Expression and the functional role of a p70/75 interleukin 2-binding molecule in human B cell

Expression of p70/75 IL-2-binding molecules and their functional roles in induction of Ig secretion by IL-2 were examined in human B cells. IL-2, at high concentrations induced higher levels of Ig secretion in Staphylococcus aureus strain Cowan I (SAC)-activated B cells than at low concentrations. About 50% of SAC-activated B cells, lacking Tac antigen, were also responsive to Ig secretion by IL-2, although the required dose of IL-2 was higher than that for Tac-positive B cells. H-31 antibody which recognizes Tac antigen did not inhibit the induction of Ig secretion by high concentrations of IL-2 in both Tac-negative and Tac-positive B cells, suggesting that IL-2 might induce Ig secretion through a receptor distinct from Tac antigens. In contrast, IL-2 was ineffective in the absence of SAC stimulation even at high concentrations. Upon analysis by SDS-PAGE, p70/75 IL-2-binding molecules were detected on Tac-negative SAC-activated B cells. Similar IL-2-binding molecules distinct from Tac antigen (p55) were detected in both Tac-positive B and T cells. However, neither p55 nor p70/75 IL-2-binding molecules could be detected in the absence of SAC stimulation. These observations suggest that p70/75 IL-2 binding molecules are induced in human B-cells in the presence or absence of Tac antigen by SAC stimulation and these determinants play an important function in the transduction of IL-2 associated signal for B cell differentiation.     

Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.     

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.     

Novel receptor-mediated internalization of interleukin 2 in B cells

Novel IL-2-binding molecules (p70 and p75) mediating internalization and degradation of IL-2 were examined by employing a human B lymphoblastoid line, SKW6-4 cells. High concentrations of IL-2 induced IgM secretion in these cells through a receptor distinct from Tac antigen. The acid-wash technique revealed that more than 60% of 125I-labeled IL-2 bound to the cells became acid-unremovable in the first 30 min of incubation at 37 degrees C and degradation products of 125I-IL-2 increased after 30 min of incubation. Treatment of the cells with NaN3 buffer inhibited the appearance of acid-unremovable 125I-IL-2, suggesting that acid-unremovable 125I-IL-2 was not due to fluid-phase pinocytosis but due to internalization. Loss of labeled bands by incubation of cells with 125I-IL-2 at 37 degrees C before affinity cross-linking demonstrated that 125I-IL-2 was internalized via novel IL-2-binding molecules. These results suggest that novel IL-2-binding molecules are responsible for internalization and may mediate signal transduction in B cells in the absence of Tac antigen.     

Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H-31 and H-A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL-2) receptor (IL-2 R), anti-Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H-31 and H-A26 antibodies both reacted with only IL-2 R-positive cells, and they precipitated IL-2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T-cell leukemia virus type I (HTLV-I)-carrying MT-2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL-2 R with anti-Tac. Antibody-binding competition assays showed that H-31 and anti-Tac, and H-A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti-Tac also inhibited the binding of HIEI but not vice versa. H-31, like anti-Tac, strongly inhibited the IL-2-dependent proliferation of normal activated T-cells, absorption of IL-2 and direct binding of IL-2 to the cells, while H-A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV-I infection were different, as revealed by immunofluorescence studies. Human IL-2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL-2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL-2 R molecules. These observations indicate that H-31 and H-A26 recognize human IL-2 R molecules and that the antigenic sites on the IL-2 R molecule defined by H-31, H-A26, anti-Tac, and HIEI are different.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Structural analysis of high- versus low-affinity interleukin-2 receptors by means of selective expression of distinct receptor classes.

Activated T cells express at least two distinct affinity classes of interleukin-2 (IL-2) receptors. The number of low-affinity receptors per cell is normally 10-30 times greater than that of the high-affinity receptors, and the difference in the dissociation constant between the two classes of receptors is in the order of 1,000-fold. In this report normal human T cells are used in a cellular system in which the number of low-affinity receptors can be manipulated. It is demonstrated that a cell population could be achieved with such low levels of low-affinity IL-2 receptors that almost half of the surface pool of anti-IL-2 receptor antibody (anti-Tac) binding sites represented high-affinity receptors. By using this cellular system it was possible to show that anti-Tac recognizes both receptor classes with similar affinity and that IL-2 inhibits Tac binding to both receptor classes in a competitive fashion. Tac antigens were purified from surface 125I-labeled cells expressing high levels of high-affinity IL-2 receptors, but low levels of the low-affinity receptor class, and this preparation was compared with another pool of Tac antigens obtained from cells expressing the normal 10- to 20-fold excess of low-affinity IL-2 binding sites over high-affinity IL-2 receptors. Biochemical characterization by peptide mapping by limited proteolysis and two-dimensional gel analysis revealed that these distinct preparations of Tac antigens were indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the beta-subunit of the IL-2 receptor induces MHC-unrestricted cytotoxicity in T acute lymphoblastic leukemia cells and normal thymocytes

Recently, several laboratories have identified a novel protein(s) of 70,000 to 75,000 Da (IL-2R beta-subunit) that, when expressed with the p55 Tac protein (alpha-subunit), imparts high affinity IL-2 binding. Expression of the beta-subunit mediates acquisition of MHC-unrestricted cytotoxicity in large granular lymphocytes. We report that thymocytes and T acute lymphoblastic leukemia cells express the beta-subunit of the IL-2R in the absence of detectable alpha-subunit expression and that IL-2 induces acquisition of MHC-unrestricted cytotoxic activity in these cells through stimulation of the beta-subunit.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.     

Impaired expression of high affinity interleukin 2 receptor on activated lymphocytes from patients with systemic lupus erythematosus

Peripheral lymphocytes stimulated with phytohemagglutinin (PHA-blasts) were examined for their responsiveness to exogenous interleukin 2 (IL-2). The proliferative response of PHA-blasts to IL-2 was significantly lower in patients with systemic lupus erythematosus (SLE) than in normal subjects. To clarify the reason for this defect, the expression of IL-2 receptor (IL-2R) on PHA-blasts was investigated using anti-Tac antibody and purified IL-2. Cytofluorometric analysis showed no statistical differences in the Tac positivity of PHA-blasts among normal subjects and patients with active and inactive SLE. Scatchard analysis using 125I-labeled anti-Tac monoclonal antibody revealed that the number of Tac epitopes on PHA-blasts was also not different among them. Next, the affinity of IL-2R expressed on PHA-blasts was determined by Scatchard analysis using radiolabeled IL-2 as a ligand. The number of high affinity IL-2R on the PHA-blasts was significantly decreased in patients with active and inactive SLE, as compared with normal subjects. The responsiveness of PHA-blasts to exogenous IL-2 was well correlated to the number of high affinity IL-2R, but not to the number of Tac epitopes or total IL-2R. Inasmuch as high affinity components of IL-2R are functionally active, the defective expression of high affinity IL-2R may be responsible for the T cell dysfunctions in SLE.     

Trypanosoma cruzi reduces the number of high-affinity IL-2 receptors on activated human lymphocytes by suppressing the expression of the p55 and p70 receptor components

We previously established that Trypanosoma cruzi, the causative agent of Chagas' disease, has the ability to suppress expression of the p55 component of the IL-2R by activated human PBMC. We explored in this work whether the parasite alters the expression of high affinity IL-2R, responsible for the internalization of IL-2 and signal transduction. Radiobinding measurements revealed that the trypanosome indeed inhibited the expression of high affinity IL-2R. Thus, a considerably smaller number of 125I-IL-2 molecules was necessary to saturate the IL-2R on PHA-stimulated PBMC cocultured with T. cruzi than those of control PBMC that had not been exposed to the organisms. Scatchard analysis of equilibrium binding data showed that, in the presence of T. cruzi, the number of high affinity IL-2R per cell was reduced by approximately 80%. The Kd for IL-2 binding to the fewer IL-2R expressed on PBMC exposed to T. cruzi was not significantly different from that of IL-2R on nonsuppressed PBMC. Independent measurements made after cross-linking 125I-IL-2 to its specific receptors with disuccinimidylsuberate showed that both the p55 and p70 components of the IL-2R were markedly suppressed and to comparable extents. These results demonstrate for the first time that T. cruzi suppresses the expression of high affinity IL-2R by human cells, including the p70 chain of the heterodimeric IL-2R. It is noteworthy that the in vitro model system we used in this work to study the mechanisms whereby T. cruzi may induce the immunosuppression that accompanies acute Chagas' disease also lends itself to the exploration of the regulatory mechanisms governing the expression of IL-2R by human PBMC.     

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: functional and structural characterization

Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the 3H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29-186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.     

Biochemical evidence for a third chain of the interleukin-2 receptor

Two receptor proteins that specifically bind interleukin-2 (IL-2) have been identified previously. The L (Tac or alpha) chain can bind IL-2 with a Kd value of 10 nM (low affinity). Although the H (beta) chain expressed on lymphocytes can bind IL-2 with a Kd value of 1 nM (intermediate affinity), transfected fibroblasts expressing the H chain cannot bind IL-2, suggesting the involvement of other lymphocyte-specific factors for the function of the H chain. To obtain direct evidence for the presence of a third component of the IL-2 receptor, we examined the IL-2 binding activity of detergent-solubilized cell membrane preparations. We found that lysates of transfected Cos7 cells expressing H chains can bind IL-2 when mixed with lysates from lymphocytes that cannot bind IL-2. Chemical cross-linking of 125I-IL-2-bound lysate mixture and subsequent immunoprecipitation with a noncompetitive anti-H chain antibody gave rise to two 125I-IL-2-bound proteins, a 56-kDa protein (p56) and the H chain, although neither the H chain nor p56 alone is able to bind IL-2. These results indicate that p56 is the IL-2 receptor third chain that is required for IL-2 binding to the H chain. A similar lysate mixing experiment also showed that p56 is involved in IL-2 binding to the high affinity IL-2 receptor by forming the quaternary complex of IL-2, p56, L chain, and H chain.     

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.