Interleukin-1 Receptor Binding Research Articles

NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models.

Aldesleukin, recombinant human IL2, is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses in approximately 10% of patients; however, severe side effects limit maximal dosing and thus the number of patients able to receive treatment and potential cure. NKTR-214 is a prodrug of conjugated IL2, retaining the same amino acid sequence as aldesleukin. The IL2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly release to generate active IL2 conjugates. We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the tumor immunology, tumor pharmacokinetics, and efficacy of NKTR-214 as a single agent and in combination with anti-CTLA-4 antibody in murine tumor models. Tolerability was evaluated in non-human primates. In a murine melanoma tumor model, the ratio of tumor-killing CD8(+) T cells to Foxp3(+) regulatory T cells was greater than 400 for NKTR-214 compared with 18 for aldesleukin, supporting preferential activation of the IL2 receptor beta over IL2 receptor alpha, due to the location of PEG molecules. NKTR-214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy as a single agent and provided durable immunity that was resistant to tumor rechallenge in combination with anti-CTLA-4 antibody. NKTR-214 was well tolerated in non-human primates. These data support further evaluation of NKTR-214 in humans for a variety of tumor types, adding to the repertoire of potent and potentially curative cancer immunotherapies.

Stabilized Interleukin-6 receptor binding RNA aptamers

Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in the progression of various inflammatory diseases, such as rheumatoid arthritis and certain cancers; for example, multiple myeloma or hepatocellular carcinoma. To interfere with IL-6-dependent diseases, targeting IL-6 receptor (IL-6R)-presenting tumor cells using aptamers might be a valuable strategy to broaden established IL-6- or IL-6R-directed treatment regimens.Recently, we reported on the in vitro selection of RNA aptamers binding to the human IL-6 receptor (IL-6R) with nanomolar affinity. One aptamer, namely AIR-3A, was 19 nt in size and able to deliver bulky cargos into IL-6R-presenting cells. As AIR-3A is a natural RNA molecule, its use for in vivo applications might be limited due to its susceptibility to ubiquitous ribonucleases.Aiming at more robust RNA aptamers targeting IL-6R, we now report on the generation of stabilized RNA aptamers for potential in vivo applications. The new 2'-F-modified RNA aptamers bind to IL-6R via its extracellular portion with low nanomolar affinity comparable to the previously identified unmodified counterpart. Aptamers do not interfere with the IL-6 receptor complex formation. The work described here represents one further step to potentially apply stabilized IL-6R-binding RNA aptamers in IL-6R-connected diseases, like multiple myeloma and hepatocellular carcinoma.

Abstract 482: Tipping the balance in the tumor microenvironment: An engineered cytokine (NKTR-214) with altered IL2 receptor binding selectivity and improved efficacy.

Abstract Introduction Immunotherapy offers the potential for durable responses in a growing list of cancer indications.1 One approach towards stimulating the immune system is to target the heterotrimeric interleukin2 receptor, IL2R. Binding of ligand to the β subunit leads to expansion of tumor killing memory effector T cells, whereas engagement of the α subunit expands undesirable regulatory T cells, promoting immune suppression in the tumor.2 NKTR-214 uses polymer technology to engineer a cytokine that has reduced affinity for the IL2Rα subunit, diminishing the expansion of regulatory T cells in the tumor, while maintaining affinity for the β subunit expands memory effector T cells. The selective immunological profile in the tumor tips the balance towards tumor killing and results in superior efficacy and tolerability in a resistant mouse melanoma model. Methods The affinity of NKTR-214 to IL2Rα and IL2Rβ was measured directly by SPR and compared to that of clinically available IL2 (Aldesleukin). C57BL/6 mice bearing subcutaneous B16F10 melanoma were treated with NKTR-214 (IV, Q9dx3), Aldesleukin (BIDx5, 2 cycles) or vehicle. Immune cell populations in the tumor microenvironment were characterized by flow cytometry. Anti-tumor efficacy was measured by monitoring tumor volumes over time. Data The affinity of the activated form of NKTR-214 to IL2Rα was reduced by ∼8x compared to clinically available Aldesleukin. Mice receiving NKTR-214 showed an increase of ∼8-fold in CD8 memory effector T cells in the tumor compared to Aldesleukin .Conversely, mice receiving NKTR-214 showed a ∼6-fold decrease of CD4 regulatory T cells in the tumor compared to Aldesleukin. Improved survival and substantial tumor growth delay were observed in the NKTR-214 treated mice as compared to Aldesleukin, despite using 10-fold fewer moles of protein. The mice in the NKTR-214 treated group were not moribund, did not shiver, and showed an 84% decrease in mortality as compared to the Aldesleukin treated group. Conclusions Engineering a cytokine through polymer modification leads to a differential IL2 receptor binding profile and an improved tumor infiltrating T cell profile. The modification increases CD8 memory effector T cells and decreases suppressive regulatory T cells in the tumor compared to a clinically available IL2 (Aldesleukin). The reduced level of immune suppression in the tumor may explain the substantially improved therapeutic index observed for NKTR-214, despite using 10-fold fewer moles of protein, and infrequent dosing (Q9Dx3). The drug was well tolerated by the mice, with an 84% decrease in mortality compared to Aldesleukin. The results demonstrate that polymer engineering of a cytokine can have a profound differentiating effect on its in vitro and in vivo behavior. 1L. Calabro, Semin Oncol 2010 37 460-467 2O. Boyman, J. Sprent Nature Reviews Immunology 2012 12 180 Citation Format: Deborah Charych, Steven Lee, Murali Addepalli, Thomas Chang, Peter Kirk, Marina Konakova, Laurie Vanderveen, Rupesh Kanhere, Dennis Fry, Cherie Ali, Stephen Harrison, Seema Kantak, Stephen Doberstein. Tipping the balance in the tumor microenvironment: An engineered cytokine (NKTR-214) with altered IL2 receptor binding selectivity and improved efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 482. doi:10.1158/1538-7445.AM2013-482

IL-6 and its receptors in coronary artery disease and acute myocardial infarction

Biomarkers such as interleukin-6 (IL-6), soluble interleukin-6 receptor (sIL-6R), and high sensitive C-reactive protein (hsCRP) have been reported to be elevated in acute myocardial infarction (AMI). The aim of this study is to determine the relationship between these markers during AMI, as well as their relationship to clinical parameters in an effort to discern their predictive potential in cardiac events.Serum was collected from 73 patients with; AMI, stable coronary artery disease (CAD), and controls during cardiac catheterization. Biomarker levels were determined and correlated with clinical data. IL-6 (11.75pg/ml, P<0.05) and sIL-6R (41,340pg/ml, P=0.05) were elevated in AMI compared with CAD and controls. At presentation, hsCRP was elevated in AMI patients (4.69mg/L) compared to controls (2.69mg/L, P<0.05); however, there was a significant decrease in hsCRP between AMI (4.69mg/L) and CAD patients (7.4mg/L, P<0.05). After 24h post-AMI hsCRP levels were increased compared to stable CAD (60.46mg/L, P<0.05) and were preceded by increased IL-6 at presentation. Soluble Gp130 (sGp130) showed no significant change between AMI, CAD, and control patients. However, sGp130 positively correlated with peak troponin in AMI (R=0.587, P<0.01), and negatively correlated with previous AMI (R=−0.382, P<0.05). Circulating monocyte mRNA expression isolated from selected AMI patients showed an increase in IL-6 mRNA (5.28-fold, P<0.01) and a decrease in both IL-6R (0.374-fold, P<0.01) and sGp130 mRNA (0.38-fold, P<0.01) as compared to CAD and controls.Results demonstrate that IL-6 and sIL-6R are associated with AMI and cardiac injury. These data support the hypothesis that trans-IL-6 receptor binding may alter intracellular signaling, and blocking of IL-6 receptor binding may be pathogenic in AMI. These data may be predictive of mechanism(s) by which plaques become unstable and rupture.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response.

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (T(reg)) and effector (T(eff)) T cells, whereby access to IL-2 can either increase the survival of T(eff) cells or the suppressive capacity of T(reg) cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of T(reg) cells, namely the specific suppression of survival signals for weakly activated T(eff) cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of T(reg) suppression.

Tocilizumab-induced erythroderma

Auteur(s) : Motonobu Nakamura, Yoshiki Tokura Department of Dermatology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Tocilizumab is a humanized anti-human interleukin 6 receptor (IL-6R) antibody, engineered by grafting the complementarily determining regions of a mouse anti-human IL-6R antibody into human IgG1 to create a human antibody with a human IL-6R binding site [1, 2]. Tocilizumab binds to the IL-6 binding [...]

Heparan sulphate recruitment to the IL-1 receptor complex

Localization of IL-1 receptors at focal adhesions was demonstrated by transfection of an EGFP containing construct and confocal microscopy. IL-1/IL-1R binding at these sites was shown to be regulated by cell matrix interaction. In addition, fibronectin binding resulted in recruitment of a novel attachment-induced component to the IL-1 receptor complex, constituting a heparan sulfate. Fibronectin attachment caused a pronounced enhancement in IL-1 receptor binding (from 2500 to 4300 receptor/cell). In addition cell–matrix interaction resulted in a decrease in binding affinity (from 1.0 × 109 to 5.6 × 108m−1) resulting from a 2 fold reduction in association rate constant. These effects could be reversed by the addition of soluble heparin. Crosslinking experiments showed that in cells on fibronectin, 75% of the radiolabeled IL-1 was associated with a highly glycosytaled high molecular weight component of about 300 kD. Enzyme digestion demonstrated the presence of a heparin sulfate with a core protein of about 90 kD. The interaction was demonstrated to involve specifically the type I IL-1 receptor and to require IL-1 binding. Further, using a mutant fibronectin, formation of the complex was shown to be dependent on cell interaction with the heparin binding region. The attachment induced involvement of heparin sulfate in regulation of IL-1/Il–1 receptor interaction correlates with data on matrix effects on signal transduction suggesting a significant role of this interaction in inflammatory responses.

IL-1 induced shape changes mediated by ras

IL-1, an inflammatory cytokine, is known to mediate a wide range of biological activities in a number of cell lines. IL-1 receptor binding causes modulation at focal adhesions and reorganisation of the actin cytoskeleton. Further, IL-1 induced signal transduction is regulated by integrin binding and fibronectin attachment is permissive in IL-1 mediated gene regulation. Members of the p21ras superfamily of GTP-binding proteins have been shown to play a central role in mediating coregulation of structural and growth factor/cytokine mediated responses. To determine the role of GTP-binding proteins in matrix regulation of IL-1 mediated responses we have made GTPase/EGFP constructs of human Ras and the dominant negative mutant Asn17-Ras. Confocal microscopy of live cells shows that IL-1 stimulation of Ha-Ras transfected cells plated on a fibronectin matrix show relocalisation of the Ras/EGFP fusion protein to the cell membrane, and subsequent rapid and pronounced shape changes, causing cell rounding to 50–70% of original section area. No such changes were seen upon stimulation with EGF, a known activator of Ras. The shape changes correlate with a transient activation of Ras in fibronectin attached cells, comparable with that of EGF. Transfection of cells with the dominant negative Ras construct (Asn17-Ras), or plating of Ras transfected cells on bare tissue culture plastic abrogates the observed IL-1 induced changes in cell morphology and activation. Automated screening of transfected cells at low magnification gave similar results, showing that IL-1 affects approximately 50% of the transfected cell population. These data indicate that IL-1 induced shape changes are due to activation of Ras, and that it is involved in regulating IL-1 mediated responses in a matrix dependent manner. The studies are supported by the STFUSH, the Medical Research Council and the Wellcome Trust.

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice.

Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN-gamma from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 microg/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10-14 and 17-21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN-gamma from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19-57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4+ and CD8+ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.

Analysis of Fish IL-1&amp;#946; and Derived Peptide Sequences Indicates Conserved Structures with Species-Specific IL-1 Receptor Binding: Implications for Pharmacological Design

A large number of IL-1 protein sequences have become available recently from a range of vertebrate species and especially from bony fish. However, 3D structures are still only known for mammalian IL-1. In this review, we use a multiple sequence alignment of all published non-mammalian vertebrate IL-1beta proteins to locate the structurally important residues critical for maintaining the beta-trefoil fold and we investigate the degree to which functionally important residues involved in receptor binding are conserved across vertebrate species. We find that although there is a high level of variability of positions involved in receptor binding, the mode of binding and overall shape of the ligand-receptor complex is probably maintained. This implies that each species has evolved its own unique interleukin-1 signalling system through ligand-receptor co-evolution. Nonetheless, the IL-1beta processing mechanism in non-mammalian vertebrates remains unclear because, with the exception of three bony fish, all non-mammalian IL-1beta sequences discovered so far lack an ICE (Interleukin Converting Enzyme) cut site. The IL-1 system has become an important drug target because of its significance in inflammatory diseases. Research on peptides derived from IL-1beta has identified peptides that possess agonist activity in humans and in trout, and peptides with antagonist activity. The agonist peptides map to two distinct loop regions of IL-1beta that are known to interact with the flexible domain III of the corresponding receptor. Further analysis of the IL-1 system may prove useful in engineering IL-1 with improved features and in suggesting new avenues for therapeutic intervention.

Integrating fragment assembly and biophysical methods in the chemical advancement of small-molecule antagonists of IL-2: an approach for inhibiting protein-protein interactions.

Fragment assembly has shown promise for discovering small-molecule antagonists for difficult targets, including protein-protein interactions. Here, we describe a process for identifying a 60 nM inhibitor of the interleukin-2 (IL-2)/IL-2 receptor (IL-2Ralpha) interaction. By use of fragment-based approaches, a compound with millimolar affinity was evolved to a hit series with low micromolar activity, and these compounds were optimized into a lead series with nanomolar affinity. Fragment assembly was useful not only for hit identification, but also for lead optimization. Throughout the discovery process, biophysical methods and structural biology demonstrated that compounds bound reversibly to IL-2 at the IL-2 receptor binding site.

The effects of endogenous interleukin-1 bioactivity on locus coeruleus neurons in response to bacterial and viral substances

In a previous study, we found that microinjection of the cytokine interleukin-1 (IL-1) into the locus coeruleus (LC) increased the electrophysiological activity of LC neurons. To determine if endogenous IL-1 similarly affects the LC, brain IL-1 was induced with lipopolysaccharide (LPS), a substance derived from Gram-negative bacteria. LPS microinjected directly into the LC increased the activity of LC neurons in anesthetized rats, and this effect was blocked by microinfusion of the IL-1 receptor antagonist (IL-1RA) protein into the LC indicating the involvement of IL-1 receptors. Similarly, intraperitoneal (i.p.) LPS injection increased the activity of LC neurons in a dose- and time-related manner that was sensitive to IL-1RA. The change in the activity of LC neurons caused by a single i.p. injection of LPS was surprisingly long-lasting, and evolved over a period of at least 3 weeks. Other microbial substances—namely, peptidoglycan from Gram-positive bacteria and poly-inosine/poly-cytosine (poly(I)/(C)), which resembles RNA viruses—were used to determine the generality of the findings with LPS. Both i.p. peptidoglycan and poly(I)/(C) increased LC activity but with lesser efficacy than LPS. IL-1RA reversed the increase in the activity of LC neurons caused by i.p. peptidoglycan treatment; however, that caused by i.p. Poly(I)/(C) was not diminished by IL-1RA. Thus, the increased activity of LC neurons caused by LPS and peptidoglycan requires IL-1 receptor binding, suggesting the involvement of endogenously-produced IL-1. In contrast, poly(I)/(C) increased the activity of LC neurons but this did not critically involve IL-1 receptors in the LC.

A New IL‐1 Receptor Inhibitor 139A: Fermentation, Isolation, Physico‐Chemical Properties and Structure.

AbstractFor Abstract see ChemInform Abstract in Full Text.

A new IL-1 receptor inhibitor 139A: fermentation, isolation, physico-chemical properties and structure.

Interleukin (IL)-1 is known to be a cytokine which plays a major role in pathological conditions like septic shock, inflammation and auto-immune disease, hence, methods that reduce the activity of IL-1 have an impact on clinical medicine. Inhibiting the binding of IL-1 to IL-1 receptors is one of the methods. A new inhibitor of IL-1 receptor, 139A, was isolated from the fermentation broth of Streptomyces sp.139. It was extracted from the broth filtrate, purified by Diaion HP-20, cation exchange resin and DEAE Sephadex A-25. 139A was identified as polysaccharide, its structure was elucidated on the basis of spectral analysis, the immobilized ligand IL-1 receptor binding assay (IL-ILRBA) proved 139A can competitively inhibits the binding of IL-1 to IL-1 receptors.

Buffalo (Bubalus bubalis) interleukin-2: sequence analysis reveals high nucleotide and amino acid identity with interleukin-2 of cattle and other ruminants.

A 4400-bp genomic sequence and a 332-bp truncated cDNA sequence of the interleukin-2 (IL-2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL-2 gene was assembled from the 5' end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL-2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL-2 with that of human and cattle IL-2 did not reveal significant differences. The putative amino acids responsible for IL-2 receptor binding were conserved in buffalo, cattle and human IL-2. The amino acid sequence of buffalo IL-2 also showed very high identity with that of other ruminants, indicating functional cross-reactivity.

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1β (IL-1β). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1α and IL-1β) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [125I] IL-1β to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1α (Ki of 341.1 pM) and IL-1β (Ki 59.9 pM), as well as the antagonist IL-1ra (Ki 257.6 pM), displaced specific [125I] IL-1β binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [125I] IL-1β binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1α and IL-1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1β (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1α by itself had no stimulatory action on NGF release. The simultaneous application of IL-1α and IL-1β elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1β. We concluded that IL-1β-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1α and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.

T-cell interleukin-6 receptor binding in interferon-beta-1b-treated multiple sclerosis patients.

Multiple sclerosis (MS) is a T-cell-mediated autoimmune demyelinating disease of the central nervous system, associated with an altered cytokine network. We previously assayed peripheral blood T-lymphocyte binding for two prototypic cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and found that T cells from MS patients had significantly more TNF-alpha and IL-6 receptors than those from healthy controls. In the present work, paralleling a previous one on T-cell TNF-alpha binding, we studied the effect of interferon (IFN)-beta-1b treatment on T-lymphocyte IL-6 binding in patients with relapsing-remitting MS. T cells from MS patients had significantly (P < 0.001) higher amounts of IL-6 receptors than those from controls [292 +/- 6 vs. 228 +/- 8 (mean +/- SEM) receptors per cell, respectively], whereas the ligand-receptor affinity values were similar in the two groups [26.2 +/- 0.7 and 25.7 +/- 0.4 (mean +/- SEM) pmoles/l, respectively]. After a 3-month IFN-beta-1b treatment, they showed a significant decrease in IL-6 binding [266 +/- 7 (mean +/- SEM) receptors per cell]. After 6 and 9 months, T-cell IL-6 B(max) values were even lower [258 +/- 8 and 251 +/- 8 (mean +/- SEM) receptors per cell]. Since an increased IL-6 binding might be linked to a lymphocyte activation, our data give further support for an enhanced immune response in patients with MS. Our data seem to demonstrate that the major effects of IFN-beta-1b treatment result in a decrease of T-cell activation.

Tollip Blooms in IL-1 Receptor Pathway

The interleukin 1 (IL-1) receptor (IL-1R) activates a proinflammatory response through the activation of several signaling proteins and the transcription factors AP-1 and NF-κB. Burns et al . have molecularly cloned Tollip, an adapter protein involved in IL-1R signaling. In transfected cells, Tollip was shown to exist in a complex with the IL-1R-associated serine/threonine kinase (IRAK) before activation by IL-1. Upon stimulation with IL-1, the Tollip-IRAK complex associated with the IL-1R accessory protein (IL-1RAcP). However, activation of IRAK by another IL-1R-associated protein, MyD88, led to the dissociation of IRAK from Tollip, suggesting that IRAK dissociation is phosphorylation-dependent. Overexpression of Tollip led to decreased activation of NF-κB and JNK1. It is uncertain whether this represents a physiological effect of Tollip or reflects occlusion of limiting IL-1R binding sites for Tollip-IRAK complexes. Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray, K., Tschopp, J., and Volpe, F. (2000) Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nature Cell Biol . 2 : 346-351. [Online Journal]

Recruitment of a Heparan Sulfate Subunit to the Interleukin-1 Receptor Complex: REGULATION BY FIBRONECTIN ATTACHMENT

In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.

T-cell interleukin-6 receptor binding in patients with myasthenia gravis.

Myasthenia gravis (MG) is a T-cell-dependent and antibody-mediated autoimmune disease of the neuromuscular junction, in which the cytokine network may be deranged. Specific receptors for interleukin (IL)-6, a cytokine with several effects on the neuroimmune system, have been found on human lymphocytes. The aim of the present study has been to assay IL-6 binding on peripheral blood T cells from MG patients. We found that T cells from MG patients have significantly more IL-6 receptors than those from controls (Bmax: 334 +/- 6 vs 251 +/- 4 (mean +/- SEM) receptors/cell). Such IL-6 binding sites are of the same type in patients and healthy subjects (Kd: 26.5 +/- 0.7 vs 25.7 +/- 0.9 (mean +/- SEM) pM). The enhanced T-cell interleukin-6 binding is due to an increased number of interleukin-6 receptors on T-helper lymphocytes. These results are discussed in terms of MG immunopathogenesis, since it has been reported that activated T cells have increased amounts of IL-6 receptors.