Báo cáo khoa học: Hodgkin Reed–Sternberg cells express 15-lipoxygenase-1 and are putative producers of eoxins in vivo Novel insight into the inflammatory features of classical Hodgkin lymphoma ppt

Hodgkin Reed–Sternberg cells express 15-lipoxygenase-1
and are putative producers of eoxins in vivo
Novel insight into the inflammatory features of classical Hodgkin
lymphoma
˚
Hans-Erik Claesson1,2, William J. Grifths3, Asa Brunnstrom2, Frida Schain4, Erik Andersson2,4,
ă
1,2
1
`
Stina Feltenmark , Helene A. Johnson , Anna Porwit5, Jan Sjoberg4 and Magnus Bjorkholm4
ă
ă
1
2
3
4
5

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Orexo AB, Uppsala, Sweden
Institute of Mass Spectrometry, School of Medicine, Swansea University, UK
Division of Hematology, Department of Medicine, Karolinska University Hospital and Institutet, Stockholm, Sweden
Department of Pathology Karolinska University Hospital and Institutet, Stockholm, Sweden

Keywords
arachidonic acid; Hodgkin lymphoma;
inflammation; L1236; lipoxygenase
Correspondence
H.-E. Claesson, Department of Medical
Biochemistry and Biophysics, Division of

Physiological Chemistry II, Karolinska
Institutet, SE-171 77 Stockholm, Sweden
Fax: +46 8 324 264
Tel: +46 8 524 87627
E-mail:
(Received 14 April 2008, revised 18 June
2008, accepted 23 June 2008)
doi:10.1111/j.1742-4658.2008.06570.x

Classical Hodgkin lymphoma has unique clinical and pathological features
and tumour tissue is characterized by a minority of malignant Hodgkin
Reed–Sternberg cells surrounded by inflammatory cells. In the present
study, we report that the Hodgkin lymphoma-derived cell line L1236 has
high expression of 15-lipoxygenase-1 and that these cells readily convert
arachidonic acid to eoxin C4, eoxin D4 and eoxin E4. These mediators were
only recently discovered in human eosinophils and mast cells and found to
be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15-lipoxygenase-1 was present mainly in the cytosol and that the enzyme translocated to the
membrane upon calcium challenge. By immunohistochemistry of Hodgkin
lymphoma tumour tissue, 15-lipoxygenase-1 was found to be expressed in
primary Hodgkin Reed–Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15-lipoxygenase-1, however, was not expressed in any of
10 biopsies representing nine different subtypes of non-Hodgkin lymphoma. In essence, the expression of 15-lipoxygenase-1 and the putative
formation of eoxins by Hodgkin Reed–Sternberg cells in vivo are likely to
contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15-lipoxygenase-1
activity in L1236 cells demonstrates that this cell line comprises a
useful model system to study the chemical and biological roles of
15-lipoxygenase-1.

Hodgkin lymphoma (HL), although a neoplastic disease, has many features of an infection ⁄ inflammatory
condition with symptoms and signs such as fever, night
sweats, itching, lymphadenopathy and splenomegaly.

The laboratory findings in this disease include neutrophilia, eosinophilia, lymphocytopenia and altered
serum phase reactants. It is now established that the
large majority of classical HL (cHL) cases are B-cell

Abbreviations
cHL, classical HL; DiHETE, dihydroxy-eicosatetraenoic acid; EBV, Epstein–Barr virus; EX, eoxin; HETE, hydroxy-eicosatetraenoic acid; HL,
Hodgkin lymphoma; IL, interleukin; LO, lipoxygenase; LT, leukotriene; MC, mixed cellularity; NHL, non-Hodgkin lymphoma; NS, nodular
sclerosis.

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H.-E. Claesson et al.

lymphomas, which are characterized by the presence of
Hodgkin Reed–Sternberg (H-RS) cells. These transformed cells appear to originate from pre-apoptotic
germinal centre B cells that have lost their capacity to
express a high-affinity B-cell receptor [1]. Importantly,
H-RS cells constitute only a small minority (approximately 1%) of the cell population in HL-affected tissue.
The inflammatory cellular infiltrate in HL tumour tissue
is rather heterogeneous, consisting of lymphocytes, macrophages, eosinophils, mast cells, plasma cells, stromal
cells and fibroblasts. There is strong evidence that these
infiltrating cells are involved in an inflammatory ⁄ reactive process creating an environment that allows, and
probably promotes, the survival of H-RS cells [2].
Cytokines and chemokines operating in a complex interaction have been suggested to be involved in the pathogenesis of HL [3]. A number of studies indicate that the
release of cytokines and other biological active mediators from H-RS cells plays an important role in the
pathophysiology of HL. Among cytokines, interleukin

(IL)-13 has been proposed to act as an autocrine growth
factor for H-RS cells [4,5].
The mammalian lipoxygenases are a family of structurally related enzymes, catalyzing the oxygenation of
arachidonic acid [6]. 5-Lipoxygenase (LO) catalyses the
conversion of arachidonic acid to leukotriene (LT)A4
which can be further converted to LTB4 or LTC4. The
latter metabolite can be further metabolized to LTD4
and LTE4 and these mediators are potent proinflammatory mediators and bronchoconstrictors [7]. There
are two forms of 15-LO, named type 1 and 2 [6,8]. It
has been shown that patients with asthma and airway
inflammation have increased expression of 15-LO-1
protein and increased activity of 15-LO-1 in the lung
compared to healthy subjects [9,10].
Only recently, we reported on the formation of eoxins (EX) in human eosinophils, cord blood derived
mast cells and surgically removed nasal polyps. The
enzyme 15-LO-1 catalyses the conversion of arachidonic acid to EXA4, which in turn can be conjugated
with glutathione, leading to the formation of EXC4
[11]. This metabolite can be further metabolized to
EXD4 and EXE4. Eoxins induce increased permeability
of the endothelial cell monolayer in vitro, indicating
that they can modulate and enhance vascular permeability, a hallmark of inflammation [11]. It has been
known for many years that 15-LO-1 can also catalyze
the formation of 15(S)-hydroxy-eicosatetraenoic acid
(HETE), 8(S,R),15(S)-dihydroxy-eicosatetraenoic acid
(DiHETE), 5(S),15(S)-DiHETE and 14(R),15(S)-DiHETE [6,12]. This latter metabolite has been reported to
inhibit natural killer cell activity [13]. Another 15-LO-1
derived mediator, 5-oxo-15-hydroxy-ETE, was found

