Roy J. Soberman

Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3.

Human eosinophils were cultured in the presence of recombinant human IL-3 for up to 14 d and their biochemical, functional, and density properties were assessed. After 3 d of culture in 10 pM IL-3, eosinophils had a viability of 70% compared with only 10% in enriched medium alone. Neither IL-1 alpha, IL-2, IL-4, tumor necrosis factor, basic fibroblast growth factor, nor platelet-derived growth factor maintained eosinophil viability. The 7- and 14-d survival of the cultured eosinophils was 55 and 53%, respectively. No other cell type, including neutrophils, was present after culture. After 7 d of culture, the normodense eosinophils were converted to hypodense cells as assessed by density centrifugation. Eosinophils exposed to 1,000 pM IL-3 for 30 min or cultured in 10 pM IL-3 for 7 d generated approximately threefold more leukotriene C4 (LTC4) in response to calcium ionophore than freshly isolated cells. Furthermore, whereas freshly isolated eosinophils killed only 14% of the antibody-coated Schistosoma mansoni larvae, these eosinophils killed 54% of the larvae when exposed to 100 pM IL-3. The enhanced helminth cytotoxicity was maintained for 7 d when eosinophils were cultured in the presence of both 10 pM IL-3 and 3T3 fibroblasts, but not when eosinophils were cultured in the presence of IL-3 alone. IL-3 thus maintains the viability of eosinophils in vitro, augments the calcium ionophore-induced generation of LTC4, enhances cytotoxicity against antibody-sensitized helminths, and induces the eosinophils to become hypodense cells. These phenotypic changes in the eosinophil may be advantageous to host defense against helminthic infections but may be disadvantageous in conditions such as allergic disease.

Regulated formation of eicosanoids

Between 1965 and the mid-1980s, investigators laid a durable foundation for understanding the regulated formation and metabolic disposition of eicosanoids. First, they sought, and found in arachidonic acid, the biosynthetic precursor for the prostaglandins. Second, they identified phospholipids as the cellular compartment that harbored arachidonic acid, and they identified phospholipases as the enzymes essential for its liberation and for the ensuing biosynthesis of prostaglandins (1). Third, they deduced the existence of transient intermediates in the prostanoid biosynthetic pathway, leading to the eventual discovery of the prostaglandin endoperoxides, PGG2 and PGH2 (2, 3). Fourth, they established that the enzyme PGH synthase, or cyclooxygenase (COX), was a molecular target of great medical significance in reproductive, cardiovascular, and inflammatory disorders (4). Fifth, they established that rapid, comprehensive pulmonary metabolism limited the steady-state levels and duration of action of prostanoids, implying that they acted as autocoid lipid mediators, not hormones. Finally, they identified new prostanoids (5, 6) and their biosynthetic enzymes, as well as new lipoxygenase enzymatic pathways and their leukotriene products (7, 8), as medically significant molecules. Over this period, the most prominent themes in eicosanoid research were the identification of novel eicosanoid mediators, the determination of their molecular structures, and the establishment of their pharmacological activities. In a typical study of the time, investigators exposed tissues or cells to 50–100 μM of exogenous arachidonic acid, a concentration five- to tenfold greater than the Km for COX or lipoxygenases (Km ≈ 10 μM) (see Brash, this Perspective series, ref. 9). The contemporary model for eicosanoid biosynthesis, circa 1985, asserted that liberation of free arachidonic acid by phospholipase was the rate-limiting step in biosynthesis. Accordingly, providing saturating amounts of exogenous arachidonic acid ought to reveal all biosynthetic products and pathways. By 1985 or thereabouts, the quest for novel eicosanoid mediators of medical significance had reached the point of diminishing returns. Simultaneously, more and more investigators sought to understand the role of eicosanoid biosynthesis in disease processes. Accordingly, investigators shifted from exogenous arachidonic acid and Ca2+ ionophore as preferred tools and embraced natural ligands, relevant to disease, to initiate receptor-coupled activation of eicosanoid formation. By the late 1980s, two lines of experimental inquiry began to provoke a reconsideration and refinement of the accepted model of eicosanoid biosynthesis. First, kinetic and quantitative aspects of eicosanoid biosynthesis initiated by growth factors or cytokines on mitotically competent cells suggested that it was an oversimplification to regard availability of arachidonic acid as the sole, rate-limiting step in cellular eicosanoid biosynthesis. Second, the discovery of the 5-lipoxygenase activating protein (FLAP) in 1990 proved unambiguously that some eicosanoids (leukotrienes) originated under conditions that were not rate-limited by arachidonic acid availability. The discovery of novel regulatory processes, particularly Ca2++-dependent redistribution of 5-lipoxygenase (5-LO) and the interaction between 5-LO and FLAP (10), offered a new framework on which to reconstruct the earlier model of eicosanoid biosynthesis. Here, we comment on several mechanisms through which cells attain more subtle control of eicosanoid biosynthesis than would be possible by simply limiting the availability of arachidonic acid. We first discuss the coordinated action of specific phospholipases, the enzymes that generate this substrate, with specific COXs and PGH isomerase enzymes. We then consider the restricted expression of eicosanoid biosynthetic enzymes and, finally, turn to the “suicide” inactivation of these biosynthetic enzymes, a general mechanism that may help terminate their proinflammatory function.

