bromelain, from pineapple stems, proteolytically blocks ... · bromelain, from pineapple stems,...

9
of July 8, 2018. This information is current as Extracellular Regulated Kinase-2 in T Cells Proteolytically Blocks Activation of Bromelain, from Pineapple Stems, Christian R. Engwerda Tracey L. Mynott, Andrew Ladhams, Pierre Scarmato and http://www.jimmunol.org/content/163/5/2568 1999; 163:2568-2575; ; J Immunol References http://www.jimmunol.org/content/163/5/2568.full#ref-list-1 , 16 of which you can access for free at: cites 48 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 1999 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on July 8, 2018 http://www.jimmunol.org/ Downloaded from by guest on July 8, 2018 http://www.jimmunol.org/ Downloaded from

Upload: dangnga

Post on 12-Jun-2018

240 views

Category:

Documents


3 download

TRANSCRIPT

Page 1: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

of July 8, 2018.This information is current as

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

Christian R. EngwerdaTracey L. Mynott, Andrew Ladhams, Pierre Scarmato and

http://www.jimmunol.org/content/163/5/25681999; 163:2568-2575; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/5/2568.full#ref-list-1

, 16 of which you can access for free at: cites 48 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

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.,

is published twice each month byThe Journal of Immunology

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on July 8, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 2: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

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-CD3 e 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: 2568–2575.

Proteases contribute to effective immune responses, al-though their role is commonly thought of as degradative,such as in the destruction of cell targets by lymphocytes

and 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 (5–7). 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). Bromelain’s

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 ofc-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 markedadvertisementin 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: [email protected] 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

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 3: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

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-dicate that bromelain is a novel inhibitor of T cell signal transduc-tion, and findings also suggest a novel role for extracellular pro-teases as inhibitors of intracellular signal transduction pathways.

Materials and MethodsAntibodies

Anti-CD3e-chain mAb (145-2C11) was purchased from PharMingen (SanDiego, CA) and goat anti-hamster IgG Ab was from Sigma (Dorset, U.K.).Mouse anti-phosphotyrosine mAb (4G10) and mouse anti-MAPK R2(ERK-2) mAb were from Upstate Biotechnology (Lake Placid, NY). Anti-pan-ras (Ab-4) mAb was from Oncogene Research Products (Cambridge,MA). Goat anti-mouse and goat anti-rabbit IgG Ab conjugated to HRPwere from Bio-Rad (Hertfordshire, U.K.). Rabbit polyclonal phospho-spe-cific MAPK IgG was from New England BioLabs (Hertfordshire, U.K.).

Reagents

Bromelain (E.C. 3.4.22.32; sp. act., 1541 nmol/min/mg) was purchasedfrom Solvay (Hannover, Germany). Trypsin (porcine pancreas, E.C.3.4.21.4; activity, 5270 nmol/min/mg), E-64 (L-trans-epoxysuccinyl-leu-cylamido(4-guanidino)butane), PMA, calcium ionophore A23187, and glu-tathione agarose beads were purchased from Sigma. Other reagents werealso from Sigma. Specific activity was determined by monitoring the re-lease ofp-nitroaniline from the peptidep-nitroanilide (pNA) substrate Z-Arg-Arg-pNA (30). MAPK activity was determined using a MAPK assaykit (New England Biolabs), which measures the in vitro phosphorylation ofthe transcription factor Elk-1.

T cells

The T cell hybridoma GA15 was a generous gift from B. Fox (ImmuLogicPharmaceutical, Boston, MA). GA15 was generated by fusing the thy-moma BW5147 with the Th2 clone F4 specific for keyhole limpet hemo-cyanin in association with I-Ab, and were maintained as previously de-scribed (31). GA15 exhibit a Th0 phenotype as they produce IL-2, IFN-g,and IL-4 following stimulation with cross-linked anti-CD3e mAb (31).Jurkat T cells (E6.1) were provided by P. Beverley (Jenner Institute, Berk-shire, U.K.).

Stimulation of T cells

Cells (2 3 107) suspended in RPMI 1640 were treated with bromelain(10–100mg/ml) diluted in PBS (0.1 M (pH 7.4)) for 30 min at 37°C. Mocktreated cells were treated with an equal volume of PBS. At high concen-trations of bromelain (50 or 100mg/ml), cell aggregation occurred, asnoted previously (9). Following treatment, cell aggregates were gently dis-persed, washed three times, and then resuspended in fresh RPMI. Cellswere stimulated via the cell surface with cross-linked mAb to the TCR, ordirectly, using combined PMA (20 ng/ml) plus ionophore (1mM) for timesshown in figure legends and the text. Stimulation via the TCR was con-ducted by first incubating T cells on ice for 30 min with anti-CD3e mAb(10 mg/ml). Excess mAb was then removed by washing once at 4°C, andanti-CD3e mAb was cross-linked with goat anti-hamster IgG (10mg/ml) at37°C. Stimulation was terminated by the addition of ice-cold lysis buffer(25 mM Tris (pH 7.4), 75 mM NaCl, 0.4 mM EDTA, 0.5% Triton X-100,0.4 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodiumpyrophosphate, 10mg/ml leupeptin, 740mM PMSF, and 10mg/ml apro-tinin) for 30 min with continual rotation at 4°C. Lysates were clarified

(14,0003 g for 10 min), and an equal volume of 23SDS-PAGE samplebuffer (50 mM Tris (pH 7), 700 mM 2-ME, 50% (v/v) glycerol, 2% (w/v)SDS, 0.01% (w/v) bromophenol blue) was added to postnuclear superna-tants. Proteins were solubilized at 100°C for 5 min, and samples containing1 3 106 cell equivalents were resolved by SDS-PAGE.

Immunoblotting

Separated proteins were transferred to nitrocellulose membranes (Bio-Rad), which were then blocked and immunoblotted (21) with the appro-priate Abs, as shown in figure legends. Immunoreactivity was determinedusing the ECL chemiluminescence detection system (Amersham, Arling-ton Heights, IL).

Detection of activated Ras

Activated p21ras was detected by a functional assay that detects the inter-action between Ras-GTP and the Ras-binding domain (RBD) of Raf-1 (32).Escherichia coliexpressing the RBD of Raf-1 fused to GST (GST-RBD)was a gift from D. Cantrell (Lymphocyte Activation Laboratory, ImperialCancer Research Fund, London, U.K.). Bromelain (0–100mg/ml)-treatedGA15 cells were washed and lysed, as described above. Cell lysates werethen incubated with GST-RBD immobilized to glutathione-Sepharose.Bound proteins were eluted with glutathione (50 mM), and subjected toSDS-PAGE and immunoblotting with anti-pan Ras mAb.

Inhibition of proteolytic activity of bromelain

Bromelain (10 mg/ml) diluted in 3mM DTT, 100 mM E-64, 60 mM so-dium acetate (pH 5), was incubated for 10 min at 30°C. The inactivatedbromelain was then dialyzed overnight in PBS at 4°C. A total of 99.5%inactivation of bromelain was achieved as assayed with the Z-Arg-Arg-pNA substrate.

Cytokine mRNA

GA15 cells diluted in RPMI were treated with bromelain (50mg/ml) orPBS at 37°C for 30 min, washed three times in fresh RPMI, and thenresuspended in culture medium. T cells were stimulated to produce cyto-kine mRNA by immobilized anti-CD3e (4 mg/ml) or combined PMA (20ng/ml) plus ionophore (1mM). Anti-CD3e mAb diluted in PBS was im-mobilized to 24-well flat-bottom microculture plates (Corning, Corning,NY) by incubation for 16 h at 4°C. Wells were then washed three times inPBS before addition of triplicate cultures of GA15 (2.5 to 53 106 cells perwell), which were incubated at 37°C in humidified 5% CO2 for 4 h.

IL-2, IFN-g, and IL-4 mRNA accumulation was measured using a semi-quantitative RT-PCR assay, as previously described (33). Significance ofdifferences was determined by Student’st test for paired observations.

T cell proliferation

GA15 cells were treated with bromelain (50mg/ml) for 30 min, washed inRPMI, and then cultured in 96-well flat-bottom plates (Nunc, Roskilde,Denmark) at 104 cells per well for 24 h. Cultures were pulsed with 0.5mCiof [3H]TdR 6 h before harvesting onto glass fiber filters.

ResultsBromelain inhibits TCR-induced tyrosine phosphorylationof ERK

Before conducting TCR signaling studies, we first confirmed anearlier report that showed bromelain does not cleave the CD3echain from T cells (9). FACs analysis of CD3e chain expression onboth naive murine T cells and GA15 that had been treated withbromelain for 30 min (0–100mg/ml) showed no cleavage ofCD3e, therefore confirming those earlier findings (data notshown). The effect of bromelain on TCR-mediated signal trans-duction was then investigated by assessing substrate tyrosine phos-phorylation of GA15 stimulated with cross-linked anti-CD3e mAb.Immunoblots of GA15 lysates with specific anti-phosphotyrosinemAb revealed increased tyrosine phosphorylation of multiple pro-teins, including those of circa 120, 100, 85, 76, 70, 42, and 40 kDa,consistent with phosphoproteins observed in other T cell lines fol-lowing TCR-ligation (15, 34) (Fig. 1A). Tyrosine-phosphorylatedproteins were readily detected between 2 and 5 min followingstimulation and remained phosphorylated for at least 10 min (Fig.

2569The Journal of Immunology

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 4: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

1A). GA15 cells stimulated with anti-CD3 mAb alone or cross-linking Ab did not induce tyrosine phosphorylation of any cellularsubstrate (data not shown). Bromelain pretreatment of GA15 for30 min caused a dose-dependent reduction in TCR-induced proteintyrosine phosphorylation of a 42-kDa phosphoprotein (Fig. 1B). Ata bromelain concentration of 100mg/ml, tyrosine phosphorylationwas reduced by 87%, as determined by densitometry. Bromelaindid not markedly affect tyrosine phosphorylation of other TCR-induced phosphoproteins, suggesting a selective mode of action.

We suspected that the 42-kDa phosphoprotein was the MAPKERK-2, so we conducted immunoblot analysis with specific anti-ERK-2 mAb and anti-phospho MAPK Abs, which specifically de-tects ERK-1 and ERK-2 only when catalytically activated by phos-phorylation at Tyr204. Immunoblotting confirmed that the p42-kDa phosphoprotein was indeed ERK-2 and that bromelaininhibited its mobility shift, which is characteristic of ERK-2 acti-vation (Fig. 1C).

