Expression of the Ryanodine Receptor Isoforms in Immune Cells Eiji Hosoi, Chiharu Nishizaki, Kathleen L. Gallagher, Hadley W. Wyre, Yoshinobu Matsuo and Yoshitatsu Sei This information is current as of September 23, 2021. J Immunol 2001; 167:4887-4894; ; doi: 10.4049/jimmunol.167.9.4887 http://www.jimmunol.org/content/167/9/4887 Downloaded from References This article cites 28 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/167/9/4887.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 September 23, 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 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Expression of the Ryanodine Receptor Isoforms in Immune Cells1 Eiji Hosoi,*† Chiharu Nishizaki,‡ Kathleen L. Gallagher,* Hadley W. Wyre,* Yoshinobu Matsuo,‡ and Yoshitatsu Sei2* Ryanodine receptor (RYR) is a Ca2؉ channel that mediates Ca2؉ release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type .RYR (RYR1) was expressed in CD19؉ B lymphocytes, but less frequently in CD3؉ T lymphocytes and in CD14؉ monocytes 1 Type 2 RYR (RYR2) was mainly detected in CD3؉ T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1␣ (MIP1␣) or TGF-␤. Type 3 RYR (RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was not associated with specific cell lineage. We showed that the RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca2؉ Downloaded from release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant (induction of RYR mRNA with Ca2؉ channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM had no effect on Ca2؉ release, whereas 4CmC (<400 ␮M) caused Ca2؉ release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1␣, or TGF-␤. Our results dem- onstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and /TGF-␤ suggests roles in regulating Ca2؉-mediated cellular responses during the immune response. The Journal of Immunology, http://www.jimmunol.org 2001, 167: 4887–4894. alcium ions play a critical role in the activation of the after surface receptor ligation (7, 8). Phospholipase C␥ is then immune cells that are responsible for cellular and hu- recruited to an upstream tyrosine kinase via its SH2 domains and C moral immunity (1–3). In the process of immune re- activated by phosphorylation. Phospholipase C␥ activation leads sponses, Ag binding to the surface receptor stimulates the immune to the hydrolysis of phosphatidylinositol 4,5-bisphosphate, yield- cells to eventually eliminate foreign Ags. Elevation of intracellular ing IP3 and diacyl glycerol. IP3 then mediates the activation of 2ϩ 2ϩ 3 2ϩ free Ca concentration ([Ca ]i) is an early and critical event in Ca release from stores in the endoplasmic reticulum through the by guest on September 23, 2021 the biochemical cascade of signal transduction pathways, which IP3 receptor. Therefore, calcium mobilization after receptor cross- include activating specific transcription factors (i.e., NF-␬B, JNK, linking in the immune cells has been explained almost solely by NF-AT, etc.), and thus in a variety of later events in the immune IP -mediated mechanisms. ϩ 3 cell activation (4). Therefore, the regulation of Ca2 signaling de- 2ϩ Although IP3 is a key messenger regulating [Ca ]i, recent stud- termines the ultimate response of an immune cell. ies have postulated the possibility that the ryanodine receptor An early manifestation of mitogen- or cell surface receptor- (RYR) contributes to the IP -insensitive component of Ca2ϩ sig- 2ϩ 3 stimulated immune cell activation is a biphasic increase in [Ca ]i naling in immune cells (9–12). The RYR was originally found in 2ϩ which is the result of rapid Ca release from intracellular stores the sarcoplasmic reticulum of skeletal muscle (type 1 receptor; 2ϩ 2ϩ followed by sustained Ca influx through store-operated Ca RYR1) and cardiac muscle (type 2 receptor; RYR2) (13–15). Ca2ϩ 2ϩ channels (SOC) (5, 6). Ca release from intracellular stores is release from the sarcoplasmic reticulum through these receptors consequent to inositol 1,4,5-trisphosphate (IP3) formation. The ac- plays a central role in regulating the contraction of skeletal and tivation of multiple protein tyrosine kinases occurs immediately cardiac muscle fibers. A third type of RYR (type 3 receptor; RYR3) has been detected in specific regions of the brain, non- *Department of Anesthesiology, Uniformed Services University of the Health Sci- muscle