IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.     

The kinetics of S phase entry by FMP2.1: effect of IL-3 and GM-CSF receptor expression and ligand affinity

FMP2.1, a cloned cell line which has morphological characteristics of mast/basophil cells, requires either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for both survival and proliferation. IL-3 and GM-CSF were equally effective as proliferative stimuli. FMP2.1 cells were sensitive to growth factor stimulation in the G1 phase, which has a duration of 9.5 h. G1 cells were selected from FMP2.1 in log phase growth on the basis of Hoechst 33324 staining using a fluorescence activated cell sorter (FACS). It was found that G1 phase cells had to be exposed to either IL-3 or GM-CSF for approximately 1 h for cells to enter S (greater than 20%); without growth factor, FMP2.1 remained in G1 unable to progress into S. Receptor expression was analyzed to further understand this rapid activation of FMP2.1 into cycle. Autoradiography using either 125I-IL-3 or 125I-GM-CSF showed that most cells express both receptor types. In the presence of saturating concentrations of IL-3, FMP2.1 have a relatively high number of IL-3 receptors (42,000/cell) compared to other cell lines (e.g., 32D cl23; 13,000 receptors/cell), and far outnumber GM-CSF receptors on the same cells (600 receptors/cell). Although average IL-3 receptor expression differed for FMP2.1- and IL-3-dependent 32D cl23, the concentration-dependent proliferative response to IL-3 was essentially identical for both cell types. Scatchard plot analysis for 125I-IL-3 and 125I-GM-CSF binding to FMP2.1 cells at 4 degrees C revealed a single type of binding site for both ligands, with dissociation constants (Kd) of approximately 1 nM for GM-CSF and 8 pM for IL-3. The relatively high affinity IL-3 binding to a large number of available IL-3 receptors was associated with a shallow dose response of the FMP2.1 cells to IL-3, compared to the steep GM-CSF dose response which was mediated through fewer receptor sites of relatively low affinity. Mitogenic stimulation of G1 phase cells was observed with either IL-3 or GM-CSF, and appeared to be unaffected by differences in receptor number or binding affinity.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

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.     

Analysis of interleukin 5 receptors on murine eosinophils: a comparison with receptors on B13 cells

The interleukin 5 receptor (IL-5R) on murine eosinophils and a mouse B cell line (B13) was investigated using iodinated murine IL-5 produced in the baculovirus system. Electrophoretic analysis of this recombinant protein identified a range of bands Mr 26,000 to 32,000 resulting from differential glycosylation. The specific activity and binding kinetics of the iodinated IL-5 (125I-IL-5) were essentially identical to unlabeled material. Both high-affinity (Kd approximately 50 pM) and low-affinity (Kd approximately 1 nM) receptor populations were identified on murine eosinophils. Approximately 50 high-affinity receptors and 10,000 low-affinity receptors were present. This was compared with approximately 2,000 high-affinity (Kd approximately 80 pM) and about 8,000 low-affinity (Kd approximately 3 nM) sites on B13 cells. An antibody that inhibits IL-5 binding to, and proliferation of, B13 cells (R52.120) was also shown to inhibit eosinophil proliferation, suggesting that eosinophils and B cells bear the equivalent IL-5 binding proteins.     

Interaction of recombinant monocyte-derived interleukin 1 receptor antagonist with rheumatoid synovial cells.

Interleukin 1 receptor antagonist (IL-1ra) is a newly described cytokine that is produced by human monocytes cultured on adherent immunoglobulin G (IgG). These studies have characterized the binding of IL-1ra to receptors on human rheumatoid synovial cells in comparison to binding of IL-1 alpha. The human synovial cells bound 35S-IL-1ra with a Kd of 213 pM and a Ki of 134 pM. 125I-IL-1 alpha bound to the synovial cells with similar values, showing a Kd of 205 pM and a Ki of 58 pM. Cross-inhibition studies were performed to examine whether IL-1ra and IL-1 alpha interacted with the same receptors and in an identical fashion. At the highest concentrations of inhibitory proteins, the binding of each ligand was inhibited 100% by the same or opposite ligand. This result indicated that IL-1ra and IL-1 alpha bound to the same receptors and not to overlapping subsets of receptors. In addition, the binding of 35S-IL-1ra was inhibited in an identical fashion by equimolar amounts of IL-1ra or IL-1 alpha. However, twofold or greater amounts of IL-1ra in comparison to IL-1 alpha were required to offer comparable inhibition of binding of 125I-IL-1 alpha. These results suggest that IL-1ra and IL-1 alpha bind with equal avidity to IL-1 receptors but may not bind identically. Additional experiments are necessary to establish whether these two ligands may bind to different regions of the extracellular portion of the IL-1 receptor.     

Presence of IL-1 receptors on human and murine neutrophils. Relevance to IL-1-mediated effects in inflammation

Inflammatory responses are characterized by the infiltration of polymorphonuclear neutrophils (PMN) at the involved site. IL-1 may have an important role in mediating this response, but whether IL-1 acts directly on PMN is controversial. In this study, we examined PMN for the presence of IL-1R and determined the effect of IL-1 on PMN migration in vivo. Thioglycollate, proteose-peptone, or IL-1 elicited peritoneal exudate cells were found to bind 125I-IL-1 alpha in a specific and saturable manner. This binding was localized to the PMN in the exudate. Scatchard plot analysis indicates the presence of approximately 1700 receptors per PMN and an apparent dissociation constant of 3.0 x 10(-10) M. Binding sites for 125I-IL-1 alpha were also found on human PMN prepared from peripheral blood. There are approximately 900 receptors per cell on human PMN with a dissociation constant similar to that observed for elicited murine PMN. Binding of 125I-IL-1 alpha to the mouse and human PMN is inhibited by both recombinant human IL-1 alpha and IL-1 beta, indicating that both IL-1 proteins bind to the same receptor on these cells. Human PMN were able to internalize radioiodinated IL-1. We conclude that PMN possess receptors for IL-1 and that these binding sites may be important in mediating IL-1 effects on granulocytes that are involved in the inflammatory response.     

Dynamics of interleukin-6 internalization and degradation in rat hepatocytes.

We have investigated the dissociation, internalization, and degradation of 125I-interleukin-6 (125I-IL-6) by primary rat hepatocytes. Temperature shift experiments following saturation binding of 125I-IL-6 to cell surface receptors in hepatocytes showed a rapid loss of surface-bound 125I-IL-6 (t1/2 = 15 min), concomitant with a rapid rise in internalized radiolabeled ligand. After reaching a maximum by 30 min at 37 degrees C, the level of internalized 125I-IL-6 decreased with time and appeared in the culture media in a non-trichloroacetic acid-precipitable (degraded) state. The addition of the lysosomotropic agent chloroquine inhibited this receptor-mediated degradation of IL-6 without affecting ligand internalization. Polyacrylamide gel electrophoresis analysis of internalized 125I-IL-6 confirms these results. Additionally, we show that the IL-6.IL-6 receptor complex is stable, and dissociation of these two molecular species occurs at a pH below 5.0. In contrast to published results, data presented in this study clearly indicate that IL-6 is rapidly internalized and degraded within hepatocytes by a receptor-mediated mechanism.     

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.     

Characterization of interleukin-13 receptor in carcinoma cell lines and human blood cells and comparison with the interleukin-4 receptor

Abstract

The interleukin-13 receptor is characterized by ligand-binding and crosslinking studies and compared with the interleukin-4 receptor. Crosslinking of radio-labeled hIL-4 and hIL-13 to the receptors on human carcinoma and mast cell lines demonstrated a predominant subunit at 130 kDa with two other minor bands of lower molecular mass (75 kDa and 65 kDa) in autoradiography. All binding of ¹²⁵I-IL-13 was specifically blocked when the carcinoma cell suspensions were incubated with an excess of unlabeled hIL-4. However, unlabeled hIL-13 was unable to completely displace ¹²⁵I-hIL-4 from the 130 kDa protein. In addition, ¹²⁵I-hIL-13 showed no binding to mouse fibroblast cells transfected with human 130 kDa hIL-4 receptor c-DNA. Using weighted nonlinear computer modeling of the data from several equilibrium binding studies with human mast cells, a model of two binding sites for IL-4 (K_d = 50 and 190 pmol/L) and one site for IL-13 (K_d=100 pmol/L) fitted better than a one site model with a very high level of significance (F = 10.66, P < 0.0001). It can be concluded that human IL-4R and hIL-13R are similar but distinct. This conclusion is supported here for the first time by a strong statistical criterion.     

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.     

Internalization of human interleukin 4 and transient down-regulation of its receptor in the CD23-inducible Jijoye cells

Human interleukin 4 (IL-4) specifically induces the low affinity receptor for IgE (Fc epsilon R2/CD23) on the surface of the Burkitt lymphoma cell line Jijoye. At 4 degrees C 125I-IL-4 specifically binds to high affinity receptors (Kd = 4-10 x 10(-11) M; Bmax, 600-1,200 sites/cell). Following a rapid temperature shift from 4 to 37 degrees C, 80% of the receptor-bound 125I-IL-4 disappeared from the cell surface within 20 min (t1/2 = 8.9 min). For every two internalized molecules of IL-4 (t1/2 = 13 min), one molecule of IL-4 dissociated from the cell surface (t1/2 = 25 min). More than 90% of the internalized IL-4 was released in a degraded form into the medium following first order kinetics (t1/2 = 68 min). Internalization was inhibited by cytoskeletal disrupting and lysosomotropic agents. Incubating cells with 1 nM IL-4 resulted in a rapid down-regulation of IL-4 receptors (75% loss after 2 h); a reexpression of receptor to control level occurred after 20 h in spite of the presence of a large excess of IL-4. Reexpression was delayed by chloroquine and blocked by cycloheximide and actinomycin D. By using the cross-linking agent bis(sulfosuccinimidyl)suberate, three polypeptides of Mr 130,000, 80,000, and 70,000 were specifically labeled with 125I-IL-4. These three polypeptides coordinately disappeared and reappeared with the 125I-IL-4 binding sites. The induction of Fc epsilon R2/CD23 required prolonged incubation (greater than 8 h) with IL-4 and thus may be dependent on the reexpression of IL-4 receptor.     

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.     

Regulation of interleukin-1 receptors on AtT-20 mouse pituitary tumour cells.

To study the cellular mechanisms of interleukin-1 (IL-1) in the pituitary corticotroph, we studied the properties of IL-1 receptors on a mouse pituitary ACTH-producing cell line, AtT-20. Scatchard plot analysis revealed a single type of receptor with a Kd (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL-1 alpha binding competed with IL-1 alpha and IL-1 beta in an equimolar fashion. A 24 h pre-incubation with either CRH, epinephrine or nor-epinephrine increased the [125I]IL-1 alpha binding sites in the AtT-20 cells and conversely, a similar pre-incubation with either dexamethasone or tumour necrosis factor-alpha (TNF alpha) decreased them without affecting the affinity of the receptors in either case.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.