Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity.

Human polymorphonuclear leukocytes (PMN) lyse antibody-coated target cells in an immunologically specific fashion--antibody-dependent cellular cytotoxicity (ADCC). PMN-mediated cytolysis is independent of complement, de novo protein synthesis, and DNA replication. Cytolysis is rapid, detectable at low PMN:target cell ratios, and exceeds lymphocyte-mediated ADCC. The interaction appears to be mediated via an Fc receptor and is inhibited by aggregated gamma-globulin. A role for PMN in host tumor cell immunity is discussed.     

Spontaneous lymphocyte-mediated cytotoxicity and antibody-dependent cell cytotoxicity of human effector cells

Antigen dependent cellular cytotoxity (ADCC) and non-killer cell activities of haematological healthy donors were investigated in the 51Cr release test. Attempts of cell fraction reveal that lymphocytes are active as killer and non-killer cells. Granulocytes were efficient effector cells of antigen dependent cellular cytotoxity (ADCC), however, they had no natural-killer activity. In testing leukocyte fractions of 11 donors, killer cell would only be found in the non-T-fraction. In contrast to that, three types could be observed in the distribution on non-killer cells: Distribution on T-lymphocyte fraction (27.3%), distribution on non-T-lymphocyte fraction (9.1%) and approximately equal distribution on T- and non-T-lymphocyte fraction 63.7%). Without any treatment patients with acute lymphocytic leukemia showed an antigen dependent cellular cytotoxity and non-killer activity only in exceptional cases. Normal activities were reached in remission, with chemotherapy having a depressive effect on non-killer activity.     

Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil-mediated antibody-dependent cellular cytotoxicity

In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5′ (or 6′) carboxyl-2′,7′-dichlorodihy-drofluorescein (H₂-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H₂-CDCF-conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H₂-CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H₂-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethyl-rosamine within tumor cells. Using two-color automated epifluorescence micros-copy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (<12) sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant-mediated damage. © 1993 Wiley-Liss, Inc.     

Early membrane rupture events during neutrophil-mediated antibody-dependent tumor cell cytolysis

Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetra-methylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.     

Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil-mediated antibody-dependent cellular cytotoxicity.

Transmitted Soret band optical microscopy has been used to image the entry and passage of reactive oxygen metabolites across target erythrocytes. Due to the rapid cytosolic diffusion of hemoglobin in comparison to video rates, it was necessary to use erythrocytes with relatively immobilized hemoglobin. To achieve this, erythrocytes from patients with sickle cell anemia were used. The movement of reactive oxygen metabolites across rabbit IgG-opsonized sickle cells was observed in real time. These observations indicate that reactive oxygen metabolites can enter and cross targets in an asymmetric fashion.     

Effector cells for antibody-dependent cell mediated cytotoxicity. I. Increased cytotoxicity after priming with BCG-SS.

Intraperitoneal injection of an aqueous extract of Bacillus Calmette-Guérin (BCG-SS) is shown to increase the cytotoxicity of murine spleen cells which mediate antibody-dependent cellular Cytotoxicity (ADCC). Three to forty-four days after in vivo stimulation with BCG-SS, spleen cells were tested for their ability to lyse antibody-coated chicken red blood cells in a ⁵¹Cr release assay. Significantly increased lysis compared to non-BCG-SS primed litter mates was observed from 3 days through 3 weeks after priming. Aqueous extracts of other bacteria including Listcria, Brucella, Salmonella, Staphlococcus and Eschcrichia did not elicit the same cytotoxic response. Separation of BCG-SS stimulated spleen cells on columns of G-10 Sephadex showed increased cytotoxicity in both the adherent (presumptive macrophage and polymorph) and nonadherent (presumptive K lymphocyte) populations. The possible relationship of these results to BCG-mediated anti-tumor effects is discussed.     

Surface properties of cells involved in antibody-dependent cytotoxicity.

Using a recently developed technique for separating cells, the two cell types that mediate the destruction of antibody coated target cells, namely K cells and macrophages, have been characterized according to a series of cell surface markers. K cells lack surface immunoglobulin, complement receptors and the theta antigen but possess Fc receptors. In contrast macrophages, although lacking surface immunoglobulin and the theta antigen, express both complement and Fc receptors on their surface. By simultaneously removing theta and immunoglobulin bearing cells, K cells were enriched by a factor of 40–50 fold. From the data presented it was calculated that not more than 0.5–0.8% of splenocytes are K cells, a finding which suggests that antibody dependent cell mediated cytotoxicity is a highly efficient mechanism of killing target cells.     

Effects of macrophage activity on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae: an in vitro cytotoxicity and ultrastructural study

The effect of the activity of macrophages on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae was studied in vitro. Macrophages present in peritoneal exudates from mice genetically selected for high and low antibody production (HL and LL, respectively) showed an inverse cytotoxic effect. Cells from HL mice were ineffective, whereas cells from LL mice had a very high killing capacity. Ultrastructural studies of cells after incubations of up to 36 h supported these observations. Furthermore, peritoneal macrophages from congenitally athymic (nu/nu) mice showed a higher killing potential than cells from thymus-bearing littermates (+/nu) mice. The activity of the latter cells could be increased by in vitro pretreatment of the mice with Calmette-Guérin bacillus, a well-known macrophage stimulating agent. The results indicate that macrophages, although not the only effector cells, may play an important role in the defence against T. spiralis newborn larvae.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.     

Inhibition of antibody-dependent cellular cytotoxicity by autologous lymph node cells.

A population of lymph node cells that lack the usual T, B, or K cell markers was found to inhibit autologous spleen cells from mediating antibody-dependent cellular cytotoxicity (ADCC) to antibody-coated chicken erythrocytes. Inhibitor cells were not susceptible to treatment with anti-Thy 1.2 or anti-Ig and C; they did not adhere to Sephadex G-10, to nylon wool, or to monolayers of sheep erythrocytes (E) or erythrocytes plus 7S antibody (EA). After a brief (4-min) exposure to 45 degrees C, the ability to inhibit was lost whereas other cellular responses remained intact. ADCC mediated by nonadherent splenic effector cells (presumptive K cells) was highly susceptible to inhibition. Possible mechanisms for and implications of lymphocyte-mediated inhibition of ADCC are discussed.     

Recent data on antibody-dependent cell cytotoxicity, or the ADCC phenomenon

A review of the recent literature on ADCC affords strong evidence that this cytotoxicity phenomenon, discovered fortuitously in 1965, can no longer be considered only a laboratory curiosity. It is, in fact, more than probable that ADCC is involved in most defense mechanisms of the individual from allograft immunity and antitumoral defense to the resistance of the host against most pathogens.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Inhibition of antibody-dependent eosinophil-mediated cytotoxicity by heparin

Killing of bloodstream forms of Trypanosoma cruzi, the unicellular parasite that causes Chagas' disease in humans, by human eosinophils in the presence of specific antibody to the parasite was inhibited by the polyanion heparin in a dose-dependent manner. The concentration of heparin required to completely abolish eosinophil-effected killing of the flagellate increased as the eosinophil to parasite ratio increased. These results suggest that antibody-dependent eosinophil-mediated cytotoxicity is mediated by basic constituents of the eosinophil granule.