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 Griffiths3, Asa Brunnstrom2, Frida Schain4, Erik Andersson2,4, ă 1,2 ` Stina Feltenmark , Helene A Johnson , Anna Porwit5, Jan Sjoberg4 and Magnus Bjorkholm4 ă ă 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: +46 324 264 Tel: +46 524 87627 E-mail: hans-erik.claesson@ki.se (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 4222 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 and [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 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 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 4223 Hodgkin lymphoma and 15-lipoxygenase H.-E Claesson et al Fig RP-HPLC chromatograms of the products formed by L1236 cells after incubation with arachidonic acid (40 lM) for 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 : 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 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 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 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 lM); and (C) NaCl ⁄ Pi without Ca2+ ⁄ Mg2+ After 10 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 at 1500 g at °C and the supernatants were transferred to new tubes for ultracentrifugation (100 000 g at °C) for 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 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 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 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 (Fig 1) A typical reverse phase HPLC chromatogram of products formed by L1236 cells after of incubation with arachidonic acid is shown in Fig 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 (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 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 : 1000) raised against recombinant human 15-LO-1 (red colour) (B) Pre-immune serum (diluted : 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 or 10 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 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 4225 Hodgkin lymphoma and 15-lipoxygenase H.-E Claesson et al Fig RP-HPLC chromatogram of the products formed by L1236 cells after incubation with arachidonic acid (40 lM) for 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 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 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 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 RP-HPLC chromatogram of the products formed by L1236 cells after incubation with EXA4 (2 lM) for 30 The numbers 1, and 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 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 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 Figure 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 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 Fig Positive ion LC-MS ⁄ MS spectrum of a metabolite produced by L1236 cells after incubation with arachidonic acid for 30 Positive ion LC-MS ⁄ MS spectrum of the material corresponding to peak V in Fig 6, formed by L1236 cells after 30 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 (data not shown) Therefore, L1236 cells were incubated with arachidonic acid for 30 followed by analysis with LC-MS ⁄ MS Figure 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 of incubation and, subsequently, the level of EXC4 declined with time Significant amounts of EXD4 were observed after of incubation with arachidonic acid and the levels increased with time, reaching a maximal level after 30 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 lm arachidonic acid (Fig 8B) The levels of these metabolites Fig 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 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 4227 Hodgkin lymphoma and 15-lipoxygenase H.-E Claesson et al Table 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 (< 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 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 36 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 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 A B Fig 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 : 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 : 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 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 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 FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 4229 Hodgkin lymphoma and 15-lipoxygenase 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 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 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 (< 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 min, 72 °C for and 72 °C for 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 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 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 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 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 (1500 g at °C) and the supernatant was transferred to new tubes for ultracentrifugation, at 100 000 g at °C for 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 h The membrane was then incubated overnight at °C with a 15-LO-1 rabbit peptide antiserum (batch 632) raised against the 15-LO-1 peptide CALDKEIEIRNAKLD MPYEY (dilution : 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 : 10000 in NaCl ⁄ Pi-Tween + 1% milk powder for h at room temperature The membrane was then FEBS Journal 275 (2008) 4222–4234 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 cells and used as standard [29] Experimental conditions and HPLC analysis The cell suspensions were centrifuged at 200 g for 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 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 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 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 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 at 90% A was followed by a 6.5 linear gradient reaching 100% of B The system was washed at 100% B for and subsequently equilibrated at 90% A for 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 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 The sections were incubated with 15-LO-1 antibody (1 : 1000), for 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 · 106 cellsỈmL)1 The cells were cytocentrifuged, fixed in 4% paraformaldehyde for 10 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 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compilation ª 2008 FEBS ... that these in? ??ltrating cells are involved in an in? ??ammatory ⁄ reactive process creating an environment that allows, and probably promotes, the survival of H-RS cells [2] Cytokines and chemokines... 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. .. 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