Internalization Of Interleukin-2 Research Articles

Activity of Outpatient Intravenous Interleukin-2 and Famotidine in Metastatic Clear Cell Kidney Cancer

Outpatient daily intravenous infusions of interleukin-2 (IL-2) have been developed to maintain anticancer activity and decrease toxicity of this agent against kidney cancer. Lymphokine activated killer cell (LAK) numbers are increased with these IL-2 schedules. Famotidine may enhance the LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. Fifteen patients with metastatic clear cell kidney cancer received IL-2 18 million IU/M² intravenously over 15-30 minutes preceded by famotidine 20 mg IV daily for 3 days for 6 consecutive weeks as outpatients. Cycles were repeated every 8 weeks. Patient characteristics were seven males/eight females, median age 59 (range: 28-70), median Eastern Cooperative Oncology Group (ECOG) performance status-1; common metastatic sites were lungs (14), lymph nodes (9), liver (4), bone (4), and pancreas (4). Prior systemic therapies were oral tyrosine kinase inhibitor (8), IL-2 (6), and mTor inhibitor (2). Most common toxicities were rigors, arthralgia/myalgia, nausea/emesis, fever, and hypotension. All episodes of hypotension were reversible with intravenous fluid. No patients required hospitalization due to toxicity. One complete response (7%) and four partial responses (26%) were seen (total response rate=33%; 95% confidence interval: 15%-59%). Responses occurred in the lungs, liver, lymph nodes, and bone. Outpatient intravenous IL-2 with famotidine has activity in metastatic clear cell kidney cancer.

Activity of dose-dense outpatient intravenous Interleukin-2 preceded by famotidine in metastatic clear cell cancer of the kidney

Daily short intravenous infusions of Interleukin-2 (IL-2) have been developed to decrease toxicity while maintaining anticancer activity of this agent against kidney cancer. Such IL-2 schedules have previously been shown to increase Lymphokine Activated Killer cell (LAK) numbers (Quan) and LAK activity (Mitchell). Famotidine may increase LAK activity by increasing Interleukin-2 internalization by the IL-2 receptor on lymphocytes (Tsunoda). We treated 15 patients with metastatic clear cell kidney cancer using IL-2 18 Million IU/M2 intravenously over 15-30 minutes preceded by famotidine 20 mg IV daily for 3 days for 6 consecutive weeks on an outpatient basis. Cycles were repeated every 8 weeks until disease progression. Patient characteristics: 7 males/8 females, median age-59 (range: 28-70), median ECOG performance status-1; common metastatic sites: lungs (14), lymph nodes (9), liver (4), bone (4), and pancreas (4). Prior systemic therapy: oral TKI (8), Interleukin-2 (6), mTor inhibitor (2), no prior (3). Prior nephrectomy (9). Most common toxicities were rigors (13), arthralgia/myalgia (12), nausea/emesis (11), fever (11), hypotension (11), and constipation (8). All episodes of hypotension were transient and alleviated with outpatient intravenous fluid. No patients have required hospitalization due to toxicity of therapy. There have been no treatment-related deaths. One complete response (7%) (in lungs lasting 13 months) and four partial responses (26%) have been seen (total response rate =33%; 95% CI:15-59%). Responses have occurred in lungs, liver, lymph nodes, and bone. Median response duration = 5 months (2-13). Median survival = 13.75 months (3-41 months). Two patients are alive at 35.7+ and 35.2+ months. Dose-dense outpatient intravenous Interleukin-2 preceded by famotidine has activity in metastatic clear cell kidney cancer.

