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The peptide TAT-I24, composed of the 9-mer peptide I24 and the TAT (48-60) peptide, exerts broad-spectrum antiviral activity against several DNA viruses. The current model of the mode of action suggests a reduction of viral entry and also a possible interaction with the viral DNA upon virus entry. To further support this model, the present study investigates the DNA binding properties of TAT-I24. DNA binding was analysed by gel retardation of a peptide-complexed DNA, fluorescence reduction of DNA labelled with intercalating dyes and determination of binding kinetics by surface plasmon resonance. Molecular dynamics simulations of DNA-peptide complexes predict high-affinity binding and destabilization of the DNA by TAT-I24. The effect on viral DNA levels of infected cells were studied by real-time PCR and staining of viral DNA by bromodeoxyuridine. TAT-I24 binds double-stranded DNA with high affinity, leading to inhibition of polymerase binding and thereby blocking of de novo nucleic acid synthesis. Analysis of early steps of virus entry using a bromodeoxyuridine-labelled virus as well as quantification of viral genomes in the cells indicate direct binding of the peptide to the viral DNA. Saturation of the peptide with exogenous DNA can fully neutralize the inhibitory effect. The antiviral activity of TAT-I24 is linked to its ability to bind DNA with high affinity. This mechanism could be the basis for the development of novel antiviral agents.

The activation of dendritic cells is marked by changes both on their cell surfaces and in their functions. We define EWI-2/CD316 as an early activation marker of dendritic cells upregulated by Toll-like receptor ligands clearly before CD86 and CD83. By expression cloning, human heat shock protein A8 (HSPA8), a member of the hsp70 family, was identified as the ligand for EWI-2. Soluble EWI-2 bound both to cells expressing HSPA8 and also to immobilized HSPA8 protein. Although heat shock proteins are evolutionarily well conserved, other members of this class, including human hsp60 and mycobacterial hsp65, did not bind to EWI-2. The ligation of EWI-2 enhanced the CCL21/SLC-dependent migration of activated mature dendritic cells but attenuated their antigen-specific stimulatory capacities. Important functions of recently activated dendritic cells are thus critically modulated by the newly discovered HSPA8-EWI-2 interaction.

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possiblein vivorole of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention1,2,3. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61β. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

The effect of orphan nuclear receptor Nur77 overexpression on activation of an interleukin-2 (IL-2) promoter-luciferase construct was analyzed in the human leukemic cell line Jurkat. Cotransfection of the IL-2 promoter construct together with the Nur77 expression plasmid resulted in a significant repression of IL-2 promoter activation compared to control cells. The repression by Nur77 requires the N-terminal activation function-1 domain. The repressive effect of Nur77 on IL-2 promoter activation is mediated through inhibition of the transcription factor complex nuclear factor-kappaB (NF-kappaB), since blocking or alteration of the IL-2 NF-kappaB binding sites resulted in abrogation of the repressive effect of Nur77. Moreover, further examination of a reporter gene construct containing multiple copies of the IL-2 CD28 response element (CD28RE) showed that Nur77 can inhibit transactivation mediated by the NF-kappaB components p65 and c-Rel. However, no effect of Nur77 was seen on p65-mediated transactivation of a construct containing multiple NF-kappaB binding sites of the HIV LTR. Our data suggest that Nur77 is able to block activation through NF-kappaB when bound to low-affinity NF-kappaB binding sites, such as those located in the IL-2 promoter.

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.

Plasminogen activator inhibitor 1 (PAI-1) is the main fibrinolysis inhibitor, and high plasma levels are associated with an increased risk for vascular diseases. Inflammatory cytokines regulate PAI-1 through a hitherto unclear mechanism. Using reporter gene analysis, we could identify a region in the PAI-1 promoter that contributes to basal expression as well as to tumor necrosis factor α (TNFα) induction of PAI-1 in endothelial cells. Using this region as bait in a genetic screen, we could identify Nur77 (NAK-1, TR3, NR4A1) as an inducible DNA-binding protein that binds specifically to the PAI-1 promoter. Nur77 drives transcription of PAI-1 through direct binding to an NGFI-B responsive element (NBRE), indicating monomeric binding and a ligand-independent mechanism. Nur77, itself, is transcriptionally up-regulated by TNFα. High expression levels of Nur77 and its colocalization with PAI-1 in atherosclerotic tissues indicate that the described mechanism for PAI-1 regulation may also be operative in vivo.

The CXC chemokine interleukin-8 (IL-8/CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the β2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3–5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing thatin vivoactivated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires thein vivoactivation of circulating PMNs.

The 5'-flanking sequences of the human macrophage inflammatory protein-3alpha/CCL20 gene were cloned and transfected into G-361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor-alpha (TNF-alpha) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF-alpha inducibility is conferred by a region between nt -111 and -77, which contains a non-standard nuclear factor-kappaB (NF-kappaB) binding site. The requirement for NF-kappaB was demonstrated as follows: (i) mutations in this NF-kappaB site abrogated TNF-alpha responsiveness; (ii) TNF-alpha activated a construct containing two copies of the CCL20 NF-kappaB binding site; (iii) overexpression of NF-kappaB p65 activated the CCL20 promoter; (iv) NF-kappaB from nuclear extracts of TNF-alpha-stimulated cells bound specifically to this NF-kappaB site.

