From the Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, January 8, 2003, and in revised form, February 13, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Docosahexaenoic acid (DHA, C22:6) is highly
enriched in brain, synapses, and retina and is a major Chemical mediators and autacoids such as local acting lipid
mediators derived from arachidonic acid are well established regulators of key events of interest in host defense, coagulation, inflammation, and cancer (1). The tight control of the enzymes that regulate conversion of unesterified arachidonic acid to key classes of mediators, including prostaglandins, thromboxane, leukotrienes, and
lipoxins highlights the importance of arachidonic acid as an essential
fatty acid and precursor of these potent bioactive eicosanoids (2). The
potential for dysregulation of each of the individual classes of
eicosanoids has been suspect as important molecular events associated
with several human diseases, including, inflammatory diseases,
atherosclerosis, cardiovascular disorders, Alzheimer's disease, and
cancer. The control in physiologic systems of these lipid mediators is
an ongoing area of intense investigation, because early results
indicated that deficiency disease can be initiated by exclusion of fat
from the diet (3), and arachidonic acid was also uncovered as the
precursor to prostanoids that play key roles in the regulation of
parturition and renal function (4, 5).
Results from recent studies indicate that arachidonic acid is not the
only fatty acid precursor that is transformed to potent bioactive
mediators in inflammation and resolution (6, 7). Both
DHA1 and eicosapentaenoic
acid (EPA), the well-known Another aspect for the many years of sustained interest in DHA lies in
the fact that the brain is lipid-rich and that DHA is highly enriched
in the membranes of brain synapses and in the retina (16). DHA declines
in brain neurons with age and may result in loss in mental function
(20). Also, it is now clear that DHA is required for fetal brain
development and is held to be critical in the newborn for appropriate
development and intelligence (23). Hence, from these and many other
recent studies (24), it is now apparent that, in humans, DHA serves a
critical role in both physiologic and pathophysiologic responses. Our
recent finding that aspirin therapy can lead to the biosynthesis of
unique series of 17R resolvins generated from DHA led us to
question whether the significant roles reported for DHA in the many
biological systems noted above were related to DHA conversion to potent
local bioactive mediators. The reason for this line of thinking is that in most experimental systems, where the actions of either DHA or EPA
were assessed either clinically or in animal models, the concentrations
required to evoke beneficial actions are usually in the high microgram
to high milligram range. In this context, it is difficult to envision
direct and specific molecular actions responsible for the many
beneficial outcomes from in vivo studies. Thus, with the
coordinates (physical and biologic properties) in hand for the new
aspirin-triggered 17R series resolvins obtained using
lipidomics and bioassays (see Ref. 7), we determined in the present
experiments whether DHA is converted to bioactive mediators de
novo. The present results indicate that (a) DHA is a
precursor to a potent family of bioactive docosanoids that include novel docosatrienes as well as the 17S epimer resolvin
series generated in human blood cells, mouse brain, and by human glial cells; (b) these compounds display potent actions on
leukocyte trafficking as well as on glial cell functions
down-regulating cytokines expression. Hence, our present results
indicate that DHA is a precursor in novel biosynthetic pathways to
previously unrecognized potent molecules.
Materials--
Zymosan A and soybean lipoxygenase
(fraction IV) were purchased from Sigma Co. (St. Louis, MO).
Docosahexaenoic acid (C22:6, DHA) was from Cayman Chemical Co. (Ann
Arbor, MI); other synthetic standards, hydroxy fatty acids, and
intermediates used for MS identification and fragment ion references
were purchased from Cascade Biochem Ltd. (Reading, UK). Authentic
docosanoids of known hydroxy-containing products from DHA used to
characterize physical properties, i.e. LC-MS-MS diagnostic
ions and MS-MS spectra, were 4S-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic
acid (4S-HDHA), and the racemate
17R/S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (denoted 17R/S-HDHA) purchased from Penn
Bio-Organics (Bellefonte, PA). For each, NMR analyses were used to
establish stereochemistry (i.e. double-bond configurations).
Using recombinant enzymes (i.e. COX-2, 5-LO, and others) and
additional reactions, we prepared and isolated
10,17S-docosatriene, 10,17R-docosatriene,
7S,17S-diHDHA, 4S,17S-diHDHA,
4S,5,17S-triHDHA,
4S,11,17S-triHDHA,
7S,8,17S-triHDHA, and
7S,16,17S-triHDHA, similar to (6, 25). Additional
materials used in LC-MS-MS analyses were from vendors reported in (6, 25).
Biogenic Synthesis of 17S Series Compounds--
The reference
materials were prepared by incubation of DHA and soybean lipoxygenase
(Type IV) and/or 5-LO from potato using different ratios of substrate
to enzyme that gave specific product profiles (see below). Each
reaction condition used was optimized according to UV spectrometric
monitoring and high performance liquid chromatography coupled with a
photodiode-array detector and a tandem mass spectrometer (LC-PDA-MS-MS,
ThermoFinnigan, San Jose, CA). In brief, soybean lipoxygenase (260 kilounits, 14 mg of protein/ml, 360 kilounits/mg of protein, Sigma, St.
Louis, MO) was incubated with DHA (100 µg) in 10 ml of Dulbecco's
PBS (phosphate-buffered saline with Ca+2/Mg+2,
pH 8.0) at 4 °C. To obtain 17S-hydro(peroxy)-DHA
(H(p)DHA), aliquots (2 ml) were removed at the initial 15-min interval.
To obtain both di- and tri-HDHA, lipoxygenase (260 kilounits), and/or potato 5-LO was added to the remaining 17S-H(p)DHA
incubations in one round-bottomed flask held at 2-4 °C. At 15-min
intervals, product profiles were assessed using LC-PDA-MS-MS following
NaBH4 reduction. The 17R series resolvins were
prepared as in a previous study (7).
Hemoglobin-catalyzed Conversion of
17-H(p)DHA--
17S-H(p)DHA was prepared and incubated with
hemoglobin (Sigma H7379A) to identify non-enzymatic transformation
products. Incubations were carried out according to those used for
hemoglobin-catalyzed transformation of
15S-hydro(peroxy)eicosatetraenoic acid (H(p)ETE) (26). In brief, 17S-H(p)DHA (30 µg/ml) was incubated with
human hemoglobin (300 µg/ml) in PBS (pH 7.4, 37 °C) for 15 min.
Incubations were terminated by acidification to pH 3.2 and taken for
immediate solid-phase extraction (SepPak). Mono-, di-, and trihydroxy
docosanoid reaction products were each separated by combined column
chromatography (CC7) followed by solid-phase extraction. Products were
identified and quantitated by LC-PDA-MS-MS lipidomic analysis.
Human Whole Blood, PMN, and Murine Brain Incubations--
Human
whole (venous) blood (10 ml) was collected with or without heparin from
healthy volunteers (that declined taking medication for ~2 weeks
before donation; Brigham and Women's Hospital protocol 88-02642). Following collection, whole blood was immediately incubated with DHA (10µg) in a covered water bath for 40 min (37 °C). Human PMN were freshly isolated from the whole blood by Ficoll gradient and
enumerated as in a previous study (7). PMN (30-50 × 106 cells/ml) were exposed to zymosan A (100µg/ml, Sigma)
followed by addition of either 17S-hydroxy-DHA (3 µg/ml)
or DHA (3 µg/ml). Cell suspensions were incubated for (37 °C, 40 min) in a covered water bath. For identification of epoxide-containing
intermediates, isolated human PMN (30-50 × 106
cells/incubation) were incubated with 17S-hydro(peroxy)-DHA
and zymosan A (100 µg/ml) in Hanks' buffer containing 1.8 mM Ca2+ and 10 mM HEPES (pH 7.4, at
37 °C for 15 min). The incubations were immediately poured into 10
volumes of cold acidified (apparent pH 3.1) methanol and kept (30 min,
4 °C) before analysis. Lipidomic and lipid mediator profile analyses
were performed as in previous studies (7, 27, 28).
