The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Putative gamma-subunit of the IL-2 receptor is detected in low, intermediate, and high affinity IL-2 receptor-bearing cells

Three forms of the IL-2R have been reported: low, intermediate, and high affinity. Each form correlates with the expression of two different chains: p55 (alpha-subunit) and p75 (beta-subunit). We report here a putative new subunit of the IL-2R, termed p95-110 or gamma-subunit. This new subunit has a molecular mass of 95 to 110 kDa and is expressed in low, intermediate, and high affinity IL-2R-bearing cells. We propose that p95-110 is the partner of p75 in the formation of intermediate affinity IL-2R, inasmuch as neither p75 nor p95-110 alone can bind IL-2.     

IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.     

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines

We have investigated the effects of IL-2 and IL-4 on different parameters of T cell activation using three T cell lines. The Th cell line L14 and the cytotoxic T cell line C30.1, both grown in IL-2-containing medium, and a line derived from C30.1 cells (line 1) cultured in IL-4 for a prolonged period were studied. All three cell lines could be activated with IL-2 or IL-4. T cell stimulation by either IL-2- or IL-4-induced identical patterns of cell size enlargement and transferrin receptor expression. However, only IL-2 up-regulated cell-surface expression of the p55 subunit of the IL-2R (p55 IL-2R) as measured by flow cytometry and RIA. This difference was also reflected by the accumulation of soluble p55 IL-2R in the culture medium. No significant increase in expression of membrane or soluble forms of p55 IL-2R was detected after IL-4 stimulation. mAb specific for p55 IL-2R which block IL-2-induced T cell growth did not affect IL-4-mediated T cell proliferation indicating that p55 IL-2R is not involved in IL-4-mediated T cell growth. Analysis of IL-4R expression performed on line 1 using biotinylated IL-4 revealed that IL-4, but not IL-2, is capable of increasing IL-4R expression. Together these results suggest that during IL-2- or IL-4-induced T cell proliferation, each lymphokine specifically up-regulates its own receptor.     

Synergistic action of monoclonal antibodies directed at p55 and p75 chains of the human IL-2-receptor

TU27, a mouse IgG1 mAb directed at the p75 chain of the human IL-2R, was analyzed for its ability to interact with IL-2 binding on isolated p75 chains (YT-2C2 cells) and high affinity p55/p75 receptors (human alloreactive T cell clone 4AS), to inhibit IL-2-induced proliferation (4AS cells) and to cooperate with an anti-p55 chain mAb (33B3.1) for inhibiting IL-2 binding and proliferation. TU27 and IL-2 bound to the isolated p75 chain expressed by YT-2C2 cells with respective dissociation constants (Kd) of 1.3 and 1 nM. They cross-inhibited each other for binding with inhibition constants (Ki) in agreement with their respective Kd values. The nature of the interaction was, however, not purely competitive and suggested nonidentical epitopes for the two ligands on the p75 chain. On 4AS cells, IL-2 bound with high affinity (Kd = 50 pM) and TU27 with an affinity similar to that found on YT-2C2 cells. The binding of TU27 and IL-2 were also mutually exclusive on 4AS cells. However, the mechanism of interaction of TU27 with IL-2 was complex since the inhibitory potency of the antibody depended on temperature, antibody preincubation and time of assay. Data obtained at 4 degrees C in the presence of suboptimal, tracer-like concentrations of IL-2 indicated that the intrinsic affinity of TU27 for the high affinity configuration was 15-fold lower than for the isolated p75 chain and argued in favor of the affinity-conversion model (as opposed to the preformed complex model) in which p55 and p75 are dissociated in the absence of IL-2. At 37 degrees C, TU27 inhibited IL-2 binding only on short time assays (6 min). Longer time (30 min) of IL-2 binding resulted in an almost complete disappearance of the effect of TU27, suggesting that internalization of the high affinity p55/p75/IL2 complex enables the cells to escape from the inhibitory effect of TU27. In the presence of the 33B3.1 mAb, the interaction of TU27 with IL-2 resembled the one observed on YT-2C2 cells, suggesting that 33B3.1 is able to inhibit the IL-2-induced association of p55 and p75. Both antibody were found to synergize on 4AS cells, as a result of a cooperative mechanism in which 33B3.1 blocks the formation of the high affinity complex hence allowing TU27 to bind with higher affinity, and TU27 blocks IL-2 binding to the p75 chain. Proliferation studies corroborated the binding experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the soluble murine IL-2R and estimation of its affinity for IL-2

