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.     

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.     

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.     

Polymorphonuclear neutrophil-mediated antibody-dependent cell cytotoxicity of herpesvirus-infected cells: ultrastructural studies

Bovine polymorphonuclear neutrophils (PMN) can mediate antibody-dependent cell cytotoxicity (ADCC) of herpesvirus-infected cells. Since cytotoxicity occurs only in the presence of PMN and specific antiviral antibody, but not until viral membrane antigens are expressed on the target cell, it is concluded that antibody must recognize viral membrane antigens before cytotoxicity can occur. Cytotoxicity also requires very close contact between the target cell and the PMN cell. These interactions occur as early as 1 h after incubating antibody, infected cells, and PMN, but the actual lysis and release of intracellular components occur over an extended period. It was assumed that degranulation was not involved in the initiation of cytotoxicity, but was involved in the final stage of destruction. The mechanism of lysis is proposed to involve the interaction of PMN membranes with target cell membranes with subsequent reorganization and activation of the PMN plasma membrane at points of contact with the target cell. This results in possible production of transmembrane channels which allows for the release of target cell contents.     

Alterations in antibody-dependent cellular cytotoxicity during the course of HIV-1 infection. Humoral and cellular defects

HIV-1-specific cell-mediated cytotoxicity (CMC) is a form of antibody-dependent cellular cytotoxicity (ADCC) in which HIV-1-specific antibodies arm NK cells directly to become cytotoxic for targets bearing HIV-1 antigenic determinants. This non-MHC-restricted cytotoxic activity is present in early stages of disease and declines markedly with disease progression. To understand the cellular and humoral factors contributing to the reduction in this activity, the conditions under which maximal arming of cells occurs was examined in vitro. With the use of a large patient cohort, a strong positive correlation was found between the capacity of a serum to direct lysis in standard ADCC assays and its ability to arm NK cells. Patients with minimal HIV-1-specific ADCC-directing antibodies exhibited low levels of CMC and were unable to arm normal effector cells in vitro. The lack of sufficient ADCC-directing antibodies was found to be one cause of defective CMC in some patients. Unlike asymptomatics, only a weak positive correlation was found between arming and ADCC with sera from AIDS patients, indicating that a factor other than absolute HIV-1 specific antibody titer was responsible for decreased CMC in this patient population. Another group of patients was found to have diminished CMC despite the presence of antibodies in the serum that were fully capable of arming normal effector cells to become cytotoxic for gp120-expressing targets. When compared with those of normal individuals, lymphocytes from seropositive patients mediated significantly reduced levels of cytotoxicity in ADCC and arming assays with the use of a high titered HIV-1-specific serum. In both assay systems, the magnitude and frequency of dysfunction in antibody-dependent cytolysis was found to be greater among AIDS patients than among asymptomatic individuals. The demonstration of both cellular and humoral defects in the ability of seropositive individuals to manifest ADCC reactivities strongly suggests that HIV-1 infection may significantly compromise the effectiveness of this potentially important cytolytic reactivity in vivo.     

The involvement of the cytoskeleton during antibody-dependent cellular cytotoxicity (ADCC)

The mechanism of initiation of antibody-dependent cellular cytotoxicity (ADCC) was analyzed using a model system consisting of sensitized chicken red blood cells (ChRBC) as targets and non-immunized Balb/c mouse splenocytes as effectors.Using a ⁵¹Cr release assay in parallel with electron and light microscopic observations, we were able to correlate the binding of target cells to effector cells with the subsequent lysis of the target cells. Untreated splenocytes are capable of binding and lysing a substantial number of target cells in the presence of anti-ChRBC. Splenocytes pretreated with the microfilament disrupting drug, cytochalasin D, display a significant reduction in their ability to bind and kill target cells. In contrast, splenocytes incubated with colchicine, a drug that disrupts microtubules, are slightly stimulated in both their binding and lysis of ChRBC. Our data suggest that microfilaments (but not necessarily microtubules) play an important role in the initial recognition and binding steps between effector and target cells which are prerequisite conditions for target killing in this ADCC sysytem.     

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.     

Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

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.     

Micro-scission of YAC tumor cells during antibody-dependent cellular cytotoxicity mediated by human neutrophils

The cellular events accompanying neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) directed against YAC erythroleukemic target cells have been studied by time-lapse fluorescence-intensified microscopy. The YAC plasma membrane and cytosol were labeled with the fluorescent probes diC18Icc and eosin Y, respectively. Fluorescently labeled and IgG-opsonized YAC cells were incubated at 37 degrees C while observed by optical microscopy. During temporal studies of neutrophil-YAC conjugates, the cytosol of YAC cells accumulated in tubular and spherical compartments of the neutrophils' vacuolar apparatuses. To distinguish between several possible mechanisms of target cytosol uptake, diC18Icc-labeled YAC cells were observed during identical conditions. The membrane label diC18Icc was found to accumulate within neutrophils in an identical fashion. At roughly 30 min, 25 and 38% of neutrophils in apparent conjugates had internalized tumor cell cytosol or plasma membrane, respectively, within a vesicular compartment. The IgG-dependent uptake of eosin Y and diC18Icc by neutrophils was diminished by exposure to 2.5 mM sodium azide. When cells were exposed to 5.5 mM sodium azide, 1 mM iodoacetamide, or 4 degrees C, conjugate formation and uptake of eosin Y or diC18Icc were abolished. An artifactual accumulation of eosin Y or diC18Icc in neutrophils was further ruled out by control studies. Non-specific exchanges of eosin Y and diC18Icc labels of YAC cells with tannic acid-treated red blood cells (RBCs) and normal neutrophils were studied. Since hemoglobin binds tightly to eosin Y, RBCs can easily detect eosin Y leakage. No exchange of eosin Y or diC18Icc from YAC cells into bound tannic acid-treated erythrocytes was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of antibody-dependent cellular cytotoxicity by protein A from Staphylococcus aureus.

Protein A, a cell wall constituent of several strains of Staphylococcus aureus, binds strongly to the Fc portion of immunoglobulins. This investigation demonstrated that such binding can inhibit antibody-dependent cellular cytotoxicity (ADCC). The degree to which ADCC was inhibited depended upon the relative concentrations of protein A and anti-target cell antiserum. Protein A also inhibited the formation of rosettes between antibody-coated sheep red blood cells and lymphoid cells with Fc receptors. We, therefore, conclude that protein A inhibits ADCC by preventing the binding of antibody-coated target cells to Fc receptors on cytotoxic effector cells.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.     

Stimulation of phagocytic processes and antibody-dependent cellular cytotoxicity of human neutrophils by cefmetazole.

Interactions between antimicrobial agents and phagocytic cells, especially neutrophils, have a potential role in the treatment of infections. The in vitro effects of cefmetazole, a novel beta-lactam antibiotic, at a therapeutic concentration reached in plasma (50 micrograms/ml) on phagocytic and cytotoxic functions of human neutrophils have been studied. In human neutrophils, adherence capacity to nylon fiber and to substrate, chemotaxis, attachment to and ingestion of Candida albicans (with serum, with decomplemented serum and without serum), ingestion of inert particles (latex beads), candidicidal activity and superoxide anion production were all stimulated by cefmetazole. Cefmetazole at this dose was a chemotactic agent for neutrophils. Antibody-dependent cellular cytotoxicity (ADCC) was also increased by this anti-microbial agent.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.