Interleukin-1 Receptor Binding Research Articles

Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions.

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261-8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100+/-3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183+/-30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P>0.2; 233+/-90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74+/-11 nM above baseline), and 1 microM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136+/-39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1beta. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 degrees C, in cells maintained for 3 h at 4 degrees C, in cells incubated with BSA beads for 3 h at 37 degrees C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1beta showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

The interleukin-2 fusion protein, DAB389IL-2, inhibits the development of infectious virus in human immunodeficiency virus type 1-infected human peripheral blood mononuclear cells.

DAB389IL-2 is a genetically engineered fusion protein that reduces human immunodeficiency virus type 1 (HIV-1) replication in activated, interleukin (IL)-2 receptor (IL-2R)-expressing human peripheral blood mononuclear cells (PBMC). The level of infectious virus released by cultured PBMC after treatment with DAB389IL-2 was measured by a quantitative microculture assay. The inhibition of p24 antigen production was also evaluated in cultures that differed in duration of infection and activation state. Although the activation state of the cell and the time of DAB389IL-2 addition to the cultures influenced its anti-HIV activity, DAB389IL-2 treatment decreased levels of infectious HIV-1 to 0.1%-6.5% of untreated cell levels. DAB389IL-2 also decreased p24 antigen expression to 10%-48% of controls, even when added as late as 8 days after acute infection. Mutational variants of DAB389IL-2 that lack catalytic activity or IL-2R binding are without anti-HIV activity.

Analysis of the Human Interleukin-6/Human Interleukin-6 Receptor Binding Interface at the Amino Acid Level: Proposed Mechanism of Interaction

AbstractThe interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6–inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their α-subunit receptors suggests that the structural location of certain binding sites are conserved.

Usefulness of flow cytometric detection of cell surface interleukin-6 receptors in human myeloma cell lines.

A flow cytometric procedure was used to analyse the cell surface interleukin-6 receptor (IL-6R) based on the principle of detecting the binding of IL-6 to IL-6R. The bound IL-6 was visualized by reacting with anti-IL-6 antibody, a second biotinylated antibody to immunoglobulins, fluorescein-conjugated avidin and biotinylated, fluorescein-conjugated bovine serum albumin. Studies with a number of human myeloma cell lines showed that the fluorescence signal from cellular binding of IL-6, and thus IL-6R, could be specifically discerned from the background. The specific IL-6R signal could be semi-quantitatively expressed as the mean channel number of the fluorescence histogram or as relative IL-6R density in relation to a reference cell line, such as U266-BL cells. The relative IL-6R density of various myeloma cell lines thus determined was found to correlate with their sensitivity to the growth inhibitory effect of glucocorticoids in vitro. Quantitatively, calibration of the staining procedure with microbeads that have a defined binding capacity for anti-IL-6 antibody allowed calculation of cellular IL-6R density that yielded results close to that reported with conventional radio-ligand binding assay. Similarly, the cytometric method was applied to the studies of kinetics of IL-6/IL-6R interaction and the cellular IL-6R changes after ligand-binding to provide estimates of IL-6R binding affinity and IL-6R turnover rate. It is suggested that flow cytometric measurement of cellular IL-6R is useful in providing biologically relevant information on myeloma cells.

Mechanism of exercise-induced augmentation of lymphokine activated killer (LAK) cell activity in the horse.

Intense exercise affects various parameters of the immune system. The overall effect of exercise on immune function is dependent upon the physical condition of the subject, the intensity and duration of the exercise period, and the immune parameter assessed. Unconditioned horses subjected to a single bout of intensive exercise exhibit multiple alterations in immune function, including an augmentation of lymphokine activated killer (LAK) cell function. This increase in LAK cell activity is not due to an increase in circulating LAK precursors. While peripheral blood mononuclear cells from exercising horses exhibit greater responsiveness to IL-2, this is not due to an increase in IL-2 receptor expression. LAK cell generation in vitro is augmented by those catecholamines and neuropeptides which are produced during exercise, suggesting a direct effect of these compounds on LAK cell generation at a step post IL-2 receptor binding.

A Cell-Free, Nonisotopic, High-Throughput Assay for Inhibitors of Type-I Interleukin-1 Receptor

A cell-free, nonisotopic assay has been developed to discover molecules that compete with the natural ligands for binding to the active site of the Type-I interleukin-1 receptor. The key reagents are the interleukin-1 receptor antagonist, a recombinant soluble form of the receptor (sIL-1R), and a specific anti-sIL-1R nonneutralizing monoclonal antibody (MAb79). With these molecules a sensitive assay has been developed using a reversed format: the ligand is immobilized and the receptor is in solution. The ligand-bound receptor is detected using MAb79 and an enzyme-linked secondary antibody. Since no cells or cell membranes are used, the assay is very robust, with no interference from membrane-perturbing agents and high resistance to the organic solvents normally used to resuspend compounds of chemical libraries. The microplate format and colorimetric detection have allowed the complete automation of the immobilized-ligand IL-1 receptor binding assay, which has been used for high-throughput screening of synthetic compounds and natural products.

DAB389 Interleukin-2 Receptor Binding Domain Mutations: CYTOTOXIC PROBES FOR STUDIES OF LIGAND-RECEPTOR INTERACTIONS

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

γc Gene Transfer Into SCID X1 Patients’ B-Cell Lines Restores Normal High-Affinity Interleukin-2 Receptor Expression and Function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL-7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL-2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R.

The therapeutic value of Interleukin 2 (IL-2) is limited by its short half life and systemic toxicity. One approach to overcoming these problems is to fuse this protein to an antibody, a protein with a long half life and the ability to target a unique antigen within the body. To examine the biochemical properties of such a molecule a fusion protein was constructed linking the N-terminus of human IL-2 to the C-terminus of IgG3. A similar fusion between IgG1 and IL-2 has previously been shown to bind antigen, generate antibody-dependent cellular cytotoxicity (ADCC) and stimulate T cell proliferation and cytotoxicity. We now extend these studies and show that the fusion protein, termed IgG3-IL2, is appropriately N-glycosylated within the IgG3 CH2 domain, binds the human high affinity Fc receptor (Fc gamma RI) with an affinity slightly lower than that of IgG3, and is able to activate complement via the classical pathway to lyse antigen coated sheep red blood cells (SRBC). When used to stimulate the proliferation of the IL-2 dependent cell line CTLL-2, IgG3-IL2 has a specific activity slightly lower than that of human recombinant IL-2 (hrIL-2). In marked contrast, when comparable unit concentrations, as defined by the standard CTLL-2 proliferation assay, are used to stimulate human peripheral blood lymphocytes (PBL), IgG3-IL2 generates significantly greater lymphokine activated killer (LAK) cell cytotoxicity than does hrIL-2. Competition studies show that IgG3-IL2 binds the intermediate affinity form of the IL-2 receptor (IL-2R), consisting of the beta and gamma subunits, with an affinity slightly less than that of hrIL-2. In contrast, IgG3-IL2 shows a greater affinity than hrIL-2 for the high affinity IL-2R, consisting of alpha, beta and gamma subunits. Our studies show that the IgG3-IL2 fusion protein possesses a combination of the biological properties of IgG3 and IL-2 including antigen binding, complement activation, Fc gamma RI binding, IL-2R binding and stimulation of both proliferation and LAK activity. This combination of activities may allow IgG3-IL2 to target humoral and cell-mediated immune activation to the site of an antigen of interest or target an antigen to IL-2R bearing cells or organs.

Ligand-induced desensitization of interleukin 1 receptor-initiated intracellular signaling events in T helper lymphocytes.