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(OH)-24,25,26,27-tetranor-23-COOH-D(3); calcitroic acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1alpha,25(OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(OH)(2)D(3) or the metabolites 1alpha,24R,25(OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,23S,25(OH)(3)D(3) resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed even for calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(OH)(2)D(3) from VDR than the parent hormone. Thus, we report that several natural metabolites of 1alpha,25(OH)(2)D(3) retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.

BACKGROUND: Strains of Helicobacter pylori carrying the virulence associated cag pathogenicity island (PAI) induce gastric epithelial synthesis of the chemokine interleukin-8 (IL-8), a neutrophil chemoattractant, and thereby a strong inflammatory response during chronic infection of the human gastric mucosa. Previous mutational analyses have shown that many genes in the cag PAI are needed to elicit IL-8 synthesis in gastric epithelial cells, and also that some genes are not involved. AIM: To test the possibility that certain genes in the cag PAI also downregulate (modulate) the inflammatory response elicited by cag+ H pylori infection. METHODS: Cells of L5F11, a derivative of the Kato-3 gastric epithelial cell line that carries an engineered IL-8 promoter-luciferase reporter gene fusion, were cocultured with H pylori strain 26695 or with an isogenic mutant in which most of the cag PAI ORF 10 gene, an Agrobacterium virD4 homologue, was deleted. Luciferase activity was measured to assess IL-8 gene transcription and secreted IL-8 was measured by enzyme linked immunosorbent assay to assess synthesis and release of IL-8 protein from gastric epithelial cells. RESULTS: Inactivation of ORF10 led to a 2.8-fold increase in IL-8 gene transcription and a 3.6-fold increase in IL-8 synthesis and secretion. CONCLUSIONS: The results suggest that this VirD4 homologue participates in the control of inflammation that H pylori infection elicits by downregulating (modulating) the strong induction of IL-8 synthesis mediated by other cag encoded proteins.

The chemokine interleukin-8 (IL-8) is frequently associated with inflammatory diseases, and autoantibodies against IL-8 are present in the periphery at elevated levels in such conditions as rheumatoid arthritis (RA). Circulating free anti-IL-8 IgG autoantibodies correlate with inflammatory parameters and disease severity in RA. In this study, correlations were sought between these disease parameters and other antibody subclasses. We assayed IgM, IgA and IgG anti-IL-8 antibodies and IL-8 immunoglobulin immune complexes in the serum of 29 healthy controls and 56 patients with defined RA, and compared the results with clinical and humoral disease parameters. IgG and IgM antibodies directed against IL-8 were present in all samples. In the disease groups, all isotypes of free anti-IL-8 antibodies correlated with increasing humoral disease parameters like CRP and CIC and their related anti-IL-8 immune complexes. Samples which contained high titers of anti-IL-8 antibody subclasses and complexes were RF subclass-positive, while IgM RF-negative sera showed low levels of anti-IL-8 and complexes. Detectable levels of IgG and IgA RF were found in all sera. Patients with extra-articular organ manifestation showed significantly increased free IgA and IgA/IL-8 complexes, with no correlation to the IgA RF titer or IgA hypergamma-globulinemia. The highest titers were seen in two RA cases with vasculitis and in one patient with colitis. Polyclonal activation of the humoral antibody system, which normally precedes the resolution of an inflammatory response, can itself lead to secondary stimulation of inflammatory processes via immune complex formation. In the immune pathology of RA, it degenerates into a persistent chronic inflammation accompanied by progressive joint destruction. The presence of elevated IgA subclass anti-IL-8 autoantibodies in RA patients with extra-articular manifestations suggests these autoantibodies as a clinically useful marker of disease severity and extra-articular manifestations.

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.

A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N-terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1.

We have studied the relationship between interleukin-8 (IL-8) and interleukin-1 alpha (IL-1 alpha) release after stimulation with all-trans retinoic acid (ATRA) and tumor necrosis factor-alpha (TNF-alpha) in the human epithelial ovarian cancer cell line HOC-7. Both IL-1 alpha and IL-8 protein release were enhanced by treatment with ATRA and TNF-alpha after 48 h exposure. Blocking of IL-1 alpha activity in HOC-7 cells with either IL-1 receptor antagonist (IL-1ra) or a neutralizing antibody directed against IL-1 alpha resulted in a dose-dependent decrease of IL-8 release by ATRA, TNF-alpha and IL-1 alpha treated HOC-7 cells. Expression of IL-8 mRNA was enhanced by the individual stimuli, whereas co-treatment with IL-1ra resulted in a loss of IL-8 specific transcripts, except in TNF-alpha treated cells. Inhibition of de novo protein synthesis by cycloheximide (CHX) and simultaneous blocking of IL-1 alpha activity by IL-1ra for 24 h revealed that ATRA controls IL-8 gene expression transcriptionally and that the extent of IL-8 protein release can be markedly influenced by cellular expressed IL-1 alpha.

Interleukin-8 (IL-8) belongs to a family of chemoattractant cytokines involved in chemotaxis and activation of neutrophils. As in vivo administration of IL-8 induces granulocytosis and the release of immature white blood cells into the circulation, we assessed a possible mobilizing effect of IL-8 on myeloid progenitor cells. IL-8 was administered at intraperitoneal doses ranging from 0.1 to 100 micrograms per mouse to female Balb/C mice (aged 8 to 12 weeks; weight, 20 to 25 g). Animals were killed at time intervals ranging from 1 to 240 minutes after IL-8 administration, and blood, bone marrow, and spleen cells were harvested. Injection of 30 micrograms IL-8 resulted in an increment from 25 +/- 9 to 418 +/- 299 granulocyte-macrophage colony-forming units (CFU-GM) per milliliter blood at 15 minutes after a single intraperitoneal injection. Sixty minutes after the injection of IL-8, the numbers of circulating CFU-GM per milliliter blood had almost returned to pretreatment values (82 +/- 39 CFU-GM per milliliter). A dose of 100 micrograms IL-8 per animal did not result in a further increment in the number of circulating CFU-GM. Transplantation of 5 x 10(5) blood-derived mononuclear cells (MNC) obtained at 30 minutes after IL-8 injection (30 micrograms) resulted in 69% survival of lethally irradiated (8.5 Gy) recipients at 60 days versus 22% for animals transplanted with an equal number of nonprimed blood-derived MNC. Transplantation of 1.5 x 10(6) MNC obtained from IL-8-treated donors resulted in 100% survival. Six months after transplantation, female recipients of MNC derived from IL-8-treated male donors were killed, and chimerism was determined in bone marrow, spleen, and thymus using a Y chromosome-specific probe and fluorescent in situ hybridization (FISH). The majority of bone marrow, spleen, and thymus cells (83% +/- 25%, 89% +/- 5%, and 64 +/- 28%, respectively) consisted of Y chromosome-positive cells, showing that the IL-8-mobilized cells had myelolymphoid repopulating ability. We conclude that IL-8 is a cytokine that induces rapid mobilization of progenitor cells and pluripotent stem cells that are able to rescue lethally irradiated mice and that are able to completely and permanently repopulate host hematopoietic tissues.

AIMS--To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS--Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS--Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS--These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration.

The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 Å, b = 39.5 Å and c = 95.3 Å. A packing density of 3.8 Å3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 Å on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.

Acute alcoholic hepatitis is characterized by a unique degree of liver neutrophil infiltration, often accompanied by marked peripheral neutrophilia in the absence of demonstrable bacterial or fungal infection. In this study we assayed plasma and tissue levels of a potent neutrophil activator and chemotaxin, interleukin-8, in patients with a spectrum of alcoholic liver diseases and in normal and diseased control subjects. Levels of circulating interleukin-8 were undetectable in normal subjects but highly elevated in patients with alcoholic hepatitis, particularly in those who died (geometric mean = 600 ng/L; confidence interval = 323 to 1,120 vs. geometric mean = 184 ng/L; confidence interval = 114 to 309 in survivors). Levels correlated with biochemical indicators of severe disease (bilirubin: R = 0.38; international prothrombin ratio: R = 0.28; white blood cell count: R = 0.35; creatinine: R = 0.34) and with tumor necrosis factor-alpha (R = 0.43) and soluble tumor necrosis factor receptors (p55; R = 0.59). In contrast, moderate elevations in the levels of circulating interleukin-8 were seen in alcoholic cirrhosis (geometric mean = 93 ng/L; confidence interval = 40 to 213) and in alcoholic patients undergoing alcohol withdrawal (geometric mean = 137 ng/L; confidence interval = 72 to 259). Levels in nonalcoholic inflammatory liver disease were comparatively low (geometric mean = 17 ng/L; confidence interval = 10 to 29).(ABSTRACT TRUNCATED AT 250 WORDS)

The neutrophil is the leukocyte most frequently recruited into the amniotic fluid in cases of microbial invasion of the amniotic cavity. Neutrophil attractant/activating peptide-1 /interleukin-8 is a newly identified cytokine that is capable of inducing selective neutrophil chemotaxis and activation. The purpose of this study was to examine the relationship between amniotic fluid concentrations of neutrophil attractant/activating peptide-1 /interleukin-8, microbial invasion of the amniotic cavity, and parturition (term and preterm). Amniotic fluid neutrophil attractant/activating peptide-1 /interleukin-8 was measured with an immunoassay validated for human amniotic fluid (sensitivity, 0.3 ng/ml). Fluid was obtained from women in the following groups: midtrimester ( n = 38), term not in labor ( n = 38), term in active labor ( n = 67), and preterm labor with intact membranes ( n = 62). Fluid was cultured for aerobic and anaerobic bacteria and Mycoplasma . Sterile amniotic fluid from most women in the midtrimester of pregnancy and women at term not in labor did not contain immunoreactive neutrophil attractant/activating peptide-1 /interleukin-8. Microbial invasion of the amniotic cavity was associated with increased concentrations of neutrophil attractant/activating peptide-1 /interleukin-8. The amniotic fluid of women with preterm labor and sterile amniotic fluid who had preterm delivery contained higher neutrophil attractant/activating peptide-1 /interleukin-8 levels than did the amniotic fluid of women who responded to tocolysis and had delivery at term. Term parturition is associated with increased concentrations of neutrophil attractant/activating peptide-1 /interleukin-8 in the amniotic fluid. We conclude that neutrophil attractant/activating peptide-1 /interleukin-8 is part of the host response to microbial invasion of the amniotic cavity and that increased amniotic fluid availability of this cytokine occurs in term and preterm parturition.

The present study was designed to investigate the capacity of human vascular smooth muscle cells (SMCs) to produce a cytokine chemotactic for monocytes (monocyte chemotactic protein [MCP]) and by way of comparison, a related polypeptide activator of neutrophils (known as interleukin-8 [IL-8] or neutrophil activating protein-1 [NAP-1]. On exposure to IL-1, SMCs released high levels of chemotactic activity for monocytes, which could be removed by absorption with anti-MCP antibodies. MCP production by activated SMCs was comparable to that of IL-1-stimulated umbilical vein endothelial cells. Activated SMCs released appreciable levels of IL-8, as determined by a specific enzyme-linked immunosorbent assay, but little chemotactic activity for neutrophils. IL-1-treated SMCs expressed high levels of both MCP and IL-8 mRNA transcripts, as assessed by Northern blot analysis. Tumor necrosis factor and bacterial lipopolysaccharide but not IL-6 also induced MCP and IL-8 gene expression in SMCs. Nuclear runoff analysis revealed that IL-1 augmented transcription of the MCP and IL-8 genes. The capacity of SMCs to produce a cytokine (MCP) that recruits and activates circulating mononuclear phagocytes may be of considerable importance in the pathogenesis of vascular diseases (e.g., vasculitis and atherosclerosis) that are characterized by monocyte infiltration of the vessel wall.

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 ± 61 vs 290 ± 64, P < .05; G-CSF: 1925 ± 1216 vs 3371 ± 1214, P < .05; FL: 562 ± 213 vs 1068 ± 528, P < .05). Treatment with ciprofloxacin eliminated only aerobic Gramnegative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 ± 66 vs 538 ± 216, P < .05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 ± 182, IL-8 germ free: 157 ± 53, P < .05). Finally, we performed reconstitution experiments adding Escherichia coli–derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8–induced mobilization (67 ± 28 vs 190 ± 98.1, P

Previously, we demonstrated that IL-8 induces rapid mobilization of hematopoietic progenitor cells (HPC) from the bone marrow of rhesus monkeys. Because activation of neutrophils by IL-8 induces the release of gelatinase B (MMP-9), which is involved in the degradation of extracellular matrix molecules, we hypothesized that MMP-9 release might induce stem cell mobilization by cleaving matrix molecules to which stem cells are attached. Rhesus monkeys were treated with a single i.v. injection of 0.1 mg/kg human IL-8, which resulted in a 10- to 100-fold increase in HPC within 30 min after injection. Zymographic analysis revealed a dramatic instantaneous increase in the plasma levels of MMP-9, followed by the increase in circulating HPC. Enzyme levels decreased at 2 h after injection of IL-8, simultaneously with the decrease in the numbers of circulating HPC. To test the hypothesis that MMP-9 induction was involved in HPC mobilization, rhesus monkeys were treated with a highly specific inhibitory monoclonal anti-gelatinase B antibody. Anti-gelatinase B at a dose of 1–2 mg/kg completely prevented the IL-8-induced mobilization of HPC, whereas a dose of 0.1 mg/kg had only a limited effect. Preinjection of inhibitory antibodies did not preclude the IL-8-induced production and secretion of MMP-9. Pretreatment with an irrelevant control antibody did not affect IL-8-induced mobilization, showing that the inhibition by the anti-gelatinase B antibody was specific. In summary, IL-8 induces the rapid systemic release of MMP-9 with concurrent mobilization of HPC that is prevented by pretreatment with an inhibitory anti-gelatinase B antibody, indicating that MMP-9 is involved as a mediator of the IL-8-induced mobilization of HPC.

Helicobacter pylori strains that contain the cag pathogenicity island (PAI) elicit increased synthesis of gastric C-X-C chemokines, promote neutrophilic infiltration into the gastric epithelium, and stimulate the synthesis of interleukin-8 (IL-8) in cultured gastric epithelial cells. To investigate the effects of cag PAI genes on the transcription of the IL-8 gene, the Kato-3 gastric epithelial cell line was stably transfected with plasmid DNA containing the IL-8 gene promoter fused to a luciferase reporter gene. The resulting reporter cell line, L5F11, was used to monitor the effects of infection in cell culture by H. pylori 26695 and isogenic derivatives with null mutations in genes in the cag PAI on transcription of the IL-8 gene. We found that null mutations in eight open reading frames, including homologs of the Agrobacterium virB9 , virB10 , and virB11 genes, in the left half of the cag PAI abrogated the induction of IL-8 gene transcription. Further studies with the L5F11 cell line showed that IL-8 gene transcription induced by H. pylori was blocked by the protein tyrosine kinase inhibitor herbimycin A but not by the protein kinase C inhibitor calphostin C or by the protein kinase G inhibitor KT5823. IL-8 gene transcription in L5F11 cells could also be induced by the cytokine tumor necrosis factor alpha (TNF-α) without exposure to H. pylori . This TNF-α-induced IL-8 transcription was inhibited by the protein kinase A inhibitor H7, which had no significant effect on H. pylori -induced IL-8 transcription. These studies show that multiple genes in the left half of the cag PAI are essential for the transcription of the IL-8 gene in gastric epithelial cells and that this depends on protein tyrosine kinase activation.

Nitric oxide (NO) is able to regulate the expression of a number of inflammatory mediators. In this study, the effect of NO on the expression of the chemokine interleukin-8 (IL-8) by primary human keratinocytes and the lines KB and HaCaT was examined. Incubation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) for 24 hr increased IL-8 protein only in HaCaT cells, partly due to the presence of constitutive interleukin-1 (IL-1). However, in combination with IL-1beta, SNAP enhanced both IL-8 mRNA and protein in all three cell types. Transfection of cells with an IL-8 promoter reporter gene construct showed that the effect of NO was at least partly due to transcriptional activation. Despite small variations in the response to NO by the three cell types, these results demonstrate that NO can up-regulate IL-1beta-stimulated IL-8 expression in human keratinocytes. This study provides a regulatory mechanism which may be important in the context of skin inflammation, and supports the role of NO as an inflammatory mediator in the skin.

Helicobacter pylori is a spiral Gram-negative bacterium that chronically infects the gastroduodenal mucosal surface of humans (Fig. 1). Since the initial successful culture of H. pylori in the early 1980s, and the appreciation that this bacterium is the causative agent of chronic gastritis (74, 111),the major role this organism plays in gastroduodenal disease has become apparent. Although many infected subjects remain asymptomatic, the very strong association with peptic ulcer disease was quickly established, and bacterial eradication markedly reduces the incidence of ulcer recurrence (18). Subsequent studies detailed an association between H. pylori infection and gastric mucosa-associated lymphoid tissue-type lymphoma (6, 56, 84, 115) and gastric adenocarcinoma (4, 25, 50, 83, 86, 106).

Regulation of interleukin-8 (IL-8) gene transcription occurs mainly through the sequences -94 to -71 of the 5'-flanking region of the IL-8 gene, involving the transcription factors nuclear factor for interleukin-6 (NF-IL-6) and nuclear factor kappaB (NF-kappaB). The human melanoma cell line A3 was derived from G-361 cells by stable transfection with an IL-8 promoter-luciferase construct containing these sequences. 1alpha,25-Dihydroxyvitamin D3 (calcitriol) repressed IL-8 promoter activity induced by tumor necrosis factor-alpha (TNF-alpha) by 50%, compared to 30% inhibition using dexamethasone, an effect consistent with its effect on TNF-alpha-induced IL-8 release and IL-8 mRNA levels. A variety of vitamin D metabolites caused the same repressive effect on IL-8 promoter activation as calcitriol. However, only those metabolites which were able to transactivate a classical vitamin D response element had the ability to repress IL-8 promoter activation, suggesting that this repression is mediated via vitamin D receptor (VDR). Furthermore, overexpression of VDR in the parental G-361 cell line enhanced the repressive effect of calcitriol on activation of the IL-8 promoter by either TNF-alpha stimulation or overexpression of the NF-kappaB subunit p65. Electrophoretic mobility shift assays using nuclear extracts from A3 cells showed that calcitriol decreased the abundance of nuclear factors bound to the NF-kappaB binding site of the IL-8 promoter and this reduced binding of NF-kappaB proteins presumably contributes to its inhibitory action.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with distinct hematopoietic activities. In vivo treatment of mice with recombinant murine LIF induces thrombocytosis and increases the number of hematopoietic progenitor cells (HPCs) in spleen and bone marrow (BM). In this study, we applied LIF to expand HPCs in vivo prior to syngeneic BM transplantation. BALB/c donor mice were treated with recombinant human LIF at a dose of 2.5 microg/day s.c. for seven days. This resulted in a 1.6-fold increment in platelet counts from 941 to 1,470 x 10(9)/l (mean, n = 20). Mean spleen weight increased from 120 mg to 160 mg (n = 5). The total numbers of HPCs in the spleen as well as in the BM, as assessed in a CFU-GM (colony forming unit-granulocyte-macrophage) assay, were significantly higher in LIF-treated donors than in saline-treated controls (30.1 +/- 14.5 versus 7.4 +/- 5.3 x 10(3) per spleen; mean +/- SD, n = 22,p < 0.001 and 74.4 +/- 17.1 versus 55.3 +/- 16.1 x 10(3) per femur, p < 0.001). Recipient mice were lethally (8.5 Gy) irradiated and transplanted with 3 x 10(5) BM cells derived from LIF- or saline-treated donors. Hematopoietic reconstitution was monitored by tail bleeding at three-day intervals. Platelet and WBC nadir counts in control animals were reached at day 9 (31 +/- 25 x 10(9)/l for platelets and 0.40 +/- 0.10 x 10(9)/l for WBC; mean +/- SD, n = 29 per treatment group); in animals transplanted with LIF-treated BM cells, these counts were 44 +/- 25 x 10(9)/l for platelets, p < 0.05 and 0.60 +/- 0.38 x 10(9)/l for WBC, p < 0.01. In addition, platelet reconstitution was faster in recipients of LIF-treated BM cells (226 +/- 118 versus 126 +/- 62 x 10(9)/l at day 12 and 633 +/- 174 versus 434 +/- 180 x 10(9)/l at day 15, p < 0.001). Similarly, the reconstitution of WBC was also significantly enhanced. The radioprotection rate of lethally irradiated recipients with increasing cell doses of BM cells derived from LIF-treated donors was higher at all cell doses tested then of control animals, but did not reach statistical significance. These results show that in vivo treatment with LIF expands the number of committed progenitor cells and BM repopulating cells that accelerate short-term hematopoietic reconstitution without increasing radioprotection. Our data do not support a major role for LIF as a single factor inducing expansion of hematopoietic stem cells in vivo.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.