Pulse Infusion Interleukin-2 with Famotidine and Cyclophosphamide Has Activity in Previously Treated Metastatic Melanoma

There is no established systemic therapy for patients with stage IV melanoma refractory to prior systemic treatment. Interleukin-2 (IL-2) is capable of inducing T-lymphocyte cytotoxicity against melanoma in vitro and in vivo. Famotidine may enhance the activity of T-cells further by allowing for increased IL-2 internalization by the IL-2 receptor on lymphocytes. Cyclophosphamide may decrease the immunosuppressive effects of regulatory T-cells. Daily short intravenous (i.v.) infusions (pulses) of IL-2 were used to treat 14 patients with metastatic melanoma, all of whom had experienced disease progression despite prior systemic therapy. The patients received 21.6 million IU/m(2) of pulse IL-2 i.v. for 15-30 minutes, preceded by 20 mg of famotidine i.v. (13) patients received 350 mg/m(2) of cyclophosphamide i.v. on day 1 (1 patient did not). Eight (8) patients were treated in an oncology inpatient unit while, most recently, 6 patients have received therapy on an outpatient basis. The cycles were repeated every 3 weeks until disease progression occurred. The patients included 10 males with a median age of 56 (range 31-87) with an Eastern Cooperative Oncology Group performance status of -1 (range 0 - -1). Common metastatice sites included lymph nodes (13), lungs (8), liver (4), and subcutaneous (4). Prior systemic therapy included IL-2 (11), interferon (7), and chemotherapy (7). The median number of cycles the patients underwent was 3 with a range of 1-7. The most common toxic reactions were fever, rigors, nausea/emesis, hypomagnesemia, and hypophosphatemia. One complete response and four partial responses were observed (response rate, 36%; 95% confidence interval: 14%-64%). Responses occurred in the lungs, liver, lymph nodes, and subcutaneous sites. The median response duration was 3.4 months, with a median survival of 8.3 months for the entire group. Six (6) patients remain alive with a median survival of 10.3 months. Pulse IL-2 with famotidine and cyclophosphamide produced activity in previously treated patients with melanoma and may be given on an outpatient basis to selected individuals.

High-Dose Intensity Pulse Interleukin-2 with Famotidine Has Activity in Metastatic Melanoma

Daily short intravenous (i.v.) infusions (pulses) of interleukin-2 (IL-2) have been developed to decrease toxicity while maintaining anticancer activity of this agent against melanoma. Such IL-2 schedules have previously been shown to promote lymphokine-activated killer (LAK) cell activity. Famotidine may increase LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. We treated 16 patients with metastatic melanoma using pulse IL-2 18 (15 patients) or 9 million IU/M2 (1 patient) i.v. over 15-30 minutes preceded by famotidine 20 mg i.v. daily for 5 days on an oncology inpatient unit. Cycles were repeated every 3 weeks until disease progression. Patient characteristics were as follows: 11 males, median age, 66, median ECOG performance status, 1; common metastatic sites: lymph nodes, lungs, subcutaneous, liver, and bone. Median number of cycles received was 3. Overall, 93% of planned doses were delivered. Most common toxicities were hypomagnesemia, fever, rigors, hypophosphatemia, and nausea/emesis. Three (3) patients had partial responses (19% response rate; 95% confidence interval: 6%-44%). A fourth patient, after resection of residual disease, remains a surgical complete responder at > 12 months. Responses occurred in lung, liver, lymph nodes, bone, and subcutaneous sites. Median response duration was 7 months. Pulse IL-2 with famotidine has activity in melanoma.

Mechanism of action of interleukin-2 (IL-2)-Bax, an apoptosis-inducing chimaeric protein targeted against cells expressing the IL-2 receptor.

