IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression Rainer Spanbroek*†, Markus Hildner*, Andre´ Ko¨ hler‡, Angelika Mu¨ ller‡, Felix Zintl‡, Hartmut Ku¨ hn§, Olof Rådmark¶, Bengt Samuelsson¶, and Andreas J. R. Habenicht* *Center for Vascular Medicine, Friedrich Schiller University Jena, Nordha¨userstrasse 78, 99089 Erfurt, Germany; ‡Children’s Hospital, Kochstrasse 2, 07740 Jena, Germany; §Institute of Biochemistry, Humboldt University Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany; and ¶Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-17177 Stockholm, Sweden Contributed by Bengt Samuelsson, February 15, 2001 Dendritic cell (DC) differentiation from human CD34؉ hematopoi- transcripts of LT receptors are expressed in lymphoid organs etic progenitor cells (HPCs) can be triggered in vitro by a combi- (unpublished data); 5-LO-deficient mice show reduced ovalbu- nation of cytokines consisting of stem cell factor, granulocyte- min-dependent cellular and humoral immune responses during macrophage colony-stimulating factor, and tumor necrosis factor antigen-dependent allergenization of the lung (7); and mice ␣. The immune response regulatory cytokines, IL-4 and IL-13, deficient in the cysteinyl LT transporter multidrug resistance promote DC maturation from HPCs, induce monocyte-DC transdif- protein-1 have a defect in DC mobilization from skin to lymph ferentiation, and selectively up-regulate 15-lipoxygenase 1 (15- nodes (8). Although these data associate the 5-LO pathway with LO-1) in blood monocytes. To gain more insight into cytokine- the adaptive immune response, the precise molecular mecha- regulated eicosanoid production in DCs we studied the effects of nisms of LTs action within the immune system remain elusive. IL-4͞IL-13 on LO expression during DC differentiation. In the DCs are members of the family of professional antigen- absence of IL-4, DCs that had been generated from CD34؉ HPCs in presenting cells that are uniquely capable of initiating immune response to stem cell factor͞granulocyte-macrophage colony- responses in naive lymphocytes (5). Because all human DC stimulating factor͞tumor necrosis factor ␣ expressed high levels of phenotypes examined so far constitutively express the 5-LO 5-LO and 5-LO activating protein. However, a small subpopulation pathway, we suggested that DC 5-LO-derived eicosanoids may of eosinophil peroxidase؉ (EOS-PX) cells significantly expressed participate in the regulation of proximal steps of the immune 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and response (3). Primary immune responses involve phenomena as selective down-regulation of 5-LO but not of 5-LO activating diverse as antigen uptake by DCs, their subsequent migration protein in DCs and in EOS-PX؉ cells and, when added at the onset through multiple tissue barriers, homing in lymphoid organs, and of DC differentiation, also prevented 5-LO up-regulation. Similar antigen presentation to T cells (reviews in refs. 2 and 3). 5-LO effects were observed during IL-4- or IL-13-dependent mono- products could play roles in each of these events; the specific cyte-DC transdifferentiation. Down-regulation of 5-LO was accom- roles of the 5-LO and other LO pathways in immunity have yet panied by up-regulation of 15-LO-1, yielding 15-LO-1؉ 5-LO-defi- to be defined. IL-4 and IL-13 (which bind to the IL-4 receptor cient DCs. However, transforming growth factor ␤1 counteracted ␣ chain) have been shown to affect DC differentiation from both ϩ the IL-4-dependent inhibition of 5-LO but only minimally affected CD34 HPCs and blood monocytes in vitro (9, 10). IL-4 is a 15-LO-1 up-regulation. Thus, transforming growth factor ␤1 plus multifunctional cytokine produced by distinct populations of T IL-4 yielded large mature DCs that coexpress both LOs. Localization helper (TH) cells (termed TH2 cells) and other lymphocytes that of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained have diverse biological activities in hematopoietic and nonhe- at all cytokine combinations in all DC phenotypes and in EOS-PX؉ matopoietic tissues (9). In particular, IL-4 is required for the ؉ cells. In the absence of IL-4, major eicosanoids of CD34 -derived execution of TH2-type immune responses (i.e., B cell expansion DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotri- and Ig isotype switching from IgM to IgE). Therefore, IL-4 is ene B4, whereas the major eicosanoids of IL-4-treated DCs were considered a central cytokine mediating allergic and autoim- 15S-HETE and 5S-15S-diHETE. These actions of IL-4͞IL-13 reveal a mune disorders. However, IL-4 also has been shown to exert paradigm of eicosanoid formation consisting of the inhibition of anti-inflammatory properties in a variety of disease conditions one and the stimulation of another LO in a single leukocyte lineage. (9). Nothing is known about the effects of IL-4 on the 5-LO pathway, but IL-4 and IL-13 up-regulate 15-LO-1 in blood transforming growth factor ␤1 ͉ arachidonic acid cascade monocytes and epithelial cells (11, 12). With the use of serial analyses of 17,000 genes it was found that 15-LO-1 is among the most abundantly expressed genes in DCs generated by treatment icosanoids produced by the 5-lipoxygenase (5-LO) pathway of blood monocytes with IL-4 for 6 days (13). Here we report that are potent mediators of inflammatory and allergic tissue E IL-4 inhibits 5-LO and concomitantly induces 15-LO-1 expres- reactions (1). Several lines of evidence indicate that 5-LO- sion in differentiating DCs derived from HPCs and blood derived eicosanoids (2, 3) participate in the regulation of adap- monocytes, that the combined activities of IL-4 and TGF␤1 tive immunity (4, 5). Human epidermal Langerhans cells and dendritic cells (DCs) in the paracortex of lymphoid organs express the 5-LO pathway at marked levels in vivo (2, 3); DCs Abbreviations: DC, dendritic cell; EOS-PX, eosinophil peroxidase; FACS, fluorescence acti- ϩ generated in vitro from CD34 hematopoietic progenitor cells vated cell sorter; FLAP, 5-LO-activating protein; GM-CSF, granulocyte-macrophage colony- (HPCs) in response to a combination of stem cell factor (SCF), stimulating factor; HETE, hydroxyeicosatetraenoic acid; HPC, hematopoietic progenitor granulocyte-macrophage colony-stimulating factor (GM-CSF), cells; LO, lipoxygenase; LT, leukotriene; RT-PCR, reverse transcription–PCR; TH lymphocyte, ␣ ␣ T helper lymphocyte; TNF, tumor necrosis factor; TGF, transforming growth factor; SCF, and tumor necrosis factor (TNF- ) up-regulate 5-LO and stem cell factor. 5-LO-activating protein (FLAP) genes, and the DC maturation- † ␤ ␤ To whom reprint requests should be addressed. E-mail: [email protected]. promoting cytokine transforming growth factor 1 (TGF- 1) The publication costs of this article were defrayed in part by page charge payment. This enhances up-regulation of 5-LO (3); leukotriene (LT) B4 has article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. been shown to support B and T lymphocyte activation in vitro (6); §1734 solely to indicate this fact. 5152–5157 ͉ PNAS ͉ April 24, 2001 ͉ vol. 98 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.091076998 Downloaded by guest on September 29, 2021 Fig. 1. IL-4 down-regulates 5-LO transcripts in CD34ϩ HPCs. HPCs were maintained in SCF͞GM-CSF͞TNF-␣ for 15 days (empty columns) or in SCF͞GM-CSF͞TNF-␣ for 8 days and additional IL-4 for 7 days (filled columns). Transcripts for 5-LO (A), FLAP (B), and 15-LO-1 (C) were determined by RT-PCR. Data are expressed as ratios of densitometry units of 5-LO (27 cycles), FLAP (25 cycles), or 15-LO-1 (30 cycles) transcripts relative to glyceraldehyde-3-phosphate dehydrogenase transcripts (23 cycles). n.d., not detectable. generate 5-LOϩ͞15-LO-1ϩ mature DCs, and that IL-4 also 1 h. Confocal laser scan microscopy was performed with a Zeiss down-regulates 5-LO in 15-LO-1ϩ eosinophil peroxidase (EOS- Axiovert 100 M microscope equipped with a LSM 510 laser ϩ PX) cells. scanner head. Additional details are available on request. Materials and Methods FACS Analyses. FACS analyses of LOs were performed as de- Materials. Avian myeloblastosis virus reverse transcriptase and scribed for 5-LO (3). For cell lineage identification of both LOs, Thermus aquaticus DNA polymerase were from Roche Diag- we combined a rabbit anti-5-LO antiserum with a guinea pig nostics. Primary monoclonal antisera against CD1a and anti-15-LO-1 antiserum, with the use of a FACSCalibur flow HLA-DR were from Coulter. EOS-PX was from PharMingen. cytometer (Becton Dickinson). Data were recorded with the use Cy2-, Cy3-, phycoerythrin (PE)-, and FITC-labeled secondary of the CELLQUEST program (Becton Dickinson). antisera were from Dianova (Hamburg, Germany). Cytokines were fromR&DSystems. All other reagents were from Sigma. Assays. Immunoblots were performed on 10 ␮g total cell protein as described (3). Eicosanoid formation in intact cells was deter- ϩ Ͼ ϩ Cell Culture. CD34 HPCs were prepared to a purity of 98% mined in response to 10 ␮MCa2 ionophore A23187 and 40 ␮M ϫ 4 ͞ from human cord blood, and 2 10 cells ml were maintained arachidonic acid or in cell-free systems in the presence of 40 ␮M in RPMI 1640 medium with supplements and cytokines as arachidonic acid as described (12). described (3). Fresh complete culture medium was added to maintain cells at a density between 3 ϫ 105 and 5 ϫ 105 cells͞ml. Results Human blood monocyte purification from peripheral blood IL-4 Down-Regulates 5-LO During Leukopoiesis of SCF͞GM-CSF͞TNF- -mononuclear cells involved two rounds of anti-CD14 antibody- ␣-Treated CD34؉ HPCs.