Renal and systemic hemodynamic responses to intravenous infusion of leukotriene C4 in the rat.

We studied the systemic and renal hemodynamic effects of leukotriene C4 (2 μg/ per min for 5 minutes, iv) in the rat. During the period of its infusion, leukotriene C4 produced a significant elevation of mean arterial pressure and reductions in cardiac output and renal blood flow, as measured by electromagnetic flow probes. These effects were abolished by FPL55712, a putative antagonist of sulfidopeptide leukotrienes, but not by saralasin or indomethacin. Leukotriene C4 also resulted in an average loss of 20% in plasma volume which, during the postinfusion period, perpetuated the low cardiac output state and thus provoked the release of angiotensin II. This vasoactive peptide sustained the elevation in systemic vascular resistance and the reduction in renal blood flow over a 70-minute postinfusion observation period. Consequently, glomerular filtration rate fell by approximately 50%. These angiotensin II-mediated effects were abolished by saralasin. Indomethacin prevented the leukotriene C4-induced loss of plasma volume and, thus, allowed for the significant recovery of cardiac output and renal blood flow during the postinfusion period, thereby preserving glomerular filtration rate. We conclude that leukotriene C4 exerts direct systemic and renal vasoconstrictor, as well as cardiodepressant effects, during the period of its infusion. By virtue of its vasopermeability enhancing effect, leukotriene C4 also results in an immediate loss of plasma volume, an effect which requires the presence of secondarily generated cyclooxygenase products and which perpetuates the hemodynamic abnormalities observed beyond the period of leukotriene C4 infusion.

The organization and consequences of eicosanoid signaling

Organization of leukotriene and prostaglandin synthesis As described in the introduction to this Perspective series (1), signaling by arachidonic acid represents a paradigm for the use of oxygen in the transmission of information. At the same time, arachidonic acid signaling can also contribute to the propagation of cellular damage. This duality is typified by a signaling cascade that (a) prevents the activation of 5-lipoxygenase (5-LO) in resting cells and (b) results in the formation and release of leukotrienes (LTs), which requires the sequential activation and interaction of at least eight different proteins. In fact, all lipoxygenases require membrane translocation to exert activity. In the case of the formation of COX products, particularly prostaglandin E2 (PGE2) and PGD2, humans have evolved two sets of biosynthetic enzymes that differ not only in their cell- and tissue-specific localization, but also in their subcellular localization and requirement for reduced glutathione, a cellular defense against oxidative damage. This review will focus on three aspects of arachidonic acid biology. First, the compartmentalization and organization of eicosanoid synthesis, specifically LTs and PGs, will be discussed. This will illustrate the elaborate mechanisms that keep unwanted lipoxygenation at arm’s length and also show that the enzymes such as glutathione-Stransferases, epoxide hydrolases, and carrier proteins that are commonly thought of as biosynthetic also belong to families that are generally considered to play a role in detoxification. Second, the potential cellular oxidative damage that is produced as a by-product of the use of oxygen and lipid substrates is examined. Finally, mechanisms that are used to amplify signaling diversity from a core of LTs and PGs are discussed. The role of leukotrienes C4 and D4 in disease

A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis.

BACKGROUND Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.

Properties of highly purified leukotriene C4 synthase of guinea pig lung.

Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The Km value of guinea pig lung LTC4 synthase for LTA4 was 3 microM and the Vmax was 108 nmol/3 min per microgram; the Km values for LTA3 and LTA5 were similar, and the Vmax values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective Ki values of 1.5, 3.3, and 2.8 microM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 microM LTA4-me with 5 mM GSH were 2.1 microM and 0.3 microM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 greater than 40 microM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.

The nuclear membrane organization of leukotriene synthesis

Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.

The expanding network of redox signaling: new observations, complexities, and perspectives

Original citation: J. Clin. Invest. 111:571–574 (2003). doi:10.1172/JCI200318099. Citation for this corrigendum: J. Clin. Invest. 111:1093 (2003). doi:10.1172/JCI200318099C. During the final stages of production, an error was introduced into the list of non standard abbreviations, and into the text. The correct list of nonstandard abbreviations and the correct sentence appear below. Nonstandard abbreviations used: reactive oxygen species (ROS); hydrogen peroxide (H2O2); hypoxia-inducible factor 1 (HIF-1); carboxy-terminal binding protein (CtBP); apoptosis-inducing factor (AIF); apoptosis signal-regulating kinase 1 (ASK1). Sensing nuclear oxygen tension. Since its original description, the study of the transcription factor hypoxia-inducible factor 1 (HIF-1) has demonstrated its central role in regulating the body’s response to changing oxygen levels (4-6).

Expression of the CYP4F3 Gene TISSUE-SPECIFIC SPLICING AND ALTERNATIVE PROMOTERS GENERATE HIGH AND LOW Km FORMS OF LEUKOTRIENE B4ω-HYDROXYLASE

Cytochrome P450 4F3 (CYP4F3) catalyzes the inactivation of leukotriene B4 by ω-oxidation in human neutrophils. To understand the regulation of CYP4F3 expression, we analyzed the CYP4F3 gene and cloned a novel isoform (CYP4F3B) that is expressed in fetal and adult liver, but not in neutrophils. The CYP4F3 gene contains 14 exons and 13 introns. The cDNAs for CYP4F3A (the neutrophil isoform) and CYP4F3B have identical coding regions, except that they contain exons 4 and 3, respectively. Both exons code for amino acids 66–114 but share only 27% identity. When expressed in COS-7 cells, the K m of CYP4F3B was determined to be 26-fold higher than the K m of CYP4F3A using leukotriene B4 as a substrate. 5′-Rapid amplification of cDNA end studies reveal that the CYP4F3A and CYP4F3B transcripts have 5′-termini derived from different parts of the gene and are initiated from distinct transcription start sites located 519 and 71 base pairs (bp), respectively, from the ATG initiation codon. A consensus TATA box is located 27 bp upstream of the CYP4F3B transcription start site, and a TATA box-like sequence is located 23 bp upstream of the CYP4F3A transcription start site. The data indicate that the tissue-specific expression of functionally distinct CYP4F3 isoforms is regulated by alternative promoter usage and mutually exclusive exon splicing.

Membrane localization and topology of leukotriene C4 synthase.

Leukotriene C4(LTC4) synthase conjugates LTA4 with GSH to form LTC4. Determining the site of LTC4synthesis and the topology of LTC4 synthase may uncover unappreciated intracellular roles for LTC4, as well as how LTC4 is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC4 synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC4 synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC4 synthase. The active site of LTC4 synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB4 and LTC4 occurs in different subcellular locations and suggests that LTC4 must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane.