PERSPECTIVE
Novel Antiinflammatory Targets for Asthma
A Role for PPAR?

Charles N. Serhan and Pallavi R. Devchand

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Pain and Perioperative Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

The search for new treatments of asthma and related airway disorders is a continuing quest of pulmonologists, pharmacologists, and pharmaceutical industries documented from as early as 1600 BC (1). The approach of modern medicine has been to identify key procontractile compounds and mediators with the hope of obtaining antagonists to a major clinical outcome, namely the loss of respiratory volume (2). New approaches are needed because many of the current treatments (e.g., antileukotriene drugs, antihistamines, and steroids) are only effective in a portion of those afflicted with asthma and the side effects of these treatments have proved to be a formidable challenge (3, 4). More recently, it has been appreciated that inflammation plays a critical role in asthma, particularly the recruitment of specialized leukocytes, neutrophils, eosinophils, and lymphocytes to lung, and eventually to airways (5). Hence, one strategy may involve the identification of novel antiinflammatory agents that could dampen the clinical sequelae in various forms of asthma. It is in this context that we consider this perspective.

	Are There Novel Approaches and Targets That Could Be of Therapeutic Benefit to This Wide, Unmet and Increasing Clinical Challenge?

From as early as 1972, evidence was presented for the antitumor activity of cyclopentenone prostaglandins (PGs) (6, 7). What are cyclopentenone prostaglandins? Following the original description of the potent actions of PGE₂ and PGF₂, a systematic total organic synthesis was undertaken to facilitate structure-function analyses (7). This resulted in a nomenclature based on the oxygenated positions around the prostanoid ring (8) (Figure 1). It was not apparent that most of these prostaglandins (A₂, B₂, and J₂) were natural products, nor were their biological functions envisioned. However, evaluation of these prostanoids as antitumor agents revealed that the cyclopentenone-type prostaglandins, such as ⁷-PGA₂ and ¹²-PGJ₂, as well as many of their derivatives and analogs, possess potent antiproliferative activities with IC₅₀s in the range of 30ng/ml (6, 7). Such compounds were maintained as synthetic lead structures until reports from two independent Japanese groups revealed that prostaglandin D₂ was converted to ¹²-PGJ₂ in the marine organism Clavularia viridis. The compounds were termed "claviridenones" or "clavulones" and appeared to be products of marine eicosanoids (9, 10). The cyclopentenone PGs also led to the identification of novel punaglandins (11, 12) in the early 1980s which, like clavulones, also displayed antitumor activity (10).

View larger version (19K): [in this window] [in a new window]   Figure 1.   (A) Structures of PGD2 and the cyclopentenone prostanoids PGA2, B2, and J2. (B) Hydrolysis of PGD2 in the presence of albumin. PGD2 can undergo nonenzymatic degradation to 12-PGJ2 via dehydration at the C9 position. Isomerization of 12 followed by dehydration of the C15 hydroxyl groups of PGD2 and 12-PGJ2 can lead to formation of 15-deoxy-12,14-PGD2 and 15-deoxy-12,14-PGJ2, respectively (see text).

Along a similar time line, studies revealed that prostaglandin D₂ is a major product of mast cells (Figure 2) and that it plays an important role as a potent regulator in mast cell degranulation (13). These and earlier findings instigated a search in human tissues for degeneration of PGD₂ and its natural metabolites. It was during the development of appropriate analytical methods (Elisa, GC-MS, LC) that it became evident that PGD₂ could be degraded to yield 15-deoxy-PGJ₂ via nonenzymatic chemical modification and elimination (14, 15) (Figure 1B). These observations led to the widely held view that 15-deoxy-PGJ₂ is not a natural biologically-generated compound in human tissues, but rather an artifactual product of the isolation and chemical degradation of prostaglandin D₂ and/or related structures. To date, this view remains to be challenged with data.

View larger version (36K): [in this window] [in a new window]   Figure 2.   Small lipid mediators that regulate cytokine production might offer novel therapeutic avenues for asthma and related airway diseases. Upon stimulation, lung airway epithelial cells produce proinflammatory cytokines such as IL-8 (a potent chemoattractant). Transcriptional upregulation of IL-8 is primarily mediated by an elaborate NFKB pathway. Release of IL-8 into the extracellular milieu facilitates recruitment of specialized leukocytes (neutrophils, eosinophils, and lymphocytes), ultimately leading to airway inflammation. Dampening of leukocyte accumulation can be achieved by downregulating the production of IL-8 or related substances and/or by direct inhibition of leukocyte chemotaxis. Wang and coworkers have shown that, when present in airway epithelial cells, PPAR can be activated by TZDs or 15-deoxy-12,14-PGJ2 and suppress production of IL-8 by decreasing transcription of the gene. This molecular mechanism is similar to that described previously in a colitis model (34). Stimulated mast cells release PGD2. One might speculate the nonenzymatic degradation of PGD2 to 15-deoxy-PGJ2. If PPAR is a target for antiinflammation in asthma, then the ligands might serve as lead compounds for drug discovery in airway related disorders. To a similar end, Gewirtz and colleagues (31) demonstrated earlier with intestinal epithelial cells the downregulation of IL-8 by endogenous antiinflammatory eicosanoids, namely lipoxin A4 and aspirin-triggered lipoxin A4, as well as their other stable analogs. Reporter-based transfection assays (35) indicate that this LXA4 activity is mediated via a PPAR-independent mechanism (Dr. Bruce Spiegelman, personal communication). The lipoxins can also act by inhibiting recruitment of neutrophils via a cell surface G protein-coupled receptor, ALX. Stable analogs of these lipoxins designed to resist rapid inactivation are biostable and show profound inhibitory actions in various murine models of inflammation (32). Thus, there is potential in the approach of using small lipid molecules based on endogenous pathways as new therapeutic avenues for asthma and related inflammatory airway diseases (36).

In the 1990s, peroxisome proliferator-activated receptors (PPARs) were identified as transcription factors that functioned as nuclear eicosanoid receptors (see (16) for recent review). Early reports describing systematic analyses of cyclooxygenase-derived products linked PPAR to 15-deoxy-^12,14-PGJ₂ and fat cell differentiation (17, 18). PPAR had previously been described as a receptor and target of the antidiabetic drugs, thiazolidinediones (19). The heightened awareness in the importance of the COX2 isoform in angiogenesis, inflammation, and cancer biology (20); the role of PPARs as drug detoxifiers (21); and the prominent role of PPAR in obesity and diabetes (22) are just some reasons for the explosion of research in PPAR and 15-deoxy-^12,14-PGJ₂ (Medline: > 1200 reports in the last five years). However, the question of whether 15d-PGJ₂ is a naturally occurring compound in human tissues, the lack of apparent stereo-selectivity, and the micromolar concentrations required for PPAR activation has led to much discussion and controversy in the literature and, needless to say, exciting and highly animated discussions amongst noted experts at national and international scientific meetings. Given that the crystal structure of the ligand binding domain of PPAR indicates a large accommodating pocket consistent with the high degree of promiscuity of this receptor (23), it has been argued that 15-deoxy-^12,14-PGJ₂ might have value as a useful lead compound in the context of drug discovery.

The PPAR subtype selective activators of different structural classes provided a powerful set of tools as chemical probes for biological functions of these receptors and their mechanisms of action (16). For example, a common experimental approach is to establish PPAR involvement by showing similar response due to activators of seemingly unrelated structures (e.g., a TZD and 15-deoxy-^12,14-PGJ₂). In utilization of these pharmacological tools, it was often noted that there was a lack of correlation between the effective concentrations required to activate PPAR and rank order potency of these compounds. This led to the notion that 15-deoxy-^12,14-PGJ₂ also acted via PPAR-independent mechanisms (24). Results from these activation-based studies have indicated roles for PPAR in different biological contexts. Wang and colleagues (25) use this chemical approach to unveil a mechanism in airway epithelial cells where activation of interleukin (IL)-4-induced PPAR downregulates expression of the proinflammatory cytokine IL-8 (Figure 2). This study beckons the question "Does PPAR offer a novel avenue for drug discovery in the treatment of airway inflammation and asthma?"

	Is There an Assigned Role for PPAR in Airway Inflammation?

The first in vivo evidence linking PPARs to inflammation came from the demonstration that PPAR knock-out mice displayed a prolonged inflammatory response when challenged with the proinflammatory mediator leukotriene B₄ (26). This study proposed a role for PPAR as a nuclear eicosanoid receptor that expedites clearance of eicosanoids as a mechanism of inflammation control. In this context, it is essential to recognize that inflammation is normally a self-resolving process with the existence of both positive and negative regulators that ultimately allow complete resolution and homeostasis (27). In the absence of resolution and clearance or in the event of dampened healing response, persistent inflammation can arise in the form of tissue damage as associated with chronic diseases, such as rheumatoid arthritis, atherosclerosis, and apparently to some extent airway inflammation, as recently appreciated in asthma. The class of antidiabetic compounds TZDs and other PPAR activators were proposed to serve as lead structures for antiinflammatory drugs and it was suggested that related libraries should be screened in traditional antiinflammatory assays (28).

The present work of Conrad and colleagues (25) demonstrates that PPAR can regulate the production of a key chemo-attractant, IL-8, from airway epithelial cells. This was also mimicked by pharmacologic levels of 15-deoxy-^12,14-PGJ₂, commercially-available material. We should emphasize again that 15-deoxy-^12,14-PGJ₂ has not yet been shown to be an endogenous product of human airway; such results remain to be obtained from rigorous mass spectrometry-based analyses. Although radioimmunoassays and immunoreactive material have been quantitated in some studies (29), the precise identity of the potential cross-reacting material has not yet been established, and it is noteworthy that Maxey and coworkers (30) clearly demonstrated the presence of additional and unrelated material in commercial preparations of 15-deoxy-^12,14-PGJ₂. Any of these contaminating substances, if in high enough levels, could be responsible for activation of PPAR. Nevertheless, despite the lack of stereo-selectivity by 15-deoxy-^12,14-PGJ₂, Wang and coworkers (25) clearly demonstrate that regulators of PPAR, whether they are xenobiotic or exogenous small molecule regulators, can inhibit IL-8 generation and, ergo, demonstrate a novel mechanism in airway epithelial cells for inhibition of leukocyte recruitment and, potentially, airway inflammation. This mechanism is reminiscent of an earlier uncovered pathway found in the host cell response to interactions with Salmonella typhimurium where endogenous antiinflammatory eicosanoids (lipoxin A₄ and aspirin-triggered lipoxin A₄) downregulate IL-8 generation by intestinal epithelial cells (31). In these studies, stable analogs of lipoxin A₄ facilitate clear demonstration of stereo-selectivity for lipoxin A₄ inhibition (in the nanomolar range) of IL-8, a process where activation of the cell surface receptor for lipoxin A₄ results in downstream events leading to inhibition of the NFB pathway. These and other findings provide the first evidence for endogenous antiinflammatory compounds emanating from lipoxygenase pathways and transcellular routes that can play a pivotal role in the resolution of inflammation (see [32] for review). The lipoxin and aspirin-triggered lipoxin stable analogs developed to date have proven to possess profound inhibitory actions in leukocyte recruitment in many different murine models of inflammation that appear relevant to several diseases, including gastroenteritis, lung injury, nephritis, and hematologic disorders (32).

	So What Have We Learned from PPAR and the 15-deoxy-12,14-PGJ2 Exercise?

In addition to having antitumor and antiviral activities, the cyclopentenone prostaglandins have been recently implicated in antiinflammation. Are these compounds found in nonmarine organisms? One argument often used against endogenous formation of 15-deoxy-PGJ₂ in higher organisms is the evidence showing in vitro nonenzymatic conversion of PGD₂ and metabolites to PGJ₂ equivalents (14, 33). In one report (33), the authors "speculate that albumin-catalyzed transformation of PGD₂ could also occur in vivo as a supplementary inactivation process." No doubt the debate on 15-deoxy-^12,14-prostaglandin J₂ as an example of cross-species chemical diversity will continue until rigorous results prove conclusively, one way or the other, whether these eicosanoids are exclusive to marine organisms and if they have associated endogenous biological functions.

The controversy on 15-deoxy-^12,14-PGJ₂ should not distract us from the fact that PPAR is considered a valid target for drug discovery in some metabolic disorders, as demonstrated by the interests of major pharmaceutical companies. If anything, the 15-deoxy-PGJ₂ exercise has taught us that we need to pay close attention to the rigor and detail of structure-function, if we are to gain knowledge of appropriate pathways for therapeutic interventions relevant to the treatment of asthma. Clearly, it is necessary to distinguish pharmacology from physiology. It is also essential to describe structure-function relationships of small molecule interactions with host tissues that could be relevant in a wide range of cell-cell interaction events in vivo. The control of cytokines is, no doubt, an important component of inflammation, whether it be pro- or antiinflammation. Novel targets have emerged that could provide many new therapeutic approaches to diseases where inflammation is held to play a key role.

Today, the treatments for asthma and related airway disorders are very different from those prescribed by the Egyptians nearly 3,500 years ago (1). Despite these advances, an estimated 7% of children aged 18 and younger have asthma, making it one of the most common chronic diseases associated with childhood (http://www.nhlbi.nih. gov). So there is every incentive to continue the quest and explore new approaches to treatment of asthma and related disorders.

	Footnotes

Address correspondence to: Charles N. Serhan, Ph.D., Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. E-mail: cnserhan{at}zeus.bwh.harvard.edu

(Received in original form April 5, 2001).

Acknowledgments: The authors thank Mary Halm Small for assistance in manuscript preparation. This work was supported in part by grant no. GM38765 (C.N.S.) from the National Institutes of Health. P.D. is the recipient of a Mentored Research Scientist Development Award no. K01-AR02218 from the National Institutes of Health.

	References

1. Ebbell, B. 1937. The Papyrus Ebers: the greatest Egyptian medical document. Levin and Munksgaard, Copenhagen.

2. Murray, J. F., and J. A. Nadel. 2000. Textbook of Respiratory Medicine, 3rd ed. W.B. Saunders Co., Philadelphia.

3. Drazen, J. M., E. Israel, and P. M. O'Byrne. 1999. Treatment of asthma with drugs modifying the leukotriene pathway. N. Engl. J. Med. 340: 197-206 [Free Full Text].

4. Barnes, P. J. 1999. Therapeutic strategies for allergic diseases. Nature 402 (6760 Suppl):B31-B38.

5. Holgate, S. T. 1999. The epidemic of allergy and asthma. Nature 402(6760 Suppl):B2-B4.

6. Fukushima, M., T. Kato, K. Ota, Y. Arai, S. Narumiya, and O. Hayaishi. 1982. 9-deoxy-delta 9-prostaglandin D2, a prostaglandin D2 derivative with potent antineoplastic and weak smooth muscle-contracting activities. Biochem. Biophys. Res. Commun. 109: 626-633 [Medline].

7. Kato, T., M. Fukushima, S. Kurozumi, and R. Noyori. 1986. Antitumor activity of delta 7-prostaglandin A1 and delta 12-prostaglandin J2 in vitro and in vivo. Cancer Res. 46: 3538-3542 [Abstract/Free Full Text].

8. Willis, A. L. CRC Handbook of Eicosanoids: Prostaglandins and Related Lipids Vol:1. CRC Press, Boca Raton.

9. Honda, A., Y. Yamamoto, Y. Mori, Y. Yamada, and H. Kikuchi. 1985. Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells. Biochem. Biophys. Res. Commun. 130: 515-523 [Medline].

10. Fukushima, M., and T. Kato. 1985. Antitumor marine icosanoids: clavulones and punaglandins. Adv. Prostaglandin Thromboxane Leukot Res. 15: 415-418 [Medline].

11. Kikuchi, H., Y. Tsukitani, K. Igushi, and Y. Yamada. 1982. Clavulones, new type of prostanoids from the Stolonifer Clavularia viridis Quoy and Gaimard. Tetrahedron Lett 23: 5171-5174 .

12. Kobayashi, M., T. Yasuzawa, M. Yoshihara, H. Akutsu, Y. Kyogoku, and I. Kitagawa. 1982. Four new prostanoids: claviridenone-a, -b, -c, and -d from the Okinawan soft coral Clavularia viridis. Tetrahedron Lett. 23: 5331-5334 .

13. Lewis, R. A., N. A. Soter, P. T. Diamond, K. F. Austen, J. A. Oates, and L. J. Roberts 2nd.. 1982. Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE. J. Immunol. 129: 1627-1631 [Abstract].

14. Fitzpatrick, F. A., and M. A. Wynalda. 1981. Albumin-lipid interactions: prostaglandin stability as a probe for characterising binding sites on vertebrate albumins. Biochem. 6129-6134.

15. Morrow, J. D., C. Prakash, T. A. Duckworth, W. E. Zackert, I. A. Blair, J. A. Oates, and L. J. Roberts. 1991. A stable isotope dilution mass spectrometric assay for the major urinary metabolite of PGD2. Adv. Prostaglandin Thromboxane Leukot. Res. 21A:315-318.

16. Kersten, S., B. Desvergne, and W. Wahli. 2000. Roles of PPARs in health and disease. Nature 405: 421-424 [Medline].

17. Kliewer, S. A., J. M. Lenhard, T. M. Willson, I. Patel, D. C. Morris, and J. M. Lehmann. 1995. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83: 813-819 [Medline].

18. Forman, B. M., P. Tontonoz, J. Chen, R. P. Brun, B. M. Spiegelman, and R. M. Evans. 1995. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83: 803-812 [Medline].

19. Lehmann, J. M., L. B. Moore, T. A. Smith-Oliver, W. O. Wilkison, T. M. Willson, and S. A. Kliewer. 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J. Biol. Chem. 270: 12953-12956 [Abstract/Free Full Text].

20. Marnett, L. J.. 2000. Cyclooxygenase mechanisms. Curr. Opin. Chem. Biol. 4: 545-552 . [Medline]

21. Kimura, S., and F. J. Gonzalez. 2000. Applications of genetically manipulated mice in pharmacogenetics and pharmacogenomics. Pharmacology 61: 147-153 [Medline].

22. Spiegelman, B. M., and J. S. Flier. 2001. Obesity and the regulation of energy balance. Cell 104: 531-543 [Medline].

23. Gampe, R. T., V. G. Montana, M. H. Lambert, A. B. Miller, R. K. Bledsoe, M. V. Milburn, S. A. Kliewer, T. M. Willson, and H. E. Xu. 2000. Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol. Cell. 5: 545-555 . [Medline]

24. Straus, D. S., G. Pascual, M. Li, J. S. Welch, M. Ricote, C. H. Hsiang, L. L. Sengchanthalangsy, G. Ghosh, and C. K. Glass. 2000. 15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc. Natl. Acad. Sci. USA 97: 4844-4849 [Abstract/Free Full Text].

25. Wang, A. C. C., X. Dai, B. Luu, and D. J. Conrad. 2001. Peroxisome proliferator-activated receptor- regulates airway epithelial cell activation. Am. J. Respir. Cell Mol. Biol. 24: 688-693 [Abstract/Free Full Text].

26. Devchand, P. R., H. Keller, J. M. Peters, M. Vazquez, F. J. Gonzalez, and W. Wahli. 1996. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature 384: 39-43 [Medline].

27. Cotran, R., V. Kumar, and T. Collins, eds. 1999. Robbins pathologic basis of disease, 6th ed. W. B. Saunders, Philadelphia.

28. Serhan, C. N.. 1996. Signalling the fat controller. Nature 384: 23-24 .

29. Gilroy, D. W., P. R. Colville-Nash, D. Willis, J. Chivers, M. J. Paul-Clark, and D. A. Willoughby. 1999. Inducible cyclooxygenase may have anti-inflammatory properties. Nat. Med. 5: 698-701 [Medline].

30. Maxey, K. M., E. Hessler, J. MacDonald, and L. Hitchingham. 2000. The nature and composition of 15-deoxy-Delta(12,14)PGJ(2). Prostaglandins Other Lipid Mediat. 62: 15-21 . [Medline]

31. Gewirtz, A. T., B. McCormick, A. S. Neish, N. A. Petasis, K. Gronert, C. N. Serhan, and J. L. Madara. 1998. Pathogen-induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A4 analogs. J. Clin. Invest. 101: 1860-1869 [Medline].

32. Serhan, C. N., T. Takano, N. Chiang, K. Gronert, and C. B. Clish. 2000. Formation of endogenous "antiinflammatory" lipid mediators by transcellular biosynthesis: lipoxins and aspirin-triggered lipoxins inhibit neutrophil recruitment and vascular permeability. Am. J. Respir. Crit. Care Med. 161(2 Pt 2):S95-S101.

33. Fitzpatrick, F. A., and M. A. Wynalda. 1983. Albumin-catalyzed metabolism of prostaglandin D2: identification of products formed in vitro. J. Biol. Chem. 258: 11713-11718 [Abstract/Free Full Text].

34. Su, C. G., X. Wen, S. T. Bailey, W. Jiang, S. M. Rangwala, S. A. Keilbaugh, A. Flanigan, S. Murthy, M. A. Lazar, and G. D. Wu. 1999. A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response. J. Clin. Invest. 104: 383-389 [Medline].

35. Wright, H. M., C. B. Clish, T. Mikami, S. Hauser, K. Yanagi, R. Hiramatsu, C. N. Serhan, and B. M. Spiegelman. 2000. A synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation. J. Biol. Chem. 275: 1873-1877 [Abstract/Free Full Text].

36. Sanak, M., B. D. Levy, C. B. Clish, N. Chiang, K. Gronert, L. Mastalerz, C. N. Serhan, and A. Szczeklik. 2000. Aspirin-tolerant asthmatics generate more lipoxins than aspirin-intolerant asthmatics. Eur. Respir. J. 16: 44-49 [Abstract].