Effector CD4+ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates This information is current as Ravi S. Misra, Dawn M. Jelley-Gibbs, Jennifer Q. Russell, of October 1, 2021. Gail Huston, Susan L. Swain and Ralph C. Budd J Immunol 2005; 174:3999-4009; ; doi: 10.4049/jimmunol.174.7.3999 http://www.jimmunol.org/content/174/7/3999 Downloaded from References This article cites 64 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/174/7/3999.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 *average by guest on October 1, 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Effector CD4؉ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates1 Ravi S. Misra,* Dawn M. Jelley-Gibbs,† Jennifer Q. Russell,* Gail Huston,† Susan L. Swain,† and Ralph C. Budd2* Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4؉ T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIPL, receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4؉ T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling. The Journal of Immunology, 2005, 174: 3999–4009. Downloaded from t is becoming increasingly evident that ligation of death re- The earlier view that caspase activation occurs only through its ceptors and activation of cellular caspases does not necessar- cleavage has been revised for certain upstream caspases. An alter- I ily always result in apoptosis. Involvement of the death re- native method of caspase activation is through a conformational ceptor, Fas, in processes other than cell death has been revealed by change. For example, when caspase-9 interacts with cytochrome c the ability of Fas ligation to enhance neural outgrowth (1), cardiac complexed to Apaf-1, a conformational change of caspase-9 ex- hypertrophy (2), liver regeneration (3), fibroblast proliferation (4), poses its active site (16, 17). In a similar manner, procaspase-8 can http://www.jimmunol.org/ dendritic cell maturation (5), and T cell activation (6). More di- induce a structural change in a neighboring procaspase-8 molecule, rectly, caspase-8 plays an integral role in T cell function, as hu- resulting in active full-length caspase-8 (10). In addition, the en- mans bearing nonfunctional caspase-8, as well as caspase-8 con- zymatically inactive casapase-8-like molecule c-FLIPL can also ditional knockout mice, exhibit severe defects in T cell activation associate with and activate full-length caspase-8 (18). As c-FLIPL (7, 8). However, there is currently no clear picture of which also contains a known caspase cleavage site, it represents poten- caspases or which substrates are involved with T cell activation tially one of the earliest substrates of caspase-8 (19). and how far down the caspase cascade these signals propagate. The significance of caspase activity in nonapoptotic effector T Traditionally, activation of the upstream initiator caspases that cells is still poorly understood. It is not known how the levels of by guest on October 1, 2021 possess large prodomains, such as procaspases-8, -9, and -10, had active caspases or cleavage of their substrates in viable effector T been thought to require proteolytic cleavage of the zymogen, resulting cells compare with those in resting T cells or those undergoing in the release of large and small subunits (9). These fragments then apoptosis after Fas ligation. We observe that intermediate levels of heterodimerize to form an active caspase molecule. Typically, the caspase activity are generated at the effector stage of T cell acti- cleavage occurs by auto- and cross-proteolysis in response to the li- vation, which are proportional to cell cycling, and that blockade of gation of death receptors, which increases the local concentrations of caspase activity inhibits cell cycling. Proximal caspase-8 sub- caspases (9–11). The upstream initiator caspase signal is propagated strates such as c-FLIPL and receptor interacting protein 1 (RIP1) to the effector procaspases, such as procaspase-3, by proteolytic cleav- are cleaved in effector T cells, but not the downstream caspase-3 age of the procaspase. As part of this caspase cascade, various caspase substrate ICAD. Furthermore, the caspase activity and cleavage of substrates are cleaved, including the DNase inhibitor inhibitor of substrates is more extensive in Th2 effector cells than in Th1 ef- caspase-activated DNase (ICAD)3 (12) and structural proteins, such fectors. Collectively, these results define the caspase activity pro- as actin (13), fodrin (14), and lamin (15). file in viable T cells and underscore the functional requirements of such activity. *University of Vermont, Immunobiology Program, College of Medicine, Burlington, Materials and Methods VT 05405; and †Trudeau Institute, Saranac Lake, NY 12983 Mice Received for publication July 14, 2004. Accepted for publication January 11, 2005. C57BL/6 mice were housed and bred in the University of Vermont animal The costs of publication of this article were defrayed in part by the payment of page facility and were used at 2–6 mo of age. B10.Br V␣11/V␤3 AND TCR charges. This article must therefore be hereby marked advertisement in accordance transgenic (pigeon cytochrome c/I-Ek responsive) mice were used at 2–3 with 18 U.S.C. Section 1734 solely to indicate this fact. mo of age and were bred in the animal facilities at the Trudeau Institute. 1 This work was supported by grants from the National Institutes of Health (AI36333 Both facilities are American Association of Laboratory Animal Care ap- and AI45666 to R.C.B.; AI26887 and AI46530 to S.L.S.). R.S.M. is supported by proved and protocols were approved by the Institutional Animal Care and National Institutes of Health Grant No. 5T32ES007122. Use Committee at the respective institutions. 2 Address correspondence and reprint requests to Dr. Ralph C. Budd, University of Vermont, Immunobiology Program, College of Medicine, 89 Beaumont Avenue, T cell purification Given Building D305, Burlington, VT 05405. E-mail address: [email protected] 3 Abbreviations used in this paper: ICAD, inhibitor of caspase-activated DNase; C57BL/6 spleens and lymph nodes were isolated and disrupted through RIP1, receptor interacting protein 1; PCCF, pigeon cytochrome c fragment; z-VAD, nylon mesh in RPMI 1640 (MediaTech) containing 5% (v/v) FCS (Hy- z-Val-Ala-Asp-fluoromethylketone; FasL, Fas ligand; WCL, whole cell lysate; XIAP, Clone). Erythrocyte lysis of splenocytes was performed using Gey’s solu- ϩ X-linked inhibitor of apoptosis protein. tion. Lymphocytes and splenocytes were combined and CD4 T cells were Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4000 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS isolated by negative selection. C57BL/6 cells were incubated with anti- Immunoblot analysis CD8 (Tib 105), anti-MHC class II (M5/114/15/2; a kind gift of M. Rinco´n, University of Vermont, Burlington, VT), anti-CD11b (M1/70), anti-NK1.1 Viable cells were lysed in buffer containing 0.2% Nonidet P-40, 20 mM (PK136), and anti-B220 (RA3-6B2) on ice for 45 min. For total T cell Tris-HCl (pH 7.4), 2 mM sodium orthovanadate, 10% glycerol, 150 mM ␮ isolation, anti-CD8 was omitted from Ab mix. Cells were washed three NaCl, complete protease inhibitor (Roche Diagnostics), and 10 M z-VAD times and rocked with goat anti-mouse and goat anti-rat conjugated mag- (Enzyme System Products). Protein concentration was determined by netic beads at a 10:1 ratio of beads to cell (Qiagen) at 4°C for 45 min. Bradford assay (Bio-Rad). Protein lysates were boiled for 5 min in loading Magnetic depletion was used to remove bead-bound cells, routinely yield- buffer containing 2-ME and were separated using SDS-PAGE on 12.5% ing Ͼ90% CD4ϩ T cells. Cells were washed and resuspended in culture gels. Proteins were transferred onto a polyvinylidene difluoride membrane medium (RPMI 1640 supplemented with 25 mM HEPES, 2.5 mg/ml glu- (Bio-Rad) and blocked using 4% milk in TBS plus 0.1% Tween 20 at room cose (Sigma-Aldrich), 10 ␮g/ml folate (Invitrogen Life Technologies), 110 temperature for 1 h. Membranes were incubated at 4°C overnight in milk Ϫ ␮ ␮g/ml pyruvate (Invitrogen Life Technologies), 5 ϫ 10 5 M 2-ME (Sig- containing one of the following primary detection Abs: 1 g/ml actin (I-19; ma-Aldrich), 292.3 ␮g/ml glutamine (Invitrogen Life Technologies), 100 Santa Cruz Biotechnology), Bid (R&D Systems), caspase-3 (a kind gift of U/ml penicillin-streptomycin (Invitrogen Life Technologies), and Y.