Although interleukin 1 (IL-1) receptor signaling events in T helper lymphocytes are incompletely characterized, events associated with translocation of the transcription factor NF-kappa B are receptor-proximal assays of ligand-initiated responses. In this report we demonstrate that the transient nature of IL-1-induced NF-kappa B nuclear translocation occurs as a consequence of ligand-induced receptor desensitization. Other receptor-initiated events including induction of I kappa B alpha phosphorylation, expression of c-jun and junB mRNA, and costimulatory effects on IL-2 synthesis also are altered by IL-1 receptor desensitization. IL-1 receptor desensitization is not initiated by tumor necrosis factor, which also stimulates NF-kappa B translocation, and is not a consequence of alterations in either IL-1 receptor expression or binding affinity. In the absence of IL-1, the effects of desensitization are completely reversed within 18 h. Since IL-1 desensitization is initiated under conditions of low receptor occupancy, it is likely that receptor desensitization results from alterations to a receptor-proximal transducer, rather than from direct modification of the IL-1 receptor. These results suggest that the cyclic nature of the events in the T helper lymphocyte activation program can be controlled, in part, by the reversible desensitization of cell surface IL-1 receptors.

Altered Gene Transcription After Burn Injury Results in Depressed T-Lymphocyte Activation

Patients with major burns and an animal model of burn injury were studied to determine the mechanism of depressed interleukin-2 (IL-2) production after thermal injury and to determine the effect of such injury on IL-2 receptor (IL-2R) expression and function. Major burn injury is known to diminish resistance to infection by altering cytokine production and prostanoic secretion and by inhibiting T-lymphocyte activation. T-cell activation requires production of regulatory cytokines, principally IL-2, and expression of the appropriate cytokine receptors. Depressed IL-2 production after major burn injury is undisputed, although the molecular mechanisms remain undefined; the effect of burn injury on IL-2R expression and function currently is controversial. The authors studied serial samples of peripheral blood mononuclear cells (PBMC) from 11 patients with 25% to 95% surface area burns and 7 age-matched volunteer control subjects. Peripheral blood mononuclear cells were stimulated by the T-cell mitogen phytohemagglutinin (PHA), and IL-2 production and mRNA expression by Northern blot were determined. Expression and function of IL-2R were determined by monoclonal antibodies to the p55 and p75 chains of the IL-2R, binding of fluorescein-labeled IL-2, and response to exogenous recombinant IL-2. We also studied a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from mice with burns (20-22 per group) were studied for IL-2 production and IL-2 mRNA expression after stimulation with the T-cell mitogen concanavalin A (ConA) and compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation also were determined and a nuclear run-on assay performed to determine IL-2 gene transcription. The mRNA expression was determined for the proto-oncogenes c-jun and c-fos, whose protein products join to form transcription factor AP1, which is necessary for activation of the IL-2 promoter. Splenocytes from mice with burns after ConA stimulation also were studied for expression and function of the IL-2R. Peripheral blood mononuclear cells from burn patients compared with healthy control subjects showed diminished (p < 0.05) IL-2 production and mRNA expression 4 to 10 days after burn injury. Burn PBMC demonstrated normal expression of IL-2R, p55, and p75 chains 0 to 7, 8 to 20, and 21 to 37 days after burn injury, normal IL-2R binding of fluorescein-labeled IL-2, and a normal proliferative response to PHA in the presence of exogenous recombinant IL-2. Splenocytes from mice 7 days after burn injury showed diminished production (p < 0.05) of IL-2 and IL-2 mRNA expression after ConA stimulation as compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation were the same for burn and control mice, indicating that reduced IL-2 mRNA expression was not caused by altered mRNA degradation. A nuclear run-on assay confirmed decreased IL-2 gene transcription in burn splenocytes. Burn splenocytes showed normal expression of mRNA for c-jun but diminished expression of mRNA for c-fos. Finally, splenocytes from mice with burns after ConA stimulation showed normal expression and function of the IL-2R 7, 10, 14, and 21 days after burn injury. These human and animal studies indicate that major burn injury depresses T-cell activation at the level of IL-2 gene transcription at least in part by inhibiting c-fos expression, whereas IL-2R expression and function remain normal and T-cell proliferation can be restored to normal levels by exogenous IL-2.

