Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

The effect of beta-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity

The ability of the central nervous system to modulate immune responsiveness has received increasing attention. A potential mechanism that would allow the central nervous system to alter the immune system is the release of neuroendocrine and neurotransmitter polypeptides into the peripheral circulation with subsequent modulation of immunocyte function. In this report, we demonstrate that the neuropeptide, beta-[D-ALA2]-endorphin augments natural cytotoxicity but does not effect antibody-dependent cellular cytotoxicity. The observations are discussed in relation to the mechanisms for natural cytotoxicity and antibody dependent cellular cytotoxicity.     

Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity.

Recent studies indicate that the herpes simplex virus type 1 (HSV-1) Fc receptor (FcR) can bind antiviral immunoglobulin G by participating in antibody bipolar bridging. This occurs when the Fab domain of an immunoglobulin G molecule binds to its antigenic target and the Fc domain binds to the HSV-1 FcR. In experiments comparing cells infected with wild-type HSV-1 (NS) and cells infected with an FcR-deficient mutant (ENS), we demonstrate that participation of the HSV-1 FcR in antibody bipolar bridging reduces the effectiveness of antibody-dependent cellular cytotoxicity.     

Human lymphocyte dependent antibody mediated cytotoxicity: Adherence of lymphocytes to antibody treated target cells

An in vitro human antibody dependent cell mediated cytotoxicity system was used to study the adherence of nonsensitized attacking lymphocytes from peripheral blood to antibody coated melanoma target cells. Specific adherence of attacking cells was documented by labeling the lymphocytes with ⁵¹Cr. The degree of specific adherence was proportional to antibody concentration and incubation time and could be detected before the lysis of target cells. Adsorption of attacking lymphocytes on immune serum treated target cells depleted B cells, enriched T cells, and removed most cytotoxic activity of nonadherent lymphocytes in this system. These results were not found when attacking lymphocytes were adsorbed on normal serum treated target cells.     

Experiments on the induction of antibody dependent macrophage-mediated cellular cytotoxicity in mixed brain cell cultures.

These examinations were based on the discussion whether in demyelinating diseases anti-lipid antibody associated with brain macrophages could have a cytotoxic effect on oligodendrocytes. We used mixed brain cell cultures of newborn rats where, among others, both oligodendrocytes and vacuolated macrophage-like cells were found. On these macrophage-like cells, the presence of Fc-receptors was proven. Besides Fc-receptor-dependent phagocytosis, these cells showed an Fc-receptor-independent type of phagocytosis. The Fc-receptor-bearing cells moved within the culture and adhered to glass fibers. In the cytoplasm of these cells, unspecific esterase, acid phosphatase and peroxidase could be visualized. The vacuolated cells showed strong autofluorescence, expressed a surface marker found on all types of rat leukocytes and were marked by Griffonia simplicifolia lectin. These results definitely characterized the vacuolised cells as macrophages. We saw globular and pleomorphic macrophages. After incubation of anti-GC serum in a highly diluted solution, significantly more macrophages bound to oligodendrocytes than in the controls. In these cases, we found target cell lysis. It could be shown in vitro that anti-GC serum together with macrophages of neonatal brains can induce a cytotoxic effect on oligodendrocytes.     

Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis

The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction.     

Cytokine effects and role of adhesive proteins and Fc receptors in human macrophage-mediated antibody dependent cellular cytotoxicity.

Mononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage-mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17-1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H-thymidine release assay occurred after culturing the monocytes for 5-7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte-macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti-FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophage-mediated antitumor ADCC.     

Lysis of uninfected HIV-1 gp120-coated peripheral blood-derived T lymphocytes by monocyte-mediated antibody-dependent cellular cytotoxicity

Abstract Previous reports from our laboratory have demonstrated that peripheral blood monocytes (PBM) from HIV-1 infected individuals are de novo activated and are cytotoxic in vitro. Significant monocyte-antibody-dependent cellular cytotoxicity (ADCC) was obtained against HIV-1 inactivated CD4⁺ CEM target cells coated with HIV-1 in the presence of autologous seropositive serum. Based on these findings, we hypothesized that in HIV-seropositive individuals the monocytes may play an important role in vivo in the autodestruction of non-infected CD4⁺ T lymphocytes. The present study was designed to test this hypothesis. Monocytes from normal donors activated with M-CSF lysed CD4+ T cells (CEM) coated with gp120 sensitized by plasma from asymptomatic HIV-1+ individuals in a 8 h ⁵¹Cr release assay. ADCC cytotoxic activity varied from one individual to another and was a function of the dilution of the individual seropositive plasma used. We then used circulating CD3+ T lymphocytes as targets for ADCC following treatment with actinomycin D to facilitate the release of radioactive ⁵¹Cr. Like CEM, ADCC was obtained with CD3⁺ T cells coated with gp120 in the presence of HIV seropositive plasma and monocytes. Lysis was specific as T cells that were not coated with gp120 were not destroyed. These findings demonstrate that activated peripheral blood derived monocytes can destroy non-infected gp120-coated circulating T lymphocytes by an ADCC-mediated mechanism. Thus, these findings suggest that ADCC may be one mechanism operating in vivo for the destruction of non-infected CD4⁺ T lymphocytes.     

The inhibitory effect of forskolin on antibody-dependent cell-mediated cytotoxicity using Chang liver cells as target cells

The effect of forskolin, a unique adenylate cyclase activator, on antibody-dependent cell-mediated cytotoxicity (ADCC) was examined. ADCC was assayed using Chang liver cells as the target cells, immuned rabbit serum as the antibody and healthy human peripheral blood mononuclear cells (PBMNC) as the effector cells. Forskolin at concentrations ranging from 1 to 20 microM significantly inhibited ADCC in a dose-dependent manner. By the addition of forskolin, cyclic AMP levels did not change in Chang liver cells but increased in PBMNC. Therefore, it appears that forskolin exerted an inhibitory effect on ADCC by increasing the intracellular cyclic AMP levels in PBMNC, the effector cells.     

The role of antibody in cellular cytotoxicity to culture human colon carcinoma cells.

The effect of autologous serum in cellular cytotoxicity was compared with the level of antibody towards the same target cells. In cancer patients, a highly significant correlation (P < 0.001) argues that enhancement of cytotoxicity by autologous serum is due to high antibody levels, and conversely an inhibition of cytotoxicity is associated with low antibody. Moreover, some antibody was detected in virtually all cancer patient and normal donor sera. It is suggested that antibody may be responsible for cytotoxicity conventionally ascribed to “natural killer” cells.     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Isolation of virally-infected insect cells from a population containing infected and uninfected cells

A method was developed that can be used to isolate virally-infected insect cells from a mixed population containing infected and uninfected cells. Specifically, Spodoptera frugiperda Sf-9 cells infected with the Autographa californica multiple nuclear polyhedrosis virus were treated with a primary antibody specific for the gp64 protein present on the surface of virally-infected cells and a secondary antibody labeled with a fluorochrome. The resulting labeled cells were isolated by using fluorescence-activated cell sorting.     

Bone marrow determination of complement dependent and complement independent cellular cytotoxicity

Adoptive transfer of both bone marrow and thymus cells is needed in lethally irradiated syngeneic mice to elicit CICC and CDCC responses against SRBC as assayed by the ⁵¹Cr-release cytotoxic test. The contribution of thymocytes to both CDCC and CICC was assessed by limiting dilution assays in BDF₁ and CDF₁ mice. Irradiated mice were reconstituted with a large number of bone marrow cells and graded limiting numbers of thymocytes, and were then immunized with SRBC. The limiting dilution plots conformed to the Poisson Model for both types of responses and both strains of mice. The numbers of thymocytes required for about 63% of the recipient spleens to be positive (i.e., the inoculum sizes containing one detectable antigen reactive unit) were similar for both CDCC and CICC ($\text{1}\text{1.8}\text{× 10}{}^7$ thymocytes) in both strains. Chi-square tests, at the 0.01 level of significance, were incompatible with the hypothesis that CICC and CDCC are independent of each other. Limiting dilutions of bone marrow cells with larger numbers of thymus cells, using CDF₁ mice, showed a non-Poisson curve for both CICC and CDCC. Chi-square values were compatible with the hypothesis of independent assortment of responses, as if the potential of the majority of precursors were restricted to either CICC or CDCC, but not to both. In contrast, BDF₁ mice showed a Poisson curve for CDCC and a non-Poisson curve for CICC. Chi-square values were also compatible with independent assortment of responses.     

Cell surface distribution of Fc receptors II and III on living human neutrophils before and during antibody dependent cellular cytotoxicity

Microscopic techniques have been employed to study the cell surface distributions of the immunoglobulin Fc receptors (FcR) II and III on living human neutrophils. Fluorescein-or rhodamine-conjugated monoclonal IgG or Fab fragments directed against FcRII (CDw32) and FcRIII (CD16) were employed to label receptors. FcRII and III were found to be uniformly distributed at neutrophil surfaces during resting conditions. During neutrophil polarization and migration FcRII but not FcRIII preferentially accumulated at the uropod. Sheep erythrocytes (SRBCs) were opsonized with IgG and then incubated with neutrophils. When neutrophils were labeled prior to target addition, FcRII but not FcRIII were found to cluster at the target-effector interface. Little or no clustering of FcRs was observed if labeling was performed after target binding. SRBC oxidation was observed using Soret band illumination during transmitted light microscopy. Time-lapse studies of FcRII distribution and target oxidation were performed. FcRII formed clusters at target effector interfaces prior to target oxidation. Three lines of evidence suggest that clustering is not a general plasma membrane response. Firstly, FcRIII do not cluster lannic acid-modified erythrocytes avidly bound to neutrophils but did not trigger clustering of FcRII. Furthermore, irrelevant neutrophil membrane labels were unaffected by the presence of IgG-opsonized erythrocytes. We suggest that FcRII clustering is one important component leading to the oxidative destruction of target cells.     

Expression of liver and placental alkaline phosphatases in Chang liver cells

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase, is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immumochemical assay, ³²P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.     

The role of antibody dependent cytotoxicity in recovery from herpesvirus infections.

In order to implicate antibody dependent cytotoxicity (ADC)¹ as playing a possible role in recovery from herpesvirus infections, target cells were infected with virus and the time of susceptibility to ADC was determined. Additional virus-host cell events were measured in parallel including the time of extracellular and intracellular transmission of infectious virus. Cells were susceptible to ADC at about the same time as intracellular transmission of virus occurred. The presence of effector cells and antibody in infected cultures markedly diminished viral transmission, indicating that ADC may play an important role in curtailing infections.