Anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit

The interleukin-2 receptor (IL-2R) is composed of one affinity-modulating subunit (IL-2Ralpha) and two essential signaling subunits (IL-2Rbeta and gammac). Although most known signaling events are mediated through tyrosine residues located within IL-2Rbeta, no functions have yet been ascribed to gammac tyrosine residues. In this study, we describe a role for gammac tyrosines in anti-apoptotic signal transduction. We have shown previously that a tyrosine-deficient IL-2Rbeta chain paired with wild type gammac stimulated enhancement of bcl-2 mRNA in IL-2-dependent T cells, but it was not determined which region of the IL-2R or which pathway was activated to direct this signaling response. Here we show that up-regulation of Bcl-2 by an IL-2R lacking IL-2Rbeta tyrosine residues leads to increased cell survival after cytokine deprivation; strikingly, this survival signal does not occur in the absence of gammac tyrosine residues. These gammac-dependent signals are revealed only in the absence of IL-2Rbeta tyrosines, indicating that the IL-2R engages at least two distinct signaling pathways to regulate apoptosis and Bcl-2 expression. Mechanistically, the gammac-dependent signal requires activation of Janus kinases 1 and 3 and is sensitive to wortmannin, implicating phosphatidylinositol 3-kinase. Consistent with involvement of phosphatidylinositol 3-kinase, Akt can be activated via tyrosine residues on gammac. Thus, gammac mediates an anti-apoptotic signaling pathway through Akt which cooperates with signals from its partner chain, IL-2Rbeta.     

IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development

Common gamma chain (gammac)-receptor dependent cytokines are required for regulatory T cell (Treg) development as gammac(-/-) mice lack Tregs. However, it is unclear which gammac-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4(+)Foxp3(+) Tregs in the thymus express the IL-2Rbeta, IL-4Ralpha, IL-7Ralpha, IL-15Ralpha, and IL-21Ralpha chains, but not the IL9Ralpha or TSLPRalpha chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4(+)Foxp3(+) Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4(+)CD25(+)Foxp3(-) thymic Treg progenitors into CD4(+)Foxp3(+) mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rbeta(-/-) x IL-7Ralpha(-/-) and IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice. We found that IL-2Rbeta(-/-) x IL-7Ralpha(-/-) mice were devoid of Tregs thereby recapitulating the phenotype observed in gammac(-/-) mice; in contrast, the phenotype observed in IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice was comparable to that seen in IL-2Rbeta(-/-) mice. Finally, we observed that Tregs from both IL-2(-/-) and IL-2Rbeta(-/-) mice show elevated expression of IL-7Ralpha and IL-15Ralpha chains. Addition of IL-2 to Tregs from IL-2(-/-) mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Ralpha and IL-15Ralpha chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.     

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.     

Signaling domains of the interleukin 2 receptor

Interleukin (IL-)2 and its receptor (IL-2R) constitute one of the most extensively studied cytokine receptor systems. IL-2 is produced primarily by activated T cells and is involved in early T cell activation as well as in maintaining homeostatic immune responses that prevent autoimmunity. This review focuses on molecular signaling pathways triggered by the IL-2/IL-2R complex, with an emphasis on how the IL-2R physically translates its interaction with IL-2 into a coherent biological outcome. The IL-2R is composed of three subunits, IL-2Ralpha, IL-2Rbeta and gammac. Although IL-2Ralpha is an important affinity modulator that is essential for proper responses in vivo, it does not contribute to signaling due a short cytoplasmic tail. In contrast, IL-2Rbeta and gammac together are necessary and sufficient for effective signal transduction, and they serve physically to connect the receptor complex to cytoplasmic signaling intermediates. Despite an absolute requirement for gammac in signaling, the majority of known pathways physically link to the receptor via IL-2Rbeta, generally through phosphorylated cytoplasmic tyrosine residues. This review highlights work performed both in cultured cells and in vivo that defines the functional contributions of specific receptor subdomains-and, by inference, the specific signaling pathways that they activate-to IL-2-dependent biological activities.     

A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit,IL-23R

IL-23 is a heterodimeric cytokine composed of the IL-12p40 "soluble receptor" subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rbeta1. However, it does not use IL-12Rbeta2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, "IL-23R." IL-23R pairs with IL-12Rbeta1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rbeta1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rbeta2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rbeta2.     

Interleukin-2 (IL-2) receptor-betagamma signalling is activated by c-Kit in the absence of IL-2, or by exogenous IL-2 via JAK3/STAT5 in human papillomavirus-associated cervical cancer

Activation of the interleukin-2 receptor (IL-2R) induces signalling cascades promoting T cell proliferation. However, signal transduction pathways triggered in IL-2R-expressing solid tumours are unknown. This report shows that human papillomavirus (HPV)-associated cervical cancer cells express an IL-2R composed of beta and gamma chains (IL-2Rbetagamma), and that IL-2-mediated activation increases the phosphorylation of JAK3 and STAT5, stimulating cell proliferation. Interestingly, endogenous IL-2 is not produced by these cells, suggesting the activation of IL-2Rbetagamma by an alternative mechanism. Accordingly, we found that Stem Cell Factor (SCF)-activated c-Kit induces phosphorylation of the IL-2Rbeta chain in the absence of IL-2. Moreover, inhibition of IL-2Rbeta phosphorylation by blocking c-Kit tyrosine kinase activity abolishes both, IL-2 and SCF-mediated proliferation. Thus, these results demonstrate that IL-2 triggers a JAK3/STAT5 cascade in HPV-associated cervical cancer cells expressing IL-2Rbetagamma, and that this receptor can be alternatively activated by SCF-activated c-Kit in the absence of IL-2.     

Endogenous IL-15 sustains recruitment of IL-2Rbeta and common gamma and IL-2-mediated chemokine production in normal and inflamed human gingival fibroblasts

We recently reported that anti-IL-15 neutralizing mAb has been shown to inhibit production of MCP-1 in response to IL-2 from normal human gingival fibroblasts (HGF), the major constituent of gingival tissue. In the present study, we examined the expression of IL-2R and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rbeta and common gamma-chain (gammac) but not IL-2Ralpha or IL-15Ralpha, whereas inflamed HGF expressed IL-2Ralpha, IL-15Ralpha, IL-2Rbeta, and gammac, as assessed by RT-PCR and flow cytometry. Exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed the 48 aa-IL-15 isoform, and the isoform was not actively secreted but rather existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb for 24 h completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Jak 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rbeta and gammac expression at mRNA and protein levels. Furthermore, excess amounts of IL-2 restored the inhibitory effect of anti-IL-15, inhibition of NF-kappaB abrogated the expression of IL-2Rbeta and gammac, and IL-2-induced-nuclear translocation of NF-kappaB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rbeta and gammac through activation of NF-kappaB in HGF.     

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.     

Crystal Structure of the interleukin-15.interleukin-15 receptor alpha complex: insights into trans and cis presentation

Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

Structural analysis and modeling of a synthetic interleukin-2 mimetic and its interleukin-2Rbeta2 receptor

Peptide p1-30, which is composed of the 30 amino-terminal residues (alpha-helix A) of human interleukin-2 (IL-2), binds as a tetramer to the dimeric IL-2Rbeta2 receptor, whereas the entire IL-2 recognizes the tricomponent receptor IL-2Ralphabetagamma. p1-30 is an IL-2 mimetic that activates CD8 low lymphocytes and natural killer cells, because these cells produce IL-2Rbeta constitutively. It also induces a strong lymphokine-activated killer cell response. A series of truncated peptides were analyzed by circular dichroism and analytical centrifugation to elucidate the role of p1-30 residues. We propose a model where residues 10-30 of the p1-30 peptide form an alpha-helix with eight hydrophobic side chains on the same surface buried in a hydrophobic core when four anti-parallel helices combine to form a bundle. IL-2Rbeta dimerization was further studied, and three-dimensional models of the free IL-2Rbeta2 receptor and the p1-304.IL-2Rbeta2 complex were built by comparative modeling based on the crystal structure of the erythropoietin receptor complex, because this belongs to the same hematopoietin family as IL-2. These models suggest that binding of the p1-30 tetramer rotates the COOH-terminal domains and brings both transmembrane regions 50 A closer together, driving the association of the two intracytoplasmic domains that would transduce the signal into the cytoplasm.     

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.     

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.     

ER Reorganization is Remarkably Induced in COS-7 Cells Accumulating Transmembrane Protein Receptors Not Competent for Export from the Endoplasmic Reticulum

The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.     

Induction of experimental autoimmune encephalomyelitis in IL-12 receptor-beta 2-deficient mice: IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system

IL-12 is thought to be involved in the susceptibility to experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. IL-12 signals through a heterodimeric receptor (IL-12Rbeta1/IL-12Rbeta2), whose beta2-chain is up-regulated on activated, autoreactive Th1 cells. Contrary to the expectation that the absence of IL-12Rbeta2 would protect from EAE, we found that IL-12Rbeta2-deficient mice developed earlier and more severe disease, with extensive demyelination and CNS inflammation. The inflammatory cells were mainly comprised of CD4(+) T cells, monocyte/macrophages, and dendritic cells. Compared to wild-type mice, IL-12Rbeta2-deficient mice exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-alpha, GM-CSF, IL-17, IL-18/IL-18Ralpha, and NO. In addition, we found significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12Rbeta2(-/-) mice compared with wild-type mice. These findings indicate that IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS, and that, in the absence of IL-12Rbeta2, increased IL-23 and other inflammatory molecules may be responsible for increased severity of EAE.     

Oligomerization of IL-2Ralpha

Interleukin (IL) 2 receptor subunit alpha (IL-2Ralpha) increases the affinity of the IL-2 receptor complex while hetero-association of IL-2Rbeta and gamma(c) chains initiates a proliferative signal. We show here that IL-2Ralpha is necessary for receptor clustering required for augmentation of IL-2 signalling. Cells expressing chimeras incorporating the extracellular domain of IL-2Ralpha demonstrated IL-2 independent homo-association of the IL-2Ralpha chimera. Singly or co-transfected IL-2Rbeta and gamma(c) chimeras showed no spontaneous or IL-2-inducible oligomerization. Co-transfection of IL-2Ralpha and IL-2Rbeta (+/- gamma(c)) chimeras diminished spontaneous IL-2Ralpha chimera oligomerization and permitted IL-2-inducible hetero-oligomerization of receptor components. Homo-association of IL-2Ralpha was also demonstrated by fluorescence resonance energy transfer (FRET). The spontaneous homo-oligomerization property of IL-2Ralpha required the membrane proximal region of the receptor (exon 6) by deletion analysis; the IL-2 inducible oligomerization property of IL-2Ralpha required the second "sushi" domain (exon 4). This work provides insight into the mechanics of this complex receptor system and to other receptor complexes in the immune system that send signals by clustering receptor subunits.     

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.     

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.