IL-2 induces cells of the cytotoxic T cell line C30.1 to express large numbers of membrane IL-2R (mIL-2R). At the height of activation, these cells also release a soluble form of IL-2R (sIL-2R). Using either crude supernatant or a semi-purified preparation of sIL-2R obtained by affinity chromatography, studies were performed to characterize murine sIL-2R. Its m.w. was determined by both gel filtration and SDS-PAGE. The affinity of sIL-2R for a panel of mAb known to recognize different epitopes of mIL-2R (p55 subunit) was assessed by saturation and competition experiments. The relationship between the various epitopes was studied by cross-inhibition experiments. The data suggest that sIL-2R and mIL-2R (p55 subunit) are structurally similar. The ability of sIL-2R to bind IL-2 was assessed by measuring the dissociation and the inhibition constant of the molecule for IL-2. Both values coincide and indicate that the affinity of sIL-2R for IL-2 is at least 10-fold lower than the that of low affinity mIL-2R. The biologic implications of these findings are discussed.     

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.     

The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.     

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.     

Study of peripheral blood lymphocyte subset distribution and IL-2 receptor (IL-2 R) p55–p75 subunit expression in patients with cancer of different sites

The aim of the study was to evaluate the subset distribution and the IL-2 R p55–p75 subunit expression on unstimulated and phytohemagglutinin (PHA)-stimulated (at 3-d) peripheral blood mononuclear cells (PBMC), of patients with solid cancers of different sites. Indeed the expression of the two subunits of IL-2R is an essential prerequisite for The action of the IL-2 on CD8⁺, CD16⁺ lymphocytes as effectors in antitumor activity (LAK-cells). The subset distribution (CD3, CD4, CD8, CD16, DR) was assessed by cytofluorometry with specific monoclonal antibodies (MAbs); the p55 (CD25) and p75 subunit expression was evaluated by specific MAb (OKT26a and anti-p75). Ninety patients with advanced cancer (mainly non-small cell lung cancer [NSCLC], head and neck cancer, and gynecological cancer; mean age 55 yr; range 27–80) were studied. Thirty-five age- and sex-matched healthy subjects were studied as controls. Our data show that there is no significant difference in the subset distribution between cancer patients and controls. Furthermore, no difference has been found in the expression of p55 subunits on unstimulated PBMC between cancer patients and controls. No difference has been found in the expression of both p55 and p75 subunits on PHA-stimulated PBMC between cancer patients and controls. Our results can support the rationale for further clinical trials with IL-2 in solid malignancies.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

Lymphokine-regulated differential expression of mRNA for p75kDa-IL-2R and p55kDa-IL-2R in a cloned B lymphoma line (BLC1-CL-3 cells)

IL-5 renders BCL1-CL-3 (CL-3) cells responsive to IL-2 by increasing the number of high affinity IL-2R, whereas IL-4 prohibits such action of IL-5 to prepare CL-3 cells responsive to IL-2. Here we have found that genes for p75kDa-IL-2R and p55kDa-IL-2R are differentially regulated by IL-4 and IL-5. Nonstimulated CL-3 cells constitutively express mRNA for p75kDa-IL-2R and p55kDa-IL-2R. IL-5 stimulation principally augments the expression of p75kDa-IL-2R mRNA (4- to 8-fold), although modestly increasing the expression of p55kDa-IL-2R mRNA. Kinetic studies have revealed a maximal increase in p75kDa-IL-2R mRNA expression at 12 h and a decline thereafter, substantiating our previous kinetic study of the expression of high affinity IL-2R after the IL-5 stimulation. By contrast, IL-4 stimulation modestly increases the expression of p75kDa-IL-2R mRNA, whereas markedly reducing the expression of p55kDa-IL-2R mRNA, irrespective of whether CL-3 cells were stimulated with IL-4 alone or together with IL-5 and IL-2. Moreover, addition of IL-4 into the culture containing IL-5 and IL-2 causes striking reduction in the level of J-chain mRNA, which otherwise is markedly induced by stimulation with IL-5 and IL-2. These results clearly illustrate the differential regulation of p75kDa- and p55kDa-IL-2R-gene expression by IL-5 and IL-4, and reinforce our notion that increased expression of high affinity IL-2R induced by IL-5 is responsible for the IL-2 competent state, and decreased expression of p55kDa-IL-2R by IL-4 is responsible for IL-2 unresponsive state.     

