Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity

A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate. This desmethionine IL-2R alpha and the wild-type IL-2R alpha genes were integrated into a eukaryotic expression vector and transferred into different cell lines. The recipient cell lines express both wild-type and mutant receptor proteins on their cell surfaces which are recognized equally by different monoclonal antibodies. It was possible to establish cell lines with high level IL-2R alpha chain expression by fluorescence-activated cell sorting. The wild-type IL-2R alpha expressed in LTK- cells is a glycoprotein with an apparent molecular size of about 60 kDa and a typical low interleukin-2 binding affinity of KD = 12 nM. Despite the fact that 11 amino acids are altered, no significant difference in the mutant IL-2R alpha is observed, exhibiting the same molecular size and a low interleukin-2 binding affinity of KD = 26 nM.     

Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac)

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.     

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.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by simultaneous activation of distinct signal transduction pathways

The human NK-like leukemic cell line YT was used to study interleukin 2 receptor (IL-2R; Tac) expression induced by activators of distinct signal transduction pathways. Tac expression was induced by active phorbol esters (12-O-tetradecanoylphorbol 13-acetate [TPA] and 4 beta-phorbol 12,13-didecanoate), which directly activate protein kinase C (PKC), as well as forskolin (FK), a stimulator of adenylate cyclase. A synergistic effect on Tac expression was obtained by simultaneous stimulation with optimal concentrations of phorbol esters and FK. Inactive phorbol esters (4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate) and the inactive analog of FK (1,9-dideoxyforskolin) had no effect on Tac expression. The active phorbol esters synergized also with interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in Tac expression. Staurosporine, a potent inhibitor of PKC in vitro, inhibited Tac expression marginally in YT cells stimulated with FK, and enhanced Tac expression in cultures treated with TPA, TNF alpha, or IL-1. Based on the assumption that synergistic effects are observed when two agonists use different signaling pathways, these findings provide evidence that IL-1, TNF, and TPA use different pathways/regulatory elements to regulate Tac expression on the cell surface. Synergistic upregulation of Tac expression by simultaneous activation of distinct pathways may be an important mechanism to modulate the immune response.     

Retention of ligand binding activity by the extracellular domain of the IL-1 receptor

The IL-1R on murine T cells is an 80-kDa cell surface glycoprotein which binds both IL-1 alpha and IL-1 beta. We have recently isolated a cDNA clone encoding this molecule. From the primary sequence mature receptor is predicted to be a 557 residue integral membrane protein with a 319 residue carbohydrate-rich extracellular region. We have constructed a cDNA clone encoding this region of the protein (residues 1 to 316). Expression of this cDNA in HeLa cells leads to secretion of a soluble IL-1 alpha binding protein into the culture medium. Quantitative binding experiments with the truncated receptor show that it possesses IL-1 binding properties which are indistinguishable from those of full length IL-1R. Gel filtration chromatography experiments show that a complex can be formed between a single truncated receptor molecule and a single IL-1 alpha molecule.     

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.     

Identification of a soluble IL-2 receptor beta-chain from human lymphoid cell line cells

A clone was isolated from the human lymphoid cell line YT that displayed IL-2R beta, and was found to express much higher levels of IL-2R beta than the original cells. Combining cell surface iodination, affinity labeling of the released soluble protein, and fluorescence sandwich-ELISA for both IL-2 and IL-2.(soluble)(s)IL-2R beta reactants revealed the presence of IL-2-binding protein in the culture supernatant as soluble forms of IL-2R beta. By using the fluorescence sandwich-ELISA elevated levels of sIL-2R beta were measured in culture supernatants of human T cell leukemia virus I positive T cell lines. In addition to this constitutive production of sIL-2R beta, normal PBMC could release low levels of IL-2R beta by stimulation with PHA. In contrast, this was not found in certain human T cell leukemia virus I negative T cell, B cell and macrophage lines. Immunoprecipitation of the soluble protein with IL-2R beta-specific mAb characterized it as an apparent 50- to 55-kDa molecule that is distinct from the 45-kDa soluble IL-2R alpha. Moreover, 10 to 15% of the total cell surface molecules were released into culture supernatants. These results suggest that the released IL-2R beta might serve as an immunoregulatory function in IL-2 dependent both normal and abnormal immune responses.     

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.     

IL-2 receptor(p55)/Tac-inducing factor. Purification and characterization of adult T cell leukemia-derived factor

Many human T cell lymphotropic virus-I (HTLV-I) transformed T cells from adult T cell leukemia (ATL) patients continuously produce a humoral factor called ATL-derived factor (ADF) which induces IL-2R/Tac expression on T and NK cells. Using gel filtration, procion red Sepharose, DEAE, and reverse phase chromatography, we have purified ADF protein to homogeneity from 15 liters of serum-free culture supernatant of an HTLV-I(+) T cell line ATL-2. Purified ADF protein had the m.w. of 14,000 by SDS-PAGE and gel filtration, and its isoelectric point is around 5.0. ADF did not react with heteroantibodies against IL-1 alpha and IL-1 beta, which have also IL-2R/Tac-inducing activity on suitable target cells. Partial N-terminal amino acid sequence of ADF is different from other cytokines such as, IFN, BSF-2, and various IL whose cDNA has been cloned. Western blot analysis using rabbit antibodies against N-terminal 10mer synthetic peptide of ADF showed that IL-1 alpha and ADF are different proteins. ADF had its IL-2R/Tac-inducing activity not only on human NK-like cell line YT, but also on HTLV-I(+) T cells, such as ED. In contrast, macrophage-derived IL-1 lacked IL-2R/Tac-inducing activity on ED cells despite their IL-2R/Tac induction on YT, indicating that ADF and IL-1 have their effect via different receptors.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

IL-1 alpha stimulates the formation of osteoclast-like cells by increasing M-CSF and PGE2 production and decreasing OPG production by osteoblasts

Interleukin-1alpha (IL-1alpha) is one of the most potent bone-resorbing factors involved in the bone loss that is associated with inflammation. We examined the effect of the inflammatory mediator IL-1alpha on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in rat osteoblasts, and the indirect effect of IL-1alpha on the formation of osteoclast-like cells. Osteoblasts were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with or without 100 units/ml of IL-1alpha for up to 14 days. The gene and protein expression of M-CSF and OPG were estimated by determining mRNA levels using the real-time polymerase chain reaction and protein levels using Western blot analysis. PGE2 expression was determined using an enzyme-linked immunosorbent assay. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from IL-1alpha-treated osteoblasts and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression in osteoblasts increased markedly in cells cultured with IL-1alpha, whereas OPG expression decreased. The conditioned medium containing M-CSF and PGE2 produced by IL-1alpha-treated osteoblasts and soluble RANKL increased the TRAP staining of osteoclast precursors. These results suggest that IL-1alpha stimulated the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production, and a decrease in OPG production by osteoblasts.     

Interleukin 2 receptor expression by T cells in human aging

Aged individuals have depressed cell-mediated immunity and diminished T cell proliferation to mitogenic and antigenic stimuli. Because T cell responses depend on the surface expression and normal function of interleukin 2 receptors, we measured the quantities and affinities of cell surface IL-2R and the amount of soluble IL-2R alpha chain (p55) release in vitro in PHA-stimulated mononuclear cells from healthy aged (greater than or equal to 65 years old) and young (less than or equal to 39 years old) donors. At the peak of the PHA response, the fraction of cells expressing IL-2R alpha chain (CD25+) was lower in the aged (43% vs 56%, P = 0.033). Relative to the lower proliferation and CD25 expression, old donor cells released unexpectedly high quantities of soluble alpha chain into culture supernatants. However, the average affinities and the mean numbers of high- and low-affinity surface receptors per CD25+ cell were equivalent in cells from eight pairs of aged and young donors (1850 vs 1586 high affinity, and 20,655 vs 23,466 low affinity, P greater than 0.2 for both). The soluble IL-2R released by stimulated cells had no effect on proliferative responses, because addition of saturating doses of exogenous recombinant IL-2 did not increase cellular proliferation, and addition of soluble anchor-minus recombinant IL-2R alpha chain did not suppress it. These results indicate that in healthy older individuals, diminished numbers of T cells can be induced to express cell surface IL-2R following mitogenic stimulation, although aged CD25+ can express a normal complement of IL-2R molecules. In the aged, either CD25+ cells release excessive quantities or a subset of cells synthesizes and releases soluble IL-2R alpha chain into the extracellular environment without expressing it on the cell surface.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by interleukin 1 or tumor necrosis factor alpha in combination with cAMP inducing agents

This study demonstrates synergistic effects on Tac expression by interleukin 1 (IL-1) or tumor necrosis factor alpha (TNF alpha) in combination with the adenylate cyclase stimulator, forskolin (FK), as well as by IL-1 with TNF alpha in the human NK-like leukemic cell line YT. The maximal expression level (greater than 80% positive cells) obtained with FK plus IL-1 or FK plus TNF alpha could not be obtained by increasing the concentration of either agent alone. Furthermore, we demonstrate that Tac protein expression is correlated with increased steady-state Tac mRNA levels. Other agents that increase intracellular cAMP, such as prostaglandin E (PGE) or isobutyl-methylxanthine (IBMX), also synergized with IL-1 or TNF alpha (but not with FK). The findings suggest that cAMP plays a role in regulating Tac expression in YT cells, and that IL-1, TNF, and FK use distinct signal transduction mechanisms, all resulting in the same end point effect, namely, induction of Tac mRNA and cell surface protein expression.     

Identification of a novel receptor/signal transduction pathway for IL-15/T in mast cells.

Interleukin-15/T(IL-15) is a growth factor that utilizes IL-2 receptor (IL-2R) components in addition to its private binding protein IL-15R(alpha) in T-cells. Here, we report that IL-15 induces mast cell proliferation in the absence of IL-2R alpha and beta. Using transfectants of these cells with a cytoplasmic-truncated mutant of gamma(c), we demonstrated that IL-15 signaling in mast cells does not involve gamma(c). Cross-linking of mast cells with [(125)I]IL-15 revealed a 60-65 kDa IL-15 binding protein that is distinct from known components of T-cell IL-15 receptors. Mast cell IL-15 receptors recruit JAK-2 and STAT-5, instead of JAK1/3 and STAT3/5 that are activated in T-cells. Thus IL-15 is a mast cell growth factor that utilizes a novel receptor and distinct signaling pathway.     

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.     

Identification, cloning, and characterization of a novel soluble receptor that binds IL-22 and neutralizes its activity

With the use of a partial sequence of the human genome, we identified a gene encoding a novel soluble receptor belonging to the class II cytokine receptor family. This gene is positioned on chromosome 6 in the vicinity of the IFNGR1 gene in a head-to-tail orientation. The gene consists of six exons and encodes a 231-aa protein with a 21-aa leader sequence. The secreted mature protein demonstrates 34% amino acid identity to the extracellular domain of the IL-22R1 chain. Cross-linking experiments demonstrate that the protein binds IL-22 and prevents binding of IL-22 to the functional cell surface IL-22R complex, which consists of two subunits, the IL-22R1 and the IL-10R2c chains. Moreover, this soluble receptor, designated IL-22-binding protein (BP), is capable of neutralizing IL-22 activity. In the presence of the IL-22BP, IL-22 is unable to induce Stat activation in IL-22-responsive human lung carcinoma A549 cells. IL-22BP also blocked induction of the suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL-22 in HepG2 cells. To further evaluate IL-22BP action, we used hamster cells expressing a modified IL-22R complex consisting of the intact IL-10R2c and the chimeric IL-22R1/gammaR1 receptor in which the IL-22R1 intracellular domain was replaced with the IFN-gammaR1 intracellular domain. In these cells, IL-22 activates biological activities specific for IFN-gamma, such as up-regulation of MHC class I Ag expression. The addition of IL-22BP neutralizes the ability of IL-22 to induce Stat activation and MHC class I Ag expression in these cells. Thus, the soluble receptor designated IL-22BP inhibits IL-22 activity by binding IL-22 and blocking its interaction with the cell surface IL-22R complex.     

Interleukin 5 up-regulates high-affinity interleukin 2 receptor expression by human resting peripheral T cells: a comparison with the effect of interleukin 4 on B cells.

The separate regulation mechanisms of cytokines on two classes of interleukin 2 receptors (IL-2R) on human peripheral T and B cells were analyzed by a flow cytometer using a double stain with IL-2R alpha (55 kilodalton Tac) and IL-2R beta (75 kilodalton mik beta 1, mik beta 3). Although the expression of IL-2R alpha by T cells was slightly enhanced by IL-2 and IL-4, expression of the beta chain was diminished by both cytokines. IL-5 by itself did not alter the expression of either IL-2R alpha or beta, but preculturing with IL-2 for 24 h followed by IL-5 for another 24 h induced an increase in IL-2R alpha expression and in simultaneous alpha/beta chain expression. Increased numbers of high-affinity IL-2R were confirmed by 125I binding assays. On B cells, IL-4 increased alpha, beta, and simultaneous alpha/beta chain expression, but IL-4-treated B cells did not show an increased number of high-affinity IL-2R.