High expression of 5-lipoxygenase in normal and malignant mantle zone B lymphocytes

RT-PCR was performed on isolated total RNA from subsets of tonsillary B cells to elucidate the gene expression of enzymes involved in the leukotriene cascade. These analyses demonstrated that 5-LO, FLAP and LTA₄ hydrolase were expressed in various degrees in tonsillary B cells (CD19⁺), memory B cells (CD19⁺, CD38⁺, IgD^-), mantle zone B cells (CD19⁺, CD38⁺, IgD⁺) and germinal centre (GC) B cells (CD19⁺, CD38⁺⁺, IgD^-). Highest relative expression of these three genes was found in mantle zone B cells and lowest expression in GC B cells (Figure 1). These results indicate differential expression of these enzymes in subpopulations of B cells. No investigated subpopulation of B cells expressed LTC₄ synthase.

Subpopulations of B cells from tonsils were separated by flow cytometry and analyzed by western blot in order to further characterize the expression of 5-LO. Whole cell lysates of purified cells were submitted to SDS/PAGE followed by western blotting using a polyclonal anti-human 5-LO antibody. A single immunoreactive band at the expected size was observed in the sample consisting of total B cells (CD19⁺) from tonsils, mantle zone B cells (CD19⁺, CD38⁺, IgD⁺) and in memory B cells (CD19⁺, CD38⁺, IgD^-) and a weak band was detected in GC B cells (CD19⁺, CD38⁺⁺, IgD^-) (Figure 2). In contrast, no band was detected in samples from plasma cells (CD19⁺, CD38⁺⁺⁺, IgD^-). There was a more marked difference in the expression of the 5-LO protein than in the 5-LO mRNA levels in the various subtypes of B cells (Figures 1 and 2).

To identify the expression of 5-LO protein in subpopulations of tissue tonsillar B cells, a panel of antibodies were used to identify mantle zone B cells (IgD⁺), germinal centre B cells (CD38⁺⁺) and plasma cells (CD38⁺⁺⁺ or CD138⁺). Figure 3A shows high expression of 5-LO in mantle zone B cells (IgD⁺). In contrast, a very weak expression of 5-LO was observed in GC cells (CD38⁺⁺) (Figure 3B). Figure 3C demonstrates no expression of 5-LO in plasma cells (CD38⁺⁺⁺) within the GC. Furthermore, no expression of 5-LO was observed in plasma cells (CD138⁺) outside the GC (Figure 3D). These results concur with the western blot analysis of 5-LO in different subsets of B cells (Figure 2).

In order to further characterize the expression of 5-LO in subsets of B cells, we investigated the expression of 5-LO in malignant cells derived from the mantle zone and the germinal centre i.e. mantle B cell lymphoma and follicular lymphoma. For comparison, lymph node biopsies were obtained from patients with reactive lymph nodes. In agreement with the data shown in figure 3, the expression of 5-LO in a reactive lymph node was mainly found in B cells from the mantle zone rather than from the germinal centre (Figure 4A). Interestingly, virtually all B cells expressed 5-LO in a biopsy obtained from a patient with MCL (Figure 4B). In contrast, corresponding analyses of biopsies from patients with follicular lymphoma, demonstrated that very few cells expressed 5-LO. These cells are most likely dendritic cells or tangible body macrophages as described earlier [27] (Figure 4C). No staining was observed with control antibody (Figure 4D). Similar results were obtained with another 5-LO antiserum (data not shown). Taken together, all investigated biopsies from patients with mantle zone B cell lymphoma clearly expressed 5-LO, whereas, little or none staining was observed in biopsies from patients with follicular lymphoma (Table 1).

As a comparison, tonsillar polymorphonuclear granulocytes expressed 5-LO in the nucleus (Figure 4E), in contrast to tonsillar CD3 positive T lymphocytes which did not express 5-LO (Figures 4F and 4G). The IHC in figure 4G was stained with human anti-CD3 antibody.

Since it was difficult to isolate large numbers of tonsillary non-malignant mantle zone B cells for chemical analysis, we used MCL cells to study the biosynthesis of LTB₄ in mantle zone B cells. For this purpose, we used both different MCL cell lines and primary MCL cells isolated from the peripheral blood of patients with leukemized MCL, so called prolymphocytic leukemia (B-PLL) with t(11;14). B-PLL is in fact a heterogenous disease and those cases that are positive for cyclin D1 and carry the t(11:14) translocation are in the current WHO classification of malignant lymphomas [22] considered to be leukemic forms of MCL [28, 29]. All three investigated MCL cell lines (Granta 519, JEKO-1 and Rec1) expressed 5-LO protein (Figure 5). Figure 6 depicts the capacity of B-PLL cells and MCL cell lines to produce leukotrienes. The challenge of B-PLL cells with ionophore A23187 plus arachidonic acid and the thiol-active compound Diamide led to the formation of similar amounts of LTB₄ as produced by human neutrophils (Figure 6A) [30]. This cocktail of compounds is known to induce LTB₄ production in other types of B lymphocytes [14]. Sonicated cells also readily produced LTB₄ (Figure 6A). Upon calcium ionophore activation only, isolated B-PLL produced low amounts of LTB₄ (about 1 pmol/10⁶ cells), showing that there were minimal amounts of myeloid cells contaminating these preparations of B-PLL cells. Figure 6B shows that intact or sonicated Granta 519, JEKO-1 and Rec1 cells also produced significant amounts of LTB₄ as B-PLL cells. The capacity of the different cell lines to produce LTB₄ correlated relatively well with the degree of expression of 5-LO protein (Figures 5 and 6B). In essence, these results demonstrate that both B-PLL cells and MCL cell lines have the capacity to produce LTB₄ and that the cells contained substantial amounts of 5-LO, which could be activated under certain conditions.

