leukotriene-b4 and Peritonitis

Polyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega (

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acid; Arthritis, Experimental; Cells, Cultured; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Humans; Leukotriene B4; Lipidomics; Macrophages; Male; Mice; Mice, Transgenic; Neutrophils; Peritoneal Lavage; Peritonitis; Primary Cell Culture; THP-1 Cells; Zymosan

Macrophage polarization is critical to inflammation and resolution of inflammation. We previously showed that high-mobility group box 1 (HMGB1) can engage receptor for advanced glycation end product (RAGE) to direct monocytes to a proinflammatory phenotype characterized by production of type 1 IFN and proinflammatory cytokines. In contrast, HMGB1 plus C1q form a tetramolecular complex cross-linking RAGE and LAIR-1 and directing monocytes to an antiinflammatory phenotype. Lipid mediators, as well as cytokines, help establish a milieu favoring either inflammation or resolution of inflammation. This study focuses on the induction of lipid mediators by HMGB1 and HMGB1 plus C1q and their regulation of IRF5, a transcription factor critical for the induction and maintenance of proinflammatory macrophages. Here, we show that HMGB1 induces leukotriene production through a RAGE-dependent pathway, while HMGB1 plus C1q induces specialized proresolving lipid mediators lipoxin A4, resolvin D1, and resolvin D2 through a RAGE- and LAIR-1-dependent pathway. Leukotriene exposure contributes to induction of IRF5 in a positive-feedback loop. In contrast, resolvins (at 20 nM) block IRF5 induction and prevent the differentiation of inflammatory macrophages. Finally, we have generated a molecular mimic of HMGB1 plus C1q, which cross-links RAGE and LAIR-1 and polarizes monocytes to an antiinflammatory phenotype. These findings may provide a mechanism to control nonresolving inflammation in many pathologic conditions.

Topics: Animals; Arachidonate 5-Lipoxygenase; Complement C1q; HMGB1 Protein; Interferon Regulatory Factors; Leukotriene B4; Macrophages; Mice, Inbred C57BL; Monocytes; Peritonitis; Receptor for Advanced Glycation End Products; Receptors, Immunologic

Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation-associated leukotriene B(4), and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A(4), and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A(4) was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

Topics: Acute Disease; Animals; Apoptosis; Goldfish; Immunity, Innate; Kidney; Leukotriene B4; Lipoxins; Macrophage Activation; Macrophages, Peritoneal; Neutrophils; Peritonitis; Phagocytosis; Reactive Oxygen Species; Respiratory Burst; Time Factors; Zymosan

Opuntia dillenii Haw (Nagphana) traditionally used against inflammation. The present study addressed the anti-inflammatory activity of O. dillenii derived methanol extract, fractions and pure compounds and their underlying mechanism of action.. O. dillenii cladode methanol extract was subjected to vacuum liquid chromatography (VLC) furnishing two main fractions viz (T-1 and -2) leading to isolation of opuntiol (aglycone) and opuntioside (O-glucoside), respectively. Anti-inflammatory activity of extract, fractions, pure compounds and reference drugs were evaluated using: (1) arachidonic acid (AA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ear edema accompanied by histological studies of mice ear sections and phospholipase A2 (PLA2)-induced mice paw edema. (2) Carrageenan and glycogen-induced peritonitis in rodents. In parallel levels of leukotriene B4 (LTB4) and reactive oxygen species (ROS) were also determined via HPLC and fluoroemetrically using 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye, respectively. Additionally, levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF-α), interleukins IL-1β and -6 were measured by ELISA assay.. O. dillenii methanol extract, fractions and pure compounds reduced AA and TPA-induced ear punch weight in a dose dependent fashion. The corresponding IC50 values obtained also suppressed inflammatory features observed histologically. Furthermore, paw edema and peritonitis were also attenuated. Similar to indomethacin and diclofenac sodium, opuntioside reduced PGE2 levels of inflamed ear which was comparatively 1.3× better than opuntiol. However, opuntiol was more potent in reducing LTB4 levels in rat neutrophils with an IC50 value of 19±3.3μΜ, while opuntioside was ineffective. Opuntiol also effectively suppressed ROS (37%) and cytokine levels (TNF-α, IL-1β and -6) by ~50% and comparable to dexamethasone.. O. dillenii cladodes possess anti-inflammatory properties via inhibition of arachidonic acid metabolites and cytokines. Opuntiol (aglycone) emerged as a dual inhibitor of cyclooxygenase (COX) and lipooxygenase (LOX) pathways. It also suppressed ROS and cytokine levels. However, opuntioside manifested its selectivity towards COX (PGE2) pathway without affecting LTB4 levels. The present report describing the anti-inflammatory activity of opuntiol and opuntioside for the first time thereby, supporting and justifying the traditional use of O. dillenii against inflammation and may serve as lead compound in designing of new anti-inflammatory agents.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acid; Capillary Permeability; Carrageenan; Coumaric Acids; Cytokines; Dinoprostone; Ear; Edema; Female; Foot; Leukotriene B4; Male; Mice; Monosaccharides; Neutrophils; Opuntia; Peritonitis; Phospholipases A2; Phytotherapy; Plant Extracts; Rats, Sprague-Dawley; Reactive Oxygen Species; Tetradecanoylphorbol Acetate

In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Arachidonate 5-Lipoxygenase; Calcium Signaling; Cathelicidins; Cells, Cultured; Eicosanoids; Humans; Inflammation; Leukotriene B4; Macrophages; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Peritonitis; Phospholipases A2, Cytosolic; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Receptors, Purinergic P2X7; Recombinant Proteins; Thromboxane A2; Tumor Necrosis Factor-alpha

