Spontaneous Cellular Cytotoxicity Research Articles

A comparison of hepatic, splenic, peritoneal and alveolar macrophages with respect to PGE 2, TXB 2, production and ADCC function

The in vitro production of PGE 2 and TxA 2, measured as TxB 2, and the antibody-dependent cellular cytotoxicity (ADCC) as well as spontaneous cellular cytotoxicity (SCC) displayed by hepatic, peritoneal, splenic, and alveolar macrophages from 12 rats was determined and compared. Kupffer cells, the fixed macrophages of the liver, were the most active cells in the production of PGE 2. Kupffer cells and peritoneal macrophages released about equal amounts of TxB 2 which was much higher than that released by splenic macrophages. Kupffer cells displayed the strongest ADCC activity, and alveolar macrophages had the lowest value. These results confirm the previously reported heterogeneity among different sources of macrophages and emphasize the enhanced activity of Kupffer cells with respect to eicosanoid production and ADCC function.

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin.

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.

Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus.

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.

Experimental polymyxin B-induced interstitial lung disease characterized by an accumulation of cytotoxic eosinophils in the alveolar structures.

A variety of lung disorders are associated with the accumulation of eosinophils in the alveolar structures. To help understand the role of eosinophils in these disorders, an animal model of eosinophilic lung disease was developed. Administration of an aerosol of polymyxin B to guinea pigs (3 times per wk for 4 wk) produced diffuse interstitial lung disease with alveolar wall thickening and an alveolitis characterized by marked increases in eosinophils and alveolar macrophages. Bronchoalveolar lavage confirmed the presence of significantly increased numbers of eosinophils and alveolar macrophages in polymyxin-B-treated animals compared with those in control animals. Using density gradient centrifugation, approximately 10(7) eosinophils could be purified from the lungs of a single polymyxin-B-treated animal. Importantly, eosinophils purified from the lungs from polymyxin B-treated animals exhibited significant spontaneous cellular cytotoxicity for human fetal lung fibroblasts. In contrast, neither eosinophils from control animals nor alveolar macrophages from either group of animals were cytotoxic. These findings demonstrate that eosinophils possess effector processes capable of injuring the lung parenchyma and suggest that eosinophils can contribute to the pathogenesis of the interstitial lung disease.

Monocyte dysfunction in human cancer.

General immunobiologic studies in cancer patients have stressed measurements of lymphocyte function and have commonly ignored the monocyte-macrophage system. A preliminary study of peripheral blood monocyte-macrophage function was undertaken in a group of 90 cancer patients (18 breast, 32 colon, 13 head and neck, 9 lung, and 18 melanoma) and 70 controls. Studies included enumeration of extractible monocytes (EM), quantitation of differentiation into macrophages (macrophage precursor test: MP), evaluation of antibody-dependent cellular cytotoxicity (ADCC), and spontaneous cellular cytotoxicity (SCC) as measured with human erythrocytes, and measurements of monocyte and serum lysozyme activity. Breast cancer patients had normal profiles. Colon cancer patients showed the most profound abnormalities with decreased EM and MP and increased ADCC and SCC. Patients with Stage I and Stage II melanoma had normal profiles, whereas those with advanced melanoma had significantly decreased MP. This defect was restored in two patients by BCG immunotherapy. Head and neck cancer and benign breast disease patients had decreased EM, whereas patients with lung cancer had increased EM. Monocyte lysozyme production was unchanged in the cancer patients compared to controls. Serum lysozyme levels, however, were significantly increased in patients with cancers of the colon, head and neck, and lung. Although patients with localized breast cancer and melanoma had normal levels, these were increased in both patient groups with advanced disease. It would appear that the source of the increased serum lysozyme is probably not the peripheral blood monocytes, but could have originated in the intra-tumoral or tissue-bound macrophages which were not examined. Selected assays of peripheral blood monocyte function were deranged in certain types of cancer patients but were fully normal in others, and did not show consistent correlations with tumor type or stage. Tissue-bound or intra-tumoral macrophages might provide a more fruitful area for study in these disease categories.

Divergence in activation by poly I:C of human natural killer and killer cells.

Interferons consistently enhance spontaneous cellular cytotoxicity (SCC) mediated by natural killer (NK) cells. More controversial is the ability of interferons to enhance antibody-dependent cellular cytotoxicity (ADCC) mediated by killer (K) cells. Since NK and K cells appear to represent overlapping subpopulations of lymphocytes, the present study was undertaken to examine in greater detail the relationship between NK and K cell functional modulation by the potent interferon inducer, poly I:C. Utilizing peripheral mononuclear cells from a panel of 21 healthy individuals, treatment in vitro with poly I:C resulted in modulation of both SCC and ADCC. SCC was significantly enhanced in 52 of a series of 55 trials (95%), whereas ADCC was significantly enhanced in parallel in only 18 of the trials (33%). Cells which mediated enhanced ADCC were plastic-nonadherent, which is characteristic of K cells. SCC was consistently enhanced in all but two of the 14 individuals who were tested two or more times. By contrast, the ability of poly I:C to enhance ADCC varied between trials in 11 of these individuals. In the other three, ADCC enhancement never occurred. No correlation existed between SCC and ADCC augmentation despite use of the same target cell to assess the two lytic activities in parallel. Poly I:C exclusively enhanced SCC in 36 trials (65%) and exclusively enhanced ADCC in two trials (4%). Discordance between SCC and ADCC enhancement also occurred in three of eight trials (38%) in which lymphocytes were treated directly with interferon a. Results in long-term (18-h) 51Cr-release assays indicated that poly I:C accelerated the kinetics of ADCC without affecting the proportion of target cells lysed by K cells. By contrast, an increased proportion of target cells was killed by poly I:C-stimulated NK cells. These results suggest that the controversy concerning relative interferon effects upon NK and K cells derives from differences both quantitative and qualitative in nature. K cell activity is enhanced but at a relatively low frequency. Enhancement of NK cell activity is selective in the sense that it occurs independently of and with greater frequency than enhancement of K cell activity. Distinct biological mechanisms may, therefore, be involved in regulation and expression of NK and K cell activation by interferons.

Effector mechanisms in the spontaneous autoimmune thyroiditis of obese strain (OS) chickens: analysis of cytotoxic cells.

Different forms of cellular cytotoxicity that may constitute potential effector mechanisms in the spontaneous autoimmune thyroiditis (SAT) arising in Obese strain (OS) chickens have been investigated. A microcytotoxicity assay (MCA) employing 51Cr-labelled-, thyroglobulin (Tg) coated-chicken red blood cells (Tg-CRBC) was used to detect cells mediating Tg-specific direct cellular cytotoxicity (DCC). Tg-CRBC presensitized with anti-Tg autoantibody (Tg-AAB) obtained from high titer OS sera served as targets for antibody-dependent cell-mediated cytotoxicity (ADCC). Tannic acid-only treated CRBC (TA-CRBC) were used simultaneously as specificity controls for DCC and also as targets for cells eliciting spontaneous cellular cytotoxicity (SCC) to surface-modified normal cells. The results demonstrated that Tg-specific cytotoxic cells exist in the OS and thus represent an effector mechanism in SAT, in addition to the previously well-documented role of antibody. This DCC appears to be unrelated to the presence and titer of circulating Tg-AAb. It is present in highest levels in the peripheral blood of OS chickens, but in only 55% of the animals tested, indicating either a secondary importance to humoral immunity in the disease process or the possibility of different effector mechanisms prevailing in different birds. There were no overall differences in ADCC between OS and normal chickens when the two strains were considered as a whole. Chronological analysis, however, revealed very low ADCC in the peripheral blood of young OS birds, followed by a later elevation above that in the normal control chickens. Destruction of the gland by ADCC may be initiated via maternally-derived of in situ-produced anti-Tg antibody. No overall significant differences in SCC were observed between OS and normal chickens.

Killer cells in the chicken: A microcytotoxicity assay using antigen-coated erythrocytes as targets

A 51chromium ( 51Cr) release microcytotoxicity assay has been established for studying cell-mediated immunity in chickens to a potentially wide variety of antigens. The system investigated in detail uses thyroglobulin-coated chicken red blood cells (Tg-CRBC) to analyse effector cell mechanisms operative in spontaneous autoimmune thyroiditis in Obese strain (OS) chickens. A variety of technical parameters were investigated in order to optimise reliable, reproducible target cell preparation and to minimise spontaneous 51Cr-release. The final method adopted used tannic acid for coupling antigen to carefully selected donor erythrocytes of uniform MHC genotype. For the study of antibody dependent, cell-mediated cytotoxicity, Tg-CRBC were pre-sensitised with OS serum containing high titre Tg-autoantibody. Tannic acid-treated CRBC (TA-CRBC) served simultaneously as controls for the Tg specificity of direct cellular cytotoxicity (DCC) to Tg-CRBC, and also as target cells for natural, or spontaneous cellular cytotoxicity (SCC). With such an assay, cells capable of mediating Tg-specific DCC were demonstrated in the OS, but not in normal chickens. No differences in ADCC or SCC were observed when the two strains were considered as a whole, i.e. regardless of age, sex, MHC genotype or extent of disease.

