Molecular characterization of interleukin 12.

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D.P. Gearing and D. Cosman (1991) Cell 66, 9-10) that IL-12 consists of a complex of cytokine and soluble 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)     

16-kilodalton heparin binding (fibroblast) growth factor type one appears in a stable 40-kilodalton complex after receptor-dependent internalization

Incubation of 16-kDa 125I-labeled heparin binding (acidic fibroblast) growth factor type one (HBGF-1) with human hepatoma cells and normal rat hepatocytes resulted in the appearance of a stable 125I-labeled complex with an apparent molecular mass of 40 kDa. The complex could be isolated with specific antibodies against HBGF-1. In contrast to membrane receptor-bound 125I-HBGF-1, the complex was resistant to dissociation by detergents, acid, heat, and reducing or denaturing agents. Formation of a stable complex did not require treatment with cross-linking agents. Appearance of the 40-kDa complex was dependent on time, temperature, and enriched culture medium. Conditions that enhanced or reduced display of specific HBGF-1 membrane receptor sites enhanced or reduced the appearance of the 40-kDa complex. Dansylcadaverine, chloroquine, and staurosporine blocked the appearance of the 40-kDa complex concurrent with the blockage of internalization of the receptor-bound HBGF-1. Two-dimensional gel electrophoretic analysis, metabolic labeling with L-[35S]cysteine, and recovery of 16-kDa HBGF-1 from the 40-kDa complex after base treatment suggest involvement of a 24-kDa cellular protein in the complex formation. These results suggest a potentially novel receptor-dependent pathway for metabolism of HBGF-1.     

High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

The ratio of P40 monomer to dimer is an important determinant of IL-12 bioactivity

IL-12 is a 75 kDa heterodimer (IL12p70) comprised of independently regulated disulfide-linked 40 kDa (p40) and 35 kDa (p35) subunits. The p40 subunit exists extracellularly as a monomer (IL12p40) or dimer (IL12(p40)2) and can antagonize the action of IL12p70. Given the disagreement in the literature over the physiologic roles for IL12p70, IL12p40, and IL12(p40)2, we asked whether the bioactivity of IL-12 depended only on the concentration of the IL12p70 subunit alone or whether the relative concentrations of IL12p70, IL12p40, and IL12(p40)2 and their competitive binding with the IL-12 receptor are essential for determining IL-12 bioactivity under simulated human physiologic conditions. A mathematical model for IL-12 bioactivity was created by incorporating the production of IL12p70, IL12p40, and IL12(p40)2 by mature human DC and the interaction of these species with the IL-12 receptor. Using this model, we explored the effects of IFN-gamma, IL-4, and PGE2 concentrations on the bioactivity of IL-12. The simulations suggest that the concentration of IL12p70 alone is not indicative of IL-12 bioactivity; rather, the bioactivity of IL-12 produced by mature DC depends on IL12p70, IL12p40, and IL12(p40)2 production and their competitive interaction with the IL-12 receptor. In addition to the typically measured quantities of total p40 (IL12p40 + IL12(p40)2) and IL12p70, the ratio of IL12p40 to IL12(p40)2 is an equally important, yet underreported, determinant of IL-12 bioactivity.     

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity.

Nineteen independently isolated hybridomas producing monoclonal antibodies to the glycoprotein of vesicular stomatitis virus were isolated and studied for their capacity to neutralize viral infectivity. By measuring competitive binding of 125I-labeled monoclonal antibodies in a radioimmunoassay. 11 different, non-cross-reacting antigenic determinants were identified on the vesicular stomatitis virus G protein. All monoclonal antibodies reacting with determinants 1, 2, 3, and 4 resulted in viral neutralization, whereas those binding to the other seven determinants did not neutralize infectivity. The mixture of two monoclonal antibodies binding to different determinants resulted in a more rapid neutralization than either antibody alone, suggesting that different antibodies can exert a synergistic effect on viral neutralization. Kinetic experiments revealed biphasic neutralization curves similar to those expected for heterologous antibody. No evidence could be obtained to relate biphasic kinetics of viral neutralization to heterogeneous populations either of antibody molecules or of virus. The possible significance of the kinetic data with monoclonal antibodies is discussed.     

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.     

