Murine EL-4 Thymoma Cells Research Articles

IL-4 and transforming growth factor-beta suppress human immunoglobulin secretion in vitro by surface IgD- B cells.

The effect of IL-4 and transforming growth factor-beta (TGF-beta) on immunoglobulin secretion in vitro by peripheral blood mononuclear cells (PBMC) or purified B cells activated with murine EL4 thymoma cells and phorbol myristate acetate (PMA) was investigated. As previously reported, IL-4 induced IgE and IgG4 secretion by B cells in PBMC preparations and B cells activated with EL4 cells and PMA. However, when B cells, either in PBMC preparations or purified and activated with EL4 cells and PMA, spontaneously secreted large quantities of immunoglobulin, IL-4 suppressed the immunoglobulin secretion of all isotypes. IL-4 also suppressed the IgE secretion by B cells from an atopic dermatitis patient. This suppressive effect was not reversed by adding IL-2 or interferon-gamma (IFN-gamma) to the cultures. We also showed that TGF-beta suppressed the immunoglobulin secretion by purified B cells activated by EL4 cells and PMA. To investigate whether IL-4 or TGF-beta suppressed immunoglobulin secretion by in vivo 'switched' and isotype-committed B cells, sIgD- B cells were isolated, activated with EL4 cells and PMA and cultured with IL-4 or TGF-beta. Such activated B cells secreted large quantities of IgG1, IgG2, IgG3, IgA1, IgA2 and IgM, and IL-4 and TGF-beta suppressed all these isotypes by greater than 80%. The data demonstrated that IL-4 and TGF-beta suppress immunoglobulin secretion in vitro by in vivo isotype-committed sIgD- B cells, suggesting that these lymphokines may play a down-regulatory role on differentiated isotype-committed B cells in an isotype-unrestricted manner. The data also showed that IL-4 and TGF-beta acted directly on isolated B cells.

A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells

It has been shown that leukemia and glioma cells are sensitive to cannabidiol (CBD)-induced apoptosis, whereas primary monocytes and glia cells are relatively insensitive. In the current study, the cellular events and sensitivity to CBD-induced apoptosis between murine thymocytes and EL-4 thymoma cells were compared. Cannabidiol markedly induced apoptosis in a time- and concentration-related manner in both cells. The efficacy of CBD to induce apoptosis was comparable between the 2 types of T cells, whereas CBD induced apoptosis in thymocytes with a slightly greater potency than in EL4 cells. Time-course analyses revealed CBD-mediated apoptosis occurred earlier in EL-4 cells than that in thymocytes. An increased level of cellular reactive oxygen species (ROS) was detected in both cells with the peak response at 2 h post CBD treatment. Concordantly, CBD triggered a gradual diminishment in the cellular thiols. The presence of N-acetyl-l-cysteine (NAC), a precursor of glutathione, markedly attenuated the induction of apoptosis, and restored the diminished levels of cellular thiols. The results demonstrated that both thymocytes and EL-4 thymoma cells were susceptible to CBD-induced apoptosis and that ROS played a critical role in the apoptosis induction.

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1

High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1beta protein. The -131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S-transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.

Functionalization and Cellular Uptake of Boron Carbide Nanoparticles. The First Step toward T Cell-Guided Boron Neutron Capture Therapy

In this paper we present surface modification strategies of boron carbide nanoparticles, which allow for bioconjugation of the transacting transcriptional activator (TAT) peptide and fluorescent dyes. Coated nanoparticles can be translocated into murine EL4 thymoma cells and B16 F10 malignant melanoma cells in amounts as high as 0.3 wt. % and 1 wt. %, respectively. Neutron irradiation of a test system consisting of untreated B16 cells mixed with B16 cells loaded with boron carbide nanoparticles were found to inhibit the proliferative capacity of untreated cells, showing that cells loaded with boron-containing nanoparticles can hinder the growth of neighboring cells upon neutron irradiation. This could provide the first step toward a T cell-guided boron neutron capture therapy.

Clostridium difficile Toxin A Induces Expression of the Stress-induced Early Gene Product RhoB

Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h, a steady state was reached, with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.

