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Enhancing IL-2 Gene Transactivation Cells by Increasing Mrna Stability

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Enhancing IL-2 Gene Transactivation Cells by Increasing Mrna Stability CD28 Costimulation Augments IL-2 Secretion of Activated Lamina Propria T Cells by Increasing mRNA Stability Without Enhancing IL-2 Gene Transactivation This information is current as of September 29, 2021. Rivkah Gonsky, Richard L. Deem, Doo Han Lee, Alice Chen and Stephan R. Targan J Immunol 1999; 162:6621-6629; ; http://www.jimmunol.org/content/162/11/6621 Downloaded from References This article cites 36 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/162/11/6621.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 • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD28 Costimulation Augments IL-2 Secretion of Activated Lamina Propria T Cells by Increasing mRNA Stability Without Enhancing IL-2 Gene Transactivation1 Rivkah Gonsky,* Richard L. Deem,* Doo Han Lee,† Alice Chen,* and Stephan R. Targan2* The pathways leading to activation in lamina propria (LP) T cells are different from peripheral T cells. LP T cells exhibit enhanced IL-2 secretion when activated through the CD2 pathway. Coligation of CD28 leads to synergistic enhancement of IL-2 secretion. Previous studies have characterized the CD28 augmentation of TCR-mediated signaling in peripheral blood T cells through transcriptional activation of an IL-2 promoter CD28 response element (CD28RE), along with enhanced mRNA stability. This study characterized molecular events involved in CD28 costimulation of IL-2 production in LP mononuclear cells (LPMC). LPMC exhibited increased IL-2 production in response to CD28 costimulation, compared with cells activated through CD2 alone. IL-2 Downloaded from secretion was paralleled by increased expression of IL-2 mRNA, resulting from enhanced IL-2 mRNA stability. In contrast to transcriptional activation in PBMC, EMSA revealed that CD28 coligation of CD2-activated LPMC does not result in increased binding of trans-factors to the CD28RE, nor did Western blots detect changes in I-kBa or I-kBb levels following CD28 coligation. Furthermore, CD28 coligation fails to enhance IL-2 promoter-reporter or RE/AP construct expression in CD2-activated LPMC. The results reported herein indicate that the molecular mechanisms involved in CD28 cosignaling and regulation of IL-2 secretion in LP T cells are unique to that compartment and differ from those seen in peripheral blood T cells. These observations suggest http://www.jimmunol.org/ a biological significance for different mechanisms of IL-2 activation in initiation and maintenance of the cytokine repertoire found in the mucosa. The Journal of Immunology, 1999, 162: 6621–6629. L-2 is secreted by activated T cells and plays an important CD28 costimulated peripheral blood (PB)3 lymphocytes is medi- role in initiation and regulation of cellular immune responses. ated transcriptionally as well as posttranscriptionally. CD28 co- I Evidence indicates that regulation of IL-2 expression by stimulation activates transcription of IL-2 through the binding of PBMC takes place largely at the level of transcriptional regulation. kB-like transcription factors to a promoter motif defined as the Transcriptional regulation of the IL-2 promoter has been exten- CD28 response element (CD28RE) (8). The Rel family of proteins, sively studied in PBMC and T cell lines following TCR activation p50, p65, and c-Rel, are components of the transcriptional complex by guest on September 29, 2021 (1). The 300-bp promoter region of the IL-2 gene upstream of the binding to the CD28RE in PBMC (9). A major component of k transcriptional start site contains multiple binding sites for tran- regulation of B-like transcription factors is the control of their k scriptional activating factors including NF-AT, NF-kB, AP-1 and intercellular localization. B-like transcription factors are nor- OCT-binding proteins (2). Cooperative binding of these trans-act- mally shielded within the cytoplasm through their physical inter- action with the I-kB inhibitory protein (10, 11). T cell activation ing factors appears to determine the balance of transcriptional ac- results in the rapid and transient decrease of I-kB, thereby releas- tivation. In addition to transcriptional regulation of IL-2 mRNA ing the transcriptionally active kB factors and increasing translo- levels, an inducible degradation mechanism has been reported (3, cation to the nucleus (10, 11). CD28-mediated IL-2 gene expres- 4). Production of IL-2 can be enhanced markedly by treatment sion in PBMC is regulated likewise by stabilization of IL-2 with translational inhibitors such as cycloheximide without di- mRNA. The IL-2 mRNA possesses AU-rich sequences within the rectly affecting the rate of IL-2 transcription (5). 