Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay.

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.

Glucocorticoid regulation of natural cytotoxicity: effects of cortisol on the phenotype and function of a cloned human natural killer cell line.

The ability of glucocorticoids to suppress cellular immune functions, including the cytotoxic activity of natural killer cells, is well known. However, the molecular mechanism(s) of glucocorticoid-mediated suppression of cellular cytotoxicity mediated by natural killer cells is not understood. We have investigated the effects of cortisol on protein expression and cytotoxic function of natural killer cells using NK3.3, a well-characterized, cloned human natural killer cell line. Cortisol, at concentrations up to 2 microM, does not significantly alter the viability or proliferative capacity of NK3.3 cells. However, micromolar concentrations of cortisol induce the expression of a small set of proteins which are not synthesized by NK3.3 cells in the absence of cortisol, and repress the synthesis of another set of proteins including several phenotypic determinants and cytokines. In the presence of added cortisol, the synthesis of perforin mRNA was partially repressed. However, the most striking effect of cortisol on this NK clone was its repression of granzyme A synthesis. In conjunction with the downregulation of adhesion proteins, NK3.3 cells cultured in the presence of cortisol exhibit a reduced capacity to form conjugates with K562 target cells. Whereas cortisol treatment of NK3.3 cells causes an approximately 50% decrease in their ability to form conjugates with K.562 target cells, the cytotoxic function of these cells is completely abolished under the same conditions. This first report of hormonal regulation of granzyme expression and the strong correlation between granzyme A repression and cytotoxic function suggests that cortisol may regulate NK function by repression of granzyme A synthesis. In addition to demonstrating the significant influence of cortisol on natural killer cell function, these studies provide a model system for elucidation of molecular mechanism(s) whereby glucocorticoids repress cellular immune function, especially with respect to natural killer cells.

Significance and immunoassay of 9- and 13-hydroxyoctadecadienoic acids.

Linoleic acid, the predominant polyunsaturated fatty acid in the diet, can be metabolized by cyclooxygenase, lipoxygenase and P450 enzymes. The monohydroxy lipoxygenation products of linoleic acid, 9- and 13-hydroxyoctadecadienoic acids (9(S)- and 13(S)-HODEs), are the most widely distributed of the known linoleic acid metabolites. These compounds exhibit interesting biological activities, including regulation of platelet function, maintenance of vascular thromboresistance and transduction of the cellular responses to certain growth factors. In view of their biological significance, we have produced polyclonal antibodies for the first time to these bioactive lipids to develop an easy, inexpensive, sensitive, specific and rapid enzyme immunoassay method for these bioactive lipids.

Neutrophil-mediated injury to gastric mucosal surface cells.

Neutrophils (PMNs) have been implicated in the pathogenesis of gastritis. This study evaluates the magnitude and mode of PMN-mediated damage to gastric mucosal surface cells (GSC) in a system independent of vascular and neural factors. Rabbit GSC were freshly isolated and preloaded with 51Cr. GSC were then incubated for 1 hr or 4 hr with freshly isolated human PMNs at varying effector-to-target cell ratios. Injury to GSC was assessed as percent specific 51Cr released and by electron microscopy. We found minimal GSC injury using nonactivated PMNs. Incubation with PMNs activated with formylmethionyl-leucyl-phenylalanine (FMLP), however, resulted in significant GSC injury at the 20:1 PMN/GSC ratio, 33.2 +/- 1.8% 51Cr release (P < 0.001 compared to nonactivated PMNs). Electron microscopy revealed well-preserved gastric surface cells after exposure to nonstimulated PMNs. GSC exposed to activated PMNs (20:1 PMN/GSC ratio) were severely injured. Proteinase inhibitors and dimethylsulfoxide failed to diminish PMN-mediated GSC injury. Conversely, superoxide dismutase (SOD) inhibited GSC injury by more than 50% (P < 0.001). In addition, glutathione peroxidase inhibited injury by 84% (P < 0.001). These data suggest that neutrophil-mediated injury to gastric surface cells in vitro involves superoxide anion and hypochlorous acid and not neutral trypsinlike proteinases or hydroxyl radicals.

Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium.

Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.

