Monocyte-mediated Cytolysis Research Articles

Human monocyte cytotoxic factor mediates cytolysis of WEHI 164 cells

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.

WEHI 164 sarcoma cells rendered resistant to monocyte-released cytotoxin are also resistant to monocyte-induced cytolysis.

WEHI 164 sarcoma cells cultured with monocyte-released cytotoxin (CF) for 4 weeks became resistant to CF-induced cytolysis and were concomitantly rendered resistant to monocyte-induced cytolysis. The resistant cell line (R-WEHI 164) has been stable with respect to resistance to monocyte- and CF-induced lysis for more than 7 months. WEHI 164 and R-WEHI 164 cells adsorbed CF and no significant difference in CF adsorption was observed. The results indicate that CF may be an effector molecule in monocyte-mediated cytolysis.

Per- and postoperative monocyte and lymphocyte functions: effects of combined epidural and general anaesthesia.

Monocyte-mediated cytolysis and thymidine uptake in phytohaemagglutinin-stimulated lymphocytes during and after hip replacement performed under combined epidural- and general anaesthesia were studied using in vitro culture techniques. Monocyte-mediated cytolysis was unchanged during operation but was significantly reduced on the first postoperative day, coinciding with an increasing serum cortisol which indicates an incomplete epidural analgesia. However, there was no significant correlation between the increased cortisol concentration and the reduction of cytolysis. Lymphocyte proliferation was unchanged both during and after operation. These findings indicate that the presence of anaesthetic agents during surgery does not influence the studied functions of monocytes and lymphocytes. This supports the hypothesis that monocyte and lymphocyte depression during surgery under general anaesthesia alone is due to the trauma-induced stress reaction. The difference between postoperative suppression of monocytes and lymphocytes in this study indicates that monocytes are more vulnerable to surgical stress than lymphocytes.

Depression of monocytes and lymphocytes by stress-related humoral factors and anaesthetic-related drugs.

The in vitro effects of six anaesthesia-related drugs and five stress-related serum factors on monocyte-mediated cytolysis and thymidine uptake in mitogen-(PHA)-stimulated lymphocytes have been studied. Thiopentone depressed both the monocyte and lymphocyte function in a dose-dependent way. However, at thiopentone concentrations which may be present in the serum after a single intravenous anaesthesia induction dose, the monocyte depression was moderate and depression of the lymphocytes was not observed. The other drugs tested, fentanyl, morphine, pancuronium, diazepam and bupivacaine, did not alter the cellular functions significantly. Prostaglandin-E2 in concentrations of 10(-6) and 10(-7) M markedly depressed monocyte-mediated cytolysis. Cortisol, catecholamines and serotonin did not alter this function. However, a synergistic depressive effect of the combination of prostaglandin-E2 and cortisol was observed. The proliferative response of PHA-stimulated lymphocytes was depressed by cortisol in concentrations of 2200 nmol/l and 1100 nmol/1 Again, there was a marked synergistic effect of the combination of cortisol and prostaglandin-E2, while prostaglandin-E2 alone, catecholamines and serotonin did not influence the PHA-response. A possible explanation for the depression of monocyte-mediated cytolysis and lymphocyte-thymidine uptake during and after surgery under general anaesthesia may be the combined effect of endocrine and local stress factors.

Human monocyte-mediated tumor cytotoxicity. I. Demonstration of an oxygen-dependent myeloperoxidase-independent mechanism.

The cytolytic capacity of monocytes per se and stimulated monocytes has been documented to only a limited extent, and when observed has been ascribed to the generation of a variety of cytolytic molecular entities. In the present study we have examined de novo human monocyte-mediated tumor cytotoxicity and that induced by the agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Cytolytic function was analyzed by reference to the release of [111In] oxine from two prelabeled tumor cell lines, K562 and U937, in a 16-hr assay in the presence of serum to more closely mimic in vivo circumstances. Observed cytolysis was clearly related to TPA concentration and effector cell number. Maximal cytolysis was obtained with TPA at 5 ng/ml, at which specific releases were 43% +/- 6 and 18% +/- 5 (mean +/- 1 SEM) at an effector cell to target cell (E:T) ratio of 2.5:1 and 65% +/- 6, and 41% +/- 12 at an E:T ratio of 20:1, for K562 and U937, respectively. In contrast, unstimulated monocytes expressed minimal cytolytic activity, or at best a low cytotoxic effect at high cellular ratios. When TPA-stimulated monocyte-mediated cytolysis was examined, catalase (2750 U/ml) inhibited K562 and U937 cytolysis by 92% and 84%, respectively; superoxide dismutase (300 U/ml) only inhibited cytotoxicity by 17% and 24%, respectively, implicating a central role of H2O2 rather than superoxide ions. Sodium azide (1 mM), an inhibitor of myeloperoxidase, did not diminish cytolysis; in contrast, it increased K562 and U937 cytolysis by 34% and 57%. This increased cytotoxicity was observed for K562 at low levels of cytotoxicity. These data tend to dismiss an essential role of the H2O2-halide-myeloperoxidase pathway of cytolysis. The OH scavengers, histidine (20 mM) and ethanol (40 mM), did not affect K562 killing; mannitol (50 mM), another OH scavenger, had only a slight inhibitory effect (23%). Finally, H2O2 generated by a glucose-glucose oxidase system directly mediated K562 killing and, to a lesser extent, U937 lysis. These results point strongly towards the role of: 1) a myeloperoxidase-independent mechanism of cytotoxicity, with 2) H2O2 as a key mediator of the cytolytic mechanism, and 3) a limited role of O2.- in synergy with H2O2 in the cytolytic activity of monocytes, and suggest that significant cytolytic function requires an inductive event.

The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: Is there selective inhibition at the level of target recognition and lytic acceptor site?

A number of different sugars were investigated for their effect on human and mouse natural killer cell (NK)-mediated cytolysis. From the pool of nonphosphorylated sugars, d-mannose, N-acetyl- d-glucosamine (NAcGlc), d-glucose, and, to a lesser extent, β-gentiobiose were found to inhibit human NK cytolysis. Mouse NK activity against YAC-1 target cells was reduced consistently in the presence of d-mannose and NAcGlc only. The sugars, NAcGlc, d-glucose, and β-gentiobiose, were specifically inhibitory against NK-mediated cytolysis with no inhibitory effects being observed against ADCC, monocyte-mediated cytolysis, or CTL activity. Pretreatment and washing at either the target or effector cell level as well as direct target binding assays using Percoll-purified NK cells indicated that at least NAcGlc and β-gentiobiose function at the recognition stage of NK cytolysis. d-Mannose, which was the most effective nonphosphorylated sugar inhibitor, was capable of inhibiting all cell-mediated cytotoxic mechanisms tested (NK, ADCC, monocyte, and CTL) and its action did not appear to be solely due to an impairment in the recognition event. All the phosphorylated sugars caused significant inhibition of human and mouse NK-mediated cytolysis, although repeated analyses of sugar titration curves consistently showed mannose-6-phosphate (Man-6-P) to be the most effective inhibitor. Inhibition with the phosphorylated sugars was apparent against all cytotoxic mechanisms investigated. It is possible that these sugars may function as general metabolic inhibitors or may activate a common signal which negatively regulates cell-mediated cytotoxic mechanisms. Nevertheless, the relative degree of inhibition with the majority of these sugars (particularly Man-6-P) was greater against NK and ADCC activity than against monocyte and CTL activity. Furthermore, studies with selected well-characterized human and mouse NK-resistant target cells strongly indicated that these sugars, particularly Man-6-P, compete at an acceptor site responsible for the uptake of the NK lytic factor, which is independent of the recognition structure(s).

The chemiluminescence response of human natural killer cells. II. Association of a decreased response with low natural killer activity

Low natural killer (NK) responders selected from a panel of 600 normal, healthy volunteers exhibited 5- to 10-fold less cytotoxicity against the human erythroleukemic cell line K562, compared with high NK responders. Antibody-dependent cellular cytotoxicity against tumor cells, which is mediated by similar or identical effectors, was also depressed in low NK donors whereas lectin-dependent T cell killing and monocyte-mediated cytolysis of tumor cells was normal. Low NK donors exhibited normal frequencies of cells expressing the HNK-1 marker of human NK cells and highly enriched NK fractions were not impaired in their ability to recognize and bind to NK-sensitive target cells. Interferon partially activated low responder NK cells but did not restore the response to normal levels. The burst of chemiluminescence that is generated by NK cells within seconds of target cell contact was markedly impaired in low NK responder donors. We have previously shown that chemiluminescence detects reactive oxygen intermediates which are necessary but not sufficient for the activation of the NK cytolytic pathway.

Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

Antibody‐Dependent Monocyte‐Mediated Cytotoxicity.

Monocyte mediated lysis of human type B-erythrocytes coated with heat-inactivated anti-B serum was measured as 51Cr-release. Preincubation of monocytes with heat-inactivated normal AB serum inhibited cytolysis while a strong stimulation of cytolysis was seen with fresh AB serum. These effects of serum were reversible upon cell washing. Preincubation of monocytes with hyperimmune human serum against type B erythrocytes did not alter the cytolysis. After incubation at 37 degrees C of monocytes with immune complexes formed of human albumin and rabbit anti-human albumin a dose-related inhibition of cytolysis was observed. This inhibition was equally expressed at different albumin to anti-albumin ratios. Pretreatment with immune complexes at 4 degrees C had the same effect on cytolysis as pretreatment at 37 degrees C. The effect of immune complexes on monocyte-mediated cytolysis was resistant to repeated cell washing. After incubation of pretreated monocytes at 37 degrees C for up to 18 h some of the inhibition disappeared, but without normalization. Platelet depletion of monocyte suspensions resulted in a significant increase of cytolysis, and addition of washed platelets reinduced a decreased of cytolysis, which correlated with the number of platelets added.

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10 3–10 4 units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.