39 untranslated region identified as the target signal for rapid Maximal activation of T cells requires two signals, the first of mRNA degradation (12). While CD28 costimulation enhances which is generated by engagement of the TCR, with a second IL-2 mRNA stability, as well as the stability of other cytokines, no signal provided by a costimulatory molecule. One major costimu- preferential enhancement in the stability of c-myc or c-fos was latory T cell surface molecule is CD28 (6, 7). IL-2 expression by detected, notwithstanding the presence of AU-rich motifs in the mRNA (13). Thus, regulation of IL-2 RNA t1⁄2 appears to be com- plex, involving specific additional pathways that may be sensitive to the events initiating T cell activation (14). *Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, Los The activation pathways of PB T cells are different from those Angeles, CA 90048; and † Seoul Surgical Clinic Banpo-4-dong 57-3, Seochogu, Seoul, Korea of lamina propria (LP) T cells (15, 16). LP T cells do not respond Received for publication August 19, 1998. Accepted for publication March 16, 1999. well to activation via the TCR/CD3 receptor. However, they do exhibit increased proliferation and cytokine production when ac- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance tivated via the CD2 pathway (16, 17). CD28 coligation further with 18 U.S.C. Section 1734 solely to indicate this fact.. enhances the activation of LP T cells. LP T cells are generally 1 This work was supported by U.S. Public Health Service Grants DK-43211 and DK-46763 and by Cedars Sinai Medical Center IBD Research Funds. 2 Address correspondence and reprint requests to Dr. Stephan R. Targan, Inflamma- 3 Abbreviations used in this paper: PB, peripheral blood; LP, lamina propria; LPMC, tory Bowel Disease Research Center, Cedars-Sinai Medical Center, 8700 Beverly LP mononuclear cells; UC, ulcerative colitis; CD, Crohn’s disease; CD28RE, CD28 Boulevard, D4063, Los Angeles, CA 90048. response element. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 6622 TRANSACTIVATION OF THE IL-2 PROMOTER thought to achieve a more intense activation state than PB T cells. intestinal specimen was washed with HBSS, and the mucosa was dissected The intensity can be amplified further as has been demonstrated in away from the underlying layers. The mucosal layer was incubated in a conditions of dysregulated inflammation such as Crohn’s disease shaking water bath (100 rpm) in calcium- and magnesium-deficient HBSS, containing 1 mM EDTA, 50 mg/ml gentamicin, 100 U/ml penicillin, 100 (CD) and ulcerative colitis (UC). These disorders are characterized mg/ml streptomycin, and 50 mg/ml fungizone, with the solution changed by widespread intestinal inflammation and an enhanced T cell ac- every 30 min until the supernatant was free of epithelial cells. The remain- tivation state with increased production of inflammatory mediators ing LP was minced into 1- to 2-mm pieces and digested for 10 min in including IL-2 (18). RPMI 1640 containing 10% FCS, 0.5 mg/ml collagenase B (Boehringer Mannheim, Indianapolis, IN), 1 mg/ml hyaluronidase (Sigma, St. Louis, We have developed a model of LP-like T cells by coculturing MO), 0.1 mg/ml DNase I (Sigma), 50 mg/ml gentamicin, 100 U/ml peni- PB T cells in the presence of irradiated Daudi B cells and IL-2, cillin, 100 mg/ml streptomycin, and 50 mg/ml fungizone in shaker water which mimics a phenotype of CD2 pathway-dominant cytokine bath (100 rpm). The supernatant was collected, filtered through 110-mm secretion similar to that seen in lamina propria lymphocytes. Pre- nylon mesh (Spectrum Laboratory Products, Houston, TX), and centri- 3 vious studies have shown that the LP-like T cell model is validated fuged at 500 g for 5 min. The cell pellet was resuspended in 15 ml and centrifuged at 30 3 g for 5 min to remove epithelial and other large cells. by its functional equivalency to LP T cells, not only with regard to The supernatant was removed and lymphocytes were isolated by separation CD2 pathway dominance, but with identical phosphorylation pat- on Ficoll-Hypaque gradients. The cells were then washed three times with terns upon CD2 pathway stimulation as well (16). Furthermore, HBSS and resuspended in RPMI 1640 containing 10% FCS.
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