DHA Uptake and Agonist-induced Release of Novel
Docosanoids--
Human glial cells (DBTRG-05MG, ATCC) were cultured as
in a previous study (7). Media were removed from semiconfluent
monolayers (~75%) and replaced with Hanks' buffer (37 °C, 10 ml)
containing HEPES, 10 mM, pH 7.4, and
[1-14C]DHA (55 mCi/mmol, American Radiolabeled Chemicals,
Inc., 10 µM), or unlabeled C22:6 (~10 µM,
Oxford Biomedical Research) was added in EtOH (5-10 µl). Cells were
placed in an incubator with an atmosphere of 5% CO2 at
37 °C for 90 min, and aliquots (100 µl) were removed at 30-min
intervals and analyzed by scintillation counting to determine cellular
[1-14C]DHA. At 90 min, buffer was removed and cells were
washed with Hanks' buffer (10 ml, 37 °C). Hanks' buffer (10 ml,
37 °C) containing HEPES (10 mM, pH 7.4) and
CaCl2 (1.6 mM) was added to the flasks, and
cells were exposed to either zymosan A (100 ng/ml), calcium ionophore
(A23187, 5 µM), or buffer (with vehicle, 0.01% EtOH) alone. Cells were placed in an incubator with an atmosphere of 5%
CO2 (37 °C for 30 min), and aliquots were removed at
initiation and 30 min post agonist addition to assess the cellular
release of [14C]DHA and 14C-labeled
oxygenated products. After 30 min buffer was removed and immediately
added to 2 volumes of MeOH (4 °C) and kept at Substrate Competition with Human rCOX-2--
Human recombinant
COX-2 was overexpressed in Sf9 insect cells (ATCC).
The microsomal fractions (~8 µl) were suspended in Tris (100 mM, pH 8.0) as done previously (33).
[1-14C]Docosahexaenoic acid (55 mCi/mmol, American
Radiolabeled Chemicals, Inc., 10 µM) was added to rCOX-2
microsomal membrane suspensions followed immediately by arachidonic
acid (C20:4, Cayman Chemical) at increasing concentrations or vehicle
alone (0.5% EtOH). Suspensions were incubated at 37 °C for 30 min
and stopped with a solution (4 °C) containing ether:MeOH:citrate
(30:4:1, v/v). The organic phase was collected and analyzed by thin
layer chromatography at 4 °C using as a mobile phase the organic
fraction of ethyl acetate:2,2,4-trimethylpentane:acetic acid:water
(110:50:20:100, v/v) as in a previous study (34). Total and
product-specific radioactivity were each quantitated using a
PhosphorImager. Human rCOX-2 conversion of DHA (10 µM) to
specific products was 95 ± 0.5% (n = 6) and was
used as the 100% value to determine the impact of arachidonic acid on
conversion of DHA.
Acute Inflammatory Exudates: Murine Dorsal Air Pouch and
Peritonitis--
In the 6- to 8-week-old male FVB mice (fed laboratory
rodent diet 5001 (Lab Diet, Purina Mills) containing 1.49%
eicosapentaenoic acid, 1.86% DHA, and <0.25% arachidonic acid,
inflammatory exudates were initiated by intra-pouch injection of
recombinant mouse TNF Novel Docosanoids via Lipidomics: Formation of 17S Series Resolvins
and Docosatrienes by Human Whole Blood and Murine Brain--
Because
brain and blood share many biochemical and signaling pathways in common
(37, 38), we compared the profiles of bioactive docosanoids produced by
murine brain, human blood, and leukocytes to those generated by glial
cells. To this end, we first determined whether human blood generates
bioactive docosanoids such as the recently described resolvins (7) by
examining the transformation of DHA in human whole blood and endogenous
products produced by murine brain on addition of agonists and utilizing LC-PDA-MS-MS-based lipidomic analysis. Fig.
1A shows representative LC-MS
selective ion monitoring for [M-1] at m/z 375 for trihydroxy-containing docosanoids and m/z 359 for dihydroxy- and m/z 343 for
monohydroxy-containing docosanoids, respectively. These results
demonstrate for the first time that, in addition to docosanoids with
physical properties similar to those of the aspirin-triggered
17R series resolvins generated in resolving murine
inflammatory exudates (7), shown in Fig. 1, human blood converts DHA to
17S series resolvins as well as novel dihydroxy-containing
docosanoids. LC-MS plots of selective ion chromatograms and mass
spectra also showed the presence of novel dihydroxy-containing
docosanoids. For example, the MS-MS spectrum of a dihydroxy-containing
DHA is reported in Fig. 1B with prominent fragment ions
consistent with the structure shown in the insert, namely
10,17S-dihydroxydocosahexaenoate and ions at
m/z 359 (M-H), 341 (M-H-H2O), 323 (M-H-2H2O), 315 (M-H-CO2), 297 (M-H-H2O-CO2), and 277 (M-H-H2O-CO2-2H). Additional diagnostic ions
consistent with the carbon 10 and carbon 17 alcohol-containing positions were observed at m/z 153, 163, 181, 189, 205, 217 (216-CO2+H), 243 (261-H2O+H),
261, and 289. The UV spectrum of this docosanoid gave a maximum
absorbance wavelength (
We questioned whether these 17-hydroxy-carrying docosanoids from human
whole blood and murine brain could be formed via 15-lipoxygenation, which is an enzymatic activity in human leukocytes and is induced during the resolution phase of acute inflammation (39) and whether these new compounds were biosynthetically interrelated. Along these
lines, earlier results with isolated soybean 15-lipoxygenase showed
that this enzyme can rapidly transform DHA to 17S-HDHA in vitro (34) that were confirmed during the course of the
present experiments with isolated enzyme and LC-PDA-MS-MS analyses
(n > 12, not shown). Results shown in Fig. 1
demonstrate that human whole blood generated
17S-hydroxydocosahexaenoic acid (
Lipidomic-based analysis employing LC-PDA-MS-MS also identified the
presence of distinct docosanoids that were consistent with the presence
of and conversion of DHA by both lipoxygenase and cyclooxygenase-like
enzymatic activities that act on DHA present in whole blood (Fig.
1A). In these analyses, we also identified 13-HDHA (UV
A Role for Cell-Cell Interactions in the Formation of 17S Series
Resolvins and Docosatrienes--
Human PMN were exposed to
17S-H(p)DHA to determine if the likely intermediates in the
formation of 17S series resolvins and docosatrienes were
transformed by leukocyte-associated lipoxygenase-like activity
(i.e. 5-LO, 15-LO) to the novel dihydroxy- and
trihydroxy-containing docosanoids identified in blood and murine brain
(see Fig. 1). Indeed, PMN freshly isolated from peripheral blood,
transformed 17S-hydro(peroxy)docosahexaenoic acid to two
distinct 5-lipoxygenase products 7S,17S-diHDHA
and the less prominent product 4S,17S-diHDHA as
indicated in the selective ion chromatogram plotted at
m/z 359 (Fig.
2A). The
4S,17S-diHDHA was identified based on the MS-MS spectrum that displayed diagnostic ions at m/z
101, 257, 239 (257-H2O), 261, 267, 287, 245 (290-CO2-H), 359 (M-H), 341 (M-H-H2O), 315 (M-H-CO2), 297 (M-H-CO2-H2O), and
277 (M-H-CO2-2H2O-2H) and on its UV spectrum
that gave a Human Glial Cells Generate and Release Novel Docosanoids:
Docosatrienes--
Next, we determined whether the S series
resolvins and docosatrienes were formed from cellular stores of DHA and
if they could also be produced by a single cell type. To this end, we
labeled human glial cells with [1-14C]DHA. As
anticipated, glial cells rapidly incorporated labeled DHA as indicated
by a statistically significant increase in total radiolabel associated
with cellular stores (p > 0.05, n = 3). Radioactivity associated with cellular stores increased from
35 ± 8% at t = 30 min to 51 ± 3% at
t = 60 min and 61 ± 4% at t = 90 min post addition of [1-14C]DHA (n = 3)
indicating that cellular uptake of labeled DHA was time-dependent. Because glial cells are involved in host
defense and inflammation in neural tissues, we exposed them to the
microbial product, zymosan A, as well as a non-selective stimulus for
cellular calcium (calcium ionophore, A23187) to test whether they were agonists for the formation of novel docosanoids from cellular DHA
stores. Activation of glial cells induced a statistically significant
release (p > 0.05, n = 4) of
cell-associated label as well as formation of the novel docosanoids.
Results in Fig. 3A indicated
release of ~4.1 ± 1.1% of total cellular DHA following exposure to zymosan A and ~5.6 ± 2.4% with calcium ionophore
when directly compared with glial cells that were exposed to vehicle alone (1.6 ± 0.6%). Reversed-phase-HPLC radioprofiles
demonstrated that zymosan A challenge of glial cells released several
distinct 1-14C-labeled compounds from cellular DHA stores
(see inset of Fig. 3A). In addition to distinct
docosanoids that carried radioactivity in the profile there was also
label associated with material that corresponded to the retention time
of native DHA that accounted for 74 ± 6% (n = 3)
of the total released radioactivity. Exposure of human glial cell to
either zymosan A or calcium ionophore gave agonist-dependent and selective loss of
[1-14C]DHA from phosphatidylethanolamine that ranged from
13 to 28% and from 3 to 22%, respectively (see Fig.
3A and "Experimental Procedures").
To identify the endogenous docosanoids formed from cellular stores of
DHA in human glial cells, we analyzed the released DHA-derived products
by LC-PDA-MS-MS based lipidomics (as in Fig. 1). Again, activation of
glial cells with either calcium ionophore (Fig. 3B) or
zymosan A stimulated the formation (not shown) of novel 17-lipoxygenation product 10,17S-diHDHA as well as its
To assess the potential pathways involved in the formation of these new
compounds, human glial cells were exposed to exogenous DHA in the
presence of calcium ionophore. Lipidomic-based analysis using
LC-PDA-MS-MS as shown in Fig. 4 yielded
products consistent with the presence of 15-LO, 12-LO, 5-LO, and COX-2
activity in glial cells. This was further supported by the formation of
stereospecific oxidation products that were also identified in murine
brain and blood (see text and Fig. 1). Formation of double oxygenation
products 7S,17S-diHDHA and
4S,17S-diHDHA was confirmed by MS-MS and UV spectra that proved to be identical to those obtained with human whole
blood (Fig. 1). The formation of Substrate Competition with Human rCOX-2--
Human COX-2 generates
stereospecific oxygenated fatty acids from multiple substrates such as
eicosatetraenoic acid, eicosapentaenoic acid (6), and docosahexaenoic
acid (7). These lipid mediators as potential COX-2 products are of
interest, because COX-2 may regulate homeostasis in the brain, kidney,
and digestive tract as well as being potentially linked to inflammatory
diseases, fetal development, and carcinogenesis (44). 13-HDHA, a COX-2 product, was identified using LC-MS-MS-based lipidomic analyses in both
blood and human glial cell incubations. We investigated the impact of
arachidonic acid, the COX-2 substrate for prostaglandins and
15-epi-lipoxin formation (45), on docosahexaenoic conversion by human
recombinant enzyme. COX-2 rapidly transformed docosahexaenoic acid to
specific docosanoids that were determined using both thin layer
chromatography and LC-PDA-MS-MS. The percentage of docosahexaenoic acid
transformation at 10 µM substrate by rCOX-2 to
specific products mainly 13-hydroxydocosanoid was 94.6 ± 0.5%
(n = 3, d = 2 in each). This conversion
of DHA was inhibited in a concentration-dependent fashion
when exposed to increasing concentrations of arachidonic acid (Fig.
5), which reached 70 ± 15%
inhibition at 1 mM arachidonic acid (n = 3). These results indicate that DHA is an effective substrate for
oxygenation and can compete for the enzyme's active site in
vitro. Hence, these findings suggest that within cells or tissues
the rate-limiting step for DHA oxygenation is substrate availability.
They do not, however, preclude the involvement per se of as
yet to be defined enzymes or ones that can specifically act on DHA to
produce bioactive local mediators (see below).
Anti-inflammatory Properties of 17S Series Resolvins and
Docosatriene: Inhibition of PMN Recruitment and Cytokine Gene
Expression--
To determine whether the 17S series
compounds in the 17S series carry biologic
properties, we tested their topical and systemic actions in established
in vivo models of acute inflammation (Fig. 6A). The novel
docosanoids proved to be potent inhibitors of TNF Properties with Glial Cells--
The 17S series DHA
compounds were also evaluated as potential regulators of
cytokine-induced gene expression in the human glioma cells (Fig.
6B). Semi-qualitative reverse transcription-PCR analyses
demonstrated that both the 17S-HDHA and
10,17S-docosatriene inhibited TNF Trapping of Epoxide Intermediates--
To evaluate the formation
of potential epoxide-containing intermediates during the biosynthesis
of 17S series resolvins and docosatrienes, we exposed PMN to
excess acidic alcohol and analyzed the extracted materials for the
presence of methoxy-trapping products as in previous work (28, 48, 49)
that could be formed as direct marker of epoxide-containing
biosynthetic intermediates using LC-PDA-MS-MS. Results shown in Fig.
7 demonstrate formation of two prominent
16-methoxy-17S-hydroxydocosahexaenoate isomers, denoted
Ia and Ib, as well as two
10-methoxy-17S-hydroxydocosahexaenoate isomers, denoted
IIa and IIb in Fig. 7A. The
16-methoxy-containing trapping products were identified based on the UV
and MS-MS (at m/z 373) spectral analysis (Fig.
7B) of the corresponding LC peaks in Fig. 7A. The
presence of a chromophore with a triple band of absorbance at
The present results indicate that DHA is a precursor to
bioactive products, the basic structures of which were established, and
the presence of previously undisclosed pathways in mammalian cells to
convert DHA to potent endogenous compounds that display anti-inflammatory properties. Results of alcohol trapping experiments provide evidence for a 16(17)-epoxide intermediate generated from the
17S-hydroperoxy precursor in both human glial cells and
leukocytes. Taken together with the results in Figs. 1-7, they support
the scheme proposed in Fig. 8. In single
cell-type systems, as for example, in glial cells, agonists stimulate
the release of unesterified DHA predominantly from
phosphatidylethanolamine (Fig. 3), which is converted via a
lipoxygenase-like reaction to 17S-hydroperoxy derivative,
which is further transformed to the
trans-16(17)-epoxide-containing intermediate (Fig.
8A).
-3 fatty
acid. Deficiencies in this essential fatty acid are reportedly
associated with neuronal function, cancer, and inflammation. Here,
using new lipidomic analyses employing high performance liquid
chromatography coupled with a photodiode-array detector and a tandem
mass spectrometer, a novel series of endogenous mediators was
identified in blood, leukocytes, brain, and glial cells as
17S-hydroxy-containing docosanoids denoted as docosatrienes
(the main bioactive member of the series was
10,17S-docosatriene) and 17S series resolvins.
These novel mediators were biosynthesized via epoxide-containing
intermediates and proved potent (pico- to nanomolar range) regulators
of both leukocytes reducing infiltration in vivo and glial
cells blocking their cytokine production. These results indicate that
DHA is the precursor to potent protective mediators generated via
enzymatic oxygenations to novel docosatrienes and 17S
series resolvins that each regulate events of interest in inflammation
and resolution.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-3 fatty acids present in fish oils,
appear to be effective as dietary supplements in the treatment of a
wide range of human disorders (8-11). For example,
-3 fatty acid
supplementation is reported to have a beneficial impact in treating
asthma, atherosclerosis, cancer, and cardiovascular disorders
(for a recent review, see Ref. 12). Of interest, the lack of
-3
fatty acid consumption has also been shown to correlate with mental
depression (13). Several clinical trials aimed at testing the
therapeutic value of
-3 supplementation have convincingly
established that
-3 fatty acids can display beneficial actions
reducing the incidence and the severity of disease (14). In many of
these, aspirin therapy was also used in conjunction, although
apparently unintentionally, along with the
-3 supplementation
(i.e. see Refs. 14 and 15). Recent results evaluating the
impact of aspirin in the transformation of
-3 in inflammatory
exudates in vivo, namely acute inflammation and spontaneous
resolution, demonstrate that DHA and EPA are each converted via
independent pathways to potent bioactive local mediators. These new di-
and tri-hydroxy-containing compounds derived from
-3 fatty acids
were termed "resolvins," because they are (a) formed
within the resolution phase of acute inflammatory response, at least in part, as cell-cell interactions products,
(b) "stop" neutrophil entry to sites of inflammation,
and (c) reduce exudates (7). These findings, together with
earlier results (16-21), suggest that
-3 fatty acids, in addition
to arachidonic acid, an n-6 fatty acid (2, 4), can serve as precursors
for potent bioactive molecules with distinct functions (22). Hence, it is likely that the resolvins and related compounds identified might
represent the active products responsible, at least in part, for the
many reported beneficial responses obtained in clinical studies with
patients given high doses of
-3 supplementation (see Refs. 6 and 7
and references within).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C for >30
min. Cells were detached, lysed with 5 N NaOH and neutralized with 1 M Tris (pH 8.0) to quantitate
cell-associated 1-14C-labeled DHA and novel docosanoids
carrying label. For the experiments aimed at determining the sites for
DHA incorporation into major classes of phospholipids, triglycerides,
and neutral lipids, incubations (37 °C, 30 min) were stopped with
CHCl3/MeOH (2:5, v/v) and cells were detached. Lipids were
extracted using the method of Bligh and Dyer (29), materials from the
organic phase were suspended in chloroform, and the identity of lipid
classes was established with authentic standards and standard
procedures (29). Phosphorimaging analysis was used for quantitation of
the 14C content and demonstrated that 80-86% of the total
cell associated DHA was in phospholipid and ~11-18% in neutral
lipid pools (range from two separate experiments). DHA was
predominantly acylated into phosphatidylethanolamine (39-41%) and
phosphatidylcholine (29-36%), findings that are consistent with
studies in human platelets (30), neutrophils (31), and a rat glioma
cell line (32). Selected samples were taken for additional
analysis and extracted, and methyl formate-eluted products were
analyzed by PDA-MS-MS for identification as in Ref. 7. Reversed-phase
HPLC radioprofiles were carried out using a 1100 series liquid
chromatography system (Agilent Technologies) as done previously (7,
27). Radioactivity in 60-s fractions was quantified with an LS6500
scintillation counter (Beckman).
(100 ng/pouch, R&D Systems). Four hours later
mice were sacrificed, in accordance with the Harvard Medical Area
Standing Committee on Animals protocol 02570, air pouch lavages were
collected, and cells were enumerated. Inhibition of TNF
(100 ng/pouch) stimulated PMN infiltration, with intravenous tail injection
of S series resolvins and docosatrienes (as prepared with
biogenic synthesis; see below) was determined with pouch lavages taken
at 4 h. To assess the impact of novel compounds in peritonitis,
mice (FVB) were anesthetized with isoflurane and docosatrienes, and
S series and R series of resolvins (suspended in
120 µl of saline) were administered intravenously and followed
(~1-1.5 min) by an intraperitoneal injection of zymosan A (1 mg) in
1 ml of sterile saline. Two hours after the intraperitoneal injections,
mice were euthanized and peritoneal lavages were rapidly collected for
enumeration. Cytosensor® analyses were evaluated using a
Cytosensor® microphysiometer (Molecular Devices) and computer
workstation as in previous work (35, 36). Also, reverse
transcription-PCR analyses of TNF
-induced IL1
was carried out
according to a previous study (7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
max) of 269 nm with shoulders at
260 and 279 nm indicating the presence of a conjugated triene chromophore (i.e. three conjugated double bonds) in the new
structure. The configuration of the alcohol at position 17 was
confirmed as being in predominately the S configuration
using materials prepared with isolated lipoxygenases and DHA as
substrate with co-elution experiments (see "Experimental
Procedures"). The predominant products, also observed from endogenous
sources of DHA in isolated murine brain incubated with ionophore, were
10,17S-docosatriene and 4S,17S-diHDHA
(n = 3, not shown). Taken together with the chromatographic behavior, these physical properties were consistent with the formation of 10,17S-diHDHA (docosatriene), a novel
carbon 17 position-oxygenated product formed from DHA.
View larger version (19K):
[in a new window]
Fig. 1.
Formation of novel docosatrienes and
17S series resolvins. Lipidomic analysis was
carried out with heparinized blood incubated (10 ml, 37 °C, 30 min)
with DHA and products identified by LC-PDA-MS-MS (see "Experimental
Procedures" and text, n = 3). A,
selected ion chromatograms of trihydroxy-carrying
(m/z 375, upper panel),
dihydroxy-carrying (m/z 359, middle
panel), and monohydroxy-carrying (m/z 343, lower panel) products from DHA. Identification of the ions
diagnostic for specific hydroxy group positions present in the
trihydroxy, dihydroxy, and monohydroxy products generated are given in
the insets (see text). B, MS-MS and UV
(inset) spectra of 10,17S-docosatriene (depicted
with tentative stereochemistry assignments). C, MS-MS
spectrum of the 17S series trihydroxy resolvin:
7S,16,17S- triHDHA.
max of 237 nm and diagnostic MS-MS ion m/z 245) from DHA.
This human product of whole blood was matched in physical properties to
those of authentic 17S-hydroxydocosahexenoate produced via
biogenic synthesis (see "Experimental Procedures"). In addition, a
novel product 7S,16,17S-trihydroxydocosahexaenoic
acid was uncovered that exhibited an MS-MS spectrum (Fig.
1C) with diagnostic ions at m/z 123 (142-H2O-H), 131(129 + 2H), 203 (246-CO2-H),
225 (263-2H2O-2H), 246, 263, 277 (276+H), 295 (M-H-2H2O-CO2), 313 (M-H-H2O-CO2), 321 (M-H-3H2O), 331 (M-H-CO2), 339 (M-H-2H2O), 357 (M-H-H2O-CO2), and
375 (M-H). The presence of this
7S,16,17S-trihydroxy-containing docosanoid suggested a pathway for its formation (see below) similar to the generation of the anti-inflammatory eicosanoid lipoxin B4,
a 5-lipoxygenase interaction product generated from arachidonic acid
(e.g.
5S,14,15S-trihydroxyeicosatetraenoic acid), and
the newly described 17R epimer termed
7S,16,17R-trihydroxydocosahexaenoic acid-resolvin
(as in 7). Of interest, the 17R series and 17S
series of di- and tri-hydroxy products appear to give different
chromatographic retention times (see "Experimental
Procedures").
max 237 nm and diagnostic ion m/z
221), a potential cyclooxygenase-2-derived product from DHA (Ref. 7 and
data not shown). Along with these compounds shown in Fig.
1A, we also observed LC-MS and MS-MS spectra consistent with
the production of both 11-HDHA and 14-HDHA the known 12-lipoxygenase
products reported earlier (17, 40), as well as the 4S- and
7S-hydroxy-containing products of DHA that can be generated
by a 5-lipoxygenase-like reaction as exemplified by the potato
5-lipoxygenase enzyme (41, 42). It is noteworthy that both clotted
whole blood (non-heparinized) as well as heparinized blood generated
the identified monohydroxydocosanoid profile (Fig. 1). In contrast, the
dihydroxy products from DHA, including the novel docosatriene
(10,17S-diHDHA), were not observed with clotted whole blood
(data not shown). In addition, incubation of
17S-hydro(peroxy)-DHA with hemoglobin as used with
15-H(p)ETE conversion (26) produced several new non-enzymatic
epoxy alcohols and trihydroxydocosanoids (i.e.
16,17S-epoxy-15-hydroxydocosapentaenoic acid
(16,17S-epoxy-15-HDPA),
13,14-epoxy-17S-hydroxydocosapentaenoic acid
(13,14-epoxy-17S-HDPA),
16,17S-epoxy-13-hydroxydocosapentaenoic acid
(16,17S-epoxy-13-HDPA),
15,16-epoxy-17S-hydroxydocosapentaenoic acid
(15,16-epoxy-17S-HDPA),
13,16,17S-tri-hydroxydocosapentaenoic acid
(13,16,17S-HDPA),
15,16,17S-tri-hydroxydocosapentaenoic acid (15,16,17S-tri-HDPA),
13,14,17S-tri-hydroxydocosapentaenoic acid (13,14,17S-tri-HDPA); see "Experimental Procedures").
These compounds were not formed in substantial quantities in whole
blood (see below) but may be relevant in other pathophysiologic
scenarios. It is likely that they were not formed in substantial
amounts in these incubations, because the 17S-H(p)DHA, once
formed, remained intracellular, limiting its ability to interact with
heme proteins in a non-selective fashion. The bioactions of these will
be reported elsewhere. Together these results indicated that human
blood and murine brain converts DHA to several previously unappreciated novel di- and tri-hydroxy-containing docosanoids. Some of these new
docosanoids resembled the physical properties of the recently identified 17R series resolvins (7), namely those denoted
here as their 17S carbon position, e.g. hydroxy
epimers or the 17S series resolvins.
max 234 nm revealing its conjugated diene
structure (Fig. 2B, inset). In addition to these
dihydroxydocosanoids, the MS-MS and UV spectral analysis also confirmed
the presence and formation of the novel docosatriene
10,17S-diHDHA (see Fig. 2A), results that are
consistent with spectra obtained from murine brain and human whole
blood lipidomic analysis (Fig. 1). The apparent ratio for
10,17S-docosatriene to the other main dihydroxy product 7,17S-diHDHA generated by human PMN was ~1.12 (calculated
based on the analysis of UV chromatographic peak areas at their
respective
max 269 and 242 nm using recorded on line PDA
spectra). Further MS-MS and UV spectral analysis revealed the formation
of
-22-hydroxy-10,17S-docosatriene (Fig. 2C),
a likely
-oxidation product of the 10,17S-docosatriene in
human leukocytes. Human leukocytes and several other tissues possess a
prominent
-oxidation apparatus that can rapidly inactivate local
bioactive lipid mediators (25, 43). The structure of
-22,10,17S-docosatriene was determined by analysis of the
MS-MS spectrum (Fig. 2C). This compound gave ions of
diagnostic value at m/z 375 (M-H), 357 (M-H-H2O), 343 (344-H), 339 (M-H-H2O), 331 (M-H-CO2), 324 (344-H2O-2H), 309 (344-2H2O+H), 300 (344-CO2), 295 (M-H-2H2O-CO2), 273 (290-H2O+H),
265 (344-2H2O-CO2+H), 261 (260+H), 217 (260-CO2+H), 193, 181, and 163 (181-H2O). Taken
together these findings suggest transformation of 17H(p)DHA via two
prominent and distinct metabolic pathways to generate 5-LO-like
interaction products (i.e. the double dioxygenase products
4S,17S-diHDHA and 7S,17S-diHDHA) and the novel
10,17S-docosatriene.
View larger version (17K):
[in a new window]
Fig. 2.
Human PMN produce both 17S
series resolvins and docosatrienes. Human neutrophils
(30-50 × 106 cells/incubation) were exposed to
zymosan A and 17S-H(p)DHA, and products were analyzed using
LC-PDA-MS-MS (see "Experimental Procedures," n = 5). A, selected ion chromatogram (m/z
359) for dihydroxy-DHA products. B, MS-MS and UV
(inset) spectra of the 17S series resolvin
4S,17S-diHDHA. C, MS-MS of
-22-hydroxy-10,17S-docosatriene. Identification of
17S series diHDHAs and 10,17S-docosatriene are
indicated with tentative stereochemistry assignments.
View larger version (27K):
[in a new window]
Fig. 3.
Human glial cells release and transform DHA.
A, human glial cells were labeled with
[1-14C]DHA and exposed to either PBS alone, zymosan A, or
calcium ionophore (see "Experimental Procedures"). Released
[1-14C]docosanoids were quantified (n = 4). The asterisk denotes significant differences from
control (PBS alone), p < 0.05 analysis of variance,
Newman-Keuls test. The inset shows a representative HPLC
radiochromatogram of released [1-14C]docosanoids (see
"Experimental Procedures") from glial cells exposed to zymosan A
(n = 3). Phospholipids from glial cells were
identified, and the 14C content in phosphatidylethanolamine
(PE) was determined. The percent loss in PE 14C
content from cells exposed to agonists was determined
(n = 2). B, lipidomic profile of human glial
cells exposed to Ca2+ ionophore: LC-MS total ion
chromatogram of released products. C, LC-MS selective ion
chromatogram of released DHA-derived products from glial cells exposed
to calcium ionophore. MS-MS and UV spectra of
-22hydroxy-4S,17S-diHDHA.
-oxidation product 10,17S,22-triHDHA (note that both were
also produced in blood; see Fig. 1). The structure of the
-22
product was determined by analysis (at m/z 375)
of the MS-MS spectrum (Fig. 3C). The material beneath the
peaks corresponded to 10,17S-docosatriene and
17S-HDHA (not shown) and gave MS-MS and UV spectra
essentially identical to the 17S-oxygenation products of DHA
that were detected with human whole blood (see
10,17S-docosatriene in Fig. 1B). Of the
theoretically possible multiple isomers, only one major peak eluted
10,17S-docosatriene produced by glial cells (see
"Experimental Procedures"). The
-22-hydroxy-4,17-diHDHA was also
formed as shown in the LC-MS selective ion chromatogram at
m/z 375 (Fig. 3C). This structure was
consistent with the MS-MS (at m/z 375) and
prominent ions of diagnostic value at m/z 216 (260-CO2-H), 236 (273-2H2O-H), 247 (303-3H2O-2H), 272 (290-H2O), 273, 293 (M-H-2H2O-CO2-2H), 300 (344-CO2), 303, 313 (M-H-H2O-CO2),
337 (M-H-2H2O-2H), 345 (344+H), 357 (M-H-H2O), and 375 (M-H) together with the UV spectrum (
max 238 nm,
Fig. 3C, inset). The formation of
-22-hydroxy-4,17-diHDHA suggests conversion of
17S-H(p)DHA by a 5-lipoxygenase-like mechanism that can
oxygenate DHA at the carbon 4 position as shown for DHA to give
7S- or 4S-HDHA (41, 42). Hence, with
17S-H(p)DHA as substrate, this LO-like reaction can generate
4S,17S-diHDHA that is subsequently
-oxidized
at carbon 22.
-22,16,17S-docosatriene was confirmed by MS/MS (at m/z 375) spectrum (Fig.
4B). Its spectrum showed ions at m/z 309 (344-2H2O+H), 345 (344+H), 217 (260-CO2+H), 243 (260-H2O+H), 259 (260-H), 271 (290-H2O-H),
111 (145-2H2O+2H), 143 (145-2H), 231, 277 (M-H-3H2O- CO2), 295 (M-H-2H2O-CO2), 311 (M-H-H2O-CO2-2H), 331 (M-H -CO2),
339 (M-H-2H2O), 357 (M-H-H2O), and 375 (M-H).
These results suggested formation of a
16,17S-epoxide-containing intermediate that opens to form
the diol 16,17S-diHDHA (see below), which is subsequently transformed by
-oxidation to give the
trihydroxy-containing product.
View larger version (20K):
[in a new window]
Fig. 4.
Glial cells generate 17S
series resolvins and 10,17S-docosatriene.
Human glial cells were exposed to DHA and calcium ionophore (37 °C,
30 min). The docosanoids were identified by LC-PDA-MS-MS analysis.
A, LC-MS-selective ion chromatograms showing generation of
specific mono-HDHAs (right) and di-HDHAs (left).
B, MS-MS of
-22-hydroxy-16,17S-docosatriene.
View larger version (11K):
[in a new window]
Fig. 5.
Substrate competition at human recombinant
COX-2. Human rCOX-2 microsomal membranes from Sf9 cells
were incubated with [1-14C]DHA (C22:6, 10
µM) in the presence of increasing concentrations of
unlabeled arachidonic acid (C20:4). COX-2 conversion of
[1-14C]DHA and specific product profiles were quantitated
(n = 3). rCOX-2 activity is expressed as the inhibition
of [14C]C22:6 product formation (95 ± 0.5%;
conversion of DHA was set to 100%).
-induced leukocyte
trafficking to the air pouch both via topical and systemic application.
The 17S series resolvins reduced PMN within the exudates by
82.2 ± 5.6% given topically and by 49.6 ± 8.2% when given
intravenously (n = 4). Systemic treatment with the
17S series resolvins also inhibited zymosan A-induced PMN
recruitment to the peritoneum by 45.1 ± 0.8% (n = 4), a value that was equipotent to the recently described
17R series resolvins (7). The direct side-by-side comparison
showed that the 17R series resolvins inhibited PMN recruitment by 42.6 ± 8.0% (Fig. 6A). Treatment with
the isolated novel docosatriene (10,17SdiHDHA), a
member of the 17S series (see Fig. 8), proved to be a potent
systemic inhibitor of PMN recruitment as evidenced by its ability to
abate PMN in the peritoneal exudates by ~42%. In this regard, it
proved to be as potent as indomethacin, a well-characterized
non-steroidal anti-inflammatory drug (46), which reduced PMN peritoneal
in exudates by ~40% (Fig. 6A).
View larger version (13K):
[in a new window]
Fig. 6.
Anti-inflammatory properties of
17S series resolvins and
10,17S-docosatriene. A, inhibition of
PMN infiltration in murine peritonitis and dorsal skin pouch following
systemic and topical application. Peritonitis: 17S series
docosanoids (100 ng) or indomethacin (100 ng) was injected
intravenously into mouse tails followed by zymosan A into the
peritoneum. Mice were sacrificed, and peritoneal lavages were collected
(2 h) and cells enumerated (n = 4). Air pouch,
17S series compounds (100 ng) were injected intrapouch or
intravenously followed by intrapouch injection of TNF . For direct
comparison, mice were treated with 17R series resolvins via
intrapouch injection followed by TNF
. At 4 h, air pouch lavages
were collected and enumerated. Values represent mean ± S.E. from three to four different mice;
all gave p < 0.05 when infiltrated PMN were compared
with vehicle control. B, inhibition of TNF
-stimulated
IL1
transcripts in human glial cells. Human glial cells were
stimulated for 16 h with TNF
in the presence of specified
concentrations of 17S-HDHA (solid line) or
10,17S-docosatriene (dotted line), and expression
of IL-1
transcripts was analyzed. Data are representative of
n = 2. C, glial cells: 17S series
docosanoids evoke ligand-operated extracellular acidification. Changes
in extracellular acidification rates (EAR) were analyzed using
Cytosensor® microphysiometry. Cells were superfused (100 µl/60 s) with DHA, 17S series docosanoids
(n = 3), or 10,17S-docosatriene
(n = 4). Values are expressed as EAR (µV/s)
normalized to baseline (100%), and ligands were added at 2 min.
-induced IL-1
transcript levels and gave a dose-dependent inhibition with
an IC50 of ~0.5 nM (Fig. 6B).
Analysis using microphysiometry can give a rapid real-time indication
of receptor ligand-operated cellular events (47). To determine if the
17S series docosanoids interact with recognition sites on
human glial cells, we evaluated whether these novel lipid mediators
could evoke ligand-operated extracellular acidification using a
four-channel Cytosensor®. Results shown in Fig. 6C
demonstrate that the novel 10,17S-docosatriene and a mixture
of both 17S series docosanoids each added to the cells
separately evoked rapid ligand-specific extracellular acidification (p < 0.03). In sharp contrast, the precursor DHA did
not evoke extracellular acidification. These findings are consistent
with the ability of both 17S-HDHA and
10,17S-docosatriene to regulate gene expression in human
glial cells (Fig. 6B) and suggest the presence of
recognition sites for 17S series docosanoids in human glial cells.
max 270 nm with shoulders at 261 and 281 nm (see Fig.
7B, inset) and diagnostic MS-MS ions of
m/z 355 (M-H-H2O) and 329 (M-H-CO2), 230, 143, 275 (274+H), 241 (274-MeOH-H), 304, 271 (304-MeOH), and 210 (304-H2O-MeOH-CO2) were
consistent with the racemic alcohol-trapping products at position 16 denoted a and b isomers, namely,
16R/S-methoxy-17S-hydroxydocosahexenoic acid. The 10-methoxy-containing trapping products were also identified by the presence of chromophores that gave a
max 270 nm
with shoulders at 260 and 281 nm (see inset in Fig. 7),
together with diagnostic ions present in their MS/MS at m/z
355 (M-H-H2O), 341 (M-H-MeOH), 329 (M-H-CO2),
323 (M-H-H2O-MeOH), 311 (M-H-H2O-CO2), 297 (M-H-MeOH-CO2), 279 (M-H-H2O-MeOH-CO2), 152, 177, 203 (221-H2O), and 119 (196-H-MeOH-CO2), 304, 275 (274+H), and 227 (304-H-MeOH-CO2). The results of
LC-PDA-MS-MS analysis also uncovered the presence of low levels
of 4S-OH-, 11-methoxy-, 17S-OH-DHA (data not
shown). The identification of these major methoxy-trapping products
indicated that both a 16,17-epoxide-containing intermediate and
4,5-epoxide intermediate were generated from 17S-H(p)DHA by
human PMN. The nucleophilic addition of excess MeOH in acidic
conditions to form two main groups of methoxy- derivatives at carbon
position 10 and/or 16 position is consistent with open 16,17-epoxy and
4,5-epoxy rings and is in agreement with the involvement of specific
epoxy intermediates as those involved in leukotriene and lipoxin
generation from arachidonic acid (28, 48, 49).
View larger version (19K):
[in a new window]
Fig. 7.
Epoxide-containing intermediates in the
biosynthesis of docosatrienes and 17S series
resolvins. PMN (30-50 × 106 cells/incubation)
were incubated with 17S-hydroperoxy-DHA and zymosan A and
stopped, and alcohol-trapping products were extracted. A,
LC-MS chromatogram obtained from selective ion monitoring for
methoxy-trapping products at m/z 373 shows two
16-OCH3 (Ia and Ib) and two
10-OCH3 (IIa and IIb) isomers.
B, MS-MS and UV (inset) spectra of
16-OCH3 product I; C, MS-MS and UV
(inset) spectra of 10-OCH3 product II.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 8.
Biosynthetic scheme proposed for
10,17S-docosatrienes and 17S series
resolvins. These transformations may involve novel enzymes in
addition to those that serve in the biosynthesis of eicosanoids (see
text for further detail). The complete stereochemistry for the
docosatrienes (A) and 17S series resolvins
(B) remain to be established and are depicted in their
tentative configurations based on biogenic total synthesis, lipidomic
analyses, and alcohol trapping profiles. The carbonium cation
intermediate shown in A is also likely to be the enzymatic
basis of the epoxide conversion in B.
This trans epoxide intermediate can undergo aqueous hydrolysis via a carbonium cation intermediate to produce 10,17S-docosatriene carrying a conjugated triene and the 17 position in the S configuration retained from 17S or the vicinol diol triene (Fig. 8A). Once the epoxide intermediate at the 16(17) position is produced via a second hydrogen abstraction mechanism, which appears to be similar to that established for human lipoxygenases and related enzymes such as leukotriene A4 hydrolase (see Refs. 43, 50-52), it gives a carbonium cation intermediate that can then be converted to the dihydroxy docosatrienes (Fig. 8A). Once produced, these dihydroxy-containing docosatrienes exert their actions, in the present studies preventing leukocyte infiltration and down-regulating the production of inflammatory cytokines.
The lipoxygenase product 17S-H(p)DHA is converted to
16(17)-epoxide, which, if generated enzymatically, is likely to be a trans epoxide by analogy with the formation of leukotriene
A4 (48, 51, 52). If 10,17-docosatriene is generated via
non-enzymatic aqueous hydrolysis of the 16(17)-epoxide, the products
would be likely to yield the position 10 racemates
(10R/S) with all-trans double-bond configurations for the resulting conjugations. We have
observed, in human cells and whole blood, production of only one
dominant 10,17-dihydroxy product, indicating the biosynthesis and most
bioactive product is likely to be enzymatically formed in
situ. This enzymatic hydrolysis could proceed via a mechanism analogous to the leukotriene A4 hydrolase. Its double-bond
geometry would likely be
10R-hydroxy-11E,13E,15Z as depicted
in Fig. 8 (48, 51, 52). Although 10,17S-docosatriene is
generated endogenously and carries potent bioactivity, the chirality of the 10 position alcohol remains to be established. Nevertheless, once
these dihydroxy products are formed and impart their bioactions, they
can undergo
-oxidation at sites of inflammation and within exudates.
They can then undergo
-oxidation at the carbon 22 position (see Fig.
8 and "Results"). This type of
-oxidation route is usually
associated with rapid inactivation of bioactive eicosanoid mediators
(for example, see Refs. 43 and 53). These are also novel trihydroxy
docosanoids and implicate that the DHAs carry bioinformation. This was
further substantiated with the results obtained from in vivo
experiments indicating that 10,17S-docosatriene is a potent
regulator of peritonitis-activated leukocyte recruitment.
The major bioactive product generated from endogenous DHA released by glial cells focused our attention on the formation and actions of 10,17S-docosatriene (see Figs. 3, 4, and 6 for bioactions). Our results obtained with glial cells, murine brain, whole blood, and human leukocytes indicate that the biosynthesis of the docosatrienes can occur in a single cell type and appears to involve a 15-lipoxygenase-like activity and/or related enzymes. Whether the newly uncovered isoforms of human 15-lipoxygenase A or B (see Ref. 54 for a recent perspective) or possibly other, yet to be discovered, novel enzymes are involved in this pathway remains to be established. The initial step to form monohydro(peroxy)-containing intermediates is not likely to occur via a classic p450 enzyme as the predominant pathway (55), because we found the production of the 16(17)-epoxide intermediate as direct evidence for formation of alcohol-trapping products, i.e. the markers of epoxides that arise from allylic epoxides (Fig. 8). From our present results, it appears that the major enzymatic route to formation in glial cells involves tightly controlled enzymatic transformations. These reactions appear to be akin to those used for eicosanoid biosynthesis and related enzymes but instead handle DHA. These novel pathways may also involve very specialized enzyme systems to produce in brain, neural systems, and cells of hematopoietic origin the docosanoids established in the present study, namely the docosatrienes and 17S series resolvins.
The results of the alcohol trapping and labeling experiments also support the scheme in Fig. 8B for the formation of 17S series resolvins. Once produced, 17-hydroperoxy-DHA can via transcellular biosynthesis be converted by human neutrophils (see Fig. 8B) to two main intermediates following the insertion of the hydroperoxy at either the 4 or 7 position in their precursor. This is rapidly transformed to both 7,8-epoxide and 4,5-epoxide intermediates as also observed with the recently elucidated 17R series resolvins produced when aspirin treatment is administered. These two pathways in leukocytes give rise to the dihydroxy- and trihydroxy-containing products termed 17S series resolvins (Fig. 8B). These are the main products derived from human leukocytes as determined here and are observed both in human whole blood and in inflammatory exudates (see Ref. 7). The 17S series compounds appear to be equally potent as the 17R series compounds in vivo as shown in Fig. 6. This is analogous to the flexibility in stereochemistry noted earlier for the carbon 15 position of arachidonic acid as R or S observed in lipoxin and aspirin-triggered 15-epi-lipoxin formation and action where the chirality of the other two alcohol positions and double bond geometries are highly critical to maintain bioactions (7). Also, there appear to be one or more specific ligand receptor systems responsible for activating glial cell receptors as indicated by the results with microphysiometry analyses (see Fig. 6C), a finding that is in line with other lipid mediators derived from arachidonic acid, namely that there are specific receptors that transduce their wide range of actions (see Refs. 43 and 56). The receptor system(s) and cell types identified in the present experiments with DHA and glial cells as well as formation and action of 10,17-docosatriene provide opportunities to uncover novel receptors signaling used by the docosatrienes and resolvins to evoke their actions in vivo.
Results from earlier studies demonstrated that DHA is released from
membrane-rich sources in retina and synaptic terminals (57, 58) and
underscores an important role for this essential fatty acid in neuronal
development (20) and function (59). Importantly, the molecular
mechanism(s) for these reported DHA actions remained of interest to
explain precise actions in many organ systems. Our present results
establish that glial cells can release and transform DHA to novel
docosatrienes. In addition, during multicellular events and cell-cell
interactions, for example, leukocytes can take up 17-HDHA and convert
it to resolvins. These results underscore the importance of cell-cell
interaction in generating products that neither cell type can generate
alone. This theme in multicellular responses appears to be a key
mechanism in the generation of a diverse array of bioactive oxygenated
lipid mediators. Because fish are rich in DHA and are able to produce essentially equal amounts of 4 and 5 series eicosanoids (49) from
arachidonic acid and EPA, it is possible that DHA transformation to
both docosatrienes and 17S series resolvins is a conserved primordial signaling pathway in certain human tissues. Hence, results
from the present experiments establish the enzymatic transformation pathways for the production of docosatrienes and 17S series
resolvins and document their actions in models in vivo.
These new pathways and structures could represent the active components
responsible for some of the beneficial actions reported with dietary
supplementation of DHA. Moreover, these results indicate that DHA is a
precursor for two novel families of bioactive mediators, namely
docosatrienes and 17S series resolvins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mary Halm Small for assistance in manuscript preparation and Katherine Gotlinger and Eric Tjonahen for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institute of Health Grants GM38765 and P01-DE13499 (to C. N. S.) and DK60583 (to K. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Center for Experimental Therapeutics and Reperfusion Injury, Thorn Medical Research Bldg., 7th Floor, Brigham and Women's Hospital, Boston, MA 02115. Tel.: 617-732-8822; Fax: 617-582-6141; E-mail: cnserhan@zeus.bwh.harvard.edu.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M300218200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DHA, C22:6, docosahexaenoic acid; 4S-HDHA, 4S-hydroxydocosa-5E,7Z,10Z,13Z,16Z,19Z-hexaenoic acid; 4S, 17R/S-diHDHA, 4S,17R/S-dihydroxydocosa-5E,7Z,10Z,13Z,15E, 19Z-hexaenoic acid; 7S, 17R/S-diHDHA, 7S,17R/S-dihydroxydocosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoic acid; 10, 17R-docosatriene, 10,17R-dihydroxydocosahexaenoic acid; 10, 17S-docosatriene, 10,17S-dihydroxydocosahexaenoic acid; 10R/S-OCH3-17S-HDHA, 10R/S-methoxy-17S-hydroxydocosahexaenoic acid; 17R/S-H(p)DHA, 17R/S-hydro(peroxy)docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid; COX-2, cyclooxygenase 2; LC-PDA-MS-MS, liquid chromatography-photodiode array detector-tandem mass spectrometry; LO, lipoxygenase; PMN, polymorphonuclear leukocytes; HPLC, high performance liquid chromatography; EPA, eicosapentaenoic acid; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; IL, interleukin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gallin, J. I., and Snyderman, R. (eds) (1999) Inflammation: Basic Principles and Clinical Correlates , Lippincott Williams & Wilkins, Philadelphia |
2. | Samuelsson, B., Dahlén, S. E., Lindgren, J. Å., Rouzer, C. A., and Serhan, C. N. (1987) Science 237, 1171-1176[Medline] [Order article via Infotrieve] |
3. |
Burr, G. O.,
and Burr, M. M.
(1929)
J. Biol. Chem.
82,
345 |
4. | Samuelsson, B. (1982) Les Prix Nobel: Nobel Prizes, Presentations, Biographies and Lectures , pp. 153-174, Almqvist & Wiksell, Stockholm |
5. | Bergström, S. (1982) Les Prix Nobel: Nobel Prizes, Presentations, Biographies and Lectures , pp. 129-148, Almqvist & Wiksell, Stockholm |
6. |
Serhan, C. N.,
Clish, C. B.,
Brannon, J.,
Colgan, S. P.,
Chiang, N.,
and Gronert, K.
(2000)
J. Exp. Med.
192,
1197-1204 |
7. |
Serhan, C. N.,
Hong, S.,
Gronert, K.,
Colgan, S. P.,
Devchand, P. R.,
Mirick, G.,
and Moussignac, R.-L.
(2002)
J. Exp. Med.
196,
1025-1037 |
8. | De Caterina, R., Endres, S., Kristensen, S. D., and Schmidt, E. B. (eds) (1993) n-3 Fatty Acids and Vascular Disease , Springer-Verlag, London |
9. |
Khair-El-Din, T. A.,
Sicher, S. C.,
Vazquez, M. A.,
Wright, W. J.,
and Lu, C. Y.
(1995)
J. Immunol.
154,
1296-1306 |
10. | McLennan, P., Howe, P., Abeywardena, M., Muggli, R., Raederstorff, D., Mano, M., Rayner, T., and Head, R. (1996) Eur. J. Pharmacol. 300, 83-89[CrossRef][Medline] [Order article via Infotrieve] |
11. | Rapp, J. H., Connor, W. E., Lin, D. S., and Porter, J. M. (1991) Arteriosclerosis and Thrombosis 11, 903-911[Abstract] |
12. | Calder, P. C. (2001) Lipids 36, 1007-1024[Medline] [Order article via Infotrieve] |
13. | Hibbeln, J. R. (1998) Lancet 351, 1213[CrossRef][Medline] [Order article via Infotrieve] |
14. | GISSI-Prevenzione Investigators.. (1999) Lancet 354, 447-455[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Marchioli, R.,
Barzi, F.,
Bomba, E.,
Chieffo, C.,
Di Gregorio, D.,
Di Mascio, R.,
Franzosi, M. G.,
Geraci, E.,
Levantesi, G.,
Maggioni, A. P.,
Mantini, L.,
Marfisi, R. M.,
Mastrogiuseppe, G.,
Mininni, N.,
Nicolosi, G. L.,
Santini, M.,
Schweiger, C.,
Tavazzi, L.,
Tognoni, G.,
Tucci, C.,
and Valagussa, F.
