interleukin-8 and Acidosis

We hypothesized that repetitive umbilical cord occlusions (UCOs) with worsening acidosis will lead to a fetal inflammatory response.. Chronically instrumented fetal sheep underwent a series of UCOs until fetal arterial pH decreased to <7.00. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1B and IL-6 levels. Animals were euthanized at 24 hours of recovery with brain tissue processed for subsequent measurement of microglia and mast cell counts.. Repetitive UCOs resulted in a severe degree of fetal acidemia. Fetal plasma IL-1B values were increased approximately 2-fold when measured at maximal fetal acidosis and again at 1-2 hours of recovery. Fetal microglia cells were increased approximately 2-fold in the white matter and hippocampus, while mast cells were increased approximately 2-fold in the choroid plexus and now evident in the thalamus when analyzed at 24 hours recovery.. Repetitive UCOs leading to severe acidemia in the ovine fetus near term will result in an inflammatory response both systemically and locally within the brain.

Topics: Acidosis; Animals; Cerebrovascular Circulation; Constriction, Pathologic; Enzyme-Linked Immunosorbent Assay; Fetal Diseases; Fetal Hypoxia; Fetus; Hippocampus; Immunohistochemistry; Interleukin-6; Interleukin-8; Mast Cells; Microglia; Sheep; Umbilical Cord

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.

Topics: Acidosis; Animals; C-Reactive Protein; Cell Line; Chlorocebus aethiops; COS Cells; Escherichia coli Infections; Ficolins; Humans; Immunity, Innate; Interleukin-8; Lectins; Macromolecular Substances; Membrane Proteins; Monocytes; Pseudomonas Infections; Receptor Cross-Talk; Receptors, Cell Surface; Salmonella Infections; Signal Transduction; Staphylococcal Infections; U937 Cells; Up-Regulation

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion.

Topics: Acidosis; Amiloride; Calcium; Cell Adhesion; Cells, Cultured; Chloride-Bicarbonate Antiporters; Cyclic AMP; Endothelial Cells; Endothelium, Vascular; Exocytosis; Guanidines; Histamine; Humans; Hydrogen-Ion Concentration; Interleukin-8; Intracellular Fluid; Kinetics; Linear Models; Methacrylates; P-Selectin; Platelet Adhesiveness; Reperfusion Injury; Sodium-Hydrogen Exchangers; Sulfones; Umbilical Veins; von Willebrand Factor

Inflammation probably plays a significant role in perinatal brain injury. To study the contribution of locally produced cytokines, the effect on cell death of addition of IL-8 and MCP-1 or antibodies to these, and the impact of acidosis, human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and reoxygenated for 21 h. After 3 h of hypoxia with neutral medium, IL-8 was significantly increased compared to controls (150 (100-250)% vs. 100 (85-115)%, p=0.023). After 21 h of neutral reoxygenation, both IL-8 (380 (110-710)% vs. 150 (85-260)%, p=0.041) and monocyte chemoattractant protein-1 (MCP-1) (650 (440-2000)% vs. 310 (230-340)%, p=0.007) were significantly increased compared to controls. After 3 h of hypoxia, both IL-8 (p=0.002) and MCP-1 (p=0.008) were significantly lower in cells with acidotic compared with cells with neutral medium. Acidosis during reoxygenation, however, significantly increased IL-8 release, whereas MCP-1 release was diminished. Similar effects of acidosis were seen in normoxic controls. The cells also secreted RANTES and IP-10, but not 8 other cytokines tested. We found no effect on cell death, measured by MTT assay, of addition of IL-8, MCP-1 or antibodies to these. We conclude that human NT2-N neurons release IL-8 and MCP-1 during 21 h of reoxygenation after 3 h of hypoxia. Acidosis led to a differential effect on IL-8 and MCP-1, with increased IL-8 and decreased MCP-1, both during reoxygenation and in normoxic controls. IL-8 and MCP-1 had no effect on cell death.

Topics: Acidosis; Antibodies; Cell Hypoxia; Cell Line; Chemokine CCL2; Chemokine CCL5; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Gene Expression Regulation; Glucose; Humans; Hypoxia; Interleukin-8; Neurofilament Proteins; Neurons; Oxygen; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles; Time Factors

We have documented the time-dependent production of chemotactic cytokine, i.e., IL-8, in the extracellular fluid of astrocyte-rich cultured rat cerebellar granule cells under acidified conditions. In this paper, the mechanism of this production was evaluated based on the production of hydrogen peroxide (H2O2). Significant and time-dependent increases of cytosolic H2O2 were detected under acidosis in astrocyte-rich cultured cell. Upon exposure to 10 microM H2O2, significant levels of IL-8 appeared in the extracellular fluid of astrocyte-rich cells, although an initial transient increase of IL-8 was also seen in the intracellular space. Concurrently, after H2O2 exposure cell injury and a delayed increase of cytosolic Ca2+ levels were detected in astrocyte-rich cells. However, in the absence of extracellular Ca2+, the cell injury and the increase of IL-8 production were significantly attenuated. A synergistic effect of cyclosporine A (an inhibitor of the Ca2+/calmodulin-regulated protein phosphatase) and trifluoperazine (an inhibitor of phospholipase A2) on the suppression of H2O2-induced IL-8 production was clearly evident. These results suggest that extracellular acidosis induced Ca2+-dependent H2O2 production, which in turn stimulated IL-8 expression. which is regulated by the cytosolic Ca2+ cascade. Thus, the production of IL-8 from glia cells may have a role in regulating in the process of cell injury.

