Expression of interleukin-1 beta and interleukin-1 receptor antagonist in oxLDL-treated human aortic smooth muscle cells and in the neointima of cholesterol-fed endothelia-denuded rabbits

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima and the proliferation of intimal VSMCs are key events in restenotic lesion development. These events, which are preceded and accompanied by inflammation, are modulated by the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), which induces vascular smooth muscle cells to express adhesion molecules and to proliferate. IL-1 beta action is complex and regulated, in part, by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Whether there was a temporal and spatial correlation between IL-1 beta and IL-1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)-stimulated human aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL-1 beta and IL-1ra expression was detected in the neointima of endothelia-denuded cholesterol-fed New Zealand white rabbits by immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL-beta and IL-1ra expression and release in a dose- and time-dependent manner. Treatment with 20 microg/ml oxLDL resulted in increased IL-1 beta release after 6 h, which peaked at 24 h, and in increased IL-1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL-beta expression showed a similar pattern to release, whereas IL-1ra expression was seen in unstimulated cells and was not increased by oxLDL treatment. Confocal microscopy showed colocalization of IL-beta and IL-1ra expression in oxLDL-stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein-1 DNA binding activity in HASMCs (6.6- and 3.3-fold, respectively). In cholesterol-fed endothelia-denuded rabbits, the notably thickened intima showed significant IL-1 beta and IL-1ra expression. These results provide further support for the role of IL-1 system in the pathogenesis of restenosis. This is the first demonstration of IL-1 beta and IL-1ra expression and secretion of oxLDL-treated HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an important source of these factors.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

IL1B gene polymorphisms influence the course and severity of inflammatory bowel disease

 There is evidence of a disbalance in the inflammatory regulation of patients with inflammatory bowel diseases (IBD). Interleukin-1β plays an important role in the pro-inflammatory response. Our aim was to study the influence which IL1B gene polymorphisms may have on the severity and course of these diseases. Ninety-six patients with ulcerative colitis (UC), 98 patients with Crohn's disease (CD), and 132 ethnically matched healthy individuals (HC) were typed for the polymorphic sites in the promoter region (position –511) and in exon 5 (position +3953) of the IL1B gene, using polymerase chain reaction (PCR)-based methods. In the CD group a significant association (P=0.009) was found in this pair of genes. Homozygotes for allele 1 at position +3953 were more often present (69% vs 31%) in the subgroup of patients carrying at least one copy of allele 2 at position –511. This association was significant in patients with non-perforating disease (P=0.002), but was not present in patients with perforating-fistulizing disease. The distribution of both allelic pairs in the non-fistulizing group proved to be significantly different from HC (P<0.05), UC (P<0.03), and the fistulizing group (P<0.05). There was a similar association in non-operated patients (P=0.024), whereas no such association was found in surgically treated patients. Among carriers of allele 2 at position –511, UC patients with more severe bleeding symptoms (P=0.006) were less frequently found. These results suggest that IL1B gene polymorphisms participate in determining the course and severity of inflammatory bowel disease and contribute to explain the heterogeneity of these diseases. Received: 16 July 1998 / Revised: 3 November 1998     

Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae

A nonapeptide from IL-1β has been reported to be an immunostimulant and adjuvant. To investigate the possibility of enhancing the immunogenicity of recombinant antigens delivered by live-attenuated Salmonella strains, we inserted an oligonucleotide coding for the nonapeptide from murine IL-1β into the genes of three model proteins: LamB, MalE, and flagellin. The hybrid proteins were expressed and delivered in vivo by Salmonella aroA strains, and serum antibody responses were analyzed. The results showed that the nonapeptide induced an increase in the immune response against Salmonella-delivered flagellin, measured on day 28 post-immunization. However, the adjuvant effect was lost by day 42. In no case was an adjuvant effect detected for Salmonella-delivered LamB or MalE. Thus, by comparing the immune responses raised by purified MalE with and without the peptide, we investigated whether the insertion of the peptide affected the immunogenicity of the protein itself. Also in this case, a modest adjuvant effect was shown only after primary immunization and when very low doses of antigen were used. In conclusion, the immunomodulatory properties of the IL-1β peptide can also be detected when it is delivered in vivo by Salmonella; however, the effect is modest and antigen-dependent. Received: 30 May 1997 / Accepted: 15 September 1997     

重组人IL-1ra在大肠杆菌中的可溶性表达

利用PCR技术从含有IL-1ra的质粒上扩增IL-1ra基因,经过序列测定后插入表达载体pTIG-Trx,并转化大肠杆菌BL21(DE3),用IPTG进行诱导表达。经SDS-PAGE分析显示,IL-1ra表达质粒在大肠杆菌中的诱导表达产物出现相对分子量大约为17000的一条新生蛋白质带,其大小与预期结果一致,经Western和ELISA分析,证明该带即为目的蛋白,SDS-PAGE显示目的蛋白全部以可溶性蛋白的形式存在。超声破碎后,上清经金属螯和层析纯化获得纯度约为98％的蛋白样品。     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

Association of interleukin-1B (IL-1B) gene polymorphisms with risk of gastric cancer in Chinese population

The incidence of gastric cancer (GC) in China is among the highest in the world. In present work, 154 patients with GC and 166 healthy controls in population of north-western China were investigated to evaluate the genetic associations of IL-1B gene single nucleotide polymorphisms (SNP) and variable number tandem repeat (VNTR) polymorphisms of IL-1RN gene with increased risk of GC. The frequency of IL-1B+3954C/T was significantly higher in GC cases group (25.97%) than that in controls (4.82%) with odds ratio (OR)=6.93 (95% confidence interval [CI] 3.13-15.36); the frequencies of IL-1B-31C/T, IL-1B-31C/C and IL-1B-511C/T genotypes were also higher in GC cases group (51.95%, 23.38% and 50.65%) than those in controls (46.99%, 19.88% and 42.77%) with OR=1.48 (95% CI 0.88-2.49), OR=1.58 (95% CI 0.84-2.95) and OR=1.39 (95% CI 0.80-2.41), respectively. The results show that these SNPs of IL-1B gene are associated with significantly increased risk of GC. This is the first report that IL-1B+3954C/T heterozygote is associated with greatly increased risk of GC. The results of this study did not support the report that IL-1RN*2+ genotypes were associated with increased risk of GC in Chinese population.     

Comparative RFLP mapping of the chlorotoluron resistance gene (Su1) in cultivated wheat (Triticum aestivum) and wild wheat (Triticum dicoccoides)

Chlorotoluron is a selective phenylurea herbicide widely used for broad-leaved and annual grass weed control in cereals. Variation in the response to chlorotoluron (CT) was found in both hexaploid bread wheat (Triticum aestivum L.) and wild tetraploid wheat (Triticum dicoccoides KöRN.). Here, we describe the comparative mapping of the CT resistance gene (Su1) on chromosome 6B in bread and wild wheat using RFLP markers. In bread wheat, mapping was based on 58 F₄ single-seed descent (SSD) plants of the cross between a genotype sensitive to chlorotoluron, ‘Chinese Spring’ (CS), and a resistant derivative, the single chromosome substitution line, CS (‘Cappele-Desprez’ 6B) [CS (CAP6B). In T dicoccoides, mapping was based on 37 F₂ plants obtained from the cross between the CT-susceptible accession B-7 and the resistant accession B-35. Nine RFLP probes spanning the centromere were chosen for mapping. In bread wheat Su1 was found to be linked to α-Amy-1 (9.84 cM) and Xpsr371 (5.2 cM), both on the long arm of 6B, and Nor2 (2.74 cM) on the short arm. In wild wheat the most probable linkage map was Nor2-Xpsr312-Su1-Pgk2, and the genetic distances between the genes were 24.8cM, 5.3cM, and 6.8cM, respectively. These results along with other published map data indicate that the linear order of the genes is similar to that found in T. aestivum. The results of this study also show that the Su1 gene for differential response to chlorotoluron has evolved prior to the domestication of cultivated wheat and not in response to the development and use of chemicals.     

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro

Summary.  Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab′)₂. We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1β inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab′)₂, or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20–30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1β. Received June 29, 2001 Accepted August 8, 2001 Published online August 9, 2002     

The interleukin-1 RN polymorphism and Helicobacter pylori infection in the development of duodenal ulcer

BACKGROUND: The host genetic factors that determine the clinical outcomes for Helicobacter pylori-infected individuals remain unclear. AIMS: To elucidate the relations among interleukin-1 locus polymorphisms, and H. pylori infection in the development of duodenal ulcers. MATERIALS AND METHODS: In a case-control study involving 168 control subjects and 147 patients with duodenal ulcer, biallelic polymorphisms of two interleukin-1 loci, IL-1B(-511) and IL-1B(+3954), as well as the penta-allelic variable number of tandem repeats of interleukin-1 receptor antagonist IL-1RN, were genotyped, and the H. pylori states of controls and patients were examined. RESULTS: Helicobacter pylori infection, male gender and the carriage of IL-1RN*2 independently increased the risk of duodenal ulcer with odds ratios of 6.4 (95% confidence interval, 3.7-11.0), 1.9 (95% confidence interval, 1.1-3.4) and 2.7 (95% confidence interval, 1.1-6.8), respectively. Statistical analysis revealed an interaction between IL-1RN*2 and H. pylori infection with the duodenal ulcer risk conferred by the H. pylori infection substantially increased (odds ratios, 22.6; 95% confidence interval, 5.9-86.5) by the carriage of IL-1RN*2. In addition, a synergistic interaction between IL-1RN*2 and blood group O existed. The combined risk of H. pylori infection, the carriage of IL-1RN*2 and blood group O for duodenal ulcer was 27.5 (95% confidence interval, 3.1-243.6). CONCLUSIONS: This work is the first to verify IL-1RN*2 as an independent factor that governs the development of duodenal ulcers. Our data indicate that H. pylori infection and IL-1RN*2 synergistically determine susceptibility to duodenal ulcer. The blood group phenotype is possibly a crucial determinant for the outcome of the impact of an interleukin-1 locus polymorphism on H. pylori-infected individuals.     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Functional analysis of the interleukin-5 receptor antagonist peptide, AF18748

AF18748 is a dimeric peptide of 2 x 19 amino acids that specifically binds to the alpha-chain of the human IL-5 receptor (IL-5R), preventing binding of IL-5 and acting as a receptor antagonist. However, the peptide acts by inducing alpha-chain dimerization, and we therefore set out to investigate whether this peptide would have any residual agonist activity. AF18748 was unable to induce activation of a number of signal transduction pathways, but was able to specifically block IL-5-dependent signal transduction. The peptide was unable to support the survival and proliferation of the cytokine dependent cell line, Ba/F3-IL-5R, and was unable to prime the respiratory burst, or induce adhesion, migration or survival of primary human eosinophils. In each case the AF18748 functioned as an antagonist. These data suggest that AF18748 may be useful to specifically modulate eosinophil function in vivo.     

Effects of β2–microglobulin mutations on the alpha-1 helical region of H2-Ld

 Beta-2 microglobulin (β₂m)has been shown to have an effect on the structural and functional constraints that facilitate proper class I antigen presentation. To date, no evidence has pinpointed the β₂m-specific amino acids that play an integral role in affecting structure in and around the peptide binding region of class I. To delineate β₂m amino acid positions that affect the alpha-1 helical region, we generated a series of mutant β₂m proteins bearing precise amino acid substitutions. The amino acid positions chosen were based upon previous results which demonstrated that human β₂m association with H2-L^d altered the structure of the alpha-1/alpha-2 super-domain. β₂m mutant proteins were used in β₂m exchange assays with cells expressing H2-L^d. Following exchange, cells were assayed to determine whether mutant β₂m association resulted in structural alteration of class I extracellular domains. The alteration in H2-L^d structure was evidenced by an increase in the binding of an antibody (34-1-2), specific for the alpha-1 helical region of H2-L^d. Results demonstrated that amino acid substitutions in β₂m positions 33 and 53 led to a dramatic increase in the reactivity of the alpha-1 domain-specific antibody 34-1-2. Identifying β₂m amino acid positions that influence the structure of the peptide binding region may allow for a better understanding of cellular immune responses that center upon class I/β₂m expression. Received: 18 December 1997 / Revised: 19 February 1998     

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996