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Modulating phosphatidic acid metabolism decreases oxidative injury in rat lungs

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 40 nM), prevented oxidant-mediated capillary leak in isolated rat lungs given interleukin-8 (IL-8) intratracheally and perfused with human neutrophils. Lung leak was prevented by lung, but not neutrophil, lisofylline pretreatment. Furthermore, although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe (fMLP)-stimulated oxidant production in vitro. Lisofylline also prevented acute capillary leak in isolated rat lungs perfused only with the oxidant generator purine-xanthine oxidase but did not scavenge Formula or H2O2 in vitro. Finally, lisofylline-mediated protection against lung leak in both models was associated with alterations in lung membrane free fatty acid acyl composition (as reflected by the decreased ratio [linoleate + oleate]/ [palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent oxidant-induced capillary leak in isolated rat lungs and that protection appears to be mediated by blocking intrinsic lung linoleoyl phosphatidic acid metabolism. We speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane composition and renders this barrier less vulnerable to oxidative damage.

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IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10−10 M) or toxin B (10−12 M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

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BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

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Cadmium Induces Interleukin-8 in Alveolar Epithelial Type II Cells (A549)

Cadmium (Cd) is a contaminant in cigarette smoke and may play an important role in the pathogenesis of smoking-related emphysema. It is reported that repeated Cd exposure causes emphysema after neutrophil infiltration in the rat lung. Interleukin-8 (IL-8) is a major neutrophil chemotactic factor commonly involved in a variety of pulmonary disorders including emphysema. The aim of this study is to elucidate the effect of Cd on IL-8 production of alveolar epithelial type II cells, using the A549 cell line. We used 1–100 mM of cadmium chloride (CdCl2) to stimulate the cells for a 48 h period. IL-8 production (2957 ± 137 pg/mL) without any cytotoxic effect was observed in the cell supernatants after 24 h exposure to 50 mM CdCl2. The Cd-removed supernatant caused neutrophil chemotaxis and was inhibited by the anti-IL-8 antibody.

Cd-induced IL-8 was inhibited by EDTA, and the inhibition was blocked by copper (II) chloride. The addition of anti-interleukin-1b or tumor necrosis factor-a antibodies did not diminish IL-8 release induced by Cd. These results suggest that Cd increases the production of IL-8 without any cytotoxic effect in alveolar epithelial cells, which may be an important factor in the developmental process of cigarette smoking-related emphysema.

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Decreased neutrophil function as a cause of retained placenta in dairy cattle.

It is unclear why some cows fail to expel the placenta following calving. One theory suggests the fetal placenta must be recognized as “foreign” tissue and rejected by the immune system after parturition to cause expulsion of the placenta. We hypothesized that impaired neutrophil function causes retained placenta (RP). We examined the ability of neutrophils to recognize fetal cotyledon tissue as assessed by a chemotaxis assay, which utilized a placental homogenate obtained from a spontaneously expelled placenta as the chemoattractant. Neutrophil killing ability was also estimated by determining myeloperoxidase activity in isolated neutrophils. Blood samples were obtained from 142 periparturient dairy cattle in two herds. Twenty cattle developed RP (14.1%). Neutrophils isolated from blood of cows with RP had significantly lower neutrophil function in both assays before calving, and this impaired function lasted for 1 to 2 wk after parturition. The addition of antibody directed against interleukin-8 (IL-8) to the cotyledon preparation used as a chemoattractant inhibited chemotaxis by 41%, suggesting that one of the chemoattractants present in the cotyledon at parturition is IL-8. At calving, plasma IL-8 concentration was lower in RP cows (51 +/- 12 pg/ml) than in cows expelling the placenta normally (134 +/- 11 pg/ml). From these data, we suggest that neutrophil function is a determining factor for the development of RP in dairy cattle. Also, depressed production of IL-8 may be a factor affecting neutrophil function in cows developing RP.

