Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the ⁵¹Cr release assay. However, measurement of target cell lysis by ⁵¹Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the ⁵¹Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.     

Killing of yeast, germ-tube and mycelial forms of Candida albicans by murine effectors as measured by a radiolabel release microassay

Candida albicans undergoes yeast to mycelial conversion under both in vivo and in vitro conditions but the relative pathogenicity of the two forms of growth is still unknown. By adapting a recently developed 51Cr radiolabel release assay, we have quantified the killing ability of different murine effector cell populations for the hyphal form of C. albicans. Up to 50% of specific 51Cr release from the mycelial form could be detected after incubation for only 1 h, with no requirement for opsonization, provided that appropriate effector: target cell ratios were used. The specific 51Cr release correlated well with viability, as assessed by dye exclusion tests, and with pathogenicity potential in cyclophosphamide-immunodepressed mice. Comparison of the activity of different murine effectors against yeast and hyphal forms showed that hyphal forms were killed by murine effectors to a similar, if not greater, extent than yeast forms. In particular, thioglycollate-induced murine polymorphonuclear neutrophils were able to kill hyphal cells extracellularly and without an opsonic requirement.     

The JAM-assay: optimized conditions to determine death-receptor-mediated apoptosis

Apoptosis induced by interaction of members of the TNF-/TNF-receptor superfamily has been considered as a major mechanism of cell-mediated cytotoxicity. For functional analysis, the 51Cr release assay has been widely used, which requires loss of membrane integrity in the apoptotic target cell. However, loss of membrane integrity is a late event during apoptosis and therefore only late apoptotic cells will be detected by this method. In contrast, the JAM-assay first described by Polly Matzinger has been demonstrated to be more sensitive than the 51Cr release assay, since this method is dependent on DNA-fragmentation which precedes loss of membrane integrity in most apoptotic cells. The JAM-assay is easier to perform, less expansive, and safer than the current standard (51)Cr release assay. Therefore, this article will focus on optimized conditions of the JAM-assay to detect and quantitate Fas (CD95/Apo-1)-induced apoptosis as an example of death-receptor-mediated cytotoxicity.     

The effect of bile salts on human vascular endothelial cells

The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective.     

Assessment of immunological parameters during tumour development in a murine model

Natural Killer activity assessed by 51Cr release assay from K-562 cells showed detectable activity from 5th day after tumour transplantation, reaching a peak on 12th day and thereafter showing a gradual decline in the activity. Antibody dependent cell mediated cytotoxicity estimated by 51Cr labelled sheep red blood cells anti SRBC system demonstrated a peak activity on 5th day. Cytotoxic T lymphocyte activity detected by 51Cr release of Dalton's lymphoma ascites target cells showed a peak on 10th day. Antibody complement mediated cytotoxicity revealed a similar pattern as natural killer cell activity.     

Spontaneous51Cr release by isolated rat hepatocytes: An indicator of membrane damage

Summary Radiochromium uptake and release by isolated rat hepatocytes in suspension was monitored under continuous-labeling conditions. Cell protein remained unchanged during the absorption phase, whereas the release of⁵¹Cr correlated well with the loss of cell viability and release of cytoplasmic protein. The results suggest that under equilibrium conditions,⁵¹Cr release represents an efflux of label from damaged or dying preparations and not an elution of radioisotope from intact cells.     

In vivo protection against syngeneic Gross virus-induced lymphoma in rats: comparison with in vitro studies of cell-mediated immunity.

Spleen cells at various times after inoculation of W/Fu rats with a syngeneic Gross virus-induced lymphoma, (C58NT)D, were tested for their in vivo activity in adoptive transfer experiments and for their in vitro reactivity in a 4-hr 51Cr release cytotoxicity assay and in a mixed lymphocyte-tumor cell interaction assay. In adoptive transfer, the best protection against tumor growth was observed with immune spleen cells taken at 30 days after tumor cell inoculation (the peak of reactivity in the mixed lymphocyte-tumor cell interaction assay) whereas cells taken at 10 days (the peak reactivity in the 51Cr release cytotoxicity assay) gave only partial protection. The protection detected in the adoptive transfer experiments was specific for (C58NT)D associated antigens, and this correlated well with the specificity observed in the in vitro cell-mediated immunity assays. T cells, but not complement receptor-bearing cells or macrophages, were essential for the protection against tumor growth in vivo, and also for the in vitro reactivity in the 51Cr release cytotoxicity and the mixed lymphocyte-tumor cell interaction assays.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Non-lytic, non-ionic detergent extraction of plasma membrane constituents from normal and transformed fibroblasts.

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cell employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, 51Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodination followed by detergent extraction permits demonstration of a 100,000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.     

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cells employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, ⁵¹Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodina- tion followed by detergent extraction permits demonstration of a 100 000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.     

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ.单克隆抗体介导细胞毒效应的研究

建立了单克隆抗体(McAb)介导细胞毒作用(ADCC)~(51)Cr释放试验的测定力法。确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同:McAb 1A12、2A8和1G8无ADCC活性;而1D10和2C5两株McAb作1:10稀释时,~(51)Cr释放率分别为27.09％和25.07％,稀释至1:100或1:1000时仍有ADCC活性。结果提示,不同的McAb抗原决定族诱导产生的抗体,在介导ADCC免疫保护作用上有差异,并为McAh治疗临床单纯疱疹病毒感染的可能性提供了实验资料。     

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations I. Comparative effects of four classes of polyene macrolides on mammalian cells

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by ⁵¹Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone®) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.     

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis, as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range of 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.     

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes (PMN) using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.     

The efficiency and linearity of the radiochromium release assay for cell-mediated cytotoxicity.

The release of radiochromium from EL-4 cells lysed by anti EL-4 peritoneal lymphocytes is a first order decay process with respect to time. The rate of ⁵¹Cr release from damaged cells was independent of the effector: target ratio but was slower from pelleted cells than cells in suspension. We conclude that under normal assay conditions the fraction of chromium released is nearly proportional to the number of cells damaged but find that the proportionality constant is influenced by assay conditions. There is a lag time of 23 ± 5 min before ⁵¹Cr is rapidly released from the cells.     

Monoclonal antibodies to reovirus sigma 1 and mu 1 proteins inhibit chromium release from mouse L cells.

Reovirus intermediate subviral particles (ISVPs) but not intact virions or cores have been shown to possess the capacity to permeabilize mouse L cells as determined by a 51Cr release assay. We used monoclonal antibodies (MAbs) directed against proteins exposed on the ISVP surface (sigma 1, mu 1, and lambda 2) to probe the role(s) of these proteins in membrane interaction and penetration. One sigma 1-specific MAb (MAb-G5) and two mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibited reovirus-induced 51Cr release when added pre- or post-ISVP attachment to L cells. MAb-G5 inhibits 51Cr release by interfering with ISVP attachment (via sigma 1) to L-cell receptor sites. The mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibit 51Cr release by interfering with an undefined post-L-cell-attachment event that involves bivalent binding of the mu 1-specific MAbs to an epitope located in a central region of the mu 1 protein.     

Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells

Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr 51Cr release or 111In release assay. Both purified monocytes (greater than 99% monocytes) and natural killer (NK)-enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NK-depleted lymphocytes did not lyse the melanoma targets. Culture supernatants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 51Cr release assay. Treatment of the monocytes with anti-Leu 1 1b and anti-Leu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leu 1 1b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-gamma (rIFN-gamma), but not lipopolysaccharide, into the 51Cr release assay or pretreatment of monocytes with rIFN-gamma significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFN-gamma from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets.     

Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes

Using ⁵¹Cr-labelled P-815 mastocytoma cells as target cells and CS7BL/6 spleen cells sensitized against DBA/2 antigens as effector cells, it is shown that the variation in the observed specific ⁵¹Cr release over a broad range of experimental conditions can be explained on the basis of a simple physical model of the interaction process. The model assumes that a target cell can be destroyed only after contact with an effector cell, contact takes place on a random basis, one contact is sufficient, and that one effector cell can kill several targets with unchanged efficiency. The fraction of target cells destroyed (f) depends only on the incubation time (t), the number of effector cells (n) and a constant interaction probability (δ). Thus f = 1 ? e^?nδt. However, the experimental measurement, the fraction of ⁵¹Cr specifically released into the supernatant during the assay, may not be the same as the fraction of target cells destroyed because it takes considerable time for the releasable ⁵¹Cr to be released from a damaged target cell. This can be overcome experimentally by following the standard 37 °C incubation with a further incubation at 45 °C during which there are no new lytic events but all previously damaged target cells release the remainder of their releasable ⁵¹Cr. The model enables one to obtain accurate measurements of relative effector cell frequency over a broad range of experimental conditions.