Increased expression of the 5-lipoxygenase pathway and its cellular localization in Barrett's adenocarcinoma

Boger P C, Shutt J D, Neale J R, Wilson S J, Bateman A C, Holloway J W, Patel P & Sampson A P
(2012) Histopathology  61, 509–517 Increased expression of the 5‐lipoxygenase pathway and its cellular localization in Barrett’s adenocarcinoma Aims: Up‐regulation of the 5‐lipoxygenase (5‐LOX) leukotriene pathway is evident in numerous tumour types, and has been linked to the promotion of cancer cell growth. The aim of this study was to evaluate the immunohistochemical expression of 5‐LOX pathway proteins in oesophageal adenocarcinoma and its premalignant lesion, Barrett’s metaplasia. Methods and results: Tissue samples were collected at endoscopy from 16 patients with Barrett’s metaplasia and from seven with oesophageal adenocarcinoma; five proximal squamous oesophagus samples were used as controls. Immunohistochemical analyses were performed on stromal and epithelial areas with optimized concentrations of primary antibodies for 5‐LOX, 5‐LOX‐activating protein (FLAP), and the distal enzymes leukotriene (LT) A₄ hydrolase (LTA₄H) and LTC₄ synthase (LTC₄S). the diagnosis was histologically confirmed from adjacent sections by a gastrointestinal pathologist. Striking increases in the stromal immunoexpression of 5‐LOX (P = 0.041), FLAP (P = 0.038), LTA₄H (P = 0.0008) and LTC₄S (P = 0.036) were seen in adenocarcinoma tissue. Stromal FLAP and LTA₄H immunostaining correlated with elevated neutrophil counts (P < 0.001). LTC₄S was also notably overexpressed within epithelial cells in both Barrett’s metaplasia (P < 0.001) and adenocarcinoma (P < 0.01) tissue. Conclusions: Key biosynthetic enzymes of the LTB₄ and LTC₄ biosynthetic pathways are incrementally expressed across the spectrum of squamous, Barrett’s metaplasia and oesophageal adenocarcinoma tissues, suggesting, for the first time, a role for both LT subfamilies in disease progression.     

Mode of action of the leukotriene synthesis (FLAP) inhibitor BAY X 1005: Implications for biological regulation of 5-lipoxygenase

Five-lipoxygenase (5-LOX) inhibition is gaining increasing importance as a novel approach to therapy of allergic asthma and other inflammatory diseases. Presently, two types of inhibitors are known, direct 5-LOX inhibitors (LOI) and the FLAP (five lipoxygenase activating protein) binding leukotriene synthesis inhibitors (LSI). The 5-LOX selective and orally active quinoline LSI, BAY X 1005, shares many mechanistic features with the indole LSI, MK-886. The binding of BAY X 1005 to FLAP correlates with LTB₄ synthesis inhibition. BAY X 1005 has been shown to bind to the 18 kD protein FLAP. BAY X 1005 inhibits 5-LOX translocation from the cytosol to membranes and reverses 5-LOX translocation. The use of BAY X 1005 has helped to elucidate part of the complex FLAP/5-LOX interaction by showing that FLAP appears to represent a 5-LOX substrate transfer protein channelling endogenous and exogenous arachidonic acid to the leukotriene synthetizing 5-LOX. This notion presented by our group in 1992 has stimulated further mechanistic studies. These findings have additionally led to the hypothesis that substrate competition is not confined to the LSI/FLAP interaction but may also be true for the LOI/5-LOX interaction and that even mixed LSI/LOI 5-LOX inhibitors are feasible, yet have not been described. Further mechanistic work on LSI will be orientated not only to further elucidate the complex FLAP/5-LOX interaction, but also to identify FLAP-related eicosanoid binding proteins.Major part presented at the 2nd International Symposium on Trends in Eicosanoid Biology: Leukotrienes as Mediators of Asthma and Inflammation: Basic and Applied Research, September 7–12, 1992, Interlaken, Switzerland     

Myrica rubra leaves as a potential source of a dual 5-LOX/COX inhibitor

Myrica rubra Sieb. et Zucc. is a valuable fruit tree that is used in Chinese, Japanese and Taiwanese traditional medicine. We investigated the anti-inflammatory activity of M. rubra leaves extracted with four different solvents. Total phenolics were determined using the Folin–Ciocalteu method. Extracts were investigated for their inhibitory activity toward the pro-inflammatory enzymes cyclooxygenase-1 and -2 (COX-1, COX-2) and 5-lipoxygenase (5-LOX). The ethanol extract of M. rubra leaves demonstrated a strong inhibition of prostaglandin E₂ (PGE₂) biosynthesis catalyzed by both COX-1 (93.42%) and COX-2 (75.71%) and leukotriene B₄ (LTB₄) formation catalyzed by 5-LOX (82.72%). Further we identified selective COX-1 inhibition by the n-butanol and aqueous fractions of the ethanol extract (with an IC₅₀ for COX-1 inhibition of 1.07 and 0.71?µg?mL^?1 _, respectively) and dual 5-LOX/COX inhibition by the ethyl acetate fraction (with an IC₅₀ of 3.29 for COX-1, 2.54 for COX-2 and 8.30?µg?mL^?1 for 5-LOX).     

Anti-CD45 antibody enhances lipoxygenase pathway of human naïve mononuclear cells and cyclooxygenase pathway of neutrophils

Objective and Design: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human na?ve MNC and PMN. Materials and Methods: Human na?ve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab’)₂ antibody or non-specific IgG F(ab’)₂ for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A₄ hydrolase (LTA₄ hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE₂ and LTB₄ in cultured supernatants was measured by EIA kits. Results: Anti-CD45 IgG F(ab’)₂ up-regulated LTA₄ hydrolase mRNA expression and LTB₄ production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of na?ve MNC compared to non-specific IgG F(ab’)₂. In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway. Conclusions: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA₄ hydrolase in MNC were enhanced. Received 4 May 2005; returned for revision 29 June 2005; returned for final revision 12 October 2005; accepted by M. Katori 16 November 2005     

Dual inhibition of 5-lipoxygenase/cyclooxygenase by a reconstituted homeopathic remedy; possible explanation for clinical efficacy and favourable gastrointestinal tolerability

Objective: In order to elucidate potential anti-inflammatory activities of Zeel comp. N and its constituents, the inhibition of the synthesis of Leukotriene B₄ (LTB₄) and Prostaglandin (PGE₂) by 5-lipoxygenase (5-LOX) and cyclo-oxygenase 1 and 2 (COX 1 and 2) respectively were examined in vitro.Materials: Human HL-60 cells, differentiated for 6–8 days with DMSO (1.2% v/v) were used for the 5-LOX assay. The COX activity assays were carried out with purified enzymes, COX 1 (ram seminal vesicles), COX 2 (sheep placenta) and with human THP-1 cells, differentiated for 24 h with PMA (50 nM).Methods: LTB₄ and PGE₂ production in the 5-LOX and COX assays respectively were determined by enzyme linked immunoassays.Results: A reconstituted Zeel comp. N combination as well as its constituent mother tinctures of Arnica montana, Sanguinaria canadensis and Rhus toxicodendron (Toxicodendron quercifolium) showed distinct inhibitory effects on the production of LTB₄ by 5-LOX (IC₅₀ values of 10, 20, 2 and 5 µg/ml respectively) and on the synthesis of PGE₂ by COX 1 (IC₅₀ values of 50, 80, 40 and 20 µg/ml respectively) and COX 2 enzymes (IC₅₀ values of 60, 110, 50 and 20 µg/ml respectively). The mother tincture of Solanum dulcamara inhibited the production of PGE₂ by COX 1 (IC₅₀ 40 µg/ml) and COX 2 (IC₅₀ 150 µg/ml) but not production of leukotriene LTB₄ by 5-LOX.Conclusions: The observed dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and the favorable gastrointestinal tolerability of the original remedy Zeel comp. N.Received 10 April 2003; returned for revision 27 May 2003; accepted by W. B. van den Berg 24 November 2003     

Impact of Selenium on the Leukotriene B4 Synthesis Pathway during Isoproterenol-Induced Myocardial Infarction in Experimental Rats

Selenium (Se), an essential micronutrient, exerts its biological functions through selenoproteins. There are evidences that show Se to have an impact on the course and outcome of a number of etiologically inflammatory diseases. Leukotriene B₄ (LTB₄) is an inflammatory mediator, and its production is mediated through two specific enzymes—lipooxygenase (LOX) and leukotriene A₄ hydrolase (LTA₄H). We examined the effect of Se on LTB₄ synthesis during isoproterenol (ISP)-induced myocardial infarction (MI) in rats. Rats were divided as: control, ISP, Se, and Se + ISP. Sodium selenite was administered at dose of 8 μg/100 g/day. ISP was injected subcutaneously twice (10 mg/100 g body weight). The rats pretreated with Se had increased concentration of phospholipids and enhanced biosynthetic enzymes compared with that of ISP. The activities of phospholipases decreased on Se treatment. The level of calcium was increased in ISP group whereas, on Se treatment, it was near normal levels. Activities of LOX and expression of LTA₄H were down-regulated in the case of Se-pretreated rats. Our study shows the anti-inflammatory mechanism of selenium during MI.     

5-Lipoxygenase in mouse cerebellar Purkinje cells

It has been suggested that the enzymatic pathway of 5-lipoxygenase (5-LOX) influences brain functioning and pathobiology. The mRNAs for both the enzyme 5-LOX and its activating protein FLAP have been found in the cerebellum. In this work, we investigated the cellular expression of 5-LOX in the adult mouse cerebellar cortex. We used the in situ mRNA hybridization assay, immunocytochemistry, laser capture microdissection, and our previously developed method for assaying the DNA methylation status of a putative mouse 5-LOX promoter. Since both 5-LOX mRNA in situ hybridization signal and FLAP immunoreactivity co-localize with calbindin 28 kD immunoreactivity (a Purkinje cell marker) but not with S-100β immunoreactivity (a Bergmann glia marker), the suggestion is that the 5-LOX pathway is expressed in cerebellar Purkinje cells. We found that methylation in the sites targeted by methylation-sensitive restriction endonucleases AciI and HinP1I but not BstUI and HpaII was greater in DNA samples obtained from a high-5-LOX-expressing cerebellar region (Purkinje cells) versus a low-5-LOX-expressing region (the molecular cell layer), suggesting a possible epigenetic contribution to the cell-specific 5-LOX expression in the cerebellum. We propose that Purkinje cell-localized 5-LOX and FLAP expression may be involved in the cerebellar synthesis of leukotrienes and/or could influence the Dicer-mediated microRNA formation and processes of neuroplasticity.     

Role of 5-lipoxygenase-activating protein in the regulation of 5-lipoxygenase activity in human neutrophils

Using intact and fractionated human polymorphonuclear leukocytes (PMNL), we provide evidence that the enantioselective leukotriene synthesis inhibitor (LSI) BAY X 1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid binds specifically to a high-affinity binding site, which is most likely identical to FLAP.BAY X 1005 blocks the translocation of 5-lipoxygenase (5-LOX) in PMNL stimulated by the calcium ionophore A23187 or chemotactic stimuli such as PAF, C5a or fMLP as does MK-886. In contrast to the direct 5-LOX inhibitors (LOI) A-64077 and AA-861, the degree of leukotriene synthesis inhibition declines with increasing duration of A23187-induced leukocyte activation in the presence of BAY X 1005 and MK-886. Kinetic studies performed with BAY X 1005 showed that this effect was not accompanied by a significant translocation of 5-LOX from the cytosol to the microsomal fraction. Because FLAP has been implicated in the transfer of arachidonic acid to 5-LOX and A23187 is a potent activator of leukocyte phospholipase A₂, we hypothesized that the observed loss of leukotriene synthesis inhibition may be due to competition of BAY X 1005 binding by endogenously released arachidonic acid. Accordingly, binding of BAY X 1005 to FLAP in intact and fractionated cells is dose-dependently inhibited by arachidonic acid and other unsaturated long-chain fatty acids, but not by saturated fatty acids. Therefore, we conclude that BAY X 1005 or MK-886 inhibit leukotriene biosynthesis by binding to FLAP, thereby preventing 5-LOX translocation and substrate transfer to the enzyme.     

Generation of leukotriene B4 by hemodialyzer membranes: A novel index of biocompatibility

Summary Leukotriene B₄ (LTB₄) plays an important role in acute and chronic inflammatory and hypersensitive reactions. We studied the time course of LTB₄ biosynthesis in whole blood in 18 patients with end-stage renal failure maintained on regular hemodialysis with two different membranes, cuprophane and polyacrylonitrile (AN 69). The basal levels of LTB₄ from dialysis patients did not differ significantly from a normal control group. Compared to predialytic values, the cuprophane membrane caused a maximal release of LTB₄ by a factor of about 4.5 (p<0.01) within the first 10 to 20 minutes. Thereafter the level fell and returned to baseline range at the end of the hemodialysis session. With the use of the AN 69 membrane no significant increase of LTB₄ could be demonstrated. The changes in LTB₄ concentration showed a close temporal correlation to the alterations in white blood cell count. We conclude that (1) LTB4 is a biologically important mediator of neutrophil activation during hemodialysis, and (2) LTB₄ may be a sensitive marker of biocompatibility in vivo.Abbreviations LTB₄ Leukotriene B₄ - 5-HETE 5-Hydroxyeicosatetraenoic acid     

Effect of 5-lipoxygenase inhibitors onin situ LTB4 biosynthesis following calcium ionophore stimulation in the rat pleural cavity

The intrapleural injection of carrageenan in the rat induces exudate formation, cellular influx and leukotriene generation in the pleural cavity. We have demonstrated that the inflammatory response (exudate volume, and LTB₄ levels) is increased,in situ by the intrapleural administration of calcium ionophore A 23187 (100 nmol/rat) at 4, 16, 24, 48, and 72 h after the injection of carrageenan and that the A 23187-induced increase is dose-dependent. The oral administration of A-64077 and MK-886, two 5-lipoxygenase inhibitors (5-LOIs), at 10 mg/kg causes marked decreases in LTB₄ release at the above-mentioned time intervals. However, A 23187-induced augmented exudate formation is not affected by the treatment with 5-LOIs. The results suggest that the use of 5-LOIs to inhibit LTB₄ biosynthesis may be beneficial in various LTB₄-dependent pathological conditions.     

Leukotriene B4 induces interleukin 5 generation from human T lymphocytes

Leukotriene B₄ (LTB₄) has been shown to affect several interleukin (IL)-linked functions of human lymphocytes. In this study, we investigated whether LTB₄ regulates IL-5 generation from human T cells and subsequently modulates eosinophil functions. Preincubation of T cells with very low concentrations (10^?12 to 10^?8 M ) of LTB₄ induced concentration-dependent IL-5 production, the event occurring after the first 24 h of cultivation. However, direct action of LTB₄ to IL-5 generation is strictly dependent on a preincubation with appropriate concentration of LTB₄. In contrast, the stereoisomer of LTB₄,5S,12S-dihydroxy-6,8,10,14-eicosatetraenoic acid showed no enhancement of IL-5 production. IL-5 released from LTB₄-primed T cells elicited sustained viability of mature eosinophils and reduced the content of eosinophil cationic protein in their crystalloid matrix by degranulation. These data suggest that LTB₄ induces bioactive IL-5 production from T cells and that the released IL-5 modulates eosinophil functions which might play a crucial role in eosinophil-linked allergic inflammatory process.     

Elevated Expressions of 15-Lipoxygenase and Lipoxin A4 in Children with Acute Poststreptococcal Glomerulonephritis

Anti-inflammatory effects of the 15-lipoxygenase (15-LO) derivatives lipoxin A₄ (LXA₄) and 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) have been documented in many experimental models of acute inflammation. However, the expression levels of 15-LO and its products in human renal diseases remain unknown. This study investigated the expression levels of LXA₄, leukotriene B₄ (LTB₄), and 15-LO in leukocytes and glomeruli obtained from 22 children with acute poststreptococcal glomerulonephritis (APSGN), and determined the modulatory effects of both 15-S-HETE and LXA₄ on LTB₄ synthesis in leukocytes and LTB₄-evoked chemotaxis of polymorphonuclear leukocytes (PMNs) obtained from children during the first 3 days after onset of APSGN. Expression levels of both LXA₄ and 15-LO in leukocytes and glomeruli were up-regulated during the acute phase of disease, further peaking between days 10 and 14, and remained increased after 6 to 8 weeks of APSGN onset. In contrast, blood and urinary levels of LTB₄ as well as the number of glomerular PMNs peaked during the acute phase of disease and then decreased during the resolution phase. Administration of both 15-S-HETE and LXA₄ in vitro inhibited LTB₄-induced chemotaxis of PMNs and production of LTB₄ from leukocytes obtained from patients with APSGN. The current study provides further support for an anti-inflammatory role for 15-LO products in human nephritis through both antagonism and inhibition of leukotriene synthesis and its biological activity.     

Effect of leflunomide on constitutive and inducible pathways of cellular eicosanoid generation

Leflunomide exerts its effects primarily via the immunomodulating and antiphlogistic activities of its major metabolite A 77 1726. Our investigation in several eicosanoid forming systems revealed that in human white blood cells the metabolite did not cause any alteration on Ca-ionophore stimulated metabolism of membrane bound arachidonic acid to cis-, trans- and epi-LTB₄. Thus, the involved enzyme systems phospholipase A₂, 5-lipoxygenase and LTA₄-hydrolase can be ruled out as a target of the drug. However, in several cellular systems the drug weakly inhibited the generation of 5-HETE and LTB₄ from exogenous arachidonic acid, possibly by interfering with the exogenous substrate's access to the 5-lipoxygenase.In order to get information about the cyclooxygenase (COX-2) which is inducible in human PMNL by inflammatory mediators viade novo protein biosynthesis, we activated the cells with LPS for 18 h. A 77 1726 and indomethacin had no influence on the enzyme activity of the newly induced COX-2. However, both drugs in low concentrations were able to blunt the long term activation process resulting in PGE₂ generation.In contrast, the prostaglandins generated by constitutive enzymes (COX-1) are probably involved in maintaining vital functions, and their inhibition by indomethacin and other nonsteroidal antiinflammatory drugs (NSAIDs) account for numerous adverse effects, for instance gastric erosion.Our study revealed that leflunomide and A 77 1726 are not to be regarded as COX-1-inhibitors, and thus cannot be associated with the typical adverse effects of the NSAIDs.     

The role of LTA4H and ALOX5AP polymorphism in asthma and allergy susceptibility

Background: Leukotrienes (LTs) have been identified as central mediators in asthma and allergy. Pharmacological inhibition of cysteinyl‐LT activity improves asthma symptoms and control. Accumulating evidence suggests a role for the dihydroxy leukotriene LTB₄ in airway disease. LTA₄ hydrolase and 5‐lipoxygenase activating protein have key roles in LTB₄ production. Single nucleotide polymorphism (SNPs) and haplotypes spanning the LTA4H and ALOX5AP genes have been associated with LTB₄ production and myocardial infarction (MI). Objective: To assess the contribution of LTA4H and ALOX5AP polymorphism to asthma and allergy susceptibility. Methods: Three hundred and forty‐one Caucasian families (two asthmatic siblings) were genotyped for eight SNPs spanning ALOX5AP and five SNPs spanning LTA4H. Association analyses of asthma and related phenotypes (total IgE, atopy, bronchial hyper‐responsiveness, FEV₁) were undertaken using the Family Based Association Test. Results: Single point analyses identified association (P < 0.05) between SNPs SG13S114, SG13S89, SG13S41 (ALOX5AP), rs1978331 (LTA4H) and asthma and/or related phenotypes. Haplotype analyses using all LTA4H SNPs identified a single key risk haplotype for the development of asthma (P = 0.006) and related phenotypes (P = 0.042–0.005). Haplotype analyses using all ALOX5AP SNPs identified several asthma and atopy risk and protective haplotypes. There was limited correlation with previously identified MI risk haplotypes in both genes. Carriers of both ALOX5AP SG13S41 and LTA4H rs1978331 alleles had an increased risk of developing asthma (OR 2.17, CI 1.41–3.32). Conclusions: These data provide evidence for the role of SNPs spanning the ALOX5AP and LTA4H genes in asthma and atopy susceptibility in the Caucasian population and support a role for LTB₄ in disease pathogenesis.     

PAF and LTB4 biosynthesis in the human neutrophil: Effects of putative inhibitors of phospholipase A2 and specific inhibitors of 5-lipoxygenase

The effect of several putative phospholipase A₂ (PLA₂) inhibitors on [³H]-acetate incorporation into platelet-activating factor (PAF) upon calcium ionophore A23187 stimulation of purified human neutrophils (PMN) were evaluatedin vitro. PLA₂ inhibitors such asp-bromophenacyl bromide (pBPB), ellagic acid, aristolochic acid and gossypol were without effect or only weakly inhibited PAF biosynthesis. Luffariellolide, a potent PLA₂ inhibitor isolated from the marine spongeLuffariella sp., dose-dependently inhibited PAF production (IC₅₀=5 M). Due to the relationship between PAF and LTB₄ biosynthesis, the effect of inhibiting LTB₄ production on PAF biosynthesis was investigated. At concentrations which reduce LTB₄ production by >95%, Wy-50,295 tromethamine and A-64,077, specific 5-lipoxygenase (5-LO) inhibitors, did not significantly effect PAF production. In contrast, L-663,536, the 5-LO translocation inhibitor, was a potent inhibitor of PAF production (IC₅₀=1 M). This activity of L-663, 536 may contribute to its pharmacological profile at higher doses. These data also suggest that PAF biosynthesis in human PMNs is not dependent on the formation or continued presence of leukotrienes.     

Novel mechanism of LTB4 metabolism in the guinea pig polymorphonuclear leukocyte

Suspensions of caseinate-elicited guinea pig PMNs actively and specifically deplete endogenous LTB₄. The depletion is initiated with and exceeds the biosynthesis of LTB₄. The depletion is inhibitable by NaCN. Human and rat PMN also show a similar depletion of endogenous LTB₄; but unlike the guinea pig PMN, exogenous LTB₄ is rapidly oxygenated to the omega hydroxy- and carboxy-LTB₄ by mechanisms unaffected by cyanide. Depletion of endogenous LTB₄ in the guinea pig PMN cannot be accounted for by specific reacylation into the phospholipid nor by the recently described enzymes capable of reducing the leukotriene triene. That the myeloperoxidase may be responsible for the depletion of LTB₄ has not been eliminated and is suspected due to the cyanide inhibitable nature of this phenomenon. Such a mechanism would require a novel utilization of an activated species of oxygen by an enzyme or nonenzymatic protein surface.     

Nasal mucosal expression of the leukotriene and prostanoid pathways in seasonal and perennial allergic rhinitis

Background Leukotrienes (LTs) and prostanoids are potent pro‐inflammatory and vasoactive lipid mediators implicated in airway disease, but their cellular sources in the nasal airway in naturally occurring allergic rhinitis (AR) are unclear. Objective To quantify cellular expression of enzymes of the 5‐lipoxygenase (5‐LO) and cyclooxygenase (COX) pathways by immunohistochemistry in nasal biopsies from patients with symptomatic perennial AR (PAR, n=13) and seasonal AR (SAR, n=14) and from normal subjects (n=12). Methods Enzymes of the 5‐LO pathway (5‐LO, FLAP, LT A₄ hydrolase, LTC₄ synthase) and the COX pathway (COX‐1, COX‐2, prostaglandin D₂ synthase) were immunostained in glycol methacrylate resin‐embedded inferior turbinate biopsy specimens, quantified in the lamina propria and epithelium, and co‐localized to leucocyte markers by camera lucida. Results In the lamina propria of PAR biopsies, median counts of cells expressing FLAP were fourfold higher than in normal biopsies (Mann–Whitney, P=0.014), and also tended to be higher than in SAR biopsies (P=0.06), which were not different from normal. PAR biopsies showed threefold more cells immunostaining for LTC₄ synthase compared with SAR biopsies (P=0.011) but this was not significant compared with normal biopsies (P=0.2). These changes were associated with ninefold more eosinophils (P=0.0005) with no differences in other leucocytes. There were no significant differences in the lamina propria in immunostaining for 5‐LO, LTA₄ hydrolase, COX‐1, COX‐2 or PGD₂ synthase. Within the epithelium, increased expression of COX‐1 was evident in PAR biopsies (P=0.014) and SAR biopsies (P=0.037), associated with more intra‐epithelial mast cells in both rhinitic groups (P<0.02). Conclusions In the nasal biopsies of PAR subjects, increased expression of regulatory enzymes of the cysteinyl‐LT biosynthetic pathway was associated with lamina propria infiltration by eosinophils. Seasonal rhinitis biopsies shared only some of these changes, consistent with transient disease. Increased intra‐epithelial mast cells and epithelial COX‐1 expression in both rhinitic groups may generate modulatory prostanoids.     

Effect of manoalide on human 5-lipoxygenase activity

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A₂(PLA₂) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB₄ and TXB₂ (IC₅₀ 1.7 and 1.4 M, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000×g and 100,000×g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 M. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its antiinflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA₂.accepted by I. Ahnfelt-Rønne     

Development and application of a membrane receptor assay for leukotriene B4

5(S),12(R)-dihydroxy-6-cis-8,10 tran-14-cis-eicosatetraenoic acid (LTB₄) is a potent inflammatory mediator generated by human cells. A receptor assay using membranes from cultured HL-60 cells has been developed to quantitate LTB₄ with a range of sensitivity from 10 pg to 5 ng. The initial metabolite of LTB₄, 20-OH LTB₄, has a cross reactivity of 28% while other, lipoxygenase products do not significantly compete. This assay has been used to study ionomyocin-induced formation of LTB₄ by human neutrophils. The use of membranes from HL-60 cells for the measurement of LTB₄ provides a sensitive and highly selective alternative to radioimmunoassay for the determination of the levels of this important eicosanoid in biological fluids and should be useful in the development of antagonists of the LTB₄ receptor.