Publications
2023
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(2023) Journal of Interferon and Cytokine Research. 43, 12, p. 539-543 Abstract
Journal of Interferon & Cytokine Research: Thank you for joining us today for this interview and congratulations on receiving the 2023 ICIS Honorary Lifetime Membership Award
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(2023) fronteirs in immnulogy. 14, 1151620. Abstract
Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the “triumph”. My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNβ proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn’s disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.
2022
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Nine receptors and binding proteins, four drugs, and one woman: Historical and personal perspectives(2022) Frontiers in Drug Discovery. 2, 1001487. Abstract
In the era of bioinformatics and high-throughput techniques, it is tempting to forget the advantage of an old yet efficient and straightforward technique, ligand affinity chromatography, in the search for unknown proteins. This type of separation is based on an interaction between the target analyte potentially present in a crude mixture of proteins and a ligand coupled covalently to a resin. This process allows thousands-fold purification in a single step, which is crucial when using an extremely rich source of naturally occurring proteins such as human urine or plasma. Before the completion of The Genome Project, this method facilitated the rapid and reliable cloning of the corresponding gene based on the partial amino acid sequence of the isolated protein. Upon completion of this project, a partial protein sequence was enough to retrieve its complete mRNA and, hence, its complete protein sequence. Ligand affinity chromatography is indispensable for the isolation of both expected and unexpected binding proteins found by serendipity. My approach of combining a rich source of human proteins (1,000-fold concentrated human urine) together with this highly specific isolation method yielded proteins from both groups. The expected proteins included the two receptors for TNF (TBPI and TBPII), type I and type II interferon receptors (IFNα/βR, IFN-γR), and IL-6 and LDL receptors. The unexpected group of proteins included IL-18 binding protein (IL-18BP), IL-32 binding protein (Proteinase 3), and heparanase binding protein, the resistin. The discovery of the type I IFN receptor was a “eureka” moment in my life since it put an end to a 35-year worldwide search for this receptor. Using chemical purification methods, the TBPII might have never been discovered. Years later, TBPII was translated into the blockbuster drug Enbrel® to treat mainly rheumatoid arthritis. IFN-beta was translated into the blockbuster drug Rebif® to treat the autoimmune disease multiple sclerosis. IL-18BP translated into the drug Tadekinig alfa™ and is in a phase III clinical study for inflammatory and autoimmune pathologies. It has saved the lives of children born with mutations (NLRC4, XIAP) and is an example of personalized medicine. COVID-19 and CAR-T cytokine storms are the recent targets of IL-18BP.
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(2022) Immunologic Research. Abstract[All authors]
Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation, severe respiratory failure, and multiple organ dysfunction caused by a cytokine storm involving high blood levels of ferritin and IL-18. Furthermore, there is a resemblance between COVID-19 and macrophage activation syndrome (MAS) characterized by high concentrations of soluble CD163 (sCD163) receptor and IL-18. High levels of ferritin, IL-18, and sCD163 receptor are associated with "hyperferritinemic syndrome", a family of diseases that appears to include COVID-19. In this retrospective cohort study, we tested the association and intercorrelations in the serum levels of ferritin, sCD163, and IL-18 and their impact on the prognosis of COVID-19. We analyzed data of 70 hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The levels of sCD163, ferritin, and IL-18 were measured and the correlation of these parameters with the respiratory deterioration and overall 30-day survival was assessed. Among the 70 patients, 60 survived 30 days from hospitalization. There were substantial differences between the subjects who were alive following 30 days compared to those who expired. The differences were referring to lymphocyte and leukocyte count, CRP, D-dimer, ferritin, sCD163, and IL-18. Results showed high levels of IL-18 (median, 444 pg/mL in the survival group compared with 916 pg/mL in the mortality group, p-value 8.54 x 10-2), a statistically significant rise in levels of ferritin (median, 484 ng/mL in the survival group compared with 1004 ng/mL in the mortality group p-value, 7.94 x 10-3), and an elevated value of in sCD163 (mean, 559 ng/mL in the survival group compared with 840 ng/mL in the mortality group, p-value 1.68 x 10-2). There was no significant correlation between the rise of ferritin and the levels sCD163 or IL-18. Taken together, sCD163, ferritin, and IL-18 were found to correlate with the severity of COVID-19 infection. Although these markers are associated with COVID-19 and might contribute to the cytokine storm, no intercorrelation was found among them. It cannot be excluded though that the results depend on the timing of sampling, assuming that they play distinct roles in different stages of the disease course. The data represented herein may provide clinical benefit in improving our understanding of the pathological course of the disease. Furthermore, measuring these biomarkers during the disease progression may help target them at the right time and refine the decision-making regarding the requirement for hospitalization.
2021
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(2021) New Innovations in Chemistry and Biochemistry, v2. p. 59-63 Abstract
My approach of combining a rich source of human proteins (500-fold concentrated human urine) with a highly specific isolation method, the ligand-affinity-chromatography, enabled rapid and efficient isolation of not only soluble receptors, but also independent binding-proteins and associated enzymes. Using this approach, the following soluble form of several receptors as well as binding proteins were isolated: IL-6R, TNFRI, TNFRII, Type I Interferon receptor (IFN-/R), Type II Interferon receptor (IFN-R), IL-18 Binding Protein (IL-18BP) and IL-32 Binding Protein. These findings enabled to coin the concept that soluble receptors and binding proteins are normal constituents of body fluids in healthy individuals and their levels in pathological situations are modulated. Moreover, the identification of soluble receptors led to the cloning of their long-sought cell surface ligand-binding counterparts. A number of the soluble proteins translated into drugs.
2017
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(2017) International Journal of Chromatography and Separation Techniques. 10, IJCST-110. Abstract
In the era of bioinformatics, big data and high throughput techniques it is quite tempting to forget the advantage of the good old techniques such as affinity chromatography, in the search of yet unknown protein molecules. Not so long ago this very efficient and straightforward technique yielded in quite a short time numerous bioactive molecules thus opening new fields of basic research. And I specifically refer to ligand affinity chromatography. This type of separation is based on unique interaction between the target analyte and a ligand coupled covalently to a resin [1,2]. It is a simple, rapid, selective, and efficient purification procedure of proteins. Tens of thousands fold purification is achieved in one step, which is crucial when an extremely rich sources of naturally occurring proteins such as human urine or plasma or spinal fluid are used [3]. [First paragraph]
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2015
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(2015) Encyclopedia of Inflammatory Diseases. Parnham M.(eds.). p. 1-7 Abstract
IL-18BP is a unique binding protein that deviates from the canonical definition of soluble receptors (Novick and Rubinstein 2007). It functions mainly as an IL-18 decoy protein and modulates the biological activity of this cytokine (Novick et al. 1999). The salient property of IL-18BP in immune responses is in downregulating Th1 activities by binding to mature IL-18 and thus reducing the induction of interferon-γ (IFN-γ) (Nakanishi et al. 2001). Since IL-18 also affects Th2 responses, its antagonist, the IL-18BP, is naturally involved in these reactions too (Nakanishi et al. 2001). Encoded by a separate gene, IL-18BP deviates from the classical definition of soluble receptors, namely, it does not correspond to the extracellular ligand-binding domain of the IL-18 receptor but rather belongs to a separate family of secreted proteins. IL-18BP is exceptionally high-affinity (400 pM) (Kim...
2014
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(2014) PLoS ONE. 9, 1, e85944. Abstract
In an attempt to isolate a heparanase receptor, postulated to mediate non-enzymatic functions of the heparanase protein, we utilized human urine collected from healthy volunteers. Affinity chromatography of this rich protein source on immobilized heparanase revealed resistin as a heparanase binding protein. Co-immunoprecipitation and ELISA further confirmed the interaction between heparanase and resistin. Importantly, we found that heparanase potentiates the bioactivity of resistin in its standard bioassay in which monocytic human leukemia cell line, THP1, differentiates into adherent macrophage-like foam cells. It is thus conceivable that this newly identified complex of heparanase and resistin exerts a stimulatory effect also in various inflammatory conditions known to be affected by the two proteins.
2013
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(2013) Seminars in Immunology. 25, 6, p. 439-448 Abstract
Together with IL-12 or IL-15, interleukin-18 (IL-18) plays a major role in the production of interferon-γ from T-cells and natural killer cells; thus, IL-18 is considered to have a major role in the Th1 response. However, without IL-12, IL-18 is proinflammatory in an IFNγ independent manner. IL-18 is a member of the IL-1 family of cytokines and similar to IL-1β, the cytokine is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. IL-18 is also present as an integral membrane protein but requires caspase-1 for full activity in order to induce IFNγ. Uniquely, unlike IL-1β, the IL-18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL-18 is balanced by the presence of a high-affinity, naturally occurring IL-18 binding protein (IL-18BP). In humans, increased disease severity can be associated with an imbalance of IL-18 to IL-18BP such that the levels of free IL-18 are elevated in the circulation. Increasing number of studies have expanded the role of IL-18 in mediating inflammation in animal models of disease using the IL-18BP, IL-18 deficient mice, neutralization of IL-18 or deficiency in the IL-18 receptor alpha chain. A role for IL-18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, macrophage activation syndrome, sepsis and acute kidney injury, although paradoxically, in some models of disease, IL-18 is protective. The IL-18BP has been used safely in humans and clinical trials of IL-18BP as well as neutralizing anti-IL-18 antibodies are being tested in various diseases.
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(2013) Proceedings of the National Academy of Sciences of the United States of America. 110, 18, p. 7306-7311 Abstract
Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.
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(2013) Frontiers in Immunology. 4, OCT, Article 28. Abstract
Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines. Similar to IL-1ß, IL-18 is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine but unlike IL-1ß, the IL-18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL-18 is balanced by the presence of a high affinity, naturally occurring IL-18 binding protein (IL-18BP). In humans, increased disease severity can be associated with an imbalance of IL-18 to IL-18BP such that the levels of free IL-18 are elevated in the circulation. Increasing number of studies have expanded the role of IL-18 in mediating inflammation in animal models of disease using the IL-18BP, IL-18-deficient mice, neutralization of IL-18, or deficiency in the IL-18 receptor alpha chain. A role for IL-18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis, and acute kidney injury, although in some models of disease, IL-18 is protective. IL-18 plays a major role in the production of interferon-γ from T-cells and natural killer cells. The IL-18BP has been used safely in humans and clinical trials of IL-18BP as well as neutralizing anti-IL-18 antibodies are in clinical trials. This review updates the biology of IL-18 as well as its role in human disease.
2011
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(2011) Cytokine Protocols. Ley M.(eds.). p. 195-214 Abstract
Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity.
2010
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(2010) Nature Immunology. 11, 11, p. 973 Abstract[All authors]
Newly cloned interleukin 1 (IL-1) family members1–3 were originally given an IL-1 family (IL-1F) designation4, but as functions have now been elucidated for several of these5,6, we propose that each now be assigned an individual interleukin designation. IL-1F6, IL-1F8 and IL-1F9 are encoded by distinct genes but use the same receptor complex (IL-1Rrp2 and AcP), are proinflammatory and deliver nearly identical signals7–12. We propose these be designated IL-36α, IL-36β and IL-36γ, respectively. IL-1F5 also binds to IL-1Rrp2 but antagonizes those cytokines in a manner analogous to that used by IL-1Ra to antagonize IL-1α and IL-1β7–9. We propose that IL-1F5 be renamed IL-36Ra (for ‘receptor antagonist’). In the IL-1 nomenclature, IL-1Ra is used for the natural product, whereas IL-1ra is used for the recombinant product; therefore, IL-36Ra is appropriate for natural IL-1F5. [first paragraph]
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(2010) Journal of Autoimmunity. 34, 2, p. 121-126 Abstract
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies particularly to nuclear antigens and by an abnormal production of proinflammatory cytokines. In the present study, we measured the levels of the proinflammatory cytokine IL-18 and its natural inhibitor, the IL-18 binding protein (IL-18BP), in sera of SLE patients at various stages of the disease. This is the first study to present IL-18BP levels in sera of SLE patients as well as the calculated, biologically active, free IL-18 concentrations that are most probably more relevant to the pathology of SLE. Sera from 48 unselective SLE patients (total of 195 samples) were obtained longitudinally with a mean follow-up period of 11.1 ± 8.9 years and were compared to sera from 100 healthy volunteers. Circulating levels of IL-18, IL-18BP and free IL-18 in the SLE patients were significantly higher than the levels of healthy controls (5 fold, 6 fold and 3 fold for IL-18, IL-18BP and free IL-18, respectively) and correlated with disease activity as scored by SLEDAI-2K. Furthermore, these levels during active disease (SLEDAI-2K ≥ 6) were higher compared to the levels measured in the sera of the same patients during remission or during mild disease (SLEDAI-2K 0-5). The high levels of IL-18 and IL-18BP in sera of active SLE patients suggest their possible role in the pathogenesis and course of the disease. However, despite the elevated levels of IL-18BP during active disease, free IL-18 remained more than 2 fold higher than the levels in healthy controls suggesting a potential benefit of administration of exogenous IL-18BP as a novel therapeutic approach for active SLE.
2009
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(2009) Contemporary Challenges In Autoimmunity. 1173, p. 301-309 Abstract
Interleukin-18 (IL-18) is an inflammation-related cytokine that plays a central role both in innate defense reactions and in Th1 activation and specific immune responses. Increased levels of IL-18 can be detected in biological fluids and organs of individuals affected by several autoimmune pathologies, as well as in autoimmune animal models. In this review, the role of IL-18 in systemic lupus erythematosus (SLE) is critically examined, including its possible role in the pathogenesis of disease. In SLE, increased levels of IL-18 have been found in serum/plasma of affected persons, which positively correlated with disease severity. The possibility that circulating IL-18 levels are predictive of renal damage has been proposed, suggesting that IL-18 may be a prognostic marker of renal involvement useful to identify patients at risk of renal failure. The evaluation of urinary levels of free active IL-18 indeed suggests a correlation with the degree of renal involvement. The possible pathogenic role of IL-18 in lupus has been studied in a mouse model of progressive disease, which makes possible the identification, at the level of the different affected organs, of IL-18 changes preceding disease development and those appearing after disease onset. It can be concluded that IL-18 has a multifaceted role in autoimmune lupus, being apparently involved both in the effector phases of the late organ damage and, in some organs, in the initial pathogenic events. Therapeutic strategies targeting IL-18 in autoimmunity are under development.
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(2009) Journal of Clinical Immunology. 29, 1, p. 38-45 Abstract
Introduction: In the present study, we examined the levels of the pro-inflammatory cytokine IL-18 and its natural inhibitor, the IL-18 binding protein (IL-18BP), in sera of Wegener's granulomatosis (WG) patients at various stages of the disease. Patients and Methods: Sera from eight consecutive biopsy-proven systemic WG patients (four men and four women; age at diagnosis 58.4∈±∈13.8 years) were obtained longitudinally with a follow-up period of 55.2∈±∈30 months. Sera obtained from 50 healthy subjects were used as controls. Results and Discussion: Serum levels of IL-18, IL-18BP, and free IL-18 obtained during an active phase of the disease (Birmingham Vasculitis Activity Score, BVAS∈>∈10) were more than twofold higher than levels in the same patients during inactive disease stages (BVAS∈
2008
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(2008) Cytokine. 43, 3, p. 249-249 Abstract
Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
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(2008) Inflammatory Bowel Diseases. 14, 1, p. 68-74 Abstract
Background: Interleukin-18 (IL-18) is increased in the inflamed mucosa of patients with Crohn's disease (CD). The balance between this pleiotropic proinflammatory cytokine and its natural inhibitor, IL-18-binding protein (IL-18BP), may contribute to the pathogenesis of inflammatory bowel disease (IBD). Methods: Serum and mucosal biopsies were collected from children with IBD, from children with celiac disease, and from controls. Biopsies were maintained in culture for 24 hours, and supernatant was collected. Serum and supernatant IL-18 and IL-18BPa concentrations were measured by immunoassay. Disease activity score (PCDAI) and standard serum inflammatory markers (albumin, platelets, ESR, and CRP) were recorded. Results: Serum IL-18 was greater in children with CD (537 pg/mL) than in controls (335 pg/mL; P
2007
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(2007) ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY. 27, 12, p. 2743-2749 Abstract[All authors]
OBJECTIVE - To investigate free interleukin-18 (fIL-18) levels, and variation within the IL-18 system genes, in heart surgery patients, and healthy men. METHODS AND RESULTS - fIL-18 was calculated from IL-18 and IL-18 binding protein (BP) levels, in 421 healthy men and 196 post-coronary artery bypass graft (CABG) patients. After surgery, fIL-18 peaked at 6 hours (from 117 to 331 pg/mL) but fell to below presurgery levels at 24 hours (99 pg/mL), because of changes in total IL-18 and IL-18BP. fIL-18 24 hours postsurgery was significantly higher in those who suffered a major complication after surgery (125 versus 80 pg/mL, P
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(2007) Israel Medical Association Journal. 9, 7, p. 504-508 Abstract
Background: Crohn's disease and ulcerative colitis are inflammatory bowel diseases with an unknown etiology. Interleukin-18 is a pro-inflammatory cytokine that is up-regulated in Crohn's disease. IL-18 binding protein neutralizes IL-18. The relationship of IL-18 and IL-18BP and disease activity in these diseases is not fully understood. Objectives: To investigate the correlation of IL-18 and IL-18BP with disease activity and other disease parameters in inflammatory bowel disease. Methods: IL-18 and IL-18BP isoform α were measured in 129 patients and 10 healthy individuals. Patients' mean age was 40.5 (range 15-70 years) and 43 were women; 58 Crohn's and 28 colitis patients were in remission and 52 and 14, respectively, were in exacerbation. Twenty-three (19 and 4 respectively) were studied in both remission and exacerbation. Results: The mean level of free IL-18 was significantly different between healthy individuals and Crohn patients, and between Crohn patients during exacerbation and remission (167 ± 32 vs. 471 ± 88 and 325 ± 24 pg/ml, respectively, P
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Antiviral and immunoregulatory activities of IFN-gamma depend on constitutively expressed IL-1 alpha(2007) Cytokine. 39, 1, p. 17-17 Abstract
Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
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(2007) Proceedings of the National Academy of Sciences of the United States of America. 104, 12, p. 5044-5049 Abstract
IFN-γ induces its immunoregulatory activities by activating genes mainly through the Jak-STAT signaling pathway. Here we show that what was considered to be intrinsic IFN-γ activities depend largely on the basal level of NF-κB, which is maintained by constitutively expressed IL-1α. The IL-1 receptor antagonist and antibodies to IL-1α, but not to IL-1β, inhibited the antiviral activity of IFN-γ by 90%, whereas no inhibition of type I IFN activity was observed. Similarly, the induction of many genes by IFN-γ, including HLA-DR, ICAM-1, IL-18BP, and genes mediating its antiviral activity, greatly depended on basal IL-1α. Furthermore, IFN-γ induced serum IL-18 binding protein in wild-type mice but not in IL-1α/β double-deficient mice. Thus, constitutively expressed IL-1α is critical for numerous IFN-γ activities.
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(2007) Cytokine & Growth Factor Reviews. 18, 5-6, p. 525-533 Abstract
Our approach of isolating proteins from a rich source of human proteins by ligand-affinity-chromatography enabled rapid and efficient isolation of not only soluble receptors corresponding to cell-associated receptors, but also independent binding-proteins and associated enzymes. No other approach would yield the latter. During the early 80's we prepared the tools and the infrastructure that enabled the subsequent 20 years of achievements. Thus we described eight soluble receptors (R) and binding proteins (BP) for various cytokines including the IL-6R, IFN-γR, TNFRI, TNFRII, LDLR, IFN-α/βR, IL-18BP and IL-32BP identified as Proteinase 3. The isolation of the soluble IFN-α/β receptor led to the cloning of its long sought cell surface ligand binding counterpart. We have established the concept that soluble receptors and binding proteins are normal constituents of body fluids in healthy individuals and that the levels of these biomarkers are modulated in various pathological situations. Each of these proteins contributed to basic science, one of them serves as a basis for therapy and some others are in various stages of clinical development.
2006
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(2006) Proceedings of the National Academy of Sciences of the United States of America. 103, 9, p. 3316-3321 Abstract
IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1β, TNF-α, IL-6, and chemokines. Here, we used ligand (IL-32α) affinity chromatography in an attempt to isolate an IL-32α soluble receptor or binding protein. Recombinant IL-32α was covalently immobilized on agarose, and preparations of concentrated crude human urinary proteins were applied for chromatographic separation. A specific 30-kDa protein eluted from the column during acid washing and was identified by mass spectrometry as proteinase 3 (PR3) and confirmed by N-terminal microsequencing. PR3, a neutrophil granule serine protease, exists in a soluble or membrane form and is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. The affinity of IL-32α to PR3 was determined by surface plasmon resonance. The dissociation constants were 2.65 ± 0.4 nM for urinary PR3 and 1.2 ± 0.05 nM for neutrophil-derived PR3. However, irreversible inactivation of PR3 enzymatic activity did not significantly change binding to the cytokine. Nevertheless, limited cleavage of IL-32 yielded products consistent with PR3 enzyme activity. Moreover, after limited cleavage by PR3, IL-32α was more active than intact IL-32α in inducing macrophage inflammatory protein-2 in mouse macrophages and IL-8 in human peripheral blood mononuclear cells. We suggest that PR3 is a specific IL-32α binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32α by PR3 enhances activities of the cytokine. Therefore, specific inhibition of PR3 activity to process IL-32 or neutralization of IL-32 by inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases.
2005
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(2005) International Journal of Sports Medicine. 26, 10, p. 836-840 Abstract
Interleukin 18 (IL-18) is an important pro-inflammatory cytokine in the early phase of human immune response to microbial infections. The influence of strenuous exercise on the intrinsic balance of IL-18 and its endogenous antagonist IL-18 binding protein (IL-18 BP) is unknown, but could be of major relevance for the athlete's immune function empirically and epidemiologically proven to be altered after exhaustive exertion. To study the effect of strenuous marathon cycling on the interaction of IL-18 and IL-18 BP we investigated 37 male, healthy, and well-trained amateur cyclists participating in the Ötztaler Radmarathon in Tyrol (distance: 230km; cumulative altitude difference: 5500m). IL-18 was measured by a commercially available ELISA-Kit and IL-18 BP by a novel IL-18 BP ELISA method. Free, unbound IL-18 was calculated according to a standard equation. The mean plasma level of IL-18 was 142.27 ± 21.85 pg/ml pre-race, remained nearly unchanged (124.35 ± 13.16 pg/ml; p = 1.0) immediately after competition (mean race time 9 h 38 min), but declined significantly 24 h afterward (62.92 ± 6.80 pg/ml; p = 0.002). The plasma levels of IL-18 BP increased considerably immediately after and kept on rising for the following 24 h (pre-race: 1.51 ± 0.20 ng/ml; immediately post-race: 3.84 ± 0.26 ng/ml, p
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(2005) Blood. 106, 10, p. 3483-3489 Abstract[All authors]
Hemophagocytic syndrome (HPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied 20 patients with HPS secondary to infections, autoimmune disease, lymphoma, or cancer and observed that the concentrations of serum interleukin 18 (IL-18), a strong inducer of Th-1 responses, interferon γ (IFN-γ) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HPS but not in control patients. In contrast, concentrations of its natural inhibitor, the IL-18 binding protein (IL-18BP), were only moderately elevated, resulting in a high level of biologically active free IL-18 in HPS (4.6-fold increase compared with controls; P
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Elevated systemic levels of free interleukin-18 (IL-18) in patients with Crohn's disease(2005) European Cytokine Network. 16, 1, p. 27-33 Abstract
Objectives. Interleukin 18 (IL-18) is a proinflammatory cytokine and a member of the IL-1 family. Animal models and investigations in humans point to an important role for this cytokine in inflammatory bowel diseases (IBD). IL-18 binding protein (IL-18BP) is a naturally occurring antagonist of IL-18. Methods. In this study, we measured IL-18 and IL-18BP plasma concentrations and spontaneous release in cultures of colonic explants from healthy subjects (n = 41), patients with Crohn's disease (CD, in = 135), and patients with ulcerative colitis (UC, n = 93). Results. Both CD and UC patients had higher IL-18BP plasma levels than controls. Plasma levels of free, unbound IL-18 were significantly elevated in CD patients compared to healthy controls, but not in UC patients. Colonic explant cultures from inflamed areas in IBD patients released significantly higher levels of IL-18 than non-inflamed areas and controls. IL-18BP levels from the same cultures were below the detection limit over a culture period of 24 h. Conclusions. Our results confirm the importance of IL-18 in the pathogenesis of IBD and suggest that especially in CD, IL-18BP might be produced in insufficient quantities to counteract the effects of endogenous IL-18.
2004
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(2004) American Journal of Obstetrics and Gynecology. 191, 6, p. S152-S152 Abstract
Keywords: Obstetrics & Gynecology
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(2004) Arthritis and Rheumatism. 50, 6, p. 1800-1805 Abstract
Objective. To study the regulation of interleukin-18 binding protein (IL-18BP) production by rheumatoid arthritis (RA) or control peripheral blood mononuclear cells (PBMCs) and by RA synovial tissue cells, and to compare the levels of IL-18BP messenger RNA (mRNA) expression in whole blood from RA patients and controls. Methods. Unstimulated or phytohemagglutinin and phorbol myristate acetate (PHA/PMA)-stimulated PBMCs from 10 RA patients and 12 healthy controls and unstimulated or PHA-PMA-stimulated synovial tissue cells from 8 RA patients were cultured with or without IL-12 (1 ng/ml) and IL-18 (5 ng/ml) alone or in combination. IL-18BP and interferon-γ (IFNγ) levels in supernatants were measured by enzyme-linked immunosorbent assay. Levels of IL-18BP and IFNγ mRNA expression in whole blood samples from 22 RA patients and 12 healthy controls were determined by quantitative reverse transcriptase-polymerase chain reaction. Results. IL-12 decreased the basal levels of IL-18BP production by freshly isolated RA or control PBMCs, but increased those of synovial cells or PHA/PMA-stimulated PBMCs. IL-18 alone had no direct effect on IL-18BP production by PBMCs or RA synovial cells, with or without stimulation. Unstimulated whole blood samples from RA patients showed lower levels of IL-18BP mRNA expression than those from healthy controls. Conclusion. The production of IL-18BP in response to IL-12 and IL-18 was regulated differently in blood and synovial cells. The difference appears to be related to the level of cell activation.
2003
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(2003) Journal of Immunology. 171, 12, p. 6574-6580 Abstract
Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R α-chain (IL-18Rα) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [125I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [125I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (Kd) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent Kd of 146 pM. Each domain of extracellular IL-18Rα was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-γ production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Rα prevented the formation of IL-18R ternary complex with IL-18R β-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Rα for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-γ in NK cells.
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(2003) Clinical Nephrology. 60, 5, p. 327-334 Abstract
Uremia is associated with suppressed cellular immune responses, manifested, in part, by impaired interferon-γ (IFNγ) production. We investigated the influence of kidney function on plasma levels of interleukin-18 binding protein (IL-18BP), the naturally occurring inhibitor of IL-18. Methods: Plasma levels of IL-18, IL-18BP and IFNγ were measured by specific immunoassays in patients with normal kidney function (NKF, n = 29), in patients with chronic renal insufficiency (CRI, n = 29), and in patients on hemodialysis (HD, n = 40). In addition, Staphylococcus epidermidis-induced production of IFNγ and IL-18 in whole blood cultures was determined in 12 patients on HD and compared to production in 9 controls with NKF. Results: Plasma IL-18 (mean ± SEM) in NKF was 17.9 ± 3.6 pg/ml, in CRI 42.6 ± 7.0 pg/ml (p 6 mononuclear cells (PBMC) but suppressed in HD to 27.3 ± 16 pg/106 PBMC (p
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(2003) Infection and Immunity. 71, 10, p. 5803-5813 Abstract[All authors]
Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo with Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1β, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killed S. aureus markedly increased IL-1β and IL-18 levels more than E. coli lipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at the P
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(2003) Experimental Gerontology. 38, 6, p. 669-672 Abstract[All authors]
Interleukin (IL)-18 is highly expressed in macrophages from human atherosclerotic plaques, suggesting its involvement in ischemic syndromes. We evaluated IL-18 and IL-18 binding protein (BP) in healthy centenarians, as longevity is characterized by a reduced incidence of ischemic events. For comparison, patients with chronic ischemic syndromes (CIS) were evaluated. Serum IL-18 and IL-18BP levels were measured by non-cross-reacting ELISA in 16 healthy centenarians and in two age-control populations, each of 18 healthy individuals aged 55.9 +/- 1.43 and 74.3 +/- 1.35, respectively, as well as in 23 CIS patients, and another cohort of 23 healthy subjects that were age- and sex-matched with CIS patients. Centenarians displayed significantly higher total IL-18 serum levels compared to each control group. Elevated IL-18 levels were also present in CIS patients. However, centenarians had a significant higher level of IL-18BP compared to the cohort of 23 controls (P = 0.0014), and compared to CIS patients (P = 0.043); as a result centenarians exhibited a lower level of free IL-18 than CIS patients. The present results indicate that quenching of IL-18 by IL-18BP may explain the apparent paradox of elevated serum IL-18 with no vascular signs in centenarians. (C) 2003 Elsevier Science Inc. All rights reserved.
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(2003) Clinical and Experimental Immunology. 131, 2, p. 355-363 Abstract
We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.
[All authors] -
(2003) Blood Purification. 21, 3, p. 258-270 Abstract
Although interleukin (IL)-18 is a member of the IL-1 family of ligands, IL-18 appears to have unique characteristics, particularly in the regulation of the T helper type 1 (Th1) response. Th1 responses are required for tumor surveillance, killing intracellular organisms, and to provide help for antibody production. In patients with chronic renal failure, the well-known immunosuppression contributes to a failure to respond to infectious challenges and vaccinations. The most salient biological property of IL-18, linking this cytokine to the Th1 response, is its ability to induce interferon gamma (IFN-γ). In fact, IL-18 was originally identified as an IFN-γ-inducing factor, and IFN-γ production is the hallmark of the Th1 response. Dysregulation of IFN-γ production resulting from reduced activity of IL-18 would explain one of the mechanisms of immunosuppression in patients with chronic renal failure. The activity of IL-18 can be regulated by the IL-18-binding protein (IL-18BP), a glycoprotein of 40,000 daltons, which is constitutively expressed and appears to be the natural inhibitor of IL-18 activity. Unlike soluble receptors for IL-18, IL-18BP does not have a transmembrane domain; IL-18BP is a secreted protein possessing a high-affinity binding and ability to neutralize IL-18. IL-18BP was discovered in human urine and is excreted in health following glomerular filtration. With decreasing renal function, the concentrations of IL-18BP in the circulation are elevated as compared with subjects with a normal renal function, and these elevated levels may result in a decreased IL-18 activity. Because of the importance of IL-18 and IFN-γ in the Th1 response, the biology of IL-18 and IL-18BP is reviewed here in the context of the immunosuppression of chronic renal failure.
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(2003) Immunology Of Diabetes Ii: Pathogenesis From Mouse To Man. 1005, p. 332-339 Abstract
Type 1 (insulin-dependent) diabetes, T1DM, is the result of an immune-mediated destruction of the pancreatic β cells dependent mainly on T helper cells and macrophages. Interleukin-18 (IL-18) is a proinflammatory cytokine produced mainly by macrophages. IL-18 is capable of inducing T lymphocyte synthesis of IFNγ, thereby skewing the T helper response toward a T helper type 1 (Th1) profile. IL-18 binding protein (IL18BP) neutralizes IL-18 and leads to a reduced Th1 response. Polymorphisms in IL18BP may affect the activity of IL-18 and the magnitude of the Th1 response and may play a role in the pathogenesis of T1DM. The aim of the study was therefore to identify polymorphisms in IL18BP and to test these for association with T1DM. We evaluated the human IL18BP gene on chromosome 11q13 as a candidate susceptibility gene for T1DM and scanned the entire IL18BP (promoter, exons 1-6, and 3′UTR) for polymorphisms using single-strand conformational polymorphism analysis and direct sequencing. We identified a total of 11 polymorphisms, all having allele frequencies ranging between 0.05 and 0.10. Four were in the 5′UTR: -257G→T,-78C→T,-65G→A, and -59A→G. Three were in intron 3: IVS3+140A→C, IVS4-147G→T, and IVS4-59G→T. The last four, 38*A→T, 48*T→A, 388*C→G, and 440*_441*insG, were in the 3′UTR of IL18BP. However, none of these were frequent enough to permit association studies in T1DM and we conclude that IL18BP does not contribute to the overall genetic susceptibility to type 1 diabetes.
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(2003) Cytokine. 21, 2, p. 65-73 Abstract
Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent. (C) 2003 Elsevier Science Ltd. All rights reserved.
[All authors] -
(2003) Blood Purification. 21, 3, p. 225-231 Abstract
Long-term hemodialysis (HD) induces an inflammatory response and is associated with a suppressed cellular immune response manifested, in part, by impaired interferon (IFN-γ) production. We investigated the effect of high-flux HD using the synthetic Helixone® membrane and ultrafiltered dialysate on plasma levels of inflammatory mediators and on the whole blood production of IFN-γ. Methods: Twelve ESRD patients were dialyzed under low-flux HD (polysulfone F6) and again after 6 weeks of high-flux HD (Helixone® FX100). Ultrafiltered bicarbonate dialysate without bacterial growth and no detectable endotoxin was used throughout the study. Plasma levels of urea, albumin, β2-microglobulin (β2-m), interleukin (IL)-6, C-reactive protein (CRP), IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-18-binding protein (IL-18BP) were measured. In addition, the Staphylococcus epidermidis-induced production of IFN-γ and IL-18 was assessed in whole blood cultures of HD patients as well as in 9 healthy subjects. Results: Plasma levels of urea, albumin, IL-6, IL-1Ra and CRP were not significantly different between high-flux and low-flux HD. In contrast, β2-m levels decreased significantly by 31% with high-flux Helixone® (p
2002
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(2002) Proceedings of the National Academy of Sciences of the United States of America. 99, 26, p. 16957-16962 Abstract
The IL-18 binding protein (IL-18BP) is a circulating inhibitor of the proinflammatory cytokine IL-18. It is constitutively expressed in mononuclear cells, and elevated expression is induced by IFN-γ. In this study, we characterized the IL-18BP promoter. We first showed that induction is at the transcriptional level and requires de novo protein synthesis. The IL-18BP promoter resides within 1.6 kb DNA upstream of the first exon and includes at least six regulatory elements. We identified in the basal promoter a gamma-activated sequence (GAS) proximal to the transcription start site (base 1), followed by an IFN regulatory factor 1 response element (IRF-E) and two CCAAT/enhancer binding protein β (C/EBPβ) sites, all of which are essential for basal promoter activity. Furthermore, GAS and IRF-E were essential for IFN-γ-induced transcription. Indeed, sera of IRF-1-deficient mice lacked basal and IFN-γ-induced IL-18BP. We found that after induction of IRF-1 by IFN-γ, it formed a complex with C/EBPβ, which bound to the IRF-E and GAS-containing proximal DNA. In contrast, the IFN-γ-induced signal transducer and activator of transcription 1 dimer did not associate with this GAS. In addition, we identified a silencer element and a distal enhancer at bases - 1081 to -1272, which was also physically associated with IRF-1. The IRF-1-C/EBPβ complex described here probably plays a fundamental role in regulating additional IFN-γ-responsive genes.
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(2002) Journal of Clinical Immunology. 22, 6, p. 331-337 Abstract
Interleukin 18 (IL-18) is a recently described proinflammatory cytokine. In mouse models it has been shown to play a key role in the development of liver injury. IL-18 binding protein (IL-18BP) is a naturally occurring antagonist of IL-18. In this study we investigated whether IL-18/IL-18BP levels are altered in patients with chronic liver disease (CLD). We measured IL-18 and IL-18BP plasma levels in 153 patients with CLD and 41 healthy controls by a specific ELISA. Plasma levels of IL-18 were significantly higher in CLD patients than in healthy controls. Cirrhotics had higher levels than noncirrhotics. IL-18 levels increased with disease progression. IL-18BP plasma levels paralled the increase of IL-18 with disease progression, except in stage Child C cirrhosis. IL-18 and IL-18BP levels were elevated independent of the etiology of CLD. IL-18 and IL-18BP correlated with laboratory parameters of inflammation and liver injury. Plasma levels of IL-18 and its antagonist, IL-18BP, are elevated in CLD and correlate with severity of disease. IL-18BP may not be sufficient to counteract the overwhelming proinflammatory response in end stage liver disease.
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IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components(2002) Journal of Leukocyte Biology. 72, 1, p. 192-198 Abstract
Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, β 1 integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-γ (INF-γ) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-γ production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites.
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(2002) Journal of Immunology. 168, 7, p. 3608-3616 Abstract
The pathogenesis of Crohn's disease (CD) remains under intense investigation. Increasing evidence suggests a role for mature IL-18 in the induction of proinflammatory cytokines and Th1 polarization in CD lesions. The aim of this study was to investigate the contribution of the IL-18-neutralizing (a and c) and non-neutralizing (b and d) isoforms of IL-18-binding protein (IL- 18BP) during active CD. Intestinal endothelial cells and macrophages were the major source of IL-18BP within the submucosa, and this IL-18BP production was also found to be relevant to other types of endothelial cells (HUVEC) and macrophages (peripheral monocytes). IL-18BP messenger transcript and protein were significantly increased in surgically resected specimens from active CD compared with control patients, correlating with an up-regulation of IL-18. Analysis of the expression of the four IL-18BP isoforms as well as being free or bound to IL-18 was reported and revealed that unbound IL-18BP isoforms a and c and inactive isoform d were present in specimens from active CD and control patients while isoform b was not detected. IL-18/IL-18BP complex was also detected. Interestingly, although most was complexed, free mature IL-18 could still be detected in active CD specimens even in the presence of the IL-18BP isoform a/c. These results demonstrate that the appropriate neutralizing isoforms are present in the intestinal tissue of patients with active CD and highlights the complexity of IL-18/IL-18BP biology.
[All authors] -
(2002) Journal of Biological Chemistry. 277, 13, p. 10998-11003 Abstract
Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds IL-18 and neutralizes its biological activities. Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor a chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor α chain does not share significant homology to IL-18BP. In the present study, Glu-42 was mutated to Lys and Lys-89 to Glu; Glu-42 and Lys-89 were also deleted separately. The deletion mutants (E42X and K89X) were devoid of biological activity, and the K89E mutant lost 95% of its activity. In contrast, compared with wild-type (WT) IL-18, the E42K mutant exhibited a 2-fold increase in biological activity and required a 4-fold greater concentration of IL-18BP for neutralization. The binding of WT IL-18 and its various mutants to human natural killer cells was evaluated by competition assays. The mutant E42K was more effective than WT IL-18 in inhibiting the binding of 125I-IL-18 to natural killer cells, whereas the three inactive mutants E42X, K89E, and K89X were unable to compete with 125I-IL-18 for binding. Similarly, WT IL-18 and the E42K mutant induced degradation of Iκ-Bα, whereas the three biologically inactive mutants did not induce degradation. The present study reveals that Glu-42 and Lys-89 are critical amino acid residues for the integrity of IL-18 structure and are important for binding to cell surface receptors, for signal transduction, and for neutralization by IL-18BP.
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(2002) Journal of Cerebral Blood Flow and Metabolism. 22, 8, p. 971-978 Abstract[All authors]
Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL- 18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP-treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.
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(2002) Clinical and Experimental Immunology. 129, 2, p. 332-338 Abstract
Interleukin-18 (IL-18), derived from macrophages and Kupffer cells, is the central pro-inflammatory cytokine leading to experimental liver failure. IL-18 binding protein (IL-18BP) is a circulating protein that binds IL-18 and neutralizes its activity. Since IL-18 production is increased in chronic HCV infection, we asked whether IFN-α might act on the IL-18/IL-18BP system in HCV patients. IL-18BP, total and free IL-18 plasma levels were determined in 13 HCV patients receiving 1 × 107 IU IFN-α subcutaneously daily for 28 days. The in vitro effects of IFN-α on macrophage IL-18BP and IL-18 were studied by enzyme-linked immunosorbent assays and Northern analysis. IFN-α administration increased IL-18BP plasma levels 3.24 fold 24 h after institution of therapy, resulting in a 67.4% reduction of free IL-18. Total IL-18 levels decreased from day +24 on. In vitro, IFN-α diminished IL-18 release from macrophages of healthy volunteers and chronic HCV patients. On top of its inhibitory effects on IL-1 and TNF-α release, IFN-α also exerts its anti-inflammatory action in vivo by induction of IL-18BP. These anti-inflammatory properties might account - together with its antiviral action - for its clinical efficacy in chronic hepatitis C.
[All authors]
2001
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(2001) Infection and Immunity. 69, 8, p. 5025-5030 Abstract
The roles of endogenous cytokines induced by either intact staphylococcal microorganisms or staphylococcal exotoxins were examined using human whole-blood cultures. To accomplish this, interleukin-18 binding protein (IL-1813P) and tumor necrosis factor binding protein (TNFbp) were used to neutralize IL-18 and TNF, respectively, whereas an anti-IL-12 monoclonal antibody was used to neutralize IL-12 and the IL-1 receptor antagonist (IL-1Ra) was used to block IL-1 receptors. Heat-killed Staphylococcus epidermidis and Staphylococcus aureus, as well as the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcus enterotoxin B (SEB) induced gamma interferon (IFN-gamma) production. Staphylococcus spp.-induced production of IFN-gamma required the presence of endogenous IL-18, IL-12, and TNF. In contrast, TSST-1-induced IFN-gamma was not significantly reduced in the presence of IL-1813P, anti-IL-12 antibodies, IL-1Ra, or anti-TNFbp. SEB-induced IFN-gamma was significantly inhibited only by anti-IL-12 antibodies, indicating that endogenous IL-18, IL-1, and TNF are not required for SEB-induced IFN-gamma. In conclusion, the mechanisms of IFN-gamma stimulation by intact staphylococcal microorganisms and by exotoxins differ, and this is likely due to the different receptors which are triggered on the cell membranes. In contrast to its role in the interactions between staphylococci and host cells, IL-18 does not appear to play a major role in superantigen-induced IFN-gamma.
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(2001) Cytokine. 14, 6, p. 334-342 Abstract[All authors]
IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 by forming a 1:1 high affinity (Kd=400 pM) complex, exhibiting a very low dissociation rate. We have developed a sandwich ELISA for IL-18BPa and determined its limit of detection (62 pg/ml). Interference by IL-18 and related cytokines, as well as cross reactivity with other IL-18BP isoforms (b, c, and d) were determined. Using this ELISA, we measured serum IL-18BPa in large cohorts of healthy individuals and in septic patients. Serum IL-18BPa in healthy individuals was 2.15 ± 0.15 ng/ml (range 0.5-7 ng/ml). In sepsis, the level rose to 21.9 ± 1.44 ng/ml (range 4-132 ng/ml). Total IL-18 was measured in the same sera by an electrochemiluminescence assay and free IL-18 was calculated based on the mass action law. Total IL-18 was low in healthy individuals (64 ± 17 pg/ml) and most of it (∼85%) was in its free form. Total IL-18 and IL-18BPa were both elevated in sepsis patients upon admission (1.5 ± 0.4 ng/ml and 28.6 ± 4.5 ng/ml, respectively). At these levels, most of the IL-18 is bound to IL-18BPa, however the remaining free IL-18 is still higher than in healthy individuals. We conclude that IL-18BPa considerably inhibits circulating IL-18 in sepsis. Yet, exogenous administration of IL-18BPa may further reduce circulating IL-18 activity.
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(2001) Proceedings of the National Academy of Sciences of the United States of America. 98, 6, p. 3304-3309 Abstract
IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.
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(2001) Journal of Hematotherapy and Stem Cell Research. 10, 6, p. 769-776 Abstract
Dysregulation of the cytokine network plays an important role in graft-versus-host disease (GVHD). Interleukin-18 (IL-18) is an obligatory cytokine for interferon-γ (IFN-γ) production and IFN-γ and sIFN-γR are elevated in patients with GVHD. Because IL-18 binding protein (IL-18BP) is an inhibitor of IL-18-mediated IFN-γ production, we evaluated IL-18BP levels in patients undergoing allogeneic peripheral blood stem cell transplantation (PBSCT). IL-18BP levels were assessed in 14 patients on day - 10 (before conditioning), on the day of transplant, on the day of engraftment, and during transplant-related complications. A comparison of the kinetics of IL-18BP and soluble(s) IL-6R, sIFN-γR, IL-18 serum levels was performed. IL-18BP levels were assessed by specific monoclonal antibodies in a double-sandwich enzyme-linked immunosorbent assay (ELISA). In all patients IL-18BP levels decreased during conditioning and increased in parallel with engraftment (p
2000
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(2000) Cytokine. 12, 4, p. 388-396 Abstract[All authors]
This is the first report of the serum profile of a glycosylated recombinant form of human IL-6 (rhIL-6) administered subcutaneously (1-10 μg/kg/day) in a phase I/II trial as a thrombopoietic agent in patients with advanced cancer. The pharmacodynamic effects of IL-6 were also examined. Detailed pharmacokinetic measurements were made in four patients. Peak concentrations at 5-8 h and a median t0.5 of ca. 5 h were similar to those previously reported for non-glycosylated IL-6. However, higher peak concentrations and apparent differences in effective dose levels to those previously reported with the non-glycosylated form were seen. Indications of an apparent attenuation in circulating IL-6 concentrations with continuing injections were seen in eight of 10 patients examined but anti-IL-6 antibody generation was seen in only two patients. Soluble interleukin 6 receptor concentrations generally decreased. No major changes in T cell subsets were seen but expression of CD25 and CD54 by T lymphocytes significantly increased, accompanied by marked increases in soluble CD25 (sIL-2R) and CD54 (sICAM-1). No consistent change in B cells, monocytes or NK cells were seen. No evidence for induction of TNF-α was found. This study demonstrates similar biological effects of glycosylated rhIL-6 to those reported for the non-glycosylated form but illustrates several apparent differences which are discussed further. (C) 2000 Academic Press.
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(2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 5, p. 2174-2179 Abstract
IL-18 shares with IL-1 the same family of receptors and several identical signal transduction pathways. Because of these similarities, IL-18 was investigated for its ability to induce prostaglandin E-2 (PGE(2)) synthesis in human peripheral blood mononuclear cells (PBMC), a prominent, proinflammatory property of IL-1. IL-18 was highly active in PBMC by inducing the synthesis of the chemokine IL-8; however, no induction of PGE(2) synthesis nor cyclooxygenase type-2 gene expression was observed in PBMC stimulated with IL-18. In the same cultures, IL-1 beta induced a 12-fold increase in PGE(2). Although IL-1 beta-induced IL-8 synthesis was augmented 3-fold by IL-18, IL-18 suppressed IL-1 beta-induced PGE(2) production by 40%. The suppressive effect of IL-18 on PGE(2) production was mediated by interferon (IFN)-gamma because anti-human IFN-gamma-antibody prevented IL-18-induced reduction in PGE(2). Consistent with these observations, IL-12, a known inducer of IFN-gamma augmented IL-1 beta-induced IFN-gamma but suppressed IL-1 beta-induced PGE(2) by 75%. IL-18 binding protein (IL-18BP) is a naturally occurring and specific inhibitor of IL-18. When recombinant IL-18BP was added to PBMC cultures, unexpectedly, spontaneous PGE(2) production increased. PGE(2) production was also increased by the addition of IL-18BP to PBMC stimulated with either IL-1 beta or IL-12 and also in whole blood cultures stimulated with Staphylococcus epidermidis. These studies demonstrate that IL-18BP decreases endogenous IL-18 activity by reducing IFN-gamma-mediated responses.
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(2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 3, p. 1190-1195 Abstract
A novel, constitutively expressed and secreted IL-18 binding protein (IL-18BP) neutralizes IL-18 and thereby suppresses the production of IFN-γ resulting in reduced T-helper type 1 immune responses. In the present study, four human and two mouse isoforms, resulting from mRNA splicing and found in various cDNA libraries, were expressed, purified, and assessed for binding and neutralization of IL-18 biological activities. Human IL-18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (K(d)) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; the K(d) of IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb anti IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modeling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which could account for its high affinity binding to the ligand. It is likely that preferential secretion of functional and nonfunctional isoforms of IL-18BP affect the immune response.
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(2000) Journal of Interferon and Cytokine Research. 20, 11, p. 971-982 Abstract
A panel of monoclonal antibodies (mAb) derived against human interferon-α/β receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-α1, IFN-α2a, and IFNβ). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-α2a and IFN-β-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-α2a and IFN-β binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-α2a and IFN-β-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation, mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-α/β to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-α/β but did not inhibit IFN-α/β-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation.
[All authors] -
(2000) Clinical and Experimental Immunology. 119, 1, p. 28-37 Abstract
This study shows that subcutaneous administration of increasing doses of IL-12, once a week, in 21 cancer patients increased the expression of cytokine genes (interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF- α), IP- 10, MIG, IL-10, IL-4) in peripheral blood mononuclear cells even at very low doses (30 ng/kg). Surprisingly, no circulating TNF-α or IL-4 could be detected in the plasma of patients treated with IL-12. However, a marked increase of soluble IL-4 receptor was demonstrated in the plasma of five of the six patients studied, which may represent an additional mechanism by which IL- 12 inhibits the development of the Th2 response in viva. A marked decline of IFN-γ and IP10 induction was recorded after repeated cycles of IL-12. In contrast, in most patients IL-12 increased IL-10 expression with no subsequent decrease during the course of therapy, and even an earlier peak of IL- 10 induction at the 6th cycle. In addition, a constant up-regulation of serum soluble IFN-γ receptor levels was observed after each cycle of IL-12 treatment with a delayed peak compared with the IFN-γ peak. The constant rise of IL-10 and soluble IFN-γ receptor during IL-12 therapy may therefore contribute to the inhibition of IFN-γ activity detected after repeated cycles of IL-12. Lastly, a marked heterogeneity of cytokine induction was observed from one patient to another, which appeared to be independent of the dose of IL-12 administered. These data may lead to a better understanding of the biological activity of IL- 12 and the in viva mechanisms of its regulation.
[All authors] -
(2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 2, p. 734-739 Abstract[All authors]
Proinflammatory cytokines, including IL-1β and tumor necrosis factor-α (TNF-α), promote cancer cell adhesion and liver metastases by up-regulating the expression of vascular cell adhesion molecule-1 (VCAM-1) on hepatic sinusoidal endothelium (HSE). In this study, hepatic metastasis after intrasplenically injected mouse B16 melanoma (B16M) cells was reduced 84-95% in mice with null mutations for either IL-1β or the IL-1β-converting enzyme (ICE, caspase-1) compared with wild-type mice. On day 12, 47% of wild-type mice were dead compared with 19% of either IL-1β or ICE-deficient mice. In vitro, conditioned medium from B16M cells (B16M-CM) induced the release of TNF-α and IL-1β from cultures of primary murine HSE. The effect of B16M-CM on HSE resulted in increased numbers of B16M cells adhering to HSE, which was completely abrogated by a specific inhibitor of ICE, anti-IL-18 or IL-18- binding protein. Exogenous IL-18 added to HSE also increased the number of adhering melanoma cells; however, this was not affected by IL-1 receptor blockade or TNF neutralization but rather by anti-VCAM-1. These results demonstrate a role for IL-1β and IL-18 in the development of hepatic metastases of B16M in vivo. In vitro, soluble products from B16M cells stimulate HSE to sequentially release TNF-α, IL-1β, and IL-18. The IL-18 cytokine increases expression of VCAM-1 and the adherence of melanoma cells.
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(2000) Cytokine. 12, 10, p. 1519-1525 Abstract
Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th1 cytokines, sharing structural features with the IL-1 family of proteins. Unlike most other cytokines, IL-18 and IL-1β lack a signal peptide, have an all β-pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1β). These precursors are cleaved by caspase-1 (IL-1β-converting enzyme, ICE) to form the biologically active mature cytokines. Direct expression of mature recombinant human IL-18 in E. coli resulted in a partially active cytokine. We tested the possibility that correct folding of huIL-18 requires its prior synthesis as pro-IL-18. Because caspase-1 is not readily available, we constructed an expression vector encoding human pro-IL-18 in which the caspase-1 cleavage site was mutated into a factor Xa site. To facilitate purification, the mutated pro-IL-18 cDNA was fused in frame to a glutathione-S-transferase (GST) coding sequence. The GST-pro-IL-18 fusion protein was expressed in E. coli, captured on glutathione agarose and mature human IL-18, exhibiting high biological activity was released upon cleavage with factor Xa. This result indicates that correct folding of huIL-18 occurs at the level of pro-IL-18 and provides a practical way to produce biologically active huIL-18. (C) 2000 Academic Press.
1999
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(1999) Journal of Experimental Medicine. 189, 3, p. 599-604 Abstract
The soluble interleukin 6 receptor (sIL-6R) circulates at elevated levels in various diseases. This suggests that inflammatory mediators control sIL-6R release. Through examination of human neutrophils, it was found that the acute phase reactant C-reactive protein (CRP) activates a threefold increase in sIL-6R production. Maximal release occurred after 30-60 min exposure to CRP (50 μg/ml), and was mimicked by peptides corresponding to amino acid residues 174185 and 201-206 of native CRP. A third peptide fragment (77-82) had no effect. Differential mRNA splicing did not account for the CRP-mediated release of sIL-6R, since this isoform was not detected in conditioned media. Furthermore, stimulation of neutrophils with CRP or with peptides 174-185 or 201-206 promoted a loss of membrane-bound IL-6R, suggesting release by proteolytic shedding. The metalloprotease inhibitor TAPI had only a marginal effect on CRP-mediated sIL-6R release, suggesting that shedding occurs via a mechanism distinct from that previously reported. It well established that IL-6 stimulates the acute phase expression of CRP. Our current findings demonstrate a novel relationship between these two mediators, since CRP may affect IL-6-mediated inflammatory events by enabling formation of the sIL-6R/IL-6 complex.
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(1999) Immunity. 10, 1, p. 127-136 Abstract
An interleukin-18 binding protein (IL-18BP) was purified from urine by chromatography on IL-18 beads, sequenced, cloned, and expressed in COS7 cells. IL-18BP abolished IL-18 induction of interferon-γ (IFNγ), IL-8, and activation of NF-κB in vitro. Administration of IL-18BP to mice abrogated circulating IFNγ following LPS. Thus, IL-18BP functions as an inhibitor of the early Th1 cytokine response. IL-18BP is constitutively expressed in the spleen, belongs to the immunoglobulin superfamily, and has limited homology to the IL-1 type II receptor. Its gene was localized on human chromosome 11q13, and no exon coding for a transmembrane domain was found in an 8.3 kb genomic sequence. Several Poxviruses encode putative proteins highly homologous to IL-18BP, suggesting that viral products may attenuate IL-18 and interfere with the cytotoxic T cell response.
1998
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(1998) European Journal of Immunology. 28, 11, p. 3514-3522 Abstract
The soluble IL-6 receptor (sIL-6R) is generated through either proteolytic shedding of the cognate receptor (PC-sIL-6R), or released as the product of differential mRNA splicing (DS-sIL-6R). Using monocytic THP-1 cells, we demonstrate that both mechanisms are independently regulated, and that each process contributes to sIL-6R production. Shedding of the IL-6R was activated by the Ca2+ ionophore, ionomycin, and inhibited by the TNF-alpha protease inhibitor (TAPI). In contrast, basal sIL-6R release was unaffected by Ca2+ depletion and largely insensitive to TAPI. Moreover although IL-6R shedding was inactivated by serum starvation, non-stimulated production remained intact. Basal sIL-6R production via differential mRNA splicing was shown through the inhibitory action of brefeldin A and an enzyme-linked immunosorbent assay specific for DS-sIL-6R. Release of this isoform was unaffected by ionomycin or TAPI, indicating that Ca2+ mobilization activates PC-sIL-6R generation, but not DS-sIL-6R. The divergent control of these sIL-6R isoforms indicates that they may independently influence the inflammatory response.
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(1998) Proceedings of the National Academy of Sciences of the United States of America. 95, 21, p. 12550-12555 Abstract
The cytokine interleukin (IL) 18 (formerly interferon γ-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL48-stimulated HIV- 1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 μM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-κB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB.
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(1998) Journal of Leukocyte Biology. 63, 6, p. 658-664 Abstract
Initially described in 1989 as interferon-γ (IFN-γ) inducing factor (IGIF), interleukin-18 (IL-18) is a novel pro-inflammatory cytokine that is clearly more than an inducer of IFN-γ. The cytokine possesses several biological properties such as activation of nuclear factor-κB (NF-κB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL-1 receptor-activating kinase (IRAK), leading to translocation of NF-κB. This property and others support the concept that IL-18 is related to the IL-1 family. Indeed, one of the IL- 18 receptor chains is the IL-1 receptor-related protein, a member of the IL- 1R family. In addition, IL-18 is structurally similar to IL-1β and like IL- 1β is first synthesized as a leaderless precursor requiring the IL-1β converting enzyme for cleavage into an active molecule. The biology of IL-18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented.
[All authors]
1997
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Soluble interferon-γ receptor and interferon-γ in patients undergoing allogeneic bone marrow transplantation for hematological malignancies(1997) Cytokines Cellular & Molecular Therapy. 3, 3, p. 153-158 Abstract
Interferon-γ (IFN-γ) is known to be involved in graft rejection of solid organs and acute graft-versus-host disease (GVHD). Its role, and especially that of soluble lFN-γ receptor (sIFN-γR), in bone marrow transplantation (BMT) has not been established. We evaluated the sera of 27 patients following BMT. Fourteen of them underwent uneventful BMT, whereas 13 developed transplant-related complications, including acute GVHD (n = 5), early rejection (n = 4), or relapse of basic disease (n = 4). Soluble IFN-γR and IFN levels were evaluated at day -10 (preconditioning), day 0 (day of BMT), day of engraftment, and during BMT-related complications using sIFN-γR-specific monoclonal antibodies (McAB) followed by double-sandwich ELISA, and a sensitive radioimmunoassay respectively. In normal controls (n = 80) sIFN-γR and IFN-γ levels in the sera were 0.5 ± 0.05 and 0.3 ± 0.04 ng/ml respectively. Soluble IFN-γR levels increased in direct correlation with engraftment (0.63 ± 0.11 ng/ml at the day of BMT to 1.43 ± 0.16 ng/ml at the day of engraftment; n = 14; P
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(1997) Gene. 196, 1-2, p. 279-286 Abstract
The human type I interferon (IFN) receptor consists of two essential subunits, huIFNaR1 and huIFNaR2; however, so far only IFNaR1 has been identified in other species. Furthermore, it has been suggested that in some species the type I IFN receptor may consist of a single subunit, since expression of murine IFNaR1 in human cells rendered them responsive to several type I murine IFNs. To resolve this issue, we screened a mouse cDNA library with a probe derived from huIFNaR2 cDNA. A cDNA clone, coding for a transmembrane protein which has 49% identity with huIFNaR2 was isolated. This level of identity suggests that this cDNA codes for a muIFNaR2. In addition, several cDNA clones, coding for two distinct soluble variants of muIFNaR2 were identified. To test whether muIFNaR2 is a functional component of the receptor, we co-expressed it with muIFNaR1 in human cells and with an IFN-responsive luciferase reporter vector. Treatment of these cells with muIFN-β induced high levels of luciferase, whereas no induction was obtained in cells expressing only one of the two subunits. We therefore conclude that the murine type I IFN receptor consists of two different subunits - a configuration shared by humans, and probably all other mammals.
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Mechanism(s) of interferon inhibitory activity in blood from patients with aids and patients with lupus erythematosus with vasculitis(1997) Research Communications in Molecular Pathology and Pharmacology. 96, 3, p. 255-265 Abstract
We have earlier reported that patients suffering from acquired immuno- deficiency syndrome (AIDS), systemic lupus erythematosus (SLE) with vasculitis, Wegner granulomatosis and certain types of late stage cancer have interferon inhibitory activity in their serum. The purpose of this study was to identify the factor(s) involved in this interferon inhibitory activity. Twenty patients with advanced AIDS, twenty patients with SLE and vasculitis and twenty normal healthy controls between ages 25 - 40 years were studied. In contrast to normal, healthy controls, significant interferon inhibitory activity was found in AIDS and SLE patients. This appears to be largely due to: (a) increased soluble circulating interferon α/β receptors, (b) increased prostaglandin E2 levels which inhibits interferon and (c) a interferon inhibitory protein. Further understanding of the nature of interferon inhibitory activity in the patient's sera and development of anti- interferon inhibitory agents would greatly enhance interferons potential as a treatment modality.
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(1997) Mediators of Inflammation. 6, 2, p. 147-153 Abstract
INTERLEUKIN-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) were detected in supernatants of cultures of B chronic lymphatic leukaemia (CLL) lymphocytes. Phorbol-12-myristate 13 acetate (PMA) caused a decrease in the levels of IL-6 in 14 out of 16 cultures and an increase in levels of sIL-6R in all 15 cases. The effect of pokeweed mitogen (PWM) was variable and not significant. The levels of IL-6 were below the detection limit (60 pg/ml) in sera of 13 CLL patients whereas sIL-6R was detected (13 ng/ml to 97 ng/ml) in the 13 sera. IL-6 was not detected in cultures of unstimulated or stimulated with PMA or PWM normal human B cells. Levels of sIL-6R were minimal in cultures of normal B lymphocytes and were increased in PMA stimulated cultures. The results are consistent with the view that B-CLL cells produce spontaneously IL-6 which could act in an autocrine fashion to cause shedding of surface IL-6R and account for the correlation found between serum levels of sIL-6R and B-CLL lymphocyte numbers. The fall in levels of IL-6 in PMA stimulated CLL cultures might express masking or degradation of IL-6 after combination with the receptor.
1996
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(1996) Science. 274, 5290, p. 1185-1188 Abstract
Leptin mediates its effects on food intake through the hypothalamic form of its receptor OB-R. Variants of OB-R are found in other tissues, but their function is unknown. Here, an OB-R variant was found in human hepatic cells. Exposure of these cells to leptin, at concentrations comparable with those present in obese individuals, caused attenuation of several insulin-induced activities, including tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, and down-regulation of gluconeogenesis. In contrast, leptin increased the activity of IRS-1-associated phosphatidylinositol 3-kinase. These in vitro studies raise the possibility that leptin modulates insulin activities in obese individuals.
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Soluble IL-6 receptors (sIL-6R) in hematological patients receiving immunotherapy with IL-2/IFN-α or donor lymphocytes following bone marrow transplantation(1996) Bone Marrow Transplantation. 18, 4, p. 721-724 Abstract
Soluble interleukin-6 receptor (sIL-6R) has previously been shown to potentiate the activity of interleukin-6 (IL-6) which may display antitumor activity. Therefore, we evaluated sIL-6R levels in the sera of 15 patients who received cytokine-mediated immunotherapy with (IL-2/IFN-α), and 15 patients who received cell-mediated immunotherapy post-BMT, in an attempt to reduce the relapse rate, sIL-6R levels were evaluated pre-, during and post-cytokine or cell-mediated immunotherapy, using IL-6R-specific monoclonal antibodies (McAb) and double-sandwich ELISA. In normal controls, sIL-6R levels were found to be 20 ± 3 ng/ml. sIL-6R levels increased significantly during IL-2/IFN-α immunotherapy in comparison to pre- or post-immunoetherapy levels (74 ± 9 ng/ml vs 46 ± 6 ng/ml, and 50 ± 9 ng/ml, respectively) (n = 15) (P
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Immunohistochemical studies of proinflammatouy cytokines and their receptors in hair follicles of normal human skin(1996) Acta Dermato-Venereologica. 76, 5, p. 348-352 Abstract
Immunoreactivity to interleukin-1 alpha, interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha and their receptors, as well as the endogenous interleukin-1 receptor antagonist, was investigated in hair follicles in paraffin-embedded normal human skin. Interleukin-1 beta- and tumour necrosis factor-alpha-like immunoreactivities mere found in the inner root sheath layer of hair follicles, at the suprapapillary level. Interleukin-1 receptor-like immunoreactivity was also found in this layer, while there was a variable immunoreactivity to the interleukin-1 receptor antagonist. In the outer root sheath there was a weak to moderate staining for the four cytokines, in addition to intense staining for their receptors and a weak staining for the antagonist. The fibrous root sheath had a moderate immunoreactivity for interleukin-1 alpha and interleukin-6. The distribution patterns suggest that these cytokines, particularly interleukin-1 beta and tumour necrosis factor-alpha, may have a protective role in hair formation, while all the investigated proinflammatory cytokines may have a role in the differentiation process.
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(1996) Transplantation. 62, 1, p. 138-142 Abstract
Soluble interleukin-6 receptor (sIL-6R) has previously been shown to potentiate the activity of interleukin (IL)-6, which may display antitumor activity. We evaluated sIL-6R and IL-6 levels in the sera of 24 patients following transplantation (allogeneic, n=17; autologous, n=7). Five patients developed acute graft-versus-host disease (AGVHD), three had early graft rejection, and three had an early relapse following bone marrow transplantation (BMT). Soluble IL-6R levels were evaluated at day -10, day 0, day of engraftment, and during BMT-related complications, using IL-6R- specific monoclonal antibodies (mAb) and double-sandwich ELISA. In normal controls, sIL-6R and IL-6 levels were 20±3 ng/ml and 0.01±0.005 ng/ml, respectively. Soluble IL-6R levels increased in direct correlation with engraftment in the uneventful allogeneic transplants (17.7±2.1 ng/ml at day 0 to 49.7±2.6 ng/ml at day of engraftment, n=6, P
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(1996) Human Pathology. 27, 7, p. 738-744 Abstract
We report the case of a patient with a clinically aggressive large cell lymphoma (LCL) which expressed several T-lymphocyte markers and, in addition, CD56 and, to a lesser degree, CD68 antigens. A marked increase in serum concentration of interleukin (IL)-2 was found (490 and 167 pg/0.1 mL in two serum samples collected 6 months apart). This increase in IL-2 appeared unique to this lymphoma because serum concentration of IL-2 was not increased in any of the cases of various types of cutaneous lymphoproliferative disorders tested: mycosis fungoides-related cutaneous T-cell lymphoma (CTCL: 28 patients), granulomatous slack-skin syndrome (GS-SS: 1 patient), anaplastic large cell lymphoma (ALCL: 2 patients), subcutaneous gamma/delta T-cell lymphoma (γ/δ-TCL: 1 patient), adult-type leukemia/lymphoma (ATLL: 1 patient), and lymphomatoid papulosis (LyP: 4 patients). Furthermore, the increase in IL-2 serum concentration appeared selective in this CD56+ large- cell lymphoma-hearing patient, because concentration of none of the five other cytokines tested (IL-4, IL-6, IFNγ, GM-CSF, and TNFα) was increased. In contrast, soluble receptors for IL-2 and two of the other cytokines (IL- 6, and TNFα) were markedly increased not only in this patient, but also in most patients with the other cutaneous lymphoproliferative disorders that we examined except for lymphomatoid papulosis. These data indicate that increased IL-2 serum concentration may help to diagnose a unique type of cutaneous CD56(+) large (T-) cell lymphoma and suggest that IL-2 may play a role of an autocrine growth factor for this lymphoma.
1995
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LIGAND-INDUCED ASSOCIATION OF THE TYPE-I INTERFERON RECEPTOR COMPONENTS(1995) Molecular and Cellular Biology. 15, 8, p. 4208-4214 Abstract
Two transmembrane polypeptides, IFNAR and IFN-alpha/beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear, To study these issues, we stably expressed and characterized the two polypeptides in host murine cells, In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected, In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein, Host cells expressing IFN-alpha/beta R bound IFN-alpha(2) with a high affinity (K-d of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding, Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha(2) was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha(2) to form an intermediate product, while IFNAR associates with this product to form a ternary complex, Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding, Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.
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(1995) Journal of Reproductive Immunology. 29, 2, p. 119-134 Abstract
The aim of this study was (a) to measure soluble tumor necrosis factor receptors (sTNF-Rs) and soluble interleukin-6 receptor (sIL-6-R) in coelomic and amniotic fluids, cord and maternal sera in pregnancy and labor, (b) to examine whether the changes in concentrations of biologically active TNF and IL-6 are related to changes in their soluble receptors, and (c) to determine if levels of soluble receptors in pre-eclamptic disorders differ from normal pregnancies at delivery. Materials collected from 206 women during pregnancy and at delivery were analyzed for soluble receptors by enzyme-linked immunosorbent assay (ELISA). All receptors were present in higher concentrations in coelomic than in the corresponding amniotic fluid. Concentrations increased in amniotic fluid from first to second trimester. The level of sIL-6-R then remained unchanged to term, but there was a decrease in the sTNF-Rs which might account for the simultaneous appearance of bioactive TNF. Labor did not affect the concentration of any receptor in amniotic fluid. In maternal serum, sTNF-Rs increased with gestational age and labor in parallel with IL-6. The origin and physiological importance of these soluble receptors are still unknown. In pre-eclamptic disorders p55 sTNF-R was elevated in maternal serum before initiation of labor compared to normal pregnancy.
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(1995) Journal of Leukocyte Biology. 57, 5, p. 712-718 Abstract
The recently cloned ligand binding component of the type I human interferon-α/β receptor (IFN-α/βR) and its soluble analogue (p40) were characterized. p40 is a potent inhibitor of type I IFNs and antibodies directed against p40 completely block the activity of type I IFNs in human cells. These antibodies immunoprecipitate cellular 102-kDa (major) and 51-kDa (minor) forms of IFN-α/βR. We find that the 51-kDa IFN-α/βR is a disulfide-linked subunit of the 102-kDa IFN-α/βR. Two types of cDNA clones were isolated and sequenced, a 1.5-kb cDNA coding for the transmembrane 51-kDa IFN-α/βR and a 4.5-kb cDNA coding for p40. In addition to ligand binding, IFN-α/βR is directly involved in signaling, because it becomes phosphorylated at Tyr residues on ligand binding and it is physically associated with the cytoplasmic tyrosine kinase JAK1.
1994
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(1994) Proceedings Of The Society For Experimental Biology And Medicine. 206, 3, p. 228-232 Abstract
Interferons (IFNs) act by inducing several intracellular antiviral proteins. We report here that IFNs also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This protein accounts for 25%-50% of the total antiviral activity elicited by IFN. The antiviral protein was purified to homogeneity from culture supernatants of IFN-treated cells by several chromatographic steps, to give a single 28-kDa active polypeptide. Upon sequencing, this novel protein corresponded to the N-terminal ligand-binding domain of the human 160-kDa low-density lipoprotein receptor (LDLR). In addition, we find that IFN induces the cell surface LDLR and this phenomenon may explain previous reports on reduction of serum cholesterol in IFN-treated patients. Viruses produce soluble cytokine receptors that inhibit their respective cytokines, thereby assisting virus infection. It appears now that host cells employ similar molecules for the opposite role of controlling virus infections.
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(1994) Journal of Clinical Investigation. 93, 5, p. 2114-2119 Abstract
By using a sandwich ELISA, soluble human IL-6 receptor (sIL-6 R) levels were measured in the sera of 20 healthy children and of 25 patients with systemic juvenile rheumatoid arthritis (JRA). In patients with systemic JRA, serum sIL-6 R levels (114.6±37.7 ng/ml) were significantly lower (P 2 = 0.51, P 2 = 0.19, P = 0.05), the large quantities of serum IL-6 present in IL-6/sIL-6 R complexes appear to be biologically relevant in vivo, at least as far as the induction by IL-6 of acute phase protein production.
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(1994) Cell. 77, 3, p. 391-400 Abstract
We describe a universal ligand-binding receptor for human interferons alpha and interferon beta (type I IFNs). A soluble 40 kDa IFN-alpha/beta receptor (p40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and immunoprecipitate both a cellular 102 kDa IFN-alpha/beta receptor and its cross-linked complexes with IFN-alpha 2. The receptor is a disulfide-linked dimer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the IFN-alpha/beta receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while its ectodomain corresponds to p40. IFN-alpha/beta receptor is physically associated with the cytoplasmic Tyr kinase JAK1, hence, in addition to ligand binding, it is directly involved in signal transduction.
1993
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(1993) Science. 262, 5131, p. 250-253 Abstract
Interferons, which induce several intracellular antiviral proteins, also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This 28-kilodalton soluble protein was purified to homogeneity and identified by protein sequencing as the ligand-binding domain of the human 160-kilodalton low density lipoprotein receptor (LDLR). The existence of an antiviral soluble LDLR was confirmed by immunoaffinity chromatography with monoclonal antibody to LDLR. This soluble receptor mediates most of the interferon-triggered antiviral activity against VSV, apparently by interfering with virus assembly or budding, and not by inhibiting virus attachment to cells.
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Interleukin-6 inhibits the proliferation of B-chronic lymphocytic leukemia cells that is induced by tumor necrosis factor-α or -β(1993) Blood. 81, 8, p. 2076-2084 Abstract
Tumor necrosis factor (TNF-α) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.
1992
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(1992) Journal of Interferon Research. 12, 6, p. 449-453 Abstract
Tobacco plants were transformed with the human gene for interferon-β (IFN-β). Transformation was determined by the polymerase chain reaction (PCR), and expression was determined by Western blot analysis, by purifying the IFN from the transgenic plants, and by bioassays indicating its activity in human cells. Plants expressing IFN-β were self-pollinated. IFN-β-expressing progeny plants were selected and produced active IFN-β, indicating stable transformation.
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(1992) FEBS Letters. 314, 3, p. 445-448 Abstract
Soluble forms of the interferon-α receptor (sIFN-αR) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-αR, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-αR and [125I]IFN-α with anti IFN-α MAb. Elevated levels or sIFN-αR were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-αR prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be its high as 70,000 Da. It is therefore concluded that sIFN-αR molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-αR, are present in human serum and in normal human urine.
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(1992) Cytokine. 4, 1, p. 6-11 Abstract
Interleukin 6 receptor soluble urinary protein (IL-6-R-SUP), a purified urinary protein binding IL-6 and identified as a truncated 50 kDa soluble form of the 80 kDa IL-6 cellular receptor, was tested for its biological activity. Addition of IL-6-R-SUP enhances the growth stimulation of mouse plasmacytoma T1165 by subliminal concentrations of human recombinant IL-6. Since this effect could be due to a lower affinity of human IL-6 for the mouse cell receptor, we tested the effect of IL-6-R-SUP on human cells. We show that the growth-inhibitory effect of IL-6 on breast carcinoma cells is enhanced by addition of IL-6-R-SUP to these human cells although they possess abundant IL-6 receptors. With IL-6-R-SUP, complete growth inhibition by IL-6 could be achieved and the cells became more sensitive to low levels of IL-6. These effects were prevented by a monoclonal antibody against IL-6-R-SUP which blocks IL-6 binding to cells. The naturally occurring IL-6-R-SUP may help to increase the growth-regulatory action of IL-6.
1991
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(1991) Hybridoma. 10, 1, p. 137-146 Abstract
Soluble IL-6 receptor (IL-6-R) purified to homogeneity from normal human urine was used for immunization of mice and rabbits. Spleen cells derived from a mouse showing a high binding titer to IL-6-R in an inverted solid phase radioimmunoassay (IsRIA) and in a Western blotting analysis were fused to mouse myeloma cells. The hybridomas were screened by the IsRIA, and 30 positive clones were isolated and characterized. They were suitable for affinity purification of the IL-6-R and for its detection by Western blot analysis, by ELISA and by sandwich type sRIA. Most of them inhibited the binding of labeled IL-6-R to IL-6 in a solid phase RIA.
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PRODUCTION, DOWNSTREAM PROCESSING AND CHARACTERIZATION OF PROTEINS FROM RECOMBINANT ANIMAL-CELLS - THE CASE OF HUMAN INTERFERON-BETA(1991) Biologicals From Recombinant Microorganisms And Animal Cells: Production And Recovery. p. 479-486 Abstract
Keywords: CHO CELLS; METHOTREXATE AMPLIFICATION; MICROCARRIER CULTURES; IMMUNOAFFINITY PURIFICATION
[All authors]
1990
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(1990) Journal of Chromatography A. 510, C, p. 331-337 Abstract
Affinity chromatography of crude human urinary proteins on either human recombinant interleukin-6 (rIL-6) or human recombinant interferon-γ (rIFN-γ) or anti IFN-γ receptor (IFN-γ-R) monoclonal antibodies (McAb) yielded the two respective soluble receptors in significant amounts. A single sequence of 30 amino acid residues was obtained by N-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reveresed-phase high-performance liquid chromatography. This sequence was identical with the predicted N-terminal sequence of IL-6-R as previously reported. The purified IL-6-R retained its biological activity. It was used for the preparation of specific anti IL-6-R monoclonal antibodies. Analysis of the eluted proteins from both IFN-γ and anti IFN-γ-R columns by inhibition of solid-phase radioimmunoassay, enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting proved the existence of soluble IFN-γ-R in normal urine. This finding together with the already known presence of soluble TNF receptors and a soluble IL-2 receptors found both in plasma and in urine indicates that release of soluble cytokine receptors into body fluids is a general phenomenon which occurs under normal physiological conditions.
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Two antiviral proteins from tobacco: Purification and characterization by monoclonal antibodies to human β-interferon(1990) Proceedings of the National Academy of Sciences of the United States of America. 87, 2, p. 588-592 Abstract
Polyclonal antibodies to human β-interferon reacted specifically with two plant proteins (gp22 and gp35) by Western blot analysis of crude protein extracts from tobacco leaves infected with tobacco mosaic virus. Immunoaffinity chromatographv of these extracts on a column of immobilized monoclonal antibodies to human β-interferon and then reversed-phase HPLC yielded gp22 and gp35 in a pure state. Both proteins reacted with the Schiff reagent and concanavalin A (indicating their glycoprotein nature) and exhibited antiviral activity (inhibiting tobacco mosaic virus replication in tobaccoleaf discs at concentrations of ng/ml). Each protein was cleaved by cyanogen bromide and the resultant peptides, separated by HPLC, were sequenced as far as the Edman degradation allowed, giving a total of 61 amino acid residues for gp22 and 105 residues for gp35, which represent 30-50% of their expected length. Computer analyses of the sequenced segments revealed no significant homology to human β-interferon, each other, or any other recorded sequence.
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EFFECTS OF RECOMBINANT HUMAN IL-6 ON BREAST-CANCER CELLS(1990) Molecular And Cellular Biology Of Cytokines. 10, p. 509-515 Abstract
Keywords: Biochemistry & Molecular Biology; Endocrinology & Metabolism; Genetics & Heredity
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SOLUBLE CYTOKINE-RECEPTORS ARE PRESENT IN NORMAL HUMAN URINE(1990) Physiological And Pathological Effects Of Cytokines. 10, p. 413-421 Abstract
Keywords: Immunology; Medicine, Research & Experimental; Pathology; Physiology
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SOLUBLE FORMS OF THE TNF RECEPTORS, PURIFIED FROM HUMAN URINE, INHIBIT TNF EFFECTS - ANTIBODIES AGAINST THE SOLUBLE RECEPTORS INDUCE TNF-LIKE EFFECTS(1990) Molecular And Cellular Biology Of Cytokines. 10, p. 299-308 Abstract
Keywords: Biochemistry & Molecular Biology; Endocrinology & Metabolism; Genetics & Heredity
[All authors]
1989
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(1989) Journal of Interferon Research. 9, 3, p. 315-328 Abstract
Monoclonal antibodies against the human interferon-γ (IFN-γ) receptor were developed by injecting mice with a preparation of receptor that was purified from solubilized placental membranes by ligand affinity chromatography. Three antibodies were identified by their ability to block the binding of 125I-labeled IFN-γ to its receptor on HeLa cells at 4°C. One of these antibodies blocked several biological activities of IFN-γ, including its antiviral activity, its ability to induce HLA-DR surface antigens, and its ability to protect cells from NK cell-mediated cytotoxicity. This antibody exhibited higher binding capacity to cells at 37°C and was significantly less displaceable by an excess of IFN-γ as compared with the other two antibodies. Immunoaffinity chromatography of solubilized crude placental membrane preparation yielded a purified receptor that exhibited a molecular weight of 88,000. The purified receptor retained its ability to bind 125I-labeled IFN-γ in solution.
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BIOLOGICAL-ACTIVITIES OF RECOMBINANT HUMAN IFN-BETA-2/IL-6 (ESCHERICHIA-COLI)(1989) Regulation Of The Acute Phase And Immune Responses : Interleukin-6. 557, p. 144-156 Abstract
Keywords: Biochemistry & Molecular Biology; Immunology; Medicine, Research & Experimental
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(1989) Journal of Experimental Medicine. 170, 4, p. 1409-1414 Abstract
Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-γ, or anti-IFN-γ-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analyis of the eluted proteins from both IFN-γ and anti-IFN-γ-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-γ-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.
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1988
1987
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The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies(1987) Journal of Biological Chemistry. 262, 18, p. 8483-8487 Abstract
The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.
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(1987) Annals of the Rheumatic Diseases. 46, 8, p. 580-586 Abstract
Labial biopsy specimens from eight patients with primary Sjögren's syndrome (SS), 10 patients with secondary SS, and three healthy controls were studied with monoclonal antibodies identifying HLA class I and class II antigens; interferon-α, β, and γ; interleukin 2 (IL2); and the IL2 receptor (Tac) among others. In the normal biopsy specimens there was evidence of HLA class I and, to a lesser extent, class II antigens in both ducts and acini, though this was much less marked than in the Sjögren's biopsy specimens. Interferon-γ staining, but not interferon-α or β, was also considerably enhanced in the biopsy specimens from the patients with Sjögren's syndrome. These data support the view that in Sjögren's syndrome the release of interferon-γ may be involved in the induction of class II determinants. Our observations were broadly similar in both primary and secondary Sjögren's syndrome except that patients with primary Sjögren's syndrome tended to have more diffusely scattered T lymphocytes.
1986
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(1986) DNA. 5, 3, p. 181-193 Abstract
A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-γ was fused through its 5′-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-γ gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-γ cell lines were obtained that produce 1.5–2 million units of IFN-γ per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-γ. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-γ with 1–2 × 108 units/mg protein. Like IFN-γ from human white blood cells, the IFN-γ from CHO-γ cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-γ/ml culture per day.
[All authors] -
Localization of interferons and interleukin 2 in polymyositis and muscular dystrophy(1986) Clinical and Experimental Immunology. 63, 2, p. 450-458 Abstract
Muscle biopsies from nine patients with polymyositis, six with muscular dystrophy, six with other muscle diseases and three controls have been studied with a panel of 10 monoclonal antibodies (MoAb) identifying T lymphocytes, HLA-class I antigens, α, β and γ interferons and interleukin 2 (IL-2). The result confirm that the staining of the sarcolemma with anti-HLA class I antibody is weak or negative, except in areas adjacent to infiltrating leucocytes or where muscle fibre damage is evident. The very similar tissue distribution of α, β and γ interferons in the polymyositis biopsies supports the hypothesis that interferons are released by the inflammatory infiltrate and induce the class I antiben expression. In contrast, little interferon was demonstrated in the dystrophic muscle implying that class I expression in these disorders must occur by a different mechanism. Little IL-2 was demonstrated in any of the biopsies though some unexplained small dense accumulations were identified by one of the anti IL-2 MoAb.
1985
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(1985) Cellular Immunology. 92, 2, p. 218-225 Abstract
The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-γ, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.
1984
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(1984) Journal of Interferon Research. 4, 2, p. 275-282 Abstract
Binding site competition studies of 125I-interferon (IFN)-γ were performed with homogeneous preparations of IFNs α2, β, and γ. It was found that only IFN-γ could compete for the specific binding site of 125I-IFN-γ in both WISH and FS11 cells. Thus, a clear distinction between the receptor of IFN- α and β on one hand, and the receptor of IFN-γ on the other hand, was obtained. This result is unambiguous because all IFN preparations which were used were purified to homogeneity by specific monoclonal antibody immunoaffinity chromatography. We propose to assign the names “Type I” for the receptor of IFN-α and IFN-β, and “Type II” for the specific receptor of IFN-γ.
1983
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(1983) The EMBO Journal. 2, 9, p. 1527-1530 Abstract
Human interferon‐gamma (IFN‐gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN‐gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN‐gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN‐gamma. Moreover, IFN‐gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN‐gamma subtypes are immunologically cross‐reactive. Immune precipitation of partially purified 125I‐labelled IFN‐gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN‐gamma gave a 50‐fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid‐phase radioimmunoassay, allowing the detection of IFN‐gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h.
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1982
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Monoclonal antibodies to human α-interferon and their use for affinity chromatography(1982) Journal of Immunology. 129, 5, p. 2244-2247 Abstract
A new screening procedure has been developed and used for the identification of three hybridomas secreting monoclonal antibodies to human leukocyte interferon (IFN-α). The screening procedure is based on immune precipitation with a second antibody, acid dissociation of the precipitate, and bioassay of the recovered interferon. This procedure selects for monoclonal antibodies that are suitable for affinity chromatography, as demonstrated by construction of immunoadsorbents from these monoclonal antibodies and purification to homogeneity in IFN-α.
1980
1978
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(1978) Nature. 273, 5663, p. 543-545 Abstract
THE variable regions of antibodies contain unique antigenic markers called idiotypes1,2. According to Jerne's network theory, antibodies reacting specifically with idiotypic determinants (anti-idiotypes) might be involved in a normal event that modulates the degree and duration of the immune response3. In view of this hypothesis anti-idiotypic antibodies may have a practical application in medicine, for example, in the treatment of autoimmune diseases or other immunological disorders. It has been suggested that the fluctuating clinical symptoms in autoimmune diseases such as myasthenia gravis or systemic lupus erythematosus are due to a regulatory effect of anti-idiotypic antibodies and that remissions are associated with an increase of specific anti-idiotypic activity4. Antibodies and antigen-binding receptors on the surface of lymphocytes share idiotypic determinants 5,6. Thus, autoimmunisation with an individual's antibodies or lymphocytes carrying specific receptors for a given antigen may be an efficient way to produce specific anti-idiotypic antibodies7-9. The acetylcholine receptor (AChR) is a major autoantigen in myasthenia gravis and immunisation of animals with AChR (isolated from electric fish) results in the induction of an experimental autoimmune disease (experimental autoimmune myasthenia gravis) similar to myasthenia gravis10,11. We have therefore prepared anti-idiotypic serum specific to AChR idiotypes to test whether it can affect the immune response against AChR and the clinical state resulting from AChR sensitisation. We describe here the preparation of specific C57BL/6J antiserum against AChR-educated lymphocytes and report its anti-idiotypic activity.
1976
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(1976) Nature. 263, 5575, p. 329-330 Abstract
EXPERIMENTAL autoimmune myasthenia gravis (EAMG) has been induced in rabbits1-4 guinea pigs5,6 rats4,6 and monkeys7 by injection of the acetylcholine receptor (AChR) isolated from electric fish. The experimental disease, together with the demonstration of humoral8-10 and cellular11 immune responses to AChR in patients with myasthenia gravis (MG), verifies hypotheses12,13 about the immunopharmacological block of AChR in MG. An experimental model disease in mice is needed for studies of the genetic aspects of MG and the role of the thymus as a specific antigen target, and as a source for immunocompetent helper and suppressor cells. We describe here the induction of EAMG in several inbred strains of mice and demonstrate different susceptibility to the disease in strains representing different haplotypes of the major histocompatibility complex (H-2). We describe some humoral and cellular aspects of the disease and demonstrate that in a susceptible mouse strain both humoral and cellular immune response to self-AChR are elicited. Finally we show that AChR behaves as a thymus-dependent antigen.
1974
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(1974) European journal of cancer. 10, 8, p. 551-553 Abstract
Recently, we reported the recovery of the murine sarcoma virus (MSV) genome after the infection of mouse cells with nuclear DNA from MSV-transformed non-virus producing cells [1]. In our initial studies the biological activity of the nuclear DNA was demonstrated by its ability to transform normal mouse cells in vitro. The MSV genome could then be "rescued" from the transformed cells with a helper Rauscher leukaemia virus (RLV), andthe pseudotype virus produced (MSV-RLV) transformed mouse cells in vitro.In this paper we wish to report the properties of 3T3-BALB/c cells which were transformed by the MSV-DNA provirus but did not contain a rescuable MSV. The properties of the transformed cells which are outlined in this paper can be summarised as follows: (a) pleomorphic cytology, (b) an abnormal karyotype containing large marker metacentric chromosomes not present in the normal mouse karyotype and (¢) highly malignant properties as evidenced by the induction of a rapidly developing sarcoma in the inoculated BALB/c mice. These three inter-related properties suggest that the cells have been transformed by the M S V - D N A provirus, in spite of the fact that a biologically rescuable virus could not be demonstrated.