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Ascaris suum-Derived Products Induce Human Neutrophil Activation via a G Protein-Coupled Receptor That Interacts with the Interleukin-8 Receptor Pathway

Infection with tissue-migrating helminths is frequently associated with intense granulocyte infiltrations. Several host-derived factors are known to mediate granulocyte recruitment to the tissues, but less attention has been paid to how parasite-derived products trigger this process. Parasite-derived chemotactic factors which selectively recruit granulocytes have been described, but nothing is known about which cellular receptors respond to these agents. The effect of products from the nematodes Ascaris suum, Toxocara canis, andAnisakis simplex on human neutrophils were studied. We monitored four parameters of activation: chemotaxis, cell polarization, intracellular Ca2+ transients, and priming of superoxide anion production. Body fluids of A. suum (ABF) and T. canis (TcBF) induced strong directional migration, shape change, and intracellular Ca2+ transients. ABF also primed neutrophils for production of superoxide anions. Calcium mobilization in response to A. suum-derived products was completely abrogated by pretreatment with pertussis toxin, implicating a classical G protein-coupled receptor mechanism in the response to ABF. Moreover, pretreatment with interleukin-8 (IL-8) completely abrogated the response to ABF, demonstrating desensitization of a common pathway. However, ABF was unable to fully desensitize the response to IL-8, and binding to CXCR1 or CXCR2 was excluded in experiments using RBL-2H3 cells transfected with the two human IL-8 receptors. Our results provide the first evidence for a direct interaction between a parasite-derived chemotactic factor and the host’s chemotactic network, via a novel G protein-coupled receptor which interacts with the IL-8 receptor pathway.

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Toll-like receptors stimulate human neutrophil function

The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10—all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection.

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Effect of Molybdenum-Induced Copper Deficiency on In Vivo and In Vitro Measures of Neutrophil Chemotaxis both Before and Following an Inflammatory Stressor

Twelve Angus Hereford heifers (avg wt=183.6kg) were allotted by initial liver copper (Cu) concentrations into one of two treatments. Control (n=6) heifers were fed a basal diet supplemented to provide a dietary Cu level of 10ppm. Molybdenum (Mo)-induced Cu-deficient heifers (n=6) were fed an identical basal diet supplemented with sodium molybdate (Cu:Mo ratio = 1:2.5), with dietary sulfur at .3% of the total diet. Dietary treatments were delivered for 120d, at which time Mo-supplemented heifers were considered Cu-deficient (286 and 49ppm liver Cu for control and Mo-induced Cu-deficient, respectively). Peripheral blood neutrophils were enumerated both before and after the administration of an inflammatory stressor, a subcutaneous injection (1.5mL) of Freund’s complete adjuvant. In vitro and in vivo measures of neutrophil chemotaxis were evaluated and the expression of two adhesion molecules, CD18 and L-selectin, were analyzed by flow cytometric procedures. Molybdenum-induced Cu deficiency increased (P<.01) the number of peripheral blood neutrophils; however, in vitro neutrophil chemotaxis was not affected. In vivo neutrophil chemotaxis tended (P<.08) to be increased in Mo-induced Cu-deficient heifers (1.55 vs 2.26 x 106 cells/sponge for control and Mo-supplemented, respectively). No differences in CD18 or L-selectin expression were detected between treatments. However, CD18 expression was decreased (P<.05) in both treatments following adjuvant injection. These data suggest that Mo-induced Cu deficiency results in an increase in peripheral blood neutrophil number, without altering chemotactic ability and adhesion molecule expression.

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LSP1 modulates leukocyte populations in resting and inflamed peritoneum

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans.

This study examines LSP1-deficient (Lsp1-/-) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen.

LSP1-/- mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5 macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in LSP1-/- mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of LSP1-/- mice are similar to those of wt mice and LSP1-/- mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the LSP1-/- mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells.

LSP1 neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis.

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Neutrophil maturation and activation determine anatomic site of clearance from circulation

The long term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using 111In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 ± 1.9 vs. 31.9 ± 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 ± 1.1%) and marrow-derived neutrophils to the marrow (65.9 ± 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence  supporting both storage and perhaps disposal functions. Furthermore, models for circulating  neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell.