Cutting Edge: Expression of the NF of Activated T Cells in Eosinophils: Regulation by IL-4 and IL-5 1 Tan Jinquan, 2 * Sha Quan,* Henrik H. Jacobi,* Claus M. Reimert,* Anders Millner,* Jens B. Hansen,* Charlotte Thygesen,* Lars P. Ryder, ² Hans O. Madsen, ² Hans-Jørgen Malling,* and Lars K. Poulsen 2 * We report that NF-AT1 and NF-AT4 are expressed cytoplas- mically in resting eosinophils, whereas NF-AT2 and NF-AT3 have not been seen. Likewise, NF-AT1 mRNA and NF-AT4 mRNA have been detected in resting eosinophils, and their levels can be significantly up-regulated by the Th2-associated cytokines IL-4 and IL-5. There is no detectable NF-AT protein expression in the nuclei of resting eosinophils. However NF- ATs appear in the nuclei of IL-4-, IL-5-, or ionomycin-stimu- lated eosinophils. Only NF-AT1 and NF-AT4, but not NF-AT2 and NF-AT3, have translocated into the nuclei in IL-4- or IL- 5-stimulated eosinophils. These findings delineate a novel path- way in the cytokine network in which Th2 lymphocytes “con- trol” eosinophils via the release of IL-4 and IL-5, and activation of NF-AT in eosinophils. The findings also suggest that a later feedback “talking” may exist between eosinophils and Th2 lymphocytes. The Journal of Immunology, 1999, 163: 21–24. N uclear factor of activated T cells is a family of transcrip- tion factors implicated in the control of cytokine and early immune response gene expression. NF-AT1-defi- cient mice are prone to developing a classically allergic phenotype characterized by eosinophilia and increased production of Th2- associated cytokines (1). Mice lacking NF-AT1 and NF-AT4 have allergic blepharitis, interstitial pneumonitis, and an increase in se- rum IgG1 and IgE levels, secondary to a selective increase in Th2- associated cytokines (2). NF-AT proteins are expressed not only in T lymphocytes but also in other cells including B lymphocytes, NK cells, mast cells, monocytes, thymus and spleen cells (3), and vascular smooth muscle cells (4). Thus far, it has not been deter- mined whether NF-AT proteins are expressed in eosinophils. In the present study, we have investigated the expression of NF-AT pro- teins and the corresponding mRNA in peripheral eosinophils and the regulation of these proteins by IL-4 and IL-5. Materials and Methods Purification of eosinophils Eosinophils were purified from healthy, nonallergic volunteers as described in detail elsewhere (5). Briefly, a Percoll gradient (1.082 g/ml; Pharmacia, Uppsala, Sweden) was used to enrich eosinophils; anti-CD16-coated MACS magnetic particles (Miltenyi Biotech, Bergisch Gladbach, Ger- many) were used to deplete neutrophils. Eosinophil purity was invariably 97% (lymphocytes 0.5%). The whole procedure was conducted at 4°C in a Ca 2+ -free and Mg 2+ -free medium. Immunocytochemistry assay The purified eosinophils were spun down on a slide, fixed, and immersed in 1% BSA blocking buffer for 10 min to avoid unspecific binding; next, primary Ab (20 g/ml of either anti-NF-AT1 (mouse mAb 4G6-G5), anti- NF-AT2 (mouse mAb 7A6), anti-NF-AT3 (goat pAb 3 C-20), or anti-NF- AT4 (goat pAb C-20); Santa Cruz Biotechnology, Santa Cruz, CA) was added. Eosinophils were then incubated overnight at 4°C, followed by the addition of secondary Ab, and were visualized by an alkaline phosphatase staining system (Dako, Glostrup, Denmark). Real time quantitative RT-PCR assay All real time quantitative RT-PCRs were performed as described elsewhere (6). Briefly, total RNA was reverse transcribed and submitted to real time quantitative PCR in an ABI Prism 7700 Sequence Detector System (Per- kin-Elmer, Norwalk, CT). By using a SYBR Green PCR Core Reagents Kit (Perkin-Elmer), fluorescence signals were generated during each PCR cy- cle via the 5' to 3' endonuclease activity of AmpliTaq Gold to provide real time quantitative PCR information. The following sequences of the specific primers (Amersham Pharmacia Biotech, Little Chalfont, U.K.) were used: NF-AT1 sense, 5'-AGAAACTCGGCTCCAGAATCC-3'; NF-AT1 anti- sense, 5'-TGGTTGCCCTCATGTTGTTTTT-3'; NF-AT2 sense, 5'-GC CGCAGCACCCCTACCAGT-3'; NF-AT2 antisense, 5'-TTCTTCCTC CCGATGTCCGTCTCT-3'; NF-AT3 sense, 5'-GGTTTCCCGGCCAGT CCAGGTCTA-3'; NF-AT3 antisense, 5'-AAGGGGCGGGGAAGGAAG GAAACT-3'; NF-AT4 sense, 5'-ACCAGCCCGGGAGACTTCAATA GA-3'; NF-AT4 antisense, 5'-AAATACCTGCACAATCAATACTGG-3'. *Laboratory of Medical Allergology, Allergy Unit, and ² Laboratory of Tissue Typing, Department of Clinical Immunology, National University Hospital, Copenhagen, Denmark Received for publication February 1, 1999. Accepted for publication April 30, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 T.J., S.Q., and A.M. are supported by generous grants from the Danish Allergy Research Center and from the Alfred Benzons Foundation. 2 Address correspondence and reprint requests to Drs. Tan Jinquan or Lars K. Poulsen, Laboratory of Medical Allergology, Finsen Center 7542, National University Hospital, Tagensvej 20, DK-2200 Copenhagen N, Denmark. E-mail address: TAN@RH.DK or lkpallgy@inet.uni2.dk 3 Abbreviations used in this paper: pAb, polyclonal Ab; EMSA, electrophoretic mo- bility shift assay; Ion, ionomycin; NF-AT huIL-2, distal NF-AT site of the human IL-2 promoter; C T , threshold cycle. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 ● ●