IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

Differential binding of IL-1 alpha and IL-1 beta to receptors on B and T cells

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.     

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.     

Heterogeneity in interleukin (IL)-1 receptors expressed on human B cell lines. Differences in the molecular properties of IL-1 alpha and IL-1 beta binding sites

IL-1 alpha and IL-1 beta although distantly related at the primary sequence level, bind to the same Mr 80,000 IL-1 receptor on various cell types. Several lines of evidence indicate, however, that the IL-1 receptor on B cells and T cells differ. By binding experiments with 125I-IL-1, marked heterogeneity in IL-1 receptor binding was observed in 13 of 24 B cell lines studied. This was classified into three categories: (I) in nine cell lines, 125I-IL-1 alpha binding revealed high (kD = 10(-10) M) and low affinity (kD = 10(-8) M) IL-1 alpha receptors, whereas 125I-IL-1 beta binding showed one class only with intermediate affinity (kD = 10(-9) M); (II) in three cell lines selective binding with 125I-IL-1 beta was observed; (III) in one cell line only, 125IL-1 alpha and 125I-IL-1 beta bind to a single class of IL-1 receptors as has been described for most cell types. Cross-linking with 125I-IL-1 alpha or 125I-IL-1 beta demonstrated their specific binding to Mr 80,000 and to Mr 68,000 in cell lines in categories I and III, whereas for those in category II, binding to the IL-1 receptor was confined to 125I-IL-1 beta. The expression of two subsets of IL-1 alpha receptors but only one class of IL-1 beta receptors was further confirmed in kinetic studies. Internalization at 37 degrees C demonstrated that only 19% of IL-1 beta was internalized and that binding with IL-1 alpha was entirely cell surface. Flow cytometry studies showed that IL-1 alpha and IL-1 beta do not influence B cell surface antigen expression, suggesting that the ability of IL-1 to influence B cell proliferation is not mediated via direct binding to the IL-1 receptor only.     

Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells

Using 125I-interleukin-1 beta (125I-IL-1 beta) as a ligand, a specific receptor of high affinity dissociation constant (1.1 +/- 0.2 x 10(-10) M) with binding sites (350 +/- 40/cell) for interleukin-1 beta (IL-1 beta) has been demonstrated on cultured porcine thyroid cells. IL-1 alpha almost equally cross-reacted with the receptor (Kd = 1.2 +/- 0.3 x 10(-10) M and 350 +/- 50 binding sites/cell). TSH, IL-2 and other peptide hormones did not inhibit the binding of 125I-IL-1 beta to thyroid cells. Crosslinking study revealed a major band (approximately 95 kD) with a corrected molecular mass of approximately 78 kD. Moreover, both IL-1 beta and IL-1 alpha stimulated prostaglandin E2 production of cultured porcine thyroid cells, although the potency of IL-1 alpha was slightly greater than that of IL-1 beta. These results suggest that IL-1 may be involved in the regulation of thyroid cell function.     

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.     

Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor.

Interleukin-15 (IL-15) is a novel cytokine of the four-helix bundle family which shares many biological activities with IL-2, probably due to its interaction with the IL-2 receptor beta and gamma (IL-2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL-15R alpha chain. IL-15R alpha alone displays an affinity of binding for IL-15 equivalent to that of the heterotrimeric IL-2R for IL-2. A biologically functional heteromeric IL-15 receptor complex capable of mediating IL-15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL-15R alpha is structurally similar to IL-2R alpha; together they define a new cytokine receptor family. The distribution of IL-15 and IL-15R alpha mRNA suggests that IL-15 may have biological activities distinct from IL-2.     

Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and Th1 cell response. Recently IL-1R-related protein (IL-1Rrp) has been cloned as the receptor for IL-18. However, the functional role of IL-1Rrp is still controversial due to its low affinity to IL-18 as well as the possibility of the presence of another high-affinity binding receptor. In the present study, we have generated and characterized IL-1Rrp-deficient mice. The binding of murine rIL-18 was not detected in Th1-developing splenic CD4+ T cells isolated from IL-1Rrp-deficient mice. The activation of NF-kappa B or c-Jun N-terminal kinase were also not observed in the Th1 cells. NK cells from IL-1Rrp-deficient mice had defects in cytolytic activity and IFN-gamma production in response to IL-18. Th1 cell development was also impaired in IL-1Rrp-deficient mice. These data demonstrate that IL-1Rrp is a ligand-binding receptor that is essential for IL-18-mediated signaling events.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective inhibition of high- but not low-affinity interleukin 2 binding by lectins and anti-interleukin 2 receptor alpha antibody

The present study demonstrated that various reagents can specifically reduce the affinity of high-affinity interleukin 2 receptor (IL-2R) but not that of low-affinity IL-2R. They included lectins such as wheat germ agglutinin (WGA), concanavalin A and phytohemagglutinin, and a chemical cross-linker, glutaraldehyde, in addition to anti-IL-2R monoclonal antibodies, HIEI and H-47. The affinity of the high-affinity IL-2R was reduced when the cells were treated with WGA or H-47 before, but not after, addition of 125I-labeled interleukin 2 (IL-2). Their inhibitory effects were also demonstrated by the chemical cross-linking method. On treatment with the reagents, the IL-2 binding to both IL-2R alpha and beta chains was inhibited and these inhibitory effects were seen only when the reagents were added before IL-2 addition, like their high-affinity reducing effects. These results support a supposition that the high affinity IL-2R is generated by assembly of the alpha and beta chains, and suggest that the IL-2 binding to IL-2R alpha and beta chains could induce stable constitution of the high-affinity state of IL-2R, but these affinity modulating reagents could perturb the optimum association between alpha and beta chains to generate the high-affinity IL-2R.     

Expression of IL-1 receptors on human peripheral B cells

The expression of IL-1R on human peripheral B cells was analyzed by the binding assay with 125I-labeled human rIL-1 alpha and by the flow cytofluorometry by the use of FITC-conjugated IL-1 alpha. The proliferation and the differentiation of B cells stimulated with Staphylococcus aureus Cowan I (SAC) in the presence of T cell-derived factors were dependent on IL-1. By the binding experiment with 125I-labeled IL-1 alpha, B cells expressed only few IL-1R without any stimulations. When they were stimulated with SAC, IL-1R on B cells began to increase by only 1 h, reached the maximum level at 6 h. The binding of 125I-labeled IL-1 alpha to B cells was inhibited by the addition of either cold IL-1 alpha or IL-1 beta suggesting that IL-1R on B cells reactive for IL-1 alpha and IL-1 beta were identical. By Scatchard plot analysis, the existence of two classes of IL-1R on B cells was found. A major class of IL-1R (320 molecules/cell) has a lower affinity (Kd = 3.8 x 10(-10) M) and a minor class of IL-1R (70 molecules/cell) has a higher affinity (Kd = 4.4 x 10(-12) M). When B cells were stimulated with SAC, both lower and higher affinity IL-1R were increased to 1960 molecules/cell and 300 molecules/cell, respectively. Furthermore, IL-1R on B cells were also detected with FITC-conjugated IL-1 alpha by a flow cytofluorometer. Only 3 to 5% of B cells expressed IL-1R without any stimulations. When B cells were stimulated with SAC, IL-1R-positive B cells were increased to 20%. The addition of anti-class II antibodies inhibited B cell proliferation and differentiation induced with SAC, IL-1, and T cell-derived factors. Anti-class II antibodies also inhibited the number of IL-1R on B cells. These results suggest that the expression of IL-1R was induced as the initial stage of B cell activation and that class II Ag play an important role for the expression of IL-1R on B cells.     

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.     

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.     

Structural elements underlying the high binding affinity of human cytomegalovirus UL18 to leukocyte immunoglobulin-like receptor-1

Human cytomegalovirus (HCMV) encodes UL18, a major histocompatibility complex (MHC) class I homologue that binds to the leukocyte immunoglobulin-like receptor (LIR)-1 (also called ILT2/CD85j/LILRB1), an inhibitory receptor expressed on myeloid and lymphoid immune cells. The molecular basis underlying the high affinity binding of UL18 to LIR-1, compared to MHC class I molecules (MHC-I), is unclear. Based on a comparative structural analysis of a molecular model of UL18 with the crystal structure of the HLA-A2/LIR-1 complex, we identified three regions in UL18 influencing interaction with LIR-1. Comparison of the relative binding affinities of mutated UL18 proteins to LIR-1 demonstrated the importance of specific residues in each region. Substitution of residues K42/A43 and Q202, localized in the alpha1 and alpha3 domains, respectively, reduced binding affinity to LIR-1 nearly by half. The model also suggested the formation of an additional disulfide bridge in the alpha3 domain of UL18 between residues C240 and C255, not present in MHC-I. Substitution of either cysteine residue prevented association of UL18 to beta2m, abolishing binding to LIR-1. All observed differences in binding affinities translated directly into functional consequences in terms of inhibition of IFN-gamma production by T cells, mediated through the UL18-LIR-1 interaction. The larger amount of interacting regions, combined with an increased stability of the alpha3 and beta2m domains allow a higher recognition affinity of UL18 by LIR-1.     

Distinctions in lymphocyte responses to IL-2 and IL-15 reflect differential ligand binding interactions with the IL-2Rbeta chain and suggest differential roles for the IL-2Ralpha and IL-15Ralpha subunits

More interleukin 15 (IL-15) than IL-2 was needed to generate comparable proliferative responses by phytohaemagglutinin (PHA) blasts and Tf-1beta cells expressing high affinity and intermediate affinity IL-2 receptor (IL-2R) complexes, respectively. The focus of these experiments was to determine the contribution of the shared IL-2 and IL-15 receptor components to these dose-response differences. Some of this difference can be attributed to the role of the IL-2Rbeta chain, in that HuMikbeta1, a monoclonal antibody recognizing the IL-2Rbeta chain, blocks 92.2+/-2.5% (mean+/-SE) of the IL-2 proliferative response by Tf-1beta cells but only inhibits 57.9+/-3.7% of the IL-15 response, indicating that IL-2 and IL-15 may physically utilize the IL-2Rbeta chain differently. Monoclonal antibody 341, which recognizes IL-2Rbeta but does not inhibit IL-2 binding to the IL-2Rbeta chain, blocks 35.4+/-2.3% of IL-15-stimulated proliferation of PHA blasts, while not affecting the IL-2-stimulated proliferation. Finally, although HuMikbeta1 does not inhibit IL-2 responses by PHA blasts bearing high affinity IL-2 receptors, HuMikbeta1 does block IL-15-stimulated proliferation by these same cells bearing high affinity IL-15 receptors (88.5+/-1.6% inhibition). This indicates that the role of IL-15Ralpha in the high affinity IL-15R complex is distinct from that of IL-2Ralpha in the high affinity IL-2R complex. Overall, these studies show that the physical interactions of the IL-2Rbetagammac complex with IL-2 are different than the interactions with IL-15.     

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.     

Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain

IL-18 and IL-12 are major IFN-gamma-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18R alpha, and IL-18R beta are expressed constitutively but IL-18 did not induce IFN-gamma unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1 beta or TNF-alpha, do not respond to IL-18, despite abundant expression of the IL-18R alpha chain. COS-1 cells lack expression of the IL-18R beta chain. The IL-18R beta cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18R beta chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18R alpha chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18R beta chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18R alpha and IL-18R beta; the production of IFN-gamma corresponded to IL-12-induced IL-18R alpha and IL-18R beta chains. We conclude that functional reconstitution of the IL-18R beta chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-gamma production is, in part, due to IL-12 up-regulation of both IL-18R alpha and IL-18R beta chains, although postreceptor events likely contribute to IFN-gamma production.