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    Extracellular Regulated Kinase-2 in T CellsProteolytically Blocks Activation of Bromelain, from Pineapple Stems,

    Christian R. EngwerdaTracey L. Mynott, Andrew Ladhams, Pierre Scarmato and; 163:2568-2575; ;J Immunol


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    Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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  • Bromelain, from Pineapple Stems, Proteolytically BlocksActivation of Extracellular Regulated Kinase-2 in T Cells

    Tracey L. Mynott,1* Andrew Ladhams,* Pierre Scarmato,* and Christian R. Engwerda

    Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteasesmay act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis,apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimu-latory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We showthat bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via theTCR with anti-CD3e mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain wasdependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However,inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelainalso inhibited PMA-induced IL-2, IFN-g, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNAproduction. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-inducedcytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstreamfrom ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cellsignal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transductionpathways. The Journal of Immunology, 1999, 163: 25682575.

    P roteases contribute to effective immune responses, al-though their role is commonly thought of as degradative,such as in the destruction of cell targets by lymphocytesand NK cells (1), or the cleavage of cellular substrates duringapoptosis (2). Recently, however, it has become apparent that pro-teases also convey hormone-like signals and stimulate intracellularsignal transduction via specific cell surface receptors (3). Throm-bin, for example, a protease involved in the blood coagulationcascade, is mitogenic in many cells and activates the Raf-1/extra-cellular regulated kinase-2 (ERK-2)2 pathway leading to DNAsynthesis, cell growth, proliferation, and differentiation (4).Thrombin activation is mediated via proteinase-activated receptor(PAR)-1, a member of the family of PARs (4). The proteases,plasmin, granzyme A, and cathepsin G also activate PAR-1 andstimulate signal transduction (57). Bromelain is a mixture of cys-teine proteases obtained from pineapple stems (Ananus comosus)(8) that has varied stimulatory effects on leukocyte populations invitro. It has been shown to increase CD2-mediated T cell activa-tion, enhance Ag-independent binding to monocytes (9), and in-crease IFN-g-dependent TNF-a, IL-1b, and IL-6 production inPBMC (10). Bromelain-treated RBC are also used in murine stud-ies of the role of CD51 B cells in autoimmunity (11). Bromelains

    effects have previously been attributed to its degradative action atcell surfaces, whereby it either removes surface molecules or, al-ternatively, reveals ones that already exist on cell membranes,thereby altering receptor-ligand interactions (9, 12, 13). Recently,however, we showed that bromelain also exerted effects indepen-dent to that mediated by the removal of cell surface molecules(14). We showed that bromelain inhibited the action of cyclicnucleotide agonists and calcium agonists that directly stimulateintracellular signaling pathways and bypass cell surface receptorsin their action (14). Therefore, to further investigate the possiblehormone-like effects of bromelain on intracellular signaling, westudied its effects on TCR/CD3 signaling and IL-2 production.

    Significant progress over recent years has led to the understand-ing of biochemical events that occur following TCR engagement(15). Therefore, TCR signaling provides an excellent model forelucidating the effects of biologically active compounds. EffectiveT cell activation requires two signals. The first signal is generatedby the TCR/CD3 complex after engagement with Ag peptide pre-sented by the MHC expressed on APC (15). The second, costimu-latory signal is generated by ligation of CD28 receptors on T cellswith the B7 family of ligands on APC (16). A key element in thesignaling pathway involved in transducing receptor-initiated sig-nals to the nucleus is the family of mitogen-activated protein ki-nases (MAPK) (17). The best studied of these kinases are ERK-1and ERK-2. ERKs are serine/threonine kinases that are activatedwhen phosphorylated on tyrosine and threonine residues (18). Invitro, this activation is reversed if either residue is dephosphory-lated (19). A relatively new member of the MAPK family is c-JunNH2-terminal kinase (JNK) (20). ERK activation is dependent onp56lck (21) and coupling of the TCR/CD3 complex to p21ras, withsubsequent activation of the Raf-1/MEK1/ERK kinase cascade(22). JNK activation also requires p21ras and signals generated bythe CD28 costimulatory receptor (20, 23). Activated ERK phos-phorylates Elk-1 (24), which, in turn, mediates induction of c-fosactivity (25). Following phosphorylation of c-jun by JNK (26),

    *Department of Biochemistry, Imperial College of Science, Technology and Medi-cine, London, United Kingdom, and Cortecs, Clwyd, United Kingdom; and Depart-ment of Infectious and Tropical Diseases, London School of Hygiene and TropicalMedicine, London, United Kingdom.

    Received for publication December 11, 1998. Accepted for publication June 22, 1999.

    The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Tracey L. Mynott, Departmentof Biochemistry, Imperial College of Science, Technology and Medicine, ExhibitionRoad, London SW7 2AZ, U.K. E-mail address: Abbreviations used in this paper: ERK, extracellular regulated kinase; MAPK, mi-togen-activated protein kinase; PAR, proteinase-activated receptor; JNK, c-Jun NH2-terminal kinase; pNA, p-nitroanilide; RBD, Ras-binding domain; E-64,(L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane.

    Copyright 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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  • activated c-fos and c-jun combine to form the AP-1 protein re-quired for IL-2 synthesis (27). All the above events require ty-rosine phosphorylation, as inhibitors of protein tyrosine kinasesinhibit many events associated with TCR stimulation (28, 29), in-cluding T cell activation and IL-2 production.

    In this report, we show that bromelain inhibits ERK-2 activationin Th0 cells stimulated via the TCR, or with combined PMA plusionophore. In association with decreased ERK activity, bromelaindecreased IL-2, IFN-g, and IL-4 mRNA accumulation in Th0 cellsstimulated with PMA plus ionophore, but did not affect cytokinemRNA accumulation in cells stimulated via the TCR. This datasuggests the existence of a TCR-activated, ERK-2-independentpathway involved in cytokine production in T cells. Bromelain didnot appear to act on ERK-2 directly, as studies indicate that it alsoinhibited p21ras activation, an effector molecule upstream fromERK-2 in the Raf-1/MEK1/ERK kinase cascade. These results in-di