IL-2 induces STAT4 activation in primary NK cells and NK cell lines, but not in T cells

IL-2 exerts potent but distinct functional effects on two critical cell populations of the immune system, T cells and NK cells. Whereas IL-2 leads to proliferation in both cell types, it enhances cytotoxicity primarily in NK cells. In both T cells and NK cells, IL-2 induces the activation of STAT1, STAT3, and STAT5. Given this similarity in intracellular signaling, the mechanism underlying the distinct response to IL-2 in T cells and NK cells is not clear. In this study, we show that in primary NK cells and NK cell lines, in addition to the activation of STAT1 and STAT5, IL-2 induces tyrosine phosphorylation of STAT4, a STAT previously reported to be activated only in response to IL-12 and IFN-alpha. This activation of STAT4 in response to IL-2 is not due to the autocrine production of IL-12 or IFN-alpha. STAT4 activated in response to IL-2 is able to bind to a STAT-binding DNA sequence, suggesting that in NK cells IL-2 is capable of activating target genes through phosphorylation of STAT4. IL-2 induces the activation of Jak2 uniquely in NK cells, which may underlie the ability of IL-2 to activate STAT4 only in these cells. Although the activation of STAT4 in response to IL-2 occurs in primary resting and activated NK cells, it does not occur in primary resting T cells or mitogen-activated T cells. The unique activation of the STAT4-signaling pathway in NK cells may underlie the distinct functional effect of IL-2 on this cell population.     

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.     

A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells

Activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been implicated in the induction of apoptosis in a variety of systems [1] [2] [3] [4] [5] [6] [7] [8]. BAF3 cells are pre-B cells that undergo apoptosis following IL-3 withdrawal or ceramide treatment [9] [10]. JNK/SAPK in BAF3 cells is stimulated by ceramide and also during cell proliferation in response to IL-3 [11], but its role in the apoptotic response is not clear. We have devised a method of selectively inhibiting JNK/SAPK activity using a dual-specificity threonine/tyrosine phosphatase, M3/6. Expression of this phosphatase in BAF3 cells prevented ceramide stimulation of JNK/SAPK activity but did not affect apoptosis following IL-3 withdrawal or ceramide treatment. IL-3-stimulated proliferation of BAF3 cells expressing the phosphatase was, however, inhibited. Hence JNK/SAPK activation is likely to be involved in the proliferative response of these cells but is not required for apoptosis. Selective ablation by dual-specificity phosphatases should be a general method for determining the functions of specific mitogen-activated kinase pathways.     

Subtype-selective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3

Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.     

Induction of IL-1 beta-converting enzyme-independent apoptosis by IL-2 in human T cell lines

Our previous study demonstrated that IL-2 suppressed growth of human T cell lines, in which the suppression was observed with members among HTLV-I-infected T cell lines independent of IL-2 for growth. In this study, we examined the molecular mechanism of IL-2-induced growth suppression with two HTLV-I-infected T cell lines; TL-OmI expressing endogenously three subunits, i.e. alpha, beta and gamma chains, of the IL-2 receptor, and an MT-1 transfectant expressing the endogenous alpha and gamma chains and exogenous beta chain. Our analysis revealed that IL-2 induced apoptosis in both T cell lines. Experiments with inhibitors for the proteases responsible for apoptosis signals showed that caspase 1 (IL-1 beta-converting enzyme) was not involved in apoptosis induced by IL-2. Other MT-1 sublines introduced with mutant beta chains demonstrated that IL-2-induced apoptosis required signals from both the serine-rich (S) region and acidic (A) region of the IL-2 receptor beta chain, which are essential but not critical for IL-2-mediated cell growth respectively. Collectively, IL-2 functions not only on growth promotion and prevention of apoptosis but also on induction of apoptosis, which may be implicated in physiological regulation of immune reactions by controlling growth and activation of T cells.     

Regulation of resting and IL-2-activated human cytotoxic lymphocytes by exogenous nucleotides: role of IL-2 and ecto-ATPases

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP, GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that the inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity of NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor, 5'-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

bcl-2 inhibits wild-type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax

We have earlier shown that wild-type (wt) p53 expressed from a temperature-sensitive construct (ts p53) triggers apoptosis in the v-myc retrovirus-induced, p53-negative T-cell lymphoma line J3D (Y. Wang et al., Cell Growth & Differ., 4: 467-473, 1993). We also found that constitutive bcl-2 expression inhibits wt p53-triggered apoptosis in these cells (Y. Wang et al., Oncogene, 8: 3427-3431, 1993). Here we demonstrate that more than 90% of the ts p53-transfected J3D cells were arrested in G1 at 18 h after induction of wt p53 expression by temperature shift to 32 degrees C. At this time, at least 80% of the cells remained viable. After 30 h at 32 degrees C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1, indicating that p53-induced apoptosis occurred following G1 arrest. The G1 cell cycle arrest at 18 h after temperature shift to 32 degrees C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37 degrees C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bcl-2 transfected cells. The kinetics of G1 cell cycle arrest at 32 degrees C was similar in both the ts p53 and the ts p53/bcl-2 double transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-2 and IL-7 induce heterodimerization of STAT5 isoforms in human peripheral blood T lymphoblasts

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface of in vitro activated CD4+ T cells. Approximately 90% of in vitro activated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% of in vitro activated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of the in vitro activated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.     

The rodent non-genotoxic hepatocarcinogen nafenopin suppresses apoptosis preferentially in non-cycling hepatocytes but also elevates CDK4, a cell cycle progression factor

Small-cell carcinoma of the lung (SCCL) is a neuroendocrine tumor characterized by having the capacity to produce and secrete a number of small neuropeptides. These peptides serve the tumor as autocrine growth factors. SCCL is known to undergo a process of dedifferentiation to a variant (drug-resistant) form, and this process is associated with loss of marker enzymes such as neuron-specific enolase (NSE) and dopa decarboxylase (DDC). The current study was designed to discover if variant SCCL, represented by cell line NCI H82, retains some capacity to generate active neuropeptides (like vasopressin) from their precursors by continuing to express the three key classes of enzymes necessary for such conversions, namely prohormone convertases (PCs), carboxypeptidases (CPs), and peptidylglycine a-amidating monooxygenase (PAM). RT-PCR for mRNAs representing PC1, PC2, CPE, and PAM was performed on total RNA extracted from NCI H82. The primers selected for PCR and partial sequencing were synthetic 20, 21, 22, and 24 oligomers designed to yield products of 533, 880, 405, and 560 base pairs (bp) for PC1, PC2, CPE, and PAM, respectively. For the conditions used, we were able to demonstrate products for all four enzymes. Each of the four products generated were of the expected size. Cloning and sequencing of these products revealed that each had a structure identical to that published for the human form of the respective enzyme. Western analysis with antibodies against PC1, PC2, CPE, and PAM, provided evidence that mRNAs for the four enzymes are translated into proteins that could represent functional forms. Our findings therefore demonstrate that key enzymes involved in the generation of active neuropeptides, unlike the marker enzymes NSE and DDC, continue to be expressed by variant SCCL.     

Control by nutrients of growth and cell cycle progression in budding yeast, analyzed by double-tag flow cytometry

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual Saccharomyces cerevisiae cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing S. cerevisiae populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.     

Control of cell cycle progression by fibronectin matrix architecture

Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNDeltaIII1-7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNDeltaIII1-7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125(FAK). The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation.     

OCI-5/rat glypican-3 binds to fibroblast growth factor-2 but not to insulin-like growth factor-2

OCI-5 encodes the rat homologue of glypican-3, a membrane-bound heparan sulfate proteoglycan that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 and glypican-3 are 95% identical. It has been recently suggested that glypican-3 interacts with insulin-like growth factor-2 (IGF-2) and that this interaction regulates IGF-2 activity. We report here that we have transfected OCI-5 into two different cell lines, and we have not been able to detect an interaction between the OCI-5 proteoglycan produced by the transfected cells and IGF-2. On the other hand, we have found that OCI-5 interacts with FGF-2, as has already been shown for glypican-1. This interaction is mediated by the heparan sulfate chains of OCI-5 because it can be inhibited by heparin or by heparitinase.     

Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.     

Regulation of neutrophil-derived IL-8: the role of prostaglandin E2, dexamethasone, and IL-4

Historically, the neutrophil has been perceived as a terminally differentiated leukocyte with limited ability to produce de novo proteins. Furthermore, in the context of acute inflammation the activated neutrophil has been appreciated only for its ability to release various proteases, reactive oxygen, and arachidonic acid metabolites. Recently, the neutrophil has been shown to have the capacity to produce a number of cytokines that may be instrumental in orchestrating the progression of acute inflammation to a more chronic and specific immune response. These cytokines include IFN-alpha, M-CSF, G-CSF, TNF, IL-1, and IL-6. Our laboratory and others have shown that neutrophils produce IL-8 in response to LPS or a phagocytic challenge. Although these studies have shown the induction of IL-8 from polymorphonuclear neutrophils (PMN), relatively little is known regarding the regulation of PMN-derived IL-8. Because PMN and monocytes share the same stem cell, and monocyte-derived IL-8 is regulated by prostaglandin E2 (PGE2), glucocorticoids (dexamethasone; DEX) and the T-Lymphocyte-derived IL-4, we postulated that PMN-derived IL-8 production may be regulated in a similar manner. To test this hypothesis, PMN were isolated (> 99% pure) from peripheral blood and cultured in media with 5% FCS in the presence or absence of LPS (10 ng/ml; a concentration of LPS that induced the half-maximal production of PMN-derived IL-8) and in the presence or absence of DEX (10(-6) M to 10(-10) M), PGE2 (10(-6) M to 10(-10) M), or IL-4 (100 ng/ml to 100 pg/ml). PMN-derived IL-8 was measured using a specific sandwich ELISA. DEX and IL-4 in the presence of LPS were found to inhibit PMN-derived IL-8 in both a dose- and time-dependent fashion. DEX and IL-4 in concentrations of 10(-6) M and 10 ng/ml resulted in maximal inhibition of LPS-induced PMN-derived IL-8, respectively. Moreover, both DEX and IL-4 administration could be delayed 4 hr post-stimulation with LPS and result in significant suppression of PMN-derived IL-8. Interestingly, in contrast to the regulation of monocyte-derived IL-8 by PGE2, PGE2 treatment of PMN failed to inhibit the generation of LPS-induced IL-8. Northern blot analysis of steady-state IL-8 mRNA demonstrated that both DEX and IL-4 treatment of PMN resulted in a 40 and 52% reduction in LPS-stimulated PMN-derived IL-8 mRNA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase

Chronic myelogenous leukemia (CML) has a progressive course but little is known about the biologic characteristics of disease progression. This study was designed to assess the changes in cell proliferative characteristics, apoptosis, the expression of the bcl-2 and c-myc genes between the time of initial diagnosis and entrance into the blastic phase of the disease. We observed that the rate of cell proliferation decreased and the cell death rate did not significantly change as the disease accelerated. The level of bcl-2 expression was significantly higher in accelerated/blastic phase cells than in the chronic phase cells in the population as a whole, however, the bcl-2 expression level did not change in blast cell subpopulation. c-myc Expression was significantly higher in the blast cell subpopulation of accelerated/blastic phase than in that of earlier phases of the disease. In conclusion, the characteristics of CML cells, namely proliferation rate, c-myc and bcl-2 change during the course of the disease. It is possible that the change in c-myc expression plays a causative role in evolution of the blastic phase from the chronic phase.     

IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression

The mechanism by which IFN-gamma inhibits tumor cell growth has not been fully understood. Here we report that IFN-gamma up-regulated the expression of Fas and Fas ligand (FasL) on HT29 cells, a human colon adenocarcinoma cell line, and subsequently induced apoptosis of these cells. The kinetics of cell death in IFN-gamma-treated HT29 cells paralleled the increase in the levels of Fas and FasL expression. We further show that IFN-gamma up-regulated the expression of Fas and FasL in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. Correspondingly, IFN-gamma induced cell death in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. IFN-gamma-induced cell death was inhibited by caspase-1 inhibitors. Our results suggest that cell growth inhibition by IFN-gamma is due to apoptosis mediated by Fas and FasL interaction.     

Dual role of ceramide in the control of apoptosis following IL-2 withdrawal

Ceramide is largely known as a lipid second messenger with pleiotropic effects. Increases in ceramide levels have been related to the onset of apoptosis, terminal differentiation, or growth suppression. In this study, addition of exogenous C2-ceramide to CTLL-2 cells is found to block IL-2-induced cell cycle entry, as well as the apoptosis triggered by IL-2 deprivation. The protective effect of C2-ceramide is achieved only in the early stages following cytokine deprivation and is related to the inhibition of bcl-xL degradation and the induction of a G0 arrest of cells. The same treatment over a longer time when, as we demonstrate, ceramide is produced physiologically, enhances cell death by apoptosis. The dual effect of ceramide both in protecting from or inducing apoptosis is discussed further.