Human Interleukin-1β Research Articles

Tumor necrosis factor-α enhances inhibitory effects of interleukin-1β on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-α (rTNFα) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-Induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNFα with human recombinant interleukin-1β (rIL-1β) enhanced the inhibitory effects of rIL-1β. The rIL-1β dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNFα had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNFα in a dose dependent manner. In the presence of both rTNFα and rIL-1β, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNFα and IL-1 may by produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.

Functional and morphological effects of interleukin-1? on the perfused rat pancreas

We recently reported a potentiating effect of recombinant human interleukin-1 beta on glucose-stimulated insulin release from the isolated perfused pancreas. With the aim of determining whether the stimulatory effect of recombinant interleukin-1 beta on the B cell in the intact gland was modulated by varying the concentration, time of exposure to recombinant interleukin-1 beta or B-cell activity, and to elucidate a possible mechanism of action, we measured in the perfused rat pancreas the release of insulin, glucagon and/or prostaglandin E2 according to the following three different protocols: (1) perfusion with 20 ng/ml of recombinant interleukin-1 beta for 92 min at 5 and 20 mmol/l D-glucose (2) perfusion with varying concentrations of recombinant interleukin-1 beta ranging from 0.1 x 10(-3) ng/ml to 100 ng/ml at 5 and 20 mmol/l D-glucose (3) perfusion with 20 ng/ml of recombinant interleukin-1 beta at 5, 11 or 20 mmol/l D-glucose. Furthermore, in a separate set of experiments we examined the influence of the cytokine on the morphology of the endocrine pancreas. Interleukin-1 beta stimulated insulin secretion at 11 and 20 mmol/l D-glucose and potentiated first as well as second phase insulin release in a dose-dependent fashion, with decreasing effect at higher concentrations. Glucagon secretion was also stimulated by recombinant interleukin-1 beta, irrespective of increasing glucose (5, 11, 20 mmol/l) and insulin concentrations. The potentiating effect of recombinant interleukin-1 beta on insulin secretion was evident even after discontinued perfusion with the cytokine, suggesting a priming effect on B-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of recombinant human interleukin-1β on cellular proliferation and the production of prostaglandin E 2, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone

There is mounting evidence implicating cytokines such as interleukin-1 in the local regulation of bone homeostasis. In this report we show that recombinant human interleukin-1β (rhIL-1β) influences several activities of osteoblast-like cells derived from human trabecular bone explants in vitro. rhIL-1β stimulated cellular proliferation and the synthesis of prostaglandin E 2 and plasminogen activator activity in the cultured human osteoblast-like cells in a dose-dependent manner. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to 1,25(OH) 2D 3, two characteristics of the osteoblast phenotype, were antagonized by rhIL-1β over a similar dose range. This study adds further support to the potential role of interleukin-1 in the physiological and pathological modulation of bone cell metabolism.

Radiolabeling of the biologically active peptide 163–171 of human interleukin-1β

N-succinimidyl[2,3- 3H]propionate was used for the radiolabeling of the biologically active peptide fragment 163–171 of human interleukin-1β (VQGEESNDK). Suitable reaction conditions were studied to obtain useful labeling of the molecule. A mixture of mono- (70%) and bi- (30%) propionyl derivatives was obtained with a total 3H specific activity of 87 Ci/mmol of peptide. The conditions for an efficient chromatographic separation of labeled peptide from unreacted reagents and by-products were established. The labeled peptide maintained the same biological activity as that of the corresponding unlabeled molecule, indicating that the labeling procedure did not alter the biological characteristics of the peptide. This thus allows the use of the radiolabeled peptide for receptor binding studies.