To investigate whether bromelain could, in addition to blockingTCR-mediated T cell signaling, also prevent TCR-independentsignaling, we examined its effect on PMA plus ionophore-inducedtyrosine phosphorylation. Phorbol ester plus ionophore stimulationof T cells act synergistically to reproduce many features of TCRstimulation, such as IL-2 secretion, IL-2 receptor expression, andT cell proliferation (35, 36). Phorbol esters mimic Ag receptortriggering and bypass TCR-induced protein tyrosine kinases to ac-tivate ERK-2 by a direct agonist action on PKC and p21ras. Iono-phore A23187 induces increased intracellular release of Ca21 andtherefore mimics the action of inositol 1,4,5-trisphosphate (IP3).Again, bromelain dose-dependently reduced tyrosine phosphory-lation of ERK-2 and prevented its mobility shift (Fig. 1C). Stim-ulation with either PMA or ionophore alone resulted in marginal,bromelain-sensitive, phosphorylation of ERK-2 (Fig. 1D). This re-sult confirms previous observations of a synergistic action of phor-bol esters plus ionophore on T cell signaling (35, 36) and alsoshows that bromelain blocks signals generated by PMA alone andionophore alone. To exclude the possibility that differences in ki-netics of the response account for our observation, longer times ofactivation were examined. Even after 60 min of stimulation, weobserved that bromelain inhibited phosphorylation of ERK-2 (datanot shown).

Bromelain inhibits ERK activity

Activation of ERK-2 induces phosphorylation and transcriptionalactivation by Elk-1 (24). Similarly, inhibitors of ERK-2 would beexpected to block activation of Elk-1. To confirm that the ability ofbromelain to block tyrosine phosphorylation of ERK-2 also cor-related with inhibition of effector function of the enzyme, the abil-ity of ERK to regulate phosphorylation of the transcription factorElk-1 was investigated. Kinase assays were conducted on brome-lain-treated GA15 cells that were then stimulated with PMA plusionophore. In control cells, combined PMA and ionophore inducedphosphorylation of Elk-1 (Fig. 2) and reduced its electrophoreticmobility (data not shown). Bromelain treatment decreased phos-phorylation of Elk-1 (Fig. 2) and its retardation in electrophoreticmobility, thereby confirming that the bromelain-mediated inhibi-tion of ERK-2 tyrosine phosphorylation results in a correspondingreduction in biological activity.

Inhibition of ERK requires proteolytic activity of bromelain

To determine whether the inhibitory effect of bromelain was de-pendent on its proteolytic activity, we used a specific cysteine pro-tease inhibitor, E-64, to inactivate its protease activity (37). GA15cells were treated with inactivated bromelain (50mg/ml) and com-pared with proteolytically active bromelain and mock-treated Tcells stimulated with PMA plus ionophore. Inactivation of the pro-tease activity of bromelain completely abrogated its inhibitory ef-fect on ERK-2 phosphorylation (Fig. 3A). E-64 treatment of GA15did not affect PMA plus ionophore-induced T cell signaling. Thesedata indicate that the inhibitory effect of bromelain on ERK isdependent on its protease activity.

We next confirmed that the inhibitory effect of bromelain onERK-2 phosphorylation was not a nonspecific enzyme effect, bytreating GA15 cells with proteolytically equivalent amounts of

FIGURE 1. The effect of bromelain on TCR-induced or PMA plus iono-phore-induced substrate tyrosine phosphorylation.A, T cells were stimu-lated with cross-linked anti-CD3e mAb for 0–20 min.B and C, T cellswere treated with bromelain (0–100mg/ml) for 30 min, washed, and thenstimulated with cross-linked anti-CD3e mAb or combined PMA plus iono-phore for 5 min.D, T cells were treated with bromelain or mock-treatedwith PBS, washed, and then stimulated with either PMA alone, ionophorealone, or PMA plus ionophore for 5 min. Following stimulation, cells werelysed, and postnuclear supernatants were subjected to SDS-PAGE and im-munoblotted using anti-phosphotyrosine mAb, phospho-specific MAPKAb, or anti-ERK-2 mAb, as indicated. Arrowheads denote anti-CD3e mAb-induced tyrosine phosphorylation. Results shown are from one experimentthat is representative of five conducted.

2570 BROMELAIN BLOCKS T CELL SIGNAL TRANSDUCTION

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 5: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

trypsin, as determined by the Z-Arg-ArgpNA substrate. Both en-zymes were tested at a specific proteolytic activity of 77 nmol/min/mg. Trypsin, a serine protease, shares common cleavage sitesto bromelain, but is not inactivated by E-64 (37). Trypsin did notinhibit tyrosine phosphorylation of ERK-2 (Fig. 3B). A specificactivity of trypsin that was two or four times that of bromelain stillhad no effect on ERK phosphorylation (Fig. 3C), indicating thatbromelain-mediated inhibition of ERK-2 is not due to a nonspe-cific degradative effect of proteolysis on the cell surface. Brome-lain, but not trypsin, also inhibited tyrosine phosphorylation ofPMA plus ionophore-induced ERK-1. In some experiments, tryp-sin appeared to increase tyrosine phosphorylation of ERK-2 (datanot shown), which has been reported earlier (38).

