rapid appearance of resolvin precursors in inflammatory exudates: novel mechanisms in resolution

Rapid Appearance of Resolvin Precursors in InflammatoryExudates: Novel Mechanisms in Resolution1

Kie Kasuga,2* Rong Yang,2* Timothy F. Porter,* Nitin Agrawal,‡ Nicos A. Petasis,†

Daniel Irimia,‡ Mehmet Toner,‡ and Charles N. Serhan3*

Resolution of inflammation is essential. Although supplementation of �-3 fatty acids is widely used, their availability at sites of inflam-mation is not known. To this end, a multidisciplinary approach was taken to determine the relationship of circulating �-3 to inflam-matory exudates and the generation of resolution signals. In this study, we monitored resolvin precursors in evolving exudates, whichinitially paralleled increases in edema and infiltrating neutrophils. We also prepared novel microfluidic chambers to capture neutrophilsfrom a drop of blood within minutes that permitted single-cell monitoring. In these, docosahexaenoic acid-derived resolvin D1 rapidlystopped neutrophil migration, whereas precursor docosahexaenoic acid did not. In second organ injury via ischemia-reperfusion,resolvin metabolically stable analogues were potent organ protectors reducing neutrophils. Together, these results indicate that circu-lating �-3 fatty acids rapidly appear in inflammatory sites that require conversion to resolvins that control excessive neutrophil infil-tration, protect organs, and foster resolution. The Journal of Immunology, 2008, 181: 8677–8687.

M ounting evidence indicates that the resolution of acuteinflammation is a highly coordinated and biochemi-cally active process that was once thought to be a pas-

sive event (for recent review, see Ref. 1). During inflammatoryresponses to infections, neutrophils first migrate into tissues toparticipate in host defense, and then, if successful, these tissuesreturn to their homeostatic functions (2). Tissue level resolutionprograms are actively initiated, and the number of tissue-associ-ated polymorphonuclear neutrophils (PMN)4 is reduced (1). Thus,it is possible that excessive inflammatory responses and their pro-gression to chronic inflammation can result from a local failure toresolve (1). The active processes by which acute inflammation isresolved to tissue homeostasis are of considerable interest becausemany widely occurring human diseases are associated with chronicinflammation. These include arthritis (3) and periodontal disease(4) as well as diseases that are recognized relatively recently to

have aberrant inflammation as a component of the disease, includ-ing asthma, cardiovascular disease, and Alzheimer’s disease (5–7).

The process of resolution is actively controlled in part by formationof newly described endogenous chemical mediators that as local au-tacoids stimulate proresolving mechanisms (for recent reviews, seeRefs. 1 and 8). These proresolving mediators are derived from essen-tial fatty acids that include lipoxins from arachidonic acid (C20:4) andresolvins and protectins from eicosapentaenoic acid (EPA; C20:5) anddocosahexaenoic acid (DHA; C22:6) (8) that are biosynthesized ininflammatory exudates during spontaneous resolution. When specificresolvins and/or protectins are administered in vivo, each stimulatesmulticellular tissue-level responses geared to bring tissues back tohomeostasis in a process coined cellular and molecular catabasis (9).

The impact of �-3 fatty acids (EPA and DHA) themselves hasbeen evaluated in numerous clinical studies (10–14). These reportsand many others emphasize the potential benefits of dietary sup-plementation with �-3 fatty acids. Along these lines, i.v. admin-istration of �-3 fatty acids leads to clinical improvements in pa-tients with rheumatoid arthritis (12). In healthy subjects, �-3 fattyacids reduce LPS-induced fever as well as inflammatory responses(13). Soy nuts, enriched with �-3 fatty acids, improve systolicblood pressure and low-density lipoprotein cholesterol levels inhypertensive postmenopausal women (14). In children at risk oftype 1 diabetes, �-3 fatty acids reduce risk (15). DHA is widelyappreciated for its neurotrophic and neuroprotection roles that re-quire esterification into phospholipids (16, 17). DHA tissue levelsalso appear to be critical because in diseases such as cystic fibrosis,the DHA tissue stores appear to be depleted (18). As noted above,�-3 fatty acids are precursors to potent families of mediators,namely resolvins and protectins that are biosynthesized in sponta-neously resolving exudates and are stereoselective in their forma-tion and actions (1). Recently, the original structural elucidation ofresolvin D1 (RvD1) (19) was confirmed by total organic synthesis,and its complete stereochemistry was established as 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (20).Thus, the relationship between circulating EPA and DHA and in-flammation resolution is of interest and a focus of this report.

The �-3 fatty acids have been measured in humans in manystudies and show a wide range. In healthy subjects, EPA levels

*Center for Experimental Therapeutics and Reperfusion Injury, Department of An-esthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Bos-ton, MA 02115; †Department of Chemistry, University of Southern California, LosAngeles, CA 90089; and ‡BioMEMS Resource Center, Massachusetts General Hos-pital, Charlestown, MA 02129, Shriners Hospital for Children, Boston, MA 02114and Harvard Medical School, Boston, MA 02115

Received for publication June 18, 2008. Accepted for publication October 7, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grant R37-GM38765 (to C.N.S.); the Specialized Center for Oral Inflammation and Resolution,P50-DE016191 (to C.N.S. and N.A.P.); and the BioMEMS Resource Center, P41-EB002503 (to M.T. and D.I.). K.K. is the recipient of a postdoctoral fellowship awardfrom the Arthritis Foundation.2 K.K. and R.Y. contributed equally to this study.3 Address correspondence and reprint requests to Dr. Charles N. Serhan, Brigham andWomen’s Hospital, 75 Francis Street, Boston, MA 02115.4 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; d5-DHA,4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic-21,21,22,22,22-d5 acid; d5-EPA,5Z,8Z,11Z,14Z,17Z-eicosapentaenoic-19,19,20,20,20-d5 acid; DHA, docosahexa-enoic acid; EPA, eicosapentaenoic acid; GC-MS, gas chromatography-mass spec-trometry; MPO, myeloperoxidase; PDMS, poly(dimethylsiloxane); PEG, polyethyleneglycol; PLA2, phospholipase A2; RvD1, resolvin D1; RvE1, resolvin E1.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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range from �0.5 to 2.8% of total fatty acids and DHA ranges from�1.3 to 5.0% (21–25). For the purpose of direct comparisons, seeSupplementary Table I (online and references within).5 This widerange of values for humans appears to reflect diet (25). Because�-3 fatty acids are now recognized as biosynthetic precursors topotent local mediators, their relationship between circulation andexudate formation may be relevant in the resolution of acute in-flammation. Given the importance of this question, a multidisci-plinary effort was undertaken to address the availability of �-3fatty acids to acute inflammatory exudates and to assess the directactions on cells and tissues compared with their potent bioactiveproduct(s), namely a resolvin.

In this study, using a multipronged and multidisciplinary ap-proach, we report the following: 1) that both labeled EPA andDHA from circulation rapidly appear at sites of evolving inflam-matory exudates in vivo; 2) in microfluidic chambers, RvD1 andnot equal concentrations of its precursor directly act on single hu-man PMN; and 3) metabolically stable resolvin mimetics havepotent organ-protective actions in murine ischemia-reperfusion in-jury. These findings emphasize the importance of circulating levelsof �-3 fatty acids and their conversion by exudates to resolvinsthat directly regulate both tissue inflammation and organ injury.

Materials and MethodsAnimals

All animals used in the present study were male FVB mice (Charles RiverLaboratories) that were 6–8 wk old (weighing 20–25 g). They were main-tained in a temperature and light-controlled environment, and had unlim-ited access to food (laboratory standard rodent diet 5001 (Lab Diet)), con-taining 1.5% EPA, 1.9% DHA of total fatty acids, and tap water.Experiments were performed in accordance with the Harvard MedicalSchool Standing Committee on Animals guidelines for animal care (Pro-tocol 02570).

Chemicals

RvD1 and 17-(R/S)-methyl RvD1 were each synthesized by total organicsynthesis for these experiments from starting materials of known chiralityin enantiomerically and geometrically pure form (N. Petasis, J. Uddin, andC. Serhan, manuscript in submission) by the Organic Synthesis Core, Na-tional Institutes of Health P50-DE-016191. Resolvin E1 (RvE1) and the19-p-fluorophenoxy-RvE1 methyl ester were prepared in an enantiomeri-cally and geometrically pure form, as described previously (26) (N. Petasis,R. Yang, G. Bernasconi, J. Uddin, and C. Serhan, manuscript in submis-sion). Physical and spectroscopic properties matching biogenic and syn-thetic RvD1 and RvE1were as reported (20, 27). The integrity of eachsynthetic resolvin and their analogues was assured by monitoring the phys-ical properties with LC-UV-tandem mass spectrometry just before evalu-ating their biological activities.

Murine peritonitis

Male FVB mice were anesthetized with isoflurane (Hospira). Both 1 �gof 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic-21,21,22,22,22-d5 acid (d5-DHA; Cayman Chemical) and 1 �g of 5Z,8Z,11Z,14Z,17Z-eicosapenta-enoic-19,19,20,20,20-d5 acid (d5-EPA; Cayman Chemical) in 100 �l of 2%ethanol (v/v) in saline or vehicle alone were administered by bolus tail veininjection. Approximately 5 min later, peritonitis was initiated with i.p.administration of 1 mg of zymosan A (Sigma Aldrich). For control exper-iments, 1 ml of saline was injected i.p. At indicated time intervals, micewere euthanized with an overdose of isoflurane, and peritoneal exudateswere collected by lavage with 5 ml of Dulbecco’s PBS without either Ca2�

or Mg2�. Exudate cells and supernatants were separated by centrifugation(10 min, 1000 rpm, 4°C), aliquots of supernatants were collected, and 4 volof cold acetone were added to precipitate proteins. Serum albumin and totalprotein levels were determined (28).

For tracer experiments, mice fasted overnight (�18 h) before 500 nCi of[1-14C]DHA (docosahexaenoic acid 4,7,10,13,16,19-[1-14C]; AmericanRadiolabeled Chemicals) were injected via the tail vein. At indicated timeintervals, exudates were collected (vide supra) and the radioactivity was

determined using a scintillation counter (multipurpose scintillation counterLS6500; Beckman Coulter).

Differential counts and FACS analysis

Aliquots of lavage cells were assessed for total and differential leukocytecounts via light microscopy to identify individual cell types (i.e., neutro-phil, monocyte, etc.). For flow cytometry analysis, aliquots of 0.5 � 106

cells were stained with 0.25 �g of FITC-conjugated anti-mouse F4/80,0.1 �g of PE-conjugated anti-mouse Ly-6G, and 0.1 �g of PerCP-Cy5.5-conjugated anti-mouse CD11b, or 0.1 �g of PE-conjugated ratIgG2c,� isotype control as a background stain. Cells were washed andanalyzed using FACSort (BD Biosciences) (29).

Mass spectrometric analysis of deuterium-labeled EPA andDHA

Upon collection, 2 vol of cold methanol containing internal standard (500ng/sample, trans-parinaric acid; Molecular Probes) were added to exu-dates, which were stored at �20°C for 1 h. After centrifugation, sampleswere diluted and applied to C18 cartridge columns (Extract-Clean EV SPE;Alltech Associates). Lipids were extracted using hexane and methyl for-mate, and each fraction was collected and its unesterified fatty acids con-verted to corresponding methyl ester using diazomethane treatment (30).The esters were suspended in hexane, and then injected in gas chromatog-raphy-mass spectrometry (GC-MS). Electron-impact GC-MS was con-ducted using an HP 6890 GC system with HP5973 Mass Selective Detector(Hewlett-Packard) equipped with an HP-5MS capillary column (0.25 mmID � 30 m, 0.25 �m; Agilent) operating at a mass range from m/z 70 to800. The ionization voltage was 70 eV, and the ion source temperature was230°C. Chromatography was conducted using a column temperature main-tained at 150°C for 2 min; programmed to increase 10°C/min up to 230°C,5°C/min up to 280°C; and then maintained at 280°C for 10 min with ahelium flow rate of 1.0 ml/min. EPA and DHA were separated, and the areabeneath each chromatographic peak of their deuterium labels was ob-tained by integration using a Chemstation integrator VersionD.02.00.275 (Agilent). For an internal standard, parinaric acid was se-lected because it exists in plants and its spectrum does not overlap withthose of endogenous fatty acids for diagnostic ions. We selected the�-3-derived ion �CH(CH�CH)CH2(CH�CH)CD2CD3 � m/z 113 forboth d5-EPA and d5-DHA methyl ester and M� (290) for methyl pari-narate. Identification was conducted by examining and retention timesas in Fig. 1 (d5-EPA-methyl ester: 11.7 min, methyl-parinarate; 13.1min, d5-DHA; 13.9 min). Calibration curves were obtained for each, asfollows: d5-EPA, y � 0.0016x � 0.0313 (r2 � 0.9954, 50 –500 ng/ml);d5-DHA, y � 0.0015x � 0.0524 (r2 � 0.9809, 50 –500 ng/ml).

Ischemia-reperfusion-induced second-organ injury

Mice were anesthetized by i.p. injection of pentobarbital (80 mg kg�1,Nembutal sodium solution NDC 0074-3778-04). Hind limb ischemia wasinitiated using tourniquets consisting of a rubber band placed on each hindlimb (as in Ref. 31). Mice were subjected to hind limb ischemia for 1 h,after which the tourniquets were removed to initiate reperfusion. Resolvinsand their analogues were each administered at 1 �g/mouse (i.e., DHA,RvD1, RvD1 methyl ester, 17-(R/S)-methyl-RvD1 methyl ester, RvE1, or19-p-fluorophenoxy-RvE1 methyl ester) in vehicle (5 �l of ethanol in 120�l of sterile saline) and compared with vehicle alone. They were admin-istered i.v. to the tail vein �5 min before the start of the reperfusion period.For postreperfusion challenge, at 60 and 90 min after tourniquet release,mice were administered 100 ng of 17-(R/S)-methyl-RvD1-methyl ester(prepared as in Ref. 20) or vehicle alone i.v. At the end of this reperfusionperiod (2 h), the mice were euthanized with an overdose of anesthetic andthe lungs were quickly harvested, frozen in liquid nitrogen, and stored at�80°C. The right lungs were homogenized from individual mice and cen-trifuged, and the tissue levels of myeloperoxidase (MPO) in the resultingsupernatants were determined using a mouse MPO ELISA (Hycult Bio-technology; Cell Sciences).

Microfluidic chambers and leukocyte motility

A new microfluidic chamber was designed for testing the actions of puta-tive novel lipid mediators on the chemotactic function(s) of neutrophils.These analyses were performed immediately after neutrophils were sepa-rated from components of whole blood within the chamber (vide infra),which routinely averaged less than 5 min, and lipid mediator (test com-pounds) adsorption to the chamber surfaces was prevented through the useof polyethylene glycol (PEG) coatings. The microfluidic chamber withmicrostructured valves was fabricated in elastomeric materials using5 The online version of this article contains supplemental material.

8678 APPEARANCE OF RESOLVIN PRECURSORS IN EXUDATES

FIGURE 1. Circulating deuterium-labeled �-3 fattyacids (d5-EPA and d5-DHA) appear in inflammatoryexudates. FVB mice (6–8 wk) received d5-EPA andd5-DHA i.v. (1 �g/mouse) just before i.p. challengewith zymosan A (1 mg/ml/mouse). Peritoneal exudateswere collected at 0, 2, 4, 12, 24, and 48 h and taken tosolid-phase extraction. Eluate fractions were treatedwith diazomethane and taken to GC-MS. Results rep-resent the mean � SEM from n � 3, separate mice.Mass spectra of d5-EPA methyl ester and d5-DHAmethyl ester. d5-EPA and d5-DHA were derivatizedinto methyl esters and injected into GC-MS. A, Massspectrum of d5-EPA methyl ester and selected ion mon-itoring chromatogram of d5-EPA in mouse exudate.The �-3-derived ion (�CH(CH�CH)CH2(CH�CH)-CD2CD3 � m/z 113) and � ion 180 were selected asdiagnostic ions. The retention time was 11.7 min. B,Mass spectrum of d5-DHA methyl ester and selectedion monitoring chromatogram of d5-DHA in mouse ex-udate. The �-3 ion 113 and � ion 160 were selected asdiagnostic ions. The retention time was 13.9 min. C,Time course of d5-EPA and d5-DHA in peritoneal ex-udates. Upper panel, Results shown as percentage ofi.v. injection: f, d5-EPA; F, d5-DHA; ‚, d5-EPA incontrol (absence of zymosan A); E, d5-DHA in control(absence of zymosan A). Results represent the mean �SEM from n � 3. �, Significantly different from 24 h,p � 0.05. Control: each two separate experiments.Middle panel, Time course of PMN infiltration. f, Zy-mosan A treatment; F, zymosan A � d5-EPA and d5-DHA. Results represent the mean � SEM from n � 3.Lower panel: Œ, Increase of total protein amount inperitoneal exudates without d5-EPA and d5-DHA. Re-sults represent the mean � SEM from n � 7�20 (35).�, Significantly different from 0 h, p � 0.05. ‡, Sig-nificantly different from 4 h, p � 0.05. F, Increase oftotal protein amount in peritoneal exudates of controlmice. Mice were injected i.v. with d5-DHA and d5-EPA,and 5 min later 1 ml of saline was administered i.p. Timezero � total protein was 1.67 mg with zymosan A, and inthe absence of zymosan A was 1.17 mg. Control exper-iment: each two separate experiments.

8679The Journal of Immunology

standard microfabrication technologies (32). Briefly, two layers of poly-(dimethylsiloxane) (PDMS), molded on two silicon wafers, werebonded together and on top of a glass slide. A network of channels wasformed between the glass and the first layer, and control channels forthe microstructured valves were formed between the first and secondlayers of PDMS. For details of the fabrication process, see Supplemen-tary Materials.5

After assembly, different sections of the chamber were isolated throughthe actuation of the valves, and each section was chemically modified forspecific functionalities. The surfaces of the network channels in the gra-dient generator section were modified with PEG to prevent absorption ofthe lipid mediators to the PDMS surfaces (see Results; Fig. 3 illustration).Briefly, the PDMS surface of the gradient generation networks was treatedwith 5% (v/v) 3-(trimethoxysilyl)-propyl acrylate (92%; Aldrich) inacetone and followed by rinsing the chamber with acetone. Next, PEGdiacrylate (Aldrich) with 1% photo initiator (2,2-dimethoxy-2-phenyl-ace-tophenone; Aldrich) was introduced to the chamber. After 15 min, thechamber was washed with 5% H2O in acetone and with pure acetone sub-sequently, and then dried with air. After this surface modification procedure,the chambers were stored in the refrigerator for up to 1 wk. The surfaces of thechemotaxis chambers, where neutrophils were captured from whole blood,were modified by physical absorption of P-selectin (10 ng/ml for 30 min; R&DSystems), followed by blocking the entire channel network with 2% humanserum albumin (Sigma-Aldrich) in HBSS (Sigma-Aldrich) immediately beforeuse. The separation between the different sections of the chamber during thesurface modification was achieved by keeping the valves between the mainchannels and the gradient generators closed.

For neutrophil chemotaxis experiments, the chambers were purged withHBSS containing 0.1% human serum albumin, with care to remove allbubbles. Four syringes, two with buffer, one with IL-8 chemoattractant(R&D Systems), and one with the lipid mediator, i.e., RvD1 or other re-lated compounds tested in HBSS were placed in syringes (1 ml), with asyringe pump set for 0.1 �l/min. The flow was allowed to stabilize for 3min, with the microscale valves between the chemotaxis chamber and thetwo gradient generators closed (see Fig. 3). Two steady-state gradients ofIL-8 (0–10 nM) were formed in the two gradient generators, one alone andone with an overlaying uniform concentration of the lipid mediator, andwere ready to be delivered to the chemotaxis chambers. Approximately5–10 �l of capillary blood was collected from healthy volunteers by fingerprick using a BD genie lancet (BD Biosciences) using Protocol 1999-P-001297 approved by the Partners Human Research Committee. The whole

FIGURE 2. Rapid exudate appearance of 14C-DHA from circulation.Each exudate was mixed with scintillation fluid, and radioactivity wascounted with a scintillation counter. Extracellular protein levels were de-termined by the Bradford method and expressed as the total amount in eachsample. Three different groups of animals and experiments wereperformed.

FIGURE 3. Microfluidic chamber for PMN chemotaxis from whole blood. A, Overview of the microfluidic chamber for isolating neutrophils from wholeblood and successive exposure of the cells to preformed chemokine gradients and lipid mediators. B, Rapid switching between chemotaxis conditions wasachieved by using microfluidic valves integrated on the chip, and pneumatically controlled from the outside. C, One drop of blood from a finger stickillustrates the loading of the chamber with whole blood.


blood was then quickly mixed with heparin (10 �l) in a syringe tip (30G;Small Parts). After opening the cell inlet valve, the suspension was slowlypushed through the tubing (Tygon; Small Parts) into the microfluidic cham-ber. The blood stayed in the main channel for 3 min, and then the valve wasopened for the two chemokine gradient generators. The flow removed themajority of RBC and other cells that were not tethered with the chamber-attached P-selectin, thus allowing direct observation of the neutrophils cap-tured on the surface of the chamber. After 10–15 min, the gradient wasswitched to the gradient containing the chemokine alone or either nativeDHA or RvD1. The migration of neutrophils in the chemokine gradient andtheir individual response(s) to addition of RvD1 or DHA were recordedwith a video and/or digital camera mounted on the microscope, and cellmigration was analyzed using the cell tracking function in Metamorph(Molecular Devices). At least a dozen cells per condition were tracked andanalyzed for displacement in the direction of the gradient and along the flow.

Statistical analysis

Results from both in vitro and in vivo experiments were analyzed by Stu-dent’s t test with p values �0.05 taken as statistically significant. Themigration of neutrophils in the microfluidic chambers is presented as av-erage and SEM displacement from the original position at the time of thegradient switching.

ResultsFrom circulation to inflammatory exudates

To determine whether circulating �-3 fatty acids in their unester-ified form are available to evolving inflammatory exudates, weadministered i.v. deuterium-labeled �-3 fatty acids, e.g., d5-EPAand d5-DHA, to mice simultaneously experiencing localized in-flammatory responses, namely peritonitis. The magnitude of theinflammatory insult was selected to be a self-limited spontaneousresolving peritonitis (33) to monitor the potential presence of deu-terium-labeled fatty acids in the inflammatory exudates (Fig. 1, Aand B). Both deuterium-labeled EPA and DHA were identified withinthe local inflammatory exudates within 2 h. As shown in Fig. 1C(upper panel), both d5-EPA and d5-DHA rapidly appeared in exu-

dates during the time course of initiation of inflammation. The levelsof both unesterified d5-EPA and d5-DHA gradually declined at 24 h.In this spontaneous resolution system, maximum inflammation as de-fined by maximum PMN infiltrates within the exudates was at �4 h,and the resolution phase ranged between 12 and 24 h.

As depicted in Fig. 1C (middle panel), PMN infiltration into theexudates was monitored. By definition, these exudates contain se-rum proteins (34) that were determined within the peritoneal ex-udates throughout the time course (Fig. 1C, lower panel; cf Ref.35). Of interest, the time course of both d5-EPA and d5-DHA par-alleled the initial appearance of edema and increases in proteinlevels in these inflammatory exudates. Their presence also coin-cided with PMN infiltration (Fig. 1C). At 48 h, both d5-EPA andd5-DHA levels were significantly greater than at 24 h. It is note-worthy that, without inflammation (i.e., peritonitis), neither d5-EPA nor d5-DHA appeared in peritoneal lavages (Fig. 1). Thus,EPA and DHA, the precursors for resolvins and protectins, rapidlyappeared within these developing inflammatory exudates from pe-ripheral circulation and are delivered to exudates during both theinitiation and resolution phases.

Next, we used a second approach to verify that DHA, i.e., a rep-resentative �-3 fatty acid, indeed appears in exudates directly fromperipheral circulation (Fig. 2). To this end, radiolabeled �-3 fatty acidtracer 14C-DHA was administered i.v., and we monitored recovery of14C from DHA and the total protein amount in murine exudates. Toreduce potential and immediate influences of diet, these experimentswere performed with mice that fasted overnight before receiving i.v.14C-DHA. Exudates were collected at 1, 2, 4, and 12 h. At 1 h, 14Clabel had already reached maximal exudate levels, and protein levelsappeared to parallel the 14C radioactivity profile. These results suggestthat 14C-DHA and its products rapidly appear coincident with in-creases in protein levels and increases in PMN infiltration within the

FIGURE 4. RvD1 stops neutrophil chemotaxis. A,Neutrophils having polarized morphology during expo-sure to IL-8 gradient (�1 min) quickly become rounded(�1 min) and were unable to regain their polarized mor-phology (�5 min) after exposure to RvD1. B, Popula-tion analysis shows that normal neutrophil migration inan IL-8 gradient is immediately and effectively blockedafter exposure to RvD1. Results represent the mean �SEM, n � 12.


exudate site of inflammation. Thus, they confirm that tracer levels of14C-DHA and its initial transformation products rapidly appearedwithin the exudates during early initiation of the inflammatory re-sponse in vivo (Fig. 2). Of note, these tracer levels of DHA did notreduce leukocyte trafficking as monitored by FACS analysis of theexudates (see Supplement5 Fig. 2).

Single-cell monitoring of real-time responses demonstrates thedirect impact of RvD1, but not its precursor DHA

The original isolation and structure elucidation of RvD1 demon-strated its presence and potent anti-inflammatory and proresolutionactions in spontaneous resolving exudates and disease models invivo (19, 20). In this study, we questioned whether RvD1 or itsprecursor DHA has direct actions with human neutrophils, andspecifically whether RvD1 can alter the directed movements ofsingle cells along chemotactic gradients. This is a central pointbecause excessive infiltration of PMN and release of proinflam-matory mediators is a well-appreciated key event in causing tissuedamage and uncontrolled inflammation. To address this, we used a1-�l microfluidic chamber that was engineered with microstruc-ture membranes (see Fig. 3). The chamber permitted the isolationof individual PMNs from one drop of whole blood (see Materialsand Methods and Fig. 3C) following a simple finger prick. Afterisolation, the microstructured valves in the chamber (Fig. 3B) wereused to control the timing of exposure of the captured PMNs to thetwo distinct chemotactic gradients (Fig. 3A) (see Materials andMethods). Images of the PMN before and during exposure to thetwo gradients were continuously monitored using a camera andrecorded (Fig. 4A).

The PMN trafficked along the IL-8 gradient (see Supplement5

Fig. 3) and displayed the typical shape change and morphology ofPMN during chemotaxis in a linear gradient (cf Ref. 36 and Fig.4A, left panel). Between 0 and 8 min, RvD1 at 10 nM was uni-formly infused in the microfluidic chamber. Before exposure toRvD1, individual PMN movements and distances were propor-tional to time and totaled �30–40 �m. Almost immediately onexposure to RvD1, PMN dramatically changed shape (see Fig. 4A,middle panel) and ceased directed chemotactic movements (Fig. 4,A and B, and see Supplementary Video online).5 Fig. 4B shows theaverage displacement, demonstrating a highly reproducible stop-

ping of PMN migration when exposed to RvD1 (n � 12). In sharpcontrast, DHA, the biosynthetic precursor to RvD1, at equimolarconcentrations did not stop PMN migration (Fig. 5). Thus, by trac-ing the displacement of single cells in the direction of an IL-8chemotactic gradient before and after RvD1 was introduced intothe chamber, we were able to record the direct actions of RvD1 onPMN. Thus, these results establish the ability of RvD1 to essen-tially completely stop PMN movements, almost immediately uponexposure, as well as the ability of RvD1 to stimulate rapid shapechanges of PMN. These single-cell-recorded PMN responses werenot shared by DHA, the RvD1 metabolic precursor in exudates,when introduced at equal molar concentrations in these microflu-idic chambers.

Resolvins are organ protective in vivo

Ischemia followed by reperfusion is a well-appreciated patho-physiologic mechanism of organ injury and local tissue damagethat can be initiated by excessively activated PMN. This systemin mice (Fig. 6) models the second organ injury and tissue dam-age observed in humans following tourniquet release of vessels

FIGURE 5. DHA, precursor of RvD1, did not stop neutrophil chemo-taxis. Direct comparisons of DHA and RvD1 in an IL-8 gradient with PMNisolated from a drop of whole blood. F, RvD1; ‚, DHA. Results representthe mean � SEM, n � 12, for each.

FIGURE 6. RvD1 protects from second organ lung injury followingischemia-reperfusion. A, Time course of experiment. B, RvD1 protectslung from ischemia/reperfusion injury. Hind limb ischemia was induced inmice by creating tourniquets using a rubber band on each hind limb. After1 h, the tourniquets were removed and reperfusion ensued. Test compounds(1 �g per mouse of DHA, RvD1, RvE1) in vehicle were administered i.v.5 min before the start of the reperfusion period. At the end of the reper-fusion period (2 h), lungs were collected and MPO levels were determined.Results indicate percentage of reduction of control, mean � SEM (n �4�6, control; n � 12). RvD1, f Values for DHA and RvE1 were notsignificant changes. �, Significantly different from values obtained withvehicle, p � 0.02. †, Significantly different from values obtained withDHA, p � 0.01. ‡, Significantly different from values obtained with RvE1,p � 0.002.


in surgery involving, for example, extremities (37, 38). Theocclusion of blood vessels creates local ischemia and remoteorgan injury that has many features of rapid, acute inflammationand tissue damage (31). On release of the tourniquet occlusion,reflow is initiated and aberrantly activated PMN rapidly perfuseand infiltrate secondary organs, causing local damage (31). Inthis study, we evaluated RvD1 in a remote organ injury system,i.e., hind limb ischemia-reperfusion, to assess whether the abil-ity of RvD1 to stop PMN migration in microchambers can in

turn reduce PMN-mediated tissue and organ damage in vivo.Using the time line illustrated in Fig. 6 (upper panel), PMNaccumulation in the lung was assessed by monitoring increasesin both tissue histology and tissue MPO, a leukocyte markerenzyme. The extent of lung tissue injury is associated withPMN activation and organ infiltration (31).

In this system, we also directly compared the actions ofRvD1 with its biosynthetic precursor DHA as well as with an-other resolvin, namely, RvE1, which is a potent product of EPAthat is anti-inflammatory in both oral inflammation (39) andcolitis (reviewed in Ref. 1). RvD1, but neither RvE1 nor DHA,was able to protect the lung tissues from excessive leukocyteinfiltration (Fig. 6). The histology and reduction in leukocyteinfiltration (data not shown) were confirmed by the reduction inthe lung-associated MPO values. At 1 �g/mouse, RvD1 sharplyreduced �50% leukocyte infiltration in the reperfusion tissues.In contrast, at equal doses, neither DHA nor native RvE1 gavesignificant reduction in PMN tissue infiltration. Because RvD1undergoes local metabolic inactivation (20), as does RvE1 (26,40), blocking of their respective metabolic sites of inactivationwithin each molecule de novo was undertaken using metabol-ically stable analog mimetics. To this end, both 17-(R/S)-methylRvD1 and RvD1 were prepared by total organic synthesis for invivo administration. Both RvD1 and the related analogues(structures shown in Fig. 7) sharply reduced MPO levels in lungtissues. Of interest, the metabolically stable analog of RvE1,namely 19-p-fluorophenoxy RvE1 at 1 �g/mouse, also signifi-cantly reduced leukocyte infiltration, whereas native RvE1 wasunable to protect lung from PMN-induced damage in this sys-tem. It is noteworthy that mouse lung tissue enzymatically con-verts RvE1 to 18-oxo-RvE1 (26), which is devoid of bioactiv-ity. The 19-p-fluorophenoxy RvE1 prevents this metabolicinactivation (Fig. 7), and as documented in this study, displayedpotent protective actions dramatically reducing PMN infiltra-tion and second organ injury of the lung. The actions of re-solvins during reperfusion were investigated. Ninety minutesafter tourniquet release, 100 ng of 17-(R/S)-methyl-RvD1methyl ester was administered. The RvD1 analog lowered theincreases in MPO levels in lung, whereas at 60 min after isch-emia-reperfusion, administration of this RvD1 analog (100 ngi.v.) did not protect from second organ injury. Thus, by pre-venting the local metabolic inactivation of resolvins, their abil-ity to reduce PMN infiltration into distal organs was sustainedfor an enhanced beneficial action when given before reflow andat 90 min after reflow compared with controls.

DiscussionDuring the course of spontaneous resolution of acute inflam-mation, �-3 fatty acids are precursors for the biosynthesis ofnewly uncovered anti-inflammatory and proresolving lipid me-diators (1, 8). These new families of mediators, coined resolvinsand protectins, were identified in vivo in resolving exudates andserve in molecular circuits that actively promote endogenousanti-inflammation and resolution of local inflammation (35).However, the mechanism of �-3 fatty acid mobilization in vivoduring inflammation resolution has not been addressed. In thispresent study, evidence for new mechanisms is presented thatindicates that unesterified �-3 fatty acids, also known as freefatty acids, rapidly appear within the inflammatory exudatesmoving directly from circulation into the site of inflammation.The movement of EPA and DHA parallels those of both albu-min and trafficking leukocytes. Also, single-cell analyses of hu-man PMN using a newly engineered microfluidics chamber pro-vide direct evidence that DHA-derived RvD1 at nanomolar

FIGURE 7. Resolvin and related analogues are protective in vivo. A,Structures and actions of compounds used in this experiment. Test com-pounds (1 �g of RvD1 carboxymethyl ester (RvD1-Me), 17-(R/S)-methyl-RvD1 carboxymethyl ester (17-(R/S)-methyl-RvD1-Me), and 19-p-fluoro-phenoxy-RvE1 methyl ester (19-p-fluorophenoxy-RvE1-Me)) in vehiclewere administered i.v. Results indicate percentage of reduction comparedwith control, mean � SEM (n � 3�5, control; n � 12). RvD1-Me, f;17-(R/S)-methyl-RvD1-Me, �; 19-p-fluorophenoxy-RvE1-Me, p. �, Sig-nificantly different from values obtained with vehicle, p � 0.02. †, Signif-icantly different from values obtained with DHA; p � 0.01. B, Postreper-fusion challenge with RvD1. At 60 and 90 min after tourniquet release, 100ng of 17-(R/S)-methyl-RvD1-Me or vehicle was administered i.v. Resultsindicate percentage of reduction compared with parallel controls, mean �SEM (n � 3). �, Significantly different from values obtained with vehicle;p � 0.02.


concentrations, and not its precursor DHA at equimolar levels,stops PMN chemotactic responses to spatial gradients of thechemokine IL-8.

Once formed, resolvins are active on target cells in their imme-diate milieu and are then inactivated by site-specific metabolism(26, 40). To enhance and prolong their actions, analogues of bothRvD1 and RvE1 were prepared that delay their local inactivationin tissues, which proved to protect organs in vivo from ischemia-reperfusion injury by reducing neutrophil tissue infiltration. Theseresults demonstrate for the first time that �-3 levels in circulatingblood are rapidly made available to sites of inflammation in vivofor their local use by exudates to generate potent bioactive localmediators, i.e., resolvins in situ. The resolvins in turn act directlyon target cells to stimulate endogenous anti-inflammation and pro-tect organs from excessive neutrophil infiltration and subsequentlocal tissue damage.

After ingestion, EPA and DHA are distributed throughout thehuman body (41). DHA is predominantly distributed in retina,sperm, cerebral cortex, spleen, and RBC, whereas EPA is foundin muscle, liver, spleen, and RBC (42). For example, DHA isesterified in phospholipids of microglial cells in culture and, onactivation of these cells, DHA is released from the phospholip-ids for enzymatic processing (43, 44). DHA is also, from recentresults, the precursor for two separate families of mediators thatare structurally distinct, namely D series resolvins and protec-tins. The protectins possess potent biological actions and a con-jugated triene structure as distinguishing features (19). EPA isthe precursor for E series resolvins that show potent actions inseveral complex disease models, including inflammatory boweldisease, periodontal diseases, and asthma (reviewed in Ref. 45).However, the availability of unesterified or free �-3 fatty acidsEPA and DHA for processing during inflammation resolutionwas of interest in the present studies and required a multidis-ciplinary approach and new tools to address these key points.

The level of total fatty acids in human blood is �343 mg/100ml plasma (46). Based on this value and Supplementary TableI,5 between 48 and 490 mg of EPA and DHA exists in humanblood as basal levels. The contribution of de novo �-3 fattyacids to the total amount in healthy human subjects, however,appears to be quite low. The proportion of �-linolenic acid con-verted to EPA is most likely on the order of 0.20 – 8.0%, and theextent of conversion of �-linolenic acid to DHA is 0.05– 4.0%(47, 48). Thus, humans require �-3 fatty acid intake via dietand/or supplementation. Currently, the FDA states that the di-etary intake of EPA and DHA should not exceed 3 g/day (49)because excess supplementation on the order of g/day wasfound to reduce the already low endogenous EPA and DHAbiosynthesis (47).

Although DHA and EPA are widely believed to possess anti-inflammatory properties themselves, the specific mechanisms re-sponsible for these actions are still evolving. The �-3 fatty acidsare generally thought to replace the sn-2 position in phospholipidstores that is usually the positional site of esterified �-6 fatty acidssuch as arachidonic acid (41). It is well appreciated that, uponactivation, cells release arachidonic acid from the sn-2 position ofphospholipids via cytosolic phospholipase A2 for conversion toeicosanoids. For example, among the potent bioactive eicosanoidsproduced by leukocytes, the PGs and leukotrienes are broadly con-sidered proinflammatory mediators (50). The sn-2 position ofphospholipids that can become substituted with �-3 fatty acids(DHA, EPA) is currently thought to simply compete for these en-zymatic reactions, thus blocking the use of arachidonate and pro-duction of specific eicosanoids that are proinflammatory and pro-thrombotic mediators. This view is consistent with results from

both cultured and isolated cells in vitro when �-3 fatty acids aresupplied to isolated cells (41). To address these points in a patho-physiologic setting in the present investigations, we determined theappearance of EPA and DHA at local sites of inflammation inexudates from circulation.

By monitoring both deuterium-labeled d5-EPA and d5-DHAlevels from circulation as well as increases in protein levelswithin exudates, we found that they appear coincident in theforming exudates. Of interest, both d5-EPA and d5-DHA wereidentified in exudates within 1 h of initiation of inflammationand maintained levels up to 48 h. At 48 h, both d5-EPA andd5-DHA levels were significantly greater within the exudatesthan their levels at 24 h. The first peak of D5 fatty acids wasdirectly delivered from the circulation. Meanwhile, the secondpeak at 48 h most likely reflects recirculation and phospholipaseA2 (PLA2) expression in the resolution of inflammation. More-over, cytosolic PLA2 and secretory PLA2 were highly expressedduring the resolution phase (51). The second peak at 48 h couldbe from esterified d5-EPA and d5-DHA and released by PLA2

mechanisms. Because in human plasma the t1/2 of EPA is 67 hand that of DHA is 20 h (48), the clearance of EPA and DHAis relatively slow compared with xenobiotic small molecules.Taken together, the present results suggest that circulating �-3fatty acids are available for sites of acute inflammation, ini-tially, directly from circulation. The presence of albumin atsites of inflammation, by definition, determines whether the in-flammatory site is considered an inflammatory exudate or tran-sudate (37). The main protein component in the inflammatory exu-dates generated in the current zymosan-initiated peritonitis is, indeed,serum albumin demonstrated by two-dimensional gel electrophoresisand proteomics (35). Albumin is a well-appreciated carrier protein ofunesterified fatty acids and particularly DHA (52).