IL-4 inhibits the expression of high affinity IL-2 receptors on monoclonal human B cells

In the presence of anti-mu antibodies (anti-microAb), monoclonal B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) can respond to IL-2. In contrast to the effect it exerts on normal B cells, IL-4 does not promote DNA synthesis by B-CLL lymphocytes. Rather this interleukin inhibits the response to IL-2 in all patients' cells that responded to this interleukin. We thus examined whether IL-4 would modulate the number and/or the affinity of IL-2 receptors. A 3-day activation of cells by anti-microAb induced a few hundred high affinity IL-2 receptors (HA-IL-2R) on B-CLL cell surface, as determined by Scatchard analysis. Treatment of cells with IL-4 caused a marked decrease in the number of HA-IL-2R without interfering with the binding ability of IL-2. In contrast with this profound suppressive effect, IL-4 did not down-regulate the expression of each chain, alpha and beta (p55 and p75, respectively), of the HA-IL-2R heterodimer. In fact, the expression of alpha and beta induced by anti-microAb was enhanced by IL-4. Altogether, IL-4 exerts a critical influence on the function and the configuration of HA-IL-2R without inhibiting the expression of two subunits, alpha and beta.     

Studies on IL-1 receptors on D10S T-helper cells: demonstration of two molecularly and antigenically distinct IL-1 binding proteins

Receptor binding studies were performed on the interleukin-1 (IL-1) sensitive T-helper cell line D10S, a stable line which proliferates to subfemtomolar concentrations of IL-1 in the absence of mitogens. IL-1 binds in a specific and saturable manner and Scatchard analysis at 4 degrees C reveals one class of binding affinity. On D10S cells, the Kd for IL-1 is 227 pM +/- 80, with 11,000 (range 3,300 to 23,800) sites per cell. EL4.6.1 cells, which are less sensitive to IL-1, bind with a single class of high affinity sites (55 pM; 4,000 sites). D10S cells incubated 18 h with IL-1 display reduced IL-1 receptor (IL-1R) numbers and affinities, consistent with reduced (75%, p less than 0.005) proliferation to subsequent IL-1; preincubation with IL-4 increases the number of IL-1R which is associated with increased (200%, p less than 0.001) proliferation to IL-1. The molecular mass of the major (80 kD) IL-1R binding [125I]IL-1 alpha on D10S cells was consistently observed at 73 kD as compared to the 80 kD molecule on the EL4 cells. On the other hand, crosslinking studies with [125I]IL-1 beta on D10S cells revealed a novel 46 kD band on gradient SDS-PAGE corresponding to a binding protein of 29 to 30 kD, which is antigenically distinct from the 80 kD IL-1R. Crosslinking of D10S or EL4 cells at 4 degrees C in the presence of phytohemagglutinin (PHA) and labeled IL-1 enhanced the appearance of the 30 kD IL-1 binding protein. The findings are consistent with a two-chain model for the IL-1R, although Scatchard analysis did not consistently indicate two classes of affinities. IL-1 binding to the 80 kD protein may form a heteroduplex with the 30 kD IL-1R which could account for the presence of the 120 to 130 kD IL-1 crosslinked proteins observed by several investigators.     

IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.     

Protein phosphorylation mediated by IL-2/IL-2 receptor beta-chain interaction

We previously found that IL-2 rapidly induced protein phosphorylation of a 67-kDa (pp67) and four 63-kDa (pp63s) cellular proteins in various T cells. Here, we show that the IL-2-stimulated phosphorylation is mediated by the IL-2R beta-chain composed of the high affinity IL-2R, and induced by activation of Ca2+/phospholipid-dependent protein kinase C (PKC). The IL-2-stimulated phosphorylation was always observed in various T cell lines bearing high affinity IL-2R, but never observed in cells which express only low affinity IL-2R consisted of alpha-chain alone. When the expression of high affinity IL-2R was modified by anti-IL-2R mAb for reducing the affinity to 8- to 10-fold lower without affecting the sites of IL-2R, the effective dose of IL-2 on phosphorylation of pp67 increased 8 to 10 times. When cells were treated with pronase, approximately 95% sites of low affinity IL-2R were selectively decreased, but the IL-2 dose dependency for pp67 phosphorylation was little affected. These data exactly suggest that protein phosphorylation in response to IL-2 such as pp67 and pp63s, is mediated by high affinity but not low affinity IL-2R. Furthermore, the IL-2-stimulated phosphorylation of these proteins was also observed in MLA 144 cells which express only low affinity IL-2R consisting of beta-chain alone. In addition, various phorbol esters and tumor promoters, which activate PKC, were also demonstrated to induce the phosphorylation of a pp67 and pp63s in these T cell lines. Therefore, the present study suggests that IL-2/IL-2R beta-chain interaction triggers the phosphorylation of pp67 and pp63s, where the PKC may have an important role.