Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN- β Anette H. H. van Boxel-Dezaire, Joana A. Zula, Yaomin Xu, Richard M. Ransohoff, James W. Jacobberger and George R. This information is current as Stark of October 1, 2021. J Immunol published online 18 October 2010 http://www.jimmunol.org/content/early/2010/10/18/jimmun ol.0902314 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/10/19/jimmunol.090231 Material 4.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 18, 2010, doi:10.4049/jimmunol.0902314 The Journal of Immunology Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN-b Anette H. H. van Boxel-Dezaire,* Joana A. Zula,* Yaomin Xu,† Richard M. Ransohoff,‡ James W. Jacobberger,x and George R. Stark* Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-b and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4+ and CD8+ T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4+ and CD8+ T cells, few B cells activated STAT1 in response to IFN-b, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in Downloaded from B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4+ T cells, IFN-b activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-b increases the survival of primary human B cells and CD4+ T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ nterferons are pleiotropic cytokines that play important roles factor 3 (ISGF3) is the major transcription factor activated in re- in infection and inflammation. Three classes of IFNs are known: sponse to IFN-a/b (3, 4). ISGF3, a complex of phosphorylated I type I IFNs includes IFN-a,-b,-v,-t,-d,-k, and -ε (1); type STAT1, STAT2, and unphosphorylated IFN regulatory factor II is IFN-g; and type III is IFN-l.IFN-a and -b use the IFNAR1 (IRF)-9, binds to the IFN-stimulated response element (ISRE) and IFNAR2c receptor subunits to signal (2), each of which binds present in the promoters of many ISGs. In response to type I IFNs, constitutively to a single member of the JAK family of kinases: activated STAT1 can also form homodimers that bind to gamma- IFNAR1 to tyrosine kinase 2 and IFNAR2 to JAK1. Ligand bind- activated sequence (GAS) elements in some ISG promoters (3, 5). by guest on October 1, 2021 ing induces the phosphorylation of JAK1, tyrosine kinase 2, intra- It is becoming clear that, in addition to ISGF3 and STAT1 cellular tyrosine residues of each receptor subunit, and STATs. homodimers, other transcription factors play important roles as cy- Activated STATs dimerize, dissociate from the receptor, and trans- toplasmic messengers between the receptor and the nucleus (4), locate to the nucleus to induce the expression of IFN-stimulated helping to explain why type I IFNs, which were discovered on genes [ISGs (3)]. Current data suggest that IFN-stimulated gene the basis of their potent antiviral activities, are now known to act much more broadly, as pleiotropic cytokines that regulate many different cellular functions. For example, STAT3 is activated in *Department of Molecular Genetics, †Statistical Genetics and Bioinformatics, De- partment of Quantitative Health Sciences, and ‡Department of Neurosciences, Neuro- response to type I IFNs in most cell lines, forming STAT3 homo- inflammation Research Center, Lerner Research Institute, Cleveland Clinic Foun- dimers or heterodimers with activated STAT1 (4). In contrast, x dation, Cleveland, OH 44195; and Case Comprehensive Cancer Center, Case West- activation of STAT4 and STAT5 by IFN-a/b is found mostly in ern Reserve University, Cleveland, OH 44106 NK and T cells (6–8). Interestingly, the activation of STAT6 in- Received for publication July 21, 2009. Accepted for publication August 27, 2010. duced by type I IFNs has only been described in B cell lines This work was supported by Pilot Grant PP1086 and Career Transition Fellowship (9). STAT homo- and heterodimers bind to GAS elements in the Award TA3032A1/1 (to A.H.H.vB-D.) from the National Multiple Sclerosis Society and by Grants P01 CA06220 (to G.R.S.) and P30 CA43703 (to Gene Expression and promoters of ISGs, but it is clear that different STAT dimers have Genotyping Facility of the Case Comprehensive Cancer Center) from the National different preferences for specific GAS elements (5). The differ- Institutes of Health. ential activation of STAT4, STAT5, and STAT6 in different cell The microarray data presented in this article have been deposited into National lines suggests the possibility of cell type-specific activation of Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE23307) under accession No. GSE23307. STATs by IFN-a/b in vivo. Of note, evidence for a cell type- Address correspondence and reprint requests to Dr. George R. Stark, Department of specific response to IFN-a was described with respect to differ- Molecular Genetics, Lerner Research Institute, Mail Code NE20, Cleveland Clinic ential ISG induction in human T cells and dendritic cells (10). Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: [email protected] In this study, we investigated how primary human leukocytes The online version of this article contains supplemental material. signal in response to IFN-b. Undiluted freshly drawn human whole Abbreviations used in this paper: D-PBS, Dulbecco’s PBS; ETS, E–twenty-six; GAS, blood was stimulated with IFN-b in vitro to mimic the situation gamma-activated sequence; HI, healthy individual; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated in vivo as closely as possible. Because the activation of STATs response element; PY-STAT, phosphotyrosine-STAT; rtPCR, real-time PCR; SHP1, occurs only transiently, the isolation of many different leukocyte Src homology region 2 domain-containing phosphatase 1; SOCS, suppressor of cy- subsets after stimulation of whole blood in combination with tokine signaling; TCP45, T cell protein tyrosine phosphatase of 45 kDa. Western blot analysis is not feasible because, by the time the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 subsets could be isolated, the optimal time point for activation of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902314 2 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS STATs would have passed. Therefore, we used a flow cytometry- Intracellular detection of PY-STATs using flow cytometry based technique that enables the detection of intracellular phos- The published method of Chow et al. (24), which was developed to measure photyrosine-STAT (PY-STAT)1, STAT3, and STAT5 at the single- intracellular phospho-ERK in whole blood cells, was adapted slightly cell level, allowing cells to be fixed at the optimal time for STAT to measure the induction of phospho-STATs in human whole blood. A activation. IFN-b–induced activation of STAT1, STAT3, and STAT5 number of commercially available anti-human CD3, CD4, CD8, CD19, was chosen because these three transcription factors regulate cell and CD14 Abs were screened. The best anti-CD3 and anti-CD14 clones were those that performed optimally after fixation and erythrocyte lysis survival in opposite directions (11–13). Furthermore, this approach but before methanol incubation. In contrast, the best anti-CD8, anti-CD4, allowed us to address whether differential activation of these STATs and anti-CD19 Abs performed best after methanol incubation. As pre- might explain how IFN-b enhances the survival of mature B cells viously published (25), IFN-b–induced phospho-STATs were optimally and T cells (14–19) while increasing apoptosis in monocytes and detected in leukocytes that were permeabilized with 90% methanol (data not shown). many cancer cell lines (20–23). Notably, we found that IFN-b in- After stimulation with IFN-b1a in vitro or leaving cells untreated for the duced significant differences in the activation of STAT1 and STAT5 same time period, whole blood or cell lines were fixed in 4 or 2% form- in different leukocyte subsets and that these differences are related aldehyde, respectively, by adding 10% prewarmed methanol-free formal- to the induction of pro- and antiapoptotic genes, respectively.