitors of prostaglandin biosynthesis, quinacrine and 5,8,11,14-eicosatetraynoic acid (ETYA), significantly reduced intracellular levels of cyclic guanosine 3', 5'-monophosphate in isolated cells treated with STa. Although these data suggested that activation of phospholipase A2 and metabolism of arachidonic acid are involved in the mechanism of action of STa, other data ruled out such a mechanism. (i) The rate of release of [3H]arachidonic acid by prelabeled intestinal cells incubated with STa was the same as control cells not treated with STa. (ii) Thin-layer chromatography of lipid extracts of intestinal cells treated with STa and untreated cells did not reveal any quantitative or qualitative differences in free fatty acids, neutral lipids, and phospholipids. (iii) Amounts of prostaglandin PGE2, prostaglandin PGF2 alpha, and thromboxane B2 in intestinal cells and BBM incubated with STa did not increase compared with controls not incubated with STa. When purified BBM preparations were incubated with phospholipase A2 inhibitors (p-bromophenacyl bromide and quinacrine) or cyclooxygenase inhibitors (ETYA and indomethacin), basal and STa-induced guanylate cyclase activities were significantly reduced. Inhibitors of calcium-calmodulin-mediated reactions (EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid], trifluoperazine, and chlorpromazine) and calcium channel blockers (verapamil and nifedipine) also nonspecifically inhibited both basal and STa-stimulated guanylate cyclase in BBM preparations. Lanthanum, a competitive inhibitor of membrane-bound calcium, did not affect either basal or STa-stimulated guanylate cyclase of BBM preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

A species-conserved NK cell antigen receptor is a novel vimentin-like molecule.

The role of a novel, evolutionarily conserved function-associated molecule (FAM) in NK cell function has been examined in several species. This molecule has previously been shown to mediate NK and NK-like recognition functions in fish NCC and human NK cells. We now show that this molecule is distributed in those tissues that contain NK cells in mice and rats. Further, we show that this molecule functions as an antigen receptor on NK cells of these species. That is, monoclonal antibodies directed against this FAM inhibit NK cell cytotoxic function and trigger signal transduction pathways in each of the species. Finally, we present evidence that this putative antigen receptor is a vimentin-like molecule which functions to mediate all NK or NK-like recognition functions in a variety of species.

Animals↗

PubMedDate not supplied

Expression of a novel function-associated molecule on cells mediating spontaneous cytolysis in swine.

In the present study, we have analyzed the effects of the anti-FAM mAb 5C6, on endogenous NK cell and lymphokine-activated killer (LAK) cell activity of swine. We report that the anti-FAM mAb 5C6 binds to fresh and cultured lymphocytes with spontaneous cytotoxic activity from swine peripheral blood. In conjunction, the anti-FAM mAb 5C6 inhibits swine cytolysis against K562 target cells. Inhibition was found to be mediated by blocking of conjugate formation. The level of spontaneous killing and mAb inhibition was correlated with the number of swine cells staining positive with the mAb 5C6 as assessed by flow cytometry.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Role of protein phosphatases in the regulation of nonspecific cytotoxic cell activity.

In vitro and in vivo experiments were conducted to determine the effects of the protein phosphatase (PPase) inhibitors sodium fluoride (fluoride), sodium orthovanadate (vanadate), and lithium chloride on nonspecific cytotoxic cell (NCC) lysis of target cells. Both vanadate and fluoride stimulated NCC activity. Lithium chloride had no effects. Optimum enhancement for "normal" NCC was at low effector:target cell ratios and at least 30 min treatment was required to achieve maximum activation effects. Fluoride, but not vanadate activation effects were largely reversible. Vanadate, 2.5-10 mM, produced a 5-10-fold increase in cytotoxicity at 25:1 E:T, whereas less than twofold increases were produced by these concentrations at 100:1. NCC activity from "stressed" fish that had essentially no cytotoxic activity were also activated by vanadate. In vitro preincubation of NCC with 10-20 mM fluoride or 2.5-10 mM vanadate produced up to a 20-fold increase in stressed cytotoxicity. Combined treatments with 2.5 mM vanadate and 20 mM fluoride produced even greater responses. In vivo responses to vanadate were also determined. Treatment of catfish by immersion in 50 microM vanadate produced significant increases in cytotoxicity by 24-48 h posttreatment. Activation of cytotoxicity was not accompanied by increases in percentage of NCC (small lymphocyte content) or in total cell numbers in anterior kidney tissue. These studies indicated that levels of NCC activity are partially regulated by control of dephosphorylation of membrane proteins. Inability of NCC from stressed fish to lyse IM-9 target cells was reversed (probably) by disruption of an equilibrium between kinase and phosphatase activities. Normal NCC were "superactivated" only under conditions were they were in limiting numbers. These data show that phosphatases must be considered as active participants in regulation of signal transduction processes.

Animals↗

PubMedDate not supplied

Activity-wheel running blunts suppression of splenic natural killer cell cytotoxicity after sympathectomy and footshock.

We used chemical sympathectomy by 6-hydroxydopamine (6-OHDA) to examine whether adaptation by the sympathetic nervous system (SNS) is a plausible explanation for our prior finding that activity-wheel running blunts the suppression of splenic natural killer cell cytotoxicity after footshock. Male Fischer rats were assigned to treatments using a group (activity wheel vs. sedentary)x treatment (6-OHDA vs. saline)x condition (footshock vs. no shock) design. After 5-6 weeks, rats were injected i.p. with saline or with 40, 80, and 80 mg/kg 6-OHDA on pre experimental days -5, -3, and -1. Half the rats received 6 min of random footshock during a 40-min period. Cytotoxicity was determined by standard 4-h 51Cr release assay. Sympathectomy reduced splenic [NE] by 72%. After 6-OHDA injection and footshock, percent lysis was 33% lower in sedentary rats compared with activity-wheel runners and home-cage controls, p=0.048. The results suggest that activity-wheel running leads to adaptations that offset an altered SNS modulation of splenic NK cell cytotoxicity in response to footshock.

Animals↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMedDate not supplied

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.

Animals↗