Structure-activity studies of human IL-1 beta with mature and truncated proteins expressed in Escherichia coli

IL-1 beta is synthesized as an inactive 31-kDa intracellular protein, which is then processed upon secretion to an active 17-kDa carboxyl-terminal fragment. To identify the minimal portion of IL-1 beta required for activity, we constructed several deletion mutants of mature IL-1 beta. These included three amino-terminal deletions of 10, 16, and 81 amino acids, two carboxyl-terminal deletions of 17 and 72 amino acids, and one internal fragment between amino acids 17 and 81. Expression of the mutants was monitored by Western blots and immunoprecipitation. With one exception, all of these mutants and the full length 17-kDa IL-1 beta were expressed as soluble protein in Escherichia coli and could be assayed for activity and receptor binding in lysates without further purification. Whereas the intact 17-kDa IL-1 beta retained full biologic activity (greater than 10(7) U/ml of lysate) and competed for binding with 125I-labeled IL-1 beta, none of the lysates containing IL-1 beta deletion mutant proteins had activity or competed for binding to receptor at significantly higher concentrations. The loss of function in the smallest C-terminal deletion mutant does not appear to be due to the direct involvement of these C-terminal residues in receptor binding because both monoclonal and polyclonal antisera directed to this region bind to IL-1 beta but do not neutralize its activity. Therefore, this region is probably indirectly involved in sustaining the structure of the receptor-binding site.     

Interleukin-18, a proinflammatory cytokine

Although IL-18 is an inducer of IFN-gamma and Th1 responses. IL-18 is a proinflammatory cytokine by several criteria. In several animal models. antibodies that neutralize endogenous IL-18 reduce the severity of disease. Endotoxin lethality is prevented by anti-IL-18. Even in models that are interferon-y independent, neutralization of IL-18 prolongs survival. Anti-IL-18 also protects the liver against cellular injury induced by toxins or activated T cells. In models of hepatic melanoma metastasis. IL-18 blockade reduces the adherence of malignant cells by preventing IL-18 upregulation of vascular endothelial adhesion-1 molecule expression. IL-18 and IL-12 act synergistically to stimulate I cells and natural killer cells to produce IFN-gamma but neutralization of IL-18 prevents IL-12 induction of IFN-gamma. IL-18. like several cytokines. can be used to enhance host defense against tumors in mice a mechanism that is most often IFN-gamma-dependent. Nevertheless. it is the proinflammatory portfolio of IL-18 which likely contributes to enhanced host defenses. In models or arthritis, lung injury or inflammatory bowel disease, neutralization of IL-18 reveals the important role of this cytokine in mediating inflammation.     

Characterization of novel peptide agonists of the alpha mating factor of Saccharomyces cerevisiae.

Alpha-factor [WHWLQLKPGQPMY], a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae (MATa, MATalpha). New analogues of alpha-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies. The Y(0)Nle(12)F(13) analogue [YWHWLQLKPGQPNleF] (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone. Binding of (125)I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native alpha-mating factor. Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (K(d) = 6.4 x 10(-8)). Specific binding of (125)I-labeled MFN5 was significantly reduced by guanosine nucleotides. Affinity cross-linking of (125)I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein. The novel synthetic alpha-factor analogue MFN5 can be easily iodinated and used as a probe for the alpha-factor receptor.     

A comparative study of IL-12 (cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-induced effects on immunomagnetically purified CD56+ NK cells.

IL-12, or cytotoxic lymphocyte maturation factor, is a recently cloned cytokine shown to influence lymphokine-activated killer cells activity in heterogeneous lymphocyte populations, proliferative activity as a costimulus in PBMC/PBL populations and IFN-gamma production in PBL. We have investigated the effects of IL-12 on immunomagnetically highly purified CD56+ lymphocytes, and compared the effects with those of IL-7 and IL-2. Our results show that IL-12 directly generated high lymphokine-activated killer cell activity in CD56+ NK cells, without the need for accessory cells. The IL-12-induced lymphokine-activated killer cell activity reached 50% of what was obtained with IL-2. In contrast, only low proliferative activity was induced by IL-12, as 10% of the IL-2-induced- and approximately 50% of the IL-7-induced proliferative activity was detected with IL-12. The CD56+ cells expressed high levels of IL-2R alpha and 75-kDa TNFR in response to IL-12, comparable to what was registered with IL-2 and IL-7. Furthermore, an extensive up-regulation of the CD56 Ag, to the level obtained with IL-2, was detected in the CD56+ NK cells in the presence of IL-12. Stimulation with IL-7 resulted in a more limited CD56 up-regulation in the CD56+ NK cells. Low concentrations of TNF-alpha were produced in response to both IL-12 and IL-7, with little or no TNF-beta production. Time course of the IL-2-induced TNF production revealed an initial TNF-alpha production, whereas significant levels of TNF-beta were detected after 72 h. The effects of both IL-12 and IL-7 on the CD56+ NK cells were inhibited by an anti-TNF-alpha mAb. Thus, IL-12 can directly influence NK cell activities in purified CD56+ cells, and endogenously produced TNF-alpha is involved in mediating the effects of both IL-12 and IL-7.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).     

