Role of epithelial interleukin-8 (IL-8) and neutrophil IL-8 receptor A in Escherichia coli-induced transuroepithelial neutrophil migration

Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8's ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.     

Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil-activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.     

Deregulated production of interleukin-8 (IL-8) in autoimmune thyroid disease studied by newly developed IL-8 radioimmunoassay

Glucagon may regulate FFA metabolism in vivo. To test this hypothesis, six healthy male volunteers were infused with somatostatin, to inhibit endogenous hormone secretion, and insulin, glucagon, and GH to replace endogenous secretion of these hormones. In the hypoglucagonemia experiments, the glucagon infusion was omitted, and in the hyperglucagonemic experiments glucagon was infused at 1.3 ng/kg.min, to produce physiological hyperglucagonemia. In two sets of control experiments, glucagon was infused at 0.65 ng/kg.min, in order to maintain peripheral euglucagonemia, and the plasma glucose concentrations were clamped at the levels observed in either the hypo- or hyperglucagonemic experiments. Rates of FFA and glycerol (an index of lipolysis) appearance (Ra) were estimated with the isotope dilution method using [1-14C]palmitate and [2H5] glycerol. Plasma glucagon concentrations decreased during the hypoglucagonemic experiments (85 +/- 12 vs. 123 +/- 22 ng/L, P < 0.05) and increased during the hyperglucagonemic experiments (186 +/- 20 vs. 125 +/- 15 ng/L, P < 0.05), whereas other hormone concentrations remained the same. Hypoglucagonemia resulted in equivalent suppression of FFA Ra (3.7 +/- 0.2 vs. 5.9 vs. 0.3 mumol/kg.min, P < 0.01) and glycerol Ra (1.2 +/- 0.2 vs. 2.2 +/- 0.5 mumol/kg.min, P < 0.05). Similarly, hyperglucagonemia resulted in equivalent stimulation of FFA Ra (5.2 +/- 0.4 vs. 3.7 +/- 0.3 mumol/kg.min, P < 0.05) and glycerol Ra (1.5 +/- 0.3 vs. 1.1 +/- 0.1 mumol/kg.min, P < 0.05). These results indicate that glucagon has a physiological role in the regulation of FFA metabolism in vivo.     

Interaction of fibronectin (FN) cell binding fragments and interleukin-8 (IL-8) in regulating neutrophil chemotaxis

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.     

Serum interleukin-6 (IL-6) and interleukin-4 (IL-4) in patients with multiple myeloma (MM)

In this study we determined, in patients with multiple myeloma (MM), serum levels of IL-4 and IL-6 at diagnosis and during the course of the disease, seeking a correlation with disease activity and prognosis. We studied 54 MM patients, 41 of whom responded to chemotherapy whilst 11 were resistant. At diagnosis, IL-6 was increased in 66% of patients (median 35.5 pg/ml) whereas IL-4 was low (median 4 pg/ml) in 75% of patients. In responding patients, IL-4 increased in remission (median 25 pg/ml), whereas IL-6 decreased (median 4 pg/ml). In chemotherapy-resistant patients, IL-6 and IL-4 values remained stable during the course of the disease.     

Endogenous interleukin-8 (IL-8) surge in granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

The relationship between stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and the endogenous production of interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was studied in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. G-CSF was administered to 20 normal donors at a dose of 10 microg/kg/d for 5 days with aphereses on days 5 and 6 of G-CSF treatment. Cytokine serum levels were measured using an enzyme-linked immunosorbent assay (ELISA) before and during G-CSF treatment. Before treatment, the average level of IL-8 was 7.1 pg/mL, increasing to 207.0 pg/mL on day 5 and 189.1 pg/mL on day 6. Serum IL-8 levels correlated CD34(+) cell numbers (P =.0151 and P =.0005 on days 5 and 6, respectively) and colony-forming unit-granulocyte-macrophage (CFU-GM) numbers (P =.0019 and P =.0010 on days 5 and 6, respectively). Furthermore, preapheresis serum IL-8 levels correlated with the yield of CD34(+) cells (P =.0027). In contrast, before treatment, the average levels of MIP-1alpha, TNF-alpha, and IFN-gamma were 70.1, 4.03, and 3.84 pg/mL, respectively, and no significant changes in the levels of these cytokines were observed during G-CSF treatment. These studies suggest that IL-8 production may be critical to G-CSF-induced stem cell mobilization, although the underlying mechanism could not be clarified.     

Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions

We analyzed the noise characteristics of two-dimensional (2-D) and three-dimensional (3-D) images obtained from the GE Advance positron emission tomography (PET) scanner. Three phantoms were used: a uniform 20-cm phantom, a 3-D Hoffman brain phantom, and a chest phantom with heart and lung inserts. Using gated acquisition, we acquired 20 statistically equivalent scans of each phantom in 2-D and 3-D modes at several activity levels. From these data, we calculated pixel normalized standard deviations (NSD's), scaled to phantom mean, across the replicate scans, which allowed us to characterize the radial and axial distributions of pixel noise. We also performed sequential measurements of the phantoms in 2-D and 3-D modes to measure noise (from interpixel standard deviations) as a function of activity. To compensate for the difference in axial slice width between 2-D and 3-D images (due to the septa and reconstruction effects), we developed a smoothing kernel to apply to the 2-D data. After matching the resolution, the ratio of image-derived NSD values (NSD2D/NSD3D)2 averaged throughout the uniform phantom was in good agreement with the noise equivalent count (NEC) ratio (NEC3D/NEC2D). By comparing different phantoms, we showed that the attenuation and emission distributions influence the spatial noise distribution. The estimates of pixel noise for 2-D and 3-D images produced here can be applied in the weighting of PET kinetic data and may be useful in the design of optimal dose and scanning requirements for PET studies. The accuracy of these phantom-based noise formulas should be validated for any given imaging situation, particularly in 3-D, if there is significant activity outside the scanner field of view.     

Biologic activity of interleukin 1 (IL-1) alpha in patients with refractory malignancies

To examine the effects of smoking and N-acetylation genetics on breast cancer risk, we analyzed data from an ongoing, population-based, case-control study of invasive breast cancer in North Carolina. The study population consisted of 498 cases and 473 controls, with approximately equal numbers of African-American and white women, and women under the age of 50 and age 50 years or older. Among premenopausal women, there was no association between current smoking [odds ratio (OR), 0.9; 95% confidence interval (CI), 0.5-1.5] or past smoking (OR, 1.0; 95% CI, 0.6-1.6) and breast cancer risk. Among postmenopausal women, there was also no association with current smoking (OR, 1.2; 95% CI, 0.7-2.0); however, a small increase in risk was observed for past smoking (OR, 1.5; 95% CI, 1.0-2.4). For postmenopausal women who smoked in the past, ORs and 95% CIs were 3.4 (1.4-8.1) for smoking within the past 3 years, 3.0 (1.3-6.7) for smoking 4-9 years ago, and 0.6 (0.3-1.4) for smoking 10-19 years ago. Neither N-acetyltransferase 1 (NAT1) nor N-acetyltransferase 2 (NAT2) genotype alone was associated with increased breast cancer risk. There was little evidence for modification of smoking effects according to genotype, except among postmenopausal women. Among postmenopausal women, ORs for smoking within the past 3 years were greater for women with the NAT1*10 genotype (OR, 9.0; 95% CI, 1.9-41.8) than NAT1-non*10 (OR, 2.5; 95% CI, 0.9-7.2) and greater for NAT2-rapid genotype (OR, 7.4; 95% CI, 1.6-32.6) than NAT2-slow (OR, 2.8; 95% CI, 0.4-8.0). Future studies of NAT genotypes and breast cancer should investigate the effects of environmental tobacco smoke, diet, and other exposures.     

Level of interleukin-6 (IL-6), soluble interleukin-6 receptors (sIL-6R) and tumor necrosis factor alpha (TNF-alpha) in untreated and progressing multiple myeloma]

The effect of original analogues of the 6-9 C-terminal AVP fragment, D-MRP and D-MPRG, on the active avoidance behaviour of rats was studied using intranasal administration in a wide range of doses. The dose in 0.1 mcg/kg was the most efficient for D-MPR and 0.01 mcg/kg for D-MPRG, which for the tripeptide was ten times, and for the tetrapeptide hundred times lower than the corresponding dose of AVP used in the similar experiments. The tri- and tetrapeptide were shown to facilitate the active avoidance behaviour affecting both the formation of the reaction and consolidation of memory trace. The peptides mostly affect the perception processes, i.e., the detection of a specific stimulus from the environment, the estimation and memorizing of its biological significance.     

Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130

The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.     

Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro

Antigen-presenting cells are thought to modulate the development of Th1 and Th2 cells by the secretion of interleukin-10 (IL-10) and IL-12. Because glucocorticoids (GC) favor the development of Th2 responses, we determined whether dexamethasone (DEX) and hydrocortisone (HC) have differential effects on lipopolysaccharide-induced IL-10 and IL-12 production in whole-blood cultures. Significant inhibition of IL-12(p40) and IL-12(p70) was found with 10(-8) mol/L and 10(-9) mol/L DEX respectively, whereas IL-10 was relatively insensitive or even stimulated. Accordingly, the expression of IL-12(p40) and IL-12(p35) mRNA was more sensitive to DEX than IL-10 mRNA. The glucocorticoid receptor (GR) antagonist RU486 enhanced IL-12 production and largely abrogated the inhibition of IL-12 by GC, indicating that this suppression was mainly GR-mediated. High concentrations of RU486 were inhibitory for IL-10, suggesting that GC may exert a positive effect on IL-10. In the presence of neutralizing anti-IL-10 antibodies, DEX was still capable of IL-12 suppression whereas RU486 still enhanced IL-12 production, indicating that GC do not modulate IL-12 via IL-10 exclusively. Taken together these results indicate that GC may favor Th2 development by differential regulation of IL-10 and IL-12.     

Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted [corrected] IL-1 and tumor necrosis factor alpha and is required for subsequent transendothelial migration of neutrophils

Previous studies have shown that Borrelia burgdorferi, the spirochetal agent of Lyme disease, promotes inflammation by stimulating endothelial cells to upregulate adhesion molecules for leukocytes and to produce a soluble agent that is chemotactic for neutrophils. We determined that interleukin-8 (IL-8) was the chemotactic agent for neutrophils present in conditioned media from cultured human umbilical vein endothelial cells stimulated with B. burgdorferi. As few as one spirochete per endothelial cell stimulated production of IL-8 within 8 h of coincubation. When 10 spirochetes per endothelial cell were added, IL-8 was detected after 4 h of coculture. Production of IL-8 continued in a linear fashion for at least 24 h. Neutralizing antibodies against IL-8 reduced migration of neutrophils across spirochete-stimulated endothelial monolayers by 93%. In contrast, pretreatment of neutrophils with antagonists of platelet-activating factor did not inhibit migration. Increases in production of IL-8 and expression of the adhesion molecule E-selectin by endothelial cells in response to B. burgdorferi were not inhibited by IL-1 receptor antagonist or a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, used either alone or in combination. These results suggest that activation of endothelium by B. burgdorferi is not mediated through the autocrine action of secreted IL-1 or tumor necrosis factor alpha. Rather, it appears that B. burgdorferi must stimulate endothelium either by a direct signaling mechanism or by induction of a novel host-derived proinflammatory cytokine.     

Epitope-labeled soluble human interleukin-5 (IL-5) receptors. Affinity cross-link labeling, IL-5 binding, and biological activity

The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5 alpha R) and a 130-kDa beta chain (IL-5 beta R). Three ectodomain constructs of the alpha chain (alpha RED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) 125I-IL-5 specifically with an ED50 of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alpha RED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alpha RED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.     

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP)

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotrophin (ACTH), beta-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 +/- 1.04 ng/g tissue, n = 9) compared to controls (2.42 +/- 0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 +/- 1.22 ng/g). beta-Endorphin levels in both the AA (10.60 +/- 1.61 ng/g) and AA/ADX (13.37 +/- 2.36 ng/g) groups were higher than controls (5.57 +/- 0.65 ng/g). Conversely, alpha-MSH spleen levels were decreased in the AA (2.89 +/- 0.22 ng/g) and AA/ADX (2.22 +/- 0.33 ng/g) groups compared to controls (4.62 +/- 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 +/- 1.41 ng/g) compared to controls (5.79 +/- 0.63 ng/g), and the same pattern was evident for thymic alpha-MSH (0.64 +/- 0.08 ng/g in AA animals compared to control levels of 0.35 +/- 0.03 ng/g). Following G50 gel filtration, ACTH and beta-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective presence of the chemokine growth-regulated oncogene alpha (GROalpha) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation

Ovulation is an inflammation-like reaction in which leukocytes are postulated to have a central role. The abundance of leukocytes in the ovary varies with the stage of the cycle and a marked influx of neutrophils and monocytes into the interior of the follicle during ovulation has been observed. The intraovarian signals causing this preovulatory influx are not known. In the present study we have investigated the presence in the ovary of two chemotactic cytokines, GROalpha (growth-regulated oncogene alpha) and RANTES (regulated upon activation, normal T-cell expressed and secreted), which have specific chemotactic activity towards neutrophils/basophils/T-cells and monocytes/T-cells/eosinophils respectively. The concentrations of these cytokines were first measured in follicular fluid and peripheral blood from a group of patients undergoing in-vitro fertilization (IVF) procedures. GROalpha was found in approximately 10-fold higher concentrations in follicular fluid than in blood plasma from the same patients (P < 0.001). The concentrations in peripheral blood of GROalpha were similar and without significant variations in women during the time of gonadotrophin stimulation for IVF and throughout the normal menstrual cycle. There was no correlation between follicular fluid concentrations of GROalpha and follicular fluid concentrations of progesterone or oestradiol. Cultured granulosa-lutein cells secreted detectable amounts of GROalpha. The concentrations of GROalpha in the medium were markedly increased by the presence of the proinflammatory cytokine interleukin (IL)-1beta, with approximately 10-fold higher concentrations in the medium, compared with the controls (P < 0.001). GROalpha was localized by immunohistochemistry predominantly in the theca layer but also in the granulosa layer of the dominant follicle during the late follicular phase. The concentrations of RANTES in follicular fluid were only 1/50 of those in blood plasma (P< 0.001). RANTES protein was not detectable in the culture medium of granulosa-lutein cells neither during basal nor IL-1beta stimulated conditions. In conclusion, these results suggest that the chemokine GROalpha is one of the chemotactic signals which cause recruitment and activation of specific leukocytes within the ovulating follicle.