The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

Promegapoietin-1a, an engineered chimeric IL-3 and Mpl-L receptor agonist, stimulates hematopoietic recovery in conventional and abbreviated schedules following radiation-induced myelosuppression in nonhuman primates.

Promegapoietin-1a (PMP-1a), a multifunctional agonist for the human interleukin 3 and Mpl receptors, was evaluated for its ability to stimulate hematopoietic reconstitution in nonhuman primates following severe radiation-induced myelosuppression. Animals were total body x-irradiated (250 kVp) to 600 cGy total midline tissue dose. PMP-1a was administered s.c. in several protocols: A) daily (50 microg/kg) for 18 days; B) nine doses (5 microg/kg) every other day for 3 weeks; C) a single high dose (100 microg/kg) at 20 hours, or D) a single high dose (100 microg/kg) at 1 hour following TBI. The irradiation controls received 0.1% autologous serum for 18 consecutive days. Hematopoietic recovery was assessed by bone marrow clonogenic activity, peripheral blood cell nadirs, duration of cytopenias, time to recovery to cellular thresholds, and requirements for clinical support. PMP-1a, irrespective of administration schedule, significantly improved all platelet-related parameters: thrombocytopenia was eliminated, the severity of platelet nadirs was significantly improved, and recovery of platelet counts to > or =20,000/miccrol was significantly reduced in all PMP-1a-treated cohorts. As a consequence, all PMP-1a-treated cohorts were transfusion-independent. Neutrophil regeneration was augmented in all treatment schedules. Additionally, all PMP-1a-treated cohorts showed an improvement in red blood cell nadir and recovery. PMP-1a in conventional or abbreviated schedules induced significant thrombopoietic regeneration relative to the control cohort, whereas significant improvement in neutrophil recovery was schedule-dependent in radiation-myelosuppressed nonhuman primates.

Synthesis and release of B-lymphocyte stimulator from myeloid cells.

B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-gamma and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-gamma. Both IFN-gamma and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.

Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium.

Megakaryocytes (MK) were expanded from purified human CD34+ cells obtained from three sources, bone marrow (BM), mobilized peripheral blood progenitor cells (PB), and umbilical cord (UC) blood. CD34+-selected cells were cultured for 12 days with 10 ng/ml thrombopoietin (TPO), 10 ng/ml IL-3, 10 ng/ml TPO + 10 ng/ml IL-3, or 200 ng/ml promegapoietin (PMP), a chimeric dual agonist of the c-Mpl and human IL-3 receptors. MK production was compared in serum-free versus human serum-supplemented liquid media. PMP and the combination of TPO and IL-3 (TPO + IL-3) increased MK production similarly. Culturing CD34+ cells with PMP in serum-free medium resulted in a twofold increase in MK yield compared with serum-supplemented medium. CD34+ cells from UC proliferated more than those from either BM or PB in liquid culture, resulting in much greater MK production under all conditions. Phenotypic analysis of the uncultured CD34+ cells showed that BM had a higher frequency of CD34+/CD41+ cells than PB or UC. TPO + IL-3 or PMP produced larger and greater numbers of BFU-MK and CFU-MK per seeded CD34+/CD41+ cell from UC than from either BM or PB. Thus, although uncultured CD34+-selected BM cells contained a higher frequency of committed mature MK progenitors, UC CD34+ cells had a greater proliferative capacity and, therefore, were more productive. PMP induced megakaryocytopoietic activity comparable to that achieved with TPO + IL-3 and may be useful for ex vivo expansion of MK for clinical trials.

Regulated expression and release of the IL-1 decoy receptor in human mononuclear phagocytes.

The aim of this study was to investigate the expression and release of the IL-1 type II decoy receptor (R) in mononuclear phagocytes, which play a central role in immune and chronic inflammatory reactions. Human monocytes expressed both type I and type II R transcripts, the latter being two- to threefold more represented. By cross-linking and Ab blocking, the predominant surface IL-1-binding molecule was the decoy RII. IL-4, IL-13, and dexamethasone induced RI and RII transcripts and augmented the number of IL-1-binding sites with no modification of Kd values. The induced surface receptor was identified as the decoy RII. These stimuli induced the release of a soluble R with a m.w. of approximately 60 kDa, of which N-glycosylation contributed 22 kDa compared with 45 kDa released from polymorphonuclear leukocytes, of which N-glycosylation contributed 15 kDa. IL-13 and dexamethasone induced a release of 24 ng/ml/2 x 10(7) cells (from 8.7 to 43.2 ng/ml) and 25.6 ng/ml/2 x 10(7) cells (from 9.7 to 36.8 ng/ml) of decoy RII in 18 h, respectively (six donors). Thus, for instance, IL-13-treated (18 h) cells expressed 3.5 x 10(3) sites/cell and released 12 x 10(3) decoy RII/cell. The released decoy RII from monocytes bound IL-1apha and IL-1 receptor antagonist 30- and 2-fold less avidly than IL-1beta, respectively. In vitro-matured, monocyte-derived macrophages showed higher levels of surface expression and release of the IL-1 decoy RII. The results show that, on exposure to diverse molecules with anti-inflammatory properties, mononuclear phagocytes express and release copious amounts of a novel version of the soluble IL-1 decoy RII.

Interaction of IL-15 with the shared IL-2 receptor beta and gamma c subunits. The IL-15/beta/gamma c receptor-ligand complex is less stable than the IL-2/beta/gamma c receptor-ligand complex.

This study was designed to compare the interactions of IL-2 and IL-15 with the IL-2R beta and IL-2R gamma c subunits, as differences in receptor interactions between IL-2 and IL-15 might contribute to the functional differences between these two cytokines. The results suggest the existence of a human IL-15R alpha subunit, although physical evidence of this molecule was not obtained. Proliferation of anti-CD3 (OKT3)-stimulated human PBL was compared for responsiveness to IL-2, IL-15, and F42K, and IL-2 mutant that does not bind the IL-2R alpha chain. F42K was more potent than IL-15 in activating a dose-dependent response. This fact, along with Scatchard binding analyses of IL-15 on OKT3 blasts and YT cells revealing both high and intermediate affinity receptors, supports the existence of IL-15R alpha on these cells. Additional characterization of the IL-15R utilized covalent cross-linking to affinity label IL-2R and IL-15R on YT cells and OKT3 blasts. Consistent with previously reported functional data, IL-2R alpha was not co-precipitated from the [125I]IL-15 receptor-ligand complex, demonstrating that IL-15 does not interact physically with the IL-2R alpha subunit. While IL-2R alpha did co-precipitate with IL-2R beta and IL-2R gamma c in the presence of IL-2, IL-15R alpha did not co-precipitate with the IL-2R beta/gamma c complex. Finally, YT cells equilibrated with IL-2 and then precipitated through IL-2R beta showed that IL-2R beta and IL-2R gamma c co-precipitate in a 1:1 ratio, while only IL-2R beta was found in the immunoprecipitates of YT cells equilibrated with IL-15. This indicates that IL-15 creates a less stable bridge between the IL-2R beta and IL-2R gamma c chains than does IL-2 on YT cells. This result was identical for both surface-iodinated YT cells and immunoprecipitates that were probed for IL-2R beta and IL-2R gamma c on Western blots.

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.

The inhibitory activity of human interleukin-1 receptor antagonist is enhanced by type II interleukin-1 soluble receptor and hindered by type I interleukin-1 soluble receptor.

Interleukin-1 (IL-1) is a major proinflammatory cytokine produced by monocytes/macrophages. At the inflammatory site, IL-1 is a potent inducer of the production of prostaglandin E2 (PGE2) and metalloproteinases on fibroblast-like cells, thus triggering tissue damage. The biological activity of IL-1 is counterbalanced by two types of inhibitors: the IL-1 receptor antagonist (IL-1Ra) which competitively binds IL-1 receptor without inducing signal transduction; and IL-1 soluble receptors (IL-1sR) which bind IL-1 and diminish the free concentration of soluble cytokine, thus hampering its binding to the cell surface receptor. Since IL-1sR can also bind IL-1Ra, we studied the simultaneous effects of both inhibitors on the production of interstitial collagenase (C'ase) and PGE2 by human dermal fibroblasts and synovial cells stimulated by either IL-1 alpha or IL-1 beta. IL-1Ra inhibited fibroblast and synovial cell stimulation by approximately 90%, with the exception of C'ase production by synovial cells which was inhibited by approximately 55%. Type I IL-1sR (IL-1sRI) preferentially inhibited IL-1 alpha, whereas type II IL-1sR (IL-1sRII) mainly inhibited IL-1 beta. When IL-1Ra was used simultaneously with IL-1sRI, the final inhibition was lower than that of either of the inhibitors. The simultaneous presence of IL-1Ra and IL-1sRII abolished the IL-1-induced production of PGE2 and C'ase on both dermal fibroblasts and synovial cells, demonstrating that concurrently these two inhibitors are able to abolish most of the inflammatory response. To our knowledge, this is the first example of two types of inhibitors that abolish each other's effects, one of which acts at the receptor level and the other at the ligand level, thus leaving ligand activity unimpaired.

IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2.

Interleukin 15 is a newly discovered cytokine that shares biological activities with IL-2 and, like IL-2, is a member of the four-helix bundle cytokine family. We have shown that IL-15 shares components of the receptor for IL-2: the alpha chain of the IL-2R is not required, but both the beta and gamma chains are needed for IL-15 mediated bioactivities. A defect in IL-15 signaling may therefore contribute to the phenotype of X-linked severe combined immunodeficiency in humans, resulting from mutations in the common gamma chain. Differential ability of cells to bind and respond to IL-2 and IL-15 suggested the existence of an additional IL-15 specific receptor component. We identified an IL-15 specific binding protein (IL-15R alpha) on a murine T cell and isolated the corresponding cDNA. The IL-15R alpha is not a member of the hematopoietin receptor superfamily, but is structurally related to the alpha chain of the IL-2R. Differences in the expression pattern of IL-15 and its receptor compared to the IL-2 system suggest unique in vivo roles for IL-15.

Elevated levels of shed type II IL-1 receptor in sepsis. Potential role for type II receptor in regulation of IL-1 responses.

Two types of cellular IL-1Rs have been characterized and cloned from both human and murine sources. The type II IL-1R has a very short cytoplasmic domain and does not seem to participate in IL-1 signaling. We demonstrate that type II IL-1Rs are released from the surface of neutrophils in response to treatment with TNF or endotoxin. In addition, serum from patients with sepsis syndrome contains elevated levels of soluble type II IL-1Rs. Neutrophils isolated from patients with sepsis have greatly enhanced expression of type II IL-1R mRNA and cell surface receptors and are therefore a likely source for the shed receptors in serum. Of the three forms of IL-1, soluble type II IL-1R binds IL-1 beta with highest affinity and also selectively inhibits IL-1 beta activity. We propose that increased cell surface expression and rapid release of preformed type II IL-1R from neutrophils, as a soluble IL-1 beta binding protein, represents a mechanism that has evolved for regulating IL-1 activity in sepsis.

Binding of IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids.

These studies have examined the binding of the three IL-1 ligands, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1 ra), to soluble forms of types I and II IL-1Rs (sIL-1RI and sIL-1RII). This interaction was measured in direct binding experiments, in which the ligands bound to immobilized sIL-1R, and in inhibition experiments, in which sIL-1R in solution inhibited the binding of IL-1 ligands to immobilized sIL-1R. In addition, the effects of sIL-1R on the detection of IL-1 ligands by ELISA were examined. Finally, levels of sIL-1R in synovial fluid samples were determined, and their effects on measurement of IL-1 in these samples were estimated. IL-1 beta bound more avidly to sIL-1RII than IL-1 alpha or IL-1ra, primarily because of a slow dissociation rate. In contrast, IL-1 ra bound more avidly than IL-1 alpha or IL-1 beta to sIL-1RI, again because of a slow dissociation rate. sIL-1RII and sIL-1RI inhibited the detection of IL-1 beta and IL-1ra, respectively, by ELISA. Low levels of sIL-1RI (approximately 1.0-2.5 ng/ml) were present in all synovial fluids, irrespective of the degree of inflammation, and were correlated inversely with the levels of measured IL-1ra. In contrast, higher levels of sIL-1RII (approximately 10-20 ng/ml) were found in inflammatory synovial fluids and were not correlated with IL-1ra levels. IL-1 beta could not be detected in any synovial fluid. These results suggest that some IL-1 beta and IL-1ra may be bound in vivo to sIL-1RII and sIL-1RI, respectively, leading to underestimations of cytokine concentrations in body fluids when measured by ELISA.

Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.

Interleukin 15 (IL-15) is a novel cytokine that has recently been cloned and expressed. Whereas it has no sequence homology with IL-2, IL-15 interacts with components of the IL-2 receptor (IL-2R). In the present study we performed a functional analysis of recombinant IL-15 on phenotypically and functionally distinct populations of highly purified human natural killer (NK) cells. The CD56bright subset of human NK cells constitutively expresses the high affinity IL-2R and exhibits a brisk proliferative response after the binding of picomolar amounts of IL-2. Using a proliferation assay, IL-15 demonstrated a very steep dose-response curve that was distinct from the dose-response curve for IL-2. The proliferative effects of IL-15 could be abrogated by anti-IL-2R beta (p75), but not by anti-IL-2R alpha (p55). The proliferative effects of IL-2 on CD56bright NK cells could be inhibited by both antibodies. CD56dim NK cells express the intermediate affinity IL-2R in the absence of the high affinity IL-2R. Activation of CD56dim NK cells by IL-15 was similar to that of IL-2 as measured by enhanced NK cytotoxic activity, antibody-dependent cellular cytotoxicity, and NK cell production of interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-15-enhanced NK cytotoxic activity could be completely blocked by anti-IL-2R beta monoclonal antibody. The binding of radiolabeled IL-2 and IL-15 to CD56dim NK cells was inhibited in the presence of anti-IL-2R beta. Scatchard analysis of radiolabeled IL-15 and IL-2 binding to NK-enriched human lymphocytes revealed the presence of high and intermediate affinity receptors for both ligands. IL-15 is a ligand that activates human NK cells through components of the IL-2R in a pattern that is similar but not identical to that of IL-2. Unlike IL-2, IL-15 is produced by activated monocytes/macrophages. The discovery of IL-15 may increase our understanding of how monocytes/macrophages participate in the regulation of NK cell function.

The two interleukin-1 receptors play different roles in IL-1 actions.

The proinflammatory cytokine interleukin-1 binds to two cell-surface receptors. The type I receptor, an 80-kDa protein with a cytoplasmic domain of approximately 215 amino acids, mediates the biological effects of IL-1. The type II receptor, a 60-kDa protein with 29 cytoplasmic amino acids, binds IL-1 and thereby prevents it from binding to the type I receptor but does not deliver a biological signal. Thus, the type II receptor acts as a negative regulator of IL-1 actions. It can do so either as a membrane-bound molecule or subsequent to shedding from the cell surface to generate a so-called "soluble" receptor. Both the naturally produced soluble type II receptor and the recombinantly generated soluble type I receptor are effective inhibitors of IL-1 action. The soluble type I receptor has shown efficacy in some preclinical models of inflammatory diseases, as well as in an initial clinical trial in a setting of cutaneous allergy.

Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15.

We have recently cloned a novel cytokine, IL-15, with shared bioactivities but no sequence homology with IL-2. We found high affinity IL-15 binding to many cell types, including cells of non-lymphoid origin. Analysis of IL-15 interaction with subunits of the IL-2 receptor (IL-2R) revealed that the alpha subunit was not involved in IL-15 binding. We demonstrated directly in cells transfected with IL-2R subunits that both the beta and gamma chains are required for IL-15 binding and signaling. Hence, IL-15, like IL-2, IL-4 and IL-7, utilizes the common IL-2R gamma subunit found to be defective in X-linked severe combined immunodeficiency in humans. IL-15 is the only cytokine other than IL-2 that has also been shown to share the beta signaling subunit of IL-2R. The differential ability of some cells to bind and respond to IL-2 and IL-15 implies the existence of an additional IL-15-specific component.

The type II "receptor" as a decoy target for interleukin 1 in polymorphonuclear leukocytes: characterization of induction by dexamethasone and ligand binding properties of the released decoy receptor.

Whereas the signaling function of the interleukin 1 (IL-1) receptor type I (IL-1R I) has been well documented, the type II "receptor" has been suggested to act as a decoy target for this cytokine. Since IL-1 may represent a key target of the immunomodulatory and antiinflammatory properties of glucocorticoids (GC), the aim of this study was to investigate the effects of dexamethasone (Dex) on IL-1R expression in human polymorphonuclear leukocytes (PMN), which express predominantly the type II molecule (IL-1R II). We found that Dex augments the levels of steady state transcripts encoding the IL-1R I and, most prominently, those of IL-1R II. Dex induced both transcripts via transcription-dependent mechanisms and by prolongation of the mRNAs half-lives. Inhibition of protein synthesis superinduced basal and Dex-augmented IL-1R II mRNA, whereas it completely inhibited the induction by Dex of IL-1R I transcripts. Induction of IL-1R II mRNA by Dex was associated with augmented membrane expression and release of the type II IL-1 binding molecule. This effect was mediated by the GC receptor. Other steroids (17 beta-estradiol, progesterone, and testosterone) were ineffective. The concentrations of IL-1 alpha and IL-1 receptor antagonist required to displace the binding of IL-1 beta to the soluble form of the decoy molecule induced by Dex from PMN were, respectively, 100 and 2 times higher compared with IL-1 beta. The induction by Dex of the type II receptor, a decoy molecule for IL-1, may contribute to the immunosuppressive and antiinflammatory activities of Dex.

Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4.

Interleukin-1 (IL-1) interacts with cells through two types of binding molecules, IL-1 type I receptor (IL-1R I) and IL-1R II. The function of IL-1R II is unknown. In studies using monoclonal antibodies, IL-1 prolonged the in vitro survival of polymorphonuclear cells (PMN) through IL-1R I, and IL-4 antagonized the action of IL-1 by inducing expression and release of IL-1R II. Dexamethasone also induced expression and release of the IL-1R II in PMN. These results, together with the effect of antibodies to IL-1R on IL-1-induced production of cytokines in monocytes, indicate that IL-1 acts on myelomonocytic cells through IL-1R I and that IL-1R II inhibits IL-1 activity by acting as a decoy target for IL-1. The existence of multiple pathways of regulation emphasizes the need for tight control of IL-1 action.

Interleukin 1 signaling occurs exclusively via the type I receptor.

Two receptors for the proinflammatory cytokine interleukin 1 (IL-1) have been cloned and characterized biochemically. While it has been well established that the type I (80-kDa) IL-1 receptor can mediate responses to IL-1, the function of the type II (60-kDa) IL-1 receptor has been unknown. In this manuscript we describe experiments designed to ask whether the type II receptor is capable of delivering a biological signal. We have examined two types of experimental situation: responses to IL-1 in cells which express predominantly the type II receptor, and responses to IL-1 which have been suggested previously in the literature to be mediated by type II receptors. In both situations we find that the responses instead are mediated via type I receptors. A blocking antibody against the type II receptor never inhibits, and in fact sometimes enhances, the responses. We conclude that a very small number of type I receptors is sufficient to mediate all of the actions of IL-1 which we have examined here and that the function of the type II receptor may not be to transduce signals.

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.

Identification of soluble interleukin-1 binding protein in cell-free supernatants. Evidence for soluble interleukin-1 receptor.

This study describes the identification and characterization of a soluble interleukin-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of 35-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.