Adoptive immunotherapy of cancer: biological response modifiers and cytotoxic cell therapy.

Immunotherapy has been developed for the treatment of metastatic cancers refractory to conventional therapies. Immunotherapy utilizes immune cells and/or biological response modifiers (BRMs) to induce an anti-tumor response mediated by the patient's immune system. BRMs, including lymphokines and cytokines, are used as single agents or in combination for cancer therapy. Some BRMs, particularly interleukin 2 (IL-2), can activate and expand in vitro lymphocytes with anti-tumor reactivity which will be adoptively transferred to the patient. To enhance the therapeutic effect of immunotherapy, gene therapy is currently under investigation and involves the insertion of cytokine genes in immune cells or in tumor cells. The development and future of cancer immunotherapy will be discussed in this review.

Hormone specific regulation of natural killer cells by cortisol. Direct inactivation of the cytotoxic function of cloned human NK cells without an effect on cellular proliferation.

Corticosteroids have previously been reported to partially inhibit the natural cytotoxic activity of peripheral blood lymphocytes. However, since only a few percent of peripheral lymphocytes are natural killer (NK) cells, it has not been possible to determine whether corticosteroids directly inhibit NK cells or mediate this effect via other cell types. This report documents direct functional inactivation, but unimpeded proliferation, of cloned human NK cells by subphysiologic levels of cortisol. In contrast, high concentrations of testosterone, progesterone or estradiol had no significant effect on proliferation or cytotoxic activity of the cloned NK cells. The kinetics of inhibition of NK function by cortisol are consistent with a transcription-dependent mechanism.

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry.

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and visual estimation of the frequency of fresh or IL-2-activated human lymphocytes that form conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E:T ratio, with extrapolated maximum conjugate frequencies (alpha max) of 40-50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.

Mutagenic response of mouse lymphoma cells after activation of benzidine and 2-aminofluorene with purified prostaglandin H synthase.

The mutagenic response of L5178Y mouse lymphoma cells to the model aromatic amine carcinogens, benzidine (BNZ) and 2-aminofluorene (2-AF) in the presence of the mammalian peroxidase prostaglandin H synthase (PHS) was examined. Standard incubation conditions for mouse lymphoma cells and the PHS system were developed. The cells were exposed to BNZ and 2-AF with purified PHS in the presence or absence of a peroxide, 5-phenyl-4-pentenyl hydroperoxide (PPHP) which is required for PHS-dependent amine oxidation. Incubations were carried out in a medium consisting of Hanks' balanced salt solution with calcium and magnesium and 0.1% pluronic F-68. BNZ by itself or in the presence of PPHP induced a weak mutagenic response in mouse lymphoma cells, but the addition of PHS or PHS and its co-factor PPHP increased the mutagenic response approximately 5-fold over that observed in the absence of PHS. A maximal mutagenic response for BNZ was observed after incubation with the complete activating system, PHS and PPHP. These data are in agreement with the fact that BNZ is an excellent substrate for PHS. When 2-AF was incubated with mouse lymphoma cells, only a minimal mutagenic response was observed. Incubation of 2-AF with either PPHP or PPHP and PHS (complete peroxidase system produced a significant enhancement in mutagenic response. Thus, the mutagenic response of the mouse lymphoma cells to 2-AF was dependent on the peroxide, PPHP but not the enzyme PHS. These data suggest that 2-AF, which is a poor PHS substrate, is oxidized by a different catalyst than PHS. This work demonstrates that BNZ and 2-AF are converted by peroxide-dependent mechanisms to mutagens that can be detected in mammalian cells.

Kinetics of cellular cytotoxicity mediated by a cloned human natural killer cell line.

The kinetics of natural killer (NK) cytotoxicity mediated by the cloned interleukin 2 (IL 2)-dependent human natural killer cell line NK3.3 has been investigated. This cloned cell line exhibits strong cytotoxic activity that is restricted to NK target cells. The initial rate of lysis of K-562 target cells by these cloned NK cells was, as anticipated, substantially greater than that previously reported for human peripheral blood NK cell preparations. However, in contrast to the kinetics of NK cytolysis mediated by fresh peripheral NK cells, the rate of 51Cr-release declined substantially within 1 to 3 h after initiation of assays involving NK3.3 cells and reached a plateau value in experiments conducted for longer periods. The data obtained suggest that NK3.3 cells cannot readily recycle and kill multiple target cells. Based on a model involving one lethal hit per active NK3.3 cell, estimates for the frequency of cytolytic cells in NK3.3 cultures were computed and compared to estimates obtained by the application of a kinetic model previously described. The cytotoxic activity of the NK3.3 cells was also found to be highly dependent on the conditions used for propagation and assay of these cells and, when cultured under "standard" conditions, only a fraction of the cloned NK3.3 cells was capable of effecting lysis of K-562 target cells. However, for the most optimal conditions developed to date, each NK cell killed an average of 1 to 2 target cells before inactivation. Although no significant differences in viability or growth rate were observed for the three different conditions described, up to 300-fold differences in lytic activity were observed. The observed strong dependence of the lytic function of NK3.3 cells on culture conditions should prove valuable for further investigations of mechanisms governing the regulation and function of human NK cells.

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells.

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.

Lysis by RNK-16 cytotoxic lymphocyte granules. Rate assays and conditions to study control of cytolysis.

Dense subcellular granules of cytolytic lymphocytes can mediate rapid lysis of erythrocytes or nucleated cells. The granules contain several different proteases and proteoglycans that regulate cytolysis. We describe a rate assay that we have already used to demonstrate the requirement for serine proteases in granule-mediated lysis. In this assay, 51Cr-labeled erythrocytes are lysed by limiting concentrations of granules from RNK-16 tumor cells. Cytolysis is initiated by the addition of calcium (1 mM final concentration) and stopped at 0.5-1 min intervals by acidification to pH 6.0. The effects of the granule protein concentration, temperature, the concentration of erythrocytes, pH, and the concentration of calcium on the rate of lysis are reported. A preliminary mathematical approach is described and suggested as a means to differentiate 'lag' or activation times from the initial burst of lysis. With this rate assay, we have found four classes of protease inhibitors that block granule-mediated lysis (Hudig et al. (1987) Biochem. Biophys. Res. Commun. 149, 882). The utility of the rate assays is underscored by the observation that reversible protease inhibitors only showed rates of cytolysis whereas irreversible protease inhibitors stopped cytolysis completely. Rate assays are essential for future analyses of the complex physiological regulation of granule-mediated cytotoxicity by proteases, endogenous protease inhibitors and proteoglycans.

Kinetics of cellular cytotoxicity mediated by cloned cytotoxic T lymphocytes.

Kinetic methods can provide significant information concerning the mechanism of cellular cytotoxicity reactions. Previous kinetic studies of cytotoxic T lymphocytes (Tc) have been hampered by the heterogeneity of the effector cell population tested. We therefore examined the kinetics of lysis mediated by cloned, IL 2 and antigen-dependent murine Tc cells with strong cytotoxic activity that is restricted to distinct tumor-associated antigens on P815 mastocytoma target cells. Initial velocity measurements for cytotoxicity mediated by these clones fit a simple Michaelian kinetic model. Specific activity values obtained from these initial rate measurements are compared to those obtained for polyclonal Tc preparations, NK cells, and activated killer cells. Whereas the initial rate of lytic programming mediated by these cloned cells was very rapid, the rate of cytolysis mediated by the cloned cells decreased significantly within one hour. Since this decrease was observed over a wide range of E:T ratios, it is unlikely to result from product inhibition or a significant decrease in the concentration of unlysed target cells, but may be due to a decrease in the rate of programming and/or effector cell recycling. These results indicate that a simple Michaelian kinetic model is not adequate for tumor cell cytolysis by Tc cells in vitro.

Tetranactin, a macrotetrolide antibiotic, suppresses in vitro proliferation of human lymphocytes and generation of cytotoxicity.

Tetranactin, a hydrophobic cyclic antibiotic produced by Streptomyces aureus, has previously been shown to suppress in vitro activation of rat lymphocytes by concanavalin A as well as the onset of experimental autoimmune uveoretinitis in Lewis rats. Here we report the effects of tetranactin on human T and NK lymphocytes in vitro. Tetranactin, at concentrations up to 100 ng/ml, was not toxic to human lymphocytes but completely abrogated the proliferation of human T lymphocytes in response to allogeneic cells in mixed lymphocyte cultures. Tetranactin also blocked the initiation of proliferation in response to interleukin-2, but did not block proliferation of interleukin-2-activated cells. Tetranactin also blocked generation of cytotoxic T lymphocytes and activated killer cells in the mixed lymphocyte culture. However, up to 100 ng/ml tetranactin did not alter the lytic activity of cytotoxic T or NK lymphocytes generated in its absence. The ability of low doses of tetranactin to block the induction of lymphoproliferation is similar to the action of cyclosporin A. Since cyclosporin A is also a cyclic hydrophobic molecule, the immunosuppressive actions of these two agents may involve a similar mechanism.

Plasminogen activator-anti-human fibrinogen conjugate.

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.

Natural cytotoxicity: recent progress and continuing controversy.

These highlights of the presentations and discussions at the Second International Workshop on Natural Killer Cells demonstrate the rapid progress that is being made in our understanding of the fundamental nature and mechanism of action of natural killer cells. Although the workshop was purposefully directed to fundamental studies, it is clear that these research efforts will soon have a substantial impact at the clinical level. For example, E. Lotzová (Houston, Tex., USA) noted that several experimental measures of NK function are altered in patients suffering from myelogenous leukemia. NK cells isolated from these patients demonstrate reduced conjugate formation, a substantial reduction in the Vmax for lysis of K-562 cells, could not recycle and were deficient in production of NKCF. She also presented preliminary data that cytotoxic activity could be restored by partially purified alpha interferon and IL-2. A book based on the proceedings of this workshop will soon be published by Academic Press.

The effect of lysed and nonviable target cells on the experimentally determined kinetic parameters for natural and antibody-dependent cytotoxicity.

In previous studies, we have demonstrated that kinetic assays of natural and antibody-dependent cellular cytotoxicity reactions can be used to obtain simultaneously estimates for both the frequency and lytic activity of the NK or K cells that lyse a given target cell. In order to generate useful information from kinetic cytotoxicity assays, it is imperative that suitable experimental conditions be used and consideration be given to factors that may alter the values of experimentally determined kinetic parameters. In this paper we derive equations to predict the effects of unlabeled inhibitor cells on the values obtained for KappM and Vmax. We further demonstrate that these equations can be used to interpret data obtained in the presence of a fixed concentration of added inhibitor cells and to correct for the apparent inhibition of cytotoxicity caused by nonviable cells present in 51Cr-labeled target cell preparations. We also predict that high concentrations of lysed target cells will cause product inhibition, present experimental evidence in support of this prediction, and present a method that can be used to test and correct for inhibition by lysed target cells. These results should provide for the more accurate determination of kinetic parameters for natural and antibody-dependent cytotoxicity reactions, and thereby more accurate quantification of effector cell frequency and activity.

Kinetic analysis of the specificity of human natural cytotoxicity.

Distribution-free analysis of kinetic data for cellular cytotoxicity reactions enables the precise determination of kinetic parameters for the lysis of target cells by individual lymphocyte preparations. This report presents results obtained when this method was used to quantitate the inhibition of human natural cytotoxicity by unlabelled (cold) target cells. In agreement with previous studies, we found that the natural cytotoxicity of a given target cell can be inhibited by heterologous as well as homologous unlabelled cells; however, the strongest inhibition was usually produced when the unlabelled inhibitor cells were homologous to the labelled target cells. The general pattern of inhibition observed for the target and inhibitor cells tested in these experiments was competitive, and when inhibitor cells were homologous to the labelled cells, the observed increase in Kappm agreed with theoretical predictions based on equations derived previously. These results support earlier reports of limited but not absolute antigenic specificity by subsets of human NK cells. Moreover, while Kappm values for cytotoxicity reactions are not simply related to antigen binding, quantitative analysis of the relative inhibition of cytotoxicity by heterologous versus homologous unlabelled cells provides useful estimates of the relative affinity of the effector cell subset(s) that kill a given target cell for various NK target cells.