The effect of medium composition on interleukin-2 production by murine EL-4 thymoma cells

Due to the role of interleukin-2 (IL-2) in the mediation of immune response, this cytokine has been used in the treatment of some types of cancer and infectious diseases. However, relatively high levels of this cytokine are required to achieve significant activity. The aim of this work was to study a culture medium composition designed to increase the production of IL-2 by suspended murine EL-4 cells. The cultivations were carried out aiming at producing IL-2 in stirred bioreactors. The effects of concentration of glutamine, phorbol-12-myristate-13-acetate (PMA), concanavalin A (Con A), Pluronic F68, and fetal calf serum (FCS) on cell viability and IL-2 production were evaluated. PMA alone was more efficient in IL-2 production than it was in association with Con A. The maximum IL-2 production was around 162 ng/mL with 856 ng/mL PMA and 1.45% (v/v) FCS.

Regulation by rho family GTPases of IL-1 receptor induced signaling: C3-like chimeric toxin and Clostridium difficile toxin B inhibit signaling pathways involved in IL-2 gene expression.

In this study the participation of Rho family GTPases in the regulation of IL-1-activated protein kinase cascades controlling IL-2 synthesis was investigated in murine EL-4 thymoma cells. The recombinant C3-like chimeric toxin, which consists of the C3 toxin of Clostridium limosum and the N-terminal part of Clostridium botulinum C2 toxin (C2IN-C3) interacting with the C2II binding subunit to facilitate uptake into cells, and selectively inactivates Rho A by ADP-ribosylation, prevented IL-1-stimulated activation of Jun-NH2-terminal-kinases (JNK) and p38 mitogen-activated-protein kinases (MAPK). UDP-monoglucosylation and concomitant inactivation of Rho A and of Rac-2 by Clostridium difficile toxin B also inhibited IL-1-induced activation of JNK and p38 MAPK, but additionally inhibited activation of the extracellular-regulated-kinase pathway and DNA binding of the transcription factor NFkappaB. Accordingly, pre-treatment of cells with C21N-C3 fusion toxin only decreased IL-1-stimulated IL-2 synthesis by 50%, while in C. difficile toxin B-treated cells IL-1-induced IL-2 secretion was reduced by 90%. These results imply that together with Rho A an additional member of the Rho family G proteins, i.e. Rac-2, is critically involved as an upstream regulator in IL-1-induced activation of different MAPK, stress-activated protein kinases, and in NFkappaB activation controlling IL-2 gene expression in response to IL-1, acting in close proximity to the IL-1-receptor complex.

C/EBPbeta enhances IL-4 but impairs IL-2 and IFN-gamma induction in T cells.

C/EBP transcription factors have been described to control the activity of the human IL-4 promoter. The C/EBP binding sites within the IL-4 promoter overlap with composite NF-AT and AP-1 binding motifs. We show here that similar binding sites are part of the murine IL-4 promoter. Retroviral overexpression of C/EBPbeta in murine EL-4 thymoma cells led to a strong induction of endogenous IL-4 and a reduction in IL-2 and IFN-gamma expression. Similarily, in primary murine T cells C/EBPbeta induction resulted in an increase in IL-4 levels, whereas in human Jurkat T cells a decrease in IL-2 RNA was detected. Like AP-1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP-1, C/EBPbeta does not act in synergy with NF-AT in the induction of the murine IL-4 promoter. Instead, both factors compete in their binding to the P4/Pu-bD site, one of the most important sequence elements of the IL-4 promoter. Whereas NF-AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL-4 promoter activity at specific developmental stages of T cells.