(2002)
Circulation
105,
1897-1903 |
16. | Bazan, N. G. (1992) Nestle Nutrition Workshop Series 28, 121-133 |
17. | Bazan, N. G., Birkle, D. L., and Reddy, T. S. (1984) Biochem. Biophys. Res. Commun. 125, 741-747[Medline] [Order article via Infotrieve] |
18. | Kim, H. Y., Karanian, J. W., Shingu, T., and Salem, N., Jr. (1990) Prostaglandins 40, 473-490[CrossRef][Medline] [Order article via Infotrieve] |
19. | Miller, C. C., Tang, W., Ziboh, V. A., and Fletcher, M. P. (1991) J. Invest. Dermatol. 96, 98-103[Abstract] |
20. | Salem, N., Jr., Litman, B., Kim, H.-Y., and Gawrisch, K. (2001) Lipids 36, 945-959[Medline] [Order article via Infotrieve] |
21. | Sawazaki, S., Salem, N., Jr., and Kim, H.-Y. (1994) J. Neurochem. 62, 2437-2447[Medline] [Order article via Infotrieve] |
22. | Serhan, C. N. (2002) Curr. Med. Chem.-Anti-Inflammatory & Anti-Allergy Agents 1, 177-192 |
23. |
Salem, N., Jr.,
Wegher, B.,
Mena, P.,
and Uauy, R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
49-54 |
24. | Lands, W. E. M. (1993) Ann. N. Y. Acad. Sci. 676, 46-59[Medline] [Order article via Infotrieve] |
25. |
Clish, C. B.,
Levy, B. D.,
Chiang, N.,
Tai, H.-H.,
and Serhan, C. N.
(2000)
J. Biol. Chem.
275,
25372-25380 |
26. | Sok, D.-E., Chung, T., and Sih, C. J. (1983) Biochem. Biophys. Res. Commun. 110, 273-279[Medline] [Order article via Infotrieve] |
27. | Gronert, K., Clish, C. B., Romano, M., and Serhan, C. N. (1999) in Eicosanoid Protocols (Lianos, E. A., ed) , pp. 119-144, Humana Press, Totowa, NJ |
28. | Serhan, C. N. (1989) Biochim. Biophys. Acta 1004, 158-168[Medline] [Order article via Infotrieve] |
29. | Gunstone, F. D., Harwood, J. L., and Padley, F. B. (1994) The Lipid Handbook , 2nd Ed. , Chapman & Hall, London |
30. | Fischer, S., Schacky, C. v., Siess, W., Strasser, T., and Weber, P. C. (1984) Biochem. Biophys. Res. Commun. 120, 907-918[Medline] [Order article via Infotrieve] |
31. | Tou, J.-s. (1986) Lipids 21, 324-327[Medline] [Order article via Infotrieve] |
32. | Garcia, M. C., and Kim, H.-Y. (1997) Brain Res. 768, 43-48[CrossRef][Medline] [Order article via Infotrieve] |
33. | George, H. J., Van Dyk, D. E., Straney, R. A., Trzaskos, J. M., and Copeland, R. A. (1996) Protein Expression Purif. 7, 19-26[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Hamberg, M.,
and Samuelsson, B.
(1967)
J. Biol. Chem.
242,
5329-5335 |
35. |
Chiang, N.,
Gronert, K.,
Clish, C. B.,
O'Brien, J. A.,
Freeman, M. W.,
and Serhan, C. N.
(1999)
J. Clin. Invest.
104,
309-316 |
36. |
Gronert, K.,
Colgan, S. P.,
and Serhan, C. N.
(1998)
J. Pharmacol. Exp. Ther.
285,
252-261 |
37. |
Moore, M. A. S.
(1999)
N. Engl. J. Med.
341,
605-607 |
38. |
Serhan, C. N.,
Fierro, I. M.,
Chiang, N.,
and Pouliot, M.
(2001)
J. Immunol.
166,
3650-3654 |
39. | Levy, B. D., Clish, C. B., Schmidt, B., Gronert, K., and Serhan, C. N. (2001) Nature Immunol. 2, 612-619[CrossRef][Medline] [Order article via Infotrieve] |
40. | Yergey, J. A., Kim, H.-Y., and Salem, N., Jr. (1986) Anal. Chem. 58, 1344-1348[Medline] [Order article via Infotrieve] |
41. | Lee, T. H., Mencia-Huerta, J.-M., Shih, C., Corey, E. J., Lewis, R. A., and Austen, K. F. (1984) J. Clin. Invest. 74, 1922-1933[Medline] [Order article via Infotrieve] |
42. | Whelan, J., Reddanna, P., Nikolaev, V., Hildenbrandt, G. R., and Reddy, T. S. (1990) Biological Oxidation Systems , Vol. 2 , pp. 765-778, Academic Press |
43. |
Yokomizo, T.,
Ogawa, Y.,
Uozumi, N.,
Kume, K.,
Izumi, T.,
and Shimizu, T.
(1996)
J. Biol. Chem.
271,
2844-2850 |
44. | Vane, J. R., and Botting, R. M. (eds) (2001) Therapeutic Roles of Selective COX-2 Inhibitors , William Harvey Press, London |
45. | Clària, J., and Serhan, C. N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9475-9479[Abstract] |
46. |
Wallace, J. L.,
Chapman, K.,
and McKnight, W.
(1999)
Br. J. Pharmacol.
126,
1200-1204 |
47. | McConnell, H. M., Owicki, J. C., Parce, J. W., Miller, D. L., Baxter, G. T., Wada, H. G., and Pitchford, S. (1992) Science 257, 1906-1912[Medline] [Order article via Infotrieve] |
48. | Borgeat, P., and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2148-2152[Abstract] |
49. | Rowley, A. F., Lloyd-Evans, P., Barrow, S. E., and Serhan, C. N. (1994) Biochemistry 33, 856-863[Medline] [Order article via Infotrieve] |
50. | Corey, E. J., and Mehrotra, M. M. (1986) Tetrahedron Lett. 27, 5173-5176[CrossRef] |
51. |
Rudberg, P. C.,
Tholander, F.,
Thunnissen, M. M. G. M.,
and Haeggström, J. Z.
(2002)
J. Biol. Chem.
277,
1398-1404 |
52. |
Rudberg, P. C.,
Tholander, F.,
Thunnissen, M. M. G. M.,
Samuelsson, B.,
and Haeggström, J.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
4215-4220 |
53. | Serhan, C. N., Fiore, S., Brezinski, D. A., and Lynch, S. (1993) Biochemistry 32, 6313-6319[Medline] [Order article via Infotrieve] |
54. |
Chanez, P.,
Bonnans, C.,
Chavis, C.,
and Vachier, I.
(2002)
Am. J. Respir. Cell Mol. Biol.
27,
655-658 |
55. |
Graham-Lorence, S.,
Truan, G.,
Peterson, J. A.,
Falck, J. R.,
Wei, S.,
Helvig, C.,
and Capdevila, J.
(1997)
J. Biol. Chem.
272,
1127-1135 |
56. |
Brink, C.,
Dahlén, S.-E.,
Drazen, J.,
Evans, J. F.,
Hay, D. W. P.,
Nicosia, S.,
Serhan, C. N.,
Shimizu, T.,
and Yokomizo, T.
(2003)
Pharmacol. Rev.
55,
195-227 |
57. | Bazan, N. G. (1970) Biochim. Biophys. Acta 218, 1-10[Medline] [Order article via Infotrieve] |
58. | Bazan, N. G. (1990) in Nutrition and the Brain (Wurtman, R. J. , and Wurtman, J. J., eds), Vol. 8 , pp. 1-22, Raven Press, New York |
59. | Bazan, N. G., and Rodriguez de Turco, E. B. (2001) in Neuroprotection (Lo, E. H. , and Marwoh, J., eds) , p. 195, Prominent Press |