Cyclophosphamide inhibits antibody-dependent cellular cytotoxicity (ADCC) suppression exerted by lymph node cells

The effect of cyclophosphamide (Cy) on suppression of antibody-dependent cellular cytotoxicity (ADCC) by lymph node cells (LNC) was evaluated. The results show that the suppression of ADCC exerted by LNC was abrogated when mice had been treated with Cy. Moreover, it was shown that ADCC inhibition induced by LNC was mediated by soluble factor(s) and that treatment with a single dose of 200 mg/kg ip of Cy, significantly decreased its release. In addition, suppressor activity of normal LNC was enriched by depletion of adherent cells and was not affected by treatment with monoclonal anti-Thy 1.2 plus complement. These observations indicate that modulatory cells are nonadherent and lack characteristic T-cell markers. Thus, we conclude that this suppressor system, which normally controls ADCC activity, can be inhibited by treatment of mice with Cy and that this effect may explain the enhancement of ADCC observed in splenocytes of Cy-treated animals.     

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.     

Effect of human adenovirus on antibody-dependent cellular cytotoxicity (ADCC) in chickens

The influence of human adenovirus type 6 on antibody-dependent cellular cytotoxicity (ADCC) in chickens has been investigated. The cytotoxic effect of peripheral blood mono-nuclear cells of chickens was studied on sheep red blood cells (SRBC) coated with chicken anti-SRBC serum. Cytotoxicity was estimated using a ⁵¹Cr release assay system. A single intravenous injection of the virus enhanced ADCC. ADCC was enhanced 14 to 24 hr after the virus injection, but then decreased and the preinjection level was reached after 36 hr. The capacity for virus-augmented activity was not removed with phagocytic cell depletion. The possible role of interferon induced by the virus in chickens in augmenting ADCC is discussed.     

Inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) by antiserum raised against brain tissue

Treatment of mouse spleen cells with a rabbit anti-mouse brain (RAMB) antiserum markedly suppressed antibody-dependent cell-mediated cytotoxicity (ADCC) on trinitrophenyl-coupled sheep erythrocyte targets. This inhibitory activity of RAMB antiserum was complement independent, absorbable with mouse brain tissue, and appeared to be separable from the anti-Thy-1 activity of this serum. Absorption studies indicated that various T- and B-lymphocyte cell lines as well as macrophage-like cell lines are not able to absorb the inhibitory activity of RAMB antiserum. In contrast, thymocytes and spleen cells, as well as the neural cell line, PC12, a chromocytoma derived from rat adrenal medulla, were capable of absorbing the inhibitory activity to some extent, suggesting that antigens characteristic for ADCC effector cells can be found on these cell populations.     

Effect of short-term ethanol intake on antibody-dependent cell-mediated cytotoxicity (ADCC) in rats.

The present study was designed to evaluate the effect of short ethanol intake on ADCC activity in blood and spleen mononuclear cells. Wistar rats were fed a standard diet and drank 0.1 M ethanol solution for three days. Glucose and water controls were used in this experiment. Increased ADCC activity was found in ethanol consuming rats in the blood and spleen as compared to both controls. Our findings support the assumption that ADCC may play an important role in liver disease of alcoholics.     

Studies on the antibody-dependent cell-mediated cytotoxicity (ADCC) of thioglycollate-stimulated and BCG-activated peritoneal macrophages

Peritoneal macrophages (mφ), activated by Bacillus Callmette-Guerin (BCG) or elicited by thioglycollate broth in vivo in C57BL/6J mice, were compared with regard to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The BCG-activated mφ had several times higher ADCC activity than did the TG-mφ against erythroid targets. When human and murine tumor cells were employed as targets in the ADCC assay, the BCG-mφ lysed each of six separate target lines considerably, while the TG-mφ had little and, occasionally no, lytic activity against the six targets. Although F_c-dependent and -independent phagocytosis of the erythroid targets by the TG-mφ exceeded that by the BCG-mφ, this rapid and extensive phagocytosis did not account soley for the decreased lysis of the erythroid targets by the TG-mφ. The results indicate that BCG-activated peritoneal mφ, in comparision with TG-elicited inflammatory mφ, are potent effectors of ADCC against both erythroid and neoplastic target cells.     

Interferon enhances the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) from healthy volunteers were tested for ADCC activity against both erythrocyte and tumor targets with and without the addition of human leukocyte interferon (IFN). It was demonstrated that IFN within 30 to 60 min enhanced the reaction in a dose-dependent manner with minimal IFN doses ranging from 1 to 100 units. Formal proof that the augmenting agent was IFN was obtained by using pure IFN proteins in combination with both mock-IFN preparations, which showed no enhancing activity, and anti-IFN antisera, which inhibited the action of the completely purified IFN proteins. In the light of data demonstrating that the IFN effect was most pronounced when the IgG antibodies in the ADCC reaction were present in suboptimal amounts, it is hypothesized that IFN may play a special role in the early nonspecific immune response against non-self antigens.     