The calcium ionophore A23187 was purchased from Calbiochem-Behring (La Jolla, CA, USA). HPLC solvents were obtained from Rathburn chemicals (Walkerburn, U.K.) and the synthetic standards of LTB₄ and prostaglandin (PG) B₁ were from Biomol (Plymouth, PA, USA). Azodicarboxylic acid bisdimethylamide (Diamide) was from Sigma-Aldrich (Stockholm, Sweden) and arachidonic acid (AA) from NU-CHEK PREP Inc. (MN, USA). The well-characterized MCL cell lines Granta 519 and JEKO-1 were purchased from Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany). The MCL cell line Rec1 [34] was a generous gift from Dr. Christian Bastard (Ronan, France). Cell lines were maintained in RPMI 1640 medium (Invitrogen Life Technologies, Stockholm, Sweden) supplemented with 2 mM L-glutamine and 10% fetal calf serum (FCS) and 50 μg/mL gentamicin (Invitrogen) under standard conditions (humidified atmosphere, 95% air, 5% CO₂, 37°C). JEKO-1 was maintained as described above but with a final concentration of 20% FCS.

Tonsils were obtained from patients undergoing tonsillectomies (HELIOS Klinikum, Erfurt, Germany). Isolated cells from tonsils were incubated with CD19 micro beads (Miltenyi, Germany) and loaded in a magnetic field on a MACS separation column LS (Miltenyi). Cells were washed three times with PBS/EDTA (2 mM)/FCS (5%), subsequently the column was removed from the magnetic field and CD19 positive cells were eluted with 4–6 mL PBS/EDTA/FCS. Isolated cells were concentrated to 10⁷ cells/50 μL and incubated with anti-CD38-PE (Pharmingen, Germany) and anti-IgD-FITC (Pharmingen) for 20 min at 4°C. Cells were washed twice, set to 2 × 10⁶ cell/mL in PBS/EDTA/FCS and sorted with a FACSVantage SE cell sorting instrument (Beckton Dickinson, Germany). B cells were classified according to the scheme described by Grammer et al. 1999 [35], mantle zone B cells (CD19⁺, IgD⁺, CD38⁺), germinal centre B cells (CD19⁺, IgD^-, CD38⁺⁺), memory B cells (CD19⁺, IgD^-, CD38⁺) and plasma cells (CD19⁺, IgD^-, CD38⁺⁺⁺). Only cell populations that were > 95% pure were used in further experiments.

Total RNA was separated according to the manufacturer's protocol with an RNeasy mini kit (Qiagen, Germany). Concentration and quality of RNA was determined with a Bioanalyzer2100 (Agilent, Germany). The reversed transcription reaction was performed on 1.8 μg of total RNA in a RT-PCR mixture (1× RT buffer, 167 μM dNTP, 0.7 U/μL RNasin, 0.1 μg/μL BSA, 0.35 U/μL AMV reverse transcriptase (Roche, Germany)).

PCR was performed with 60 ng of cDNA for each reaction. The PCR reaction mixture contained; PCR buffer, dNTP mix (0.2 mM each), BSA (0.1 μg/μL), SybrGreen (1:50) (Roche), MgCl₂ (1.8 mM), Platinum Taq DNA polymerase (1.5 U) (Invitrogen, Germany) and primer mix (0.2 μM each). Primers that were used are described in table 2.

Tonsillary B cell samples were washed in PBS (2×) and re-suspended to 4 × 10⁴ cells/μL in 5× sample buffer (250 mM Tris/HCl pH 6.8, 30% Glycerol (v/v), 10% SDS (w/v), 5% β-mercaptoethanol (v/v), 0.02% Bromphenolblue (w/v), complete mini protease inhibitor (Roche)). The suspension was heated to 95°C (5–10 min) and run on a regular 10% SDS-PAGE with SDS running buffer (25 mM Tris/HCl; 190 mM Glycine; 0.1% SDS (w/v)). HybondC nitrocellulose membrane (GE Healthcare, Germany) and semi-dry blotting apparatus (LMS labortechnik, Germany) was used for western blot transfer. The detection was performed with ECL kit (GE Healthcare, Germany). Primary antibody used for the immunoblot detection was anti-5-LO polyclonal rabbit antiserum [36] and secondary antibody was an anti-rabbit-IgG coupled with HRP (GE Healthcare, Germany).

