Superantigen-Induced Steroid Resistance Depends on Activation of Phospholipase C β2 Auke P. Verhaar, Manon E. Wildenberg, Marjolijn Duijvestein, Anne Christine W. Vos, Maikel P. This information is current as Peppelenbosch, Mark Löwenberg, Daniel W. Hommes and of September 26, 2021. Gijs R. van den Brink J Immunol 2013; 190:6589-6595; Prepublished online 20 May 2013; doi: 10.4049/jimmunol.1202898 Downloaded from http://www.jimmunol.org/content/190/12/6589 References This article cites 17 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/190/12/6589.full#ref-list-1 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 by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication *average 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 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Superantigen-Induced Steroid Resistance Depends on Activation of Phospholipase Cb2 Auke P. Verhaar,*,† Manon E. Wildenberg,† Marjolijn Duijvestein,† Anne Christine W. Vos,* Maikel P. Peppelenbosch,‡ Mark Lo¨wenberg,† Daniel W. Hommes,*,x and Gijs R. van den Brink† The glucocorticoid receptor is present in a TCR-associated complex, which includes the Src family tyrosine kinase Lck. Gluco- corticoids rapidly dissociate this complex, resulting in the inhibition of canonical Lck-phospholipase C (PLC)g–dependent TCR signaling. The relative importance of this nongenomic role for the glucocorticoid receptor compared with its direct transcriptional effects is not known. Superantigens induce a state of steroid resistance in activated T cells. It was reported that, in addition to canonical Lck-PLCg signaling, superantigens can activate a noncanonical G protein–PLCb–dependent signaling pathway. In this study, we show that staphylococcal enterotoxin B activates a Gaq and PLCb2–dependent pathway in human T cells. We find that Downloaded from this pathway bypasses the need for canonical Lck-PLCg signaling in T cell activation and renders superantigen-stimulated T cells insensitive to glucocorticoids in vitro. We show that the PLCb inhibitor U-73122 sensitizes staphylococcal enterotoxin B–treated mice to dexamethasone in vivo. In conclusion, we find that effects of glucocorticoids on TCR-induced T cell proliferation are mainly nongenomic and can be bypassed by the activation of an Lck-independent signaling pathway. The Journal of Immunol- ogy, 2013, 190: 6589–6595. http://www.jimmunol.org/ lucocorticoids bind the glucocorticoid receptor (GR), TCR-associated GR and the resulting inactivation of Lck versus which subsequently dimerizes and translocates from the transcriptionally mediated effects of the GR is not known. If G cytosol to the nucleus. The GR can bind to specific DNA the dissociation and inactivation of Lck are an important part of sequences and alter the transcriptional activity of a wide range of the mechanism of action of steroids, this predicts that signals genes, many of which play a role in the immune system. However, that bypass Lck render T cells relatively insensitive to steroids. the GR also can affect cell signaling in its DNA-unbound state Superantigens activate T cells by directly cross-linking MHC (so-called “nongenomic” or “nontranscriptional effects”) (1). We class II to the TCR as unprocessed proteins. Therefore, super- found previously that the GR is present in a TCR-associated antigens bypass the specificity of conventional Ag recognition and by guest on September 26, 2021 complex that contains the Src-like tyrosine kinase Lck (2). Glu- can activate up to 20% of the total number of T cells (5). The cocorticoids rapidly dissociate the GR from this complex, leading massive activation of the adaptive immune system that ensues to the inactivation of Lck and downstream signaling pathways (2, helps the pathogen to evade an appropriate immune response. The 3). Lck mediates canonical TCR signaling via activation of ZAP- release of cytokines, such as IL-1, TNF-a, and IFN-g, causes the 70, phosphorylation of LAT, and the assembly of a multiprotein signs and symptoms of toxic shock syndrome (TSS) (6). Super- complex. A key component of this complex is phospholipase C antigen signaling is an excellent model to address the question of (PLC)g, which converts phosphatidylinositol 4,5-bisphosphate to the relevance of glucocorticoid-mediated inhibition of upstream inositol 3,4,5-triphosphate and diacylglycerol. Generation of ino- T cell signaling. Superantigens were reported to cause steroid sitol 3,4,5-triphosphate activates Ca2+ signaling and leads to the resistance in T cells (7). More importantly, it was shown that nuclear translocation of NFAT, whereas diacylglycerol activates superantigens can activate a noncanonical G protein–PLCb sig- PKC and RAS-MAPK signaling (see Fig. 1A) (4). Glucocorticoids naling pathway in addition to canonical Lck-PLCg signaling (8). inhibit T cell activation, but the functional relevance of loss of If steroid-mediated inhibition of T cell proliferation depends mainly on nongenomic inhibition of canonical T cell signaling, this pre- dicts that superantigen-mediated glucocorticoid resistance depends *Department of Gastroenterology and Hepatology, Leiden University Medical † on PLCb signaling. Center, 2300RC Leiden, The Netherlands; Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Academic Medical In this study, we find that superantigen-mediated steroid re- Center, 1105AZ Amsterdam, The Netherlands; ‡Department of Gastroenterology and sistance depends on Gaq-mediated activation of PLCb2, which Hepatology, Erasmus Medical Center, 3015CA Rotterdam, The Netherlands; and xCenter for Inflammatory Bowel Diseases, University of California Los Angeles, bypasses dependence on Lck-PLCg signalinginprimaryhuman Los Angeles, CA 90095 T cells in vitro. We go on to show the efficacy of glucocorticoid Received for publication October 18, 2012. Accepted for publication April 16, 2013. therapy in combination with a PLCb inhibitor in a mouse model of This work was supported by an unrestricted research grant from Giuliani Pharma TSS in vivo. These findings have implications for our understanding (Milan, Italy). of the mechanism of action of glucocorticoids. They may point to Address correspondence and reprint requests to Dr. Gijs R. Van den Brink, Academic novel therapeutic strategies to counter glucocorticoid resistance. Medical Center, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands. E-mail address: [email protected] Abbreviations used in this article: GR, glucocorticoid receptor; PBL, peripheral Materials and Methods blood leukocyte; PLC, phospholipase C; RT, room temperature; SEB, staphylococcal Abs and reagents enterotoxin B; siRNA, small interfering RNA; TSS, toxic shock syndrome. The following Abs were purchased from Santa Cruz Biotechnology Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (Heidelberg, Germany): anti-Lck (3A5), anti–b-actin, anti-PLCb1 (D-8), www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202898 6590 SUPERANTIGEN-INDUCED STEROID RESISTANCE anti-PLCb2 (Q-15), anti-PLCb3 (C-20), and anti-pTyr (PY20). Anti– Small interfering RNA transfection phospho-Zap70 (Tyr319)/Syk (Tyr352), anti–phospho-Src (Tyr410), anti– phospho-LAT (Tyr171), and the PI3K inhibitor LY924002 were purchased Two independent small interfering RNAs (siRNAs) targeting GNA11 from Cell Signaling Technology (Beverly, MA). Anti-PLCg1 (2B1) was (siRNA #1: CGACAAGAUCAUCUACUCAtt, siRNA #2: GCAUCAUC- purchased from Abcam (Cambridge, MA). Anti-CD3 and anti-CD28 were GAGUACCCUUUtt), GNAq (siRNA #1: UCGUCUAUCAUAGCAUUC- purchased from Sanquin (Amsterdam, The Netherlands). PMA, ionomycin, Ctg, siRNA #2: UAUCUUCAUCAGAGUAUCCtg), and PLCb2 (siRNA staphylococcal enterotoxin B (SEB), and the PLCb inhibitor U-73122 were #1: GGCUACUACUUAUACUGGAtt, siRNA #2: CCAUGACCACAGA- purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). PHA-M CAUCUUtt) were purchased from Ambion Applied Biosystems (Austin, was purchased from Roche (Mannheim, Germany). TX). Lymphocytes were transfected by electroporation using Amaxa’s Nucleofector (Lonza, Cologne, Germany). 6 Mice A total of 10 3 10 lymphocytes was electroporated per condition, accordingtoprotocol,using30pmolsiRNAandtheHumanTCell Eight-week-old female C57BL/6 wild-type mice were purchased from Nucleofector Kit for unstimulated T cells (Lonza, Cologne, Germany). Harlan (Boxmeer, The Netherlands) and housed in pathogen-free con- The media were replaced after 4 h. Experiments were performed 48 h ditions. All mice received a single i.p. injection with a total volume of posttransfection. 500 ml. Depending on the group, mice received 20 mg dexamethasone and/or 10 mg U-73122. All mice received 8.8 ml DMSO, 100 mgSEB. Quantification and data analysis Twenty-four hours later, the mice were euthanatized with