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.     

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.     

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.     

A comparison of antibody-dependent cellular cytotoxicity (ADCC) mediated by murine and human lymphoid cell populations

Mouse lymphoid cells are known to lyse chicken red blood cells (CRBC) in the presence of antibody and in the absence of complement. They have also been reported to effect lysis of mouse tumor cells and other nucleated targets, although this has been disputed. Using a 4-hr ⁵¹Cr-release assay, we have compared the activity of mouse effector cells from the thymus, spleen, bone marrow, peritoneal cavity, and mesenteric and subcutaneous lymph nodes of many strains of mice to the activity of human lymphoid cell effectors against CRBC and a number of murine targets. Human effectors mediate lysis of all targets tested. Mouse effectors lyse CRBC, but usually less well than human effectors. Mouse cells from lymphoid organs were either very inefficient or completely inactive against nucleated mammalian targets under a range of test conditions. Interestingly, in experiments where cells from solid lymphoid organs or the peritoneal cavity were ineffective, peripheral blood lymphocytes from one subline of DBA/2 consistently gave significant lysis of EL4 targets, while cells from another subline of the same strain did not.     

Simultaneous development of antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity in irradiated mice reconstituted with bone marrow cells

Spleen cells from irradiated, bone marrow-reconstituted mice were tested for their ability to mediate antibody-dependent cellular cytotoxicity against P815 target (ADCC-P815), ADCC against sheep red blood cells (ADCC-SRBC), and natural killer (NK) activity judged as YAC-1 lysis at different times after bone marrow reconstitution. Donor-derived ADCC-P815 effectors were found to appear in the spleens 10-12 days after bone marrow reconstitution simultaneously with the appearance of donor-derived NK cells. NK cells recently derived from bone marrow are known to express the Thy-1 antigen; the phenotype of the "early" ADCC-P815 effectors was found to be the same as that of NK cells, i.e., Thy-1+, asialo-GM1+. These data suggest that ADCC-P815 effector cells belong to the NK cell population. ADCC-SRBC, in contrast to ADCC-P815 and NK activity, was already high on Day 7 after bone marrow reconstitution. However, it was mediated partly by recipient-derived effectors. ADCC-SRBC effectors were characterized to be different from ADCC-P815 effectors.     

Spontaneous cell-mediated cytotoxicity in patients with transitional cell carcinoma of the urinary bladder

Summary Lymphocytes isolated from the blood of TCC patients, like those of control patients, were capable of mediating spontaneous cell-mediated cytotoxicity against K-562 cells. When this natural cytotoxicity was analyzed with regard to the effector cell type it was found that in TCC patients the SLMC was mostly displayed by E-rosetting T lymphocytes, whereas compared with controls, a significant decline in the SLMC of the non-T lymphocytes was observed. The SLMC of the T lymphocytes derived from TCC patients was further demonstrated on a T leukemia target cell (Peer). When the SLMC on K-562 and on Peer target cells was compared, a specificity difference was observed between TCC and the control patients' effector cells. The SLMC activity of the TCC patients' T cells was not abolished after depletion of Fc receptor-positive cells or following treatment with monoclonal antibodies OKT 8 or OKT 4 and complement (C'). These NK-like cells are therefore distinguished from cytotoxic T lymphocytes and NK cells.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in specific pathogen-free (SPF) miniature swine and germfree piglets. III. Two distinct effector cells for NK and ADCC

Effect of "anti-NK" sera on NK and ADCC activities was examined to study further the relationship between the effector cells for NK and ADCC in specific pathogen-free miniature swine. An appropriate dilution of "anti-NK" sera that had no effect on ADCC activity not only inhibited NK activity but also eliminated NK cells in the presence of C which supports the presence of NK cells distinct from K cells for ADCC.     

Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells

Bombesin and the two mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) and neuromedin C, at physiological concentrations have been previously shown to stimulate significantly in vitro the antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activities in BALB/c mouse leukocytes from axillary nodes, spleen and thymus. In the present work we have shown that adherent cells are required in leukocyte samples for stimulation of cytotoxicity by the neuropeptides, which suggests that this effect may be mediated by those cells. Here we demonstrate the specificity of the effects by reversing them in the presence of the bombesin-antagonist (Leu¹³-ΨCH₂NH-Leu¹⁴)-BN, and by detecting specific receptors for GRP on macrophages of high and low affinity. Using the same binding technics, no receptors for this neuropeptide were found in non-adherent leukocytes.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.