MCL cell lines were washed in PBS (w/o calcium and magnesium) and suspended in PBS (w/o calcium and magnesium) supplemented with complete mini protease inhibitor (Roche, Sweden). Cells were sonicated on ice (3 × 5 seconds) (Sonics Vibra cell, 40% amplitude, CiAB, Sweden) and centrifuged (1 × 10⁵ × g, 60 min, 4°C). Supernatant was collected and protein concentration was determined with Bradford. Protein (7 μg) was loaded and separated on a 7% tris-acetate gel. PVDF membrane (GE Healthcare, Sweden) and semi-dry blotting apparatus (GE Healthcare, Sweden) was used in the western blot transfer.

Confocal laser scan microscopy was performed with 7 μm cryostat sections embedded in Tissue Tec (Sakura, Japan). Specimens were placed on glass slides and fixed with acetone for 15 min at 4°C. After rehydration in PBS supplemented with BSA (2%) for 30 min at 20°C, specimens were incubated for 1 h at room temperature with rabbit anti-5-LO antiserum and for double immunofluorescence analyses with unlabeled primary antibodies against IgD and CD38 (Pharmingen), CD19 (Coulter Immunotec) or CD138 (Serotec). Nuclei were counterstained with DAPI. Secondary antibodies (goat anti-mouse-IgG F(ab')2-Cy2 and donkey anti-rabbit-IgG F(ab')2-Cy3; Dianova) were applied for 1 h at room temperature. Sheets were mounted on coverslips with Permafluor (Beckman, Munich, Germany) and viewed on a Zeiss Axiovert 200 M microscope equipped with a confocal laser scanning head (LSM510). Pictures were taken and analyzed using LSM510 Image Examiner software (Zeiss, Jena, Germany).

Formalin-fixed, paraffin-embedded tumor (lymph nodes) tissue samples and hyperplastic tonsils were obtained from the Department of Pathology, Karolinska University Hospital Huddinge. Before constructing a TMA block, serial 5 μm sections were cut from each donor block. One of these sections was stained with H&E for marking morphologically representative areas of the tumor. One representative area was targeted. Using Beecher Instruments Tissue Arrayer (Silver Springs, MD, USA), tissue cylinders with a diameter of 1.0 mm were punched from the targeted area in each donor block and deposited into a recipient TMA block. The cores of each TMA included duplicate cores from all cases and two cores of normal human heart muscles as a control for orientation of the sections.

Multiple serial 5 μm-thick TMA sections containing samples were cut de-paraffinized in xylene and hydrated in a series of graded alcohols. Enzyme digested antigen retrieval was carried out with Protease (Sigma P-5147, Sweden) at 20°C for 10 min. Anti-5-LO antibody (LO-32, Merck Frosst, Canada) was used at a dilution of 1:300 and anti-CD3 antibody was used at a dilution of 1:100 (DAKO). All staining were semiautomatic and performed on a TechMate 500 plus (DAKO, Glostrup, Denmark) by using the Dako REAL™ Detection System, PeroxidaseDAB, RabbitMouse kit as recommended by the manufacturer.

The results of the staining were evaluated by three persons (XW, BC and BS). Images of immunohistologic staining were acquired using an Olympus BX45 microscope (Olympus, Stockholm, Sweden) and Sony digital camera (DXC-S500, Sony, Stockholm, Sweden). Digitized images were processed using Picsara 8.9 Rev 5 software (Bildanalyssytem AB, Stockholm, Sweden).

Intact cells (10 × 10⁶) were suspended in 1 mL phosphate buffered saline (PBS) and pre-incubated for two min with azodicarboxylic acid bisdimethylamide (Diamide) (100 μM) prior to stimulation with arachidonic acid (40 μM) and calcium ionophore A23187 (1 μM). The cells were stimulated for five min and the reaction was terminated with 1 mL methanol. The A23187 incubation was performed as described above but without any addition of Diamide and arachidonic acid. Alternatively, cells (10 × 10⁶) were re-suspended in 1 mL calcium free PBS including EDTA (2 mM) and sonicated for 3 × 5 seconds. The cells were pre-incubated for two min at 37°C in the presence of ATP (1 mM) prior addition of calcium chloride (2 mM) and arachidonic acid (40 μM). The reaction was terminated after five min of incubation with 1 mL methanol.

After addition of PBS (0.5 mL) and the internal standard PGB₁ (50 pmol), the samples were centrifuged (1250 × g, 5 min). The supernatants were subsequently subjected to solid phase extraction on Oasis HLB Extraction Cartridges (10 mg, Waters, Sweden). The methanol fractions were analyzed on a Waters Alliance 2695 reverse phase HPLC and detected with Waters PDA 996. Methanol:water:trifluoroacetic acid (70:30:0.007, v/v) was used as mobile phase at a flow rate of 1.2 mL/min. Qualitative analysis was performed by comparison of retention times of synthetic standards and quantitative determinations were performed by computerized integration of the area of eluted peaks. Mean and standard deviation was calculated.

This study was approved by the local ethic committee of Friedrich-Schiller-University of Jena, Germany, and Karolinska University Hospital, Stockholm, Sweden.