Dorothy Hudig

Assessment of a role for phospholipase A2 and arachidonic acid metabolism in human lymphocyte natural cytotoxicity

The reagents quinacrine, hydrocortisone, and dexamethasone have been assumed to affect phospholipase A2 (PA2) when they reduce natural killer (NK) activity. However, these reagents did not reduce lymphocyte incorporation of [14C]arachidonate, which implies that they are not acting as PA2 inhibitors in this lymphocyte system. However, p-bromophenacyl bromide (BPB), which is an active site inhibitor of PA2, irreversibly abrogated NK activity of pretreated lymphocytes, disrupted target cell binding, and reduced [14C]arachidonic acid incorporation by 70-80% as compared to controls. Other observations contrary to expectations for PA2 inhibitors were: (1) quinacrine inhibited NK lysis when lymphocytes were pretreated and (2) the glucocorticoids only inhibited NK activity when continuously present in the assay. Furthermore, NK inhibition by hydrocortisone did not require protein synthesis. The lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosotetraynoic acid (ETYA), and hydroxyphenylretinamide, and not cycloxygenase inhibitors, reduced NK activity. These data suggest that arachidonate must be metabolized through the 5-lipoxygenase pathway in order to function in NK.

The mechanism of cell-mediated cytotoxicity. III. Protease-specific inhibitors preferentially block later events in cytotoxic T lymphocyte-mediated lysis than do inhibitors of methylation or thiol-reactive agents.

Highly active mouse cytotoxic T lymphocytes (CTL) generated in secondary mixed-lymphocyte responses were used to examine the manner in which adenosine derivatives, thiol-specific reagents, or protease-specific probes affected CTL-mediated lysis (CML). The adenosine deaminase inhibitor deoxycoformycin (dCF) enhanced inhibition by adenosine (AR) or by deoxyadenosine (AdR), but not by 7-deazaadenosine (tubercidin). L-Homocysteinethiolactone (L-Hcy) acted synergistically with AR, but not with AdR or tubercidin, to block CML. Thus, AR derivatives may act both by affecting cellular methylation reactions, as demonstrated by the synergism between AR and L-Hcy, and by inhibiting other events required for CML. Conditions were then established to determine whether these reagents preferentially affected either the Ca2+-independent initial stage of cytolysis or the subsequent Ca2+-dependent events. Methylation inhibitors blocked lysis most effectively if added before effector-target binding. Similarly, the nonpenetrating thiol-specific reagent quaternary ammonium monobromobimane (qBBr) was more inhibitory when added prior to the Ca2+-dependent stage. Protease inhibitors such as alpha-1-antichymotrypsin and protease substrates such as acetyltyrosine ethyl ester (ATEE) or tyrosine ethyl ester (TEE) also inhibited CML. But, in contrast to qBBr or methylation inhibitors, neither TEE nor ATEE was more effective when added prior to the initial effector-target interaction. Furthermore, TEE did not appreciably affect CTL binding to target cells at concentrations that nearly abrogated CML. Thus, the implicated protease step is unique in that it does not appear to participate in recognition or binding.

Ethanol activation of human natural cytotoxicity

Human lymphocytes cultured with ethanol and subsequently assayed for natural killer (NK) activity to K562 cells have enhanced NK activity compared to lymphocytes cultured without exposure to ethanol. Optimal enhancement occurred at 0.64% (v/v) ethanol, and required several hours of culture. Lymphocytes retained their enhanced cytolytic ability for several hours after removal from the ethanol-containing medium. The enhancement correlated with a faster rate of cytolysis by ethanol-treated lymphocytes, rather than recruitment of an increased number of killer cells, as measured with single cell assays. Inclusion of ethanol directly in the NK assays was inhibitory. Cells that had been cultured with ethanol were less sensitive to inhibition of NK activity by the proteinase substrate acetyl tyrosine ethyl ester than were control cells cultured without ethanol. Although this observation and the increased rate of cytolysis in the single cell assays are consistent with increased production of a chymotrypsin-like proteinase involved in cell-mediated cytotoxicity, no alteration in protein synthesis was detected concomitant with ethanol treatment. This report demonstrates that even without hepatic metabolism, ethanol can produce effects on lymphocyte function which remain after exposure to the reagent is discontinued.

Effect of unsaturated fatty acids on human lymphocytes. Disparate influences of oleic and linolenic acids on natural cytotoxicity

Abstract This study examined whether unsaturated fatty acids induced enhancement of human natural cytotoxicity as has been observed to occur in mouse T-cell killing. Alterations in unsaturated-to-saturated fatty acid ratios in human lymphocyte phospholipids resulted from incubation of the cells in medium supplemented with oleic acid (18:1) or γ-linolenic acid (18:3). Major changes in unsaturated-to-saturated fatty acid ratios in phospholipids were accompanied by less than twofold enhancement or no change in natural killing (NK) after oleic acid supplement and by less than twofold depression after γ-linolenic acid supplement. Thus when enhancement of NK responses occurred, the effects were not a function of simple incorporation of unsaturated fatty acids in cellular phospholipids.