Modulation of Transcription Factor AP-1 Activity in Murine EL-4 Thymoma Cells by Vomitoxin (Deoxynivalenol)

Trichothecene mycotoxins have been reported to suppress or superinduce cytokine mRNA expression by leukocytes both in vitro and in vivo. Modulation of transcription factor activities may be critical for these observations. Here, the effect of trichothecene vomitoxin (VT, deoxynivalenol) on activator protein-1 (AP-1) activity was determined in the murine EL-4 thymoma. Electrophoretic mobility shift assay (EMSA) revealed that VT modulated AP-1 binding activity in a concentration- and time-dependent manner when using a synchronous model in which VT was added concurrently with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) to EL-4 cells. Induction of AP-1 binding activity by PMA/ION was suppressed in the presence of VT for a short period (1 to 12 h), but was enhanced upon prolonged VT exposure (48 to 72 h). VT also enhanced AP-1 binding activity when added to the cell culture 12 h after PMA/ION activation (delayed synchronous model). Using specific antibodies against AP-1 complex proteins, it was demonstrated by gel supershift assay that VT preferentially affected phosphorylated c-Jun, Jun B, c-Fos, and Fra-2 binding activities, whereas it did not alter Jun D and Fra-1 binding. A transient transfection assay demonstrated that these increased binding activities are associated with enhanced AP-1 transactivation potential. Elevation of AP-1 activity may contribute to cytokine dysregulation and immunotoxic effects associated with exposure to trichothecene mycotoxins such as VT.

Superinduction of IL-2 Gene Expression by Vomitoxin (Deoxynivalenol) Involves Increased mRNA Stability

To better understand molecular mechanisms by which the trichothecene vomitoxin (VT) superinduces cytokine gene expression, we studied the posttranscriptional effects of this mycotoxin on interleukin-2 (IL-2) gene expression in murine EL-4 thymoma cells stimulated with phorbol 12-myristate 13-acetate and ionomycin (PMA + ION). Northern analysis revealed that doses of 50 to 500 ng/ml VT superinduced IL-2 mRNA expression in a dose- and time-dependent manner in a synchronous model where VT was added at onset of PMA + ION stimulation. In accordance with the mRNA levels, IL-2 production was significantly elevated in the presence of 50 to 250 ng/ml VT. Superinduction of IL-2 mRNA was also observed in a delayed synchronous model (VT added 20 hr after PMA + ION stimulation) and an asynchronous model (VT added 20 hr after PMA + ION stimulation and removal). To assess the effects of VT (500 ng/ml) on IL-2 mRNA half-life, three transcriptional inhibitors were used in the delayed synchronous model. Actinomycin D (ActD) had a pronounced stabilizing effect on IL-2 mRNA but not on mRNA for the housekeeping gene GAPDH. VT did not affect IL-2 mRNA levels in ActD-treated cells. Although 5,6-dichloro-β- d-ribofuranosyl-benzimidazole (DRB) also had a stabilizing effect on IL-2 mRNA, IL-2 mRNA half-life t 1/2in VT-treated cells was three times that of control. In contrast, inclusion of cyclosporin A (CsA) into the cultures specifically arrested IL-2 transcription in EL-4 cells without any stabilizing effect. VT exposure in the presence of CsA markedly prolonged the half-life of IL-2 mRNA in a dose-dependent manner. The t 1/2for IL-2 mRNA in the control culture was 2.1 hr, whereas t 1/2was 3.1, 3.4, 4.2, and 10.5 hr in cultures containing 50, 100, 250, and 500 ng/ml VT, respectively. These results suggest that VT can superinduce IL-2 at both the mRNA and the protein level and that this superinduction can be explained, in part, by posttranscriptional mechanisms such as enhanced mRNA stability.

Terminal Differentiation of Human Germinal Center B Cellsin Vitro

In order to define anin vitroculture system allowing growth of single human germinal center B cells (GC-B), we have studied the proliferation and differentiation of human tonsillar GC-B, and subsets thereof, when cultured together with murine EL-4 thymoma cells in the “EL-4 system.” The cells were analyzed and compared to resting tonsillar B cells with respect to phenotypic changes, proliferation, Ig secretion, intracellular Ig levels, and growth abilities under limiting dilution conditions. It was found that GC-B differentiated terminally to Ig-secreting cells with the phenotypic features of plasma cells in a similar manner to tonsillar resting B cells. The GC-B proliferated for 4–5 days, followed by a loss of GC-B phenotype and an increase in intracellular immunoglobulin levels. Over a 10-day culture period a larger proportion of the Ig produced by GC-B was IgG and IgA, as compared to resting B cells, indicating that these cells switched isotype more easily or had already switched in the germinal center prior to the culture period. Analysis of frequencies of Ig-producing cells revealed that 1/3.8 of GC-B and less than 1/10 of the centroblast B cell subpopulation (CB-B) differentiated toward Ig-producing cells when cultured in the EL-4 system whereas 1/1.25 and 1/1.5 of peripheral blood B cells (PBL-B) and resting tonsillar B cells did so, respectively. Taken together, these findings show that tonsillar GC-B differentiate in a similar manner to resting B cells when cultured in the EL-4 system, and we conclude that these conditions allow manipulation of GC-B in single cell culturesin vitro.

Protein Kinase C-θ Isoenzyme Selective Stimulation of the Transcription Factor Complex AP-1 in T Lymphocytes

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Expression of naloxone-resistant β-endorphin binding sites on A20 cells: effects of concanavalin A and dexamethasone

β-Endorphin affects mononuclear cell proliferation, cytokine production and calcium uptake in a naloxone-resistant manner. The presence of naloxone-insensitive binding sites for β-endorphin have been demonstrated on murine EL4-thymoma cells, transformed human mononuclear cells and normal murine splenocytes. Since murine splenic B cells have been shown to express naloxone-resistant receptors for β-endorphin in response to the mitogen, concanavalin A (Con A), the A20 B-cell lymphoma line was used to further study regulation of this site by Con A and dexamethasone. Analyses showed two sites: a high-affinity site, Kd1 = (8.7 ± 2.3) × 10−11 M and binding capacity (Bmax1) of (2.6 ± 2.0) × 103 receptors/cell; and a low-affinity site, Kd2 = (2.2 ± 0.8) × 10−8 M with Bmax2 of (1.5 ± 0.8) × 105 receptors/cell. Competition studies showed that N-acetyl-β-endorphin was approx. 5-fold and β-endorphin6−31 10-fold less potent than β-endorphin1−31. Neither β-endorphin1−27 nor naloxone, morphine or other opioid receptor agonists displaced [125I]β-endorphin. Con A (20 μg/ml) significantly increased the Bmax (3.5-fold; expressed per cell) and resulted in a loss of the higher-affinity site. However, the increased Bmax occurred in proportion to the Con-A-induced increase in protein/cell. Dexamethasone (Dex) also increased Bmax, primarily by increasing (2–3-fold) the number of lower affinity sites. In contrast to Con A, two binding sites persisted after treatment with Dex, which exerted a minimal effect on protein/cell. Therefore, binding/cell and binding/protein/cell were both significantly enhanced by Dex. The combined effects of Dex and Con A on binding failed to show additivity or synergy. When binding was analyzed per protein/cell, the effect of Con A appeared to dominate; the Dex-enhanced binding/protein/cell was no longer evident in the presence of Dex plus Con A. Thus, Dex and Con A may enhance binding by independent mechanisms.

Expression and Biochemical Characterization of Human Protein Kinase C‐θ

In this study, the recently identified human protein kinase C-theta (PKC-theta) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-theta identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-theta was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-theta with a representative member of the Ca(2+)-dependent PKC enzymes, full-length cDNAs encoding PKC-theta or PKC-alpha were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-theta and PKC-alpha. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-alpha, however, full activation of PKC-theta did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin-basic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-theta. Similar to PKC-alpha, transient PKC-theta overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-theta suggest that it may play a specialized role in T-cell signaling pathways.

