Serum and effector-cell antibody-dependent cellular cytotoxicity (ADCC) activity remains high during human immunodeficiency virus (HIV) disease progression

The activity of both serum and effector cell antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus (HIV-1, HIV) was assessed in HIV-infected individuals. The goal was to relate ADCC levels with the stage or progression of HIV disease. Serial serum samples, usually collected at 6-month intervals, from individuals at defined stages of HIV disease (sero-conversion, the HIV-seropositive period before AIDS, and around the time of clinical AIDS diagnosis) were tested. HIV-coated CEM tumor cells were used as targets. Effector-cell ADCC activity was evaluated using fresh peripheral blood mononuclear cells (PBMC) from HIV-infected individuals at different stages of HIV disease. Samples were obtained from male homosexual participants in the Multicenter AIDS Cohort Study (MACS). In seroconverters, ADCC-inducing HIV-specific antibodies were detected at the time that the ELISA antibody test was first positive. Within several months, serum ADCC activity stabilized in each individual. In 29 HIV-seroprevalent individuals (HIV seropositive on their first visit), serum ADCC activity remained constant regardless of whether the individual's HIV disease was stable (high stable CD4;n=9) or rapidly deteriorating (sharply declining CD4,n=10; AIDS progressors,n=10). With respect to effector-cell activity, PBMC from HIV-infected individuals with or without AIDS were capable of mediating ADCC with heterologous and usually with autologous sera. Although the level of NK cytotoxic activity and the level of antibody-armed effector cell activity have been reported to decline as disease progresses, our results support previous observations that ADCC effector-cell activity against antibody-coated targets does not decline in HIV infection. These results indicate that both serum and effector cells with ADCC activity are present in HIV-infected individuals shortly after seroconversion and are maintained throughout HIV disease. Although levels of serum and effector-cell ADCC activity do not predict whether an individual will develop AIDS, CD4 cells which express HIV antigens (either produced endogenously or adsorbed onto the surface) could serve as targets for anti-HIV-mediated ADCCin vivo. ADCC could, thereby, contribute to CD4 T-cell depletion in infected individuals. However, since serum and effector-cell ADCC levels do not seem to relate to disease stage or progression, the protective or pathogenic role that ADCC plays in HIV-disease remains unresolved.     

Circulating soluble factor-inhibiting natural killer (NK) activity of fresh peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.     

Azathioprine suppression of natural killer activity and antibody-dependent cellular cytotoxicity in renal transplant recipients

The relative effects of azathioprine (AZA) and prednisone (PRED) on natural killer (NK) activity and the antibody-dependent cellular cytotoxicity (ADCC) of K (killer) cells and the number of FcR and other lymphoid cells were examined in renal transplant recipients. In addition to both long-term (>6 months) and short-term (<6 months) transplant recipients receiving conventional AZA-PRED therapy, an important group of long-term recipients receiving PRED but not AZA was studied for the first time. Both NK activity and ADCC are profoundly reduced in the long-term AZA-PRED group but are normal in the long-term PRED-alone (no-AZA) group. The short-term AZA-PRED group exhibits NK and ADCC levels significantly lower than normal but not as low as those of the long-term AZA-PRED group. Patient groups with low NK and ADCC also have low circulating Fc receptor-bearing (FcR) cells. A single patient in the long-term AZA-PRED group was removed from AZA therapy, and approximately 3 months was required for the patient's suppressed NK and ADCC to return to normal. These findings indicate that AZA rather than PRED is the major drug important in suppressing ADCC and NK activity in renal transplant recipients. Several months are required for combination AZA-PRED therapy to reduce these cytotoxic activities. Similarly, several months are required for suppressed ADCC and NK activity to return to normal upon discontinuation of AZA.     