Hodgkin lymphoma and 15-lipoxygenase

to be a potent chemotactic agent for human eosinophils [14]. Thus, mediators formed via the 15-LO-1
pathway can induce inflammatory reactions and
influence the immune system in man. There are also
indications, however, that 15-LO-1 may have an antiinflammatory role because this enzyme can be involved
in the formation of lipoxins [15].
The enzyme 15-LO-1 not only metabolizes free fatty
acids, but also can oxygenate phospholipids located in
the cell membrane, and these oxidated phospholipids
might contribute to 15-LO-1 signaling in inflammation
[6,16]. The enzyme has been proposed to play a role in
reticulocyte maturation through breakdown of mitochondria membranes [6,12]. The enzyme may, however, play a more general role in the differentiation of
cells, including the maturation of keratinocytes and the
eye lens [17]. 15-Lipoxygenase is predominantly
expressed in human eosinophils, activated monocytes,
airway epithelial cells, reticulocytes and mast cells
[6,18–20]. The Th2 cytokines IL-4 and IL-13 induce
the expression of 15-LO-1 in monocytes, airway epithelial cells and mast cells [20–23]. Demethylation of the
15-LO-1 promoter is a prerequisite for gene activation
[24].
In light of the characteristic inflammatory features
of cHL, it was of interest to investigate the expression
of lipoxygenases in H-RS cells and the formation of
arachidonic acid metabolites by these cells.

Results
Expression and localization of 15-LO-1 in the HL
cell lines
The large majority of H-RS cells are derived from
germinal centre B lymphocytes [1]. Because human B
lymphocytes express 5-LO [25], it was of interest to

determine whether HL cell lines also expressed this
enzyme. For that purpose, the metabolism of arachidonic acid was examined in the HL cell lines L1236,
L428, KMH2 and L570. These cell lines produced no
or very low amounts of 5-HETE or LTs after incubation with arachidonic acid in the presence or absence
of calcium ionophore (data not shown). Incubation of
L1236 cells with arachidonic acid for 5 min, however,
led to the formation of a major product that cochromatographed with synthetic 15-HETE (Fig. 1). In
addition, a minor peak (UV absorbance maximum at
236 nm) that coeluted with synthetic 12-HETE was
observed. The UV spectra of the materials in these
peaks are in agreement with the reported spectra for
15-HETE and 12-HETE, respectively. Chiral chromatography analysis demonstrated that the formed

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H.-E. Claesson et al.

Fig. 1. RP-HPLC chromatograms of the products formed by L1236
cells after incubation with arachidonic acid (40 lM) for 5 min. UV
monitoring was carried out at 236 nm. The retention times for synthetic 15(S)-HETE, 12(S)-HETE and 5(S)-HETE are indicated. Inset:
UV spectra of the materials that coeluted with synthetic 15(S)-HETE
and 12(S)-HETE, respectively. Solid line, cells incubated with arachidonic acid; broken line, cells incubated without arachidonic acid.

products were exclusively 15(S)-HETE and 12(S)HETE (data not shown). The ratio of approximately
9 : 1 between 15(S)-HETE versus 12(S)-HETE was

also in agreement with a 15-LO-1 catalyzed formation
of these products. Incubation of these cells
(40 · 106 cellsỈmL)1) with arachidonic acid (final concentration 40 lm) for 10 min led to the formation of
294 ± 172 pmol 15-HETE per 106 cells (n = 9;
mean ± SD). No formation of 12- or 15-HETE was
observed in the other examined HL cell lines after
incubation with arachidonic acid with or without the
calcium ionophore A23187. Linoleic acid is an excellent substrate for 15-LO-1 and L1236 cells also
efficiently metabolized linoleic acid to 13-hydroxy-octadecadienoic acid (data not shown), which also is in
agreement with the expression of 15-LO-1 in these
cells.
RT-PCR analysis revealed mRNA expression of
15-LO-1 but not of 15-LO-2 in L1236 cells (data not
shown). To demonstrate the expression of the 15-LO-1
protein in L1236 cells and the cellular localization of
the enzyme in the presence and absence of calcium, the
4224

Fig. 2. Western blot and assay of 15-LO-1 activity after subcellular
fractionation of L1236 cells. The cells were washed twice in
NaCl ⁄ Pi without Ca2+ ⁄ Mg2+ and resuspended in 1 mL of NaCl ⁄ Pi
without Ca2+ ⁄ Mg2+. Five million L1236 cells were added to three
Eppendorf tubes and the buffer was changed to: (A) NaCl ⁄ Pi with
Ca2+ ⁄ Mg2+ (0.9 and 0.5 mM, respectively); (B) NaCl ⁄ Pi with
Ca2+ ⁄ Mg2+ plus calcium ionophore A23187 (final concentration
5 lM); and (C) NaCl ⁄ Pi without Ca2+ ⁄ Mg2+. After 10 min of incubation at 37 °C, the samples were homogenized by sonication three
times for 10 s on ice, using a Sonics vibracell VC750 with 30%
amplitude. The cell suspensions were centrifuged for 10 min at
1500 g at 4 °C and the supernatants were transferred to new
tubes for ultracentrifugation (100 000 g at 4 °C) for 1 h. The supernatants were collected and the pellets were resuspended by sonication in the same buffer as used during the incubation. Lower

panel: western blot analysis. An aliquot from each fraction equal to
40 000 cells was loaded on the NuPAGE 4–12% Bis-Tris gradient
gel (1 mm) with running buffer followed by western blotting using
antibodies raised against of 15-LO-1 (see Experimental procedures).
Upper panel: an aliquot of each fraction was incubated with 40 lM
arachidonic acid for 10 min at room temperature. The reaction was
terminated by the addition of three volumes of methanol and the
amounts of 15(S)-HETE and 12(S)-HETE were analysed by
RP-HPLC. The results are the mean ± SD of three separate experiments. P, 100 000 g pellet; S, 100 000 g supernatant.

cells were incubated with NaCl ⁄ Pi with Ca2+ ⁄ Mg2+;
NaCl ⁄ Pi with Ca2+ ⁄ Mg2+ plus calcium ionophore
A23187 (final concentration 5 lm); or calcium-free
NaCl ⁄ Pi. Subsequently, the cells were sonicated
followed by subcellular fractionation. The separate
fractions were analyzed by SDS ⁄ PAGE followed by
western blotting (Fig. 2, lower panel). In the presence
of Ca2+ ⁄ Mg2+, with or without calcium ionophore,
the majority of 15-LO-1 was found in the membrane
fraction, although significant amounts were also
detected in the supernatant fraction. In the absence of

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H.-E. Claesson et al.

