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 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.     

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.     

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.     

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.     

Binding, internalization, and intracellular localization of interleukin-1 beta in human diploid fibroblasts

This study demonstrates internalization of interleukin-1 (IL-1) via its cell surface receptor on human diploid fibroblasts and shows intracellular localization of IL-1 beta. Binding experiments at 8 degrees C using confluent fibroblast monolayers revealed 5,000-15,000 IL-1 receptors/cell that bound both IL-1 alpha and IL-1 beta. Incubation of monolayers with 125I-IL-1 beta (10(-9) M) at 8 degrees C and then at 37 degrees C for various times up to 8 h revealed a t1/2 for internalization of receptor-bound IL-1 beta of about 1.5 h. In addition, it was shown that IL-1 beta internalized via receptors was undegraded and retained binding activity. Electron microscopic autoradiography of monolayers incubated with 125I-IL-1 beta, as above, showed a progressive increase in the ratio of cytoplasmic to cell surface-associated grains. Grains at the cell surface were primarily localized at cell processes or attachment sites, frequently close to intra- and extracellular filamentous material. During incubation at 37 degrees C, most grains were free in the cytoplasm, with few present in lysosomes or vesicles. After 1 h, approximately 15% of the grains were over nuclei. Control cultures incubated at 37 degrees C with 125I-IL-1 beta and 100-fold excess unlabeled IL-1 beta showed increased uptake of label into lysosomes and little into nuclei. This study shows that IL-1 receptors are primarily located at fibroblast processes and that receptor-mediated internalization of the ligand is slow. Nuclear localization apparently requires IL-1 receptor-specific internalization of IL-1 beta, suggesting a possible role for this process in eliciting the IL-1 signal.     

Identification of cell surface receptors for murine macrophage inflammatory protein-1 alpha.

We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line. The 125I-rMIP-1 alpha binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor, with a Kd of approximately 1.5 x 10(-9) M and approximately 1200 binding sites/Con A-stimulated CTLL-R8 cell and a Kd of 0.9 x 10(-9) M and approximately 380 binding sites/RAW 264.7 cell. 125I-rMIP-1 alpha binding was inhibited by unlabeled rMIP-1 alpha in a dose-dependent manner, but not by IL-1 alpha or IL-2. rMIP-1 alpha inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti-rMIP-1 alpha antibodies blocked the growth-inhibitory effect of the rMIP-1 alpha on CTLL-R8 cells.     

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.     

Studies on IL-1 receptors on D10S T-helper cells: demonstration of two molecularly and antigenically distinct IL-1 binding proteins

Receptor binding studies were performed on the interleukin-1 (IL-1) sensitive T-helper cell line D10S, a stable line which proliferates to subfemtomolar concentrations of IL-1 in the absence of mitogens. IL-1 binds in a specific and saturable manner and Scatchard analysis at 4 degrees C reveals one class of binding affinity. On D10S cells, the Kd for IL-1 is 227 pM +/- 80, with 11,000 (range 3,300 to 23,800) sites per cell. EL4.6.1 cells, which are less sensitive to IL-1, bind with a single class of high affinity sites (55 pM; 4,000 sites). D10S cells incubated 18 h with IL-1 display reduced IL-1 receptor (IL-1R) numbers and affinities, consistent with reduced (75%, p less than 0.005) proliferation to subsequent IL-1; preincubation with IL-4 increases the number of IL-1R which is associated with increased (200%, p less than 0.001) proliferation to IL-1. The molecular mass of the major (80 kD) IL-1R binding [125I]IL-1 alpha on D10S cells was consistently observed at 73 kD as compared to the 80 kD molecule on the EL4 cells. On the other hand, crosslinking studies with [125I]IL-1 beta on D10S cells revealed a novel 46 kD band on gradient SDS-PAGE corresponding to a binding protein of 29 to 30 kD, which is antigenically distinct from the 80 kD IL-1R. Crosslinking of D10S or EL4 cells at 4 degrees C in the presence of phytohemagglutinin (PHA) and labeled IL-1 enhanced the appearance of the 30 kD IL-1 binding protein. The findings are consistent with a two-chain model for the IL-1R, although Scatchard analysis did not consistently indicate two classes of affinities. IL-1 binding to the 80 kD protein may form a heteroduplex with the 30 kD IL-1R which could account for the presence of the 120 to 130 kD IL-1 crosslinked proteins observed by several investigators.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

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.     

Interaction of bovine uterine luminal protein with interleukin-2 and the interleukin-2 receptor of T lymphocytes

Bovine uterine luminal proteins (ULP) collected on Day 17 of pregnancy were tested for inhibition of binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) of bovine (CLC) and human (HLC) T lymphocytes and for binding to IL-2. Additional experiments assessed IL-2 binding to the p55 alpha chain (Tac protein) of the IL-2R of HLC. High- and low-molecular weight (Mr) ULP components (H-ULP greater than 248,000 Mr and L-ULP 21,000 Mr, respectively) inhibited (p less than 0.05 and 0.01, respectively) the binding of 125I-IL-2 to the IL-2R of CLC, whereas only H-ULP inhibited (p less than 0.05) binding to the IL-2R (presumably, the p75 beta chain) of HLC. H-ULP failed (p greater than 0.05) to bind to the p55 alpha chain of the IL-2R of HLC. For IL-2 binding, L-ULP failed (p greater than 0.05) to bind 125I-IL-2 in short (2 h)-term and long (45 h)-term experiments, whereas binding was evident (p less than 0.05) for H-ULP at 2 h of incubation. For H-ULP, mean (+/- SEM) percentages for bound and unbound 125I-IL-2 were 70.1 +/- 11.4 and 29.9 +/- 11.4, respectively. Further purification of H-ULP yielded a component (1.76 x 10(6) Mr) that bound 11.7% of 125I-IL-2 and inhibited (p less than 0.01) thymidine uptake and binding of 125I-IL-2 to the IL-2R of CLC. H-ULP-mediated suppression of lymphocyte proliferation may result from blocking IL-2R recognition of IL-2 as well as binding to IL-2, whereas suppression by L-ULP may predominantly result from blocking IL-2R.     

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.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.     

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.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.