The Effect of N-Terminal Extension on the Structure and Function of Human Interleukin-1β

Interleukin-1 beta (IL-1 beta) and N-terminally extended Met-Glu-Ala-Glu-IL-1 beta (MEAE-IL-1 beta) were cloned and expressed in E. coli. Extension of the chain results in a limited conformational change reflected by the CD spectrum in the far ultraviolet, while the aromatic side chains responsible for the CD in the near ultraviolet are not affected. No difference in immunoreactivity between IL-1 beta and MEAE-IL-1 beta is observed in the IL-1 beta ELISA. Like IL-1 beta, MEAE-IL-1 beta exhibits biological activity tested in the costimulatory mouse thymocyte (LAF) assay. The specific biological activity of IL-1 beta is 3 x 10(8) U/mg and that of MEAE-IL-1 beta 3 x 10(6) U/mg. Like IL-1 beta, MEAE-IL-1 beta displaces [125I]IL-1 beta from mouse thymocytes and the binding affinities of the two forms differ by a factor of 10(2). Finally the inhibitory effect of the two IL-1 beta forms on in vitro insulin secretion from isolated rat islets of Langerhans was measured. Again MEAE-IL-1 beta is 10(2) times less potent than IL-1 beta. The structure-activity relationship for IL-1 beta and MEAE-IL-1 beta is discussed.

High level expression of interleukin-1β in a recombinant Escherichia coli strain for use in a controlled bioreactor

A recombinant bacterial strain for the large scale production of human interleukin-1β (IL-1β) was constructed. The λP r and the tryptophan systems were compared for efficiency of transcription and regulation. The efficiency of IL-1 protein production from these constructs was analyzed. Enhanced protein synthesis was achieved by the fusion of λP r promoter sequences with trp leader sequences which included the trp RBS. A strain (JM101) was selected for use as a host and tested in a one liter bioreactor. A growth and induction regimen was established for use in bioreactors which results in the accumulation of 0.75–0.95 g 1 −1 of recombinant IL-1.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1β

Expression plasmids carrying the coding sequence of mature human interleukin 1β(ILIβ) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1β from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1β fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1βmaturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1β fusion. Immuno-electron microscopy revealed that the sOmpA-IL 1β fusion was targeted to the inner membrane, whereas the ThoA-IL 1βfusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1β. Moreover, these E. coli

Preliminary studies of cytokine-induced functional effects on the visual pathways in the rabbit

Epidural visual evoked potentials (VEP) were used to study the role of cytokines in the induction of pathophysiologic changes associated with inflammation in the central nervous system (CNS) of the rabbit. In normal rabbits, intraocular injection of human recombinant interferon-γ (IFN-γ) and tumor necrosis factor (TNF) increased the peak latency of the cortical VEP by more than 2 ms within 3 h of injection; equal volume injections of control subtances had no effect. Alterations in conduction induced by IFN-γ and TNF reversed within 24 h and could be reinduced by reinjection. Intraocular injection of recombinant human interleukin-1β (IL-1) induced a more progressive delay in conduction that peaked 24 h after intraocular challenge and reversed over the ensuing 48 h. Pathologic examination of the tissues indicated that the primary effect of these cytokines is on the vasculature and induces changes associated with inflammation. The results suggest that the acute reversible effects of cytokines on CNS function are associated with vascular events; further they support the sensitivity of the ‘rabbit eye model’ for studies on the pathophysiologic effect of inflammatory mediators on the CNS in vivo.

Multiple charge isomers of human recombinant interleukin-1β

Human interleukin-1β (rhuIL-1β), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high-voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL- 1β molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5–9.5, it was resolved into two broad bands appearing in the pH range of 6.2–5.8 and 5.5–5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5–8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL-1β. However, all these components reacted to the antiserum to rhuIL-1β and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL-1β.

Biotinylation of reactive amino groups in native recombinant human interleukin-1β

Recombinant human interleukin-1β (rIL-1β) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1β products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92–94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1β by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1β was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1β.

Interleukin-1β stimulates phospholipase A 2 mRNA synthesis in rabbit articular chondrocytes

A cDNA that codes for human rheumatoid synovial fluid phospholipase A 2 (PLA 2) hybridised with a RNA of the same size (900 base pairs) isolated from rabbit articular chondrocytes (RAC). Treatment of RAC with 100 ng/ml recombinant human interleukin-1β (IL-1) for 24 hours caused a 13-fold increase in mRNA for this PLA 2. Timecourse studies demonstrated that maximal induction of PLA 2 mRNA occurred by 16 hours post addition of IL-1 (100 ng/ml). Augmentation of RAC PLA 2 mRNA levels was dose-dependent; half-maximal induction was estimated to require 0.15 ng/ml IL-1. Actinomycin D inhibited IL-1 effects on PLA 2 mRNA levels. Coordinate effects of IL-1 on RAC PLA 2 activity were observed with respect to time and dose dependence as well as actinomycin D sensitivity.

Crystal structure of recombinant human interleukin-1β at 2·0 Å resolution

The crystal structure of recombinant human interleukin-1β (IL-1β) has been determined at 2·0 Å resolution and refined to a crystallographic R-factor of 0·19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel β-strands and no α-helix. The single 153-residue polypeptide chain is folded into a six-stranded β-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the β-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of β-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1β receptors.

Fate and biological action of human recombinant interleukin 1β in the rat in vivo

The plasma half life of recombinant human interleukin 1 beta (rhIL 1 beta) was determined in rats by measuring the disappearance of the radioactivity of 125I-labeled rhIL 1 beta from the circulation. The plasma clearance showed a biphasic behavior: an initial fast disappearance (half life of about 3 min) was followed by a second slower one (half life of about 4 h). Twenty minutes after a single-dose injection of 125I-labeled rhIL 1 beta most of the radioactivity was concentrated in kidneys, liver and intestine. rhIL 1 beta induced the synthesis of alpha 1-acid glycoprotein (AGP), alpha 1-cysteine proteinase inhibitor (CPI) and beta-fibrinogen mRNA in liver. Half maximal stimulation was elicited by approximately 3000 U of rhIL 1 beta per animal. The mRNA changes for AGP and CPI were followed by corresponding protein increases in serum. Twenty hours after rhIL 1 beta injection, serum AGP rose from 0.7 to 2.5 mg/ml. CPI increased from 0.3 to 1.9 mg/ml 25 h after administration of rhIL 1 beta. Within 20 h after rhIL 1 beta injection, albumin serum concentration showed a strong decrease, preceded by a reduction in hepatic albumin mRNA levels. Neither changes in albumin synthesis nor degradation can explain this decrease suggesting that other mechanisms such as increased transvascular permeability are involved.

Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta.

The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of generated acetyl-coenzyme-A residues undergoing oxidation. However, in the presence of IL-1 there was a significant increase in L-[U-14C]glutamate oxidation. These combined observations suggest that exposure to IL-1 induces a preferential decrease in glucose-mediated insulin release and mitochondrial glucose metabolism. This mitochondrial dysfunction seems to reflect an impairment in proximal steps of the Krebs cycle. It is conceivable that the IL-1-induced suppression and shift in islet metabolism can be an explanation for the beta-cell insensitivity to glucose observed in the early phases of human and experimental insulin-dependent diabetes mellitus.

Different conformation of purified human recombinant interleukin 1β from Escherichia coli and Saccharomyces cerevisiae is related to different level of biological activity

Human recombinant interleukin 1β produced in Escherichia coli and in Saccharomyces cerevisiae was purified to homogeneity by a combination of ion exchange, gel filtration and hydroxylapatite column chromatography. The two proteins, both expressed in the mature form, differ in that the protein secreted from yeast is glycosylated and lacks the first four amino acids. The biological activity of IL-1 obtained from E. coli is comparable to that of the natural protein, while the protein produced from yeast showed very low specific activity. The analysis of the state of oxidation of the two cysteine residues present in the IL-1 molecule and the evaluation of the immunoreactivity of the two proteins have proved that a different conformation is at the basis of the different biological activity of the two proteins.

Cytokine regulation of interleukin 6 production by human endothelial cells

The influence of recombinant (r) human tumor necrosis factor α (rTNF-α), r human interleukin 1β (rIL-1β), and r human interferon γ (rIFN-γ) on the production of interleukin 6 (IL-6) by human endothelial cells (HEC) was investigated. The addition of 1–100 U/ml of either rTNF-α or rIL-1β to cultures of HEC monolayers caused a dose-related increase in IL-6 production as detected after 24 hr of incubation. In contrast to rIL-1β and rTNF-α, the use of up to 1000 U/ ml of rIFN-γ caused only a moderate increase in IL-6 production. However, significantly greater quantities of IL-6 were produced by HEC monolayers subjected to 1000 U/ml of rIFN-γ in combination with 1–100 U/ml of rTNF-α. Furthermore, the addition of graded concentrations of human transforming growth factor β (TGF-β) to cultures resulted in a dose-related inhibition of rIL-1β- and rTNF-α-induced IL-6 production by HEC. The results demonstrate that rIL-1β and rTNF-α share the ability to stimulate HEC for production of IL-6 and indicate that TGF-β may act as an immunosuppressive agent, at least partially, through its ability to inhibit the action of TNF-α and IL-1 on endothelial cells.

Interleukin-1β inhibits acetylcholine synthesis in the pituitary corticotropic cell line AtT 20

Pituitary corticotropic AtT 20 cells were found to synthesize [ 3H]acetylcholine, when they were incubated with [ 3H]choline. Treatment of the cells with picomolar concentrations of human interleukin-1β inhibited [ 3H]acetylcholine production. This effect was specific, since two antisera, directed against human interleukin-1β neutralized the inhibitory action of interleukin-1β. A possible implication of the inhibitory action of interleukin-1 on acetylcholine production in neuronal transmission disorders is discussed.

Interleukin 1 induces new protein formation in isolated rat islets of Langerhans.

It is hypothesized that interleukin 1 induces toxic free radical formation in pancreatic beta-cells leading to beta-cell degeneration and destruction. Therefore, isolated rat pancreatic islets were examined for interleukin 1 and heat shock induced proteins. Afer exposure to human interleukin 1 beta (0.015 to 10 micrograms/1) or heat (40 degrees C) for up to 24 h, islets were labelled with 35S-methionine and solubilized. Islets proteins were analysed by SDS-polyacrylamide gel electrophoresis. By autoradiography it was shown that both interleukin 1 and heat exposure induced the formation of a 70 kD protein. Further, interleukin 1 induced the formation of two proteins of 32 and 80 kD, respectively, which was not seen after heat exposure. Possibly, the 70 kD protein is a member of the heat shock protein 76 family, participating in unspecific cellular defence and maybe free radical scavenging. Other candidates are the superoxide radical scavenging enzyme manganous superoxidedismutase, MHC class II molecules, and heme oxygenase.

In vivo studies on the effects of human recombinant interleukin-1 beta on articular cartilage.

Interleukin-1 (IL-1) is a cytokine produced by a number of connective-tissue and inflammatory cells which has been shown in organ culture to stimulate the breakdown of cartilage proteoglycans and inhibit their synthesis. Intraarticular injection of human recombinant IL-1 beta into the knee joints of rabbits induced a dose-related decrease in cartilage proteoglycan content and increased infiltration of cells into the synovial fluid. Following a single intraarticular injection, the loss of proteoglycan was maximal at 3 days. By 7 days, proteoglycan content began to return toward control levels. IL-1 also resulted in a dose-related decrease in the ability of cartilage to synthesize new proteoglycan as measured by 35S incorporation. These in vivo effects of IL-1 on articular cartilage closely reflect those effects observed in vitro in organ culture and are consistent with the hypothesis that IL-1 may play a role as a mediator of the loss of cartilage in some arthritic diseases.

Recombinant human interleukin-1β decreases serum carboxypeptidase N and modifies serum amino acid concentrations in rats

The effects of recombinant human interleukin-1β (rhIL-1β) on various serum constitutents were studied following subcutaneous injection (12.5 or 125 μg/kg) in female Wistar rats. Protein electrophoresis and the determination of the serum concentrations of carboxypeptidase N (CPN), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, aldolase, total proteins, iron, urea, creatinine, and several amino acids were performed 12, 24, and 72 hr after injection. With both doses of rhIL-1β, iron, albumin, CPN, and lysine were significantly decreased whereas α 2-globulin, urea, and creatinine were significantly increased 12 hr after administration. Iron and CPN were still low after 24 hr but returned to normal levels after 72 hr. With the higher dose of rhIL-1β, only alanine and phenylalanine levels were increased after 12 and 72 hr, taurine after 12 hr, and methionine after 24 hr. There were no biochemical or histological signs of hepatotoxicity. The findings indicate that rhIL-1β produces a reversible alteration of various biochemical plasma constituents without any apparent signs of cytotoxicity. Moreover, the decrease in CPN observed may influence the degradation of inflammatory peptides.