Ca2+ ⁄ Mg2+, almost all 15-LO-1 protein was detected
in the supernatant fraction. The translocation of
15-LO-1 to the membrane fraction, upon calcium

challenge, is in accordance with the findings reported
in eosinophils [26]. The activity of 15-LO-1 in the
separate fractions, measured as conversion of arachidonic acid to 15- and 12-HETE (Fig. 2, upper panel),
did not correlate well with the approximated amounts
of 15-LO-1 detected by immunoblotting (Fig. 2, lower
panel). This might be due to the presence of more free
(cytosolic) enzyme in samples incubated without calcium and the cytosolic enzyme converting exogenously
added arachidonic acid to 15-HETE and 12-HETE
more efficiently than the membrane bound enzyme.
Immunostaining of L1236 cells revealed a heterogeneous cell population consisting of small to
medium-sized mononuclear cells, large blasts and
multinucleated giant cells. Immunocytochemical
15-LO-1 staining of non-activated L1236 cells was in
agreement with the western blot results and showed
diffuse strong cytoplasmic staining of 15-LO-1 in both
small cells and in multinuclear large cells (Fig. 3).
Taken together, the results demonstrated that L1236
cells contain abundant amounts of active 15-LO-1.

Hodgkin lymphoma and 15-lipoxygenase

A

B

Formation of eoxins by L1236 cells
To determine whether L1236 cells could produce
eoxins, an acetonitrile based mobile phase was used to
improve separation of peaks with retention times of
approximately 5–8 min (Fig. 1). A typical reverse

phase HPLC chromatogram of products formed by
L1236 cells after 5 min of incubation with arachidonic
acid is shown in Fig. 4. Four peaks (1–4) were
observed in a cluster, all containing a conjugated triene
spectrum and a UV absorbance maximum at 268 nm.
These peaks had elution times corresponding to synthetic standards of the four 8(R,S),15(S)-DiHETE (the
two double oxygenation metabolites and the two
derived from non-enzymatic degradation of EXA4;
also named 14,15-LTA4) [12,19]. The pattern formed
by these metabolites was almost identical to that
reported for human airway epithelial cells and eosinophils incubated with arachidonic acid [11,19]. The
material in peak 5 (Fig. 4) had the same retention time
as 14(R),15(S)-DiHETE. The cells were also found to
produce 5(S),15(S)-DiHETE (data not shown), which
can be converted to the chemotactic metabolite 5-oxo15-ETE [14,27].
The two major peaks in the chromatogram also
possessed a conjugated triene spectrum but had a UV
absorbance maximum at 282 nm (peaks II and IV).
This indicated that these cells also produced eoxins

Fig. 3. Immunocytochemical analysis of 15-LO-1 expression in
L1236 cells. Cytocentrifuged, paraformaldehyde-fixed L1236 cells
were analyzed for 15-LO-1 expression by the avidin-biotin complex
alkaline phosphatase method. (A) L1236 cells were stained with an
antiserum (diluted 1 : 1000) raised against recombinant human
15-LO-1 (red colour). (B) Pre-immune serum (diluted 1 : 1000) was
used as a negative control. Original magnification ·46.

due to the high 15-LO-1 activity in these cells and the
chemical structures of eoxins [11]. The material in

peaks II and IV also coeluted with synthetic EXC4
and EXD4, respectively. Furthermore, the UV spectra
of the materials in peaks II and IV were in agreement
with the UV spectra for eoxins (Fig. 4, inset) [11]. To
further analyze the identity of the materials in peak II
and IV, the cells were incubated with arachidonic acid
for 2 or 10 min followed by analysis with positive ion
LC-MS ⁄ MS. The analysis showed that the material in
peak II and IV had identical MS ⁄ MS spectra to synthetic EXC4 and EXD4 (Fig. 5) [11]. In addition, two
other minor peaks with conjugated triene spectra and
a UV absorbance maximum at 278 nm were observed
(peaks I and III). Metabolite III was analyzed by
positive ion ESI-MS and MS ⁄ MS and the spectrum of

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H.-E. Claesson et al.

Fig. 4. RP-HPLC chromatogram of the products formed by L1236
cells after incubation with arachidonic acid (40 lM) for 5 min. The
numbers 1–4 correspond to the retention times of synthetic standards for the four isomers of 8(S,R),15(S)-DiHETE and number 5
corresponds to 14(R),15(S)-DiHETE, respectively. The material in
peaks I–IV was further analysed by LC-MS ⁄ MS. Inset: UV spectra
for the material in peaks II and IV which also coeluted with
synthetic standards of EXC4 and EXD4, respectively.

Fig. 5. Positive ion LC-MS ⁄ MS spectra of metabolites produced by
L1236 cells after incubation with arachidonic acid. (A) MS ⁄ MS
spectrum of the material corresponding to peak II in Fig. 4, produced by L1236 cells after incubation with arachidonic acid (40 lM)
for 2 min, compared to the spectrum of synthetic EXC4 ([M+H]+
626). (B) MS ⁄ MS spectra of the material corresponding to peak IV
in Fig. 4, produced by L1236 cells after incubation with arachidonic
acid (40 lM) for 10 min, compared to the spectrum of synthetic
EXD4 ([M+H]+ 497).

this metabolite was identical to the spectrum for EXD4
(data not shown). The exact structure of this metabolite has not been established but the MS ⁄ MS spectrum
4226

Fig. 6. RP-HPLC chromatogram of the products formed by L1236
cells after incubation with EXA4 (2 lM) for 30 min. The numbers 1,
3 and 5 correspond to retention times of synthetic standards for
8(R),15(S)-DiHETE, 8(S),15(S)-DiHETE and 14(R),15(S)-DiHETE,
respectively. Peaks I–IV had identical retention times to the peaks
shown in Fig. 4. Inset: UV spectrum of the material in peak V.

