Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons.     

Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils

The regulation of monocyte-derived neutrophil chemotactic factor (MDNCF)/interleukin 8 (IL 8) receptor expression by the MDNCF/IL 8 ligand was examined using freshly isolated human peripheral blood neutrophils. MDNCF/IL 8 down-regulated greater than 90% of its own receptor expression within 10 min at 37 degrees C. This down-regulation was associated with internalization of the ligand. The radiolabeled MDNCF/IL 8 molecules after internalization were proteolytically degraded, and trichloroacetic acid-soluble molecules were released into the culture medium starting at 60 min. Lysosomotropic agents could inhibit this degradation of ligand suggesting the involvement of lysosomal enzymes in this proteolytic digestion. MDNCF/IL 8 receptors reappeared on the cell surface within 10 min after removal of free ligands from the culture medium. Cycloheximide did not alter the reappearance of the receptor suggesting that de novo protein synthesis of MDNCF/Il 8 receptors is not involved in this event and that receptors probably recycled. The addition of lysosomotropic agents partially inhibited the reappearance/recycling of the receptors, although none of these agents inhibited the binding of ligand to the surface receptors or ligand internalization. Ammonium chloride reduced the MDNCF/IL 8-induced neutrophil chemotactic response in a dose-dependent fashion. These data suggest that MDNCF/IL 8 receptor expression is dynamically regulated by MDNCF/IL 8 and that the rapid recycling of MDNCF/IL 8 receptors may be essential for the chemotactic response of neutrophils.     

The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exosn and span about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activiation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank nucleotide sequence databases and have been assigned the accession number X86564 (exon 1), X86565 (exon 2), X86566 (exon 3 and 4), and X86567 (exon 5)     

Optimal techniques for the immunocytochemical demonstration of beta-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets

Summary The distribution of -thromboglobulin, platelet factor 4, and fibrinogen in unstimulated platelets was investigated by several immunocytochemical techniques. All three substances were found to be localized in the majority of platelet alpha granules either by immunoperoxidase methods on saponin-treated platelets or by colloidal gold immunoconjugates on frozen thin sections. The optimal conditions for preparing and fixing platelets for immunocytochemistry were also determined. Platelets obtained from blood dripped directly into fixative or anticoagulated blood were compared systematically with respect to shape. Temperature was found to be the most important variable. Immediately fixed platelets were generally disc-shaped, regardless of the temperature of the fixative. Reducing the temperature of blood (stored with anticoagulant) before fixation resulted in more swollen and fewer disc-shaped platelets. However, if the blood was mixed with an anticoagulant and maintained at 37° C for 1 h before fixation, the same number of disc-shaped platelets were present as in samples from blood fixed immediately. The intracellular localization of -thromboglobulin, platelet factor 4, and fibrinogen was consistent regardless of platelet preparatory procedure, but several technical problems were encountered with respect to plasma membrane labelling when control experiments were analysed. Immediately fixed, non-permeabilized platelet plasma membranes were always labelled, no matter which control substances or immunoperoxidase markers were used. However, when platelets were washed by centrifugation, the plasma membranes were negative. Exposure to saponin markedly diminished labelling of the plasma membranes. Optimal techniques for the immunocytochemical demonstration of these alpha granule proteins in platelets are presented in this report.This paper is based in part on theHistochemical Journal Lecture for 1983 given by Dr Bainton at a Symposium on Haematological Cytochemistry in Cambridge on 29 September 1983 at the invitation of the Royal Microscopical Society.     

Interleukin-8 gene polymorphism is associated with acute coronary syndrome in the Chinese Han population

Background

Acute coronary syndrome (ACS) is one of the most common forms of heart disease. Recent studies have shown that interleukin (IL)-8 plays a key role in the development of atherosclerotic plaques, but the relationship between the common genetic variants of IL-8 and ACS has not been extensively studied.

Methods

This case-control study in the Chinese Han population included 675 patients with ACS and 636 age- and sex-matched controls. We investigated IL-8 polymorphisms and their association with susceptibility to ACS. The investigation was replicated in the second study comprising 360 cases and 360 control subjects. The plasma concentration of IL-8 was measured by enzyme-linked immunosorbent assay.

Results

IL-8 −251 A/T polymorphism was associated with increased susceptibility to ACS (P = 0.004; odds ratio = 1.30; 95% confidence interval: 1.12–1.53). The second study yielded similar results. An increased IL-8 level was found in the plasma of acute myocardial infarction patients, suggesting that IL-8 −251 A/T may affect the expression of IL-8.

Conclusion

IL-8 −251 A/T polymorphism is associated with ACS risk in the Chinese Han population and the A allele of IL-8 −251 A/T may be an independent predictive factor for ACS.     

Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures

A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 g l^–1 after 10 d' cultivation.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli

Summary Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for th subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30°C) but do not grow at high temperature (42°C) due to the inability to synthesize factor. In one mutant studied in detail (rpoD40), the rate of -factor synthesis at 30°C is about half that of the wild type and is decreased to 10%–15% within 1 h of incubation at 42°C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD, supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42°C) exhibit different thermostabilities attributable to alterations in the factor. The reduced synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as amber fragment. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the factor and directly affects synthesis upon inactivation of the suppressor at high temperature.     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

The effects of recombinant interleukin-1 and recombinant tumor necrosis factor on non-specific resistance to infection

Natural and synthetic immunomodulators that increase non-specific resistance to infection induce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, we investigated the effect of IL-1 and of TNF on the survival of lethally-infected mice. Mice were injected with 1 × 10⁶ Klebsiella pneumoniae in the thigh muscle. When recombinant human IL-1 was given as a single i.p. injection 24 h before the infection, survival was increased. Using 80 ng IL-l per mouse, survival compared to control animals was 80% versus 20% 48 h after the infection (p < 0.001). No effect of IL-1 was observed when it was given &frac; h before or 6 h after the infection. IL-l proved to be at least as potent as IL-1.Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Protection against death by IL-1 was also investigated in granulocytopenic mice with aPseudomonas aeruginosa infection. Administration of the cyclooxygenase-inhibitor, ibuprofen, did not affect the beneficial effect of IL-1. In this model human recombinant TNF was at least tenfold less active than IL-1.Pretreatment with IL-1 also had a significant effect on survival of mice that received a high dose of bacterial lipopolysaccharide.     

The effect of methylmercury (CH3HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells based on its anti-aggregatory effect on human platelets

The effect of methylmercury (CH₃HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells (HUVECs) based on its antiaggregatory effect on human platelets was examined. HUVECs were harvested from umbilical veins by collagenase treatment. The platelet aggregation test was performed with cuvettes lined with HUVECs. Platelet aggregation induced by 0.05 units thrombin/ml was inhibited in the presence of HUVECs. This HUVEC-dependent anti-platelet aggregatory effect was enhanced by the addition of bradykinin (10 nmol/L), which stimulates the production of EDRF. Indomethacin (IND, 1 mol/L) reduced the HUVEC-dependent anti-platelet aggregatory effect. The effect ofN ^G-monomethyl-L-arginine (L-NMMA, 100 mol/L), an inhibitor of nitric oxide synthase (NOS) in endothelial cells, on HUVECs pretreated with IND showed almost complete platelet aggregation similar to results without HUVECs. The anti-platelet aggregatory effect of HUVECs pretreated with IND seemed to depend mainly on EDRF. Methylmercury (MeHg) (20–50 mol/L) induced dose-dependent platelet aggregation in cuvettes, without HUVECs. Methylmercury (30 mol/L) induced less platelet aggregation in the presence of HUVECs than in their absence. The degree of inhibitory effect by HUVECs on MeHg-induced platelet aggregation was reduced dose-dependently (30–50 mol/L MeHg). Methylmercury-induced platelet aggregation at 50 mol/L MeHg with or without HUVECs was similar. These findings suggest that this simple new experimental system is useful for assessing the production of EDRF by HUVECs, and show that MeHg inhibits the production of EDRF by HUVECs, which may be involved in the etiology of cardiovascular diseases such as hypertension and arteriosclerosis.Abbreviations BK bradykinin - EDRF endothelium-derived relaxing factor - HUVECs human umbilical vascular endothelial cells - IND indomethacin - L-NMMA N ^G-monomethyl-L-arginine - MeHg methylmercury     

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Assignment of the human tissue-type plasminogen activator gene (PLAT) to chromosome 8

Summary Using 1.2kb 3-terminal Pst-I fragment of a full length tissue-type plasminogen activator (t-PA) cDNA clone (ptPA-8FL) and a set of rodent human somatic cell hybrids, the corresponding human gene PLAT was localized on chromosome 8.This work was submitted as an abstract entitled: Provisional assignment of human tissue-type plasminogen activator (PLAT) to chromosome 8, by R. Visse, G. T. G. Chang, J. Th. Wijnen, J. H. Verheijen, C. Kluft, and P. Meera Khan, for a poster presentation at the 8th International Conference on Human Gene Mapping, Helsinki, August 4–10, 1985     

Cloning and nucleotide sequence of the gene for an archaebacterial protein synthesis elongation factor Tu

Summary A restriction fragment enrichment procedure was devised for the identification and cloning of the gene for protein synthesis elongation factor Tu (EF-Tu) from Methanococcus vannielii, employing hybridisation with an internal tufB gene probe from Escherichia coli. Methanococcus contains a single tuf gene on its chromosome; it is expressed in E. coli and it codes for a polypeptide of 46.5 kDa. The overall architecture of the protein bears a striking resemblance to that of eukaryotic elongation factor 1 (EF-1). The close similarity to EF-1 is supported by the sequence homology values which are in the range of 34% to 35% with eubacterial, plastid and mitochondrial EF-Tu sequences and as high as 52% to 54% with those from eukaryotic EF-1.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.     

Anti-(tumor necrosis factor) alters the response of human monocytes to liposomal muramyl tripeptide

The purpose of this study was to examine the mechanisms by which liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) stimulates monocytes to produce tumor necrosis factor (TNF) and interleukin-1 (IL-1). We have previously shown that secretion of TNF protein occurred 2–4 h following incubation of monocytes with L-MTP-PE and that this stimulation of TNF production was associated with an increase in TNF mRNA. Increased intracellular interleukin-1 (IL-1) and IL-1 were not detected until 8 h after exposure to L-MTP-PE. To determine whether TNF played a role in the stimulation of IL-1 production by L-MTP-PE, normal human monocytes were incubated with L-MTP-PE or medium in the presence or absence of anti-TNF or anti IL-1 plus anti IL-1. Enhanced expression of IL-1 and IL-1 mRNA was inhibited at 4 h but not 24 h when monocytes were incubated with L-MTP-PE plus anti-TNF compared with L-MTP-PE alone. By contrast, enhanced expression of TNF mRNA wasnot inhibited at any time when monocytes were incubated with L-MTP-PE and anti-IL-1 plus anti-IL-1. These data indicate that the up-regulation of IL-1 seen in monocytes following L-MTP-PE exposure may be due in part to the production of TNF. The up-regulation of TNF, however, appears to be independent of IL-1 production.     

The murine leukemia inhibition factor gene (Lif) is located on proximal Chromosome 11, not Chromosome 13

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding human interleukin in DA cells (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing. To address this issue, blot hybridization methods have been used to establish the position of the structural gene sequence unambiguously. DNAs from somatic cell hybrids, recombinant inbred mice, and backcross mice have been probed with a sequence that encodes LIF/HILDA. The results support the assignment of this sequence to proximal Chr 11. These studies also establish a synteny group, including Lif and Tcn-2, the structural gene for transcobalamin 2, that is conserved between man and mouse.     

The T-cell receptor in primates: identifying and sequencing new owl monkey TRBV gene sub-groups

The New World primate Aotus nancymaae (owl monkey) has been shown to be an excellent experimental model when studying malarial parasites. Characterising the T-cell receptor (TR) repertoire by means of the different variable beta (TRBV) genes displayed contributes to a better understanding of these lymphocytes role in the response against several malarial antigens. This study describes identifying and characterising eleven new TRBV gene sub-groups in cDNA from Aotus nancymaaes peripheral blood lymphocytes; these 11 gene sequences displayed homology to the previously reported human TRBV3, TRBV10, TRBV11, TRBV14, TRBV18, TRBV19, TRBV20, TRBV25, TRBV27, TRBV29 and TRBV30 sub-groups, resulting in 83% overall homology at the amino acid level. An additional Aotus sequence was found having similarity with the human TRBJ-2–7*01 gene. Evolutionary relationships amongst these sequences and the homologous genes from both New and Old World primates have shown that the TRBV repertoire has been maintained in the species being studied, displaying varying association patterns and substitution rates, depending on the sub-group being studied. The degree of identity observed when comparing human and Aotus genes suggests that these species might have a similar TRBV repertoire.     

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression

Summary The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from –982 to –634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 F1 gene and of several highly expressed plant genes.