Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional 51 Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.Cytotoxicity assays provide an in vitro evaluation of the lytic activity of natural killer (NK) and T cells against tumors or transformed target cells (3,16,25). In in vitro experimental conditions, lytic activity is evaluated using isotopes or dyes either released from dead cells or retained by living ones. The 51 chromium ( 51 Cr) release assay is the most widely used method although it has several significant drawbacks. The major disadvantage results from the use of a radioactive compound, with the related problems of handling and disposal due to the short half-life of the isotope.To overcome these problems, several nonradioactive methods have been developed, but none have yet found a broad acceptance, probably due to a lack of comparability of their results with the results obtained using the 51 Cr release assay, which still remains the most popular. Some alternative methods consist of measuring endogenous or transfected reporter enzymes released in the supernatant by dead targets (1,11,19,24). Other assays using nonradioactive compounds (dimethylthiazol-diphenyl bromide tetrazolium bromide, methylumbelliferyl heptanoate, Alamar blue) evaluate the variations in metabolic activity, which is directly proportional to the number of viable cells (6, 18). Alternative methods using fluorochromes (e.g., europium, D275, rhodamine-123, carboxyfluorescein diacetate, bis-carboxyethyl-carboxyfluorescein) (7,9,12,14,23,26,28) measure the amount of dye released from or remaining in prelabeled target cells (2, 8, 13). The major drawbacks for these methods appear to be the high spontaneous release of the fluorescent dye, the slow specific release of the fluorescein dye, and the low intensity of the fluorescence signal, all of which decrease the overall sensitivity of the test.Among fluorescent dyes, the use of calcein-acetoxymethyl (calcein-AM) in cytotoxicity assays has already been described (13,21,22). This dye has good retention in targets and low pH sensitivity, and there is no stain transfer among cells (21). Acetoxymethyl ester of calcein is a lipid-soluble diester fluorogenic esterase substrate that passively crosses the cell membrane and that is frequently used to stain viable cells. Inside the cells it is converted by intracellular ...

The progressive increase in the number of peripheral NK cells found in the elderly does not correlate with a corresponding increase in lytic activity. On the contrary, a decreased function of circulating NK cells purified from old subjects was observed on a per cell basis. Most of the studies on NK cells have focused on late events such as lytic activity. In view of this, little is currently known about the modification of the early signalling pathways of NK cells in elderly people. This study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of these cells in the elderly. NK cells were negatively purified by immunomagnetic depletion from the peripheral blood of selected old and young healthy subjects. Hydrolysis of inositol phospholipids was measured following incubation with K562 target cells and/or CD16 mAb for different times. Our data show that there is a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover is preserved following Fc triggering (antibody-dependent cell-mediated cytotoxicity). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs and may aid the development of experimental and therapeutic strategies to counteract declines in cell mediated immune functions associated to advancing age.

Increasing evidence has demonstrated that the immune system closely interacts with other physiological systems, whose communications are mediated by circulating cytokines and hormones. The aim of our study was to test whether the number and cytolytic activity of NK cells in a group of relatively healthy Italian nonagenarians and centenarians were affected by the modifications of endocrine, metabolic and functional parameters that occur during ageing. Because of the extreme age of the study population, a cross-sectional analysis was performed. This study revealed that the group of oldest subjects with the highest number of NK cells and the best preserved cytolytic function also presented a preserved metabolism of thyroid hormones and vitamin D and integrity of muscle mass. In fact, the NK cell number and/or cytolytic activity of healthy subjects > 90 years old was positively associated with serum levels of vitamin D, while T3, FT4, i-PTH hormones and lean body mass were associated only with NK cell number. In conclusion, our results stress the paramount importance of nutritional evaluation in the clinical assessment of elderly people.

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In this study we investigated whether purified NK cells, derived from a group of nonagenarian healthy subjects, were able to produce the chemokines MIP‐1α, RANTES and IL‐8, and also characterized the effect of IL‐12 or IL‐2 immunomodulatory cytokines (that are among the most effective inducers of NK lytic activity and soluble factor secretion) on the induction, in vitro, of these chemokines and on the modulation of the corresponding receptors. This study provides evidence that human NK cells from healthy subjects over 90 years old retain the ability to synthesize MIP‐1α, Rantes and IL‐8 chemotactic cytokines, that NK cells isolated from these subjects can be activated to significantly up‐regulate the production of these chemokines in response to stimulation by IL‐12 or IL‐2 cytokines (even though production remains lower than that observed in young subjects), and that NK cells express the corresponding chemokine receptors.