HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/15/2269 02-1162fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GILROY, D. W. Articles by LAWRENCE, T. Search for Related Content PubMed PubMed Citation Articles by GILROY, D. W. Articles by LAWRENCE, T.

Inducible cyclooxygenase-derived 15-deoxy^12-14PGJ₂ brings about acute inflammatory resolution in rat pleurisy by inducing neutrophil and macrophage apoptosis¹

Department of Experimental Pathology, William Harvey Research Institute, St. Bartholomew’s & The Royal London School of Medicine and Dentistry, London EC1M 6BQ, UK

²Correspondence: E-Mail: d.w.gilroy{at}qmul.ac.uk

BACKGROUND AND SPECIFIC AIMS

Two isoforms of cyclooxygenase (COX) have been identified and characterized. In general, COX 1 is constitutively expressed in most tissues, where it synthesizes prostaglandins (PGs) in an immediate manner at low levels to maintain physiological functions. COX 2, on the other hand, is highly inducible in response to proinflammatory stimuli, resulting in delayed but exaggerated PG release, leading to the suggestion it contributes significantly to the development of an inflammatory response. However, recent studies have suggested that COX 2 may also be protective in wound healing and tissue injury. We have previously shown that COX 2 mediates acute inflammatory resolution in a rat carrageenin-induced pleurisy through the generation of anti-inflammatory PGs, PGD₂, and 15-deoxy-^12-14 PGJ₂. In the current paper we uncover the mechanism by which COX 2 and its fatty acid metabolites bring acute inflammation under control and mediate resolution.

PRINCIPAL FINDINGS

1. Inflammatory cell profile and apoptosis in an acute pleurisy
After carrageenin injection into the pleural cavity of rats, total inflammatory cell numbers and edema peaked at 24 h and were cleared by 72 h. Flow-assisted cell sorting in combination with specific cell surface markers for rat inflammatory cells as well as histological analysis of cell smears revealed that polymorphonuclear leukocytes (PMNs) predominated at the onset of the lesion and were replaced by influxing monocytes, which differentiated into macrophages. These macrophages persisted until resolution at 72 h (Fig. 1 ). Having established the inflammatory cell profile in this model, we identified the cell types undergoing apoptosis. We used microscopy to discern morphological features of apoptosis, annexin V labeling to identify phosphatidylserine, which is expressed on the surface of apoptosing cells, and finally TUNEL, which identifies DNA fragmentation, a later stage of programmed cell death. We found that the natural reduction in PMN numbers resulted from apoptosis, which occurred 36/48 h after carrageenin injection. Macrophages also underwent programmed cell death, leading to an abatement of the inflammatory response and eventual resolution (Fig. 2 ).

View larger version (19K): [in this window] [in a new window]   Figure 1. Inflammatory cell profile from onset to resolution in rat carrageenin-induced pleurisy. Two separate experimental approaches were used to discern the cell types present. First, cell smears were stained with hematoxylin and eosin to identify the cells by morphology. Second, inflammatory cells were stained with either FITC-labeled anti CD11B (which binds PMNs, monocytes and macrophages but not lymphocytes), FITC-labeled ED1 (monocytes and macrophages), or FITC-labeled ED2 (only macrophages) and analyzed by flow-assisted cell sorting.

View larger version (46K): [in this window] [in a new window]   Figure 2. Characterization of inflammatory cell apoptosis in the rat carrageenin-induced pleurisy by annexin V. Inflammatory cells were double labeled with PE-labeled annexin V and FITC-labeled anti CD11B, -ED1, or -ED2 and analyzed by FACS. Samples from each time point were halved: one-half was incubated in a calcium naïve binding buffer (A) and the other in a calcium-rich buffer (B); both samples received annexin V (representative scatter grams are shown). This method was used as the binding of annexin V to phosphatidylserine on the surface of apoptosing cells is calcium dependent. The absence of calcium in the binding buffer corrected for nonspecific annexin V binding and was used as the most appropriate control. Therefore, all cells that shifted beyond 102 (FL2-H) were considered to be apoptosing (gated area).