Antibody-dependent cellular cytotoxicity (ADCC)-mediated destruction of human immunodeficiency virus (HIV)-coated CD4+ T lymphocytes by acquired immunodeficiency syndrome (AIDS) effector cells

The acquired immunodeficiency syndrome (AIDS) is defined in clinical terms by the development of Kaposi's sarcoma and/or severe opportunistic infections in persons without predisposing conditions. A hallmark of the syndrome has been a decrease in the number of CD4⁺ T helper cells. The reduction in the frequency of the CD4⁺ lymphocytes has been postulated to be primarily the result of human immunodeficiency virus (HIV) tropism and cytophathogenicity for the T-cell subset. Yet only a small percentage of cells is actually infected with HIV. Recently, we provided evidence indicating that AIDS patients' natural killer cells can mediate normal levels of antibody-dependent cellular cytotoxicity (ADCC) despite exhibiting a defect in natural killer (NK) effector function (J Immunol 139:55, 1987). This finding prompted us to investigate whether AIDS patients' effector cells could mediate ADCC against circulating CD4⁺ T cells infected with or expressing HIV antigen. The findings reported herein demonstrate that AIDS effector cells can mediate lysis of CEM (CD4⁺ T-cell line) coated with HIV protein in the presence of HIV-specific antibody. Lysis was specific, as non-HIV-coated CEM or the addition of HIV-negative serum resulted in no lysis. We then examined HIV-coated peripheral blood-derived CD4⁺ T lymphocytes as targets in ADCC. We demonstrate that in the presence of HIV-specific antibody, HIV-coated CD4⁺ T lymphocytes serve as targets for ADCC by AIDS effector cells. The lytic activity obtained with AIDS effector cells was comparable to that obtained with normal effector cells. These results demonstrate that AIDS effector cells can mediate ADCC against HIV-coated CD4⁺ T lymphocytes and suggest that ADCC may play a rolein vivo in the pathogenesis of AIDS.     

Labelling of lymphocytes with indium 111 oxine: effect on cell surface phenotype and antibody-dependent cellular cytotoxicity

Indium 111 oxine is currently used to label peripheral lymphocytes in order to study the kinetics of these cells in vivo. Since the quantity of radioisotope for labelling is still a matter of controversy, we have investigated in vitro the effect of increasing the concentration of indium 111 oxine on the lymphocyte surface phenotype and the antibody-dependent cellular cytotoxicity (ADCC) using lymphocytes from normal subjects. The cell surface phenotype, as evaluated by 2 monoclonal antibodies, was not affected whereas ADCC, at any of the doses used, was significantly reduced compared to the baseline value. The implications of these results for the use of indium 111 oxine for the in vivo studies are discussed.     

Effect of auranofin on antibody-dependent cellular cytotoxicity (ADCC) mediated by rat peripheral blood polymorphonuclear leukocytes and mononuclear cells

Auranofin and other clinically used gold compounds were evaluated in vitro for effects on antibody-dependent cellular cytotoxicity (ADCC)of L929 fibroblast target cells mediated by adjuvant rat peripheral blood PMNs or mononuclear cells. Auranofin (10M) was found to be a potent inhibitor of PMNADCC. In contrast, gold sodium thiomalate (10–100M), gold thioglucose (10–1000M, and nongold substructures of auranofin (10M) were not inhibitory. In continuous culture, gold sodium thiomalate and relatively low concentrations of auranofin (1 M) significantly enhanced PMNADCC. Results of pretreatment studies indicate that auranofin's inhibitory activity of PMNADCC is caused by a noncytotoxic effect on PMN function which is not associated with alteration of PMN-target cell contact. In contrast to its inhibitory activity on PMNADCC, auranofin pretreatment of mononuclear cells resulted in enhanced target cell destruction which appeared to correlate with increased mononuclear cell-target cell contact.SK&F D-39162 (2,3,4,6-tetra-O-acetyl-l-thio--d-glucopyranosato-S)triethylphosphine gold.     

Measurement of antibody-dependent cell-mediated cytotoxicity by Hb-enzyme release assay (Hb-ERA)

A non-isotope method, hemoglobin-enzyme release assay (Hb-ERA), for measuring antibody-dependent cell-mediated cytotoxicity (ADCC) has been successfully developed. The assay is based on the fact that hemoglobin has peroxidase activity capable of catalyzing the oxidation of o-phenylenediamine to give coloration which can be measured by spectrophotometer. In comparison to the conventional 51Cr release assay, Hb-ERA is economic, sensitive, reproducible and does not have the shortcomings associated with manipulation of radioactive substances. It is applicable in clinical and research laboratories of any scale. The optimum conditions for the assay are as follows: an effector cell: target cell ratio of 10:1 and an incubation period of 14-16 h at 37 degrees C in 5% CO2. The splenic ADCC activity of mice was measured. The cytotoxic index of mice aged 10-14 weeks (39.71 +/- 11.19) was significantly higher than that of 20-24 week-old mice (18.42 +/- 10.31) (P less than 0.001). The ADCC activity is not influenced by sex. The merits and drawbacks of Hb-ERA are objectively evaluated.     

Natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities can be differentiated by their different sensitivities to interferon and prostaglandin E1

Though purported to be identical cells (or in identical populations of cells), the natural killer (NK) cell mediating spontaneous natural cytotoxicity and the killer (K) cell mediating antibody-dependent cellular cytotoxicity (ADCC) may not be totally identical, at least in susceptibility to regulation by the immunomodulators prostaglandin E₁ (PGE₁) and interferon (IFN). We demonstrate here that NK cells are always enhanced by IFN, while K cells are inhibited from binding targets, resulting in fewer effectors at optimal concentrations of antibody. Only at 10- to 100-fold suboptimal concentrations of antibody is ADCC activity enhanced. As measured by magnitude of inhibition and dose-response titration, ADCC activity is less sensitive to the effects of PGE₁ than is NK activity in the⁵¹Cr release assay and single-cell assay. After overnight incubation with or without PGE₁, whatever sensitivity ADCC activity had to PGE₁ is lost. However, NK cells incubated in the presence of PGE₁ overnight are still sensitive to inhibition. Indomethacin boosts NK activity without having any effect on ADCC activity. Finally, NK activity is substantially reduced by overnight incubation of cells at room temperature, which has no effect on K cells.     

Colchicine derivatives inhibit neopterin production in human peripheral blood mononuclear cells (PBMC)

Colchicine is a microtubule disrupting agent, mostly used as treatment in various kinds of inflammatory diseases such as acute familial Mediterranean fever and Behcet's disease, as well as gout. In patients with familial Mediterranean fever treatment with colchicine induces a decline of urinary neopterin concentrations which indicates a decrease of cell-mediated immune activation. In this study, we investigated a potential effect of colchicine on the T cell/macrophage system in vitro. The human myelomonocytic cell line THP-1 and PBMC were treated with colchicine or the colchicine derivative, colcemide, in the presence or absence of 250 U/ml interferon-gamma (IFN-γ) or 10 μg/ml lipopolysaccharide (LPS) for 48 h or 96 h. Colchicine and colcemide increased neopterin/protein production in unstimulated THP-1 cells, but no such effect was apparent in cells stimulated with IFN-γ. By contrast, when PBMC were treated with colchicine or colcemide a significant reduction in neopterin formation was evident in cells without and with prestimulation by IFN-γ or LPS. In parallel, reduced production of IFN-γ was observed in PBMC. These data suggest that colchicine and colcemide are able to inhibit T cell activation within the cellular immune response.     

