Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Signaling and Microglial Proliferation by Anti-CD45RO: Role of Hck and This information is current as Phosphatidylinositol 3-Kinase/Akt of October 7, 2021. Hyeon-Sook Suh, Mee-Ohk Kim and Sunhee C. Lee J Immunol 2005; 174:2712-2719; ; doi: 10.4049/jimmunol.174.5.2712

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Signaling and Microglial Proliferation by Anti-CD45RO: Role of Hck Tyrosine Kinase and Phosphatidylinositol 3-Kinase/Akt1

Hyeon-Sook Suh, Mee-Ohk Kim,2 and Sunhee C. Lee3

Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macro- phage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced mi-

croglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 Downloaded from (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 http://www.jimmunol.org/ tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases. The Journal of Immunology, 2005, 174: 2712–2719.

icroglia are resident brain macrophages essential for produced by activated astrocytes, macrophages and microglia, and the maintenance of normal brain physiology as well as inflammatory cells (12–15). M response to injury (1, 2). Microglia are activated in Although GM-CSF is an important growth factor for microglia several CNS disorders of varying etiologies such as degenerative, and macrophages, little is known about the mechanism by which inflammatory, infectious, and ischemic disorders. Mounting evi- GM-CSF induces proliferation of primary human macrophages. dence indicates that activated microglia contribute to neuronal in- The GM-CSF receptor consists of a low-affinity ␣ subunit by guest on October 7, 2021 jury by producing inflammatory and neurotoxic mediators (1, 3, 4). (CD116) specific to GM-CSF and a high-affinity ␤c subunit Suppression of microglial activation may thus lead to reduction of (CD131) that is shared with IL-3 and IL-5 (16–19). ␤c lacks in- neural damage. The attributes of activated microglia in vivo in- trinsic kinase activity but ligand binding induces tyrosine phos- clude ameboid transformation, increased (or de novo) expression phorylation of ␤c as well as a number of cytoplasmic proteins of activation Ags such as class II MHC, scavenger receptors, including various kinases and adaptor proteins. This results in the CD11b/c and CD45 (5, 6), and proliferation. Although microglia in activation of several cell signaling pathways including the Jak/Stat, mature CNS are considered terminally differentiated, they can re- Ras/Erk MAPK, and the PI3K/Akt pathways (for review, see Refs. enter the cell cycle following injury. In the rat facial axotomy 16, 20, and 21). In addition, serine phosphorylation (S585) of the model, microglia proliferate in the regenerating facial nucleus co- common ␤ chain (␤c chain)4 has been shown to mediate cell sur- inciding with the increase in CSF receptor expression (7, 8). Mi- vival through activation of NF-␬B and induction of the antiapop- croglia are the main dividing CNS cell type in experimental aller- totic protein bcl-2 (22, 23). Because many of these studies are gic encephalomyelitis (9), cortical stab wound model (10), and performed in murine cells or cell lines with overexpression of re- experimental Wallerian degeneration (11). Microglia proliferate in ceptor proteins or signaling molecules, we examined the GM-CSF response to growth factors such as GM-CSF and M-CSF that are signal transduction pathway involved in proliferation of primary human microglial cells. CD45 is a transmembrane protein tyrosine phosphatase (PT- Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461 Pase) expressed in all hemopoietic cells including lymphocytes, Received for publication August 18, 2004. Accepted for publication December monocytes, and granulocytes (24–26). Several isoforms of CD45 14, 2004. exist as a result of the alternative splicing of exons that encode the The costs of publication of this article were defrayed in part by the payment of page extracellular domain. All CD45 isoforms possess identical trans- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. membrane domains and a cytoplasmic tail with two tandem PT- 1 This study was supported by National Institutes of Health RO1 MH55477 and Pase homology domains (D1 and D2). CD45 enhances T cell and AI44641 to S.C.L. 2 Current address: Department of Neurology, Massachusetts General Hospital, Bos- ton, MA 02129. 3 Address correspondence and reprint requests to Dr. Sunhee C. Lee, Department of 4 Abbreviations used in this paper: ␤c chain, common ␤ chain; PTPase, protein ty- Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, rosine phosphatase; WT, wild type; KE, kinase inactive; p, phosphorylated; T, total; NY 10461. E-mail address: [email protected] Grb2, -bound protein 2.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2713

BCR signaling and plays an important role in lymphocyte devel- Stock solutions were made in DMSO and further dilutions were made in opment and function, as shown in humans and mice with altered DMEM-containing serum. Vehicle controls were used to determine the CD45 expression that develop SCID (27–30). The best specificity of the drug. known molecular targets of CD45 are Src family tyrosine kinases Cell treatment (such as and Lyn) that play crucial roles in development a