2. Role of COX 2 in inflammatory cell apoptosis
Next we sought to determine the involvement, if any, of COX 2 and its fatty acid metabolites in the induction of PMN and macrophage apoptosis during resolution. The nonselective COX inhibitor indomethacin as well as the selective COX 2 inhibitors NS-398 and SC-58125 were administered orally 24 h after carrageenin injection and, as already published, were found to impair resolution. This impairment of resolution, however, was associated with a concomitant reduction in PMN and macrophage apoptosis. The selective COX 1 inhibitor, SC-560, was without effect on either resolution or inflammatory cell apoptosis. By carrying out experiments where animals were dosed orally with COX 2 inhibitors, but had 15-deoxy^12-14PGJ₂ instilled intrapleurally to rescue locally inhibited levels of the cyclopentenone PG, we found that both PMN and macrophage apoptosis was increased compared with NSAID-treated animals and resolution was restored. We concluded that through the release of 15-deoxy^12-14PGJ₂, COX 2 brought about the resolution of an acute pleurisy through the induction of both PMN and macrophage apoptosis.

CONCLUSION AND SIGNIFICANCE

Acute inflammatory resolution may be described as the clearance of inflammatory cells and edema as well as the initiating stimulus from an inflamed site leading to restoration of the tissue to its original physiological function with little or no evidence of lasting tissue damage. A number of factors may control the fate of inflammatory cells during inflammatory resolution. First, inflammatory cells may die locally by apoptosis and/or necrosis, followed by their phagocytosis by monocyte-derived macrophages or local tissue histiocytes. Alternatively, inflammatory cells may be cleared from the inflamed site through the draining lymphatics to local lymph nodes or by emigration back into systemic circulation. Regarding the mechanisms and mediators that govern resolution of an acute pleurisy initiated with carrageenin, the novel finding reported in this paper is that PMNs and macrophages both die locally during acute inflammatory resolution by programmed cell death that is mediated at least in part by COX 2-derived cyclopentenone PGs (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic representing the changes in inflammatory cell influx and death by apoptosis in the background of changing profile of COX 2 protein expression in a resolving pleurisy. PMNs are the first cell types to migrate into the pleural cavity in response to carrageenin injection and are progressively replaced by monocytes, which differentiate into macrophages. Using a combination of annexin V labeling, cell morphology and TUNEL we found that PMNs and subsequently macrophages underwent apoptosis leading to resolution, and that this inflammatory cell apoptosis was brought about by COX 2-derived 15-deoxy12-14PGJ2.

PMN programmed cell death is well described in various clinical and experimental settings; the emerging concept regarding PMN apoptosis during inflammation is that not only is it a means of cell death that does not aggravate an existing inflammatory response, but that apoptotic cells themselves possess a range of anti-inflammatory signals on their surface that may hasten resolution. Thus, there seems to be every benefit to the host that inflammatory cells like PMNs die by programmed cell death. Although PMN apoptosis is well understood, the observation that macrophages apoptose during resolution of acute inflammation is less well described and provides further insight into mechanisms that switch off acute inflammation. This is particularly important as it was originally thought that macrophage clearance in self-limiting inflammatory lesions was via lymphatic drainage. Here we provide evidence that apoptosis is an additional route. Macrophages can damage tissues by the release of histotoxic enzymes and proinflammatory cytokines, and initiate an immune response by the presentation of antigens to T cells. Through the sustained release of TGFß1, macrophages can also contribute significantly to fibrosis, collagen, and fibronectin synthesis as well as granulation tissue formation. Thus, while macrophages are essential to clear up effete cells and debris, their persistence at an inflammatory site must be tightly controlled in order to prevent the development of chronic inflammation. In this report we show that macrophages undergo apoptosis locally at the end phase of an acute resolving pleurisy. While little is known about the etiology of chronic inflammation, we suggest that COX 2-mediated macrophage apoptosis may represent an important endogenous means of preventing the transition of acute inflammation to chronic nonresolving inflammatory diseases.

In summary, in an acute pleurisy, PMNs predominated at the onset of the lesion but decreased in number as a result of undergoing apoptosis. Populations of PMNs were progressively replaced by monocytes, which differentiated locally into macrophages. As with PMNs, macrophages also underwent programmed cell death leading to resolution. We found that apoptosis of both these inflammatory cell types was mediated by COX 2-derived 15-deoxy^12-14PGJ₂.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1162fje; doi: 10.1096/fj.02-1162fje

This article has been cited by other articles:

				R. Rajakariar, T. Lawrence, J. Bystrom, M. Hilliard, P. Colville-Nash, G. Bellingan, D. Fitzgerald, M. M. Yaqoob, and D. W. Gilroy Novel biphasic role for lymphocytes revealed during resolving inflammation Blood, April 15, 2008; 111(8): 4184 - 4192. [Abstract] [Full Text] [PDF]

				W.-L. Song, M. Wang, E. Ricciotti, S. Fries, Y. Yu, T. Grosser, M. Reilly, J. A. Lawson, and G. A. FitzGerald Tetranor PGDM, an Abundant Urinary Metabolite Reflects Biosynthesis of Prostaglandin D2 in Mice and Humans J. Biol. Chem., January 11, 2008; 283(2): 1179 - 1188. [Abstract] [Full Text] [PDF]

				R. Rajakariar, M. Hilliard, T. Lawrence, S. Trivedi, P. Colville-Nash, G. Bellingan, D. Fitzgerald, M. M. Yaqoob, and D. W. Gilroy From the Cover: Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxy{Delta}12 14 PGJ2 PNAS, December 26, 2007; 104(52): 20979 - 20984. [Abstract] [Full Text] [PDF]

				M. Joo, M. Kwon, R. T. Sadikot, P. J. Kingsley, L. J. Marnett, T. S. Blackwell, R. S. Peebles Jr, Y. Urade, and J. W. Christman Induction and Function of Lipocalin Prostaglandin D Synthase in Host Immunity J. Immunol., August 15, 2007; 179(4): 2565 - 2575. [Abstract] [Full Text] [PDF]

				H. Sandig, J. E. Pease, and I. Sabroe Contrary prostaglandins: the opposing roles of PGD2 and its metabolites in leukocyte function J. Leukoc. Biol., February 1, 2007; 81(2): 372 - 382. [Abstract] [Full Text] [PDF]

				R. Soares, I. Azevedo, D. A. Sawatzky, and A. G. Rossi Apigenin: Is It a Pro- or Anti-Inflammatory Agent? Am. J. Pathol., May 1, 2006; 168(5): 1762 - 1763. [Full Text] [PDF]

				A. S. Damazo, S. Yona, R. J. Flower, M. Perretti, and S. M. Oliani Spatial and Temporal Profiles for Anti-Inflammatory Gene Expression in Leukocytes during a Resolving Model of Peritonitis J. Immunol., April 1, 2006; 176(7): 4410 - 4418. [Abstract] [Full Text] [PDF]

				S. G. Trivedi, J. Newson, R. Rajakariar, T. S. Jacques, R. Hannon, Y. Kanaoka, N. Eguchi, P. Colville-Nash, and D. W. Gilroy Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity PNAS, March 28, 2006; 103(13): 5179 - 5184. [Abstract] [Full Text] [PDF]

				O. Millan, D. Rico, H. Peinado, N. Zarich, K. Stamatakis, D. Perez-Sala, J. M. Rojas, A. Cano, and L. Bosca Potentiation of tumor formation by topical administration of 15-deoxy-{Delta}12,14-prostaglandin J2 in a model of skin carcinogenesis Carcinogenesis, February 1, 2006; 27(2): 328 - 336. [Abstract] [Full Text] [PDF]

				D. A. Sawatzky, D. A. Willoughby, P. R. Colville-Nash, and A. G. Rossi The Involvement of the Apoptosis-Modulating Proteins ERK 1/2, Bcl-xL and Bax in the Resolution of Acute Inflammation in Vivo Am. J. Pathol., January 1, 2006; 168(1): 33 - 41. [Abstract] [Full Text] [PDF]

				S. Appel, V. Mirakaj, A. Bringmann, M. M. Weck, F. Grunebach, and P. Brossart PPAR-{gamma} agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-{kappa}B pathways Blood, December 1, 2005; 106(12): 3888 - 3894. [Abstract] [Full Text] [PDF]

				M. D. Morgan, L. Harper, X. Lu, G. Nash, J. Williams, and C. O. S. Savage Can neutrophils be manipulated in vivo? Rheumatology, May 1, 2005; 44(5): 597 - 601. [Full Text] [PDF]

				X. Zhang, M. C. Rodriguez-Galan, J. J. Subleski, J. R. Ortaldo, D. L. Hodge, J.-M. Wang, O. Shimozato, D. A. Reynolds, and H. A. Young Peroxisome proliferator-activated receptor-{gamma} and its ligands attenuate biologic functions of human natural killer cells Blood, November 15, 2004; 104(10): 3276 - 3284. [Abstract] [Full Text] [PDF]

				N. Jiang and D. S. Pisetsky The Effect of Dexamethasone on the Generation of Plasma DNA from Dead and Dying Cells Am. J. Pathol., May 1, 2004; 164(5): 1751 - 1759. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/15/2269 02-1162fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GILROY, D. W. Articles by LAWRENCE, T. Search for Related Content PubMed PubMed Citation Articles by GILROY, D. W. Articles by LAWRENCE, T.