tissues, and also skeletal muscle (16–18). We recently ences, Bethesda, MD 20814; †Department of Medical Technology, School of Medical demonstrated that human B cells express a RYR that is identical Sciences, University of Tokushima, Tokushima, Japan; and ‡Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan with skeletal muscle type I by RFLP studies and sequencing anal- Received for publication May 30, 2001. Accepted for publication August 23, 2001. ysis of partially cloned cDNA (12). In addition, 4-chloro-m-cresol (4CmC), a potent activator of the RYR (19), induced Ca2ϩ release The costs of publication of this article were defrayed in part by the payment of page 2ϩ charges. This article must therefore be hereby marked advertisement in accordance after depleting IP3-sensitive Ca pools in B cells (12). These with 18 U.S.C. Section 1734 solely to indicate this fact. results suggested that human B cells express functional RYR1 that 1 This work was supported by grants from the Uniformed Services University of the is involved in regulating Ca2ϩ signaling, perhaps in conjunction Health Sciences (R08078) and the Malignant Hyperthermia Association of the United States (G18090). with the IP3 receptor. For T cells, expression of the RYR has been found in human Jurkat T cells (10, 11) and murine T lymphoma 2 Address correspondence and reprint requests to Dr. Yoshitatsu Sei, Department of 3 Anesthesiology, Uniformed Services University of the Health Sciences, 4301 Jones cells (9). In both T cell lines, cyclic ADP-ribose increased [ H]ry- Bridge Road, Bethesda, MD 20814-4799. E-mail address: [email protected] anodine binding and induced Ca2ϩ release from intracellular Ca2ϩ 3 2ϩ 2ϩ Abbreviations used in this paper: [Ca ]i, intracellular free Ca concentration; stores (9, 10). The isoform of the RYR expressed in Jurkat T cells 4CmC, 4-chloro-m-cresol; IP3, inositol 1,4,5-trisphosphate; mIg, membrane Ig; RYR, ryanodine receptor; SOC, store-operated Ca2ϩ channel; SDF-1, stromal cell-derived was identified to be type 3 (10, 11). Therefore, the RYR3 has been ␣ ␣ 2ϩ factor 1; MIP1 , macrophage-inflammatory protein-1 . proposed to control [Ca ]i in response to cyclic ADP-ribose Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 4888 RYR ISOFORMS IN IMMUNE CELLS during T cell activation (10). These findings of the RYR in T and Selective RT-PCR followed by RFLP analysis B cells allowed us to hypothesize that the RYRs are more widely 2ϩ Total RNA was extracted using the SV Total RNA Isolation System (Pro- expressed and responsible for regulating [Ca ]i in immune cells mega, Madison, WI), and reverse transcription was performed to the first than currently thought. strand of cDNA using a cDNA synthesis kit (Promega). Synthesized cDNA In this study, we have investigated expression of all three iso- was then amplified by RT-PCR using a primer set which selectively am- forms of the RYR in human primary T cells, B cells, and mono- plifies specific isoform of the RYR. Using the same downstream primer, 5Ј-dC-AGATGAAGCATTTGGTCTCCAT-3Ј, and an isoform-specific cytes using selective RT-PCR followed by RFLP analysis. We also upstream primer: JBR1, 5Ј-dG-ACATGGAAGGCTCAGCTGCT-3Ј; examined a total of 164 human hemopoietic cell lines (36 T cell, JBR2, 5Ј-dAAGGAGCTCCCCACGAGAAGT-3Ј; and JBR3, 5Ј-dAA 92 B cell, 19 myelomonocytic, 11 megakaryocytic, 3 erythrocytic, GAGGAAGAAGCGATGGT-3Ј,anϳ1200-bp product was recognized and 3 nonlymphocytic, nonmyelocytic) to determine the lineage from the 3Ј-regions of RYR1, RYR2, and RYR3, respectively. PCR am- plifications were conducted using the Expand Long PCR system (Boehr- and differentiation specificity of the expression of the 3 RYRs. The inger Mannheim, Indianapolis, IN). PCR was performed in a 50-␮l reaction possibility that any isoform of RYR is induced by stimulation with mixture containing 100 ng DNA, 15 pmol of each primer, 0.5 mM dNTPs, mitogens, chemokines, and other stimuli were investigated to gain 2.5 U Expand Long polymerase mixture and Expand Long PCR buffer 3 insight of the roles of this Ca2ϩ release channel in immune func- (Boehringer Mannheim). The PCR amplification conditions were 95°C for tion. Finally, to verify the functional expression of the RYRs, 2 min, followed by 40 cycles of 95°C for 1 min, 55°C for 2 min, and 68°C 2ϩ for 3 min, followed by a 7-min extension at 68°C.