Bromelain inhibits ERK-2 tyrosine phosphorylation in JurkatT cells

To confirm that the effect of bromelain was not restricted to theGA15 T cell hybridoma, we also showed inhibitory effects on ty-rosine phosphorylation of ERK-2 in human Jurkat T cells (Fig. 4).In addition, bromelain also inhibited tyrosine phosphorylation ofERK-2 in murine EL-4 and 2B4 T cell lines (data not shown).

Bromelain inhibits p21ras activity

To begin to explore the mechanism of action of bromelain onERK-2, we investigated its effect on p21ras, an upstream effector of

the Raf-1/MEK1/ERK-2 kinase cascade. GA15 cells were treatedwith bromelain (0–100mg/ml) and then stimulated with combinedPMA plus ionophore, as described earlier. Cell lysates were thenincubated with a GST-Ras-binding domain of Raf-1, known tospecifically interact with the active Ras-GTP complex. Bromelaindose-dependently inhibited the affinity precipitation of activep21ras, thereby inhibiting p21ras activation (Fig. 5). Further studiesalso showed that bromelain blocked activation of Raf-1 (data notshown). These data indicate that the inhibitory effect of bromelain

FIGURE 2. ERK-2 from bromelain-treated T cells is reduced in its abil-ity to phosphorylate Elk-1. T cells were treated with bromelain (0–100mg/ml) for 30 min, washed, and then stimulated with PMA plus ionophorefor 5 min. Cells were then lysed, and postnuclear supernatants were im-munoprecipitated with phospho-specific MAPK Ab. ImmunoprecipitatedERKs were then incubated with an Elk-1 fusion protein and assessed forElk-1 phosphorylation using phospho-specific Elk-1 Ab. Phosphorylationof the Elk-1 substrate was quantitated by densitometry of bands revealedby chemiluminescence. Results shown are from one experiment that isrepresentative of three conducted.

FIGURE 3. Inhibition of ERK-2 phosphorylation is dependent on spe-cific proteolytic activity.A, T cells were treated with bromelain (50mg/ml),E-64-inactivated bromelain (50mg/ml), or mock treated for 30 min. Cellswere then washed and stimulated with PMA plus ionophore for 5 min.B,GA15 cells were treated with bromelain (50mg/ml; sp. act., 77 nmol/min/mg), proteolytically equivalent amounts of trypsin (sp. act., 77 nmol/min/mg), or mock-treated for 30 min. Cells were then washed and stimulatedwith PMA plus ionophore for the times indicated.C, GA15 cells weretreated with bromelain (50mg/ml; sp. act., 77 nmol/min/mg; Br.), trypsin(sp. act., 77–308 nmol/min/mg), or mock treated for 30 min (Cont.). Cellswere washed and stimulated with PMA plus ionophore (1) or media alone(2) for 5 min. Cells were lysed, and postnuclear supernatants were sub-jected to SDS-PAGE and immunoblotted with anti-phosphotyrosine mAbor phospho-specific MAPK Ab as indicated. Results shown are from oneexperiment that is representative of four conducted.

2571The Journal of Immunology

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 6: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

on ERK-2 in GA15 cells is mediated by a block in the p21ras/Raf-1/MEK1 cascade, upstream from ERK-2.

Bromelain inhibits IL-2 mRNA accumulation in GA15

Activation of p21ras and the Raf-1/MEK-1/ERK pathway is essen-tial for induction of IL-2 transcription in T cells (22). The inhibi-tion of p21ras and ERK activation shown here could therefore beexpected to inhibit IL-2 production. Therefore, we investigatedwhether bromelain could effect a functional outcome of GA15 cellsignaling, namely IL-2 mRNA accumulation induced by anti-CD3e mAb or PMA plus ionophore-induced stimulation.

Bromelain treatment of GA15 cells significantly inhibitedmRNA accumulation of IL-2 when the ERK pathway was stimu-lated directly with combined PMA and ionophore (p , 0.01). Incontrast, however, bromelain did not block IL-2 cytokine mRNAaccumulation when cells were stimulated via the TCR with anti-CD3e mAb (Fig. 6). Although not statistically significant, brome-lain appeared to increase IL-2 production when cells were stimu-lated via the TCR (p , 0.09). To assess whether the defect in ERKactivation could affect production of other cytokines, we also ex-amined the effect of bromelain on IL-4 and IFN-g mRNA accu-mulation. We found that bromelain similarly inhibited PMA plusionophore-, but not TCR-induced IL-4 and IFN-g mRNA accu-mulation (p , 0.01 andp , 0.05, respectively). IL-4 productionseemed to be most sensitive to bromelain treatment with bromelainblocking IL-4 expression by 94%. Bromelain blocked PMA plusionophore-induced IL-2 and IFN-g by 68% and 56%, respectively(Fig. 6A).

Cytokine mRNA production was dependent on cell stimulation,as negligible cytokine mRNA was detected in cells cultured in

media alone (Fig. 6B). In addition, the culture period of 4 h en-sured that cytokine mRNA accumulated was a result of the specificstimuli used, rather than an autocrine effect of newly synthesizedcytokine. However, at present, it is unknown whether bromelainaffects cytokine mRNA transcription or stability. These resultsshow that bromelain can inhibit PMA plus ionophore-induced cy-tokine production in GA15 cells, but does not affect TCR/CD3e-induced cytokine production. In addition, this data suggests thatTCR signals can bypass the requirement for p21ras and ERK-2 inIL-2 production.

Bromelain is not toxic to T cells

To ensure that the effects of bromelain were not simply due to atoxic effect, we investigated its effect on GA15 proliferation. Bro-melain pretreatment (0–100mg/ml for 30 min) did not decreaseGA15 proliferation, nor affect their viability, as determined bytrypan blue staining (data not shown). Similar results were ob-tained when we investigated the proliferation of purified murineCD41 and CD81 T cells (C. R. Engwerda and T. L. Mynott,manuscript in preparation) and human PBMCs (9). These dataindicate that bromelain does not significantly affect the viability ofT cells.

FIGURE 4. Bromelain inhibits ERK-2 phosphorylation in Jurkat Tcells. Jurkat T cells were treated with bromelain (50mg/ml) for 30 min,washed, and then stimulated with PMA plus ionophore for 5 min. Cellswere then lysed, and postnuclear supernatants were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine mAb. Results shownare from one experiment that is representative of four conducted.

FIGURE 5. Bromelain inhibits p21ras. T cells were treated with brome-lain (0–100mg/ml) for 30 min, washed, and then stimulated with PMAplus ionophore for 5 min. Cell lysates were incubated with a GST-fusionprotein coupled to the RBD of Raf-1. Affinity-precipitated active p21ras-GTP proteins were detected by immunoblotting with anti-Ras mAb. Re-sults shown are from one experiment that is representative of fourconducted.

2572 BROMELAIN BLOCKS T CELL SIGNAL TRANSDUCTION

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 7: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

DiscussionRecently, it has emerged that extracellular proteases have specificregulatory roles in modulating immune responses (reviewed inRefs. 3, 39, 40). Cysteine and serine proteases may act as signalingmolecules via an interaction with specific PARs leading to activa-tion of the Raf-1/ERK-2 pathway to participate in mitogenesis,apoptosis, and cytokine production. Most reports on the role ofprotease-mediated cell signaling focus on the stimulatory effects ofproteases on signal transduction. In this study, we show for the firsttime that extracellular proteases may also block signal transduc-tion. We show that bromelain, a mixture of cysteine proteases frompineapple stems, inhibits signal transduction and cytokine produc-tion in a Th0 cell hybridoma. The inhibitory action of bromelain is

not simply caused by a degradative action on cell surface mole-cules to alter receptor-ligand interactions, because bromelainblocked signaling by combined PMA plus ionophore, membrane-permeable agonists, which do not rely on extracellular receptors intheir action. To our knowledge, bromelain is the first example ofan extracellular protease that blocks p21ras and ERK-2 signaling.Other compounds that act as inhibitors of MAPK pathways, forexample, the pyrrolo[2,3-d] pyrimidines (41) and flavones (42), arevery small organic molecules of,1000 Da. The precise mecha-nism of action of bromelain remains to be determined. Preliminarystudies suggest that bromelain acts upstream in the MAPK path-way and, at least, acts at the level of p21ras. Since p21ras is aneffector for multiple MAPK pathways, it is likely that bromelain

FIGURE 6. Bromelain blocks PMA plus ionophore, but not TCR-induced cytokine gene expression. GA15 cells were treated with bromelain (50mg/ml)or mock treated for 30 min. Cells were washed, resuspended in fresh culture medium, and then stimulated with PMA plus ionophore, cross-linked anti-CD3emAb, or mock treated with PBS for 4 h. IL-2, IFN-g, and IL-4 mRNA accumulation was measured using a semiquantitative RT-PCR assay and standardizedwith the hypoxantine-guanineohosphoribosyl transferase (HPRT) house-keeping gene.A, The intensity of signals was quantitated by densitometry of bandsrevealed by chemiluminescence. Results from individual experiments (n 5 5) are represented by unique symbols, and bars represent the mean. Closedsymbols represent cytokine mRNA levels in bromelain-treated cells; open symbols represent cytokine levels in mock-treated cells. †,p , 0.05;p, p , 0.01;‡, p 5 0.09, paired Student’st test.B, RT-PCR results (in duplicate) of a representative experiment (symbolE from A) from five performed.

2573The Journal of Immunology

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 8: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

also affects other MAPK signaling cascades, such as the JNK path-way or p38 MAPK pathway. Studies are in progress to investigatethe action of bromelain on other p21ras effector pathways.

Bromelain inhibition of ERK-2 can be abrogated by a selectiveprotease inhibitor. Therefore, bromelain most likely exerts its in-hibitory effect via a proteolytic action on a T cell surface mole-cule(s), possibly via an, as yet, unidentified PAR. The bromelaintarget remains to be elucidated and studies are in progress. We do,however, thus far, rule out the possibility that bromelain cleavescertain T cell surface molecules. Earlier, Hale and Haynes (9) re-ported that bromelain cleaved the CD45 RA isoform and selec-tively removed other surface molecules from human PBMCs, in-cluding partial removal of CD4. Since CD45 and CD4 play anobligate, stimulatory role in TCR-mediated T cell activation (43,44), bromelain could interfere with TCR signaling by affectingthese molecules. However, this seems unlikely because it is pos-sible to bypass the requirement for CD45 and CD4 in T cell acti-vation by the use of phorbol ester plus ionophore. Use of combinedphorbol esters and ionophore restores normal function to T cellsthat have been made refractory to TCR stimulation by tyrosinekinase inhibitors (29), or which are CD45- (45) or p56lck (21)-deficient. In the current study, PMA plus ionophore did not restoreERK-2 activity; therefore, the inhibitory effect of bromelain is notthought to be mediated via effects on CD45 or CD4 in GA15. It isinteresting to note, that unlike other substances that inhibit TCRstimulation upstream of p21ras activation and block tyrosine phos-phorylation of several substrates activated via the TCR (28, 29),bromelain appears to only affect tyrosine phosphorylation of ERK.

Activation of ERKs is essential for cytokine gene transcription(27); therefore, ERK inhibition would be expected to block cyto-kine mRNA accumulation and cytokine production. Bromelain didinhibit cytokine mRNA accumulation induced by PMA plus iono-phore, as predicted, but did not block cytokine mRNA induced byCD3e-mediated TCR signals. This data would suggest the exis-tence of a TCR signaling pathway in GA15 cells that induces cy-tokine mRNA production independent of ERK-2 activation. Theexistence of an ERK-independent pathway in CD3e/TCR stimu-lation of cytokine production and other T cell functions has alreadybeen proposed (46, 47). Our data is consistent with this view andsupports the hypothesis that T cells have developed alternativemechanisms for up-regulating the IL-2 gene independent of ERKactivity. Interestingly, we found that IL-4 and IFN-g productionare also linked to the alternate pathway, as both cytokines wereinhibited by bromelain treatment following GA15 cell stimulationwith PMA plus ionophore, but not with anti-CD3e mAb. Theseresults indicate that, similar to IL-2 gene transcription, the ERK-2signal transduction cascade is not the only effector pathway forIL-4 and IFN-g production.

An interesting observation in this study concerns the action ofbromelain on cytokine production. We noted that bromelain had amore marked effect on IL-4 production than on IL-2 and IFN-gproduction. IL-4 is produced by Th2 cells, and its expression wasinhibited by 94%. IL-2 and IFN-g are produced by Th1 cells, andtheir production was reduced by 68% and 56%, respectively. Ear-lier, Egerton et al. (48) showed that a hierarchy of cytokine re-sponsiveness to ERK activation existed in T cells that expressedconstitutively active MEK. It may also be possible that bromelaininhibition of the MAPK pathway may lead to a more selectiveeffect against Th2-specific cytokine expression patterns than Th1-specific expression.

In summary, we have shown that bromelain is a novel, nontoxicinhibitor of Th0 cell signaling and cytokine production. Furtherstudies conducted with purified murine (BALB/c) CD41 T cellsshow that bromelain also blocks IL-2 production but does not af-

fect CD41 T cell proliferation, thereby confirming these findings(C. R. Engwerda and T. L. Mynott, manuscript in preparation). Itis interesting to note that bromelain has been used therapeuticallyfor the treatment of inflammation and trauma (49). It has also beenproposed to control tumor growth (50). Despite these reports, noconvincing data are available on its mechanism of action. Theantiinflammatory effects of bromelain may be mediated, in part, bythe inhibition of thromboxane synthesis (49) or by inhibition ofbradykinin production (51). ERK-2 and p21ras have a pivotal rolein the integration of many extracellular stimuli, allowing for cellgrowth, differentiation, cytokine, and stress responses. Targetingof p21ras and the MAPK pathways is proposed as one approach todevelop therapeutic agents to treat chronic inflammation or cancer.Therefore, the discovery that bromelain inhibits signaling byERK-2 and p21ras may account for its antiinflammatory and anti-tumor action. Many reports about the role of proteases in diseasehave centered around their potential to cause damage (reviewed inRef. 39). However, it is possible that cysteine proteases may, incontrast, have a therapeutic role in certain diseases.

AcknowledgmentsWe thank Barbara Fox for her support in the early stages of the work.

References1. Smyth, M. J., M. D. O’Connor, and J. A. Trapani. 1996. Granzymes: a variety of

serine protease specificities encoded by genetically distinct subfamilies.J. Leu-kocyte Biol. 60:555.

2. Vaux, D. L., S. Wilhelm, and G. Hacker. 1997. Requirements for proteolysisduring apoptosis.Mol. Cell. Biol. 17:6502.

3. Dery, O., C. U. Corvera, M. Steinhoff, and N. W. Burnett. 1998. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases.Am. J. Physiol. 274:C1429.

4. Molloy, C. J., J. E. Pawlowski, D. S. Taylor, C. E. Turner, H. Weber, andM. Peluso. 1996. Thrombin receptor activation elicits rapid protein tyrosine phos-phorylation and stimulation of the raf-1/MAP kinase pathway preceding delayedmitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligateautocrine mechanism promoting cell proliferation induced by G-protein-coupledreceptor agonist.J. Clin. Invest. 97:1173.

5. Molino, M., N. Blanchard, E. Belmonte, A. P. Tarver, C. Abrams, J. A. Hoxie,C. Cerletti, and L. F. Brass. 1995. Proteolysis of the human platelet and endo-thelial cell thrombin receptor by neutrophil-derived cathepsin G.J. Biol. Chem.270: 11168.

6. Suidan, H. S., J. Bouvier, E. Schaerer, S. R. Stone, D. Monard, and J. Tschopp.1994. Granzyme A released upon stimulation of cytotoxic T lymphocytes acti-vates the thrombin receptor on neuronal cells and astrocytes.Proc. Natl. Acad.Sci. USA 91:8112.

7. Vouret-Craviari, V., D. Grall, J. C. Chambard, U. B. Rasmussen, J. Pouyssegur,and E. Van Obberghen-Schilling. 1995. Post-translational and activation-depen-dent modifications of the G protein-coupled thrombin receptor.J. Biol. Chem.270:8367.

8. Rowan, A. D., D. J. Buttle, and A. J. Barrett. 1990. The cysteine proteinases ofthe pineapple plant.Biochem. J. 266:869.

9. Hale, L. P., and B. F. Haynes. 1992. Bromelain treatment of human T cellsremoves CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surfacemolecules and markedly enhances CD2-mediated T cell activation.J. Immunol.149:3809.

10. Desser, L., A. Rehberger, E. Kokron, and W. Paukovits. 1993. Cytokine synthesisin human peripheral blood mononuclear cells after oral administration of poly-enzyme preparations.Oncology 50:403.

11. Andrew, E. M., W. Annis, M. Kahan, and R. N. Maini. 1990. CD5 (Ly-1)-negative, conventional splenic B cells make a substantial contribution to thebromelain plaque-forming cell response in CBA and BW mice.Immunology69:515.

12. Hale, L. P., K. H. Singer, and B. F. Haynes. 1989. CD44 antibody against In(Lu)-related p80, lymphocyte-homing receptor molecule inhibits the binding of humanerythrocytes to T cells.J. Immunol. 143:3944.

13. Payes, J., and A. E. Buzzard. 1975. Precommitment of normal mouse peri-toneal cells by erythrocyte antigens in relation to auto-antibody production.Nature 257:316.

14. Mynott, T. L., S. Guandalini, F. Raimondi, and A. Fasano. 1997. Bromelainprevents secretion caused byVibrio choleraeandEscherichia colienterotoxins inrabbit ileum in vitro.Gastroenterology 113:175.

15. Cantrell, D. 1996. T cell antigen receptor signal transduction pathways.Annu.Rev. Immunol. 14:259.

16. Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system ofT cell costimulation.Annu. Rev. Immunol. 14:233.

17. Nishida, E., and Y. Gotoh. 1993. The MAP kinase cascade is essential for diversesignal transduction pathways.Trends Biochem. Sci. 18:128.

2574 BROMELAIN BLOCKS T CELL SIGNAL TRANSDUCTION

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 9: Bromelain, from Pineapple Stems, Proteolytically Blocks ... · Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells Tracey

18. Boulton, T. G., S. H. Nye, D. J. Robbins, N. Y. Ip, E. Radziejewska,S. D. Morgenbesser, R. A. DePinho, N. Panayotatos, M. H. Cobb, andG. D. Yancopoulos. 1991. ERKs: a family of proteins-serine/threonine kinasesthat are activated and tyrosine phosphorylated in response to insulin and NGF.Cell 65:663.

19. Anderson, N. G., J. L. Maller, N. K. Tonks, and T. W. Sturgill. 1990. Require-ment for integration of signals from two distinct phosphorylation pathways foractivation of MAP kinase.Nature 343:651.

20. Derijard, B., M. Hibi, I. -H. Wu, T. Barret, B. Su, T. Deng, M. Karin, andR. J. Davis. 1994. JNK1: A protein kinase stimulated by UV light and Ha-Rasthat binds and phosphorylates the c-Jun activation domain.Cell 76:1025.

21. Gupta, S., A. Weiss, G. Kumar, S. Wang, and A. Nel. 1994. The T cell antigenreceptor utilises Lck, Raf-1, and MEK-1 for activating mitogen-activated proteinkinase: evidence for the existence of a second protein kinase C-dependent path-way in an Lck-negative Jurkat cell mutant.J. Biol. Chem. 269:17349.

22. Izquierdo, M., S. J. Leevers, C. J. Marshall, and D. Cantrell. 1993. p21ras couplesthe T cell antigen receptor to extra signal regulated kinase in T cells.J. Exp. Med.78:1199.

23. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNKis involved in signal integration during costimulation of T lymphocytes.Cell77:727.

24. Marais, R., J. Wynne, and R. Treisman. 1993. The SRF accessory protein Elk-1contains a growth factor-regulated transcriptional activation domain.Cell 73:381.

25. Gille, H., A. Sharrocks, and P. Shaw. 1992. Phosphorylation of p62TCF by MAPkinase stimulates ternary complex formation at c-fos promoter.Nature 358:414.

26. Hibi, M., A. Lin, T. Smeal, A. Minden, and M. Karin. 1993. Identification of anoncoprotein and UV responsive protein kinase that binds and potentiates thec-Jun activation domain.Genes Dev. 7:2135.

27. Angel, P., and M. Karin. 1991. The role of Jun, Fos and the AP-1 complex in cellproliferation and transformation.Biochim. Biophys. Acta 1072:129.

28. Mustelin, T., K. M. Coggeshall, N. Isakov, and A. Altman. 1990. T cell antigenreceptor-mediated activation of phospholipase C requires tyrosine phosphoryla-tion. Science 247:1584.

29. June, C. H., M. C. Fletcher, J. A. Ledbetter, G. L. Schieven, J. N. Siegal,A. F. Phillips, and L. E. Samelson. 1990. Inhibition of tyrosine phosphorylationprevents T-cell receptor-mediated signal transduction.Proc. Natl. Acad. Sci. USA87:7722.

30. Napper, A. D., S. P. Bennett, M. Borowski, M. B. Holdridge, M. J. Leonard,E. E. Rogers, Y. Duan, R. A. Laursen, B. Reinhold, and S. L. Shames. 1994.Purification and characterization of multiple forms of the pineapple-stem-derivedcysteine proteinases ananain and comosain.Biochem. J. 301:727.

31. Fox, B. S. 1993. Antigen presenting cell-derived co-stimulatory signals can se-lectively regulate IL-2 and IL-4 production from a Th0 cell hybridoma.Int. Im-munol. 5:323.

32. Taylor, S. J., and D. Shalloway. 1996. Cell cycle-dependent activation of Ras.Curr. Biol. 6:1621.

33. Engwerda, C. R., S. C. Smelt, and P. M. Kaye. 1996. An in vivo analysis ofcytokine production duringLeishmania donovaniinfection in scid mice.Exp.Parasitol. 84:195.

34. June, C. H., M. C. Fletcher, J. A. Ledbetter, and L. E. Samelson. 1990. Increasesin tyrosine phosphorylation are detectable before phospholipase C activation afterT cell receptor stimulation.J. Immunol. 144:1591.

35. Truneh, A., F. Albert, P. Golstein, and A -M. Schmitt-Verhulst. 1985. Early stepsof lymphocyte activation bypassed by synergy between calcium ionophores andphorbol esters.Nature 313:318.

36. Rayter, S. I., M. Woodrow, S. C. Lucas, D. A. Cantrell, and J. Downward. 1992.p21ras mediates control of IL-2 gene promoter function in T cell activation.EMBO 11:4549.

37. Barrett, A. J., A. A. Kembhavi, M. A. Brown, H. Kirschke, C. G. Knight,M. Tamai, and K. Hanada. 1982. L-trans-Epoxysuccinyl-leucylamido(4-guanidi-no)butane (E-64) and its analogues as inhibitors of cysteine proteinases includingcathepsins B, H and L.Biochem. J. 201:189.

38. Belham, C. M., R. J. Tate, P. H. Scott, A. D. Pemberton, H. R. P. Miller,R. M. Wadsworth, G. W. Gould, and R. Plevin. 1996. Trypsin stimulates pro-teinase-activated receptor-2-dependent and independent activation of mitogen-activated protein kinases.Biochem. J. 320:939.

39. Chapman, H. A., R. J. Riese, and G-P. Shi. 1997. Emerging role for cysteineproteases in human biology.Annu. Rev. Physiol. 59:63.

40. Altieri, D. C. 1995. Proteases and protease receptors in modulation of leukocyteeffector functions.J. Leukocyte Biol. 58: 120.

41. Hanke, J. H., J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette,E. J. Weringer, B. A. Pollok, and P. A. Connelly. 1996. Discovery of a novel,potent, and Src family selective tyrosine kinase inhibitor.J. Biol. Chem. 271:695.

42. Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, and A. R. Saltiel. 1995. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinasekinase in vitro and in vivo.J. Biol. Chem. 270:27489.

43. Glaichenhaus, N., N. Shastri, D. R. Littman, and J. M. Turner. 1991. Requirementfor association of p56lck with CD4 in antigen-specific signal transduction in Tcells.Cell 64:511.

44. Trowbridge, I. S., and M. L. Thomas. 1994. CD45: An emerging role as a proteintyrosine phosphatase required for lymphocyte activation and development.Annu.Rev. Immunol. 12:85.

45. Ihle, J. N., B. A. Wittuhn, F. W. Qelle, K. Yamamoto, and O. Silvennoinen. 1995.Signaling through the haematopoietic cytokine receptors.Annu. Rev. Immunol.13:369.

46. Genot, E., S. Cleverley, S. Henning, and D. A. Cantrell. 1996. Multiple p21ras

effector pathways regulate nuclear factor of activated T cells.EMBO J. 15:3923.47. Alberola-IIa, J., K. A. Forbush, R. Seger, E. G. Krebs, R. M. Perlmutter. 1995.

Selective requirement for MAP kinase activation in thymocyte differentiation.Nature 373:620.

48. Egerton, M., D. R. Fitzpatrick, A. D. Catling, and A. Kelsoe. 1996. Differentialactivation of T cell cytokine production by the extracellular signal-regulated ki-nase (ERK) signaling pathway.Eur. J. Immunol. 26:2279.

49. Taussig, S., and S. Batkin. 1988. Bromelain, the enzyme complex of pineapple(Ananus comosus) and its clinical application: an update.J. Ethnopharmacol.22:191.

50. Batkin, S., S. J. Taussig, and J. Szekerczes. 1988. Anti-metastatic effect of bro-melain with or without its proteolytic and anticoagulant activity.Cancer Invest.6:241.

51. Kumakura, S., M. Yamashita, and S. Tsurufuji. 1988. Effect of bromelain onkaolin-induced inflammation in rats.Eur. J. Pharmacol. 150:295.

2575The Journal of Immunology

by guest on July 8, 2018http://w

ww

.jimm

unol.org/D

ownloaded from