Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.     

Two distinct mechanisms of cytotoxicity by porcine alveolar macrophages in antibody-dependent and immobilized immune complex-dependent cellular cytotoxicity

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.     

Monocyte antibody-dependent cellular cytotoxicity in splenectomized subjects

Monocyte antibody-dependent cellular cytotoxicity (ADCC) was determined in normal subjects by using human A1 erythrocytes and immune human anti-A1 antiserum. The experimental data were fitted to a mathematical model and the values for maximal cytotoxicity and monocyte numbers required to produce 15% specific cytotoxicity (MD15) were subsequently computed and compared with the values for these two parameters estimated from the dose response curves. The values for both were significantly associated, and the mathematical model permitted precise quantitation of cytotoxicity in instances where this was impossible by standard methods. Maximal monocyte ADCC was significantly reduced in a group of splenectomized subjects, whereas the MD15 was increased. These findings emphasize the possible influences of the method of quantitation of ADCC and provide one explanation for the apparent conflict of published data.     

Monocyte antibody-dependent cellular cytotoxicity in cancer patients

Summary Antibody-dependent cellular cytotoxicity (ADCC) mediated by peripheral blood monocytes was determined in 120 patients who had gastrointestinal tract (GIT), lung and breast cancer, melanoma, or Hodgkin's and non-Hodgkin's lymphoma. Results were expressed in terms of maximum cytotoxicity and cytotoxicity at E : T=1 : 10 and were compared with the results obtained in 63 normal subjects. There was a significant decrease in maximal cytotoxicity for both the GIT cancer and the melanoma patient groups, but not for any of the other groups. These differences were not confirmed when results were expressed at low effector: target cell ratios, e.g., cytotoxicity at E : T=1 : 10. The relationship between monocyte ADCC and disease extent was examined in those groups with sufficient numbers. Monocyte ADCC was higher in patients with GIT cancer of limited extent than in patients with extensive GIT cancer and in the control group.     

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.     

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.     

Effects of inhibitors of C1q biosynthesis on macrophage Fc receptor subclass-mediated antibody-dependent cellular cytotoxicity and phagocytosis

We examined the effects of the inhibitors of C1q or collagen biosynthesis, 2,2'-dipyridyl (DP), and 3,4-dehydro-DL-proline (DHP) on murine macrophage (M phi) FcR subclass-mediated antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of sheep erythrocyte targets. Oil-elicited peritoneal M phi from C3HeB/FeJ mice which were cultured for 24 hr with DP (0.08 or 0.10 mM) or DHP (0.8 or 1.0 mM) showed a significant decrease in FcR subclass-mediated ADCC for murine monoclonal IgG2a (FcRI) and IgG2b/IgG1 (FcRII) as well as for heterologous polyclonal IgG. These collagen inhibitors also blocked phagocytosis mediated by both IgG2a- and IgG2b-opsonized erythrocytes. DP was more potent than DHP in blocking FcR effector functions in a reversible fashion and neither inhibitor affected M phi C3b receptor function. Pretreatment of M phi with collagenase resulted in significant reduction in FcR-mediated ADCC and phagocytosis. The inhibition of M phi FcR subclass-mediated ADCC and phagocytosis by collagen C1q synthetic inhibitors or by collagenase treatment further confirms a functional relationship between cell-associated C1q and FcR-dependent functions.     

Natural killer and antibody-dependent cellular cytotoxicity in cervical carcinoma patients

Summary Natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC) was measured in 62 untreated cervical carcinoma patients and 25 normal healthy women, using a short-term chromium release assay. A significant reduction in NK and ADCC activity was observed in disseminated disease than in localized disease, when compared with normal donors. The majority of the patients received radiotherapy and both NK and ADCC activity recovered after therapy. Furthermore, interferon- was demonstrated to augment NK activity of peripheral blood mononuclear cells from healthy donors as well as patients. Also large granular lymphocytes separated on Percoll density gradient were the same in number in both the populations studied, although in cervical cancer there seemed to be a defect in killing activity.     

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.     

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.     

Indomethacin up-regulates the generation of lymphokine-activated killer-cell activity and antibody-dependent cellular cytotoxicity mediated by interleukin-2

Prostaglandins can inhibit the generation of lymphokine-activated killer (LAK) cells by interleukin-2 (IL-2) whereas indomethacin augmented the induction of LAK cells by inhibiting prostaglandin synthesis. In the present study we demonstrate that prostaglandin E2 substantially inhibited the generation of both LAK and antibody-dependent cellular cytotoxicity (ADCC) activity by IL-2. In addition, indomethacin enhanced the induction of LAK activity and ADCC in splenocytes exposed to IL-2 in vitro. The effect of indomethacin was dose-dependent, reaching an optimal effect at 1 microM when 100-1000 units/ml IL-2 were employed. The effect of indomethacin on the generation of ADCC was seen in cells taken from both tumor-bearing mice and normal mice. ADCC induced by IL-2 was augmented by culturing cells from the spleen, liver and lungs, in the presence of indomethacin. ADCC induced in the presence of IL-2 and indomethacin was mediated by cells that were mainly plastic non-adherent cells and expressed the asialo-GM1 glycolipid. The potential of indomethacin in combined therapy with cytokines and specific anti-tumor monoclonal antibodies is discussed.     

The effect of various cytokines on the in vitro induction of antibody-dependent cellular cytotoxicity in murine cells. Enhancement of IL-2-induced antibody-dependent cellular cytotoxicity activity by IL-1 and tumor necrosis factor-alpha

We have previously demonstrated that incubation with IL-2 can induce ADCC activity in murine cells and that this activity was mediated by asialo GM1+, FcR+ cells. In the present study we show that the cytokines IFN-alpha and IFN-gamma, TNF-alpha, and IL-1 alpha are unable to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells; however, TNF-alpha and IL-1 alpha could substantially augment the ADCC induced by IL-2. IL-1 increased the IL-2-induced ADCC activity in a dose-dependent fashion and in cells isolated from the thymus and spleen. The precursors of the ADCC induced by the combination of IL-1 and IL-2 were asialo GM1+ cells, similar to the precursor cells of IL-2-induced ADCC. The effect of IL-1 and TNF on ADCC was not the result of an increase in the FcR density on the cell surface or the result of an increase in the number of FcR+ cells although IL-1 increased the recovery of viable cells in culture. The main effect of IL-1 and TNF was the enhancement of the lytic ability of the IL-2 cultured cells as indicated by increased intra-cellular benzyloxycarbonyl L-lysine thiobenzylester-esterase activity. These results suggest that lymphokines such as IL-1 and TNF may synergize with IL-2 in the induction of ADCC and could thus potentially be useful for the immunotherapy of established tumors when combined with the administration of specific anti-tumor antibodies.     

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.     

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.