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-␬B␣ or I-␬B␤ 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. ␬B-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 ␬ transcriptional start site contains multiple binding sites for tran- regulation of B-like transcription factors is the control of their ␬ scriptional activating factors including NF-AT, NF-␬B, 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 interaction with the I-␬B 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-␬B, thereby releas- tivation. In addition to transcriptional regulation of IL-2 mRNA ing the transcriptionally active ␬B 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). 3Ј 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 complex, 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.

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 ␮g/ml gentamicin, 100 U/ml penicillin, 100 (CD) and ulcerative colitis (UC). These disorders are characterized ␮g/ml streptomycin, and 50 ␮g/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 ␮g/ml gentamicin, 100 U/ml peni- PB T cells in the presence of irradiated Daudi B cells and IL-2, cillin, 100 ␮g/ml streptomycin, and 50 ␮g/ml fungizone in shaker water which mimics a phenotype of CD2 pathway-dominant cytokine bath (100 rpm). The supernatant was collected, filtered through 110-␮m secretion similar to that seen in lamina propria lymphocytes. Pre- nylon mesh (Spectrum Laboratory Products, Houston, TX), and centri- ϫ 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 ϫ 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. PBMC were recent studies have provided evidence that the LP-like T cell ac- isolated from normal healthy volunteers by separation on Ficoll-Hypaque gradients. tivation model is most representative of the activated state of T cells present in inflamed CD mucosa, as evidenced by CD2-acti- Induction of LP-like T cells

vated up-regulation of AP-1-transactivating factors with similar Downloaded from Mononuclear cells from Ficoll-Hypaque gradients were cultured in RPMI kinetic profiles (19). The LP-like T cell model has very similar 1640 with 10% FCS with a 1Ϻ5 ratio DaudiϺlymphocytes plus 10 U/ml activation properties to those expressed by LP mononuclear cells rIL-2 for 5 days (16). Daudi were irradiated with 3000 rad and washed (LPMC) from inflamed CD mucosa, and allows investigations of three times in HBSS before addition to cultures. Following a 5-day culture, the mechanism of cytokine gene regulation to be performed ini- LP-like cells were washed with HBSS to remove dead Daudi cells (at this point, there were virtually no live Daudi cells in the culture as determined tially on a more easily manipulated experimental system. by flow cytometry).

Little is known regarding the underlying mechanisms regulating http://www.jimmunol.org/ IL-2 secretion in LP T cells. Recent studies have suggested that Stimulation of mononuclear cells regulation of IL-2 production in LPMC following CD2 activation For stimulation through the CD2 receptor, LPMC and LP-like T cells were may involve a distinct transcriptional regulatory mechanism dif- stimulated with 0.1 ␮g anti-CD2 Abs (both CB6 and GD10 clones)/106 ferent from that observed in T cell lines (19). The purpose of this cells at 37°C for the times indicated for each experiment. CD28 costimu- ␮ study was to determine whether enhanced IL-2 secretion by CD2 lation was carried out with 0.1 g anti-CD28 Ab. Stimulation of T cells with anti-CD2 Abs did not require further cross-linking, since the combi- and CD28 costimulation was the product of unique events that nation of two anti-CD2 Abs directed against different epitopes was suffi- regulate transactivation of the IL-2 gene in LP T cells. The data in cient to induce activation. this study support the hypothesis that enhanced CD28-mediated IL-2 secretion following coligation with CD2 does not result from IL-2 bioassay by guest on September 29, 2021 increased transactivational activity of the CD28RE or other ele- IL-2 activity was determined using a CTLL-2 bioassay (21). rIL-2 stan- ments within the first 600 bp of the IL-2 promoter. The results dards and dilutions of samples were added to 96-well flat-bottom microtiter ϫ 3 suggest that the molecular mechanisms involved in CD28 cosig- plates (Costar, Cambridge, MA) containing 5 10 CTLL-2/well and incubated for 48 h at 37°C. Wells were then pulsed with 2 ␮Ci/well naling and regulation of IL-2 secretion in LP T cells are unique [3H]thymidine for 4 h. Wells were harvested using a Harvester 96 (Tomtec, from those involved in the peripheral compartment. Orange, CT) microplate harvester and counted using a Microbeta 1450 liquid scintillation counter (Wallac, Gaithersburg, MD). Data acquisition and analysis was performed using the ELISA Master program for Macin- Materials and Methods tosh computers, developed by R. L. Deem. Target cells and culture media Northern blot analysis The murine T cell line, CTLL-2, was obtained from American Type Cul- ture Collection (Manassas, VA) and was maintained at 37°C in a humid- Total cellular RNA was extracted using the Qiagen RNeasy kit (Chats- ified atmosphere of 5% CO2 in RPMI 1640 supplemented with 10% FCS worth, CA). RNA was electrophoretically separated on a denaturing 1% with 10 U/ml rIL-2 (R&D Systems, Minneapolis, MN). The human B cell agarose gel containing 7% formaldehyde. Gels were transferred to nylon line, Daudi, was obtained from American Type Culture Collection and membrane (Amersham, Arlington Heights, IL) and hybridized to 32P-la- maintained at 37°C in a humidified atmosphere of 5% CO2 in RPMI 1640 beled DNA probe. Isolated cDNA insert was labeled by random priming supplemented with 10% FCS. and used at 106 cpm/ml of hybridization buffer. Blots were prehybridized (50% formamide, 0.75 M NaCl, 75 mM sodium citrate, 1ϫ Denhardt so- Monoclonal Abs lution, 25 mM sodium phosphate (pH 6.5), and 100 ␮g/ml of sheared Anti-CD2 mAbs (clones CB6 and GD10) were a gift from Chris Benjamin salmon sperm DNA) at 42°C for 2 h and hybridized overnight in prehy- (Biogen, Cambridge, MA). Anti-CD28 mAB ascites, clone 9.3, was ob- bridization solution containing labeled probe and 10% dextran sulfate. tained from Bristol-Meyers Squibb Pharmaceutical Research Institute Nuclear run-on (Princeton, NJ). The ascites was purified over a protein G column and quantified by ELISA. A total of 5 ϫ 107 LPMC were stimulated and nuclei isolated as previously described (22). In vitro transcription was carried out at 26°C for 20 min in Purification of LPMC and PBMC transcription buffer (50 mM HEPES (pH 7.9), 100 mM KCl, 2 mM DTT, 30 ␮M EDTA, 1 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 2 mM MnCl ,35 Intestinal specimens were obtained from patients undergoing surgical re- 2 mM (NH ) SO , 8.8 mM creatine phosphate, 40 ␮g/ml creatine phosphoki- section of the colon (with colon carcinoma or treatment-resistant CD or 4 2 4 nase), and 100 ␮Ci of [␣-32P]UTP. Labeled mRNA transcripts were puri- UC) at Cedars-Sinai Medical Center, Los Angeles, CA. Approval for the fied using Qiagen Rneasy kit for liquid samples and hybridized to 2 ␮gof use of human subjects was obtained from the Institutional Review Board at cDNA insert immobilized on a nylon membrane. Cedars-Sinai Medical Center. In this study, all tissue specimens were taken from an uninvolved area of resected colon from patients with colonic car- Preparation of nuclear protein extracts cinoma (normal), involved areas of patients with UC, as well as from uninvolved and involved areas of patients with CD. LPMC were isolated Nuclear protein extractions were carried out with 5 to 10 ϫ 106 LPMC or using a technique modified from that described previously (20). Briefly, the LP-like T cells. Following activation, cells were centrifuged, washed in The Journal of Immunology 6623