Effect of vernolide-A,a sesquiterpene lactone from Vernonia cinerea L., on cell-mediated immune response in B16F-10 metastatic melanoma-bearing mice

One of the major reasons for the rapid progression of cancers is the ability of tumor cells to escape from the immune surveillance mechanism of the body. Modulation of immune responses is highly relevant in tumor cell destruction. Effect of vernolide-A on the cell-mediated immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of vernolide-A enhanced natural killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared with the metastatic tumor-bearing control. Administration of vernolide-A significantly enhanced the production of interleukin (IL)-2 and interferon-gamma (IFN-γ) in metastatic tumor-bearing animals. In addition, vernolide-A significantly down-regulated the serum levels of proinflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and granulocyte–macrophage colony-stimulating factor (GM-CSF) during metastasis. All these results demonstrate that vernolide-A could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.     

Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells

We studied the in vitro effect of testosterone on spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Testosterone inhibited IgG and IgM production by PBMC both from males and females. The inhibitory effect of testosterone was revealed at doses more than 1 nm, increased dose-dependently, and reached a plateau at 100 nm. At doses <1000 nm, testosterone did not reduce cell viability. Testosterone treatment reduced IgG production by 59.0% and that of IgM by 61.3% compared with control. Immunoglobulin production by B cells was also suppressed by testosterone, though the magnitude of the suppressive effect on B cells was lower than that on whole PBMC; testosterone-induced decrease of IgG production compared with control was 26.9% and that of IgM was 24.9%. Exogenous IL-6 partially restored the impaired immunoglobulin production of testosterone-treated PBMC; IgG production in testosterone culture was increased by IL-6 from 35.6% to 66.5% of control and that of IgM was also increased from 38.9% to 71.2%, respectively. Testosterone treatment reduced IL-6 production of monocytes by 78.4% compared with control, but neither affected that of T cells or B cells. These results suggest that testosterone may suppress immunoglobulin production of human PBMC directly by inhibiting B cell activity and indirectly by reducing IL-6 production of monocytes. It is thus indicated that this hormone may have protective and therapeutic effects on human autoimmune diseases.     

Natural killing and antibody-dependent cytotoxicity by lymphocyte subpopulations in young and aging humans

Natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) were examined in the peripheral blood lymphocytes and their major subpopulations from young and aging subjects. Monocyte-depleted unseparated lymphocyte-mediated NK activity (against cells of K-562) and ADCC (against IgG-coated chicken erythrocytes) were comparable between young and aging subjects. Similarly no significant difference was observed in T cell-mediated NK and ADCC and non-T cell-mediated ADCC between young and aging subjects. Non-T cell-mediated NK activity, however, was significantly (P<0.025) greater in aging humans compared to that of young subjects. When the data were analyzed according to gender, T cell-mediated ADCC in aging males was significantly (P<0.05) greater than that found in young males. No significant difference was observed between T-cell ADCC among young and aging females. T cell-mediated NK was comparable among young and aging males and young and aging females. Non-T cell-mediated NK as well as ADCC activity was significantly (P<0.025 or <0.05) greater in aging males compared to that in young males. Both non-T-cell NK and ADCC were comparable among young and aging females. This study demonstrates an increase in NK and ADCC activity in aging subjects that is primarily shared by males and not by females. No correlation was observed between the proportion of T cells and T-cell NK or ADCC activity.     

Homocysteine accumulates in supernatants of stimulated human peripheral blood mononuclear cells

Moderate hyperhomocysteinaemia is associated with atherosclerosis, thrombosis and also with stroke and dementia. Elevated homocysteine concentrations are related to deficiency of folate and also vitamin-B12, as these two vitamins are essential co-factors in the remethylation of homocysteine to methionine. A causal role of homocysteine in the pathogenesis of vascular disease has been discussed over years. Immune activation appears to be involved strongly in atherogenesis as well as in other diseases found to be associated with moderate hyperhomocysteinaemia. To study a possible influence of immune stimulation on homocysteine metabolism, in vitro experiments were performed using peripheral blood mononuclear cells upon stimulation with mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen. In stimulated cells a dose-dependent increase of homocysteine concentrations was found. When cells were kept in medium supplemented with methionine, homocysteine concentrations increased further, while supplementation with folate had only a slight effect. We conclude that in supernatants of stimulated peripheral blood mononuclear cells homocysteine is accumulating. T cell activation could be involved in the development of moderate hyperhomocysteinaemia.     

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100 % homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab ′ )₂ γ specifically bound to psTf. Neutralization by psTf abolished that binding. Although α1, 3-linked galactose residues (Galα1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Galα1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.     

Cryopreservation of human mononuclear cells for quality control in clinical immunology. I. Correlations in recovery of K- and NK-cell functions,surface markers,and morphology

Cryopreserved human peripheral blood mononuclear cells (PBMC) were tested for natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) and for high-affinity (29°C) and total (4°C) rosette formation with sheep erythrocytes. PBMC produced variable NK activity following freezing and thawing, but consistently reacted well in ADCC. A significant correlation was found between low NK activity and a decreased percentage of low-affinity rosette-forming cells. On the contrary, the number of large granular lymphocytes (LGL), among which NK cells are restricted, and the reactivity with the monoclonal antibody OKT10, which recognizes the majority of LGL in the peripheral blood, were not significantly altered by cryopreservation. Cryopreserved cells proved to be excellent controls for determining the day-to-day variability of the NK assay and for selecting optimum conditions for this test in the clinical immunology laboratory.     

A rapid method for isolation of human mononuclear cells free of significant platelet contamination

In order to obtain mononuclear cells from peripheral human blood for the study of cell surface receptors, it was necessary to effectively eliminate contaminating platelets. The usual Hypaque-Ficoll isolation procedures were found to produce mononuclear cells contaminated with 10–1000 platelets per mononuclear cell (by phase microscopy). Multiple slow speed centrifugations reduced the contamination to 5–10 platelets per mononuclear cell. However, centrifugation of EDTA-anticoagulated blood through Hypaque (D₂₀²⁰ 1.060) at 400 × g for 5 min at 22°C followed by the usual Hypaque-Ficoll gradient reduced platelet contamination to < 1 platelet per 2 mononuclear cells. Thus, a rapid and simple gradient procedure is capable of significantly reducing platelet contamination of mononuclear cell preparations and should facilitate the analysis of mononuclear cell receptors and functions.     

A simple allogeneic test system for the study of antibody-dependent cellular cytotoxicity (ADCC) in man.

A simple allogeneic test system for the study of ADCC activity of human mononuclear peripheral blood leucocytes (MPBL) is presented. The test uses human Rh-positive erythrocytes as target cells coated with specific human IgG antibody. The effector function of human MPBL is demonstrated after depletion of most of the monocytes and B lymphocytes on nylon wool column. The test system seems to be useful in the clinical study of ADCC activity in man.     

Antibody-dependent cellular cytotoxicity and neutralizing activity in sera of HIV-1-infected mothers and their children.

The prognostic and protective role of antibodies mediating cellular cytotoxicity (ADCC) and neutralization was evaluated in sera of HIV-1-infected mothers and their consecutively followed children. The presence and titres of ADCC mediating and/or neutralizing antibodies in maternal sera did not predict HIV-1 infection in their respective children. No significant difference in the sera from the children was seen when comparing the presence of neutralizing antibodies between the uninfected and infected children. Stratification of the infected group according to clinical status revealed differences. Only one of 24 AIDS patients had a high neutralizing titre against IIIB. Four patients had a very low titre and the remaining had no detectable neutralizing antibodies at all. In contrast, 10/17 infected non-AIDS children had neutralizing antibodies. Similarly, no significant difference was seen when comparing the presence of ADCC-mediating antibodies between the uninfected and the infected group of children. However, a significantly higher frequency of ADCC was seen in the seropositive non-AIDS children compared with the AIDS children. This study clearly shows that the presence of antibodies mediating ADCC and neutralization in infected children, 0-2 years old, is associated with a better clinical status and delayed disease progression.