Studies in acute leukemia. I. Antibody-dependent and spontaneous cellular cytotoxicity by leukemic blasts from patients with acute nonlymphoid leukemia

Leukemic blasts from patients with acute nonlymphoid leukemia were examined for the presence of Ig, receptors for IgGFc, and for their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC) against chicken red blood cells (RBC) coated with IgG and spontaneous cell-mediated cytotoxicity (SCMC) against cells of K562 cell line. Leukemic blasts from acute myeloblastic leukemia (AML) patients lacked both Fc receptors and Ig on their surface, had no SCMC activity and majority, but not all of them, lacked ADCC activity. Leukemic blasts from patients with acute monocytic leukemia (AMOL) had Fc receptors, and 50% had IgG on their surface. IgG was cytophilic and appeared not to be directed against cell-surface antigens. This antibody did not interfere with the ADCC activity of leukemic cells. Leukemic blasts from majority of patients with AMOL mediated ADCC, but had no SCMC activity. An association between ADCC and presence of Fc receptor was observed.

Studies in Acute Leukemia. I. Antibody-Dependent and Spontaneous Cellular Cytotoxicity by Leukemic Blasts From Patients With Acute Nonlymphoid Leukemia

AbstractLeukemic blasts from patients with acute nonlymphoid leukemia were examined for the presence of Ig, receptors for IgGFc, and for their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC) against chicken red blood cells (RBC) coated with IgG and spontaneous cell-mediated cytotoxicity (SCMC) against cells of K562 cell line. Leukemic blasts from acute myeloblastic leukemia (AML) patients lacked both Fc receptors and Ig on their surface, had no SCMC activity and majority, but not all of them, lacked ADCC activity. Leukemic blasts from patients with acute monocytic leukemia (AMOL) had Fc receptors, and 50% had IgG on their surface. IgG was cytophilic and appeared not to be directed against cell-surface antigens. This antibody did not interfere with the ADCC activity of leukemic cells. Leukemic blasts from majority of patients with AMOL mediated ADCC, but had no SCMC activity. An association between ADCC and presence of Fc receptor was observed.

Comparison of the effector cells in human spontaneous cellular cytotoxicity and antibody-dependent cellular cytotoxicity: differential sensitivity of effector cells to in vivo and in vitro corticosteroids.

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.

Human peripheral Null lymphocytes I. Isolation, immunological and functional characterization

AbstractA Null lymphocyte‐enriched population was isolated from human peripheral blood of healthy donors using a combination of Ficoll density gradient centrifugation followed by elimination of mononuclear phagocytes, passage through Ig‐anti‐Ig columns and sedimentation of E rosettes. After each separation step lymphocyte fractions were examined for morphology, cell surface markers, mitogen responsiveness and effector functions in antibody‐dependent (ADCC) and spontaneous cellular cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line. The final Null lymphocyte preparation was recovered at a rate of 1 to 3 % from the population passed through Ig‐anti‐Ig columns (fraction FFF‐C). The marker analysis revealed over 99 %of surface Ig‐negative lymphoid cells; 50 to 60 % of these cells were ‘real Null’ cells lacking immunological cell surface markers, 7 % formed EA, 13 % EAC and 24 %E rosettes.Regarding the mitogen responses, passage through Ig‐anti‐Ig columns drastically reduced concanavalin A (Con A), pokeweed mitogen (PWM)and tuberculin (PPD) responses, whereas the phytohemagglutinin (PHA) response was reduced in absolute counts but not in the stimulation index. Compared to the T cell‐enriched lymphocyte fraction, the Null cells showed significantly diminished proliferative responses to PHA and Con A and slightly increased reactivity to PWM and PPD.Although depleted of high affinity Fc receptor lymphocytes, the Null cell fractions exhibited good ADCC and SCMC activities being about 4 to 6 times higher than in the T cell fraction.