Leukotrienes (LTs) are 5-lipoxygenase (5-LO) metabolites which are implicated in sex-dependent inflammatory diseases (asthma, autoimmune diseases, etc.). We have recently reported sex differences in LT biosynthesis in in vitro models such as human whole blood, neutrophils and monocytes, due to down-regulation of 5-LO product formation by androgens. Here we present evidences for sex differences in LT synthesis and related inflammatory reactions in an in vivo model of inflammation (mouse zymosan-induced peritonitis). On the cellular level, differential 5-LO subcellular compartmentalization in peritoneal macrophages (PM) from male and female mice might be the basis for these differences. Sex differences in vascular permeability and neutrophil recruitment (cell number and myeloperoxidase activity) into peritoneal cavity were evident upon intraperitoneal zymosan injection, with more prominent responses in female mice. This was accompanied by higher levels of LTC4 and LTB4 in peritoneal exudates of female compared to male mice. Interestingly, LT peritoneal levels in orchidectomized mice were higher than in sham male mice. In accordance with the in vivo results, LT formation in stimulated PM from female mice was higher than in male PM, accompanied by alterations in 5-LO subcellular localization. The increased formation of LTC4 in incubations of PM from orchidectomized mice confirms a role of sex hormones. In conclusion, sex differences observed in LT biosynthesis during peritonitis in vivo may be related, at least in part, to a variant 5-LO localization in PM from male and female mice.

Topics: Animals; Arachidonate 5-Lipoxygenase; Ascitic Fluid; Capillary Permeability; Female; Leukotriene B4; Leukotriene C4; Macrophages, Peritoneal; Male; Mice; Orchiectomy; Peritonitis; Peroxidase; Sex Characteristics; Testosterone; Zymosan

Mechanisms underlying delays in resolution programs of inflammation are of interest for many diseases. Here, we addressed delayed resolution of inflammation and identified specific microRNA (miR)-metabolipidomic signatures. Delayed resolution initiated by high-dose challenges decreased miR-219-5p expression along with increased leukotriene B(4) (5-fold) and decreased (~3-fold) specialized pro-resolving mediators, e.g. protectin D1. Resolvin (Rv)E1 and RvD1 (1 nM) reduced miR-219-5p in human macrophages, not shared by RvD2 or PD1. Since mature miR-219-5p is produced from pre-miRs miR-219-1 and miR-219-2, we co-expressed in human macrophages a 5-lipoxygenase (LOX) 3'UTR-luciferase reporter vector together with either miR-219-1 or miR-219-2. Only miR-219-2 reduced luciferase activity. Apoptotic neutrophils administered into inflamed exudates in vivo increased miR-219-2-3p expression and PD1/NPD1 levels as well as decreased leukotriene B(4). These results demonstrate that delayed resolution undermines endogenous resolution programs, altering miR-219-2 expression, increasing pro-inflammatory mediators and compromising SPM production that contribute to failed catabasis and homeostasis.

Topics: 3' Untranslated Regions; Acute Disease; Animals; Apoptosis; Arachidonate 5-Lipoxygenase; Cells, Cultured; Dinoprostone; Exudates and Transudates; Gene Expression; Humans; Inflammation; Inflammation Mediators; Leukotriene B4; Lipid Metabolism; Macrophages; Male; Mice; MicroRNAs; Neutrophils; Peritonitis; Prostaglandin D2; RNA Interference; Zymosan

Lysophosphatidylcholines (lysoPCs) with polyunsaturated acyl chains are known to exert anti-inflammatory actions. 15-Lipoxygeanation is crucial for anti-inflammatory action of polyunsaturated acylated lysoPCs. Here, the anti-inflammatory actions of 1-(15-hydroxyeicosapentaenoyl)-lysoPC (15-HEPE-lysoPC) and its derivatives were examined in a mechanistic analysis.. Anti-inflammatory actions of 15-HEPE-lysoPC in zymosan A-induced peritonitis of mice were examined by measuring plasma leakage and leucocyte infiltration, and determining levels of lipid mediators or cytokines.. When each lysoPC, administered i.v., was assessed for its ability to suppress zymosan A-induced plasma leakage, 15-HEPE-lysoPC was found to be more potent than 1-(15-hydroperoxyeicosapentaenoyl)-lysoPC or 1-eicosapentaenoyl-lysoPC. Separately, i.p. administration of 15-HEPE-lysoPC markedly inhibited plasma leakage, in contrast to 15-HEPE, which had only a small effect. 15-HEPE-lysoPC also decreased leucocyte infiltration. Moreover, it reduced the formation of LTC₄ and LTB₄, 5-lipoxygenation products, as well as the levels of pro-inflammatory cytokines. The time-course study indicated that 15-HEPE-lysoPC might participate in both the early inflammatory phase and resolution phase. Additionally, 15-HEPE-lysoPC administration caused a partial suppression of LTC₄-induced plasma leakage and LTB₄-induced leucocyte infiltration. In the metabolism study, peritoneal exudate was shown to contain lysoPC-hydrolysing activity, crucial for anti-inflammatory activity, and a system capable of generating lipoxin A from 15-hydroxy eicosanoid precursor.. 15-HEPE-lysoPC, a precursor for 15-HEPE in target cells, induced anti-inflammatory actions by inhibiting the formation of pro-inflammatory leukotrienes and cytokines, and by enhancing the formation of lipoxin A. 15-HEPE-lysoPC might be one of many potent anti-inflammatory lipids in vivo.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cytokines; Eicosapentaenoic Acid; In Vitro Techniques; Inflammation Mediators; Injections, Intraperitoneal; Leukocytes; Leukotriene B4; Leukotriene C4; Lysophosphatidylcholines; Male; Mice; Mice, Inbred ICR; Oligopeptides; Oxidation-Reduction; Peritonitis; Structure-Activity Relationship; Zymosan

Antibodies to the pan-leukocyte adhesion-GPCR CD97 efficiently block neutrophil recruitment in mice, thereby reducing antibacterial host defense, inflammatory disease, and hematopoietic stem cell mobilization. Here, we investigated the working mechanism of the CD97 antibody 1B2. Applying sterile models of inflammation, intravital microscopy, and mice deficient for the CD97L CD55, the complement component C3, or the FcR common γ-chain, we show that 1B2 acts in vivo independent of ligand-binding interference by depleting PMN granulocytes in bone marrow and blood. Granulocyte depletion with 1B2 involved FcR but not complement activation and was associated with increased serum levels of TNF and other proinflammatory cytokines. Notably, depletion of granulocytes by CD97 antibody required acute inflammation, suggesting a mechanism of conditional, antibody-mediated granulocytopenia.

Topics: Animals; Antibodies, Blocking; Antibody Specificity; CD55 Antigens; Cell Adhesion; Cell Movement; Cytokines; Granulocytes; Humans; Inflammation; Leukotriene B4; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neutropenia; Peritonitis; Receptors, Fc; Receptors, G-Protein-Coupled; Tumor Necrosis Factor-alpha

We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis.. Wistar rats received intra-articular (i.art.) zymosan (30-1000 microg) or LPS (1-10 microg). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl)benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art.. Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis.. Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus- and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Topics: Acute Disease; Animals; Arthritis; CD18 Antigens; Cell Movement; Intercellular Adhesion Molecule-1; Interleukin-10; Joints; Leukotriene B4; Lipopolysaccharides; Male; Mice; Mice, Knockout; Neutrophil Infiltration; Nitric Oxide; Nitric Oxide Synthase; Peritoneal Cavity; Peritonitis; Rats; Rats, Wistar; Species Specificity; Tumor Necrosis Factor-alpha; Zymosan

Cytosolic phospholipase A2alpha (cPLA2) plays an important role in the development of several inflammatory diseases. The aim of the present study is to determine whether inhibition of cPLA2 expression, using specific antisense oligonucleotides against cPLA2 (antisense), is efficient in reducing inflammation after its development. Two mouse models of inflammation were included in the study: thioglicolate peritonitis and collagen-induced arthritis (CIA). The antisense was found to be specific and efficient in inhibiting cPLA2 expression and NADPH oxidase activity ex vivo in peritoneal phagocytes. Immunoblotting and immunohistochemistry analysis showed a significant elevation in cPLA2 expression in the inflamed joints of collagen-induced arthritis mice localized in cell infiltrate, chondrocytes and the surrounding skin and skeletal muscle. Similarly, the cPLA2 metabolite, leukotriene B4, accumulated in the peritoneal cavity of mice with peritonitis. Inhibition of elevated cPLA2 expression after development of inflammation by intravenous administration of antisense resulted in a dramatic reduction in inflammation and a significant reduction in neutrophils recruitment to the site of inflammation in both mouse models of inflammation. Our results demonstrate the critical role of cPLA2 for the duration of inflammation and suggest that inhibition of cPLA2 expression by antisense oligonucleotides may serve as an efficient treatment of inflammatory diseases.

Topics: Animals; Arthritis, Experimental; Collagen Type II; Disease Models, Animal; Group IV Phospholipases A2; Leukotriene B4; Mice; Mice, Inbred DBA; Neutrophil Infiltration; Neutrophils; Oligonucleotides, Antisense; Peritonitis; Superoxides; Thioglycolates

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.

Topics: Animals; Cell Movement; Complement Activation; Injections, Intraperitoneal; Leukocytosis; Leukotriene B4; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Peritonitis; Receptor, Anaphylatoxin C5a; Receptors, Complement; Toll-Like Receptor 2; Zymosan

Leukotriene A(4) hydrolase (LTA(4)H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B(4), which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA(4)H (IC(50), approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA(4)H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB(4) production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED(50), 1-3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB(4) production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A(4). The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB(4), LTC(4), and LXA(4) production. Although zileuton inhibited LTB(4) production in the peritonitis model more effectively than the LTA(4)H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA(4) levels in the presence of the LTA(4)H inhibitor. The selective inhibition of LTB(4) production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA(4), may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB(4)-mediated inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Ascitic Fluid; Benzothiazoles; Dogs; Ear; Edema; Eicosanoids; Enzyme Inhibitors; Epoxide Hydrolases; Female; Humans; Hydroxyurea; Inflammation; Leukotriene B4; Leukotriene C4; Lipoxins; Lipoxygenase Inhibitors; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Neutrophil Infiltration; Peritonitis; Piperidines; Recombinant Proteins

4-(2',4'- Difluorobiphenyl-4-yl)-2-methylbutyric acid (deoxoflobufen, VUFB 19053, CAS 847475-35-8) has been developed as a new omega-biphenyl-alkanoic acid and studied in comparison with the racemic form of 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (flobufen, CAS 112344-52-2). The compounds were tested in a series of models including acute inflammation induced by carrageenan, adjuvant arthritis, in vitro inhibition of the leuktotriene B4 (LTB4) production, reaction of the graft versus the host (GVHR), production of specific antibodies against ovalbumin, peritoneal exudate formation induced by thioglycollate and phagocytosis of thioglycollate-stimulated mouse peritoneal macrophages. Deoxoflobufen exhibited strong anti-inflammatory, antiarthritic and immunomodulatory effects in most of the performed tests. Anti-inflammatory and antiarthritic effects are fully comparable with those of flobufen, however, the compound is less toxic and has apparently stronger immunomodulating effects.

Topics: Animals; Anti-Inflammatory Agents; Antibody Formation; Area Under Curve; Arthritis, Experimental; Biphenyl Compounds; Butyrates; Carrageenan; Cell Adhesion; Exudates and Transudates; Female; Graft vs Host Disease; Hypersensitivity, Delayed; Inflammation; Leukotriene B4; Macrophages; Mice; Ovalbumin; Peritonitis; Phagocytosis; Pleurisy; Rats; Structure-Activity Relationship; Thioglycolates

Signaling pathways instrumental in the temporal and spatial progression of acute inflammation toward resolution are of wide interest. Here a transgenic mouse with myeloid-selective expression of human lipoxin A4 receptor (hALX) was prepared and used to evaluate in vivo the effect of hALX expression. hALX-transfected HEK293 cells transmitted LXA4 signals that inhibit TNFalpha-induced NFkappaB activation. Transgenic FvB mice were generated by DNA injections of a 3.8 kb transgene consisting of the full-length hALX cDNA driven by a fragment of the hCD11b promoter. When topically challenged via dermal ear skin, hALX transgenic mice gave attenuated neutrophil infiltration (approximately 80% reduction) in response to leukotriene B4 (LTB4) plus prostaglandin E2 (PGE2) as well as approximately 50% reduction in PMN infiltrates (P<0.02) to receptor-bypass inflammation evoked by phorbol ester. The hALX transgenic mice gave markedly decreased PMN infiltrates to the peritoneum with zymosan and altered the dynamics of this response. Transgenic hALX mice displayed increased sensitivity with >50% reduction in PMN infiltrates to suboptimal doses (10 ng/mouse) of the ligand lipoxin A4 stable analog compared with <10% reduction of PMN in nontransgenic littermates. Soluble mediators generated within the local inflammatory milieu of hALX mice showed diminished ability to activate the proinflammatory transcription factor NFkappaB. Analyses of the lipid-derived mediators from exudates using LC-MS tandem mass spectroscopy indicated an altered profile in hALX transgenic mice that included lower levels of LTB4 and increased amounts of lipoxin A4 compared with nontransgenic littermates. Together these results demonstrate a gain-of-function with hALX transgenic mouse and indicate that ALX is a key receptor and sensor in formation of acute exudates and their resolution.

Topics: 3T3 Cells; Animals; Cell Line; Chromatography, Liquid; Dinoprostone; Female; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Leukotriene B4; Lipoxins; Male; Mass Spectrometry; Mice; Mice, Transgenic; Neutrophil Infiltration; NF-kappa B; Peritoneum; Peritonitis; Plasmids; Receptors, Cell Surface; Receptors, Formyl Peptide; Receptors, Lipoxin; Skin; Transfection; Tumor Necrosis Factor-alpha; Zymosan

Leukotriene B4 (LTB4) is a potent chemoattractant for myeloid leukocytes, which express BLT1, the high-affinity receptor for LTB4. We report here that BLT1 is induced substantially in CD8+ effector T cells and at lower amounts in CD8+ central memory T cells. LTB4 elicited BLT1-dependent chemotaxis in effector cells, but not in naive or central memory cells. Intravital microscopy showed that BLT1 signaling induced rapid integrin-mediated arrest of rolling effector and central memory cells in postcapillary venules. In competitive homing experiments, wild-type effector cells were three times more efficient at migrating to the inflamed peritoneal cavity than were BLT-deficient effector cells. These results identify LTB4-BLT1 as a potent nonchemokine pathway for cytotoxic effector cell traffic.

Topics: Acute Disease; Animals; Chemotaxis, Leukocyte; Flow Cytometry; Immunologic Memory; Integrins; Leukotriene B4; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Peritonitis; Receptors, Leukotriene B4; Receptors, Lymphocyte Homing; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

Leukotrienes (LTs) producing capacity was investigated in calcium ionophore A23187-stimulated peripheral white blood cells and peritoneal inflammatory cells suspension isolated from the same rat. A reverse phase high performance liquid chromatography technique and computerized UV spectroscopy were employed to isolate and quantitate the released LTs namely, LTC(4) and LTB(4). Preincubation of rat peritoneal inflammatory cells at 37 degrees C for 5 min followed by calcium ionophore A23187 stimulation for another 5 min produced significantly elevated amounts of LTB(4) as compared to peripheral white blood cells isolated from the same rat (103+/-12.7 versus 40+/-3.6 pmol/10(7) cells, respectively; mean+/-SEM). Enhanced generation of LTB(4) was associated with production of similar amounts of LTC(4) as compared with LTC(4) produced by peripheral white blood cells (15.2+/-4.2 versus 14.6+/-2 pmol/10(7) cells, respectively). In subsequent experiments, when peritoneal inflammatory cells and white blood cells suspension isolated from the same rats were stimulated with calcium ionophore A23187 (1 micro M) after preincubation with different concentrations of exogenous arachidonic acid (1, 3 and 10 micro M), significantly higher amounts of LTB(4) were produced by the peritoneal inflamed cells while a similar amounts of LTC(4)were produced by both types of cells. Increased LTB(4) formation by rat peritoneal inflammatory cells may prove to be of pathophysiological relevance, since this compound has been described to play an important role in acute inflammatory reaction.

Topics: Animals; Arachidonic Acid; Calcimycin; Carrageenan; Chromatography, High Pressure Liquid; In Vitro Techniques; Ionophores; Leukocyte Count; Leukocytes; Leukotriene B4; Male; Peritoneal Cavity; Peritonitis; Rats; Spectrophotometry, Ultraviolet

Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4. Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection. In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance. We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro. To test the functional significance of mast cell-derived LTs during an E. coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice. Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice. Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.

Topics: Adhesins, Bacterial; Adhesins, Escherichia coli; Animals; Cells, Cultured; Escherichia coli; Escherichia coli Infections; Fimbriae Proteins; Leukotriene B4; Leukotriene C4; Male; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neutrophils; 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.

Topics: Animals; Calcium; Cell Adhesion; Chemotactic Factors; Chemotaxis, Leukocyte; Disease Models, Animal; Eosinophils; Gene Targeting; Leukotriene B4; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscles; Neutrophils; Peritonitis; Receptors, Leukotriene B4; Thioglycolates; Venules

We investigated the involvement of monocyte chemoattractant protein (MCP)-1 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that CLP induced a dramatic increase in MCP-1 production in the peritoneum, followed by an increase in the recruitment of leukocytes. MCP-1 blockade with anti-MCP-1 antiserum significantly decreased the survival rate following CLP, which was accompanied by an enhanced recovery of viable bacteria from the peritoneum. This was likely due to the reduction in the recruitment and activation of both macrophages and neutrophils. To understand the mechanisms whereby MCP-1 may influence neutrophil infiltration, levels of chemokines known to attract neutrophils were monitored, which showed that peritoneal levels of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1alpha were not altered with anti-MCP-1 Abs. However, anti-MCP-1 Abs reduced the peritoneal levels of leukotriene B4 (LTB4) by 59%. The i.p. injection of MCP-1 into normal mice resulted in elevated levels of LTB4 in the peritoneum. In vitro, MCP-1 stimulated the production of LTB4 from peritoneal macrophages, in a dose-dependent manner. A specific LTB4 receptor antagonist (CP-105, 696) inhibited CLP-induced recruitment of both neutrophils and macrophages, which was accompanied by a reduced level of MCP-1 in the peritoneum. Finally, administration of CP-105,696 was extremely detrimental to the survival of mice following CLP. These experiments demonstrate that endogenous MCP-1 serves as an indirect mediator to attract neutrophils via the production of LTB4, and suggest the cross-talk can occur between MCP-1 and the lipid mediator LTB4 during septic peritonitis.

Topics: Animals; Ascitic Fluid; Benzopyrans; Carboxylic Acids; Cecum; Chemokine CCL2; Chemokines; Female; Leukotriene B4; Mice; Peritoneal Cavity; Peritonitis; Receptor Cross-Talk; Receptors, Leukotriene B4; Sepsis

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.

Topics: Acute Disease; Anaphylaxis; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Cell Movement; Crosses, Genetic; Cysteine; Dermatitis, Contact; Ear; Epoxide Hydrolases; Fluorescein-5-isothiocyanate; Immunoglobulin E; Inflammation Mediators; Leukotriene B4; Leukotrienes; Lipopolysaccharides; Mice; Mice, Knockout; Neutrophils; Peritonitis; Platelet Activating Factor

Intraperitoneal administration of zymosan to mice resulted in marked biosynthesis of eicosanoids and influx of neutrophils with distinct time course profiles. 6-Keto-prostaglandin-F1 alpha (6-KPA) increased between 30 and 60 min and rapidly decreased thereafter. Leukotriene (LT)C4 levels showed similar patterns, but were sustained for several hours. LTB4 increased in a biphasic manner with peak increases between 2 to 3 hr. Repeated injections with zymosan suggested that incoming neutrophils generate most of the LTB4. Myeloperoxidase (MPO), an enzyme marker for neutrophils, continued to increase throughout the time course. Mast cells regulate LTB4 biosynthesis and neutrophil trafficking, whereas resident macrophages contribute to 6-KPA and LTC4 biosynthesis. The complement fragment C5a has a minimal role in zymosan-induced inflammation. Selective 5-lipoxygenase (5-LO) inhibitors, zileuton [N-(1-benzo[b]thienyl-2yl-ethyl)-N-hydroxyurea], TZI-41127 [2-(4-hydroxy-3,5-dimethylphenyl)-5-methoxy-3-methylindole] and cyclooxygenase (CO) inhibitors selectively modulated eicosanoid biosynthesis. Both 5-LO and CO inhibitors attenuated influx of neutrophils to varying degrees. A LTB4 receptor antagonist, SC-41930 [7-(3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid) and an LTD4 receptor antagonist, LY-171883 [1-(2-hydroxy-3-propyl-4-(4-1H-tetrazol-5-yl)butoxy-phenyl) ethanone)] (i.v.) attenuated influx of neutrophils and associated LTB4 biosynthesis. These results suggest that both 5-LO and CO metabolites regulate neutrophil influx in this model. Marked eicosanoid biosynthesis and cellular influx in response to zymosan provides an attractive experimental paradigm to evaluate anti-inflammatory effects of inhibitors of arachidonate CO or 5-LO pathways.

Topics: Acetophenones; Animals; Arachidonate 5-Lipoxygenase; Benzopyrans; Complement C5a; Leukotriene B4; Male; Mast Cells; Mice; Peritonitis; Peroxidase; Tetrazoles; Zymosan

1. The intraperitoneal (i.p.) injection of 1 or 10 micrograms ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose-dependent increase in the amounts of immunoreactive leukotriene (LT) C4 and LTB4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 micrograms ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non-immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC4 and LTB4, but not histamine, release in mouse peritoneum 1 h after its injection. 2. Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels therefore. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed. 3. Ovalbumin (1 microgram)-induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg-1, s.c.) or with the 5-lipoxygenase inhibitor, BWA4C (20 mg kg-1, s.c.), whereas indomethacin (2 mg kg-1, s.c.) and the platelet-activating factor (PAF)-antagonist SR 27417 (10 mg kg-1, s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo-oxygenase derivatives or PAF, mediate antigen-induced eosinophil accumulation in the mouse peritoneum. 4. The histamine HI receptor antagonist drug, cetirizine (15-30 mg kg-1, s.c.) markedly reduced ovalbumin-induced eosinophil accumulation under conditions where terfenadine was ineffective, suggesting that the effect of cetirizine was not related to the inhibition of the H1 receptor effects of histamine.5. The immunosuppressive agent, FK-506 (1-2 mg kg-1, s.c.) and the protein synthesis inhibitor,cylcoheximide, when administered either in situ (0.06 ng/cavity) or systemically (5 mg kg-1, s.c.),prevented antigen-induced eosinophil accumulation in the mouse peritoneum, contributing to the concept that substances (probably cytokines) originating from lymphocytes may be involved in the modulation of the eosinophilotactic response in this model.6. The results of the present study indicate that the i.p. administration of ovalbumin to actively sensitized mice induced late eosinophil accumulation in t

Topics: Animals; Benzeneacetamides; Cetirizine; Cycloheximide; Dexamethasone; Eosinophils; Histamine Release; Hydroxamic Acids; Indomethacin; Kinetics; Leukocyte Count; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Peritoneal Cavity; Peritonitis; Platelet Activating Factor; T-Lymphocytes; Tacrolimus; Terfenadine; Thiazoles; Zymosan

A novel series of N-[(2-benzothiazolylthio)alkyl]-N'-hydroxyurea derivatives (9-25) was synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase both in vivo (mouse zymosan peritonitis assay) and in vitro (Ca2+ ionophore-stimulated human peripheral blood leukocyte model). The compounds of this series were based on the corresponding hydroxamic acid derivatives (1, 3, 4, and 5) which were moderately active in vitro but inactive in vivo. A number of compounds in the hydroxyurea series exhibited oral activity for 5-lipoxygenase inhibition. Results of studies relating structure to in vivo and in vitro 5-lipoxygenase activity are reported.

Topics: Animals; Benzothiazoles; Calcimycin; Dogs; Humans; Hydroxamic Acids; Hydroxyurea; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Methemoglobin; Mice; Molecular Structure; Peritonitis; SRS-A; Structure-Activity Relationship; Thiazoles

Alterations of leukotriene (LT) productivity in peritoneal macrophages (PM) from untreated rats (control) as well as from rats treated i.p. with thioglycollate broth (TG) were investigated on days 3, 7 and 14 after TG administration. The resident PM from the untreated rats produced mainly LTB4 and 5-HETE with small amounts of 12-HETE and LTD4 with only a trace of LTC4 when stimulated with the calcium ionophore A23187. The PM elicited from rats on days 3 and 7 produced more LTC4 than did the resident PM but fewer other lipoxygenase metabolites. On day 14, however, the elicited PM resembled the resident PM in terms of lipoxygenase metabolite production. Similar results were achieved in the presence of arachidonic acid and A23187. A decrease in lipoxygenase metabolism in the elicited PM was also suggested by using opsonized zymosan. Catabolism studies indicated a reduction in r-glutamyl transpeptidase activity in the elicited PM and suggested a reduction in catabolism for LTB4 in the former cells. The authors conclude that the TG-elicited PM generate fewer lipoxygenase metabolites than the resident PM following stimulation, but show a preferential conversion of LTA4 to sulfidopeptide LTs rather than to LTB4. The elicited PM also show a reduced catabolism for LTC4 and LTB4.

Topics: Animals; Arachidonic Acid; Calcimycin; Cell Count; gamma-Glutamyltransferase; Leukotriene B4; Macrophage Activation; Macrophages; Male; Membrane Lipids; Peritonitis; Rats; Rats, Inbred Strains; SRS-A; Thioglycolates; Time Factors; Zymosan

A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activity in vitro: sideritoflavone, cirsiliol, hypolaetin-8-O-beta-D-glucoside, hypolaetin, oroxindin, quercetagetin-7-O-beta-D-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E2 (PGE2) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retained in vivo since they behave as dual inhibitors of PGE2 and leukotriene B4 (LTB4) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Arachidonic Acids; Carrageenan; Dinoprostone; Edema; Flavonoids; Inflammation; Leukocytes; Leukotriene B4; Male; Mice; Peritonitis; Plants, Medicinal

Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25-0.5 h) and a secondary increase (2-3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB4, C4, D4, E4 and 6-oxo-PGF1 alpha) which was maximal at 0.125-0.25 h. Intra-articular injection led to an initial peak of LTB4 production (maximal at 0.25 h) and a secondary peak of LTB4 and PGE2 production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO) inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB4 production in A23187 stimulation blood ex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB4 production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid production in vivo but the relative importance of these mediators varies depending on the inflammatory site.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arthritis; Benzeneacetamides; Calcimycin; Colchicine; Dexamethasone; Dinoprostone; Disease Models, Animal; Hydroxamic Acids; Inflammation; Kinetics; Knee Joint; Leukocytes; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Peritonitis; Pyrazoles; Rats; Zymosan

Leukotriene induction of the fluid and cellular phases of the inflammatory response in the mouse was evaluated. Intraperitoneal injection of leukotriene C4 (LTC4 250 ng) led to dye extravasation but not polymorphonuclear leukocyte (PMN) infiltration, whereas injection of leukotriene B4 (LTB4 250 ng), led to PMN infiltration but not dye extravasation. The injection of both leukotrienes did not result in synergy. LTC4 did not appear to induce significant release or formation of chemotactic mediators, but the dye extravasation induced by LTC4 was inhibited by the vasoactive amine antagonist cyproheptadine and not by the eicosanoid inhibitors phenidone or naproxen. The response was markedly inhibited by the cytokine and eicosanoid inhibitors SK&F 86002 and SK&F 104493. PMN infiltration induced by LTB4 was not inhibited by SK&F 86002 or phenidone but was abrogated by colchicine treatment. LTB4 in this model did not appear to cause release or formation of vasoactive mediators. These leukotrienes appeared to be independent, complementary, and sufficient to mount a complete inflammatory response in the mouse.

Topics: Animals; Ascites; Capillary Permeability; Chemotaxis, Leukocyte; Colchicine; Drug Interactions; Imidazoles; Inflammation; Injections, Intraperitoneal; Leukotriene B4; Male; Mice; Mice, Inbred BALB C; Neutrophils; Peritonitis; Pyrazoles; SRS-A; Thiazoles

The metabolism of arachidonic acid, by isolated peritoneal macrophages in response to stimulation with microorganisms isolated from continuous ambulatory peritoneal dialysis (CAPD) patients, is not specific to the individual strain but is determined by species. Isolates of Staphylococcus aureus (n = 7) and Staphylococcus epidermidis (n = 13) produced a significant increase in leukotriene B4 generation by peritoneal macrophages compared with unstimulated cells (P less than .001). The coincubation of peritoneal macrophages with gram-negative organisms (n = 3), however, did not result in an increase in leukotriene B4 synthesis. In contrast, all the organisms tested significantly inhibited the formation of prostaglandin E2 and thromboxane B2 but the degree of inhibition was species dependent. The above results were reproduced using bacteria-free supernatants, indicating that the interaction was dependent on a secreted bacterial product. Thus in response to certain bacterial species, the recruitment of neutrophils may be facilitated by the generation of leukotriene B4 from peritoneal macrophages. In addition, the reduction in synthesis of prostaglandin E2 may remove a regulatory effect that this metabolite has on the immune response.

Topics: Adult; Aged; Eicosanoids; Female; Hemolysin Proteins; Humans; Leukotriene B4; Macrophages; Male; Middle Aged; Peritoneal Cavity; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Virulence

To evaluate the role of bacterial peritonitis in peritoneal macrophage (PMO) Beta-2 Microglobulin (B2M) production and its relationship with PMO Interleukin-1 (IL-1) and Leukotriene B4 (LTB4) release we analyzed in 20 CAPD patients (10 with peritonitis): 1. in vivo plasma and peritoneal dialysis effluent (PDE) B2M, IL-1 and LTB4 levels; 2. in vitro B2M, IL-1 and LTB4 release by PMO. Values were compared with those seen in the plasma or with peripheral blood monocytes of 30 hemodialysis (HD) patients (10 treated with Cuprophan-CU-, 10 with Polyacrylonitrile - PAN, and 10 with Cellulose Acetate - CA). Results showed that in CAPD patients with bacterial peritonitis B2M, IL-1 and LTB4 concentrations in the PDE were significantly higher than those seen in CAPD patients without peritonitis or in the plasma of HD patients treated with PAN or CA, but were similar to those seen in HD patients treated with CU. At the same time, in vitro, PMO from CAPD patients with bacterial peritonitis produced more B2M, IL-1 and LTB4 than did PMO from CAPD patients without peritonitis or peripheral blood monocytes from HD patients treated with PAN or CA. We conclude that in CAPD patients bacterial peritonitis is able to induce PMO B2M production, probably via a cytokine-mediated process, which may be analogous to what occurs with peripheral blood monocytes of HD patients treated with CU.

Topics: Adult; Bacterial Infections; beta-Thromboglobulin; Female; Humans; Interleukin-1; Leukotriene B4; Macrophages; Male; Membranes, Artificial; Middle Aged; Peritoneal Cavity; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Renal Dialysis

Our previous in vitro studies have shown that Ca++ and 1,25(OH)2D3 modulate peritoneal macrophage (PMO) antimicrobial activity in CAPD patients. We thus evaluated in vivo in 24 CAPD patients (12 who had never had peritonitis and 12 with an overall peritonitis incidence of more than one episode per 8 patient/month), the effects of different peritoneal dialysis fluid (PDF) Ca++ concentrations (1.25, 1.75 and 2.25 mmol/L) on PMO: 1. cytoplasmic Ca++ concentration; 2. superoxide generation; 3. Leukotriene B4 (LTB4) release; 4. bacterial killing for staphylococcus epidermidis. The same parameters were also evaluated after adding 1,25(OH)2D3 (0.25 microgram/L) to the PDF. Results showed a direct correlation between the PDF Ca++ concentration and PMO Ca++ levels, superoxide and LTB4 generation, and bacterial killing, such that with 2.25 mmol/L of Ca++ these values were significantly higher than those seen with 1.75 mmol/L. The addition of 1,25(OH)2D3 potentiated the Ca++ - induced effects. On the contrary, with PDF Ca++ levels of 1.25 mmol/L, an inhibition of the aforementioned parameters was seen. However, this effect was reversed by the addition of 1,25(OH)2D3. These in vivo results confirm the importance of Ca++ and 1,25(OH)2D3 in PMO antibacterial functions in CAPD patients and may be useful in the prophylaxis and therapy of peritonitis.

Topics: Adult; Calcitriol; Calcium; Dialysis Solutions; Female; Humans; Leukotriene B4; Macrophages; Male; Peritoneal Cavity; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Phagocytosis; Staphylococcus epidermidis; Superoxides

The mode of action of the dual inhibitors of eicosanoid metabolism, SK&F 86002 and SK&F 104493 was evaluated on inflammatory cell infiltration induced in mice by carrageenan, monosodium urate crystals, and arachidonic acid. The results were compared to those seen with standard antiinflammatory compounds. Inflammatory cell infiltration was inhibited by SK&F 86002. SK&F 104493, colchicine, and phenidone but not naproxen. In vivo, PMN infiltration induced by LTB4 was inhibited by colchicine but not by SK&F 86002, SK&F 104493, or phenidone treatment. Similarly, in vitro chemotaxis to LTB4 was not inhibited by SK&F 86002. The 5-lipoxygenase inhibitors, SK&F 86002, SK&F 104493, and phenidone inhibited LTB4 production in vivo as well as inflammatory cell infiltration induced by arachidonic acid. The data are consistent with the suggestion that the bicyclic imidazoles inhibit PMN infiltration by virtue of inhibition of LTB4 production.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Chemotaxis, Leukocyte; Cyclophosphamide; Dinoprostone; Ear Diseases; Edema; Eicosanoids; Imidazoles; Inflammation; Leukotriene B4; Male; Mice; Mice, Inbred BALB C; Neutrophils; Peritonitis; Pyridines; Thiazoles

Ca++ has been proposed as an intracellular second messenger for the activation of immune cells. An immune regulatory role for 1,25(OH)2D3 has also been suggested. We therefore evaluated the role of Ca++ and 1,25(OH)2D3 in the depressed antibacterial functions of 8 CAPD patients with relapsing bacterial peritonitis by evaluating in vitro the effects of escalating concentrations of 1,25(OH)2D3 and/or Ca++ on: 1. peritoneal macrophage (PMO) cytoplasmic Ca++; 2. PMO superoxide generation; 3. PMO leukotriene B4 release, 4. PMO bacterial killing. Results showed a dose-dependent increase in all parameters for Ca++ concentrations from 500 to 3,000 microM while with both a CA(++)-free medium and with Ca++ concentrations of 5,000 microM of medium all the aforementioned functions were abrogated. Addition of low doses of 1,25(OH)2D3 strongly potentiated the stimulatory effect of Ca++ on cell functions, while high doses were inhibitory. These in vitro data underline the importance of Ca++ and 1,25(OH)2D3 in PMO antibacterial functions in CAPD patients, and may be useful in the prophylaxis and therapy of peritonitis.

Topics: Adult; Calcitriol; Calcium; Colony Count, Microbial; Female; Humans; Leukotriene B4; Macrophage Activation; Macrophages; Male; Middle Aged; Peritoneal Cavity; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Recurrence; Staphylococcus epidermidis; Superoxides; Uremia

Specific-pathogen-free kittens experimentally infected with feline infectious peritonitis virus (FIPV) subsequently demonstrated increased plasma levels of the arachidonic acid metabolites, leukotriene (LT) B4 and prostaglandin (PG) E2. Significant increases (P less than 0.025) in LTB4 plasma levels occurred in all (5/5) FIPV-inoculated kittens on postchallenge-exposure days (PCD) 7 and 14 vs PCD 0. Significant increases (P less than 0.05) in PGE2 plasma levels occurred in 80% (4/5) of FIPV-infected kittens on PCD 7 and 14. Maximal mean plasma levels of LTB4 and PGE2 occurred on PCD 7 (502.5 +/- 45.6 pg/ml and 1108.0 +/- 247.9 pg/ml, respectively). A positive correlation was found between LTB4 plasma levels and body temperature (r = 0.609, P less than 0.01). Mean survival time in FIPV-inoculated kittens was 19.4 +/- 3.2 days. Gross lesions, including peritoneal or pleural effusions (or both) and connective tissue edema, indicated an increased vascular permeability in the FIPV-infected kittens. Histologically, lesions were characterized by vasculitis or perivasculitis, vasodilatation, perivascular edema, and fibrinonecrotizing and pyogranulomatous inflammation. Immunofluorescent studies of tissues from FIPV-infected kittens demonstrated foci of polymorphonuclear leukocytes and FIPV-positive macrophages oriented around dilated blood vessels. Seemingly, arachidonic acid metabolites, including LTB4 or PGE2 released from macrophages, neutrophils or other cells, may be involved in the pathogenesis of FIP vascular and inflammatory lesions and in some of the clinical disease manifestations.

Topics: Animals; Antibodies, Viral; Cat Diseases; Cats; Coronaviridae; Coronaviridae Infections; Dinoprostone; Leukotriene B4; Peritoneum; Peritonitis; Specific Pathogen-Free Organisms; Time Factors

Ten frogs (Xenopus laevis) were injected with mixed bacteria to produce a septic peritonitis. Peritoneal inflammatory cells of eight animals were studied for monohydroxyeicosanoid and leukotriene production from exogenous arachidonic acid. Large amounts of 12-hydroxyeicosatetraenoic acid were produced; smaller amounts of 5- and 15-hydroxyeicosatetraenoic and leukotriene B4 were produced. Identifications were confirmed by retention times on HPLC, ultraviolet spectroscopy on all products, and gas chromatograph/mass spectrometry in the case of 12-hydroxyeicosatetraenoic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Chromatography, High Pressure Liquid; Female; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Peritoneal Cavity; Peritonitis; Xenopus laevis

Eicosanoid production by inflammatory cells which resulted from infection of the peritoneal cavity of Rana catesbeiana and Bufo americanus was studied after addition of exogenous arachidonic acid and for metabolites generated in vivo. From exogenous substrate, the cells of Rana catesbeiana produced substantial amounts of 5-hydroxyeicosatetraenoic acid, leukotriene B4, the non-enzymatic isomers of leukotriene B4 and leukotriene C4. From endogenous substrate, 5-hydroxyeicosatetraenoic acid and leukotriene B4 were produced. Cells from Bufo americanus produced leukotriene B4 and 5-hydroxyeicosatetraenoic acid, from both exogenous and endogenous substrate. These observations of in vivo eicosanoid production confirm the participation of 5-lipoxygenase activity in the inflammatory response to infection.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Bufonidae; Chromatography, High Pressure Liquid; Inflammation; Leukotriene B4; Peritonitis; Rana catesbeiana; SRS-A

Macrophages, isolated from dialysis fluid of three patients with continuous ambulatory peritoneal dialysis (CAPD) at different times during peritonitis were labelled with 14C-arachidonic acid and stimulated with the calcium ionophore A23187. The main metabolites formed by 5-lipoxygenase activity were leukotriene B4 (LTB4) and 5-hydroxy-6, 9, 11, 14-eicosatetraenoic acid (5-HETE). Smaller amounts of cyclooxygenase metabolites were present and also a major compound with an elution time between 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TxB2). This substance was isolated, analyzed by GC-MS and identified as 20-hydroxy-leukotriene B4 (20-OH-LTB4). This indicates that human peritoneal macrophages obtained from CAPD not only produce leukotrienes and prostaglandins, but also the omega-hydroxylase product of LTB4, which has been demonstrated to be present in polymorphonuclear leucocytes. The activity of this enzyme was not correlated with the severity of the peritonitis.

Topics: Aged; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Humans; Kidney Diseases; Leukotriene B4; Macrophages; Male; Middle Aged; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis

Inflammatory peritoneal exudates from 11 patients with purulent peritonitis or non-perforative appendicitis were analyzed by gas chromatography mass spectrometry (GC-MS) for their content of 5S, 12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) and its omega-oxidized catabolite, 5S,12R,20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-OH-LTB4). Eleven samples of peritoneal exudates were examined. LTB4 and 20-OH-LTB4 were demonstrated only in the samples from patients with purulent peritonitis. LTB4 was detected in 4 samples, and 20-OH-LTB4 was detected in 6 samples by GC-MS with selected ion monitoring. In addition, a nearly full spectrum corresponding to that of synthetic 20-OH-LTB4 was obtained with 3 samples. Neither LTB4 nor 20-OH-LTB4 were detected in samples from patients with non-perforative appendicitis. LTB4 or 20-OH-LTB4 may be related to the pathophysiological mechanism of purulent inflammations.

Topics: Appendicitis; Ascitic Fluid; Gas Chromatography-Mass Spectrometry; Humans; Inflammation; Leukotriene B4; Peritonitis