The chimaeric protein interleukin-2 (IL-2)-Bax was designed to target and kill specific cell populations expressing the IL-2 receptor. However, it is not well understood how IL-2-Bax causes target cells to die. In the present study, we investigated the pathway of apoptosis evoked by IL-2-Bax and the possible involvement of endogenous Bax in this process. We report here that, upon internalization of IL-2-Bax into target cells, it is localized first mainly in the nucleus, and only later is it translocated to the mitochondria. Similarly, endogenous Bax is also partially localized in the nucleus, and accumulates mainly in this compartment soon after physiological triggering of apoptosis. Despite the fact that Bax has no nuclear localization sequence, our data suggest that Bax has one or more physiological roles and/or substrates within the nucleus. Indeed, a dramatic repression of nuclear Tax protein expression was induced following treatment of HUT-102 cells with IL-2-Bax, similar to what occurs following serum deprivation of these cells. Unexpectedly, induction of apoptosis using IL-2-Bax was preceded by enhanced expression of newly synthesized Bax protein and suppression of Bcl-2. This imbalance between the pro- and anti-apoptotic genes was associated with p53 induction, although IL-2-Bax activity was also evident in cells lacking p53 expression. By studying the mechanism of action of IL-2-Bax, we were able to follow the intrinsic events and their cascade that culminates in cell death. We have shown that the ability of IL-2-Bax to affect the intracellular apoptotic machinery within the target cells, and to cause the cells to die, uses a mechanism similar to that induced following a normal apoptotic signal.

The Proteasome Regulates Receptor-mediated Endocytosis of Interleukin-2

Recent studies have increasingly implicated the proteasome in the regulation of cell surface receptors. In the present study, we investigated the role of the proteasome for ligand-dependent endocytosis and degradation of the interleukin-2 (IL-2)-interleukin-2 receptor (IL-2R) complex. Proteasome inhibitors impaired internalization of IL-2.IL-2R and prevented the lysosomal degradation of this cytokine. Based on time-course studies, proteasome activity is primarily required after initial endocytosis of the IL-2.IL-2R. Proteasome function was also necessary for the lysosomal degradation of IL-2 internalized by IL-2R that were comprised of cytoplasmic tailless beta- or gamma c-subunits, suggesting that the target protein for the proteasome is independent of either the cytoplasmic tail of the IL-2R beta- or gamma c-subunits and their associated signaling components. Therefore, a functional proteasome is required for optimal endocytosis of the IL-2R/ligand complex and is essential for the subsequent lysosomal degradation of IL-2, possibly by regulating trafficking to the lysosome.

Computational model for effects of ligand/receptor binding properties on interleukin-2 trafficking dynamics and T cell proliferation response.

Multisubunit cytokine receptors such as the heterotrimeric receptor for interleukin-2 (IL-2) are ubiquitous in hematopoeitic cell types of importance in biotechnology and are crucial regulators of cell proliferation and differentiation behavior. Dynamics of cytokine/receptor endocytic trafficking can significantly impact cell responses through effects of receptor down-regulation and ligand depletion, and in turn are governed by ligand/receptor binding properties. We describe here a computational model for trafficking dynamics of the IL-2 receptor (IL-2R) system, which is able to predict T cell proliferation responses to IL-2. This model comprises kinetic equations describing binding, internalization, and postendocytic sorting of IL-2 and IL-2R, including an experimentally derived dependence of cell proliferation rate on these properties. Computational results from this model predict that IL-2 depletion can be reduced by decreasing its binding affinity for the IL-2R betagamma subunit relative to the alpha subunit at endosomal pH, as a result of enhanced ligand sorting to recycling vis-à-vis degradation, and that an IL-2 analogue with such altered binding properties should exhibit increased potency for stimulating the T cell proliferation response. These results are in agreement with our recent experimental findings for the IL-2 analogue termed 2D1 [Fallon, E. M. et al. J. Biol. Chem. 2000, 275, 6790-6797]. Thus, this type of model may enable prediction of beneficial cytokine/receptor binding properties to aid development of molecular design criteria for improvements in applications such as in vivo cytokine therapies and in vitro hematopoietic cell bioreactors.

Signal for IL-2 internalization located in the endocellular domain of IL-2R γ subunit only

Interleukin-2(IL-2) is an important immunoregulatory factor. After the interaction between IL-2 and its receptor, intracellular signal molecules were activated and pro-oncogenes, e.g.c-Myc, c-Fos, c-Jun andBcl-2 were induced, then cells stimulated with IL-2 would proliferate or differentiate. During these processes, IL-2 were internalized. The internalization of IL-2 mediated by IL-2R decreases the number of IL-2R on cell membranes, and makes extracellular IL-2 enter cells and degrade. It will down-regulate IL-2 biological activities. In order to identify the IL-2R subunit responsible for IL-2 internalization, chimeric IL-2Rs which consisted of different endocellular domains and extracellular domains from various IL-2R subunits were constructed. After they were transfected into L929 cell line, the IL-2 internalization was detected. Results showed that intracellular domains from IL-2R a and β subunlts could not mediate IL-2 internalization alone; the signal for IL-2 internalization was located only in the endocellular domain of IL-2R γ subunit. Furthermore, IL-2 internalization extent would be affected by affinity between IL-2 and IL-2R.

Microtubule depolymerization inhibits clathrin coated-pit internalization in non-adherent cell lines while interleukin 2 endocytosis is not affected.

The microtubule cytoskeleton is generally not considered to be essential for the first steps of clathrin-mediated endocytosis of membrane receptors. Its role in clathrin-independent endocytosis has not been investigated. We have previously shown that the cytokine interleukin 2 (IL2) is internalized in lymphoid cells expressing its receptors when clathrin-dependent endocytosis is inhibited. Here we compare the internalization of IL2 and of transferrin, a marker of clathrin-dependent endocytosis, after microtubule disruption. In hemopoietic cell lines, which express IL2 receptors, transferrin receptor entry was inhibited by about 40%. However, in adherent cell lines, transferrin entry was unaffected by microtubule disruption, as previously reported. Unlike the case for transferrin, internalization of IL2 receptors was not affected by depolymerization of the microtubule cytoskeleton in hemopoietic cell lines. These results show that IL2 and transferrin receptors do not have the same endocytic properties and support our previous conclusion that these receptors follow different pathways of endocytosis.

Interleukin-2 (IL-2) receptor alpha, beta and gamma subunit expression as a function of B-cell lineage ontogeny: the use of IL-2-PE66(4Glu) to characterize internalization via IL-2 receptor subunits.

Interleukin-2 (IL-2) is a pluripotent cytokine which plays a crucial role in the immune system response. Although the IL-2/IL-2 receptor (IL-2R) system has been well characterized in cells of the T lineage it is less known in B lymphocytes. The authors therefore studied the expression of the IL-2R alpha, beta and gamma subunits in human B-cell lines at different stages of maturation, by the polymerase chain reaction technique. The authors found that the alpha and beta subunits are expressed in the final stages of B-cell lineage maturation, whereas the gamma subunit is constitutively expressed during B-lymphocyte differentiation. The results indicate that the IL-2/IL-2R system, most probably, does not have a role in the early stages of B-cell differentiation, but may be involved only in the final stages of B-cell lineage ontogeny. Moreover, the ability of the different forms of IL-2R to internalize the IL-2 ligand was investigated, using the chimeric protein IL-2-PE66(4Glu). Cell lines bearing the alphagamma, betagamma and alpha betagamma forms of IL-2R were inhibited by the chimeric protein, while those bearing the gamma subunit alone did not respond to the chimera. Thus, internalization of IL-2 is most likely mediated via the alphagamma form of the IL-2R, as shown here for the first time, as well as through the betagamma and alpha betagamma IL-2R forms. However, IL-2 cannot be internalized through the IL-2R gamma subunit alone.

Control of Cell Cycle Progression in Human Natural Killer Cells Through Redox Regulation of Expression and Phosphorylation of Retinoblastoma Gene Product Protein

AbstractUsing thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2–dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-γ activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.

Elevated intracellular glutathione levels in CD4 + T cells of BB rats

GLUTATHIONE (GSH) is a dominant intracellular redox buffering compound which is essential for survival and several functions of peripheral T cells. It is the main free radical scavenger in T cells, protecting them from oxidative stress-induced and antigen-driven apoptosis. 1,2 In addition, GSH is important for T cell proliferation, 3,4 mitogen-induced activation, 5 cytotoxicity, 6 interleukin-2 (IL-2)-dependent DNA synthesis, 7 and it enhances IL-2 binding, internalization, and degradation by cytotoxic T cells. 8 GSH depletion in vitro was shown to inhibit mouse T cell proliferation; however, this was shown to exist predominantly for CD8 + T cells and to a much lesser extent for CD4 + T cells, thereby markedly increasing the CD4/CD8 ratio in GSH-depleted cultures. 9 Peripheral T cells of the T lymphocytopenic diabetes-prone BB (DPBB) rat display many phenotypic and functional abnormalities. After thymic emigration, a majority of these cells dies very rapidly 10,11 by apoptosis (S. Ramanathan, this issue). The remaining cells proliferate poorly on allogenic and mitogenic stimulation, 12,13 and IL-2 production has been reported to be decreased. 14 CD8 + T cells are virtually absent in DPBB rats, 15 which is partly due to a more severely reduced life span for CD8 + than for CD4 + T cells, 11 and cytotoxic T lymphocytes exhibit reduced cytotoxic capacity. 16,17 It is presently largely unknown why T cells in DPBB rats display these abnormalities; however, it can be hypothesized that they are a reflection of decreased intracellular GSH levels. For this reason, we investigated whether peripheral T cells in the DPBB rat, and CD8 + T cells in particular, have reduced intracellular GSH levels.

A Point Mutation in Interleukin-2 That Alters Ligand Internalization

In previous studies, we have identified an interleukin-2 (IL-2) analog containing a point mutation at position 51 (T51P) that expresses nearly wild-type bioactivity, yet has approximately 10-fold lower receptor binding affinity. Since ligand-dependent receptor internalization may be the rate-limiting step controlling the duration of IL-2 receptor signaling, a reduction in the receptor internalization rate could contribute to the observed response enhancement for this analog. To evaluate this possibility, we compared the internalization of IL-2 and T51P in three separate assays. While the internalization rate for IL-2 agreed with values determined by others, the internalization of T51P was markedly reduced. The receptor binding rate constants for this analog were only slightly different; thus, altered binding kinetics could not explain the decreased internalization rate. The effects of reduced internalization were also observable in bioassays, where T51P maintained T-cell proliferation for a longer period compared with IL-2. These results indicate that the T51P point mutation reduces the receptor internalization rate compared with IL-2 in a fashion that is independent of the dissociation rate. This analog may represent a new approach to the preparation of cytokine analogs with potentiated agonist and antagonist properties.

Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited

The cytokine interleukin 2 (IL2) is produced by activated helper T lymphocytes and modulates the growth and activity of cells expressing high-affinity surface IL2 receptors that transduce its signaling. After ligand binding to receptors on the plasma membrane, receptor-ligand complexes are rapidly endocytosed and IL2 is degraded in acidic compartments. The best known receptor-mediated endocytosis pathway involves clathrin-coated pits. Receptors that carry an internalization signal recognized by adaptors on the cytosolic side of the plasma membrane are clustered into the coated pits and enter cells very efficiently. Many receptors use this pathway, but other endocytic pathways have also been reported, for ricin, EGF and insulin, for instance, which seem to be less efficient than the coated one. We compared the endocytosis of IL2 and its receptors to that of transferrin, a marker of the coated pit pathway. Under normal conditions, the kinetics of entry of IL2 was two times slower than that of transferrin. When internalization via coated pits was inhibited by two different methods, potassium depletion and cytosol acidification, endocytosis of IL2 and its receptors was only partly inhibited, while transferrin entry was strongly affected. Treatment with the cationic amphiphilic drug chlorpromazine, which induces a redistribution of a clathrin-coated pit component, AP-2, to endosomes, reduced transferrin, but not IL2 internalization. Thus, unexpectedly, this cytokine and its receptors can still be rapidly endocytosed in the absence of functional clathrin-coated structures. We propose a model for receptor-mediated endocytosis that may account for these results and published data on other receptors.

EXPRESSION OF THE INTERMEDIATE AFFINITY INTERLEUKIN-2 RECEPTOR BY A HUMAN SOLID TUMOR-CELL LINE - EVIDENCE FOR FUNCTIONAL-ACTIVITY

Interleukin 2 (IL2) has well recognized pleiotropic effects on the activation, differentiation and proliferation of lymphoreticular cells, mediated via its specific membrane-bound receptors, but its effect on human solid tumour cells is poorly characterised. This study has used the A549 tumour cell line, derived from a human lung carcinoma, to demonstrate the presence of the interleukin 2 receptors (p70/75 beta and/or p55 alpha components) and to document functional activity. Immunohistochemical techniques demonstrated large amounts of the p70/75 beta chain of the interleukin 2 receptor, which is necessary for IL2 internalization, receptor signal transduction and mediation of the biological effects of IL2. In contrast, the p55 alpha chain was detected minimally and sparsely. In addition we documented the presence of IL2 in the nucleus of the A549 cells. The addition of exogenous rIL2 (10-500 IU/ml), to the culture medium of A549 cells over 48 hours resulted in a significant stimulation of DNA synthesis, as assessed by tritiated thymidine uptake. The results were; 9335+/-365, 12669+/-271, 12889+/-255, 19448+/-1427, 20189+/-1004 and 22586+/-1334 CPM, at 0 IU/ml, 10 IU/ml, 50 IU/ml, 100 IU/ml, 250 IU/ml and 500 IU/ml, respectively (means+/-SEM), and were significantly increased when compared with control cultures, p<0.001). These results may have important implications in our understanding of tumour-host interactions and in future strategies involving immunotherapy.

Endocytosis of the β chain of interleukin‐2 receptor requires neither interleukin‐2 nor the γ chain

Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL-2R is a multisubunit receptor which contains at least three chains, IL-2R alpha (p55), IL-2R beta (p70) and IL-2R gamma (p64). Some components of high-affinity receptors (alpha beta gamma) are continuously internalized in the absence as well as in the presence of IL-2. From studies on other receptors, it is known that endocytosis of ligand-receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high-affinity IL-2 receptors have not been fully elucidated. IL-2R gamma has been reported to be necessary for IL-2 internalization, based on the fact that fibroblasts transfected with IL-2R alpha and -beta do not internalize IL-2. However, IL-2 dissociates too rapidly from IL-2R alpha beta receptors to allow for its internalization. From the reported results on IL-2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL-2R beta and/or IL-2R gamma in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization. In this work, we have studied the endocytic properties of IL-2R beta. We demonstrate that IL-2R beta is constitutively endocytosed in a B cell line, derived from a X-linked severe combined immunodeficiency patient, which lacks expression of IL-2R gamma. IL-2R beta was also constitutively internalized in T and natural killer cell lines independently of IL-2R gamma. These results suggest that IL-2R beta is endowed with endocytic capacity and carries internalization signals.

Mechanism of the impaired T-cell proliferation in adult rats exposed to alcohol in utero

Although attempts have been made to assess the effect of ethanol on the immune responses in individuals with fetal alcohol syndrome, there is no consensus as to the effect of ethanol on the immune system. Evidence that fetal alcohol-exposed (FAE) humans and animals have diminished proliferative response of T-cells to mitogenic lectins is well established. However, little is known about the mechanism of a toxic effect of ethanol on T-cell growth. Thus, a rat model was used to delineate the mode of ethanol action on T-cell proliferation. We found that the diminished T-cell proliferation in young adult FAE rats was due to a decreased responsiveness to interleukin 2 (IL2), but not to an impaired production of IL2 and expression of IL2 receptors (IL2R). Furthermore, the decreased proliferative response did not result from the presence of an excessive suppressor T-cell activity. Measurements of [Ca +2] i and T-cell proliferation were concurrently performed in batches of cells from the same animals. It was demonstrated that an increase in [Ca +2] i induced by Concanavalin A (Con A) in T-cells from FAE rats was not impaired, although the T-cell proliferation induced by Con A was significantly diminished. The results of the IL2-binding study showed that the K d values and the number of both high- and low-affinity IL2R binding sites on the T-cells of FAE rats were comparable to those of pair-, or chow-fed rats. Finally, the results of the kinetics and rate of the internalization of IL2 showed that (1) the amount of the internalized IL2 was significantly reduced in T-cells from FAE rats, and (2) the half-time (t 1 2 ) for dissociation of IL2 from the receptors in the T-cells from FAE rats was also greater than that of the control rats. These results taken together indicate that ethanol suppresses T-cell proliferation by interfering with events following the IL2-IL2R interaction.

Selection of internalization‐deficient cells by interleukin‐2‐Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin‐2 receptor β chain does not contribute to internalization of interleukin‐2

To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) alpha- and beta-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2R beta cDNA led to surface expression of IL-2R beta and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the beta subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated beta cDNA, several CX1 transfectants were produced that expressed a beta-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type beta-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the beta subunit and raise the possibility that such signals may be entirely contained within the gamma subunit.

Immobilized IL-2 preserves the viability of an IL-2 dependent cell line

The mouse T-cell line CTLL-2 is known to be dependent on interleukin-2 (IL-2) for both growth and viability. These cells possess high affinity IL-2 receptors and have been shown to internalize IL-2 after binding. To determine if internalization of IL-2 is required for the mediation of its signal, IL-2 was covalently coupled to an insoluble matrix via glutaraldehyde cross-linking and CTLL-2 cells were incubated with the immobilized lymphokine matrix. This covalent cross-linking prevents the free lateral diffusion and internalization of the bound IL-2 receptors (IL-2R) while still permitting specific binding between the cells and the immobilized ligands. Although only very limited proliferation was observed during the incubation as assessed by 3H-thymidine incorporation, the viability of the CTLL-2 cells on the immobilized IL-2 matrix was preserved. Cells incubated on the immobilized IL-2 surface could proliferate in response to exogenous soluble IL-2 that was added to the cultures after 36 hours whereas control cultures incubated with an immobilized BSA matrix had died. This indicates that immobilized IL-2 can mediate some of the activity of soluble IL-2 and that internalization of the IL-2 receptor may not be required for at least part of the IL-2 mediated effect.

Reconstitution of functional interleukin 2 receptor complexes on fibroblastoid cells: involvement of the cytoplasmic domain of the gamma chain in two distinct signaling pathways.

We have previously shown that the interleukin 2 (IL-2) receptor gamma chain is a member of the cytokine receptor superfamily and is indispensable for the formation of receptor complexes with high and intermediate affinities for IL-2. The present study demonstrates that the alpha beta gamma heterotrimer and beta gamma heterodimer complexes of IL-2 receptor reconstituted on murine fibroblast L929 cells can transduce IL-2-mediated signals for activation of tyrosine kinase and for induction of c-myc, c-fos, and c-jun expression. A mutant of the gamma chain lacking the C-terminal 68 amino acids in its cytoplasmic region showed a loss of such signal-transducing ability when incorporated into the IL-2 receptor complexes but brought no effect on IL-2 binding and IL-2 internalization. Another mutant, with a C-terminal deletion of 30 amino acids, retained the ability to activate a tyrosine kinase and to induce c-myc expression but lost the ability to induce c-fos and c-jun expression. These results suggest that at least two distinct signals, one for c-myc induction, which parallels tyrosine kinase activation, and the other for c-fos and c-jun induction, can be transduced from the IL-2 receptor complexes reconstituted on fibroblastoid cells.