Activation of a Functionally Distinct 80-kDa STAT5 Isoform by IL-5 and GM-CSF in Human Eosinophils and Neutrophils Eric Caldenhoven, Thamar B. van Dijk, Jan A. M. Raaijmakers, Jan-Willem J. Lammers, Leo Koenderman, and Rolf P. de Groot 1 Department of Pulmonary Diseases, University Hospital Utrecht, 3584 CX Utrecht, The Netherlands Received February 23, 1999 Interleukin-5 (IL-5), IL-3, and granulocyte macro- phage colony-stimulating factor (GM-CSF) are hema- topoietic cytokines which signal through a common  subunit (c) of a heterodimeric receptor. Among the intracellular signaling pathways activated via c is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosino- phils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the -casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl- terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT re- porter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might con- tribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells. © 1999 Academic Press Key Words: granulocytes; cytokine signaling; tran- scription. Interleukin-3 (IL-3), IL-5 and granulocyte macro- phage colony-stimulating factor (GM-CSF) are related cytokines which exert similar biological functions in their target cells (1-3). Whereas IL-3 and GM-CSF are pleiotropic cytokines stimulating the development of various lineage’s of haematopoietic cells (4), IL-5 is lineage specific and plays a major role in the develop- ment of eosinophils and basophils in the human system (5,6). These cytokines signal through a heterodimeric receptor consisting of a cytokine-specific  chain and a shared c chain (reviewed in 1,2). Receptor binding leads to the activation of multiple sigan transduction pathways, including the Ras/Raf/Erk, the PI3K and the JAK/STAT pathways (reviewed in 7). Recently a mem- ber of the Janus kinase family, JAK2, was found to be activated in response to IL-3, IL-5 and GM-CSF (8). The observation that overexpression of a dominant negative form of JAK2 abrogates GM-CSF signaling suggests that this kinase plays a major role in the activation of downstream signals (9). One of the substrates of the JAK kinases is a family of SH2-containing transcription factors called STATs (signal transducers and activators of transcription) (10). Currently seven members of the STAT family have been reported, which are involved in signal trans- duction by different cytokines (11). Although STAT1 and STAT3 are activated in a cell type specific manner (12-14), STAT5 seems to be one of the major targets for IL-3, IL-5 and GM-CSF signaling (15-18). Unlike the other STAT proteins, STAT5 consists of two different but highly homologous isoforms referred to as STAT5A and STAT5B (15,19). These two proteins are encoded by two different genes and have a molecular weight of 96 (STAT5A) and 94 kDa (STAT5B). Furthermore, two shorter STAT5 homologues of 77 and 80 kDa were found to be activated by IL-3 in immature murine myeloid cell lines (20). Recently, evidence has been provided that these shorter STAT5 isoforms are gen- erated by a specific protein-processing event of the full-length STAT5 proteins (21). In this study we present evidence that the induced DNA-binding proteins by IL-5 in human eosinophils This work was supported by a research grant from Glaxo Wellcome bv. 1 To whom correspondence should be addressed at Department of Pulmonary Diseases, G03.550, University Hospital Utrecht, Heidel- berglaan 100, 3584 CX Utrecht, The Netherlands. Fax: 31-30- 2505414. E-mail: R.deGroot@hli.azu.nl Molecular Cell Biology Research Communications 1, 95–101 (1999) Article ID mcbr.1999.0114, available online at http://www.idealibrary.com on 95 1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.