Elevated circulating levels of soluble interleukin-1 receptor type II during interleukin-2 immunotherapy

Interleukin-1 (IL-1) is a critical mediator of inflammation. Two naturally occurring IL-1 antagonists have been described, namely the IL-1 receptor antagonist (IL-1Ra) and the IL-1 receptor type II (IL-1RII). IL-1RII does not transmit a signal upon binding of IL-1, but competes with the signaling of IL-1RI for binding of IL-1. Shedding of IL-1RII yields the soluble IL-1 receptor type II (IL-1sRII) which retains the ability of membrane-bound IL-1RII to bind IL-1beta avidly, but binds IL-1Ra and IL-1alpha with low affinity. In contrast, IL-1sRI retains the ability of membrane-bound IL-1RI to bind IL-1Ra and IL-1alpha with high affinity, but binds IL-1beta poorly. We have previously shown that immunotherapy with IL-2 or IL-6 in cancer patients is associated with a dramatic increase in IL-1Ra plasma levels. In the present study, plasma levels of soluble IL-1 receptors were monitored in healthy individuals and cancer patients. In healthy controls, the mean IL-1sRII level was 4.76 0.16 ng/ml. IL-1sRII levels in cancer patients were comparable to those measured in healthy controls. IL-1sRII levels did not vary during the first 52 hours after initiation of IL-2 therapy, but increased significantly thereafter to reach 9.56 1.16 ng/ml on day 5. In contrast, IL-6 immunotherapy with a 5-day continuous infusion did not trigger an increase in IL-1sRII levels. IL-1sRI levels did not increase during immunotherapy with IL-2 or IL-6. Our results indicate that IL-1sRII, unlike IL-1Ra, remains a modest, natural, anti-inflammatory mechanism triggered by immunotherapy with IL-2, but not with IL-6.     

Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils.

We have examined the binding of human and rodent interleukin-1 receptor antagonist (IL-1ra) to the type II IL-1 receptor on the human B cell line, Raji, on the mouse pre-B cell line, 70Z/3, and on human polymorphonuclear leukocytes (PMNs). Human IL-1ra binds to the receptors on the human B cells with an affinity (KD = 15 +/- 3 nM) equal to that of IL-1 alpha and only 15-fold lower than that of IL-1 beta and, likewise, binds to human PMNs with an affinity (KD = 8 +/- 4 nM) 15-fold lower than that of IL-1 beta. Mouse and rat IL-1ra bind to these two human cell types with an affinity similar to that of the human protein. Human IL-1ra binds very weakly to the type II receptor on the mouse pre-B cells with an affinity (KD = 1.4 +/- 0.2 microM) about 1500-fold lower than human IL-1 beta. Mouse and rat IL-1ra also bind to the mouse pre-B cells with low affinity. The weak binding of the three IL-1ra proteins to these mouse cells appears to be more a consequence of the cell type rather than species specificity. There may be a population of cells for which the actions of IL-1 cannot be effectively opposed by IL-1ra, although this group does not include mature B cells and PMNs.     

Protein kinase C-linked inactivation of the interleukin-1 receptor in a human transformed B-cell line

The effect of tumor-promoting phorbol ester treatment on the binding of interleukin-1 beta (IL-1 beta) to specific cell surface receptors was investigated. A 1 h exposure of Raji human B lymphoma cells with the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu), reduced IL-1 beta binding by up to 90% of control cells. This effect was dose-dependent and was not observed with 4-alpha-phorbol, an inactive tumor promoter. Analysis of 125I-labeled IL-1 beta binding to intact cells revealed that PDBu caused a 91% decrease in high-affinity cell-surface receptor number without an effect on receptor affinity. The phorbol ester response was rapid (30 min), observed both at 4 and 37 degrees C, and was preceded by the rapid translocation (t much less than 6 min) of protein kinase C (PKC) from the cytosol to the cell membrane. The PDBu-induced decrease in IL-1 beta receptor number was inhibited by prior incubation of cells for 30 min with the PKC inhibitor 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine (H7). The decrease in receptor binding was not due to enhanced IL-1 beta receptor internalization or shedding into the extracellular medium, since a similar effect was observed with solubilized IL-1 beta receptor. The most likely explanation for the phorbol ester effect appears to be cell surface inactivation of IL-1 receptors. These data suggest that modulation of PKC activity could play a role in the regulation of the IL-1 beta receptor.     

Measurement of biologically active interleukin-1 by a soluble receptor binding assay

A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants.     

A biochemical and kinetic analysis of the interleukin-1 receptor. Evidence for differences in molecular properties of IL-1 receptors

This study describes the biochemical characterization and kinetic analysis of the interleukin-1 (IL-1) receptor in Raji human B-lymphoma and EL4 murine T-lymphoma cells. The internalization of 125I-IL-1 was studied in both cell types by an acid extraction technique which removes surface bound ligand. At 37 degrees C, binding to Raji IL-1 receptors was almost entirely cell surface (91%). EL4 cells, in contrast, internalized 59% of ligand at this temperature and this was almost totally inhibited by sodium azide. Receptor binding studies showed that the B-cells had a lower binding affinity but much higher receptor density per cell (KD = 2.1 nM, Ro = 7709) than the T-cells (KD = 0.4 nM, Ro = 241). The receptor binding affinity of two IL-1 analogs, Glu-4 and clone 18, was determined in competitive binding studies. In the B-cells the analogs had binding affinities of 25 and 90%, respectively, whereas in the T-cells the affinities were 0.2 and 200%, respectively. Chemical cross-linking studies showed that the IL-1 receptor in B-cells had a lower molecular weight than that in T-cells (68 kDa compared to 80 kDa). In summary these studies demonstrate that structural differences exist between IL-1 receptors in Raji and EL4 cells.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor.

The interleukin-1 receptor antagonist (IL-1ra) inhibits the binding of interleukin-1 (IL-1) to T-cell lines possessing the type I IL-1 receptor; evidence has been published (Carter, D. B., Deibel, M. R. J., Dunn, C. J., Tomich, C. S., Laborde, A. L., Slightom, J. L., Berger, A. E., Bienkowski, M. J., Sun, F. F., McEwan, R. N., Harris, P. K. W., Yem, A. W., Waszak, G. A., Chosay, J. G., Sieu, L. C., Hardee, M. M., Zurcher-Neely, H. A., Reardon, I. M., Heinrickson, R. L., Truesdell, S. E., Shelly, J. A., Eessalu, T. E., Taylor, B. M., and Tracey, D. E. (1990) Nature 344, 633-638; Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., Dripps, D. J., Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg, S. P., and Thompson, R. C. (1990) Nature 343, 336-340) that IL-Ira does not bind to the type II IL-1 receptor (IL-1RtII). In this study we examined the ability of human recombinant IL-1ra to block the binding of IL-1 to the IL-1RtII on human polymorphonuclear leukocytes (PMN) and Raji human B-lymphoma cells. The binding of 125I-IL-1 beta to PMN was competively inhibited by IL-1ra. IL-1 beta was more potent in inhibiting the binding of 125I-IL-1 beta than IL-1ra. Incubating PMN with 125I-IL-1ra in the presence of increasing concentrations of IL-1 beta or IL-1ra showed that IL-1 beta was an approximately 40-fold more potent inhibitor of binding of 125I-IL-1ra than unlabeled IL-1ra. The IL-1ra was approximately 500-fold less potent in inhibiting the binding of 125I-IL-1 alpha than IL-1 alpha. IL-1ra was also able to competitively inhibit binding of 125I-IL-1 beta to Raji cells. PMN or Raji cells were also incubated with 125I-IL-1 in the absence or presence of IL-1 or IL-1ra. After cross-linking of IL-1 to cells followed by specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 kDa corresponding to the 68-kDa IL-1RtII. However, in the presence of an excess of either unlabeled IL-1 or IL-1ra, the 85-kDa IL-1.IL-1RtII complex was not present. These findings demonstrate that the IL-1ra recognizes and blocks IL-1 binding to the IL-1RtII.     

Regulation by interleukin-1beta of gene expression of bradykinin B1 receptor in MH-S murine alveolar macrophage cell line.

The effects of recombinant murine interleukin (IL)-1beta on gene expression of murine bradykinin B1 receptor (BDKRB1) in MH-S murine alveolar macrophage cell line were evaluated. BDKRB1 mRNA expression in MH-S cells was increased by IL-1beta (1, 3, and 10 ng/ml) in a time-dependent manner, peaking at 3-4 h by 100-1000 fold. IL-1beta (5 ng/ml, 24h) also induced significant binding to [3H]-des-Arg10-kallidin with a dissociation constant (Kd) of 2.95 nM and a maximal binding density (Bmax) of 670 sites/cell. Des-Arg10-kallidin (10 microM), a BDKRB1 agonist, increased intracellular calcium ion ([Ca2+]i) in IL-1beta (5 ng/ml, 24 h)-exposed cells, an increase not observed in the cells not exposed to IL-1beta. A significant increase of tumor necrosis factor (TNF)-alpha secretion occurred in the IL-1beta (5 ng/ml, 24 h)-exposed cells following addition of des-Arg10-kallidin (the IL-1beta-exposed group: 57. 8 +/- 13.7 vs. the vehicle-exposed group: 16.7 +/- 4.3 pg/ml, p < 0.05 after a 100 nM des-Arg10-kallidin for 8 h), with an optimal effect at 3-100 nM. These data suggest that IL-1beta may up-regulate BDKRB1-mediated functions of alveolar macrophages via an induction of BDKRB1 gene expression.     

A soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection.

Vaccinia virus gene B15R is shown to encode an abundant, secretory glycoprotein that functions as a soluble interleukin-1 (IL-1) receptor. This IL-1 receptor has novel specificity since, in contrast with cellular counterparts, it binds only IL-1 beta and not IL-1 alpha or the natural competitor IL-1 receptor antagonist. The vaccinia IL-1 beta receptor is secreted when expressed in a baculovirus system and competitively inhibited binding of IL-1 beta to the natural receptor on T cells. Deletion of B15R from vaccinia virus accelerated the appearance of symptoms of illness and mortality in intranasally infected mice, suggesting that the blockade of IL-1 beta by vaccinia virus can diminish the systemic acute phase response to infection and modulate the severity of the disease. The IL-1 beta binding activity is present in other orthopoxviruses.     

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.     

The interleukin-1 receptor in Raji human B-lymphoma cells. Molecular characterization and evidence for receptor-mediated activation of gene expression.

In a previous paper [Horuk, Huang, Covington & Newton (1987) J. Biol. Chem. 262, 16275-16278] we reported that there were fundamental differences in the biochemical properties of the interleukin-1 (IL-1) receptor between Raji and EL4 cell lines. In the present study we have investigated the basis for these differences. Kinetic studies measuring the on and off rates of IL-1 receptor binding revealed that the low-affinity IL-1-binding sites observed in Raji cells, compared with EL4 cells, result from a combination of a lower association rate and a higher dissociation rate in the Raji cells. The turnover of the Raji IL-1 receptor, measured by inhibiting protein synthesis with cycloheximide, was much faster than that of the EL4 IL-1 receptor, with a half-time of 2 h as against 5 h. Treatment of 125I-IL-1-labelled IL-1 receptors in Raji and EL4 cells with neuraminidase decreased their molecular mass by approx. 2-5 kDa as assessed by SDS/polyacrylamide-gel electrophoresis (PAGE). The covalently labelled IL-1 receptors in both cell types were sensitive to treatment with endoglycosidase F, which decreased their molecular mass on SDS/PAGE by 12-13 kDa. Incubation of Raji cells with maximally stimulating doses of IL-1 resulted in an increase in the nascent RNA levels of several genes, including the IL-2 receptor and the proto-oncogenes c-Ha-ras and c-myc.     

Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines

A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappaB ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral factors. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting, but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta1 suppressed the expression of RANKL and/or augmented OPG expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG in osteoblast/stromal lineage cells.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

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.     

Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor.

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.     

Identification of a soluble GM-CSF binding protein in the supernatant of a human choriocarcinoma cell line.

We identified two forms of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) made by the human choriocarcinoma cell line JEG-3 using an affinity-labeling technique. The protein was identified in the detergent-extract was 78 kDa, very similar to that of the membrane-bound GM-CSF receptor alpha chain expressed in a wide variety of hematopoietic and nonhematopoietic cells, including JEG-3. In contrast, a 62-kDa GM-CSF binding protein, or the soluble GM-CSF receptor, was identified in the supernatant of JEG-3 cells. Utilizing the same affinity labeling technique, we did not detect the soluble GM-CSF binding protein in the supernatant of several hematopoietic cell lines, such as U-937 and KG-1, which express membrane bound alpha chain as well as beta chain. The 62-kDa soluble GM-CSF receptor is produced in abundant amounts by JEG-3, but in very small amounts, if any, by hematopoietic cell lines.     

Purification and characterization of interleukin 1 receptor level antagonist proteins from THP-1 cells

Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.33-0.64 nM) and for inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by 1A5 cells (IC50 values 25-100 pM). Amino-terminal sequence analysis of the two major forms (25 kDa/pI 5.1 and 22 kDa/pI 5.8) revealed complete identity for the first 27 residues in both forms. Based on the results of peptide mapping, amino acid compositional analysis and immune blotting, all of the forms were deduced to be variants of a common protein. Deglycosylation of the antagonist proteins with N-glycanase converted them to a common form (22 kDa/pI 5.8), indicating that the four isoforms represent glycosylation variants of a common protein and that asparagine-linked oligosaccharides are responsible for the observed size and charge heterogeneity.