Topics: Acidosis; Animals; Animals, Newborn; Astrocytes; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Culture Media; Cyclosporine; Cytoplasmic Granules; Cytosol; Drug Synergism; Hydrogen Peroxide; Interleukin-8; Rats; Rats, Wistar; Trifluoperazine

The role and regulation of interleukin 8 (IL-8) in the growth and metastasis of SG, FG, and L3.3 variants derived from COLO 357 human pancreatic cancer cells were determined. After orthotopic implantation in the pancreas of nude mice, SG cells produced the smallest tumors, whereas L3.3 cells produced the largest tumors. SG cells produced no liver metastasis, whereas FG cells produced numerous liver metastases, and L3.3 cells produced more and larger liver metastases. In vitro analysis of IL-8 expression indicated that SG cells expressed the lowest level of IL-8 gene expression as determined by both Northern blot analysis and ELISA, whereas L3.3 cells expressed the highest level of IL-8. Immunohistochemical analysis of tumor lesions indicated that IL-8 overexpression was predominant in the regions surrounding necrotic areas, where cells were exposed to low oxygen tension (hypoxia) and acidic pH. In vitro treatment of FG tumor cells with hypoxia or acidosis led to an increased expression of IL-8. To directly determine the role of IL-8 in the growth and metastasis of pancreatic cancer, FG cells were transfected with IL-8 sense or antisense oligonucleotide expression vectors. The neo-resistance gene-transfected FG cells were used as controls. Decreased IL-8 expression after transfection with IL-8 antisense oligonucleotide expression vector retarded the growth of FG cells in mice after intrapancreatic implantation, which correlated with decreased tumor angiogenesis. Our data demonstrated that hypoxia and acidosis contribute to the overexpression of IL-8, which in turn plays an important role in tumor angiogenesis and contributes significantly to the aggressive biology of human pancreatic cancer.

Topics: Acidosis; Animals; Cell Division; Cell Hypoxia; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Oligodeoxyribonucleotides; Oligodeoxyribonucleotides, Antisense; Pancreatic Neoplasms; Recombinant Proteins; Transfection

In order to evaluate the functional role of chemotactic cytokines in the regulation of brain function, we examined the effects of acidosis on the production of IL-8 in cultured neurons and/or astrocyte-rich cerebellar granule cells as assessed by the ELISA method. A time-dependent and significant production of IL-8 was detected in the extracellular fluid of astrocyte-rich cultured cells at 2, 3 and 6 hrs after treatment with acidified Krebs-HEPES buffer (pH 6.9), although such production did not appear in the fluid of neuron-rich cells. Additionally, microglia were detected by microscopic examination in both cultured cells under acidotic conditions. Only astrocyte-containing cultured cells produced a marked increase in intracellular IL-8 under acidotic conditions, although this production was much less than that seen in the extracellular fluid at 6 hrs under acidosis. The increase of IL-8 in astrocyte-rich cultures induced by acidosis was potentiated by treatment with glutamate, which enhanced the increase of cytosolic Ca2+ levels under acidosis, and was affected by extracellular Ca2+ conditions, by cyclosporine A, an inhibitor of calcineurin, and by trifluoperazine, an inhibitor of phospholipase A2. Significant inhibition of IL-8 production was detected after 6 hrs of pretreatment with trifluoperazine. Furthermore, the production of IL-8 under acidosis was associated with the appearance of astrocyte damage. These results suggest that Ca2+-dependent IL-8 is produced by astrocytes, but not neuronal cells, under acidosis, and that this production may be related to the process of cell dysfunction resulting from membrane destruction induced by acidosis.

Topics: Acidosis; Animals; Astrocytes; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Cyclosporine; Cytoplasmic Granules; Cytosol; Dopamine Agonists; Extracellular Space; Glutamic Acid; Immunosuppressive Agents; Interleukin-8; Rats; Trifluoperazine

Our purpose was to investigate the participation of cytokines and neutrophils in fetal distress.. Umbilical cord serum interleukin-6 (IL-6), interleukin-8 (IL-8), and plasma granulocyte elastase (GEL) were measured in 30 normally grown newborns without fetal distress (Group A), 10 growth-retarded newborns without acute fetal distress (Group B), 5 normally grown newborns with fetal distress (Group C), and 5 growth-retarded newborns with fetal distress (Group D). Umbilical arterial blood pH and PO2 were also measured.. Umbilical arterial blood pH and PO2 in either Group C or Group D were significantly lower than those in either Group A or Group B. The concentration of IL-6 in Group D was significantly higher than that in either Group A, B, or C. The level of IL-8 in either Group C or Group D was significantly higher than that in either Group A or Group B. The concentrations of GEL in Group D was significantly higher than that in either Group A or Group B.. This study suggests that fetal distress in utero causes an elevation of immune factors such as IL-6, IL-8 and GEL.

Topics: Acidosis; Fetal Blood; Fetal Distress; Fetal Growth Retardation; Fetal Hypoxia; Humans; Infant, Newborn; Interleukin-6; Interleukin-8; Leukocyte Elastase