Induction of Heat-Shock Protein Synthesis and Thermotolerance in EL-4 Ascites Tumor Cells by Transient ATP Depletion after Ischemic Stress

The effect of a short-term energy deprivation (ischemia) on thermoresistance and heat-shock protein (HSP) synthesis in murine ascites EL-4 thymoma cells was studied in vitro. The incubation of the cells in glucose-free medium with rotenone (respiratory inhibitor) for 10 min caused rapid ATP depletion (to 9% of the initial level). After recovery, the synthesis of HSP70 and HSP90 was stimulated in the cells and they became greatly more resistant to hyperthermia than the control cells. The simultaneous rotenone and thermal treatment significantly decreased cell viability. The transition of HSP70 to Triton X-100-insoluble cell fraction was found in the ATP-depleted cells as well as in the heat-shocked cells, and 1 m M ATP fully reversed such insolubilization when it was added in Triton extraction buffer. The data obtained reveal that transient ATP depletion per se is sufficient to result in the HSP70 insolubilization, thus being conducive to induction of HSP synthesis and thermotolerance in the cells which recovered after energy deprivation. A novel mechanism of protein aggregation in ATP-deficient cells and a possible role of transient ischemia in development of tumor thermotolerance in vivo are discussed.

Production of Interleukin-2 by EL4 Tumor Cells Induces Natural Killer Cell- and T-Cell-Mediated Immunity

Systemic administration of recombinant interleukin (rIL)-2 to cancer patients has met with limited clinical success since, despite significant antitumor effects, its use is associated with severe toxicity. Local production of IL-2 by IL-2 gene transfected tumor cells in murine model systems has been reported to induce specific immunity--devoid of toxicity--to the parental non-IL-2-producing tumor cells. We now report enhanced resistance in nonimmunized mice to murine EL4 thymoma cells, producing murine IL-2 following gene transfer (EL4pIL-2). This effect is mediated by activated natural killer (NK) cells, since we observed the same effect in nude mice but not in NK-depleted mice. Additionally, in mice repeatedly vaccinated with irradiated EL4pIL-2 cells, we observed immunity to challenge with a tumorigenic dose of EL4 cells transfected with a control vector, EL4p. EL4-specific cytotoxic T-lymphocytes (CTLs) were detected in these mice. Mice vaccinated with irradiated EL4p cells were less protected against challenge with a tumorigenic dose of EL4p cells. This study indicates that although some IL-2-producing autologous tumor cells elicit NK-mediated responses and not CTL responses upon inoculation, tumor-specific CTL responses are generated upon repeated vaccinations with these cells. This strategy has potential application for treating a wide variety of cancer patients with autologous IL-2 producing tumor cells.

Binding of human β2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells

A variety of murine tumor cell lines was studied for its binding of exogeneously added human β 2-microglobulin (h β 2m). Three T lymphomas and one IL-2-dependent T-cell line (HT-1) bound substantial amounts of h β 2m, whereas P815 mastocytoma cells, an Abelson virus-infected pre-B cell line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h β 2m. In two of the T lymphomas, EL4 and BW5147, binding of h β 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis. EL4 cells which had bound h β 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest that binding of h β 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both effector and target functions of the cell.

Binding of sequence-specific proteins to the adenosine- plus uridine-rich sequences of the murine granulocyte/macrophage colony-stimulating factor mRNA.

Adenosine+uridine (AU)-rich sequences in the 3' untranslated region (3'UTR) of the mRNA of many cytokines and oncogenes play an important role in mediating RNA degradation. Among the cytokines containing such AU-rich sequences in their 3'UTR is the hematopoietic growth factor granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF gene expression in T cells is regulated by modulation of mRNA half-life. Transfection studies using murine EL-4 thymoma cells have demonstrated that degradation depends on the presence of specific elements in the 3'UTR, including the AU-rich sequences. A number of AU-binding factors have recently been discovered, suggesting that specific regulation may occur through specific protein-mRNA interaction(s). We present evidence from gel-shift analyses and label-transfer experiments that murine cells contain proteins that bind specifically to AU-rich sequences. Three major proteins of 33, 39.5, and 42 kDa are detected. Phorbol ester treatment of cells does not alter the abundance or apparent binding affinity of the proteins. The 33-kDa protein is present in the cytoplasm of murine and human cells, whereas the 39.5- and 42-kDa proteins are present in murine extracts only. Constitutively expressed AU-binding proteins of the type that we describe may function by directing mRNA degradation in the absence of a stimulus to the contrary.

Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins.

The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24-membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model.

Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.