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.
It is not yet clear whether nuclear phospholipase C (PLC) signaling is implicated in the response of primary cells to cytokines or not and, in particular, whether lymphocytes utilize the nuclear phosphoinositides breakdown as a step in the signal transduction generated by cytokine receptor triggering. Therefore, we have studied the repertoire and functional regulation of nuclear PLC, the key enzyme of phosphoinositides turnover, by interleukin‐2 (IL‐2) in primary human natural killer (NK) cells and its effects on the IL‐2‐driven NK cell proliferation. We found that 1) IL‐2 stimulates nuclear PLC activity within 60 min of treatment; 2) NK cells express only the β family of PLCs in the nucleus, and the isoform of this enzyme regulated by IL‐2 is PLCβ1b; 3) IL‐2 induces nuclear translocation of extracellular signal‐related kinase (ERK)‐2, or p42, and to a lesser extent, of ERK‐1, or p44, of mitogen‐activated protein kinase (MAPK), whose specific inhibition prevents the IL‐2‐driven nuclear PLCβ1 activation; 4) PLCβ1b is serine‐phosphorylated after IL‐2, and the phosphorylation is abolished after MAPK inhibition; and 5) inhibition of nuclear PLC activation leads to the inhibition of the IL‐2‐induced proliferation of NK cells. Altogether, our data indicate that nuclear PLC signaling is a downstream target of the ERK/MAPK pathway stimulated by IL‐2 receptor triggering in human primary NK cells, and that the nuclear phosphoinositides turnover is a key step in proliferative response of NK cells to IL‐2.
Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKC and PKC-a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKC and PKC activities; 2) whereas PKC activity is induced by cytokine stimulation, PKC activity is inhibited; and 3) both the induction of PKC and the inhibition of PKC functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation. Anat Rec 266:87-92, 2002.
SUMMARYA cloning technique was used lo estimate (he frequency of proliferating Tcell precursors, the growth capacity of clone-forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8^ clones. In contrast, CD4 * and CDl 6 *" clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4 ' clones from both groups failed to show any cytolylic activity, while CDS ' clones exerted cytolysis against K562 and in antibody-dependent cell-mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CDIG"^ clones from the elderly showed a decreased activity at some effcctor-to-target cell ratios. We conclude that the impaired functional activity (T or NK-dcpendent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.
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