Hence, the present results indicate that circulating DHA andEPA are directly used by developing inflammatory exudates anddo not require specialized mobilization from complex lipids orspecific phospholipase activation to initially impact inflamma-tion and its timely resolution. Both EPA and DHA appearedrapidly in exudates in their unesterified form coincident withedema generation and movement of circulating albumin andleukocyte trafficking into the evolving inflammatory exudates.Lundy et al. (53) also indicated that edema formation and timecourse of arachidonic acid in mouse peritonitis were parallel.These findings in mice suggest that EPA and DHA are directlymobilized for resolvin production from the circulation via al-bumin as the most abundant and likely main carrier into theinflammatory sites. In humans, 13C-DHA in phosphatidylcho-line was rapidly hydrolyzed and available as a free fatty acid inplasma (54). Furthermore, after ingestion of supplement, non-esterified EPA is detected in plasma (55). Taken together, thesefindings imply that in humans the circulating levels of EPA andDHA do not require storage and subsequent release from com-plex lipid precursors to have an important contribution to con-trolling inflammation and its resolution.

Next, we questioned the relationship between circulating �-3fatty acids and their anti-inflammatory properties in vivo. Usinga new microfluidic chamber approach to rapidly isolate humanneutrophils directly from circulating whole blood via capture onP-selectin-coated surface, we assessed the direct actions of bothprecursor DHA and RvD1 on single neutrophil chemotaxis re-sponses. Earlier procedures required time-consuming isolationof neutrophils from whole blood before in vitro analyses. Theseprotocols involved several steps of centrifugation and RBC ly-sis that usually take several hours (56) to perform that could


lead to changes in the responsiveness and characteristics of theisolated neutrophils. By contrast, the P-selectin-based captureof neutrophils from a single drop of whole blood (�5–10 �l) iscapable of performing the PMN separation in less than 5 min.This short time interval is ideal for assessing the activationand/or inhibition status of neutrophils from the blood of healthydonors as well as patients. The combination of separation andassessment of shape and migration responses in the same cham-ber is closely akin to in vivo scenarios in which neutrophils rollon the endothelial surfaces (37), stick to the endothelium inregions of higher selectin expression, and respond via chemo-taxis in the gradient of a chemokine, e.g., IL-8, and migrate intotissues (see Ref. 37). The small amount of blood used in themicrofluidic chamber is an additional advantage for performingthese analyses with human PMN because they circumvent theneed for venous phlebotomy and associated risks.

Another key feature of this microfluidic chamber system isthe ability to record real-time changes in morphology of PMNupon exposure to chemokines, DHA, and lipid mediators, suchas RvD1, as well as to track migration through switches. Thefast gradient switches in the chamber allowed visual assessmentand recording of the earliest events after exposure of cells toRvD1 or native DHA as well as precise measurement of thesechanges in migration direction and velocity. Currently, no otherchemotaxis systems are available that allow this level of pre-cision or time resolution. For instance, in Boyden chambers(57), one cannot directly observe the cells, the information ob-tained is indirect, and the system requires a large number ofcells and separate controls. The Boyden chamber is an end pointassay, and one would not be able to assess whether neutrophilsmigrated before or after the addition of the inhibitor, or byadding the inhibitor before cells start moving if the effect wouldextrapolate to chemotaxis. In the Dunn and Zigmond chambers(58, 59), although one could visualize the cells in real time, itis not possible to swiftly switch between cell incubation andexposure conditions as with the present microfluidics chambers.The engineered microstructured valves for switching betweenindependent gradients allowed the same neutrophils to serve aspositive controls for migration in a chemoattractant gradientand then probed with either RvD1 or its precursor native DHA.The preservation of chemoattractant gradient before and afterthe switch is important for avoiding neutrophil responses tosudden changes of chemoattractant gradient. Of interest, suddendecrements in the concentration of chemokines alone have thepotential to stop neutrophil migration for 3–5 min (60). Thus,the present direct assessment of DHA with PMN indicates thatDHA itself is not a potent bioactive stop signal for PMN, butrather requires exudate conversion to RvD1 to evoke its signal-ing properties on these cells. Hence, following their actions,local tissues inactivate resolvins and permit organs to return tohomeostasis.

Along these lines, ischemia-reperfusion is an event of signif-icant clinical importance. Reperfusion-related tissue injury oc-curs during surgical procedures, particularly those involving ex-tremities, causing both local and remote organ injury as well asincreasing costs associated with prolonged postoperative recov-ery (38). Briefly, when vessels are surgically clamped or oc-clude, stasis of blood at the occlusion site leads to local isch-emia and the neutrophils in that blood become activated. Uponrelease of the occlusion, activated leukocytes give rise to sec-ond organ or remote organ injury. For example, when neutro-phils from the occluded vessels of the hind limb reach the lung,they cause local tissue damage (31). In many respects, the ab-errant activation of neutrophils in this scenario exhibits features

similar to those in uncontrolled acute inflammation and neutro-phil-mediated tissue damage (31, 37, 38). Given the clinicalimportance and pathophysiology of this type of organ injury,we investigated the direct actions of DHA, resolvins, and re-lated stable analogues (i.e., directly comparing the actions ofRvD1, its 17-(R/S)-methyl analog, RvE1, and its 19-p-fluoro-phenoxy analog) in ischemia-reperfusion second organ injury.At equivalent doses, DHA was not protective, whereas RvD1and its analog, as well as the stable analog of RvE1, showedpotent antileukocyte actions, each reducing infiltration into lungtissues.

Native RvE1 itself was not able to protect the lung at these lowdoses most likely because of local inactivation. Of interest, RvE1itself is both anti-inflammatory and proresolving in several inflam-matory disease models (45). Recently, RvE1 was also shown tohave potent actions in preventing joint damage and cartilage de-struction in collagen-induced rodent arthritis (61) and protectsfrom inflammation-induced bone loss in periodontal disease (39).Both RvD1 and RvE1 undergo site-specific metabolic inactivation(26, 40). Thus, the RvD1 and RvE1 analogues that display potentorgan protective actions, as demonstrated in this study (Fig. 7),may provide new approaches to reduce organ damage character-ized by excessive PMN infiltration. PMN are now appreciated toplay an important role in the pathogenesis of many lifelong chronicinflammatory diseases such as arthritis (62). Recurring bouts ofunresolved local acute inflammation may underlie many chronicinflammatory diseases. Thus, new means of monitoring neutro-phils from peripheral whole blood within minutes, as demonstratedin this study with these newly designed microfluidic chambers,may have implications in selecting appropriate therapies for acuteand chronic inflammatory diseases.

In summation, we tracked the movements of labeled EPA andDHA from circulation to inflammatory exudates and recorded inreal time the direct actions of RvD1 on human PMN migrationusing a newly engineered microfluidic chamber (32, 60). Usingthis approach, we visualized individual cellular responses withneutrophils obtained from a single drop of peripheral blood in lessthan 5 min. This new system provided a unique opportunity toinvestigate the actions of RvD1 at the single-cell level. As shownin the Supplementary Video (online),5 PMN moving along thechemotactic gradient stopped movement essentially immediatelyafter exposure to RvD1, but not with equimolar amounts of itsexudate precursor DHA.

We also demonstrate new organ protective actions of re-solvins in a murine model of second organ lung injury follow-ing hind limb ischemia-reperfusion. These results emphasizethe substantial contribution of unesterified �-3 fatty acid levelsin circulation and their rapid availability via edema formation inexudates to mount resolution and assist in the return of localtissues to homeostasis. Recent review of the evidence for ben-eficial effects of fatty acids in human inflammation indicates apoor relationship and predictive value suggesting specific dose-response relationships for dietary �-3 fatty acids, and fatty acidprofiles of immune cells differ between species (63). These areconsistent with our results and suggest that circulating �-3DHA and EPA levels and their transfer by edema rather thanphospholipid-esterified precursors are an initial direct sourcefor exudate biosynthesis of resolvins, and thus should be takeninto account in evaluating the impact of nutrition in humans.The new pathways uncovered in this study are likely to be rel-evant in maintaining health, as well as in the many diseasescharacterized by excessive uncontrolled inflammation.


AcknowledgmentsWe thank Dr. Birgitta Schmidt (Department of Pathology, Children’s Hos-pital, Boston, MA) for microscopic evaluation of tissue histology, Dr.Jasim Uddin (University of Southern California) for preparation of some ofthe synthetic resolvins and their analogues, and Mary Halm Small for ex-pert assistance with manuscript preparation.

DisclosuresC.N.S. is inventor on patents assigned to The Brigham and Women’s Hos-pital on resolvins that are subjects of licensing agreements and consultantarrangements for C.N.S.

References1. Serhan, C. N. 2007. Resolution phases of inflammation: novel endogenous anti-

inflammatory and pro-resolving lipid mediators and pathways. Annu. Rev. Im-munol. 25: 101–137.

2. Majno, G., and I. Joris. 2004. Cells, Tissues, and Disease: Principles of GeneralPathology. Oxford University Press, New York.

3. Weissmann, G. 2004. Pathogenesis of rheumatoid arthritis. J. Clin. Rheumatol.10(Suppl. 3): S26–S31.

4. Kantarci, A., and T. E. Van Dyke. 2005. Resolution of inflammation in peri-odontitis. J. Periodontol. 76(Suppl. 11): 2168–2174.

5. Nathan, C. 2002. Points of control in inflammation. Nature 420: 846–852.6. Libby, P. 2002. Inflammation in atherosclerosis. Nature 420: 868–878.7. Bazan, N. G., V. Colangelo, and W. J. Lukiw. 2002. Prostaglandins and other

lipid mediators in Alzheimer’s disease. Prostaglandins Other Lipid Mediat. 68–69: 197–210.

8. Gilroy, D. W., T. Lawrence, M. Perretti, and A. G. Rossi. 2004. Inflammatoryresolution: new opportunities for drug discovery. Nat. Rev. Drug Discov. 3:401–416.

9. Schwab, J. M., N. Chiang, M. Arita, and C. N. Serhan. 2007. Resolvin E1 andprotectin D1 activate inflammation-resolution programmes. Nature 447:869–874.

10. GISSI-Prevenzione Investigators. 1999. Dietary supplementation with n-3 poly-unsaturated fatty acids and vitamin E after myocardial infarction: results of theGISSI-Prevenzione trial. Lancet 354: 447–455.

11. Marchioli, R., F. Barzi, E. Bomba, C. Chieffo, D. Di Gregorio, R. Di Mascio,M. G. Franzosi, E. Geraci, G. Levantesi, A. P. Maggioni, et al. 2002. Earlyprotection against sudden death by n-3 polyunsaturated fatty acids after myocar-dial infarction: time-course analysis of the results of the Gruppo Italiano per loStudio della Sopravvivenza nell’Infarto Miocardico (GISSI)-Prevenzione. Circu-lation 105: 1897–1903.

12. Leeb, B. F., J. Sautner, I. Andel, and B. Rintelen. 2006. Intravenous applicationof omega-3 fatty acids in patients with active rheumatoid arthritis: the ORA-1trial: an open pilot study. Lipids 41: 29–34.

13. Pluess, T. T., D. Hayoz, M. M. Berger, L. Tappy, J. P. Revelly, B. Michaeli,Y. A. Carpentier, and R. L. Chiolero. 2007. Intravenous fish oil blunts the phys-iological response to endotoxin in health subjects. Intensive Care Med. 33:789–797.

14. Welty, F. K., K. S. Lee, N. S. Lew, and J. R. Zhou. 2007. Effect of soy nuts onblood pressure and lipid levels in hypertensive, prehypertensive, and normoten-sive postmenopausal women. Arch. Intern. Med. 167: 1060–1067.

15. Norris, J. M., X. Yin, M. M. Lamb, K. Barriga, J. Seifert, M. Hoffman,H. D. Orton, A. E. Baron, M. Clare-Salzler, H. P. Chase, et al. 2007. Omega-3polyunsaturated fatty acid intake and islet autoimmunity in children at increasedrisk for type 1 diabetes. J. Am. Med. Assoc. 298: 1420–1428.

16. Lefkowitz, W., S. Y. Lim, Y. Lin, and N. N. Salem, Jr. 2005. Where does thedeveloping brain obtain its docosahexaenoic acid: relative contributions of di-etary �-linoleic acid, docosahexaenoic acid, and body stores in the developingrat. Pediatr. Res. 57: 157–165.

17. Bazan, N. G. 2007. Omega-3 fatty acids, pro-inflammatory signaling and neuro-protection. Curr. Opin. Clin. Nutr. Metab. Care 10: 136–141.

18. Freedman, S. D., P. G. Blanco, M. M. Zaman, J. C. Shea, M. Ollero, I. K. Hopper,D. A. Weed, A. Gelrud, M. M. Regan, M. Laposata, et al. 2004. Association ofcystic fibrosis with abnormalities in fatty acid metabolism. N. Engl. J. Med. 350:560–569.

19. Serhan, C. N., S. Hong, K. Gronert, S. P. Colgan, P. R. Devchand, G. Mirick, andR.-L. Moussignac. 2002. Resolvins: a family of bioactive products of omega-3fatty acid transformation circuits initiated by aspirin treatment that counter pro-inflammation signals. J. Exp. Med. 196: 1025–1037.

20. Sun, Y.-P., S. F. Oh, J. Uddin, R. Yang, K. Gotlinger, E. Campbell, S. P. Colgan,N. A. Petasis, and C. N. Serhan. 2007. Resolvin D1 and its aspirin-triggered 17Repimer: stereochemical assignments, anti-inflammatory properties and enzymaticinactivation. J. Biol. Chem. 282: 9323–9334.

21. Gong, J., B. Rosner, D. G. Rees, E. L. Berson, C. A. Weigel-DiFranco, andE. J. Schaefer. 1992. Plasma docosahexaenoic acid levels in various geneticforms of retinitis pigmentosa. Invest. Ophthalmol. Visual Sci. 33: 2596–2602.

22. Newcomer, L. M., I. B. King, K. G. Wicklund, and J. L. Stanford. 2001. Theassociation of fatty acids with prostate cancer risk. Prostate 47: 262–268.

23. Albert, C. M., H. Campos, M. J. Stampfer, P. M. Ridker, J. E. Manson,W. C. Willett, and J. Ma. 2002. Blood levels of long-chain n-3 fatty acids and therisk of sudden death. N. Engl. J. Med. 346: 1113–1118.

24. Kew, S., M. D. Mesa, S. Tricon, R. Buckley, A. M. Minihane, and P. Yaqoob.2004. Effects of oils rich in eicosapentaenoic and docosahexaenoic acids on im-

mune cell composition and function in healthy humans. Am. J. Clin. Nutr. 79:674–681.

25. Wakai, K., Y. Ito, M. Kojima, S. Tokudome, K. Ozasa, Y. Inaba, K. Yagyu,A. Tamakoshi, and JACC Study Group. 2005. Intake frequency of fish and serumlevels of long-chain n-3 fatty acids: a cross-sectional study within the JapanCollaborative Cohort Study. J. Epidemiol. 15: 211–218.

26. Arita, M., S. Oh, T. Chonan, S. Hong, S. Elangovan, Y.-P. Sun, J. Uddin,N. A. Petasis, and C. N. Serhan. 2006. Metabolic inactivation of resolvin E1 andstabilization of its anti-inflammatory actions. J. Biol. Chem. 281: 22847–22854.

27. Arita, M., F. Bianchini, J. Aliberti, A. Sher, N. Chiang, S. Hong, R. Yang,N. A. Petasis, and C. N. Serhan. 2005. Stereochemical assignment, anti-inflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1.J. Exp. Med. 201: 713–722.

28. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72: 248–254.

29. Ariel, A., G. Fredman, Y.-P. Sun, A. Kantarci, T. E. Van Dyke, A. D. Luster, andC. N. Serhan. 2006. Apoptotic neutrophils and T cells sequester chemokinesduring immune response resolution via modulation of CCR5 expression. Nat.Immunol. 7: 1209–1216.

30. DeBoer, T. J., and H. J. Backer. 1954. A new method for the preparation ofdiazomethane. Rec. Trav. Chim. Pays-Bas. 73: 229–234.

31. Qiu, F.-H., K. Wada, G. L. Stahl, and C. N. Serhan. 2000. IMP and AMP deami-nase in reperfusion injury down-regulates neutrophil recruitment. Proc. Natl.Acad. Sci. USA 97: 4267–4272.

32. Irimia, D., and M. Toner. 2006. Cell handling using microstructured membranes.Lab Chip 6: 345–352.

33. Winyard, P. G., and D. A. Willoughby. 2003. Inflammation protocols. In Methodsin Molecular Biology. J. M. Walker, ed. Humana, Totowa, p. 378.

34. Meyers, D. G., R. E. Meyers, and T. W. Prendergast. 1997. The usefulness ofdiagnostic tests on pericardial fluid. Chest 111: 1213–1221.

35. Bannenberg, G. L., N. Chiang, A. Ariel, M. Arita, E. Tjonahen, K. H. Gotlinger,S. Hong, and C. N. Serhan. 2005. Molecular circuits of resolution: formation andactions of resolvins and protectins. J. Immunol. 174: 4345–4355.

36. Geiser, T., B. Dewald, M. U. Ehrengruber, I. Clark-Lewis, and M. Baggiolini.1993. The interleukin-8-related chemotactic cytokines GRO�, GRO�, and GRO�activate human neutrophil and basophil leukocytes. J. Biol. Chem. 268:15419–15424.

37. Cotran, R. S., V. Kumar, and T. Collins. 1999. Robbins Pathologic Basis ofDisease. W. B. Saunders, Philadelphia, p. 1425.

38. Gelman, S. 1995. The pathophysiology of aortic cross-clamping and unclamping.Anesthesiology 82: 1026–1060.

39. Hasturk, H., A. Kantarci, E. Goguet-Surmenian, A. Blackwood, C. Andry,C. N. Serhan, and T. E. Van Dyke. 2007. Resolvin E1 regulates inflammation atthe cellular and tissue level and restores tissue homeostasis in vivo. J. Immunol.179: 7021–7029.

40. Hong, S., T. F. Porter, Y. Lu, S. F. Oh, P. S. Pillai, and C. N. Serhan. 2008.Resolvin E1 metabolome in local inactivation during inflammation-resolution.J. Immunol. 180: 3512–3519.

41. Lands, B. 2008. A critique of paradoxes in current advice on dietary lipids. Prog.Lipid Res. 47: 77–106.

42. Arterburn, L. M., E. B. Hall, and H. Oken. 2006. Distribution, interconversion,and dose response of n-3 fatty acids in humans. Am. J. Clin. Nutr. 83(Suppl. 6):1467–1476.

43. Hong, S., K. Gronert, P. Devchand, R.-L. Moussignac, and C. N. Serhan. 2003.Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid inmurine brain, human blood and glial cells: autacoids in anti-inflammation. J. Biol.Chem. 278: 14677–14687.

44. Marcheselli, V. L., S. Hong, W. J. Lukiw, X. Hua Tian, K. Gronert, A. Musto,M. Hardy, J. M. Gimenez, N. Chiang, C. N. Serhan, and N. G. Bazan. 2003.Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infil-tration and pro-inflammatory gene expression. J. Biol. Chem. 278: 43807–43817.

45. Serhan, C. N., and N. Chiang. 2008. Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus. Br. J. Pharmacol.153(Suppl. 1): 200–215.

46. Patil, V. S., and N. G. Magar. 1960. Fatty acids of human blood. Biochem. J. 74:427–429.

47. Burdge, G. C., and P. C. Calder. 2005. Conversion of �-linolenic acid to longer-chain polyunsaturated fatty acids in human adults. Reprod. Nutr. Dev. 45:581–597.

48. Pawlosky, R. J., J. R. Hibbeln, J. A. Novotny, and N. N. Salem, Jr. 2001. Phys-iological compartmental analysis of �-linolenic acid metabolism in adult humans.J. Lipid Res. 42: 1257–1265.

49. U.S. Food and Drug Administration Center for Food Safety and Applied Nutri-tion Office of Nutritional Products Labeling and Dietary Supplements (FDA/CSFAN). 2000. Letter regarding dietary supplement health claim for omega-3fatty acids and coronary heart disease (Docket No. 91N-0103).

50. Samuelsson, B., S. E. Dahlen, J. A. Lindgren, C. A. Rouzer, and C. N. Serhan.1987. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.Science 237: 1171–1176.

51. Gilroy, D. W., J. Newson, P. Sawmynaden, D. A. Willoughby, and J. D. Croxtall.2004. A novel role for phospholipase A2 isoforms in the checkpoint control ofacute inflammation. FASEB J. 18: 489–498.

52. Huang, B. X., C. Dass, and H. Y. Kim. 2005. Probing conformational changes ofhuman serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry. Biochem. J. 387: 695–702.


53. Lundy, S. R., R. L. Dowling, T. M. Stevens, J. S. Kerr, W. M. Mackin, andK. R. Gans. 1990. Kinetics of phospholipase A2, arachidonic acid, and eicosanoidappearance in mouse zymosan peritonitis. J. Immunol. 144: 2671–2677.

54. Lemaitre-Delaunay, D., C. Pachiaudi, M. Laville, J. Pousin, M. Armstrong, andM. Lagarde. 1999. Blood compartmental metabolism of docosahexaenoic acid(DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidyl-choline. J. Lipid Res. 40: 1867–1874.

55. Surette, M. E., I. L. Koumenis, M. B. Edens, K. M. Tramposch, and F. H. Chilton. 2003.Inhibition of leukotriene synthesis, pharmacokinetics, and tolerability of a novel dietaryfatty acid formulation in healthy adult subjects. Clin. Ther. 25: 948–971.

56. Boyum, A. 1968. Isolation of leukocytes from human blood: further observations:methylcellulose, detran, and Ficoll as erythrocyte aggregating agents. Scand.J. Clin. Lab. Invest. Suppl. 97: 31–50.

57. Boyden, S. 1962. The chemoactic effect of mixtures of antibody and antigen onpolymorphonuclear leukocytes. J. Exp. Med. 115: 453–466.

58. Zicha, D., G. A. Dunn, and A. F. Brown. 1991. A new direct-viewing chemotaxischamber. J. Cell Sci. 99: 769–775.

59. Zigmond, S. H., and J. G. Hirsch. 1973. Leukocyte locomotion and chemotaxis:new methods for evaluation, and demonstration of a cell-derived chemotacticfactor. J. Exp. Med. 137: 387–410.

60. Irimia, D., S.-Y. Liu, W. G. Tharp, A. Samadani, M. Toner, andM. C. Poznansky. 2006. Microfluidic system for measuring neutrophil migratoryresponses to fast switches of chemical gradients. Lab Chip 6: 191–198.

61. Carillo-Rivas, B., S. Qin, A. Savinainen, C. Chen, T. Crandall, D. Dodge,A. Dubrovskiy, D. Lau, M. Lin, M. Shumway, et al. 2007. 10th InternationalConference on Bioactive Lipids in Cancer, Inflammation and Related DiseasesMontreal, September 16–19, 2007 (Abstr. 123).

62. Tanaka, D., T. Kagari, H. Doi, and T. Shimozato. 2006. Essential role of neu-trophils in anti-type II collagen antibody and lipopolysaccharide-induced arthri-tis. Immunology 119: 195–202.

63. Fritsche, K. 2007. Important differences exist in the dose-response relationshipbetween diet and immune cell fatty acids in humans and rodents. Lipids 42:961–979.


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