We investigated the role of nitric oxide (NO) in the expression of interleukin-8 (IL-8) in the human melanoma cell line, G361. Three NO donors, 3-morpholinosydnonimine hydrochloride (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP). and S-nitroso-L-glutathione (SNOG), all caused an increase in both IL-8 protein secretion and promoter activity. Truncation of the promoter showed that 101 bp of the 5′ flanking region proximal to the transcription start site are sufficient for the response to NO. Furthermore, mutation of the NF-κB and NF-IL-6 binding sites led to a significant decrease in NO-stimulated promoter activity. The nitric oxide synthase inhibitor, NG-amino-L-homoarginine (NAHA), inhibited TNF-α-stimulated IL-8-promoter activity by 60%. Addition of excess L- but not D-arginine partially reversed the NAHA-mediated inhibition, These results demonstrate that NO is an endogenous regulator of IL-8 production in G361 melanoma cells.

AIMS--To investigate the expression of interleukin-8 (IL-8) in Helicobacter pylori infected normal and neoplastic gastroduodenal mucosa, and in established gastric cancer cell lines. METHODS--Immunofluorescence techniques were used to localise IL-8 in cryosections of gastric (n = 25) and duodenal (n = 17) endoscopic biopsy specimens an in resected gastric tumour tissue samples from 16 patients. Two gastric cancer cell lines (Kato 3 and MKN 45) were examined for IL-8 protein expression by immunofluorescence and for the presence of IL-8 mRNA by reverse transcription followed by the polymerase chain reaction (RT-PCR). RESULTS--IL-8 was localised to the epithelium in histologically normal gastric mucosa, with particularly strong expression in the surface cells. IL-8 expression was also a feature of surface epithelium in the duodenal bulb, but was much reduced in the second part of the duodenum. In chronic H pylori-associated gastritis gastritis gastric epithelial IL-8 expression was increased and expression of IL-8 within the lamina propria was evident. By contrast, large areas of IL-8 negative epithelium were observed in the body mucosa of a subject with Menetrier's disease. In gastric carcinoma the tumour cells were positive for IL-8. IL-8 was also detected by immunofluorescence in unstimulated Kato 3 and MKN 45 cells, and constitutive IL-8 gene expression in these cell lines was confirmed by detection of IL-8 mRNA by RT-PCR. CONCLUSIONS--Immunoreactive IL-8, a potent neutrophil chemotactic and activating factor, is present in the epithelium of both normal and inflamed gastric mucosa with increased expression in the latter. There is site dependent variation in epithelial IL-8 expression within the gastroduodenal mucosa. The expression of the pro-inflammatory cytokine IL-8 in gastric carcinoma cells may influence peritumoural cellular infiltrates.

After its identification in 1987 as a novel neutrophil-activating cytokine, interleukin-8 (IL-8) was the center of interest of a large variety of interdisciplinary scientists for study of its action in vitro and in vivo (1,2,3,4). Soon it became clear that interleukin-8 plays a critical role in a series of diseases which are all characterized by strong neutrophil involvement. Rheumatoid artrithis, psoriasis, polytrauma, septicemia, adult respiratory distress syndrome (ARDS), asthma, emphysema, myocardial infarction, nephritis, inflammatory bowel disease, and gout are diseases which affect millions of people, and IL-8 is implicated in all these disease states. Therefore, inhibition of interleukin-8 itself, or blocking of the interaction with its seven transmembrane domain cellular receptor represents an attractive and logical approach for treatment of these inflammatory states. Possibilities for interference include neutralising antibodies, small molecules binding to the chemokine at critical positions, (e.g. domains involved in dimerization, heparin binding or receptor binding) or molecules which bind to the receptor. An additional approach would be the use of peptides based on the IL-8 structure or mutant IL-8 molecules to act as antagonists. As an alternative to screening for inhibitors in a series of biological assays, a rational way of finding an inhibitory principle involves the elucidation of structure/function relationships. This is usually done by production of mutant proteins of the target molecule and subsequent determination of biological activities. As only the primary structures of the mutants are known this might be called a “one-dimensional structure activity relationship”. We are attempting to go one step further and actually determine the three-dimensional structure of mutant IL-8 derivatives with interesting and clear changes in biological activities and, based on structure/function relationships in three dimensions, get a more detailed insight into structural requirements for specific biological activities.

Cytokine-activated human mesangial cells generate the neutrophil chemoattractant, interleukin 8. Human mesangial cells (MC) in culture, when stimulated by interleukin 1α(IL-1α) or tumour necrosis factor (TNFα), but not with lipopolysaccharide (LPS), express interleukin 8 (IL-8) mRNA, and both cell associated and extracellular IL-8. Dexa-methasone treatment of mesangial cells induced partial inhibition of the release of extracellular IL-8, while cell-associated IL-8 and IL-8 mRNA were not significantly altered. We propose that the mesangial cell has a direct role in the initiation and propagation of inflammatory events within the glomerulus via the generation of the chemotactic cytokine IL-8.

Neutrophil activating peptide 1, or interleukin-8 (NAP-1, IL-8), was isolated almost simultaneously by several groups as a neutrophil chemotactic or activating peptide from supernatants of endotoxin stimulated monocytes (1–5). The major form with 72 amino acids, and an approximate molecular weight of 8.4 kD, has since been shown to have activities on a variety of different cell types. It is chemotactic for neutrophils (PMN) and lymphocytes both in vitro and in vivo (6,2,4,7), and has been shown to stimulate PMN in vitro to degranulate and to exhibit respiratory burst (8,9), and thus has the basic attributes of an inflammatory mediator. Further actions which could be of great importance in inflammatory processes are chemotactic attraction of basophils, the triggering of IL-3 primed basophils to release leukotrienes and histamine (10), spasmogenic activity on airway smooth muscle (11), and effects on PMN adherence to endothelial cells (12).

Previously, we have shown that IL-8 and G-CSF-induced hematopoietic stem cell (HSC) mobilization is inhibited in mice that underwent low dose (0.5 Gy) total body irradiation (TBI), whereas the number of progenitor cells in the bone marrow remained similar in all groups. The mechanism underlying this inhibition remains unknown. Since the release of granular proteases by neutrophils is well known to play a role in HSC mobilization, we also considered a possible role for serine protease inhibitors in the induction of HSC mobilization. Serine proteases, such as elastase and cathepsin G, are irreversibly inhibited by serine protease inhibitors including alpha-1 antitrypsin (alpha-1 AT) and alpha2-macroglobulin. In-vitro tests revealed that addition of bone marrow extracellular extracts, that were obtained from murine femurs 24 hours following low dose (0.5 Gy) TBI, inhibited the activity of exogenous elastase in a chromogenic substrate conversion assay up to 78.1 % compared to extracts obtained from sham irradiated controls (p

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

1Alpha,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), the active metabolite of vitamin D, can inhibit NF-kappaB activity in human MRC-5 fibroblasts, targeting DNA binding of NF-kappaB but not translocation of its subunits p50 and p65. The partial inhibition of NF-kappaB DNA binding by 1,25-(OH)2-D3 is dependent on de novo protein synthesis, suggesting that 1,25-(OH)2-D3 may regulate expression of cellular factors which contribute to reduced DNA binding of NF-kappaB. Although NF-kappaB binding is decreased by 1,25-(OH)2-D3 in MRC-5 cells, IL-8 and IL-6 mRNA levels are only moderately downregulated, demonstrating that inhibition of NF-kappaB DNA binding alone is not sufficient for optimal downregulation of these genes.

A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.

Interleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 Å. The crystals belong to the space group P3121 or P3221 and have unit cell dimensions a = b = 40.9 Å, c = 90.3 Å.

Oligonucleotide probes based on the known amino acid sequence of salmon calcitonin were used to screen a cDNA library obtained from ultimobranchial glands of salmon for clones encoding salmon calcitonin. From the cDNA sequence of strongly hybridizing clones the complete primary structure of the calcitonin precursor could be deduced. Its overall structure is identical with the structures of procalcitonins from other vertebrates and has the highest homology with the chicken precursor.

The family of peptides illustrated in Fig. 1 has attracted much attention following the discovery of the neutrophil-activating peptide, NAF/NAP-1 (Walz et al.1987; Yoshimura et al.1987b; Gregory et al.1988). Alignment according to their four cysteine residues differentiates the sequences into two groups. The two first cysteines are separated by one amino acid in the peptides of the upper group and are adjacent in those of the lower one. NAF/NAP-1 has considerable sequence homology with peptides from human platelet alpha-granules, platelet basic protein (PBP) (Holt et al.1986), connective tissue activating peptide III (CTAP-III) (Castor et al.1983; Niewiarowski et al.1980) and platelet factor 4 (PF-4) (Deuel et al.1977). NAP-2 is a neutrophil-activating truncation product of PBP (Walz and Baggiolini 1989). NAF/NAP-1, has structural and biological similarities with macrophage inflammatory protein 2 (MIP-2) (Wolpe et al.1989) which could represent the murine homologue of NAF/NAP-1. Identical relative positions of the cysteines and sequence homology is also found in two proteins obtained from human cell lines, gamma-IP-10 (Luster et al.1985) and gro or MGSA (Anisowicz et al.1987; Richmond et al.1988). Two murine macrophage inflammatory proteins (MIP-1-a and -13) (Sherry et al.1988), and a novel peptide that is chemotactic for human monocytes (MCP-1) (Furutani et al.1989; Robinson et al.1989) belong to the species with adjacent cysteines 1 and 2. The peptides of the upper group presumably have a two-loop configuration resulting from the linkage of the first to the third and the second to the fourth cysteine (Begg et al.1978) as shown for NAF/NAP-1 in Fig. 2. The disulfide bridges are essential for NAF/NAP-1 activity (Peveri et al.1988).

Previously, we have shown that interleukin (IL)-8 induces the rapid (15 to 30 minutes) mobilization of hematopoietic progenitor cells (HPC) in mice. Because integrins are essential for adhesion and transendothelial migration of HPC, we studied the involvement of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) in IL-8–induced mobilization. After a single injection of blocking anti–LFA-1 antibodies, no mobilization of colony-forming cells was observed. In addition, when mice were pretreated with anti–LFA-1 or saline and subsequently injected with 30 μg of IL-8, mobilization of HPC was completely blocked. We showed that this was not due to anti–LFA-1 antibodies affecting colony formation, as addition of anti–LFA-1 antibodies to colony cultures in semisolid medium had no inhibitory activity. Also, anti-intercellular adhesion molecule (ICAM)-1 antibodies, directed to the main ligand of LFA-1 significantly inhibited the IL-8–induced mobilization. Furthermore, IL-1–induced mobilization was significantly inhibited by anti–LFA-1 antibodies. Because LFA-1 is reported to be expressed on more differentiated HPC, it was considered that the IL-8–induced mobilization of more primitive HPC would not be blocked by anti–LFA-1 antibodies. Transplantation of blood-derived mononuclear cells (MNC) from IL-8–mobilized animals pretreated with anti–LFA-1 antibodies protected only 25% of lethally irradiated recipient mice, whereas the radioprotection rate of control mice transplanted with MNC derived from IL-8-mobilized animals was 86% (P < .01). Anti-LFA–1 antibodies did not interfere with stem cell homing, as transplantation of IL-8-mobilized blood MNC, incubated in vitro with these antibodies resulted in 100% radioprotection. We conclude that anti–LFA-1 antibodies completely prevent the rapid mobilization of colony-forming cells and of cells with radioprotective capacity induced by IL-8. These results indicate a major role for the β2-integrin LFA-1 in the IL-8–induced mobilization of hematopoietic stem cells.

AIMS--To investigate: (1) whether Helicobacter pylori directly induces interleukin-8 (IL-8) message expression and protein secretion in established gastric epithelial cell lines; and (2) if CagA/cytotoxin positive and negative strains of H pylori differ in their ability to induce epithelial IL-8. METHODS--Gastric epithelial cell lines were co-cultured with H pylori NCTC 11637 and 10 clinical isolates (four cytotoxic, six non-cytotoxic) and secreted IL-8 was measured by enzyme linked immunosorbent assay (ELISA). Specific induction of gastric epithelial IL-8 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR) amplification. RESULTS--H pylori (NCTC 11637) induced IL-8 secretion from three gastric epithelial cell lines (KATO-3, ST42, AGS) but not from MKN 45 (gastric) or intestinal (SW480, HT29) cell lines. H mustelae did not stimulate IL-8 secretion from KATO-3, ST42, and AGS cells. H pylori induced IL-8 secretion was reduced by heat killing, sonication, freeze thawing or formalin fixation of the bacteria. CagA/cytotoxin positive strains of H pylori induced significantly higher IL-8 secretion than CagA/cytotoxin negative strains in the three positive gastric epithelial cell lines (KATO-3, ST42: p < 0.01; AGS: p < 0.02). A significant increase (p < 0.01) in the expression of IL-8 mRNA relative to G3PDH mRNA was observed in KATO-3 cells after three hours of co-culture with CagA/cytotoxin positive strains. CONCLUSIONS--H pylori directly increases gastric epithelial IL-8 mRNA expression and IL-8 protein secretion in a strain specific manner. Induction of epithelial IL-8 by CagA/cytotoxin positive strains is likely to result in neutrophil chemotaxis and activation and thus mucosal damage. These observations on epithelial IL-8 may explain the association between CagA/cytotoxin positive strains and gastroduodenal disease.