Monoclonal antibodies to either domain of ovotransferrin block binding to transferrin receptors on chick reticulocytes

Monoclonal antibodies produced to both chicken ovotransferrin and to the isolated N- and C-terminal half-molecule domains of ovotransferrin have been used to probe the interaction of ovotransferrin with its specific receptor on chick embryo red blood cells. Two antibodies to epitopes on the N-terminal domain and one antibody to an epitope on the C-terminal domain were able to block the binding of 125I-labeled diferric ovotransferrin to the receptor. When the cellular surface receptors were first saturated with ovotransferrin at 0 degrees C, none of these antibodies bound to the cell-associated ovotransferrin. This suggests that the antibodies are to epitopes which lie very near to, or in the regions of, the two domains which interact with receptor. The same three antibodies also blocked the binding to the receptor of ovotransferrin associated in situ from the isolated N- and C-terminal half-molecule domains. A fourth antibody did not block binding to receptor of 125I-labeled diferric ovotransferrin or the associated domains; furthermore, it was able to bind to ovotransferrin bound to the cell surface at 0 degrees C. This antibody thus appears to recognize an epitope remote from the receptor binding region of ovotransferrin. Additional evidence for the requirement of the presence of both domains of ovotransferrin to effect binding to the transferrin receptor on chick reticulocytes was obtained with a fifth antibody which recognized only the N-terminal half-molecule domain but not holo-ovotransferrin. Although this antibody had no effect on the binding of 125I-labeled ovotransferrin to cells, it blocked binding to receptor of the associated domains of ovotransferrin, presumably by inhibiting the association of the two domains.     

Low density lipoprotein receptor-related protein mediates endocytosis of monoclonal antibodies in cultured cells and rabbit liver

Monoclonal antibodies that bound to the external domain of the rabbit low density lipoprotein receptor-related protein (LRP) were taken into rabbit fibroblasts by receptor-mediated endocytosis. Uptake occurred in fibroblasts from Watanabe-heritable hyperlipidemic rabbits, which lack low density lipoprotein receptors, as well as in normal rabbit fibroblasts. The fate of the internalized antibodies differed, depending on the domain of LRP that was recognized. LRP is synthesized as a single polypeptide chain that is cleaved to form a heterodimer of two noncovalently bound proteins, 1) a 515-kDa subunit that contains the binding domain, and 2) an 85-kDa subunit that contains the membrane-spanning region and cytoplasmic tail. A monoclonal antibody directed against the 515-kDa subunit (anti-LRP 515) rapidly dissociated from LRP at pH 5.2. After uptake by cells this antibody dissociated from the receptor and was degraded in lysosomes. A second antibody directed against the external portion of the 85-kDa subunit (anti-LRP 85) failed to dissociate at acid pH. After uptake by cells this antibody was not degraded, but instead was released from the cells in an acid-precipitable form. When administered intravenously to rabbits, both 125I-labeled antibodies were rapidly cleared from the circulation, 75-95% of the uptake occurring in the liver. The anti-LRP 515 antibody was degraded and acid-soluble products appeared in the plasma. No significant acid-soluble products appeared when the anti-LRP-85 antibody was infused. We conclude that LRP can carry out receptor-mediated endocytosis and that its ligand-binding domain, like the similar domain of the low density lipoprotein receptor, undergoes an acid-dependent conformational change that ejects ligands within the endosome. We also conclude that in the body this endocytotic function is expressed primarily in the liver. Both of these conclusions lend support to the hypothesis that LRP may function in humans and animals as a receptor for apolipoprotein E-enriched lipoproteins, such as chylomicron remnants.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Phosphorylation of a cytosolic 65-kDa protein induced by interleukin 1 in glucocorticoid pretreated normal human peripheral blood mononuclear leukocytes

The authors have previously observed that glucocorticoids dramatically increase the number of interleukin 1 (IL-1) receptors on normal human peripheral blood mononuclear cells (PBMC) (from approximately 100 to 2000 receptors/cell) without significant change in the binding affinity (Kd = approximately 2.6 x 10(-10) M). We, therefore, used such a receptor-enriched glucocorticoid-pretreated PBMC to investigate whether IL-1 induces/increases the phosphorylation of any cell-associated proteins, including possible autophosphorylation of IL-1 receptors. Extraction of 125I-labeled IL-1 alpha cross-linked to IL-1 receptor on steroid-treated PBMC yielded two bands estimated to be 60 and 70 kDa in molecular mass. No molecules were significantly cross-linked with 125I-labeled IL-1 alpha on untreated PBMC. Carrier-free recombinant human IL-1 alpha induced phosphorylation of an acidic 65-kDa protein (pp65) at serine residues within 5 min more effectively in glucocorticoid-treated PBMC than in untreated PBMC. Fractionation of extracts of IL-1-stimulated prednisolone-pretreated PBMC by ultracentrifugation showed that pp65 is located in the cytosol, suggesting that pp65 is not the IL-1 receptor itself. Protein kinase inhibitors, HA1004 and W-7, but not H-7, significantly inhibited the induction of the phosphorylation of 65-kDa protein by IL-1. These data indicate that the glucocorticoid-induced IL-1 receptor is functional and either contains or is closely associated with a serine kinase that is distinct from protein kinase C.     

Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.     

Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.