cold PBS, and kept on ice for subsequent extraction steps. The cell pellet was resuspended in 0.9 ml of RSB (10 mM Tris (pH 7.4), 10 mM NaCl, 3 ␮ ␮ mM MgCl2, 0.5 mM DTT, 2 M leupeptin, 1 g/ml aprotinin, 1 mM PMSF, and 0.1 mM EGTA), and 0.1 ml of 5% NP-40 was added. Samples were mixed by gentle inversion and kept on ice for 10 min followed by centrifugation. The pellet was resuspended in 25–60 ␮l (volume is dependent on the starting number of cells) of cold buffer C (20 mM HEPES (pH

7.4), 0.42 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM DTT, 20 ␮M leupeptin, 10 ␮g/ml aprotinin, and 1 mM PMSF). Samples were incubated on ice for 30–40 min during which time they were pipetted twice. Cellular debris was removed by centrifugation and nuclear proteins were diluted with an equal volume of buffer D (20 mM HEPES (pH 7.4), 50 mM KCl, 0.2 mM EDTA, 20% v/v glycerol, 0.5 mM DTT, 20 mM leupeptin, 10 ␮g/ml aprotinin, and 1 mM PMSF). Protein concentra- tions were determined by Coomassie Plus assay (Pierce, Rockford, IL).

