Signal transduction of interleukin 2 in human natural killer cells: involvement of the p56lck tyrosine kinase.

Despite numerous reports, the role of the protein tyrosine kinase p56lck in IL-2 signal transduction has remained controversial. We show here, using IL-2-dependent human natural killer cell lines, that p56lck is regulated by IL-2 in two different ways: (1) IL-2 induces a rapid increase of p56lck kinase activity as assessed in vitro; and (2) following IL-2 stimulation, p56lck undergoes phosphorylation on serine residues that is reflected by a modification of its electrophoretic mobility in SDS-PAGE. Furthermore, dose response experiments, and blocking studies performed with anti-IL-2R alpha antibodies, indicated that binding of IL-2 to the IL-2R beta chain was sufficient to produce these modifications of p56lck. In contrast, activation of the CD2 pathway stimulated the kinase activity of p56lck, but did not induce a significant shift in NK cells, as opposed to T lymphocytes. Western blot analyses, and immunoprecipitations of cell lysates from 32P-preloaded NK cells demonstrated that seven major proteins are tyrosine phosphorylated in response to IL-2. These phosphoproteins, with apparent molecular weights of 190, 150, 120, 110, 85, 65 and 56, which may not all be p56lck substrates, undergo phosphorylation and dephosphorylation with different kinetics. Furthermore, pp120 was identified as rasGAP, by Western blot and immunoprecipitation experiments. rasGAP and some of its co-precipitating molecules become phosphorylated in response to IL-2, presumably by p56lck, which would thus provide a link between IL-2R and downstream events critical for NK cell proliferation and function.

Cyclic AMP upregulates mRNA and surface expression of IL-2R alpha p55(Tac) in normal human NK cell clones.

The effects of cAMP upon cell proliferation, cytotoxic activity, and regulation of IL-2R expression was investigated in normal human, IL-2-dependent natural killer (NK) cell clones. We report here that addition to the cultures of Bt2cAMP, a cell permeant analogue of cAMP, results in inhibition of IL-2-dependent proliferation, as assessed by [3H]Thymidine incorporation, in both NK and T cell clones. In addition, Bt2cAMP was shown to block the cytotoxic activity of NK cell clones at the level of the lytic phase. Contrasting with these inhibitory effects, cAMP induces an upregulation of the membrane expression of the IL-2R alpha chain (p55, Tac) in normal NK cell clones, which correlates with an accumulation of Tac mRNA. This is clearly at variance with T cell clones in which no such effect of cAMP alone is observed. In both cell types however, cAMP appears to synergize with IL-2 to increase IL-2R alpha mRNA expression. In addition, we demonstrate, using a cDNA probe to the IL-2R beta, that expression of this second component of the high affinity IL-2R, does not appear to be co-regulated together with IL-2R alpha in response to cAMP or/and IL-2 in cultured NK cells. Thus the effects of cAMP on human NK cell clones are complex. cAMP is inhibitory of proliferation and cytolytic function, whereas it is stimulatory of IL-2R alpha expression in these cells.

Functional consequences of cAMP accumulation in human natural killer cells. Implications for IL-2 and IL-4 signal transduction.

To approach the mechanisms whereby IL-2 activates human NK cells, we have compared the effects of IL-4 and of Bt2cAMP on this activation. Both agents block completely the proliferation induced by IL-2 on highly purified CD3-negative human NK cells. We also report that the net LAK response of PBL is inhibited by IL-4 and cAMP. However, kinetics analysis showed that IL-4 appears to inhibit an early stage of IL-2-induced activation of NK cells. IL-4 does not affect the cytotoxicity of freshly isolated NK cells against the K562 target and is ineffective on IL-2-preactivated cells. In contrast, cAMP primarily blocks the lytic effector phase, whether cells have been cultured in IL-2 or not, and its effect appears independent of time of addition. These differences between the activity of IL-4 and cAMP suggested that cAMP was not directly involved in IL-4 signal transduction in human NK cells. Consistent with this interpretation, we did not observe any variation in the level of intracellular cAMP concentrations when NK cells were stimulated with IL-4, or when they are stimulated with IL-2 or IL-2 plus IL-4. In addition, we also demonstrate that NK cell cytotoxic activation induced by IL-2 is still demonstrable in the presence of Bt2cAMP under conditions in which NK cell proliferation is blocked. These results clearly indicate that the differentiative effect of IL-2 on NK cells is independent of cell proliferation. Furthermore, the p70-75 IL-2R-initiated signal transduction pathway that leads to increased cytotoxicity appears not to be susceptible to inhibition by cAMP in human NK cells.

Differential effects of glucocorticoids on the proliferation of a murine helper and a cytolytic T cell clone in response to IL-2 and IL-4.

The proliferation of murine T cell clones can be supported by IL-2 or by IL-4. We present here evidence that glucocorticosteroids differentially affect these two pathways of proliferation. Dexamethasone (DEX) and other corticosteroids were observed to induce autocrine proliferation of the D10.G4.1 Th cell clone (D10) in the presence of the anti-clonotypic antibody 3D3. This effect was inhibited by the anti-murine IL-4 antibody 11B11, indicating that it is mediated by IL-4. Furthermore, on this cell line, representative of the Th2 group of helper cells, DEX had little effect on the proliferation induced by exogenous IL-4 but completely inhibited the growth-promoting effects of IL-2. In contrast, the effects of DEX on the proliferation of the cytotoxic IL-2-dependent CTLL-2 cell line are completely opposite. DEX blocked the IL-4-driven proliferation of CTLL-2 cells, while leaving unaffected their response to IL-2. It is also shown in this study that the effects of glucocorticoids in this system are totally antagonized by the high affinity anti-glucocorticosteroid RU 38486, indicating that they are mediated through the described intracellular glucocorticoid receptor. These data suggest that the growth effects of IL-2 and IL-4 may be mediated by distinct pathways that are strikingly different in their sensitivity to glucocorticoids. In addition, the regulation of lymphokine-dependent proliferation and the response to glucocorticoids appeared very different in helper and cytotoxic cells.

B-cell-derived human interleukin 1.

This review addresses the questions of the molecular nature and of the physiological role of interleukin-1 (IL-1)-like activities produced by B lymphocytes. IL-1 was originally described as an exclusive product of activated monocytes/macrophages. The recent cloning of two genes for IL-1 (IL-1 alpha and beta), together with the availability of specific antibodies to these two species of IL-1 have allowed their identification as secretory products of a number of other cell types, including B cells. B cells secrete a variety of other autostimulatory factors and of IL-1-like molecules, the identification of which is still pending. In addition, B cells express receptors for IL-1, which has been shown to enhance proliferation and immunoglobulin synthesis. An important issue is that of whether B-cell-derived IL-1 serves a purpose in the physiology of the immune response. Inasmuch as IL-1 is required for T-cell response, it has been suggested that B-cell-derived IL-1 may contribute to the amplification of the immune response, particularly where B lymphocytes serve as antigen-presenting cells.

B-cell line-derived interleukin 1 is cytotoxic for melanoma cells and promotes the proliferation of an astrocytoma cell line.

We have previously described a unique molecular species of Interleukin 1 spontaneously produced by a human EBV-transformed B-cell line. This IL 1 shares a number of biological activities with monocytic IL 1 although its NH2-terminal amino-acid sequence is different from both IL 1 alpha and beta. In the present study we have investigated the activity of this B-cell IL 1 in two recently reported new assays for interleukin 1. We found that this B-cell IL 1 is able to promote the proliferation of the human astrocytoma cell line U373 in a dose dependent manner. We also describe that B-cell IL 1 is directly cytotoxic for a melanoma cell line, A375, but not for the tumor necrosis factor target cell, the murine transformed fibroblast line L929. These studies should prove useful in analysing structure-function relationships of the various IL 1 species, when the primary sequence of B-cell IL 1 becomes available.

The reactivity of frozen B lymphocytes to B cell mitogens and human B cell growth factor: a study of step 1 and step 2 activators.

Human B lymphocytes, purified from the peripheral blood of several different donors can be pooled, frozen, and stored in liquid nitrogen to provide an easy and reproducible source of cells for mitogenic assays. These B cell preparations did not show any reactivity to T cell mitogens, but responded to Staphylococcus aureus Cowan strain 1 (SAC) and anti-IgM antibodies to the same extent as freshly purified B cells. When stimulated with either anti-IgM antibodies or SAC, these B cells became responsive to B cell growth factor (BCGF), allowing a quantitative measurement of this important lymphokine activity. In addition, we have studied the reactivity of frozen B lymphocytes to various combinations of activators. We have confirmed that phorbol myristate acetate (PMA) was a very potent mitogenic agent for preactivated human B cells and shown that bacterial lipopolysaccharide (LPS), although not mitogenic by itself, could synergize with anti-IgM antibodies to yield increased levels of stimulation. Furthermore experiments using the lysosomotropic agent leucine methyl ester showed that the action of LPS on anti-IgM-stimulated B cells did not require the presence of functional monocytes. Neither PMA nor LPS could induce BCGF responsiveness and thus these two compounds can be considered exclusive step 2 activators for human peripheral blood B cells.

Monocyte-independent stimulation of human B lymphocytes by phorbol myristate acetate.

Phorbol myristate acetate (PMA) induces a low level of proliferation in purified human B lymphocytes when added in nanomolar concentrations to the culture medium. Much higher levels of thymidine incorporation, however, are obtained in the presence of other B cell stimuli such as anti-IgM antibodies or Staphylococcus aureus Cowan Strain 1 (SAC). The peak activity of PMA was observed on day 3 of B cell cultures containing either anti-IgM or SAC. The rigorous depletion of monocytes as well as add-back experiments indicate that the effect of PMA on anti-IgM-stimulated B cells is not mediated by the stimulation of accessory cells. Thus, PMA acts as a very potent mitogenic agent for human B cells under culture conditions that are commonly used to assess B cell growth factor activity.