Human macrophage inflammatory protein-3alpha/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-alpha via a non-standard NF-kappaB site.

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.

Natural metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor.

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.

Prevention of interleukin-8-induced mobilization of hematopoietic progenitor cells in rhesus monkeys by inhibitory antibodies against the metalloproteinase gelatinase B (MMP-9).

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.

Multiple genes in the left half of the cag pathogenicity island of Helicobacter pylori are required for tyrosine kinase-dependent transcription of interleukin-8 in gastric epithelial cells.

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-alpha) without exposure to H. pylori. This TNF-alpha-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.

Gastric mucosal interleukin-8 and IL-8 antibody concentrations related to prevailing Helicobacter pylori infections. A Danish-Albanian study.

OBJECTIVES: Helicobacter pylori (H. pylori) appears to initiate an inflammatory cascade. Thus, phagocytes are accumulated in the gastric mucosa, in inflammatory conditions. Further, a potent chemotactic mediator, interleukin 8 (IL-8) is synthesized at such sites. The recently described IL-8 autoantibodies may, however, counteract the pro-inflammatory actions of IL-8. The aim was to study the correlation between H. pylori infection and IL-8, together with IL-8 autoantibodies in two different populations from a developed and a developing country. METHODS: Two different endoscopically characterized populations (65 Danes and 89 Albanians) were examined. IL-8 and IL-8 autoantibodies were detected by ELISA techniques, and H. pylori was identified by histological examinations. RESULTS: Significantly more Albanian controls and dyspeptic patients (80 out of 89 persons) were H. pylori positive as compared to 24 of 65 Danes (p < 0.001). The median IL-8 level among Albanian controls 349 pg/mg protein was significantly higher than among Danes < 61 pg/mg protein (p < 0.001), and was at the same level as found in Danish peptic ulcer patients (p > 0.05). Further, H. pylori positive patients from both countries had significantly higher levels of IL-8 as compared to H. pylori negative patients (p < 0.001). However, significantly higher levels of IL-8 autoantibodies were found in the Albanian sub-population (median 138 O.D. units versus 52 O.D. units among Danes) (p < 0.001). CONCLUSIONS: In H. pylori related disorders, a high mucosal IL-8 production has been found. However, this investigation further demonstrates higher levels of IL-8 autoantibodies among dyspeptic patients from a developing country, which might possibly counteract the pro-inflammatory actions of IL-8 by binding the molecule. The physiological significance of an altered immune response as described here needs to be elucidated in future studies.

Up-regulation of interleukin-1beta-stimulated interleukin-8 in human keratinocytes by nitric oxide.

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.

Anti-IL-8 autoantibodies and complexes in rheumatoid arthritis: polyclonal activation in chronic synovial tissue inflammation.

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.

Modulation of Helicobacter pylori induced interleukin-8 synthesis in gastric epithelial cells mediated by cag PAI encoded VirD4 homologue.

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.

1Alpha,25-dihydroxyvitamin D3 decreases DNA binding of nuclear factor-kappaB in human fibroblasts.

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.

Anti-LFA-1 blocking antibodies prevent mobilization of hematopoietic progenitor cells induced by interleukin-8.

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 beta2-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 microg 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 beta2-integrin LFA-1 in the IL-8-induced mobilization of hematopoietic stem cells.

Free and complexed interleukin-8 in blood and bronchial mucosa in asthma.

We have tested the hypothesis that the expression of interleukin-8 (IL-8) is increased in bronchial tissue and circulating leukocytes of atopic asthmatics, indicating a role for this chemokine in asthma. The concentration of IL-8 in its free form and complexed with IgG or IgA was measured by ELISA in bronchial tissue, serum, and lysates of freshly isolated peripheral blood mononuclear cells and granulocytes from subjects with mild or severe asthma and nonatopic nonasthmatic subjects. Serum ECP was measured by fluorescent enzyme immunoassay. Free IL-8 was detected in the sera (n = 44) and bronchial tissue (n = 9) of all subjects with severe atopic asthma, but it was undetectable in normal subjects and subjects with mild atopic asthma, suggesting that free IL-8 is a marker of severe asthma. A positive correlation between free IL-8 and serum ECP levels found in severe disease suggests that IL-8 is associated with eosinophil activation. Complexes of IL-8 with IgA and IgG were detected in all serum and tissue samples. However, the levels of the IL-8-IgA complex were increased in the bronchial mucosa in asthma, and in blood were related to disease activity. Together, these results point to upregulation of IL-8 production in asthma and the induction of IL-8 binding immunoglobulins of the IgA class in the inflamed mucosa. We suggest a proinflammatory role for these complexes in lung tissue.

Intensity-independent fluorometric detection of cellular nitric oxide release.

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.

Leukemia inhibitory factor induces in vivo expansion of bone marrow progenitor cells that accelerate hematopoietic reconstitution but do not enhance radioprotection in lethally irradiated mice.

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.

1alpha,25-dihydroxyvitamin D3 and a variety of its natural metabolites transcriptionally repress nuclear-factor-kappaB-mediated interleukin-8 gene expression.

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.

Intradermal challenge with interleukin-8 causes tissue oedema and neutrophil accumulation in atopic and non-atopic human subjects.

BACKGROUND: Interleukin-8 (IL-8) is a cytokine with potent neutrophil chemotactic and activating properties and is active in inflammatory conditions in man. It has been identified in human inflammatory skin conditions where it is likely to be responsible for both neutrophil recruitment from the circulation and possibly T-lymphocyte chemoattraction. Studies in animals also suggest that IL-8 may augment skin oedema. OBJECTIVE: To study the effects of intradermally administered IL-8 in humans on tissue oedema and cellular recruitment in atopic and non-atopic volunteers. METHOD: Interleukin-8 (1.2 x 10(-7) M) in the presence and absence of histamine was administered by intradermal injection. Wheal and erythema area were measured at regular intervals and 3 h following challenge punch biopsies were taken for immunocytochemistry. Cellular infiltrate was measured by immunocytochemical identification of neutrophils, eosinophils and T-lymphocytes in glycol-methacrylate-embedded sections. RESULTS: In the presence of histamine, IL-8 provoked a significantly greater wheal area when compared to that produced by histamine alone (P < 0.001). In the presence of histamine, IL-8 produced a significantly greater neutrophil infiltrate (P < 0.05), however, neither lymphocyte or eosinophil infiltration was found to be increased with IL-8 challenge. There was no difference observed between atopic and non-atopic subjects, nor were any effects of IL-8 demonstrated in the absence of histamine. CONCLUSION: This study demonstrates that in human skin, IL-8 induces increased microvascular permeability and neutrophil infiltration, but not eosinophil or T-lymphocyte chemoattraction.

Synergistic activation of interleukin-8 gene transcription by all-trans-retinoic acid and tumor necrosis factor-alpha involves the transcription factor NF-kappaB.

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.

Improved survival of lethally irradiated recipient mice transplanted with circulating progenitor cells mobilized by IL-8 after pretreatment with stem cell factor.

We have demonstrated previously that a single bolus-injection of interleukin (IL)-8 induces instant mobilization of hematopoietic progenitor cells (HPC) in mice and primates. To further improve the mobilization of HPC, we treated mice with hematopoietic growth factors (HGF) before IL-8-administration. The mobilized HPC were transplanted into lethally irradiated recipient mice to study the effects on survival. Male donor mice (age 8-12 weeks, weight 20-25 grams) were pretreated intraperitoneally (ip) with a fixed dose of 2.5 micrograms of either granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, stem cell factor (SCF), or saline administered twice daily for 2 to 4 days. Then a fixed dose of 30 micrograms of IL-8 was administered ip at various time intervals before harvesting blood, bone marrow, and spleen. Cell counts and numbers of colony-forming units granulocyte/macrophage (CFU-GM) of these organs were assessed. Donor mice pretreated with HGF for 2 days and subsequently injected with IL-8 showed an increase in the numbers of circulating CFU-GM per mL blood from 168 +/- 98 to 402 +/- 201 (mean +/- SD, CFU-GM/mL blood) when GM-CSF was used, 314 +/- 133 to 2502 +/- 513 with G-CSF, and 27 +/- 15 to 524 +/- 339 with SCF compared with saline-pretreated controls (28 +/- 17 to 462 +/- 335 CFU-GM/mL blood, mean +/- SD; n = 42 and 40 per interval). Donor-mice pretreated for 4 days with IL-3 or GM-CSF showed an increase in the numbers of circulating HPC from 62 +/- 52 to 368 +/- 118 and 859 +/- 387 to 1034 +/- 421, respectively (CFU-GM/mL, mean +/- SD, n = 4 per group). Lethally irradiated (8.5 Gy) female Balb/c mice were then injected with decreasing numbers of peripheral blood mononuclear cells (PBMNC). Transplantation of 1.5 x 10(5) MNC obtained from donors pretreated with SCF for 2 days prior to IL-8 mobilization resulted in a significantly enhanced survival of 100% of the recipients, whereas recipients of PBM-NCs derived from donors treated with SCF only or IL-8 as a single injection had a survival rate at day 60 of only 50% and 60% respectively. When equal numbers of IL-8 mobilized MNCs from G-CSF, GM-CSF, or IL-3 pretreated donors were transplanted into lethally irradiated recipients, no such survival-advantage was observed. We conclude that pretreatment with SCF for 2 days improves the mobilizing effect induced by IL-8 and that transplantation of these cells enhances survival of lethally irradiated recipients.

Neutrophil attractant protein-1 interleukin 8 and its autoantibodies in IgA nephropathy.

Human neutrophil attractant protein-1/interleukin 8 (IL-8) has been shown to activate neutrophils to degranulate in vitro and to be a potent chemotactic agonist for neutrophils and lymphocytes in vitro and in vivo. There is accumulating evidence that neutrophils are involved in inflammatory injury in IgA nephropathy (IgAN). We studied the serum levels of IL-8 and its autoantibodies of the IgA or IgG class in 36 patients with IgAN in comparison with 31 healthy controls and 26 patients with other primary glomerulonephritides (CGN). Interleukin 8 was more frequently detected in sera of patients with IgAN and their serum levels were significantly higher than those of healthy controls. The free IL-8 autoantibodies of the IgA, but not IgG class, were more frequently detected in patients with IgAN and their serum levels were significantly elevated compared with both groups of controls. The complexed IL-8 autoantibodies of either class were not different among the three groups of subjects. Again the ratio of free to complexed IL-8 autoantibodies of the IgA class was raised in patients with IgAN. Histologic examination revealed increased polymorphs and monocyte/macrophage infiltration in IgAN compared with other glomerulonephritides. When the serum levels of IL-8 and IL-8 autoantibodies were compared between IgAN patients with milder pathology and those with more severe pathology, the latter group had significantly higher serum levels of free and complexed IL-8 autoantibodies of the IgA class. These observations suggest a possible role for IL-8 and its autoantibodies of the IgA class in the inflammatory process of IgAN. These autoantibodies may provide a clinically useful marker for the diagnosis of disease severity.

Structure and activity of a chimeric interleukin-8-melanoma-growth-stimulatory-activity protein.

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.

Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8.

Interleukin-8 (IL-8) is a chemoattractant cytokine involved in chemotaxis and activation of neutrophils. Because in vivo administration of IL-8 induces mobilization of hematopoietic stem cells in mice, we assessed the mobilizing properties of IL-8 in rhesus monkeys. Recombinant human IL-8 was administered as a single intravenous injection at doses of 10, 30, and 100 micrograms/kg to rhesus monkeys (age, 2 to 3 years; weight, 2.5 to 4.5 kg). Venous blood samples were obtained at time intervals ranging from 1 to 480 minutes after IL-8 administration. Cell counts, colony-forming unit-Mix assays, and fluorescence-activated cell sorter analysis were performed. Plasma was harvested to assess IL-8 levels. A time-controlled bolus intravenous injection of 100 micrograms IL-8 per kilogram of body weight resulted in peak IL-8 plasma levels up to 5 micrograms/mL. The calculated half-time life of free IL-8 was 9.9 +/- 2.2 minutes. IL-8 injection resulted in instant neutropenia that was due to pulmonary sequestration, as shown using 99mTc-labeled leukocytes. Within 30 minutes after IL-8 injection, neutrophilia developed with counts up to 10-fold greater than baseline levels. The numbers of hematopoietic progenitor cells (HPCs) increased from 45 +/- 48/mL to 1,382 +/- 599/mL of blood at 30 minutes after injection of 100 micrograms IL-8 per kilogram of bodyweight (mean +/- SD, n = 8). Individual animals showed 10- to 100-fold increase in numbers of circulating HPCs that returned to almost pretreatment values (92 +/- 52 CFU/mL) at 240 minutes after the injection of IL-8. Immunophenotyping showed no significant changes in lymphocyte (sub)populations. A second bolus injection of IL-8 with an interval of 72 hours resulted in similar numbers of mobilized stem cells as observed after the first injection, showing that no tachyphylaxis had occurred. We conclude that IL-8 induces mobilization of HPCs from the bone marrow of rhesus monkeys in a rapid and reproducible fashion. Therefore, IL-8 may be a potentially useful cytokine in the setting of blood stem cell transplantation.