Solution structure of human interleukin-1 receptor antagonist protein

Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. In contrast to IL-1β IRAP binds to the IL-1 receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153-residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL-1 receptor binding are conserved between IRAP and IL-1β. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.

Interleukin-2 (IL-2) receptor gamma chain mutations in X-linked severe combined immunodeficiency disease result in the loss of high-affinity IL-2 receptor binding.

Interactions of interleukin-2 (IL-2) with its high-affinity, heterotrimeric receptor (IL-2R alpha beta gamma) play a pivotal role in the autocrine pathway of T lymphocyte expansion required in an immune response. Mutations in the IL-2R gamma chain-encoding gene have been found in SCIDX1, a primary immunodeficiency characterized by the absence of T cell and NK cell development. We have investigated six unrelated SCIDX1 patients for molecular abnormalities of the IL-2R gamma gene. A variety of defects were identified, including the absence of transcripts, frame-shift deletions and point mutations within canonical cytokine receptor motifs (conserved cysteines and the "WS" box). The ability of these mutated IL-2R gamma chains to participate in the function of a high-affinity IL-2R complex was examined by radiolabeled IL-2 binding studies using Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) derived from SCIDX1 patients. Although normal control B-LCL express high-affinity IL-2 binding sites (Kd = 60 pM, 150 sites/cell), B-LCL derived from SCIDX1 patients failed to bind IL-2 under high-affinity conditions. These SCIDX1 mutations confirm the critical role of the IL-2R gamma chain in T cell and NK cell development. In addition, these data provide insight into the structure/function relationship of the IL-2R gamma chain by identifying residues required for the formation of a high-affinity IL-2R complex.

Interleukin 1 alpha stimulates nuclear phospholipase C in human osteosarcoma SaOS-2 cells.

Interleukin 1 (IL-1) is one of the most potent stimulators of bone resorption. However, the early biochemical events elicited by IL-1 receptor binding are not fully understood. Here we show that in human osteosarcoma SaOS-2 cells the treatment with IL-1 alpha is able to evoke a rapid and transient increase of nuclear phospholipase C (PLC) activity. A parallel decrease of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate is observed. All these events are strictly confined to the nuclear compartment without affecting the cytoplasmatic inositol lipid pool. In addition we show that by Western blot analysis with specific monoclonal antibodies the PLC gamma is located both in the cytoplasm and in the nucleus, while PLC beta appears exclusively localized in the nucleus. Moreover, the increase of PLC activity in response to IL-1 alpha is completely neutralized by monoclonal antibody against the beta-form. While confirming the existence of an autonomous nuclear phosphoinositide signaling system, our data clearly indicate that in SaOS-2 cells one of the earliest events following IL-1 alpha treatment is the breakdown of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate because of the activation of a specific nuclear PLC isoform.

Development of a human interleukin-6 receptor antagonist.

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.

Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo.

Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia. Recently, the involvement of interleukin-6 (IL-6) in the wasting of colon-26 adenocarcinoma-bearing mice was demonstrated. The research presented here establishes an anticachectic role for the experimental drug suramin, since it partially blocks (up to 60%) the catabolic effects associated with the growth of this tumor in vivo. Suramin prevents the binding of IL-6 to its cell surface receptor subunits, as demonstrated by radioreceptor binding assay and affinity crosslinking experiments. Furthermore, the uptake of radioactive IL-6 by the liver is significantly reduced in suramin-treated mice. On the other hand, the drug is approximately 10-fold less potent in inhibiting the binding of tumor necrosis factor-alpha to indicator cell line in vitro and fails to block liver uptake of this cytokine in vivo. Collectively, these results suggest that suramin inhibits cancer-associated wasting, in part by interfering with the binding of IL-6 to its receptor. Whether suramin inhibits the action of other factors/cytokines that may also participate in colon-26-mediated cachexia is not yet known.

Structure-function analysis of the C-terminal segment of human interleukin-6.

