Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors.

A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.     

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.     

Receptor binding and internalization of mouse interleukin-2 derivatives that are partial agonists.

Mouse interleukin-2 (mIL-2) mutant proteins with subunit-specific receptor binding defects have been previously described. Some of these mutant proteins are unable to trigger a maximum proliferative response of T cells. In this study, mIL-2 and mIL-2 mutant proteins were labeled with 32P, and their association and dissociation kinetics with the high affinity IL-2 receptor (IL-2R) were investigated. A mIL-2 mutant protein with a partial defect in binding to the low affinity component of IL-2R had a slower on-rate than mIL-2. On the other hand, a mIL-2 antagonist with a binding defect to the intermediate affinity component of IL-2R had a normal on-rate, whereas its off-rate at 37 degrees C was faster than mIL-2. This fast off-rate at physiological temperature interfered with mIL-2 internalization. When three mIL-2 partial agonists, each inducing a different maximal response, were examined, no difference was found between their dissociation rates or their internalization properties. The significance of these findings for the function of each receptor subunit in the IL-2R complex, as well as for the mechanism of activation of the receptor, is discussed.     

Identification and cellular distribution of distinct proteins forming human GM-CSF receptor.

Two proteins forming the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF)1 were identified and characterized. One with apparent Mr of about 80,000 was defined as alpha-chain and has Kd of 0.7-2.8 nM. The other binding molecule with apparent Mr of about 135,000 was defined as beta-chain and is related to the high-affinity binding with Kd of 10-40 pM. The binding kinetic studies confirmed that the 125I-GM-CSF associated slower to and dissociated more rapidly from the alpha-chain than the beta-chain. The alpha-chain is expressed not only on hemopoietic cells but also on full-term placental tissues, choriocarcinoma cells, and other solid tumor cells. In contrast, the distribution of the beta-chain is restricted on hemopoietic cells. The alpha-chain probably corresponds to the low-affinity GM-CSF receptor whose cDNA has been cloned and sequenced.     

Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors.

The molecular basis for binding of alpha-macroglobulin-proteinase complexes to the human two-chain 500/85-kDa (alpha/beta) alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein was analyzed. Ligand blotting experiments showed that a 40-kDa protein, present in the affinity-purified alpha 2MR preparation, is bound to the alpha 2MR alpha-chain and released by heparin. Removal of the 40-kDa protein resulted in a 3-5-fold increase in binding of alpha 2M-trypsin. Nitrocellulose-immobilized pure two-chain alpha 2MR was incubated with human alpha 2M-trypsin, containing four identical subunits, and two monovalent ligands: rat alpha 1-inhibitor-3-chymotrypsin and the 18-kDa receptor binding fragment of the alpha 2M subunit. Binding of alpha 2M-trypsin to the alpha-chain of immobilized alpha 2MR was composed of a high (Kd = 40 pM at 4 degrees C) and a low (Kd = 2 nM) affinity component. alpha 1-Inhibitor-3-chymotrypsin bound to the same sites but with one component (Kd = 0.4 nM). Competition-inhibition experiments and dissociation experiments, using ligands with different valences, as well as experiments with alpha 2MR immobilized at different densities, led to the following model. The low (Kd = 2 nM) affinity of alpha 2M-proteinase is prevalent when only one of the four domains binds to alpha 2MR, i.e. when the receptor density is low or when neighboring receptors are occupied. The high (Kd = 40 pM) affinity is achieved by binding of at least two domains to adjacent receptors.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.     

Differential coupling of smooth muscle and liver vasopressin (V1) receptors to guanine nucleotide binding proteins

We report the reconstitution of the smooth muscle vasopressin V1 receptor functionally coupled to a pertussis toxin-insensitive guanine nucleotide-binding protein. This V1 receptor was spontaneously coupled to this guanine nucleotide-binding protein upon solubilization in the absence of agonist, in contrast to our earlier report on the liver V1 receptor, which required agonist for coupling. The smooth muscle V1 receptor was reconstituted as a high affinity receptor (Kd = 5 nM), with a slow rate of agonist dissociation. Upon the addition of guanosine 5'-thiotriphosphate, there was a decrease in receptor affinity (Kd = 30 nM) concomitant with an increase in the rate of ligand dissociation. The ability of the smooth muscle V1 receptor to spontaneously couple to a guanine nucleotide-binding protein(s) suggests that in the absence of agonist it exists as a high affinity receptor. The smooth muscle V1 receptor may, therefore, be more sensitive to plasma concentrations of vasopressin than its liver homologue.     

Purification of the murine interleukin 3 receptor.

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.     

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.     

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.     

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.     

Studies on adrenocorticotropic hormone receptor using isolated rat adrenocortical cells.

To clarify the function of ACTH receptors, the actions of ACTH on cyclic AMP formation, Ca2+-influx across cell membrane, and corticoidogenesis were examined using dispersed adrenocortical cells prepared from the rat adrenal gland. 1) There are two types of ACTH receptors from Scatchard analysis of 125I-ACTH1-24 binding to the cell, the one receptor is of high affinity and low capacity (dissociation constant (Kd1) = 2.6 x 10(-19) M and 7,350 sites per cell), and the other one is of low affinity and high capacity (dissociation constant (Kd2) = 7.1 x 10(-9)M and 57,400 sites per cell). 2) Both apparent dissociation constants derived from the effects of ACTH on corticoidogenesis and Ca2+ influx well correspond with Kd1 of the high affinity receptor, 3) Apparent dissociation constant obtained from the effect of ACTH on cyclic AMP formation is in good agreement with Kd2 of the low affinity receptor. Thus it could be deduced from these data that the high affinity receptor is concerned with an increased Ca2+-influx to regulate corticoidogenesis at physiological levels of ACTH, whereas the low affinity receptor is coupled to adenylate cyclase at supraphysiological concentrations of ACTH.     

An associated molecule, p64, with IL-2 receptor beta chain. Its possible involvement in the formation of the functional intermediate-affinity IL-2 receptor complex.

We identified previously a membrane molecule, p64, which co-precipitates with the IL-2R beta-chain in human T cells. We have now investigated the biologic significance of p64 in the formation of the functional IL-2R complex with cell lines transfected with cDNA of IL-2R alpha- and/or beta-chains. Two functional parameters associated with IL-2R, IL-2 binding ability and association of p64 with the beta-chain, were examined. Two subclones, MOLT beta-11 and MOLT beta-12, of an IL-2R beta cDNA-transfected MOLT4 clone expressed similar numbers of IL-2R beta molecules on cell surfaces and bound to IL-2 with intermediate affinity. However, the numbers of IL-2 binding sites were significantly lower than those of IL-2R beta molecules and considerably different between the two subclones. The amount of p64 co-precipitated with IL-2R beta was proportional to numbers of the IL-2 binding sites in the two subclones. In addition, neither p64 co-precipitation nor IL-2 binding was detected in HeLa and COS7 cells transfected with IL-2R beta, and no p64 precipitation was seen even in those transfectants with both IL-2R alpha and beta cDNAs, which bind to IL-2 with high affinity but are not able to transduce intracellular signals. These results suggest the possibility that p64 associates with IL-2R beta and has an important role in formation of the functional IL-2R complex.     

Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs.     

Endothelins activate Na+/H+ exchange in brain capillary endothelial cells via a high affinity endothelin-3 receptor that is not coupled to phospholipase C.

Endothelial cells from brain microvessels (BCEC) express high affinity receptor sites for endothelin-1 that recognize endothelin-3 with a low affinity (Vigne, P., Marsault, R., Breittmayer, J.P. & Frelin, C. (1990) Biochem. J. 266, 415-420). Binding experiments using 125I-endothelin-3 showed the presence in BCEC of a new class of receptor sites that had a high affinity for endothelin-3 (Kd = 0.8 nM), endothelin-1 (Kd = 0.8 nM), and sarafotoxin S6b (Kd = 0.3 nM). Endothelins activated phospholipase C in BCEC and produced transient increases in intracellular Ca2+ with properties of a low affinity endothelin-3 receptor. Endothelins also increased 22Na+ uptake via the Na+/H+ antiporter in BCEC. Concentrations for half-maximum activation (endothelin-1, 0.5 nM; sarafotoxin S6b, 1 nM; endothelin-3, 2 nM) were close to the Kd values determined in 125I-endothelin-3-binding experiments. The action of endothelins on Na+/H+ exchange was not mimicked by phorbol myristate acetate, it was not reversed by staurosporine, and it did not correlate with the phosphorylation of the 80-kDa protein. These results indicated that the action of endothelins on Na+/H+ exchange did not involve protein kinase C. It is concluded that BCEC coexpress two types of functional receptor sites for endothelins: (i) a high affinity endothelin-1, low affinity endothelin-3 receptor that is coupled to phospholipase C and to intracellular Ca2+ mobilization, and (ii) a high affinity endothelin-1, high affinity endothelin-3 receptor that controls Na+/H+ exchange activity via a protein kinase C-independent mechanism.     

Selective availability of IL-2 is a major determinant controlling the production of CD4+CD25+Foxp3+ T regulatory cells

The development and maintenance of T regulatory (Treg) cells critically depend on IL-2. This requirement for IL-2 might be due to specificity associated with IL-2R signal transduction or because IL-2 was uniquely present in the niche in which Treg cells reside. To address this issue, we examined the capacity of IL-7R-dependent signaling to support Treg cell production and prevent autoimmunity in IL-2Rbeta(-/-) mice. Expression of transgenic wild-type IL-7R or a chimeric receptor that consisted of the extracytoplasmic domain of the IL-7R alpha-chain and the cytoplasmic domain of IL-2R beta-chain in IL-2Rbeta(-/-) mice did not prevent autoimmunity. Importantly, expression of a chimeric receptor that consisted of the extracytoplasmic domain of the IL-2R beta-chain and the cytoplasmic domain of IL-7R alpha-chain in IL-2Rbeta(-/-) mice led to Treg cells production in the thymus and periphery and prevented autoimmunity. Signaling through the IL-2R or chimeric IL-2Rbeta/IL-7Ralpha in vivo or the culture of thymocytes from IL-2Rbeta(-/-) mice with IL-7 led to up-regulation of Foxp3 and CD25 on Treg cells. These findings indicate that IL-7R signal transduction is competent to promote Treg cell production, but this signaling requires triggering through IL-2 by binding to the extracytoplasmic portion of the IL-2R via this chimeric receptor. Thus, a major factor controlling the nonredundant activity of the IL-2R is selective compartmentalization of IL-2-producing cells with Treg cells in vivo.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.