Expression of 5-lipoxygenase (5-LOX) in T lymphocytes

Abstract

5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Figure 1

Expression of 5-LOX mRNA by MOLT4 and Jurkat T cell lines as well as peripheral blood T cells. (a) Amplification of 5-LOX mRNA by RT-PCR (35 cycles). Lane 1, molecular weight ladder; lane 2, negative control – cDNA from chloramphenicol acetyl transferase mRNA with 5-LOX primers; lane 3, positive control – same cDNA with corresponding primers; lane 4, Jurkat mRNA run without reverse transcriptase; lane 5, MOLT4 mRNA run without reverse transcriptase; lane 6, T-lymphocyte mRNA run without reverse transcriptase; lane 7, 5-LOX cDNA-positive control; lane 8, Jurkat cells; lane 9, MOLT4 cells; lane 10, purified peripheral T lymphocytes. (b) Amplification of 5-LOX mRNA by RT-PCR in situ. Panel i, negative control: peripheral blood T lymphocytes submitted to RT-PCR in situ without reverse transcriptase; panel ii, same field visualized by fluorescence microscopy after incubation with anti-CD3 antibodies; panel iii, negative control: peripheral blood T lymphocytes submitted to RT-PCR in situ with primers specific for chloramphenicol acetyl transferase; panel iv, same field visualized by fluorescence microscopy after incubation with anti-CD3 antibodies, panel v, peripheral blood T lymphocytes submitted to RT-PCR in situ with specific 5-LOX primers; panel vi, same field visualized by fluorescence microscopy after incubation with anti-CD3 antibodies. Magnification 65×.

Figure 2

5-LOX expression in Jurkat and MOLT4 cell lines and peripheral blood T lymphocytes by indirect immunofluorescence. (a and c) Jurkat and MOLT4 cells, respectively, incubated with a negative isotypic control serum; (b and d) Jurkat and MOLT4 cells, respectively, incubated with anti-5-LOX monoclonal antibody; (e and f) peripheral blood T lymphocytes incubated with either a negative isotypic control serum (e) or anti-5-LOX monoclonal antibody (f) followed by staining with anti-CD3 monoclonal antibodies (magnification × 40); (g) peripheral blood T lymphocytes stained with anti-5-LOX and anti-CD3 monoclonal antibodies and (h) after 3 days incubation in Chang MF medium and stained with anti-5-LOX and anti-CD3 monoclonal antibodies (arrow indicates localization of 5-LOX at nuclear periphery).

Figure 3

Analysis of peripheral blood T lymphocyte populations by flow cytometry. The acquisition panels represent T cells gated on the CD19-negative, CD45-positive population. Cells were incubated with anti-5-LOX monoclonal antibody and F(ab)′₂ goat anti-mouse fluorescein isothiocyanate (FITC) followed by staining with anti-CD3 (a), CD4 (b), CD8 (c), CD2 (d), CRTH2 (e), CD45RA (f), CD45RO (g), TCR-αβ (h), TCR-γδ (i), CD1a (j), CD7 (k) and CD10 (l) antibodies. The x-axis represents 5-LOX FITC and the y-axis represents the antibodies defining the different subpopulations. Results are from one experiment and are representative of five.

Figure 4

5-LOX protein expression and metabolite production. (a) Western blot of 5-LOX in MOLT4 and Jurkat T-cell lines and peripheral blood T lymphocytes. Cells were lysed and the cytosolic fraction was retrieved by ultracentrifugation. Equal amounts of protein (25 μg) were subjected to SDS–PAGE on 4–15% gradient gels, then transferred and immunoblotted using anti-5-LOX monoclonal antibody. Lane 1, molecular weight standard; lane 2, Jurkat cells; lane 3, MOLT4 cells; lane 4, peripheral blood T cells; lane 5, neutrophils. (b) Synthesis of LTB₄ and LTC₄ by Jurkat cells. Cells were activated by CD2–CD3–CD28 crosslinking. Supernatants were harvested at different times and production of LTB₄ and LTC₄ was quantified by EIA.