It has been hypothesized that interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF) may fold as four-alpha-helix bundle proteins. To probe the functional role of the putative fourth helical segment of IL-6 (D-helix), a chimeric IL-6/G-CSF analog containing the predicted D-helix of G-CSF as well as a panel of IL-6 D-helix point mutants were analyzed for their respective secondary structure, antigenicity, and receptor binding and biological activities. The putative D-helix of IL-6 could not be replaced by its G-CSF counterpart in spite of their high degree of similarity and thus is indispensable for the antigenic and functional integrity of the IL-6 receptor binding site. Conversely, the grafting of the G-CSF D-helix did not confer any G-CSF activity to IL-6. A synthetic helical peptide containing the IL-6 D-helix was inactive, even when mixed with or linked to a peptide from the A-helix known to be involved in the active site. However, the conserved residues F173, R179, and R182 found in the D-helices of both IL-6 and G-CSF critically contribute to the architecture of the IL-6 active site. Indeed, mutation of F173 or R179 markedly affected IL-6 receptor binding and biological activities, but not the conformation of a major neutralization epitope. Furthermore, substitution of R182 resulted in a significant unfolding of the D-helix accompanied by a drastic loss in IL-6 antigenicity and functional activities. Nevertheless, residues other than F173, R179, and R182 also contribute to IL-6 specificity.

Evidence for an acute phase response in human intestinal epithelial cells

During the host response to inflammation/tissue injury there are many changes in intermediary metabolism including a dramatic change in the concentrations of many "acute phase" plasma proteins. Although many of these acute phase proteins are predominantly derived from the liver and the response can be elicited from liver cells incubated in tissue culture with cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor-alpha, interferon-gamma, leukemia inhibitory factor, interleukin-11 (IL-11), and oncostatin M, there is now evidence that the response can also be elicited in extrahepatic tissues and cell types. In this study, we show that many of the acute phase plasma proteins are expressed in human intestinal epithelial cell lines Caco2 and T84 and that their expression is induced or regulated by cytokines IL-6, IL-1, interferon, and tumor necrosis factor in a manner characteristic of the acute phase response. In fact, effects of IL-1 and IL-6 which are additive, synergistic, and antagonistic in liver cell lines are also observed in these intestinal epithelial cell lines. Responses to IL-6 and IL-1 are seen at all stages of differentiation of Caco2 cells from crypt-like enterocytes to villus-like enterocytes. Caco2 cells express binding sites for IL-6 at both poles, for IL-1 at the basolateral pole and, to a lesser extent, at the apical pole. T84 cells have IL-1 and IL-6 receptor binding sites only at the basolateral pole. IL-6 and IL-1 also regulate the expression of enterocyte-specific integral membrane proteins as exemplified by down-regulation of sucrase-isomaltase gene expression in response to IL-6. These data raise the possibility that enterocytes are involved in a local response to injury/inflammation at the epithelial surface and establish a model system for examining coordination of the acute phase response in a bipolar cell.

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.

Suppression of lymphocyte proliferation by a retroviral p15E-derived hexapeptide.

CKS-17 (LQNRRGLDLLFLKEGGL), a synthetic peptide derived from a conserved region of retroviral transmembrane proteins, has previously been shown to suppress several different immune effector mechanisms. The present study was undertaken to further delineate immunosuppressive site(s) of CKS-17. Overlapping hexapeptides covering the complete sequence of CKS-17 were synthesized. One CKS-17-derived hexapeptide, LDLLFL, suppressed ligand [CD3, interleukin (IL)-2]-induced lymphocyte proliferation. Spontaneous proliferation of transformed lymphoid cell lines, as well as cell lines from myeloid or epitheloid origin, was not inhibited by LDLLFL. Full suppression required the continuous presence of LDLLFL during culturing, and did not involve interference with monocyte function. Radiolabeling studies showed that the hexapeptide did not compete with IL-2 for IL-2 receptor binding. Most likely the LDLLFL motif interferes with steps shared by the IL-2 and CD3 receptor-induced signaling pathways. Since LDLLFL displays multiple immunosuppressive activities, it may constitute a biologically relevant immunosuppressive site of retroviral transmembrane proteins.