The cysteinyl leukotrienes: Where do they come from? What are they? Where are they going?K Frank Austen

Cysteinyl leukotrienes are established mediators of bronchial asthma and have agonist roles analogous to those of histamine in allergic rhinitis. We now know that the substance originally termed slow-reacting substance of anaphylaxis was composed of three cysteinyl leukotrienes that act in the inflammatory response via receptors on smooth muscle and on bone marrow–derived inflammatory cells. K. Frank Austen describes the work culminating in the identification, biosynthesis and functional characterization of these moieties.

I was introduced to the substance originally termed slow-reacting substance of anaphy-laxis (SRS-A) in l959 in the laboratory of Walter Brocklehurst at the National Institute of Medical Research (NIMR) in Mill Hill, England. Brocklehurst had developed a bioas-say to measure SRS-A based on the contractile response of guinea pig ileal smooth muscle sus-pended in an organ bath. We first measured the rapid contractile responses induced by the vasoactive amine histamine and then added an H1 histamine receptor antagonist to ren-der the muscle unresponsive to histamine. Even in the presence of the H1 histamine receptor antagonist, the muscle responded to SRS-A with slow contractions in a dose-response fash-ion. Abundant in the diffusate from allergen-challenged human lung fragments obtained from tissue resected for a malignancy, SRS-A induced contraction of human bronchioles in an organ bath in the presence of a histamine blocker. I knew that histamine blockers were efficacious for the management of allergic rhinitis, conjunctivitis and hives but not for bronchial asthma, and I had a ‘gut feeling’ that SRS-A might be a mediator of bronchial

asthma. Together, ‘Brock’ and I sought to iden-tify and understand the pathways responsible for the release of SRS-A relative to those for histamine1–3.

When I started my own laboratory at the Massachusetts General Hospital, I collabo-rated with Kurt Bloch in an effort to identify the cellular source of SRS-A. We showed that hapten-specific IgG1, but not IgG2, antibodies passively sensitized guinea pig lung fragments to antigen-elicited SRS-A, thereby implicating the mast cell as a source of SRS-A4. Additional findings made with Kimishige and Teruko Ishizaka indicated that atopic human serum IgE mediated allergen-induced generation of SRS-A in monkey lungs and human lung frag-ments, thus firmly establishing mast cells as a source of SRS-A and strengthening my feel-ing that SRS-A was a mediator of bronchial asthma5.

At this point, my colleagues and I shifted our attention to efforts to purify and characterize SRS-A. As agonists other than SRS-A could contract the guinea pig ileum in an antihis-tamine-resistant fashion, we felt that it was important to study SRS-A by producing the spasmogenic activity in an immune reaction. To develop a chromatographic procedure for isolation of the activity, Robert (Bob) Orange produced SRS-A in the peritoneal cavities of rats and pooled material from several hun-dred rats. Because SRS-A behaved as a polar, acidic lipid, Robert Murphy evaluated bioac-

tive material after each purification step by gas chromatography, gas chromatography–mass spectroscopy and/or high-resolution mass spectroscopy to ascertain the removal of likely contaminants. This initial purification sequence was later improved by the addition of a final reverse-phase high-performance liquid chromatography step. Rat and human SRS-A behaved identically during isolation; the final products were of low molecular weight (400–1,400) and were bioactive in nanogram or smaller quantities6. Surprisingly, the par-tially purified final product was not amenable to spectrometric analysis because it did not leave the probe. Nonetheless, spark-source mass spectroscopy and electron probe analysis revealed the sulfur atom to be abundant in the bioactive samples and provided an improved UV spectrum. Murphy integrated these find-ings with those subsequently obtained by others and, during a sabbatical with Bengt Samuelsson at the Karolinska Institute in Sweden, generated double-labeled SRS-A–like material from mouse mastocytoma cells that were exposed to [3H]arachidonic acid and [35S]cysteine and then were stimulated with a calcium ionophore7. After the sulfur bond was broken to remove the peptide adduct, the lipid backbone of the labeled substance was char-acterized as a triene metabolite of arachidonic acid. By synthesizing the likely stereochemical candidates with various peptide adducts for parallel bioassays with biologically generated

K. Frank Austen is with the Department of

Medicine, Harvard Medical School and the Division

of Rheumatology, Immunology and Allergy, Brigham

and Women’s Hospital, Boston, Massachusetts

02115, USA.

e-mail: fausten@rics.bwh.harvard.edu

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material, E.J. Corey established the structure of leukotriene C4 (LTC4) as 5(S)-hydroxy-6(R)-glutathionyl-7,9-trans-11,14-cis-eicosatetrae-noic acid8. Using synthetic LTC4 and related structures, we found that SRS-A consisted of three bioactive cysteinyl leukotrienes (cys-LTs)9. In addition, we noted that each cys-LT triggered the contraction of guinea pig trachea and lung parenchymal strips with a distinct potency, which was most likely due to hetero-geneity among their receptors10; however, the receptors of these distinct cys-LTs would not be cloned until almost two decades later11.

Two of my colleagues, Bob Lewis and Nick Soter, joined me in a study of our own intra-dermal responses to the cys-LTs. We learned that these moieties were much more potent than histamine in eliciting a wheal-and-flare reaction and that the reaction triggered by the cys-LTs was of longer duration than that induced by histamine. Tissue exposed to the cys-LTs showed marked edema without cellular infiltration, reflecting a microvascular leak12. When Jeffrey Drazen, a former postdoctoral fellow with our group, and others inhaled the cys-LTs, LTC4 and LTD4 were 1,000-fold more potent than histamine in reducing the maxi-mum expiratory flow rate. Furthermore, the onset of the response was slow, and the response was prolonged and associated with an audible wheeze; in contrast, the response induced by histamine was rapid, brief, and accompanied by a cough but not a wheeze13. Final validation

of the role of the cys-LTs in bronchial asthma came from the establishment of the clinical efficacy of antagonists14,15. These agents were developed empirically based on inhibition of cys-LT–mediated smooth muscle contraction and were later recognized to block only one of the known cys-LT receptors, namely the CysLT1 receptor.

Others clarified the key steps in the bio-chemical pathway responsible for synthesis of cys-LTs and leukotriene B4 (LTB4). Arachidonic acid, released from the outer nuclear mem-brane by cytosolic phospholipase A2 (cPLA2), is converted by 5-lipoxygenase (5-LO) in the presence of the 5-lipoxygenase–activating pro-tein (FLAP) to 5-hydroperoxyeicosatetraenoic acid (HPETE) and, in a second reaction, to an epoxide, LTA4. Human eosinophils loaded with LTA4-methyl ester at 4 °C produced LTC4 but did not release it until the temperature was increased. This finding revealed a carrier-mediated, energy-dependent export step that preceded any extracellular metabolism of LTC4 to the other receptor-binding bioactive cys-LTs, LTD4 and LTE4.

The enzyme LTC4 synthase (LTC4S) con-jugates glutathione (GSH) to LTA4, thereby converting it to LTC4. Partial purification of LTC4S solubilized from rat basophilic leuke-mia-1 cells or guinea pig lung revealed that it is an integral membrane protein of 18 kDa that lacks the substrate specificity characteristic of the glutathione S-transferases (GSTs)16. Bing

Lam developed a high-throughput fluores-cence-linked competitive cytofluorographic immunoassay to detect LTC4 generation in Cos cells transfected with a cDNA library from human KG-1 cells, a transformed cell line that expresses LTC4S in abundance17. The full-length cDNA that transferred LTC4S activity encoded the limited number of residues that were identified by amino acid sequencing in the purified protein. The N-terminal two-thirds of LTC4S amino acids were 44% identi-cal to FLAP. Both LTC4S and FLAP are integral perinuclear membrane proteins, and genomic cloning revealed that their intron-exon junc-tions align identically, suggesting that they may have evolved through gene duplication18.

The gene encoding human LTC4S (LTC4S), identified by fluorescence in situ hybridiza-tion, is on chromosome 5q35 distal to the gene cluster encoding interleukin 4 (IL-4), IL-5 and IL-13, the cytokines central to T helper type 2 (TH2) functions that have been implicated in asthmatic and allergic inflammation. The cDNAs encoding mouse and human LTC4S have 87% identity, and genomic cloning revealed identical intron-exon boundaries. In addition, the gene encoding mouse LTC4S (Ltc4s) is located in a region syntenic to human 5q35 on chromosome 11 near a TH2 gene clus-ter. Working with Joshua Boyce, we examined the regulation of LTC4S expression in human cord blood–derived mast cells. IL-4 priming induced LTC4S transcription and production of functional LTC4S protein; these effects were unique to LTC4S, as IL-4 stimulation did not induce transcription of genes encoding other pathway components, such as cPLA2, FLAP or 5-LO19.

Yoshihide Kanaoka overexpressed and puri-fied LTC4S in fission yeast and generated the initial crystals for electron and X-ray crystal-lography. Electron crystallography by Ingeborg Schmidt-Krey at 4.5 and 7.5 Å showed that LTC4S is a trimer and that each monomer contains at least four α-helices that insert into the membrane20. Analysis of the atomic structure of LTC4S at 3.3-Å resolution by X-ray crystallography revealed that each trimer contains three GSH residues positioned within the interface between adjacent monomers21. LTA4 fits into the interface so that one mono-mer activates GSH to the thiolate anion that attacks at C6 to form the thioether bond while the neighboring monomer donates a proton to form the hydroxyl group at C5 to create LTC4. Overall, the trimeric structure with binding of GSH between adjacent monomers provides a hydrophobic environment in which the labile LTA4 can dock after its stimulus-initiated gen-eration.

To understand the range of pathobiologi-

114 VOLUME 9 NUMBER 2 FEBRUARY 2008 NATURE IMMUNOLOGY

K. Frank Austen engaged in a bioassay of rat SRS-A some 30 years ago. The guinea pig ileum is suspended in an organ bath, and the contractions elicited by an agonist are recorded on a smoked drum with a steel needle attached by a string to the ileum.

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cal functions of the integrated LTC4S-cys-LT system, we sought to investigate the particu-lar actions of exported LTC4 and its metabolic products, LTD4 and LTE4, each of which is generated in distinct sites within tissues and has distinct stability and receptor potency. To this end, we used gene targeting techniques to produce mice lacking LTC4S and each of the known cys-LT receptors, CysLT1 and CysLT2. Disruption of Ltc4s abrogated the capacity of tissues to conjugate LTA4-methyl ester to GSH, thereby establishing the dominant role of this enzyme, rather than other GSTs, in LTC4 bio-synthesis22. IgE-dependent passive cutaneous anaphylaxis in the ears of the LTC4S-deficient, CysLT1-deficient and CysLT2-deficient mice was reduced by more than half compared to that observed in wild-type counterparts; these findings indicated that cys-LTs had a role at least equal to that of mast cell secretory gran-ule amines in eliciting plasma leakage at this anatomical site23,24.

Next we assessed the role of cys-LTs in the more complex pathology of bleomycin-induced pulmonary fibrosis. Twelve days after intratracheal administration of bleomycin, the extent of macrophage and fibroblast accumu-lation and of deposition of extracellular matrix proteins, including collagen, was much less in the LTC4S-deficient and CysLT2-deficient mice than in their wild-type counterparts. Digital image analysis revealed that the septal thicken-ing of the lower lobes in LTC4S-deficient and CysLT2-deficient mice was reduced to one-half of that observed in wild-type littermates. The observation that fibrosis is prevented by disruption of the pathway at different points, namely ligand biosynthesis and receptor-ligand binding, and by mutation of genes located on different chromosomes provides strong evidence for the role of cys-LTs in this form of chronic inflammation.

More than four decades after I first encoun-tered SRS-A, I am gratified that it has proven to

be relevant to human disease. I feel privileged to continue to participate in the definition of the biology of the three cys-LTs and in the structural definition of the enzyme respon-sible for the generation of these moieties. At the same time, the production of mouse strains harboring targeted mutations in Ltc4s and in the genes encoding each cys-LT recep-tor has begun to reveal an array of cell-based functions for cys-LTs that were not remotely anticipated by the original term ‘SRS-A’.

1. Austen, K.F. & Brocklehurst, W.E. Anaphylaxis in chopped guinea pig lung. I. Effect of peptidase sub-strates and inhibitors. J. Exp. Med. 113, 521–539 (1961).

2. Austen, K.F. & Brocklehurst, W.E. Anaphylaxis in chopped guinea pig lung. II. Enhancement of the anaphylactic release of histamine and slow reacting substance by certain dibasic aliphatic acids and inhi-bition by monobasic fatty acids. J. Exp. Med. 113, 541–557 (1961).

3. Austen, K.F. & Brocklehurst, W.E. Anaphylaxis in chopped guinea pig lung. III. Effect of carbon mon-oxide, cyanide, salicylaldoxine, and ionic strength. J. Exp. Med. 114, 29–42 (1961).

4. Stechschulte, D.J., Austen, K.F. & Bloch, K.J. Antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-A) in the guinea pig and rat. J. Exp. Med. 125, 127–147 (1967).

5. Ishizaka, T., Ishizaka, K., Orange, R.P. & Austen, K.F. The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J. Immunol. 104, 335–343 (1970).

6. Orange, R.P., Murphy, R.C., Karnovsky, M.L. & Austen, K.F. The physicochemical characteristics and puri-fication of slow reacting substance of anaphylaxis. J. Immunol. 110, 760–770 (1973).

7. Murphy, R.C., Hammarstrom, S. & Samuelsson, B. Leukotriene C: a slow-reacting substance from murine mastocytoma cells. Proc. Natl. Acad. Sci. USA 76, 4275–4279 (1979).

8. Marfat, A. & Corey, E.J. Synthesis and structure eluci-dation of leukotrienes. in Advances in Prostaglandin, Thromboxane, and Leukotriene Research Vol. 14 (eds. Pike, J.E. & Morton, D.R. Jr.) 155–228 (Raven Press, New York, 1985).

9. Lewis, R.A. et al. Identification of the C(6)-S-con-jugate of leukotriene A with cysteine as a naturally occurring slow reacting substance of anaphylaxis (SRS-A). Importance of the 11-cis-geometry for bio-logical activity. Biochem. Biophys. Res. Commun. 96, 271–277 (1980).

10. Lee, T.H., Austen, K.F., Corey, E.J. & Drazen, J.M.

Leukotriene E4-induced airway hyperresponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three separate sulfidopeptide leukotriene receptors. Proc. Natl. Acad. Sci. USA 81, 4922–4925 (1984).

11. Lynch, K.R. et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399, 789–793 (1999).

12. Soter, N.A., Lewis, R.A., Corey, E.J. & Austen, K.F. Local effects of synthetic leukotrienes (LTC4, LTD4, LTE4 and LTB4) in human skin. J. Invest. Dermatol. 80, 115–119 (1983).

13. Weiss, J.W. et al. Bronchoconstrictor effects of leukotriene C in humans. Science 216, 196–198 (1982).

14. Drazen, J.M., Israel, E. & O’Byrne, P. Treatment of asthma with drugs modifying the leukotriene pathway. N. Engl. J. Med. 340, 197–206 (1999).

15. Bisgaard, H. Leukotriene modifiers in pediatric asthma management. Pediatrics 107, 381–390 (2001).

16. Yoshimoto, T., Soberman, R.J., Spur, B. & Austen, K.F. Properties of highly purified leukotriene C4 syn-thase of guinea pig lung. J. Clin. Invest. 81, 866–871 (1988).

17. Lam, B.K., Penrose, J.F., Freeman, G.J. & Austen, K.F. Expression cloning of a cDNA for human leukotriene C4 synthase, a novel integral membrane protein con-jugating reduced glutathione to leukotriene A4. Proc. Natl. Acad. Sci. USA 91, 7663–7667 (1994).

18. Penrose, J.F. et al. Molecular cloning of the gene for human leukotriene C4 synthase: organization, nucleo-tide sequence, and chromosomal localization to 5q35. J. Biol. Chem. 271, 11356–11361 (1996).

19. Hsieh, F.H. et al. T helper cell type 2 cytokines coordi-nately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C4 syn-thase expression by interleukin 4. J. Exp. Med. 193, 123–133 (2001).

20. Schmidt-Krey, I. et al. Human leukotriene C4 syn-thase at 4.5 Å resolution in projection. Structure 12, 2009–2014 (2004).

21. Ago, H. et al. Crystal structure of LTC4 synthase, the membrane protein for cysteinyl leukotriene biosynthe-sis. Nature 448, 609–612 (2007).

22. Kanaoka, Y. et al. Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase. J. Biol. Chem. 276, 22608–22613 (2001).

23. Maekawa, A., Austen, K.F. & Kanaoka, Y. Targeted gene disruption reveals the role of cysteinyl leukotri-ene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses. J. Biol. Chem. 277, 20820–20824 (2002).

24. Beller, T.C. et al. Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary fibrosis in mice. J. Biol. Chem. 279, 46129–46134 (2004).

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