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Chronic obstructive pulmonary disease (COPD) is a worldwide threat. Cigarette smoke (CS) exposure causes cardiopulmonary disease and COPD and increases the risk for pulmonary tumors. In addition to poor lung function, patients with COPD are susceptible to bouts of dangerous inflammation triggered by pollutants or infection. These severe inflammatory episodes can lead to additional exacerbations, hospitalization, further deterioration of lung function, and reduced survival. Suitable models of the inflammatory conditions associated with CS, which potentiate the downward spiral in patients with COPD, are lacking, and the underlying mechanisms that trigger exacerbations are not well understood. Although initial CS exposure activates a protective role for vascular endothelial growth factor (VEGF) functions in barrier integrity, chronic exposure depletes the pulmonary VEGF guard function in severe COPD. Thus, we hypothesized that mice with compromised VEGF production and challenged with CS would trigger human-like severe inflammatory progression of COPD. In this model, we discovered that CS exposure promotes an amplified IL-33 cytokine response and severe disease progression. Our VEGF-knockout model combined with CS recapitulates severe COPD with an influx of IL-33-expressing macrophages and neutrophils. Normally, IL-33 is quickly inactivated by a post-translational disulfide bond formation. Our results reveal that BAL fluid from the CS-exposed, VEGF-deficient cohort promotes a significantly prolonged lifetime of active proinflammatory IL-33. Taken together, our data demonstrate that with the loss of a VEGF-mediated protective barrier, the CS response switches from a localized danger to an uncontrolled long-term and long-range, amplified, IL-33-mediated inflammatory response that ultimately destroys lung function.

IL-33 and its receptor ST2 play important roles in airway inflammation and contribute to asthma onset and exacerbation. The IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. MyD88 forms a complex with IL-R–associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88–IRAK4–IRAK2). The myddosome subsequently activates downstream NF-κB and MAPKs p38 and JNK. We established an asthma-like mouse model by intratracheal administration of IL-33. The IL-33 model has a very similar phenotype compared with the OVA-induced mouse asthma model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knockout mice, which were protected from the IL-33–induced asthma. We synthesized small molecule mimetics of the α-helical domain of IRAK2 with drug-like characteristics based on the recent advances in the designing of α-helix compounds. The mimetics can competitively interfere in the protein–protein interaction between IRAK2 and IRAK4, leading to disruption of Myddosome formation. A series of small molecules were screened using an NF-κB promoter assay in vitro. The lead compound, 7004, was further studied in the IL-33–induced and OVA-induced asthma mouse models in vivo. Compound 7004 can inhibit the IL-33–induced NF-κB activity, disrupt Myddosome formation, and attenuate the proinflammatory effects in asthma-like models. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases.

What is the central question of this study? Non-invasive, quantitative methods to assess right cardiac function in mice with pulmonary hypertension have not been demonstrated. What is the main finding and its importance? This study shows the potential of magnetic resonance imaging to estimate right ventricular ejection fraction and measure spatial, dynamic changes in cardiac structure in the Sugen 5416/hypoxia mouse model of pulmonary hypertension. Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery pressures and right heart failure. Mouse models of PAH are instrumental in understanding the disease pathophysiology. However, few methods are available to evaluate right cardiac function in small animals. In this study, magnetic resonance imaging was used to measure in vivo cardiac dimensions in the Sugen 5416/hypoxia mouse model. Pulmonary hypertension (PH) was induced in C57BL/6 mice by 3 weeks of exposure to 10% oxygen and vascular endothelial growth factor receptor inhibition (20 mg kg

Pulmonary arterial hypertension (PAH) is a condition characterized by elevated mean pulmonary artery pressure, pulmonary vascular remodeling and secondary right heart failure. Mouse models are inst...

Background Intranasal administration of aspirin-lysine has been used in Europe for the diagnosis of aspirin-exacerbated respiratory disease. It has a low adverse effect profile and is believed to be safer for asthmatic patients, particularly those with a low baseline forced expiratory volume in 1 second, in whom oral aspirin challenge would be contraindicated. Ketorolac, a nonsteroidal anti-inflammatory drug useful for severe pain, is available in the United States in parenteral form. Objectives To determine whether ketorolac nasal challenge has acceptable specificity and sensitivity for diagnosing aspirin-exacerbated respiratory disease. Methods Twenty-nine patients with suspected aspirin-exacerbated respiratory disease were challenged with nasal ketorolac before oral challenge and desensitization with aspirin. Symptoms, objective changes in nasal examination findings, and peak nasal inspiratory flow values were recorded. Nasal lavage fluid for cysteinyl leukotriene analysis was collected. Ketorolac doses of 2.1, 5.2, or 7.8 mg were administered and compared with the results of oral aspirin challenge. Results Eighteen patients had a positive challenge reaction to oral aspirin. Ketorolac nasal inhalation had a sensitivity of 78% and a specificity of 64%. Patients in the reactor group had significantly higher levels of cysteinyl leukotrienes after ketorolac challenge than in the nonreactor group. Mild bronchospasm occurred in 3 patients, and 2 of these occurred at higher starting doses of ketorolac. Conclusions Nasal ketorolac administration is a reasonably accurate and safe method for diagnosing aspirin-exacerbated respiratory disease.

We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. We conclude that LPS enhances FLAP gene expression via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes.

While effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty-four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS-ODN or control oligodeoxynucleotide (C-ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS-ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS-ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C-ODN. In addition, splenocytes from ISS-ODN but not C-ODN treated mice displayed attenuated OVA-specific interleukin (IL)-4, IL-5, and IL-13 but increased interferon-gamma responses. Finally, ISS-ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2-biased immune parameters towards Th1 dominance. As ISS-ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS-ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.

Significant glomerular vasoconstriction and production of reactive oxygen species has been known to occur with exposure to anti-glomerular basement membrane antibody (AGBM-Ab) in the rat model. Previously published studies have demonstrated that such effects can be reduced by therapy with phentolamine, an alpha-adrenergic antagonist. It was hypothesized that antioxidant pretreatment with water-soluble probucol would improve glomerular hemodynamics 60 to 90 min after the administration of AGBM-Ab. These relationships were examined with both in vivo renal micropuncture and in vitro studies in rats. Single-nephron GFR (SNGFR) decreased markedly in untreated rats after AGBM-Ab as a result of afferent and efferent arteriolar vasoconstriction with consequent reductions in nephron plasma flow (SNPF) and decreases in the glomerular ultrafiltration coefficient (LpA). Basal SNGFR was increased, and SNGFR was significantly higher after AGBM-Ab in probucol-treated versus untreated rats. This finding was due solely to higher values for SNPF and prevention of afferent arteriolar constriction. A reduction in LpA after AGBM-Ab was not prevented by probucol treatment. In vitro analyses of glomeruli revealed reduced myeloperoxidase activity in antioxidant-treated rats. Lipoxygenase activity and leukotriene products, however, were not changed by antioxidant therapy, yet vasoconstriction was prevented. H(2)O(2) generation before and after formyl-methionyl-leucyl-phenylalanine stimulation was significantly reduced before and after AGBM-Ab in glomeruli harvested from rats that were treated with the antioxidant. Antioxidant therapy in this model of AGBM-Ab injury did not prevent reductions in LpA, an index of glomerular membrane damage, but did prevent afferent arteriolar vasoconstriction. Reactive oxygen species generation was reduced by probucol. The specific mechanisms whereby antioxidant therapy ameliorates glomerular hemodynamic effects will be defined in additional studies and is likely to involve either enhanced vasodilator or diminished vasoconstrictor activity.

We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance that leads to right ventricular failure. PAH can be a primary disease (idiopathic, IPAH) or secondary to conditions such as connective tissue disease. Interleukin-33 (IL-33), which is the ligand for the ST2 receptor, is a cytokine that acts as an alarmin. Since PAH is associated with the infiltration of inflammatory cells and increased cytokines, we aimed to investigate the role of IL-33 in PAH. Using an IL-33 ELISA we found that IL-33 is increased in the serum of PAH patients, in particular IPAH [1798 +/-846 pg/ml (n=6) vs. 24 +/- 13 pg/ml (n=6), p

The cysteinyl leukotrienes (LT), LTC 4 , LTD 4 , and LTE 4 , are oxygenated metabolites of arachidonic acid derived from the 5-lipoxygenase (5-LO) pathway (Figure 1) (1). The term leukotriene was originally proposed by Samuelsson as a trivial name to describe these compounds that are derived from leukocytes and are conjugated trienes (2). 5-LO is an iron-containing enzyme most often found free in the cytosol of cells of myeloid origin (3). With cellular activation, this enzyme translocates to the nuclear envelope (4). Once translocated, it is responsible for the first committed steps of the 5-LO pathway in association with the 5-LO–activating protein (FLAP). The precise role of FLAP remains obscure; however, it is required for appreciable 5-LO activity in intact inflammatory cells (5). 5-LO inserts molecular oxygen at the carbon 5 position of arachidonic acid forming the hydroperoxy-intermediate, 5-hydroperoxyeicosatetraenoic acid. This intermediate is further metabolized by 5-LO via a dehydration reaction to form the epoxy intermediate, LTA 4 . LTA 4 can be further metabolized to LTB 4 by the enzyme LTA 4 hydrolase, an epoxide hydrolase enzyme with a ubiquitous distribution (6). The role of LTB 4 in asthma remains to be clarified. LTA 4 can also be metabolized to LTC 4 by the enzyme LTC 4 synthase (7). This enzyme is a microsomal glutathioneS -transferase that belongs to a larger family of membrane-bound proteins involved in eicosanoid and glutathione metabolism (8). LTC 4 synthase appears to have a more restricted distribution than 5-LO and appears to be limited to eosinophils, mast cells, basophils, mononuclear phagocytes, and some leukemic cells lines such as THP-1 (9) and KG-1 (10). The concept that lipid soluble mediators may play a role in asthma is not new. Van Leeuwen and Zeydner demonstrated that alcohol extracts of blood obtained from subjects with asthma, but not healthy individuals, contained a substance that caused contraction of smooth muscle (11). Moreover, Harkavy reported in 1930 that alcohol extracts of sputum obtained from asthmatics during exacerbation also contained a substance that could induce sustained smooth muscle contraction, which he termed “spasm-producing substance” (12). In studies performed by Feldberg and Kellaway (13, 14), and subsequently by Kellaway and Trethewie (15), a substance that could induced sustained smooth muscle contraction was present in the blood of antigen-challenged guinea pigs. These authors coined the term “slow-reacting smooth-muscle stimulating substance in anaphylaxis,” which ultimately was refined to “slow-reacting substance of anaphylaxis” (SRS-A) by Brocklehurst (16). The subsequent forty years of research and major efforts of many investigators culminated in the characterization of SRS-A as the cysteinyl leukotrienes C 4 , D 4 , and E 4 (17). These compounds mediate sustained smooth muscle contraction, mucus hypersecretion, airway edema (18), and eosinophil recruitment (19). Since 1979 a substantial body of evidence has accumulated demonstrating that the cysteinyl LTs are increased in the sputum (20), lung lining fluid (21), blood, and urine (22) of asthmatics when compared with healthy subjects. Aspirin-intolerant asthma (AIA) is a syndrome of aspirin sensitivity, asthma, and nasal polyposis (23). Individuals with this syndrome often have profound systemic reactions when challenged with aspirin. Reactions to aspirin may include cutaneous and gastrointestinal manifestations, in addition to potentially life-threatening acute airway obstruction. The onset of AIA is usually beyond the third decade, and these individuals commonly have severe asthma, which may require systemic corticosteroids. We now recognize that this is not an allergically mediated phenomenon, but is instead a disorder that can occur with any nonsteroidal anti-inflammatory drug that acts via inhibition of cyclo-oxygenase. However, the molecular mechanism(s) involved in the pathogenesis of AIA remains unclear (23). The initial hypothesis suggested that AIA was due to a shunt of arachidonic acid from the cyclo-oxygenase pathway to the lipoxygenase pathway (24). We now understand that the pathogenesis of AIA is more complex. Utilization of arachidonic acid within the cell is a highly ordered process with multiple regulatory steps, and simple shunting of substrate does not appear to play a major role. Additional hypotheses have been put forth to explain AIA. For example, studies suggest that a failure of cyclooxygenase-2 expression in the nasal mucosa of patients with AIA may be a feature (25). The largest body of evidence, however, supports a central role for cysteinyl LT synthesis and an enzyme responsible for this synthesis, LTC 4 synthase, in AIA. AIA patients have markedly elevated cysteinyl LT levels at baseline that increase dramatically with aspirin challenge (26). Moreover, studies have demonstrated that LTC 4 synthase is strikingly increased in eosinophils within the airways of aspirin-intolerant asth( Received in original form July 10, 2000 )

The goal of this study was to determine whether cytokines modulate leukotriene C4 (LTC4) synthase expression in mononuclear phagocytes. A panel of cytokines was surveyed for changes in LTC4 synthase mRNA in THP-1 cells. TGF-β1, -2, and -3 had significant stimulatory effects. The addition of TGF-β resulted in a time-dependent increase in LTC4 synthase mRNA at 6 h, which persisted through 48 h. Furthermore, this conditioning resulted in an increase in immunoreactive protein for LTC4 synthase through 7 days. TGF-β conditioning of cells resulted in a time- and dose-dependent increase in stimulated LTC4 synthase activity. Following transient transfection of THP-1 cells with a promoter-reporter construct containing 1.2 kb of the LTC4 synthase promoter, TGF-β treatment resulted in a 2-fold increase in reporter activity. Conditioning with TGF-β did not prolong the half-life of LTC4 synthase mRNA, as assessed by RNase protection assays in actinomycin D-treated cells. Cycloheximide exposure experiments revealed that new protein synthesis was not required for the observed stimulatory effect of TGF-β on LTC4 synthase mRNA. We conclude that LTC4 synthase expression is increased at a transcriptional level by TGF-β in mononuclear phagocytes.

The aim of this study was to assess the effect of dexamethasone on 5-lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP-1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium-ionophore-stimulated 5-lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and its activating protein (5-LAP) was also assessed. Dexamethasone increased the stimulated release of 5-lipoxygenase products from both monocytes and THP-1 cells in a dose-dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4-hydrolase-immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5-lipoxygenase pathway expression in both monocytes and in THP-1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP. These results suggest a role for glucocorticoids in the regulation of 5-lipoxygenase pathway expression in mononuclear phagocytes.

The goal of this investigation was to assess the effect of leukotriene B4 (LTB4) on 5-lipoxygenase activity and to examine the possible mechanisms of this effect. Exogenous LTB4 significantly increased the release of endogenous LTB4 from A-23187-stimulated neutrophils. The 5-lipoxygenase product release from A-23187-stimulated neutrophils decreased in the presence of an LTB4 receptor antagonist, suggesting that LTB4 has a receptor-mediated, autocrine effect on 5-lipoxygenase activity. Neutrophil 5-lipoxygenase activity increased significantly as cell density increased. In the presence of exogenous LTB4, no significant change in [14C]arachidonic acid release from neutrophils was observed. Exogenous LTB4 increased the amount of immunoreactive 5-lipoxygenase protein detected in the nuclear fraction of disrupted cells. LTB4 receptor antagonism decreased the amount of immunoreactive 5-lipoxygenase detected in the nuclear fraction. Thus LTB4 exerts an autocrine, receptor-mediated, costimulatory effect on 5-lipoxygenase activity. This feedback appears to have biological significance and involves enhanced 5-lipoxygenase translocation to the nuclear membrane.

Cysteinyl leukotrienes (LT) are mediators involved in inflammatory and allergic disorders. LTC4 synthase catalyzes the first committed step in the synthesis of these inflammatory mediators, and its cellular distribution appears to be unique. A human genomic library was screened by polymerase chain reaction (PCR) with primers that were designed based on the reported cDNA sequence for the LTC4 synthase gene. The gene was identified in one clone by Southern blotting of restriction enzyme digests, subcloning of fragments containing regions of interest, and DNA sequencing of these sub-clones. The transcription initiation site was detennined by primer extension analysis. Chromosome location was determined by fluorescent in situ hybridization and screening of somatic cell hybrids by PCR. The LTC4 synthase gene is ∼2.5 kb in length, consisting of five exons (136, 100, 71, 82, and 257 bp, respectively) and four introns (1,447, 102, 84, and 230 bp, respectively). Transcription initiation occurs at a single site 78 bp upstream of the coding region. The 5′-flanking region contains neither a TATA nor a CAAT box. The first 1 kb of the 5′-flanking region, however, contains putative DNA binding motifs for SP-1, AP-1, AP-2, ets factors, and CREB/ATF. A STAT binding motif is present in the first intron. The LTC4 synthase gene is located in the distal region of the long arm of chromosome 5 in 5q35. The LTC4 synthase gene does not contain elements of a typical regulated gene and may therefore contain novel regulatory elements. This gene is also located in a region on chromosome 5 that appears to play a role in allergic and inflammatory disorders, such as asthma.

The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5-lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5-lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3.

We have previously shown that human airway epithelial cells contain leukotriene A4 (LTA4) hydrolase activity. To characterize this activity further, airway epithelial cells, cultured to confluence, were disrupted by sonication and were fractionated at 15,000 and 100,000 x g. Enzymatic activity was assessed by incubating fractions with 15 microM LTA4 at 37 degrees C for 15 min. LTA4 hydrolase activity was present in the 15,000 x g and the 100,000 x g supernatants and was inactivated by heating at 56 degrees C or by pronase, as is the case for neutrophil LTA4 hydrolase. However, the epithelial cell enzyme had a slower time course for product generation and demonstrated a different dose-response relationship to substrate when compared with the neutrophil. Kinetic analysis revealed nonlinear plots for epithelial data, most consistent with an enzyme that has multiple active sites. Immunoblotting, performed with anti-neutrophil LTA4 hydrolase antibody, recognized two bands in epithelial cell 15,000 x g supernatant (M(r) of 69,000 and 110,000-115,000). When resolved by gel filtration chromatography, only the M(r) 69,000 protein had enzymatic activity. Anion exchange chromatography of epithelial cell samples revealed that LTA4 hydrolase and aminopeptidase activity did not co-elute, whereas one of three peaks of aminopeptidase activity did co-elute in chromatograms of neutrophil samples. Immunoblots of proteolytic digests of partially purified M(r) 69,000 protein from epithelial cells and neutrophils revealed different immunoreactive bands. The digest of the M(r) 110,000-115,000 protein revealed no immunoreactive bands. Repeat kinetic analysis on 179-fold purified epithelial LTA4 hydrolase again revealed that it lacked significant aminopeptidase activity and retained its unique kinetic properties.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined cell-free human bronchoalveolar lavage fluid (BALF) for enzymes of the 5-lipoxygenase pathway. BALF was obtained from six patients who were active smokers and six nonsmokers. Enzymatic activity in cell-free BALF was assessed by specific assays for leukotriene (LT) A4 hydrolase, 5-lipoxygenase, and LTC4 synthase using HPLC. Only LTA4 hydrolase enzymatic activity was found. This activity ranged from 101 to 667 when expressed as picomoles of LTB4 produced per milliliter BALF. Enzymatic activity in smokers vs nonsmokers was 484 +/- 120 vs 129 +/- 32 pmol LTB4/ml BALF (mean +/- SD, P < 0.0001). There were no leukotrienes found in BALF before assay. Immunoblot analysis revealed an immunoreactive band at a relative molecular mass of 69,000 D in all samples, consistent with LTA4 hydrolase, but no evidence of 5-lipoxygenase. BALF had greater LTA4 hydrolase activity per milligram of protein than neutrophil cytosol, epithelial cell cytosol, plasma, or serum. The synthesis of LTB4 was significantly increased when neutrophils were stimulated in BALF. These data indicate the selective presence of LTA4 hydrolase in BALF which is significantly increased in smokers. This enzyme in BALF may contribute to the inflammatory response in tobacco-related lung disease.

Context. Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties. Objective. We are developing two new cyanide antidotes cobinamide, a vitamin B12 analog, and sulfanegen, a 3-mercaptopyruvate prodrug. Both drugs can be given by intramuscular administration, and therefore could be used to treat a large number of people quickly. We now asked if the two drugs would have an augmented effect when combined. Materials and methods. We used a non-lethal and two different lethal models of cyanide poisoning in mice. The non-lethal model assesses neurologic recovery by quantitatively evaluating the innate righting reflex time of a mouse. The two lethal models are a cyanide injection and a cyanide inhalation model. Results. We found that the two drugs are at least additive when used together in both the non-lethal and lethal models: at doses where all animals died with either drug alone, the combination yielded 80 and 40% survival in the injection and inhalation models, respectively. Similarly, drug doses that yielded 40% survival with either drug alone, yielded 80 and 100% survival in the injection and inhalation models, respectively. As part of the inhalation model, we developed a new paradigm in which animals are exposed to cyanide gas, injected intramuscularly with an antidote, and then re-exposed to cyanide gas. This simulates cyanide exposure of a large number of people in a closed space, because people would remain exposed to cyanide, even after receiving an antidote. Conclusion. The combination of cobinamide and sulfanegen shows great promise as a new approach to treating cyanide poisoning. © 2011 Informa Healthcare USA, Inc.

Asthma is a major health problem worldwide that has increased. Much of the prior focus in asthma research has been on adaptive, antigen-dependent immune responses. Recent work suggests that the innate, non-antigen-dependent immune system responses play a critical role in asthma pathogenesis. Here we will focus on key pathways within the innate immune system and their interface with adaptive immunity, highlighting how the innate immune system modulates adaptive allergic immune responses. In particular, we review data on the role of toll-like receptor signaling, including accessory pathways and TRIF-IRF, in modulating allergic pulmonary responses. We will briefly review the role of the growing field of bioinformatics in identifying networks of immune genes involved in allergic responses.

The inflammatory mediator, adenosine, induces chloride secretion from the human colonic epithelial cell line, T84, in a manner apparently independent of increases in adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, or cytoplasmic Ca2+. This prompted a search for other messengers that might account for the secretory response. A possible role for arachidonic acid or a metabolite in the response to adenosine has been demonstrated 1) by showing a relationship between arachidonic acid mobilization and chloride secretion induced by the adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and 2) by determining that exogenous arachidonic acid affects T84 cell function. Addition of NECA to T84 cells induces chloride secretion and release of radioactivity from cells preloaded with [3H]arachidonic acid with similar dose dependencies. The effect of NECA on chloride secretion is inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide or the diglyceride lipase inhibitor RG80267 but is unaffected by inhibitors of lipoxygenase or cyclooxygenase. Arachidonic acid has a small but significant effect on chloride secretion when added alone to T84 cells and synergistically enhances, as does NECA, responses to calcium-dependent secretogogues. Thus receptor-stimulated release of arachidonic acid in T84 cells may provide a second-messenger system promoting chloride secretion, in addition to calcium and cyclic nucleotides.

In view of conflicting reports concerning the effect of macrophage activation on arachidonic acid metabolism, we examined the effect of the macrophage activator, interferon-gamma (IFN-gamma), on the 5-lipoxygenase pathway in rat lung macrophages. Rat lung macrophages were conditioned in the presence or absence of 10(2) U/ml IFN-gamma for 4 h before stimulation with 1 microM A23187 for 15 min or 100 micrograms/ml opsonized zymosan for 60 min at 37 degrees C as well as other stimuli. Lipoxygenase products in extracted cell supernatants were identified and analyzed by high-pressure liquid chromatography and ultraviolet spectroscopy. The predominant lipoxygenase products included leukotriene (LT) B4, LTC4, and 5-hydroxyeicosatetraenoic acid (5-HETE). These products were not qualitatively altered by conditioning with IFN-gamma. However, 5-lipoxygenase pathway activity, as measured by LTB4 release, was maximally increased 2-fold after conditioning with IFN-gamma and stimulating with either A23187 or opsonized zymosan. IFN-gamma-conditioned macrophages, stimulated with A23187, released greater quantities of lipoxygenase products in comparison with control cells (307.6 +/- 13.3 versus 167.6 +/- 3.9 pmol LTB4/10(6) cells) (mean +/- SEM) (P less than 0.05). Similar results were obtained with the less potent stimulus, opsonized zymosan. IFN-gamma had no direct stimulatory effect on the 5-lipoxygenase pathway. No effect was observed with a variety of other stimuli with or without IFN-gamma conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Asthma remains a major health problem worldwide that has increased in developed countries. Much of the focus in asthma research in the past has been on adaptive, antigen-dependent immune responses. Recent work suggests that the innate, non-antigen-dependent immune system plays a critical role in asthma pathogenesis. Here we will highlight innate receptors and cells in the context of allergic responses. Reviewing animal models and human studies, we focus on interactions of innate and adaptive immunity.

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi–stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC4 synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC4 release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC4 synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC4 synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC4 synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-α and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-κB activation resulted in inhibition of the LPS effect, while activation of NF-κB and p50/p65 overexpression down-regulated the LTC4 synthase gene. LPS down-regulates cysteinyl LT release and LTC4 synthase gene expression in mononuclear phagocytes by an NF-κB-mediated mechanism.

We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.

The 5-lipoxygenase pathway is the source of potent pro-inflammatory mediators. More than 20 years ago, the details of the 5-lipoxygenase pathway began to emerge from seminal work performed by Borgeat and Murphy while they were working in the laboratory of Bengt Samuelsson ([Borgeat and Samuelsson

A large body of experimental evidence has implicated the cysteinyl leukotrienes in a variety of inflammatory responses201C;2. Leukotriene C4 (LTC4) synthase (Mr of 16,568; EC#2.5.1.37) is a selective, membrane-bound glutathione-S-transferase that catalyzes the conversion of LTA4 to LTC4. Although LTC4 synthase enzymatic activity is found in various cell types, mRNA for LTC4 synthase is present only in eosinophils, basophils, mast cells, and cells of monocyte/macrophage lineage. Previous studies suggest that LTC4 synthase expression is increased in the bronchial mucosa of patients with aspirin-sensitive asthma3. The gene for LTC4 synthase has been cloned, sequenced, and mapped to the distal region of chromosome 54’5. The LTC4 synthase 5’ flanking region lacks a TATA box but has known consensus sites for ets, AP-1, AP-2, and Sp14’5. Previous work from our laboratory indicates that LTC4 synthase gene expression is upregulated by transforming growth factor-s (TGF-s) through a transcriptional mechanism in the monocyte-like cell line, THP-16. The purpose of this study was to begin to investigate the molecular mechanisms of regulation of transcription of the LTC4 synthase gene in mononuclear phagocytes.

The goal of this study was to examine the mechanisms of leukotriene C(4) (LTC(4)) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC(4) synthase promoter-reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between -92 and -23 bp, which contains a signal protein (Sp)1 consensus site at -42 to -37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from -56 to -17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 cell line with a construct containing the -92 to -23 bp promoter region and Sp expression vectors revealed that Sp1 and Sp3 transactivate gene transcription. We conclude that the Sp1 site is a necessary element for LTC(4) synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC(4) synthase gene is transcriptionally regulated in mononuclear phagocytes.

The aim of this study was to investigate the regulation of the 5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the monocyte-like cell line, THP-1. When THP-1 cells were incubated over 4–7 days in 10% supernatant from lectin-activated human lymphocytes, their capacity to synthesize 5-lipoxygenase products was significantly increased. In contrast, the supernatant from nonactivated lymphocytes had no effect. The increase in capacity to synthesize 5-lipoxygenase products was mimicked by the addition of either granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3. These increases in synthetic capacity reflected increased enzymatic activity. Increased immunoreactive protein and mRNA for the enzymes 5-lipoxygenase and 5-lipoxygenase-activating protein were also found in cells conditioned with activated lymphocyte supernatants. Furthermore, the increase in mRNA for both enzymes was not blocked by cycloheximide, suggesting that the effect on steady-state mRNA levels does not require the synthesis of new protein. The increase in mRNA could be reproduced by GM-CSF. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in THP-1 cells over a period of days via the release of soluble factors.

Previous studies in mononuclear phagocytes have shown that macrophages have substantially greater 5-lipoxygenase activity than monocytes and that this is associated with greater amounts of 5-lipoxygenase and its activating protein (FLAP). The aim of this study was to examine the effect of mononuclear phagocyte maturation in vitro on 5-lipoxygenase expression. At baseline, monocytes had significant 5-lipoxygenase activity, but then lost all detectable 5-lipoxygenase activity over 7 days. Immunoblot and Northern blot analysis revealed that immunoreactive protein and mRNA for both 5-lipoxygenase and FLAP were significantly decreased over time. These studies demonstrate that in vitro differentiation of monocytes into a macrophage phenotype is not accompanied by the enhanced expression of 5-lipoxygenase and FLAP seen in macrophages derived from in vivo sources. In fact, baseline expression of 5-lipoxygenase and FLAP by monocytes is lost in vitro. These studies have clear implications for the use of cultured monocytes as a model of macrophages, and they also further our understanding of the regulation of the 5-lipoxygenase pathway.

Leukotrienes are a family of oxygenated metabolites derived from the 5-lipoxygenase pathway of arachidonic acid metabolism via the unstable epoxide intermediate 5(S)5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid or leukotriene A4 (LTA4). Leukotrienes have potent effects on inflammatory cell Chemotaxis, adherence, and activation. They also have effects on noninflammatory cells and tissues thus increasing vascular permeability1, inducing smooth muscle contraction, modulating T- and B-cell function2, augmenting natural killer activity3, and inducing fibroblast Chemotaxis4. In humans, the distribution of the 5-lipoxygenase enzyme appears to be limited to inflammatory cells1. Within these cells, the 5-lipoxygenase enzyme is stimulated by millimolar calcium concentrations and ATP 5, associating with the cell membrane via the 5-lipoxygenase-activating protein6. The enzyme then inserts molecular oxygen at the C5 position of unesterified arachidonic acid and, in turn, further converts the resultant hydroperoxide, 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE), into LTA4. This epoxide is the pivotal intermediate in the 5-lipoxygenase pathway and can be metabolized within inflammatory cells to 5(S),12(R)-dihydroxy-6, 14-cis, 8, 10-trans-eicosatetraenoic acid (leukotriene B4, LTB4) by LTA4 hydrolase or to 5(S)-hydroxy-6(R)-S-glutathionyl-7, 9-trans-ll, 14-cis-eicosatetraenoic acid (leukotriene C4, LTC4) by glutathione-S-transferase 1.

Pinkie is a recessive ENU-induced mutation, homozygotes develop premature graying and subsequently, progressive alopecia. Histologically, the skin is characterized by the destruction of hair follicle architecture, formation of dermal cysts and appearance of black papules throughout the ventral skin late in life. The Pinkie mutation was mapped to the retinoid X receptor alpha (Rxra) locus (chromosome 2) on 700 meioses and positionally cloned. As a component of the nuclear receptors for retinoic acid and other bioactive molecular ligands, RXRa plays an irreplaceable role during embryogenesis and a null allele of RXRa invariably causes embryonic lethality. Pinkie, which results from the amino acid substitution N273I in the ligand-binding domain, is the first viable germline hypomorphic mutation of RXRa. Transfection studies suggest that the abnormal protein retains only 10 to 20% of the activity of the wild type receptor. Pinkie homozygotes develop a progressive phenotype, part of which entails a severe, age-dependent imbalance of Th1/Th2 differentiation upon antigenic challenge. When immunized with ovalbumin (OVA), pinkie homozygotes displayed markedly decreased IgG1 levels in serum as compared with normal C57BL/6 mice; in vitro OVA re-stimulation of pinkie splenocytes causes greatly enhanced IFN-g production and decreased IL-4 production. Upon MCMV infection, pinkie homozygotes showed significantly higher type I interferon levels in serum than normal C57BL/6 mice. The in vitro differentiation of naïve CD4 T cells demonstrated skewed Th1 development in pinkie mouse compared to that of C57BL/6 control animal. However, real-time PCR didn’t reveal significant difference in mRNA level of GATA-3 and T-bet, which are believed to be critical transcription factors in Th1/Th2 development. These data collectively imply that RXRa is required for Th2 development. This requirement might reflect an influence of RXRa on the expression of pertinent transcription factors (e.g., GATA-3, T-bet), cytokines (IL-4, IFN-gamma) and/or cytokine receptors that are involved in Th1/Th2 differentiation. RXRa represents a likely interface between the nutritional environment and the immune status of the host. Consumption of retinoids and vitamin D may therefore be expected to influence the development of the allergic/atopic (Th2) phenotype, and provide a plausible explanation for the increasing frequency of atopic disease observed in developed countries.

The effect of lung macrophages stimulated with calcium ionophore on parasympathetic contractile response of canine bronchial rings was studied. Macrophages augmented the contraction induced by electrical field stimulation, an effect that was inhibited by indomethacin and by SQ29548, a thromboxane A2 receptor antagonist, but had no effect on the contractile response to exogenous acetylcholine. These results suggest that macrophage-derived thromboxane A2 facilitates cholinergic neurotransmission prejunctionally in airway smooth muscle.

Pulmonary mucormycosis is an uncommon, but important, opportunistic fungal pneumonia which is often diagnosed post-mortem. This review emphasizes clinical and pathologic characteristics of pulmonary mucormycosis that differentiate this infection from other fungal pneumonias. The most common clinical presentation of pulmonary mucormycosis is a rapidly progressive pneumonia with diffuse infiltrates on chest radiographic examination of a patient with an underlying hematologic malignancy treated with immunosuppressive drugs. Other immunocompromised hosts at risk for pulmonary mucormycosis include patients with diabetes mellitus who may develop a distinctive endobronchial form of this disease. Early consideration of this diagnosis, along with aggressive diagnostic evaluation, are critical to effective therapy and patient survival. While treatment with amphotericin B is the mainstay of therapy for pulmonary mucormycosis, diabetics with endobronchial disease may benefit from early, aggressive surgical resection of the involved lung tissue.

Human tracheal epithelial cells were incubated with LTA4 and metabolic products were identified in extracted supernatants by high pressure liquid chromatography, ultraviolet spectroscopy, and gas chromatography-mass spectrometry. In the presence of epithelial cells, LTA4 was converted to LTB4, but not to LTC4 or LTD4. Maximum LTB4 was released at an LTA4 concentration of 3 μM and had occurred by 30 min. LTB4 release was increased in the presence of albumin, but was not affected by extracellular calcium or A23187. This LTA4 hydrolase activity had a slower time course and could not be clearly inactivated by repeated exposure to substrate as is the case for previously described LTA4 hydrolase enzymes. This hydrolase appears to have novel biochemical characteristics.