and UV profile of this metabolite are consistent with
an all-trans triene structure because the spectrum is
shifted 4 nm hypsochromically compared to the spectrum of EXD4 [28]. The material in peak III is therefore probably 8-trans-EXD4. The material in peak I
also had a conjugated triene spectrum and a UV
absorbance maximum at 278 nm but the amount of
the material was insufficient to obtain an interpretable
ESI-MS ⁄ MS spectrum. In line with the formation of
8-trans-EXD4, it is likely, however, that this metabolite
is 8-trans-EXC4. Furthermore, incubation of the cells

with synthetic EXC4 and EXD4 led to the formation
of metabolites I and III, respectively, in agreement
with the postulated all-trans trienes, formed from the
parent EXC4 and EXD4, respectively (data not
shown).
To determine the mechanism of formation of EXC4
and EXD4, as well as the putative formation of EXE4,
the cells were incubated with synthetic 14,15-epoxy5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acid (EXA4) for
30 min. Figure 6 shows that EXA4 was converted to
EXC4 (peak II) and EXD4 (peak IV). In addition, the
material in peak V also contained a conjugated triene
and had a UV absorbance maximum at 282 nm. This
metabolite was not detected when the cells were incubated with arachidonic acid because the material in
this peak V coeluted on HPLC with the double dioxygenation product 8(S),15(S)-DiHETE (Fig. 4). This
product is not formed from EXA4 and metabolite V
was now visible on the HPLC chromatogram and
no longer hidden behind the double dioxygenation
product. Metabolite V was also formed when the cells

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H.-E. Claesson et al.

Hodgkin lymphoma and 15-lipoxygenase

Fig. 7. Positive ion LC-MS ⁄ MS spectrum of a metabolite produced
by L1236 cells after incubation with arachidonic acid for 30 min.
Positive ion LC-MS ⁄ MS spectrum of the material corresponding to
peak V in Fig. 6, formed by L1236 cells after 30 min of incubation

with arachidonic acid (40 lM). The lower panel shows the MS ⁄ MS
spectrum of authentic EXE4 ([M+H]+ 440).

were incubated with synthetic EXD4 for 30 min (data
not shown). Therefore, L1236 cells were incubated with
arachidonic acid for 30 min followed by analysis with
LC-MS ⁄ MS. Figure 7 demonstrates that the MS ⁄ MS
spectrum corresponding to peak V contains the characteristic 205 m ⁄ z fragment ion of eoxins and is identical
to synthetic 14(R)-cysteinyl-15(S)-hydroxy-5,8,10,12(Z,Z,E,E)-eicosatetraenoic acid (EXE4). Metabolite V
also had an identical retention time as synthetic EXE4
on HPLC. In addition, the materials in peaks II, IV and
V were separately collected and analyzed by positive ion
ESI-MS ⁄ MS with a triple quadrupole mass spectrometer, as well as by negative ion ESI-MS ⁄ MS using a
hybrid magnetic sector ⁄ TOF instrument. These analyses
also demonstrated that the materials in peaks II, IV and
V were EXC4, EXD4 and EXE4, respectively (data not
shown). Taken together, these results show that L1236
cells convert arachidonic acid to EXC4, which is readily
converted to EXD4 and EXE4.
The time courses of the formation of EXC4 and
EXD4 after incubation of L1236 cells with arachidonic
acid are shown in Fig. 8A. A substantial amount of
EXC4 (37 pmol per 106 cells) was produced already by
10 · 106 cells after 30 s of incubation with arachidonic
acid (40 lm). The maximal level of EXC4 was observed
after 5 min of incubation and, subsequently, the level
of EXC4 declined with time. Significant amounts of
EXD4 were observed after 2 min of incubation with
arachidonic acid and the levels increased with time,
reaching a maximal level after 30 min. It was not possible to measure the level of EXE4 in this experiment

because 8(S),15(S)-DHETE co-chromatographed with
EXE4 in this HPLC system. The dose–response curves
of the formation of eoxins from arachidonic acid show
that significant amounts of EXC4 and EXD4 were
formed already at a concentration of 1 lm arachidonic
acid (Fig. 8B). The levels of these metabolites

Fig. 8. Time course (A) and dose–response (B) curves of the
formation of EXC4 and EXD4 by L1236 cells after incubation with
arachidonic acid. (A) The concentration of arachidonic acid was
40 lM. (B) Five minutes of incubation with the indicated concentration of arachidonic acid. One typical experiment out of three is
shown. d, EXC4; s, EXD4.

increased with the concentration of arachidonic acid
and a plateau was reached at a concentration of 40 lm
arachidonic acid.
Expression of 15-LO-1 in tumour biopsies from
HL and non-Hodgkin (NHL) patients
To determine whether H-RS cells also expressed
15-LO-1 in vivo, diagnostic biopsies from HL lymph
nodes were stained with an antibody raised against
15-LO-1. In most HL tumours, there was a distinct
cytoplasmic positivity for 15-LO-1 in tissue macrophages and a strong staining in eosinophils. In 17 of
20 tumours, 15-LO-1 expression could also be detected
in H-RS cells (Table 1). Figure 9A shows a typical
H-RS cell, strongly stained by an antibody raised
against human recombinant 15-LO-1. No staining was

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Table 1. Patient and tumor characteristics. The degree of eosinophilia and the number of H-RS cells in HL tumors were determined by random selection of 10 consecutive HPFs. In each HPF, the number of eosinophils and H-RS cells was determined and the sum of ten HPF
was calculated. The biopsies were then classified as low eosinophilia (< 50 eosinophils per 10 HPF), medium eosinophilia (50–120 eosinophils per 10 HPF) or high eosinophilia (> 120 eosinophils per 10 HPF). The number of H-RS cells were classified as few (< 5 H-RS cells per 10
HPFs), medium (5–10 H-RS cells per 10 HPFs) or many (> 10 H-RS cells per 10 HPFs). Macrophages and eosinophils were used as internal
positive controls for 15-LO-1 staining. ), no H ⁄ RS cells expressed 15-LO-1; +, < 20% of the H ⁄ RS cells expressed 15-LO-1; ++, > 20% of
the H ⁄ RS cells expressed 15-LO-1; +++, the majority of the H ⁄ RS cells strongly expressed 15-LO-1. PTCL, peripheral T-cell lymphoma;
DLBCL, diffuse large B-cell lymphoma; MALT, mucosa-associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; B-CLL, B-cell
chronic lymphocytic leukemia; BL, Burkitt lymphoma; FCDL, follicular centre derived lymphoma; PMBCL, primary mediastinal B-cell
lymphoma; NA, not applicable; ND, not determined.
Patient
number
HL
1
2
3
4
5
6
7
8
9
10
11
12