Serum concentrations of α-macrofeto-protein (acute-phase α2-macroglobulin), a proteinase inhibitor, in pregnant and neonatal rats and in rats with acute inflammation

Alpha macrofetoprotein (AMF) or acute-phase alpha2 macroglobulin serum concentrations are elevated in pregnant, fetal, and newborn rats and in rats with experimentally induced acute inflammation when quantitated using a double-antibody radioimmunoassay. Concentrations of AMF are 17±2μg/ml and 32±6μg/ml in normal adult rat sera. Both maternal (1.2 mg/ml) and neonatal (9.3 mg/ml) concentrations are maximal at term. AMF serum concentrations of rats injected with croton oil into the hind footpads are unchanged for the first 4 h after injection, then increase to a maximum of 10.5 mg/ml 36 h after injection. Serum AMF concentrations correlate directly with the dose of croton oil and the increase in size (swelling) of the injured foot, and remain elevated during the course of inflammation for at least two weeks. The possible function of AMF as an antiproteinase limiting the extent of inflammation after acute tissue injury is discussed.

The mechanism of cell-mediated cytotoxicity: IV. K-76 COONa, Which inhibits the activity of factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation☆

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.

EVIDENCE FOR PROTEASES WITH SPECIFICITY OF CLEAVAGE AT AROMATIC AMINO ACIDS IN HUMAN NATURAL CELL-MEDIATED CYTOTOXICITY

Publisher Summary This chapter examines the evidence for proteases with specificity of cleavage at aromatic amino acids in human natural cell-mediated cytotoxicity. In a study described in the chapter, lymphocyte-K562 conjugates were formed by centrifuging 2:1 mixtures of peripheral blood mononuclear cells and K562 cells for 3 minutes at 40g at room temperature. The inhibition of NK by plasma antiproteinases implies that a protease of the serine-dependent type is required for killing because these antiproteinases inactivate only this class of protease. The marked inhibitory activity of a-1-X to both slow and fast NK implies that at least one serine-dependent protease with chymotrypsin-like activity is crucial to NK. It is likely that the protease activity is either membrane-associated or released upon activation of the lytic mechanism because plasma antiproteinases are large acidically charged molecules which are unlikely to cross lymphocyte membranes. It was found that because pretreatment of lymphocytes with plasma antiproteases followed by washing has no subsequent effect, this indicates that the NK protease is either not activated and/or sequestered prior to killing. This conclusion that a protease with aromatic amino acid specificity is required for NK is further supported by the data from the experiments using ester and amide derivatives of aromatic amino acids to inhibit NK to K562 cells.

Isolation, characterization and radioimmunoassay of rat alpha-macrofetoprotein (acute phase A2 macroglobulin)

Abstract Rat alpha-macrofetoprotein (AMF)§ has been isolated and purified by antibody affinity chromatography and quantitated using a double antibody radioimmunoassay (RIA) with a sensitivity of 20 ng. Two forms of AMF, differing in reference mobilities ( R ) in 5% polyacrylamide gels and in isoelectric focusing points (pI), are present in serum. The R f 0.10 AMF has a pI of 4.35 and R f 0.25 AMF a pI of 4.65. R f 0.25 AMF competes 2.2 times better than R f 0.10 AMF in an RIA with 125 I R f 0.25 AMF as the predominant antigen. The two forms do not differ substantially in mol. wt. AMF has a mol. wt of 700,000 ± 70,000 determined by SDS polyacrylamide gel electrophoresis. Dithiothreitol reduction of AMF resulted in six different chains, the largest with mol. wt 175,000. It is suggested that the five smaller chains are the result of interaction of AMF with endoproteases. The E 1% 280 of AMF is 9.16 ± 0.3 by the biuret method using bovine serum albumin as a standard and 10.6 ± 0.3 by microKjeldahl N analysis. Amino-acid analysis of AMF is compared with the analyses recently reported by others. Normal adult rat serum AMF concentrations determined for two groups of rats are 17 ± 2 and 32 ± 6 μg/ml.

CELL SURFACE THIOLS IN HUMAN NATURAL CELL-MEDIATED CYTOTOXICITY

Publisher Summary This chapter examines the role of cell surface thiols in human natural cell-mediated cytotoxicity. The data that indicate that a lymphocyte cell surface thiol is required for target binding and killing. Several types of reagents were used as inhibitors of cellular thiols. Although chloromethyl ketone derivatives of amino acids specifically inactivate serine-dependent proteases with affinity for the amino acid component of the compound by alkylating the histidine the active site of the enzyme, these chloromethyl ketones can also nonspecifically alkylate thiol residues. Substrate specificity is expected among the chloromethyl ketone derivatives of different amino acids when a specific protease is inactivated but not when −SH groups of most proteins are alkylated. Iodoacetamide can alkylate glyceraldehyde-3-phosphate dehydrogenase, thereby blocking oxidative phosphorylation as a cellular source of energy. An additional series of experiments also suggests that these chloromethyl ketones were inhibiting killing by alkylating −SH groups as opposed to inactivating proteases. TosLysCH2Cl inhibition of isolated proteases can be prevented by the inclusion of excess substrate or inhibitor for the protease.