EMSA FIGURE 1. Effect of costimulation by CD28 on IL-2 secretion in LPMC and LP-like T cells activated via the CD2 pathway. A, LPMC from ␥ 32 Double-stranded oligonucleotide was end-labeled with [ - P]ATP and T4 inflamed mucosa stimulated with 1 ␮g anti-CD2/106 cells or 1 ␮g anti- polynucleotide kinase. A total of 3–6 ␮g of nuclear extract protein was CD2/106 cells ϩ 1 ␮g anti-CD28/106 cells. B, PBL cultured 5 days in IL-2 incubated at 25°C with 0.25 mg/ml poly(dI-dC), in 20% glycerol, 5 mM MgCl , 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 50 mM Tris (pH with irradiated Daudi cells (LP-like T cells) prior to stimulation with anti- 2 ϩ ■ F ϩ Downloaded from 7.5) for 10 min. The oligonucleotide was then added (20,000 cpm) and the CD2 or anti-CD2 anti-CD28. , Anti-CD2 alone; , anti-CD2 anti- binding reactions were incubated for an additional 30 min. Specificity was CD28. Representative of three experiments with similar results is shown. determined by the addition of 100-fold excess unlabeled oligonucleotide as competitor. The DNA-protein complexes were separated from unbound probe on a prerun native 5% polyacrylamide gel in low ionic strength buffer (22.3 mM Tris (pH 7.4), 22.3 mM borate, and 0.5 mM EDTA (pH Results 8.0)). After 2 h, the gel was dried under vacuum and exposed to x-ray film. Costimulation by CD28 augments IL-2 secretion and increases The oligonucleotide used was the composite IL-2 CD28RE/AP site: (5Ј- http://www.jimmunol.org/ GTTTAAAGAAATTCCAAAGAGTCATCAG-3Ј). Nonspecific competi- mRNA accumulation in LPMC and LP-like T cells activated via tor oligonucleotide used was: (5Ј-GAGCCTGATTTCCCCGAAATGA the CD2 pathway Ј TGAGC-3 ). We have described previously a model developed using in vitro- activated PB T cells, known as LP-like T cells, which reproduce DNA constructs the CD2-driven cytokine secretion phenotype seen in LP T cells The human IL-2 cDNA clone and ␤-actin were obtained from American (16, 19). LP T cells and LP-like T cells are significantly more Type Culture Collection. A human IL-2 luciferase reporter plasmid con- responsive than PB T cells to activation via the CD2 pathway than taining a 600-bp fragment of the IL-2 promoter subcloned immediately 5Ј the CD3 pathway, as exhibited by the secretion of IL-2 and other to the luciferase gene has been described previously (23). The human cytokines (16). Coligation of CD2, but not CD3, with CD28 re- by guest on September 29, 2021 RE/AP and mutant RE/AP promoter-reporter constructs have been previously described (24) and were a generous gift from Virginia Smith-Shapiro sulted in a marked increase of IL-2 secretion in LPMC and LP-like and Arthur Weiss (University of California, San Francisco, CA). The plas- T cells (16). mid TRE2 luciferase, used to determine AP-1-dependent trans-activation, To assess the molecular mechanisms of CD28-enhanced IL-2 was generated by subcloning three copies of the collagenase AP-1 binding secretion, we first examined the kinetics of IL-2 production fol- sites into a luciferase reporter plasmid (a gift from M. Karin) (25). lowing activation of LPMC and LP-like T cells via the CD2 pathway in the presence or absence of CD28 costimulation. As shown Transfection in Fig. 1A, measurable amounts of IL-2 were detected in superna- Freshly isolated LPMC and PBMC were primed for transfection compe- tants of LPMC isolated from inflamed mucosa as early as 3 h tence by culturing for 16 or 20 h, respectively, in RPMI 1640 medium following CD2 activation. IL-2 levels continued to increase over containing 10% FCS, 50 mM 2-ME, and 1 ␮g/ml PHA-L (Sigma) as pre- 24 h. CD28 costimulation resulted in a striking increase of IL-2 viously described (19, 23). Cells were then washed and resuspended in 250 production at all time points measured. CD28 coligation of LP-like ␮ ϫ 7 l fresh medium at 2 10 cells/ml and electroporated in the presence of T cells likewise resulted in enhancement of IL-2 secretion at all 50 ␮g of reporter construct (250 V, 2250 microfarads, 48 ohms) using 4-mm (gap width) cuvettes in a BTX Electro Cell Manipulator (Genetron- time points tested (Fig. 1B). ics, San Diego, CA). After electroporation the cells were diluted in fresh To determine the molecular events associated with CD28-aug- medium, allowed to rest for 1 h prior to plating, and then stimulated with mented IL-2 production, mRNA was measured at time points fol- ϩ anti-CD2 or anti-CD2 CD28 mAbs for 4 h. Since transfection of a single lowing CD2 or CD2 ϩ CD28 activation of LPMC and LP-like T promoter was assayed for expression under different activation conditions, normalization for transfection efficiency was not necessary. However, mul- cells. Northern blot analysis of IL-2 mRNA from LPMC revealed tiple transfections with different preparation of reporter constructs gave that following activation by CD2, cytokine secretion was preceded similar results. Luminescence was measured using a Promega (Madison, by an increased expression of IL-2 mRNA. IL-2 mRNA was de- WI) luciferase assay kit and counted on a 6-detector Wallac (Gaithersberg, tectable within 1 h, peaked at 4 h, and then declined to barely MD) 1450 Microbeta liquid scintillation counter with coincidence counting detectable levels by 6 h (Fig. 2A). CD28 costimulation resulted in turned off. a striking elevation of IL-2 mRNA expression, as well as a pro- longed period of detectability. The early kinetics of IL-2 mRNA Western blotting accumulation appeared similar to that seen with CD2 activation Samples containing 10–15 ␮g of nuclear protein extracts were separated alone. IL-2 mRNA was detectable at 1 h following activation and on a SDS-polyacrylamide gel (10% polyacrylamide) and transferred by peaked at 4 h. However, while no IL-2 mRNA was detectable from electroblotting to nitrocellulose membrane (Hybond C, Amersham). The the CD2-activated cells at 24 h following activation, IL-2 mRNA membrane was blocked for 2 h with 5% nonfat milk in TBS with 0.1% Tween-20 and analyzed for immunoreactivity with antisera to I␬B␣ and was still detectable from cells costimulated with CD28. I␬B␤ (Santa Cruz Biotechnology, Santa Cruz, CA) with an enhanced Figs. 1B and 2B demonstrate that IL-2 secretion and mRNA chemiluminescence detection system (Pierce). levels in the LP-like T cell model activated by Abs to CD2 or 6624 TRANSACTIVATION OF THE IL-2 PROMOTER Downloaded from http://www.jimmunol.org/

FIGURE 2. Effect of costimulation by anti-CD28 on IL-2 mRNA accumulation in LPMC and LP-like T cells activated via the CD2 pathway. EtBr represents ethidium bromide staining of the ribosomal RNA from equalized RNA samples used to prepare the Northern blot. A, LPMC from inﬂamed mucosa stimulated with 1 ␮g anti-CD2/106 cells or 1 ␮g anti-CD2/106 cells ϩ 1 ␮g anti-CD28/106 cells. B, PBL cultured 5 days in IL-2 with irradiated Daudi cells (LP-like T cells) prior to stimulation with anti-CD2 or anti-CD2 ϩ anti-CD28. Representative of three experiments with similar results is shown.

CD2 ϩ CD28 are similar to those seen in LPMC from inflamed CD28 costimulation. Thus, CD28 costimulation increases the sta- mucosa. Activation of LP-like T cells via the CD2 pathway re- bility of IL-2 mRNA in both LPMC and LP-like T cells activated by guest on September 29, 2021 sulted in a steady increase in IL-2 secretion within 3 h. The in- via the CD2 pathway. crease in IL-2 persisted throughout 24 h. IL-2 mRNA was detectable within 2 h, and persisted over 6 h, but declined by 10 h (Fig. CD28 costimulation of CD2-activated LPMC does not affect the 2B). In contrast, the levels of IL-2 mRNA expressed in LP-like T transcriptional rate of IL-2 gene expression cells costimulated with CD28 were significantly elevated com- Previous studies carried out with activated PBMC have reported pared with those stimulated with CD2 alone. Increased IL-2 increased transcription after CD28 costimulation of the CD3 path- mRNA expression in CD2- and CD28-costimulated LP-like T cells way (26). To determine the contribution of CD28 signaling on was still detectable at 10 h and remained elevated at the 24-h time initiation of IL-2 gene transcription in LPMC, run-on assays were point. CD28 alone was unable to induce cytokine production or performed. Nuclei were isolated from LPMC activated by CD2 in expression of IL-2 mRNA (data not shown). Thus, CD28 costimu- the presence or absence of CD28 costimulation. As seen in Fig. 4, lation of the CD2 activation pathway leads to an increase in IL-2 while IL-2 gene transcription was undetectable in unstimulated secretion that is paralleled by enhanced level and duration of IL-2 LPMC, following CD2 activation induction of IL-2 transcription mRNA expression in both LPMC and LP-like T cells. was evident. CD28 costimulation, however, did not result in an increased transcriptional rate above that observed by CD2 alone Costimulation by CD28 enhances mRNA stability in LPMC and (Fig. 4). Under all conditions, close to equivalent levels of ␤-actin LP-like T cell activated via the CD2 pathway were detected. No increase in signal was detected hybridizing to The increased IL-2 mRNA accumulation induced by CD2 and the plasmid vector alone (Fig. 4B). To further study transcriptional CD28 coligation could be related to an increase in mRNA tran- activation, a wild-type IL-2 promoter reporter luciferase construct scription, processing, and/or enhanced mRNA stability. To deter- encompassing the CD28RE, known to be involved in CD28 en- mine whether this augmentation was related to mRNA stability, hanced gene transcription in PBMC, was transfected into LPMC LPMC from inflamed mucosa were preactivated by CD2 or CD2 activated by CD2, and LPMC activated by CD2 and CD28. Fig. 5 and CD28 for 2 h (maximum for mRNA expression in LPMC is shows that in contrast to results in PBMC, CD2 ϩ CD28 coligation 2–4 h). Actinomycin D was added to prevent further transcription, did not significantly increase IL-2 transcription in LPMC from and mRNA decay was monitored for the time periods indicated in normal or inflamed mucosa over that induced by CD2 alone. As

Fig. 3A. CD28 coligation extended the t1⁄2 of IL-2 mRNA from 70 reported recently, CD2 signaling in LPMC is transmitted in part by to 180 min. A strikingly similar result was obtained with LP-like induction of AP-1-transactivating factors (19). Fig. 6 demonstrates T cells. LP-like T cells were preactivated by CD2 or CD2 and that while CD2 ligation results in transactivation of a multimeric CD28 for 5 h (to maximize mRNA expression in LP-like T cells). AP-1-binding TRE2 reporter construct in LPMC from normal or

As seen in Fig. 3B, the t1⁄2 of IL-2 mRNA from CD2-activated inﬂamed mucosa, coligation of CD28 was unable to further en- LP-like T cells was 60 min compared with over 200 min following hance promoter activity. These results indicate that while there is The Journal of Immunology 6625 Downloaded from http://www.jimmunol.org/

FIGURE 3. Half-life of IL-2 mRNA following anti-CD2 activation. LPMC from inﬂamed mucosa (A) or LP-like T cells (B) were preactivated by 1 ␮g 6 6 6 anti-CD2/10 cells or 1 ␮g anti-CD2/10 cells ϩ 1 ␮g anti-CD28/10 cells for2hor5h,respectively, followed by addition of actinomycin D (10 ␮g/ml) by guest on September 29, 2021 to prevent further transcription. Northern blots were prepared and the amount of mRNA was determined at indicated time points by scanning densitometry (left panel). EtBr represents ethidium bromide staining of the ribosomal RNA from equalized RNA samples used to prepare the Northern blot. Repre- sentative of three experiments with similar results is shown.

an increase in the IL-2 promoter activity following CD2 pathway activation of LPMC, costimulation in the presence of CD28 does not further increase transcriptional activity.

Activation of LPMC via the CD28 pathway does not enhance binding of nuclear proteins to the CD28RE, or decrease I-␬B function in LPMC and LP-like T cells Previous studies have identified a CD28RE within the IL-2 promoter region that is required for transcriptional induction of IL-2 gene expression by CD28 costimulated PBMC and T cell lines (8, 9, 26). It is believed that the complexes binding to the CD28RE are members of the NF-␬B/Rel family, the function of which is regulated by sequestration of binding of inhibitory I-␬B proteins (10, 11). The results depicted in Fig. 5 suggest that while the IL-2 promoter containing the CD28RE responds to CD2 activation, the CD28RE is not the site at which transactivation is enhanced following CD28 coligation. To assess the effect of CD28 coligation on functional regulation of the CD28RE in our system, LP-like T cells were activated with CD2 in the presence or absence of CD28 FIGURE 4. Nuclear run-on analysis of IL-2 mRNA. LPMC were preactivated by 1 ␮g anti-CD2/106 cells or 1 ␮g anti-CD2/106 cells ϩ 1 ␮g costimulation and nuclear proteins were analyzed for binding to anti-CD28/106 cells for 2 h and nuclei were isolated. A, Run-on assays the composite CD28RE/AP-1 cis regulatory element by EMSA. As were performed and transcripts were hybridized to filter containing 2 ␮gof shown in Fig. 7, a very modest increase in binding of complexes plasmid inserts specific for human IL-2 or ␤-actin. B, Densitometric read- to the CD28RE/AP-1 element was detected following CD2 acti- ings of run-on in A. Densitometric units were calculated from ␤-actin and vation. No additional alteration in the binding of this complex was used to correct the readings for IL-2 mRNA and empty vector. Represen- detectable following CD28 costimulation. Furthermore, Western tative of two experiments with similar results is shown. 6626 TRANSACTIVATION OF THE IL-2 PROMOTER