13
14
15
16
17
18
19
20
NHL
21
22
23
24
25
26
27
28
29
30

Sex

Age
(years)

Clinical
stage

Tumor

EBV
status

H-RS
cells

Eosinophilia

15-LO-1
expression

Male
Female
Male
Female
Female
Female
Male
Female
Male
Female
Male
Male
Male
Male
Male
Male
Female
Female
Female

Male

66
45
27
25
61
14
13
28
19
31
89
46
24
23
8
36
5
77
70
37

IIIA
IVA
IVB
IIA
IIIA
IIIA
IIB

IIB
IIB
IIIB
IA
IA
IVB
IIB
IIA
IIIB
IIA
IA
IA
IIIB

HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL
HL

HL
HL
HL
HL

ND
ND
Pos
Neg
Neg
Neg
Neg
ND
ND
Neg
Pos
ND
ND
ND
Pos
Pos
Pos
ND
Neg
Neg

Few
Medium
Few
Medium

High
Medium
Medium
Many
Many
Many
Few
Many
Many
Many
Medium
Few
Many
Medium
Medium
High

Low
Medium
Low
Low
Low
High
High
High
High
High
Low
High
High

Medium
High
Low
High
Low
Medium
High

++
++
)
++
++
++
)
++
++
++
+
+
+++
++
+
)
+
+
+
++

Female

Male
Female
Female
Female
Male
Male
Male
Female
Male

39
80
60
54
75
81
63
32
72
28

IIA
IIA
IVB
IA
IVB
IVA
Rai IV
IIA
IVA

IVB

PTCL
DLBCL
DLBCL
MALT
Immunocytoma
MCL
B-CLL
BL
FCDL
PMBCL

ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

NA
NA
NA
NA
NA
NA

NA
NA
NA
NA

ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

)
)
)
)
)
)
)
)
)
)

NSI
NSI
NSI

NSI
NSI
NSI
NSI
NSII
NSII
NSII
MC
MC
MC
MC
MC
MC
MC
MC
MC
MC

observed with the pre-immune sera (Fig. 9B). The
strongest staining was noted in biopsies from three
cases of nodular sclerosis (NS) subtype II HL and in
one case of mixed cellularity (MC) HL, all with
marked high eosinophilia. However, there were also
cases with high eosinophilia and weak staining of
15-LO-1 in H-RS cells (Table 1). By contrast, no staining of 15-LO-1 was observed in tumour biopsies from
ten patients with nine different subtypes of NHL
(Table 1). We have not yet, however, examined the
expression of 15-LO-1 in all different entities of human
4228

lymphoma. Thus, H-RS cells express 15-LO-1 in vivo
and it is therefore very likely that these cells can
produce eoxins and other 15-LO-1 derived metabolites
in vivo, contributing to the inflammatory features of
HL.

Discussion
The present study shows that the cHL-derived cell line
L1236 possesses high 15-LO-1 activity and readily
converts arachidonic acid to eoxins, a recently

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H.-E. Claesson et al.

Hodgkin lymphoma and 15-lipoxygenase

A

B

Fig. 9. Immunohistochemical analysis of 15-LO-1 expression by primary H-RS cells. Paraffin sections from formalin-fixed HL tumours
were stained with 15-LO-1 antibody (diluted 1 : 1000), raised
against recombinant 15-LO-1, using the avidin-biotin complex alkaline phosphatase method. (A) HL biopsy of NS subtype with a typical 15-LO-1 positive H-RS cell in the middle (red colour),
surrounded of by several macrophages positive for 15-LO-1 (red
colour). (B) Pre-immune serum (diluted 1 : 1000) was used as a
negative control. Original magnification ·46.

identified group of proinflammatory cysteinyl-containing arachidonic acid metabolites produced by human

eosinophils and mast cells [11]. The structures of the
metabolites produced by L1236 cells were determined
by positive ion LC-MS ⁄ MS. The interpretation of the
spectra showed that the structure of these metabolites
corresponded to EXC4, EXD4 and EXE4 (Figs 5 and
7). These metabolites also co-chromatographed with
synthetic EXC4, EXD4 and EXE4 on RP-HPLC,
respectively. In addition, two minor products were
formed which were postulated to be 8-trans-EXC4 and
8-trans-EXD4. Furthermore, the cells produced a series
of 8(R,S),15(S)-DiHETEs and 14(R),15(S)-DiHETE
(Fig. 4). The configurations of the double bonds in
EXC4, EXD4 and EXE4 were not determined with

Fig. 10. Overview of the metabolic pathway for the formation of
eoxins in L1236 cells.

NMR in the present study. However, because
these metabolites were also formed from synthetic
14,15-epoxy-5,8,10,12 (Z,Z,E,E)-eicosatetraenoic acid
(EXA4) and had identical retention times on HPLC
as the corresponding synthetic metabolite (Figs 4 and
6), the structures of these metabolites are likely to
be 14(R)-glutathionyl-15(S)-hydroxy-5,8,10,12-(Z,Z,E,
E)-eicosatetraenoic acid (EXC4), 14(R)-cysteinyl-glycyl15(S)-hydroxy-5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acid
(EXD4) and 14(R)-cysteinyl-15(S)-hydroxy-5,8,10,12(Z,Z,E,E)-eicosatetraenoic acid (EXE4), respectively.
Taken together, the UV profile and mass spectra show
that L1236 cells can metabolize arachidonic acid to
EXA4 which in turn can be readily converted to
EXC4, EXD4 and EXE4 (Fig. 10). Eoxin C4 was more

rapidly converted to EXD4 and EXE4 in L1236 cells
than in human eosinophils [11], indicating that L1236

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H.-E. Claesson et al.

cells express higher amounts of c-glutamyltransferase
and dipeptidase, which probably catalyse the
conversion of EXC4 to EXD4 and EXD4 to EXE4,
respectively.
Western blot analysis demonstrated that 15-LO-1
was mainly cytosolic under calcium-free conditions
and in non-activated cells (Figs 2 and 3). The enzyme
was, however, mainly located to the cell membrane in
the presence of calcium (Fig. 2). There was higher
15-LO-1 activity in the cytosolic fraction, although
western blot analysis demonstrated a higher amount of
15-LO-1 protein in the membrane fraction. This was
an unexpected finding because membrane bound
15-LO-1 has been found to possesses higher activity
than the cytosolic enzyme [26,29,30] This might be due
to the orientation of the enzyme in the membrane of
L1236 cells and the exogenous addition of arachidonic
acid reaching the active site of the cytosolic enzyme to

a greater extent than that of the membrane enzyme in
L1236 cells [31]. Alternatively, the membrane associated enzyme in L1236 cells undergoes suicidal inactivation during the oxygenation of membrane lipids. In
addition, 15-LO-1 might be inactivated by 15-HPETE,
which is probably rapidly inactivated in the cytosol,
whereas 15-HPETE generated by membrane bound
15-LO may persist and inactivate the membrane-bound
enzyme.
Immunohistochemistry analysis of diagnostic HL
lymph node biopsies demonstrated the expression of
15-LO-1 in H-RS cells in 17 of the 20 (85%) examined
biopsies (Table 1). By contrast, 15-LO-1 was not
expressed in biopsies derived from ten patients representing nine different entities of NHL (Table 1). Thus,
the expression of 15-LO-1 might be a useful biomarker
to distinguish HL from NHL. Extensive studies on the
expression of 15-LO-1 in biopsies derived from various
subtypes of NHL are ongoing.
Of all the HL cell lines investigated, the L1236 cell
line was the only one that expressed 15-LO-1 like
primary H-RS cells in vivo. Among all the established
HL cell lines, however, only the L1236 cell line is clonally related to original tumour tissue [32,33]. The transcription of 15-LO-1 is dependent on the transcription
factor STAT6 [6,23]. Through autocrine IL-13 stimulation, several HL cell lines, such as L1236 and L428,
possess constitutively activated STAT6 [34,35]. Studies
are now ongoing to clarify whether the epigenetic
status of the 15-LO-1 promoter region in L1236 cells,
which express 15-LO-1, and L428 cells, which do not
express 15-LO-1, is different because only L1236
express 15-LO-1.
The expression of 15-LO-1 and putative formation
of eoxins by H-RS cells in vivo might contribute to the
4230

inflammatory features of HL. The eoxins induce
increased vascular permeability and 5-oxo-15-hydroxyETE is chemotactic for eosinophils, which infiltrate
many HL tumours [2]. In addition, H-RS cells can
produce 15-HETE and this metabolite can exert either
proinflammatory or anti-inflammatory effects in various model systems [6,12]. Thus, H-RS cells in vivo have
the putative capacity to produce biological active arachidonic acid metabolites that might play an important
role in the pathophysiology of HL.
The formation of the giant H-RS cells in HL is still
a mystery although many attempts have been made to
clarify the formation of these cells. The enzyme
15-LO-1 has been proposed to induce breakdown of
mitochondria membranes during the differentiation of
reticulocytes to erythrocytes and to degrade intracellular membranes during the differentiation of keratinocytes and the eye lens [6,17]. It is therefore tempting to
speculate that 15-LO-1 might be involved in the formation of the giant H-RS cells through remodelling of
intracellular membranes.
A cell line with high 15-LO-1 activity has been
sought subsequent to the discovery of this enzyme
more than 30 years ago. The identification of 15-LO-1
in L1236 cells, which possess high 15-LO-1 activity
without addition of exogenous IL-4 or IL-13, opens up
great possibilities for all researchers studying the function of 15-LO-1.
In summary, the present study demonstrates that the
HL cell line L1236 produces eoxins and that primary
H-RS cells in vivo express 15-LO-1 and also have the
putative capacity to produce eoxins that might contribute to the inflammatory features of HL. These findings
may have important diagnostic and therapeutic implications.

Experimental procedures
Materials

Synthetic 14,15-epoxy-5,8,10,12-(Z,Z,E,E)-eicosatraenoic acid
(EXA4), 14(R)-glutathionyl-15(S)-hydroxy-5,8,10,12-(Z,Z,
E,E)-eicosatetraenoic acid (EXC4), 14(R)-cysteinyl-glycyl-15
(S)-hydroxy-5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acid (EXD4),
14(R)-cysteinyl-15(S)-hydroxy-5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acid (EXE4), 8(R),15(S)-dihydroxy-5,9,11,13-(Z,E,
E,E)-eicosatetraenoic acid [8(R),15(S)-DiHETE], 8(S),15(S)dihydroxy-5,9,11,13-(Z,E,E,E)-eicosatetraenoic acid [8(S),
15(S)-DiHETE], 8(R),15(S)-dihydroxy-5,9,11,13-(Z,E,Z,E)eicosatetraenoic acid [8(R),15(S)-DiHETE], 8(S),15(S)-dihydroxy-5,9,11,13-(Z,E,Z,E)-eicosatetraenoic acid [8(S),15
(S)-DiHETE], 14(R),15(S)-dihydroxy-5,8,10,12-(Z,Z,E,E)eicosatetraenoic acid [14(R),15(S)-DiHETE], 15(S)-hydroxy-5,

FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS


H.-E. Claesson et al.

8,11,13-(Z,Z,Z,E)-eicosatetraenoic
acid
[15(S)-HETE],
12(S)-hydroxy-5,8,10,14-(Z,Z,E,Z) eicosatetraenoic acid
[12(S)-HETE] and 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid [13(S)-HODE] were from Biomol Inc. (Plymouth
Meeting, PA, USA). Cell culture medium RPMI 1640, fetal
bovine serum, penicillin, streptomycin and glutamine were
obtained from Gibco BRL (Gaithersburg, MD, USA).
HPLC solvents were obtained from Rathburn Chemicals
(Walkerburn, UK). Complete mini EDTA-free protease
inhibitor cocktail was purchased from Roche (Indianapolis,
IN, USA).

Cell lines, biopsies and patients
The human HL cell lines L1236, HDLM2, KMH2 and
L428 (kind gifts from V. Diehl, Department of Internal

Medicine, University Hospital of Cologne, Germany) were
cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum, l-glutamine (2 mm),
penicillin (100 mL)1) and streptomycin (100 lgỈmL)1)
(Gibco, Paisley, Scotland, UK) at 37 °C in an atmosphere
containing 5% CO2. The examined cell lines were of B-cell
phenotype (L1236, L428 and KMH2) and T-cell phenotype
(HDLM2), and derived from cHL of MC (L1236 and
KMH2) and NS (L428 and HDLM2) subtypes, respectively. All cell lines were negative for Epstein–Barr virus
(EBV). Diagnostic HL-involved lymph node biopsies were
collected from 1994 to 2004 at the Department of Pathology and Cytology, Karolinska University Hospital Solna,
Stockholm, Sweden. Routine morphological and immunohistochemical stainings of tumour biopsises were performed
on paraffin sections. EBV expression was investigated by
immunohistochemistry (latent membrane protein 1) and
in situ hybridization (EBV encoded RNA) as described
previously [36].
The degree of eosinophilia and the number of H-RS cells
were determined by random selection of ten consecutive
high power fields (HPFs) in hematoxylin and eosin stained
paraffin sections. In each HPF, the number of eosinophils
or H-RS cells was determined and the sum of ten HPF was
calculated. The biopsies were then classified as low eosinophilia (< 50 eosinophils per ten HPF), medium eosinophilia (50–120 eosinophils per 10 HPF) or high eosinophilia
(> 120 eosinophils per ten HPF). The number of H-RS
cells were classified as few (< 5 H-RS cells per ten HPF),
medium (5–10 H-RS cells per ten HPF) or many (> 10
H-RS cells per ten HPF). This study was approved by the
local ethic committee of Karolinska University Hospital.

RT-PCR
Total RNA was extracted from L1236 cells using RNeasy

Mini kit (Qiagen GmbH, Hilden, Germany). One microgram of RNA was reverse-transcribed with reverse transcriptase and random hexamers. For full length 15-LO-1

Hodgkin lymphoma and 15-lipoxygenase

amplification forward 5¢-GAA GTT ATC AGT CGA CAT
GGG TCT CTA CCG CA-3¢ and reverse 5¢-ATG GTC
TAG AAA GCT TTT AGA TGG CCA CAC TGT TTT
CCA CCA C-3¢ primers were used. For partial 15-LO-2
amplification, forward 5¢-AAT CTC GGC AAGGAG TTC
ACT-3¢ and reverse 5¢-AGT CAA ACT GCC CTG CAC
T-3¢ primers were used. b2-microglobulin was included as
an internal control for RT efficiency and RNA integrity.
AmpliTaq GoldÒDNA Polymerase (Applied Biosystems,
Foster City, CA, USA) was used for DNA amplification.
The PCR conditions were: 95 °C for 10 min, 95 °C for
15 s, 58 °C for 1 min, 72 °C for 2 min and 72 °C for
6 min. b2-microglobulin was run for 24 cycles and 15-LO-1
and 15-LO-2 for 34 cycles.

Translocation assay and western blot
L1236 cells were washed twice in NaCl ⁄ Pi without
Ca2+ ⁄ Mg2+and resuspended in 1 mL of NaCl ⁄ Pi without
Ca2+ ⁄ Mg2+. Five million L1236 cells were added to three
Eppendorf tubes and the buffer was changed by spinning
down the cells for 5 min at 600 g, removing the supernatant
and resuspending the cells in respective buffers
[buffer A: NaCl ⁄ Pi with Ca2+ ⁄ Mg2+ (0.9 and 0.5 mm,
respectively); buffer B: NaCl ⁄ Pi with Ca2+ ⁄ Mg2+ plus calcium ionophore A23187 (final concentration 5 lm); and
buffer C: NaCl ⁄ Pi without Ca2+ ⁄ Mg2+]. One tablet complete mini protease inhibitor without EDTA was added to
10 mL of the respective buffers. After 10 min of incubation

at 37 °C, the samples were homogenized by sonication,
using a Sonics vibracell VC750 (Chemical Instruments AB,
Lidingo, Sweden) with 30% amplitude, three times for 10 s
ă
on ice. The homogenate was centrifuged for 10 min (1500 g
at 4 °C) and the supernatant was transferred to new tubes
for ultracentrifugation, at 100 000 g at 4 °C for 1 h. The
supernatant was collected and the pellet was resuspended by
sonication in the same buffer as used during the incubation.
Western blot analysis was performed according to the
NuPAGE reduced sample protocol (Invitrogen, Carlsbad,
CA, USA). An aliquot from each fraction equal to 40 000
cells was loaded on the NuPAGE 4–12% Bis-Tris gradient
gel (1 mm) with running buffer. After transfer to a polyvinylidene difluoride membrane, it was blocked in 5% milk
powder in NaCl ⁄ Pi-Tween at room temperature for 1 h.
The membrane was then incubated overnight at 4 °C with
a 15-LO-1 rabbit peptide antiserum (batch 632) raised
against the 15-LO-1 peptide CALDKEIEIRNAKLD
MPYEY (dilution 1 : 5000) (antibodies made by Innovagen
AB, Ideon, Lund, Sweden) in NaCl ⁄ Pi-Tween + 1% milk
powder. These antibodies did not detect human 5-LO,
platelet 12-LO or 15-LO-2 (data not shown). After washing
three times in NaCl ⁄ Pi-Tween, the membrane was incubated with the anti-rabbit horseradish peroxidase serum,
diluted 1 : 10000 in NaCl ⁄ Pi-Tween + 1% milk powder for
5 h at room temperature. The membrane was then

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4231

Hodgkin lymphoma and 15-lipoxygenase

H.-E. Claesson et al.

washed three times in NaCl ⁄ Pi-Tween before detection with
ECL-plus. Recombinant human 15-LO-1 was expressed in
Sf 9 cells and used as standard [29].

Experimental conditions and HPLC analysis
The cell suspensions were centrifuged at 200 g for 5 min,
suspended in NaCl ⁄ Pi and washed twice with NaCl ⁄ Pi
(37 °C) and subsequently solubilized in NaCl ⁄ Pi at a
concentration of 10–20 · 106 cellsỈmL)1. The sample was
pre-warmed for 2 min prior to addition of arachidonic acid,
EXA4 or other indicated metabolites and subsequently
incubated for the period indicated. The incubations were
terminated by addition of three volumes of ice-cold methanol. Precipitated proteins and cell fragments were removed
by centrifugation and excess water and methanol were
evaporated under reduced pressure. The residues were
solubilized in methanol and transferred to a test tube and
evaporated to dryness under a stream of nitrogen. Subsequently, the residues were reconstituted in the appropriate
chromatographic solvent described below. The samples
were applied to an octadecyl reverse-phase column (NovaPak C18 4 lm, 150 mm, Waters AB, Sollentuna, Sweden or
Chromabond, C18, 100 mm, Macherey-Nagel, Duren,
ă
Germany for monohydroxy acids and cysteinyl-containing
arachidonic acid metabolites, respectively), and eluted
isocratically at a flow rate of 0.4 mLỈmin)1. The mobile
phase was methanol ⁄ water ⁄ triflouroacetic acid (69 : 31 :

0.07, v ⁄ v ⁄ v) or acetonitrile ⁄ methanol ⁄ water ⁄ acetic acid
(28 : 18 : 54:1, v ⁄ v ⁄ v, pH adjusted to 5.6 with NH3) for
analysis of monohydroxy acids or cysteinyl-containing
arachidonic acid metabolites and dihydroxy acids, respectively [11]. Eluted metabolites were detected and quantified
utilizing a programmable Waters 991 diode array spectrophotometer connected to the HPLC system.

LC-MS ⁄ MS
L1236 cells were incubated at 37 °C with exogenous arachidonic acid (40 lm) for the indicated time. The incubations
were terminated by adding one volume of methanol.
Samples were centrifuged (1400 g for 6 min) and the supernatants were diluted with water to a maximum of 25%
methanol and then transferred to a washed and equilibrated
extraction cartridge (Oasis HLB 1 mL, 10 mg; Waters AB).
The columns were washed with water and eluted with
200 lL of methanol to retrieve the metabolites. The
samples were analyzed by LC-MS ⁄ MS on a Surveyor MS
coupled to a TSQ Quantum Ultra triple quadrupole mass
spectrometer (Thermo Finnigan AB, Stockholm, Sweden).
Half a volume of water was added to samples prior to injection. RP-LC was performed using a Sunfire 3.5 lm C18
column 2.1 · 30 mm (Waters AB) with the flow rate
constantly held at 400 lLỈmin)1. Mobile phase A consisted
of 2% acetonitrile in water and 0.1% acetic acid, and

4232

mobile phase B consisted of 80% acetonitrile in water and
0.1% acetic acid. All water used was of MilliQ grade (Millipore, Billerica, MA, USA). Starting isocratically for
1.5 min at 90% A was followed by a 6.5 min linear gradient
reaching 100% of B. The system was washed at 100% B for
4 min and subsequently equilibrated at 90% A for 4 min.
The mass spectrometer was operated using an electrospray atmospheric pressure ionization source in positive

mode. The spray voltage was set to 4500 V, the capillary
temperature was 375 °C and sheath and auxiliary gas was
optimal at 60 and 5 arbitrary units, respectively. Skimmer
offset was at 10 V and tube lens was 99 V. MS ⁄ MS product ions of 626, 497 and 440 m ⁄ z, corresponding to the
[M+H]+ of EXC4, EXD4 and EXE4, were recorded.
Collision energy was set to 21 for MS ⁄ MS at 626 m ⁄ z and
to 18 for MS ⁄ MS at both 497 and 440 m ⁄ z. The metabolite
spectra were compared with those of synthetic standards.

Immunostaining
Formalin-fixed and paraffin embedded lymph node biopsies
were obtained for diagnostic purposes and used to study
15-LO-1 expression by the avidin-biotin complex alkaline
phosphatase method [37]. The tumours studied were cHL
NS (n = 10), cHL MC (n = 10) and ten NHL including
nine different entities (Table 1) according to the WHO classification [38]. A polyclonal 15-LO-1 antibody was raised in
rabbit against recombinant human 15-LO-1 (the enzyme
was expressed in Sf9 cells in house and purified prior to
immunization, > 95% pure enzyme preparation was used).
This antiserum (made by Innovagen AB) did not detect
human 5-LO, platelet 12-LO or 15-LO-2 (data not shown).
Briefly, antigen retrieval was performed by boiling deparaffinized and rehydrated tissue sections in citrate buffer
(pH 6.0) in a microwave oven for 20 min. The sections were
incubated with 15-LO-1 antibody (1 : 1000), for 1 h at
room temperature. Subsequently, the biopsies were incubated with biotinylated goat anti-(rabbit IgG) and alkaline
phosphatase conjugated avidin-biotin complex. VectorÒ
Red Alkaline Phosphatase Substrate (Vector Laboratories,
Burlingame, CA, USA) was used for 15-LO-1 visualization.
Endogenous alkaline phosphatase activity was inhibited by
levamisole (Vector Laboratories). Macrophages and eosinophils were used as internal positive controls for 15-LO-1

staining.
For immunocytochemistry, L1236 cells were resuspended
in NaCl ⁄ Pi with 10% fetal bovine serum at a final concentration of 1 · 106 cellsỈmL)1. The cells were cytocentrifuged,
fixed in 4% paraformaldehyde for 10 min and washed in
NaCl ⁄ Pi. The 15-LO-1 staining was performed as described
above, with the exceptions of deparaffinization, rehydration
and antigen retrieval. Pre-immune serum (1 : 1000) was
used as a negative control. For analysis, a microscope
(BX60, Olympus, Tokyo, Japan) equipped with a digital
camera (DKC-5000, Sony, Tokyo, Japan) was used.

FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS


H.-E. Claesson et al.

Hodgkin lymphoma and 15-lipoxygenase

Acknowledgements
We wish to thank Dr Mats Hamberg for help with the
chiral chromatography analysis. This work was
supported by grants from the Swedish Cancer Society,
Karolinska Institutet, Stockholm County Council,
Orexo AB and European Commission Sixth Framework Programme Grant LSHM-CT-2004-005033.

12

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FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS