John F. Penrose

MOLECULAR CLONING OF THE GENE FOR HUMAN LEUKOTRIENE C4 SYNTHASE : ORGANIZATION, NUCLEOTIDE SEQUENCE, AND CHROMOSOMAL LOCALIZATION TO 5Q35

Leukotriene C (LTC) synthase catalyzes the conjugation of LTA with reduced GSH to form LTC, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase superfamily. Genomic cloning from a P1 library now reveals that the gene for LTC synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC synthase align identically with those of FLAP; however, the small size of the LTC synthase gene contrasts with the >31-kilobase pair size reported for FLAP. Confirmation of the LTC synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A) RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5′ of the ATG translation start site. The 5′-flanking region revealed a GC-rich promotor sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5′ from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5′ and 3′ oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.

Rhinovirus infection increases 5-lipoxygenase and cyclooxygenase-2 in bronchial biopsy specimens from nonatopic subjects.

Rhinovirus infections cause wheeze, cough, and bronchial hyperresponsiveness. To investigate the involvement of cysteinyl-leukotrienes and prostanoids in these symptoms, bronchial biopsy specimens from 9 normal subjects (nonatopic and with no history of chronic lung disease) were immunostained for 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathway enzymes 2 weeks before and 4 days after experimental infection with human rhinovirus serotype 16. 5-LO-positive cell counts increased 9-fold (from 0.48 to 4.4 cells/mm(2); P <.05), and 5-LO-activating protein (FLAP)-positive cell counts increased 3.6-fold (from 1.8 to 6.5 cells/mm(2); P =.09). Levels of leukotriene A(4) hydrolase and leukotriene C(4) synthase were unchanged. COX-2--positive cell counts increased from 0 to 2.6 cells/mm(2) (P =.009), with no change in COX-1 levels. Increases of 3-4-fold were seen in levels of macrophages (P =.02) and mast cells (P =.07) but not of eosinophils (P >.4), and bronchoalveolar lavage fluid cysteinyl-leukotriene levels doubled (from 11.2 to 20.4 pg/mL; P =.13). Cold symptom scores correlated with bronchial immunostaining for FLAP (rho = 0.93; P =.001). In normal subjects, rhinovirus colds induce bronchial inflammation with markedly enhanced expression of 5-LO pathway proteins and COX-2.

Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases

Background Leukotrienes derived from the 5‐lipoxygenase pathway are proinflammatory lipid mediators that possibly play a role in inflammatory bowel diseases. The expression of 5‐lipoxygenase pathway proteins has not previously been examined in colonic mucosa in inflammatory bowel disease. Results Quantitative immunohistochemical analyses showed that, compared to those of the control subjects (n = 9), colonic biopsies from patients with active inflammatory bowel disease (n = 17) had 3‐ to 7‐fold higher mean counts of cells expressing 5‐lipoxygenase (P = 0.03), 5‐lipoxygenase‐activating protein (P = 0.005), and the leukotriene A4 hydrolase (P = 0.004), which make up the biosynthetic pathway of the potent neutrophil chemotaxin leukotriene B4. Immunoexpression of the leukotriene C4 synthase was unaltered (P > 0.2). The increased representation of leukotriene B4–pathway enzymes was associated with higher counts of neutrophils (P = 0.0001), macrophages (P = 0.03), eosinophils (P = 0.0004), CD8+ T cells (P < 0.001), activated T cells (P < 0.05), and B cells (P < 0.05) but not of mast cells (P > 0.9). These eicosanoid and cellular changes were most marked in the subgroup of patients with ulcerative colitis (n = 9), and were absent in patients with quiescent disease (n = 6). The anomalies in the 5‐lipoxygenase pathway were accompanied as expected by more cells immunostaining for cytokine‐inducible COX‐2 (P = 0.004, n = 17), but this study also revealed a greater number of cells expressing COX‐1 in the samples from the patients in the ulcerative colitis subgroup (P = 0.03, n = 9). Conclusions The 5‐lipoxygenase data provide a cellular basis for increased tissue synthesis of the leukotriene B4, as reflected in the colonic mucosa and rectal dialysates of patients with active inflammatory bowel disease, which contributes to neutrophil influx and colonic injury. The COX‐1/COX‐2 data highlight the ambiguous functional role of prostanoid pathways in inflammatory bowel diseases. (Inflamm Bowel Dis 2007)

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

Background Human bronchial epithelial cells synthesize cyclooxygenase and 15‐lipoxygenase products, but the 5‐lipoxygenase (5‐LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro‐inflammatory mediators is thought to be restricted to leucocytes.

Human bronchial fibroblasts express the 5-lipoxygenase pathway

BackgroundFibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Fibroblast activity is influenced by members of the leukotriene family of bronchoconstrictor and inflammatory mediators, but it is not known whether human bronchial fibroblasts can synthesize leukotrienes.MethodsThe expression of leukotriene biosynthetic enzymes and receptors was investigated in primary fibroblasts from the bronchi of normal and asthmatic adult subjects using RT-PCR, Western blotting, immunocytochemistry and flow cytometry.ResultsThese techniques revealed that human bronchial fibroblasts from both subject groups constitutively express 5-lipoxygenase, its activating protein FLAP, the terminal enzymes leukotriene A4 hydrolase and leukotriene C4 synthase, and receptors for leukotriene B4 (BLT1) and cysteinyl-leukotrienes (CysLT1). Human bronchial fibroblasts generated immunoreactive leukotriene B4 and cysteinyl-leukotrienes spontaneously and in increased amounts after calcium-dependent activation. Flow cytometry showed that human bronchial fibroblasts transformed to a myofibroblast-like phenotype by culture with transforming growth factor-β1 expressed 320–400% more immunofluorescence for leukotriene C4 synthase and CysLT1 receptors, with 60–80% reductions in leukotriene A4 hydrolase and BLT1 receptors.ConclusionThese results indicate that human bronchial fibroblasts may not only respond to exogenous leukotrienes but also generate leukotrienes implicated in narrowing, inflammation and remodeling of the asthmatic airway.

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.

Leukotriene C4 synthase

Leukotriene C4 synthase polypeptide identified by photoaffinity labeling, and purification of leukotriene C4 synthase to homogeneity is described.

LTC4 synthase: A key enzyme in cysteinyl leukotriene formation

The cysteinyl leukotrienes (cysteinyl LTs) LTC4, LTD4 and LTE4, are a family of receptor-active bioactive molecules. These proinflammatory lipid mediators were first described as the slow-reacting substance of anaphylaxis (SRS-A) [1] with the observation that perfusates from antigen-sensitized and activated lung tissue released a spasmogenic activity [1]. Bioassay of SRS-A with the antihistamine mepyramine revealed it to be distinct from histamine [2,3], and physiologic studies clearly implicated SRS-A in anaphylaxis and asthma [4-6]. The chemical structures of the three components of SRS-A were not completely elucidated for more than 40 years [7-11]. After their identification (Fig. 1), the leukotrienes were shown in several model systems to cause the characteristic pathophysiologic features of asthma - bronchoconstriction, microvasculature permeability, epithelial mucus secretion and recruitment of inflammatory cells [12-16]. The levels of the cysteinyl LTs are increased in the bronchoalveolar lavage fluid, serum and urine of patients with asthma [17-20] and are further elevated during disease exacerbations [18-20]. The most compelling evidence for the role of cysteinyl LTs in asthma has been provided by studies in which their biosynthesis was blocked or their receptor-mediated actions were antagonized with anti-leukotriene drugs. These agents inhibit the asthmatic responses in patients with spontaneous or induced exacerbations [21-23], and they also induce bronchodilatation in patients with mild to moderate asthma, implying that chronic overproduction of cysteinyl LTs is responsible for basally elevated bronchial tone in these individuals [24]. With the critical role of the cysteinyl LTs in asthma in mind, we will briefly review the pathway for their generation and then focus on the only enzyme committed to their biosynthesis, LTC4 synthase (LTC4S).

An efficient approach to the synthesis of LTB4 and ω-substituted LTB4 metabolites

Abstract The first total synthesis of the methyl ester of 20-oxo-LTB 4 26 is described. The key synthon 6 is an advanced new intermediate which has been used in the synthesis of LTB 4 1 , 20-oxo-LTB 4 methyl ester 26 , and 20-hydroxy-LTB 4 2 . The synthetic 26 has been used to study the cytosolic aldehyde dehydrogenase-catalyzed oxidation of LTB 4 to its ω-carboxy metabolite.