FIGURE 7. EMSA analysis of 5 ␮g nuclear protein isolated from LPMC binding to the CD28RE/AP-1 element following stimulation with by 1 ␮g anti-CD2/106 cells or 1 ␮g anti-CD2/106 cells ϩ 1 ␮g anti-CD28/ 106 cells. Lanes: C, competition with 100-fold excess CD28RE oligo; NS, competition with 100-fold excess nonspeciﬁc oligonucleotide. Represen- tative of three experiments with similar results is shown. FIGURE 5. Transfection of LPMC with an IL-2 promoter-luciferase sequence. Freshly isolated LPMC were cultured in the presence of 1 ␮g/ml PHA-L for 20 h and electroporated with the IL-2 promoter construct. Elec- Activation of LPMC via the CD28 pathway does not troporated cells were rested for1hat37°C and then stimulated with either signiﬁcantly enhance transcriptional expression of composite 6 20 g/ml PMA plus 400 nM calcium ionophore, 1 ␮g anti-CD2/10 cells CD28/AP promoter-reporter construct Downloaded from 6 plus or minus 1 ␮g anti-CD28/10 cells for 4 h. Luminescence was mea- Previous studies carried out in T cell lines have demonstrated that sured using a Promega luciferase assay kit, counted on a six detector Wal- transactivation of IL-2 gene expression following CD28 coligation lac 1450 Microbeta liquid scintillation counter (Wallac, Gaithersburg, MD) is conferred through a composite element, RE/AP, consisting of with coincidence counting turned off. Fold increase was calculated as the ratio of cpm stimulated divided by cpm unstimulated. Unstimulated cpm the synergistic interaction of both the CD28RE and the adjacent was 41. Representative of four experiments with similar results is shown. nonconsensus AP-1-binding site (24). Mutation of either one of

these components was sufﬁcient for abolishing the CD28 response http://www.jimmunol.org/ of RE/AP promoter-reporter constructs transfected into T cell lines. The data presented above supported the hypothesis that fol- blot analysis revealed that CD2 activation precipitated a striking lowing CD28 costimulation of LPMC no transcriptional up-regu- loss of I␬B␣ within 2 h, which was then rapidly restored (Fig. 8A). lation of the IL-2 promoter could be detected over that observed in A similar decrease from baseline of I-␬B␣ was generated by CD28 the presence of CD2 alone. However, most of the information costimulation. I␬B␤ proteins were not signiﬁcantly altered follow- regarding our understanding of transcriptional regulation of IL-2 ing CD2 activation whether or not CD28 was present (Fig. 8B). expression has been performed in T cell line systems rather than primary T cells. Therefore, it seemed possible that the failure to

This data are consistent with transfection functional analysis and by guest on September 29, 2021 the EMSA data, suggesting that CD28 costimulation does not re- detect transcriptional up-regulation following CD28 coactivation sult in an increase in either binding of trans-acting factors to the of LPMC might not be unique for mucosal T cells but, rather, CD28RE promoter region or increased promoter activation over might be due to unique transcriptional regulatory mechanisms re- that induced by CD2 alone. lated to primary T cells compared with T cell lines. In order to investigate this possibility, PBMC were transfected with multim- erized RE/AP and mutant RE/AP reporter constructs, and expression compared with that of LPMC following CD2 and CD2 ϩ CD28 costimulation. Fig. 9 shows that there is approximately a 10-fold activation of the composite RE/AP construct in response to CD2 stimulation in both peripheral T cells and LPMC. Moreover,

FIGURE 6. Transfection of LPMC with an IL-2 TRE2 promoter-luciferase sequence. Freshly isolated LPMC were cultured in the presence of 1 ␮g/ml PHA-L for 20 h and electroporated with the TRE2 promoter construct. Electroporated cells were rested for1hat37°C and then stimulated with 1 ␮g anti-CD2/106 cells plus or minus 1 ␮g anti-CD28/106 cells for 4 h. Luminescence was measured using a Promega luciferase assay kit, counted on a six detector Wallac 1450 Microbeta liquid scintillation FIGURE 8. Kinetics of I␬B␣ (A) and I␬B␤ (B) binding following CD2 counter with coincidence counting turned off. Fold increase was calculated activation. PBL were cultured 5 days in IL-2 with irradiated Daudi cells as the ratio of cpm stimulated divided by cpm unstimulated. Mean un- (LP-like T cells) and stimulated with anti-CD2 or anti-CD2 ϩ anti-CD28. stimulated cpm was 380,000. Means (ϮSEM) of three experiments is Western blot was used to determine the presence of I␬B␣ and I␬B␤ nuclear shown. proteins. Representative of three experiments with similar results is shown. The Journal of Immunology 6627

the LP-like T cell model as a system with analogous activation properties to those expressed by LPMC, which allows investigations of mucosal cytokine gene regulation to be performed initially on a more easily manipulated experimental system. Numerous studies have been carried out in PBMC and tumor T cell lines activated through the TCR and CD28 costimulation, which indicated that CD28 synergism was due to both increased IL-2 mRNA transcription as well as enhanced mRNA stability (8, 13, 27, 28). CTLA-4 expression is likewise enhanced in a synergistic fashion following TCR and CD28 costimulation involving a mechanism contributed by both transcriptional and posttranscriptional components (29). However, studies performed with murine T cell clones suggest that in these cells increased IL-2 mRNA FIGURE 9. Transfection of LPMC and PBMC with human IL-2 pro- production is mediated by a more complex series of events (30). moter-reporter constructs. Freshly isolated LPMC and PBMC were cul- Regulation on a nuclear level does not involve modiﬁcation of tured in the presence of 1 ␮g/ml PHA-L for 16 or 20 h, respectively, and transcriptional activity of the IL-2 enhancer, but rather is the result electroporated with the RE/AP and mutant RE/AP promoter constructs. Electroporated cells were rested for1hat37°C and then stimulated with of increased expression of unspliced IL-2 pre-mRNA (30). A sim- 1 ␮g/ml anti-CD2/106 cells plus or minus 1 ␮g/ml anti-CD28/106 cells for ilar mechanism of posttranscriptional regulation of IL-2 expres- 4 h. Luminescence was measured using a Promega luciferase assay kit, sion, at the level of pre-mRNA splicing and processing, has been Downloaded from counted on a six detector Wallac 1450 Microbeta liquid scintillation reported in PBMC activated with PHA but without CD28 costimu- counter with coincidence counting turned off. Fold increase was calculated lation (5). Thus, it remains uncertain as to whether the nuclear as the ratio of cpm stimulated divided by cpm unstimulated. Unstimulated effects of CD28 costimulation involve transcriptional activation of ᮀ ■ cpm was 256 for LPMC and 116 for PBMC. , Anti-CD2 alone; , anti- the IL-2 promoter in different cell systems. CD2 ϩ anti-CD28. Representative of ﬁve experiments with similar results The vast majority of information acquired regarding transcrip- is shown. tional activation of the IL-2 promoter by CD28 costimulation has http://www.jimmunol.org/ been obtained from experiments using human and murine T cell similar to up-regulation of the RE/AP element described for T cell lines. It should be noted that, although primary T cells must be lines, in peripheral T cells transfected with the RE/AP reporter suboptimally activated with PHA in order to achieve transfection construct following CD28 costimulation, there is enhanced pro- competency, no transcription is detectable in the absence of stim- moter transactivation (Fig. 9, from 14- to 300-fold); however, in ulation (19). Recent studies have indicated that regulation of IL-2 contrast to peripheral T cells, in LPMC from normal mucosa fol- gene expression in primary T cells differs from that observed in lowing CD2 ϩ CD28 coligation, only a marginal 2-fold increase in tumor T cell lines. For example, NF-AT sites are much less im-

RE/AP reporter activation was noted over that induced by CD2 portant in primary T cells than in tumor T cell lines, while the by guest on September 29, 2021 alone. CD28 stimulation alone did not activate promoter-reporter proximal AP-1 and NF-␬B sites are of critical importance (23). In constructs in all cells tested (data not shown). Likewise, mutation addition, expression of an IL-2 promoter construct mutated at the of either the CD28RE or adjacent nonconsensus AP-1 site abol- CD28RE site and transfected into primary T cells resulted in re- ished CD2 responsiveness. These results indicate that the regula- duced expression following CD28 costimulation; however, muta- tory mechanisms involved in IL-2 gene expression following tion of this site did not affect expression following CD2 activation. CD28 pathway stimulation in LPMC are distinct from those ob- Moreover, the appearance of components of cis-regulatory factors served in peripheral T cell and T cell lines. binding to the CD28RE, particularly c-rel, has been reported to be closely linked with CD28 costimulation in tumor T cell lines, Discussion which has not been the case in primary T cells (31). In PBMC In this study, we examined molecular events and mechanisms in- following PMA stimulation alone, c-rel binding to the CD28RE volved in the regulation of IL-2 production in CD2-activated was detected in the absence of IL-2 secretion. While costimulation LPMC and LP-like T cells costimulated with CD28. IL-2 gene by CD28 did result in enhanced c-rel binding, the presence of expression in LPMC and LP-like T cells is highly sensitive to CD2 CD28 cis-binding factors in cells not actively expressing IL-2 in- activation (16). Coligation of the CD28 molecule leads to a syn- dicate that binding to the CD28RE is not exclusively a response to ergistic effect, further enhancing IL-2 secretion in these cell pop- CD28 costimulation (31). A similar finding of CD28-independent ulations (16). Our data show that, in LPMC and LP-like T cells, transcriptional activation was reported following CD3 plus PMA CD28 costimulation enhanced mRNA stability, but did not in- costimulation and reporter gene transactivation of a CD28RE pro- crease the rate of transcriptional activation. Nuclear run-on data as moter-reporter construct (32). well as transfection of a 600-bp IL-2 promoter-reporter construct The CD28RE has been reported to bind ␬B-like proteins, and into LPMC revealed significant promoter activity following CD2 CD28 costimulation results in the rapid down-regulation of I␬B␣ activation, but failed to exhibit augmented transcriptional activa- and concordant translocation of c-rel to the nucleus in PBMC and tion following CD2 ϩ CD28 costimulation above that observed T cell lines (33). These reports conflict with our observations of with CD2 alone. Likewise, expression of the IL-2 RE/AP com- LPMC costimulated with CD28. While CD2 activation modestly posite element was responsive to CD2 stimulation but failed to increases expression of factors binding to the CD28RE, CD28 co- exhibit enhanced activity following CD28 costimulation. EMSA stimulation did not further enhance this effect. Likewise, a rapid and Western blot analysis of the CD28RE cis and trans-acting decrease in the level of I␬B␣ was detected following CD2 activa- elements support the conclusion that CD2 signaling in LPMC tion, however, no further or sustained decrease was detected fol- leads to up-regulation of NF-␬B-like factors paralleled by rapid lowing CD28 costimulation. decrease of I␬B␣. Costimulation with CD28 does not alter this Recent evidence suggests that the CD28RE does not function as pattern of expression. These results highlight again the utility of a discrete element but rather is part of a composite RE/AP site, 6628 TRANSACTIVATION OF THE IL-2 PROMOTER consisting of the CD28RE and the adjacent AP-1 (24). The im- production paralleled by increased expression of IL-2 mRNA due portance of AP-1 involvement in regulation of c-rel activity and in part to enhanced IL-2 mRNA stability. However, in contrast to IL-2 expression is supported by the finding that overexpression of CD28-mediated transcriptional activation in PBMC, EMSA anal- c-rel greatly up-regulates AP-1 expression. Not only is binding to ysis revealed that CD28 coligation of LPMC did not augment the consensus AP-1 sequence increased, but more importantly, the binding of trans-acting factors to the CD28RE or alterations in binding to the nonconsensus AP-1 sequence immediately adjacent I␬B␣ or I␬B␤ levels. Although CD2-enhanced IL-2 secretion is to the CD28RE is enhanced as well (34). The requirement for a reflected by transactivation of IL-2 promoter reporter constructs composite regulatory site is further supported by studies demon- transfected into LPMC, there was no enhanced CD28-mediated strating that the HIV tax responsiveness region of the IL-2 pro- promoter activation following coligation. In addition, these studies moter requires both the CD28RE and the adjacent AP-1 regions represent the first reports of transcriptional activation of the RE/AP (35). These findings suggest that the CD28RE might be function- composite element in response to CD2 stimulation. These obser- ally redundant and thereby mediate more than one signaling path- vations suggest a biological significance for different mechanisms way. Our data suggest that, at least in regard to the composite of IL-2 gene activation in initiation and maintenance of cytokine RE/AP, peripheral T cells behave in a manner similar to that of T production in the mucosa. Furthermore, these results strengthen cell lines while transactivation of LP T cell entails unique cis- the use of LP-like T cells as a model for LP T cell activation and regulatory elements. It is quite likely that the cell system as well as the analysis of mucosal cytokine gene regulation. the mode of T cell activation may influence regulation of IL-2 gene transcription. Acknowledgments

Interpretation of the data obtained through functional expression We thank Rachel Kramer for providing cultured LPMC and PBMC and Dr. Downloaded from of transfected IL-2 promoter-reporter construct are subject to an Howard Young for his helpful discussions. important bias in that they are reflective of the sequences selected, while elements residing outside of the chosen promoter region References would not be detected. In this study, transfection experiments were 1. Durand, D. B., M. R. Bush, J. G. Morgan, A. Weiss, and G. R. Crabtree. 1987. carried out utilizing a 600-bp IL-2 promoter construct that encom- A 275 basepair fragment at the 5Ј end of the interleukin 2 gene enhances expression from a heterologous promoter in response to signals from the T cell passes the highly conserved region defined as the critical IL-2 http://www.jimmunol.org/ antigen receptor. J. Exp. Med. 165:395. promoter element. However, DNase I hypersensitive sites have 2. Serfling, E., R. Barthelmas, I. Pfeuffer, B. Schenk, S. Zarius, R. Swoboda, been identified within 3 kb upstream of the IL-2 transcriptional F. Mercurio, and M. Karin. 1989. Ubiquitous and lymphocyte-specific factors are start site (36, 37). Additionally, the EMSA analyses were con- involved in the induction of the mouse interleukin 2 gene in T lymphocytes. EMBO J. 8:465. ducted utilizing the well-studied CD28RE/AP-1 composite ele- 3. Efrat, S., S. Pilo, and R. Kaempfer. 1982. Kinetics of induction and molecular ment. Likewise, transcriptional run-on assays, although the meth- size of mRNAs encoding human interleukin-2 and ␥-interferon. Nature 297:236. 4. Efrat, S., and R. Kaempfer. 1984. Control of biologically active interleukin 2 ods of choice in analyzing fluctuations in the rate of transcription messenger RNA formation in induced human lymphocytes. Proc. Natl. Acad. Sci. initiation are susceptible to attenuation through DNA sequences USA 81:2601. within a gene (37). It is possible that sequences other than those 5. Gerez, L., G. Arad, S. Efrat, M. Ketzinel, and R. Kaempfer. 1995. Post-transcriptional regulation of human interleukin-2 gene expression at processing of by guest on September 29, 2021 studied might play a role in transcriptional regulation following precursor transcripts. J. Biol. Chem. 270:19569. CD28 costimulation in LPMC, albeit unlikely considering that in- 6. Hansen, J. A., P. J. Martin, and R. C. Nowinski. 1980. Monoclonal antibodies hibitory elements have been reported residing upstream of the identifying a novel T cell antigen and Ia antigens of human lymphocytes. Immu- nogenetics 10:247. 600-bp promoter element. Studies are underway to identify other 7. June, C. H., J. A. Ledbetter, M. M. Gillespie, T. Lindsten, and C. B. Thompson. regions that might respond to CD28 costimulation and regulate 1987. T cell proliferation involving the CD28 pathway is associated with cyclos- porine-resistant interleukin 2 gene expression. Mol. Cell. Biol. 7:4472. transcriptional activation in LPMC. 8. Fraser, J. D., B. A. Irving, G. R. Crabtree, and A. Weiss. 1991. Regulation of An increase in mRNA stability in PBMC and T cell tumor cell interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. lines following CD28 costimulation has been reported for numer- Science 251:313. ␥ ␣ 9. Ghosh, P., T. Tan, N. R. Rice, A. Sica, and H. A. Young. 1993. The interleukin ous cytokines, including IL-2, IFN- , GM-CSF, and TNF- (13). 2 CD28-responsive complex contains at least three members of the NF-␬B fam- Little is known about posttranscriptional regulation of IL-2 expres- ily: c-Rel, p50, and p65. Proc. Natl. Acad. Sci. USA 90:1696. sion, however, several cytokines and transiently expressed proto- 10. Henkel, T., T. Machleidt, I. Alkalay, M. Kronke, Y. Ben-Neriah, and P. A. Baeuerle. 1993. Rapid proteolysis of I␬B␣ is necessary for activation of oncogenes contain one or more highly conserved AUUUA motifs transcription factor NF-␬B. Nature 365:182. in the 3Ј-UT region of their mRNA (12). These consensus ele- 11. Liou, H., and D. Baltimore. 1993. Regulation of the NF-␬B/rel transcription factor and I␬B inhibitor system. Curr. Opin. Cell Biol. 5:477. ments are determinants for rapid degradation, probably through 12. Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shiner, and A. Cerami. interaction with specific proteins that can be cross-linked to these 1986. Identification of common nucleotide sequence in the 3Ј-untranslated region AU-rich domains (38). Nonetheless, while CD28 costimulation re- of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:1670. sults in the stabilization of IL-2 mRNA, the proto-oncogenes c-fos, 13. Lindsten, T., C. H. June, J. A. Ledbetter, G. Stella, and C. B. Thompson. 1989. and c-myc were not further stabilized, despite the fact that the Regulation of lymphokine messenger RNA stability by a surface-mediated T cell mRNAs of these factors possess multiple AUUUA sequences (13). activation pathway. Science 244:339. 14. Sachs, A. B. 1993. Messenger RNA degradation in eukaryotes. Cell 74:413. Recent studies of regulation of IL-2 mRNA stability in T cell lines 15. Pirzer, U. C., G. Schu¨rmann, S. Post, M. Betzler, and S. C. Meuer. 1990. Dif- indicate that multiple mechanisms regulate IL-2 mRNA stability ferential responsiveness to CD3-Ti vs. CD2-dependent activation of human in- Ј Ј testinal T-lymphocytes. Eur. J. Immunol. 20:2339. and suggest that both 5 as well as 3 sequences within the un- 16. Targan, S. R., R. L. Deem, M. Liu, S. Wang, and A. Nel. 1995. Definition of a translated region of the IL-2 mRNA are critical for stabilization lamina propria T cell responsive state: enhanced cytokine responsiveness of T (39). Furthermore, in activated T cell lines, activation of the c-jun cells stimulated through the CD2 pathway. J. Immunol. 154:664. 17. Boirivant, M., I. J. Fuss, C. Fiocchi, J. S. Klein, S. A. Strong, and W. Strober. amino-terminal kinase is involved in IL-2 mRNA transcription and 1996. Hypoproliferative human lamina propria T cells retain the capacity to se- stabilization. Thus, it would appear that multiple posttranscrip- crete lymphokines when stimulated via CD2/CD28 signaling pathway. Proc. As- tional mechanisms other than the AUUUA motifs regulate IL-2 soc. Am. Physicians 108:55. 18. Mulin, G., Z. R. Maycon, L. Braun-Elwert, R. Cerchia, S. P. James, S. Katz, mRNA stability. G. Weissman, M. J. McKinley, and S. E. Fisher. 1996. Inflammatory bowel In summary, the studies presented here show that regulation of disease mucosal biopsies have specialized lymphokine mRNA profiles. Inflamm. Bowel Dis. 2:16. IL-2 expression in LPMC and LP-like T cells differs from that 19. Gonsky, R., R. L. Deem, C. C. W. Hughes, and S. R. Targan. 1998. Activation observed in PBMC. CD28 costimulation of LPMC increased IL-2 of the CD2 pathway in lamina propria T cells up-regulates functionally active The Journal of Immunology 6629

AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription on plus CD28 signals mediated by increased transcription and messenger ribonucleic IL-2 secretion. J. Immunol. 160:4914. acid stability. J. Immunol. 158:4074. 20. Shanahan, F., M. Brogan, and S. R. Targan. 1987. Human mucosal cytotoxic 30. Umlauf, S. W., B. Beverly, O. Lantz, and R. H. Schwartz. 1995. Regulation of effector cells. Gastroenterology 92:1951. interleukin 2 gene expression by CD28 costimulation in mouse T cell clones: both 21. Gillis, S., M. M. Ferm, O. Winny, and K. A. Smith. 1978. T cell growth factor: nuclear and cytoplasmic RNAs are regulated with complex kinetics. Mol. Cell parameters of production and quantitative microassay for activity. J. Immunol. Biol. 15:3197. 120:2027. 31. Bryan, R. G., Y. Li, J. Lai, M. Van, N. R. Rice, R. R. Rich, and T. Tan. 1994. 22. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells. J. A. Smith, and K. Struhl, eds. 1990. Current Protocols in Molecular Biology, Mol. Cell. Biol. 14:7933. Greene Publishing Associates and Wiley-Interscience, New York, pp. 32. Civil, A., M. Geerts, I. A. Aarden, and C. L. Verweij. 1992. Evidence for a role 4.10.1–4.10.8. of CD28RE as a response element for distinct mitogenic T cell activation signals. 23. Hughes, C. C. W., and J. S. Pober. 1996. Transcriptional regulation of the inter- Eur. J. Immunol. 22:3041. leukin-2 gene in normal human peripheral blood T cells. J. Biol. Chem. 271:5369. 33. Beg, A. A., and A. S. Baldwin, Jr. 1993. The I␬B proteins: multifunctional reg- 24. Shapiro, V. S., K. E. Truitt, J. B. Imboden, and A. Weiss. 1997. CD28 mediates ulators of Rel/NF-␬B transcription factors. Genes Dev. 7:2064. transcriptional upregulation of the interleukin-2 (IL-2) promoter through a com- 34. Shapiro, V. S., M. N. Mollenauer, W. C. Greene, and A. Weiss. 1996. c-Rel posite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol. Cell. Biol. regulation of IL-2 gene expression may be mediated through activation of AP-1. 17:4051. J. Exp. Med. 184:1663. 25. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J. Rahmsdorf, C. Jonat, 35. Curtiss, V. E., R. Smilde and K. L. McGuire 1996. Requirements for interleukin P. Herrlich, and M. Karin. 1987. Phorbol ester-inducible genes contain a common 2 promoter transactivation by the Tax protein of human T cell leukemia virus type cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729. 1. Mol. Cell. Biol. 16:3567. 26. Verweij, C. L., M. Geerts, and L. A. Aardon. 1991. Activation of interleukin-2 36. Novak, T. J., P. M. White, and E. V. Rothenberg. 1990. Regulatory anatomy of gene transcription via the T cell surface molecule CD28 is mediated through the the murine terminal kinase pathway interleukin-2 gene. Nucleic Acids Res. NF-␬B-like response element. J. Biol. Chem. 266:14179. 18:4523. 27. Fraser, J. D., and A. Weiss. 1992. Regulation of T cell lymphokine gene tran- 37. Wright, S., and J. M. Bishop. 1989. DNA sequences that mediate attenuation of scription by the accessory molecule CD28. Mol. Cell. Biol. 12:4357. transcription from mouse protooncogene myc. Proc. Natl. Acad. Sci. USA 28. Fraser, J. D., M. E. Newton, and A. Weiss. 1992. CD28 and T cell antigen 86:505. Downloaded from receptor signal transduction coordinately regulate interleukin 2 gene expression 38. Sachs, A. 1993. Messenger RNA degradation in eukaryotes. Cell 74:413. in response to superantigen stimulation. J. Exp. Med. 175:1131. 39. Chen C. Y., F. D. Gatto-Konczak, Z. Wu, and M. Karin. 1998. Stabilization of 29. Finn, P. W., H. He, Y. Wang, Z. Wang, G. Guan, J. Listman, and D. L. Perkins. interleukin-2 mRNA by the c-jun NH2-terminal kinase pathway. Science 280: 1997. Synergistic induction of CTLA-4 expression by costimulation with TCR 1945. http://www.jimmunol.org/ by guest on September 29, 2021