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1hck the Phosphorylation and Activation of p59IL-2 Signaling in Human Monocytes Involves

Malabarba, John R. Ortaldo and Igor Espinoza-DelgadoMaria C. Bosco, Rafael E. Curiel, Arnold H. Zea, Maria G.

http://www.jimmunol.org/content/164/9/4575doi: 10.4049/jimmunol.164.9.4575

2000; 164:4575-4585; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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IL-2 Signaling in Human Monocytes Involves thePhosphorylation and Activation of p59hck 1

Maria C. Bosco,* Rafael E. Curiel,† Arnold H. Zea,‡ Maria G. Malabarba, § John R. Ortaldo,¶

and Igor Espinoza-Delgado2†

The activating properties of IL-2 and the structure of the IL-2R on human monocytes are well characterized. However, relativelylittle is known about the biochemical mechanisms involved in IL-2 signal transduction in these cells. We investigated the role ofprotein tyrosine kinases (PTKs) in the activation of monocytes by IL-2. Incubation of monocytes with the PTK inhibitor herbi-mycin A (HA) resulted in the dose-dependent suppression of IL-2-induced monocyte tumoricidal activity. This inhibition wasrather potent, as a concentration of HA as low as 0.5mM caused a complete abrogation of cytolytic activity. Furthermore, HAmarkedly suppressed the ability of IL-2 to induce IL-1b, TNF-a, IL-6, and IL-8 mRNA expression and protein secretion bymonocytes. Anti-phosphotyrosine immunoblotting demonstrated that IL-2 induced a rapid and time-dependent increase in ty-rosine phosphorylation of several cellular proteins of molecular masses ranging from 35 to 180 kDa. Interestingly, IL-2 caused asignificant up-regulation of the constitutive levels ofhck PTK mRNA and protein relative to medium-treated cells as well as anincrease in p59hck tyrosine phosphorylation. Finally, we demonstrated by in vitro kinase assay that the specific activity of p59hck

PTK was also induced by IL-2 in monocytes. Thus, these data show that the activation of PTKs is required for the triggering ofmonocyte effector and secretory functions by IL-2 and strongly suggest that p59hck is a key participant in IL-2 signaling in humanmonocytes. The Journal of Immunology,2000, 164: 4575–4585.

T he IL-2R complex has been extensively characterized in Tlymphocytes and is now known to comprise at least threedistinct, noncovalently associated, and independently reg-

ulated membrane components, thea-chain (IL-2Ra), theb-chain(IL-2Rb), and theg-chain (IL-2Rg). These components can beexpressed in various combinations, resulting in receptors with dif-ferent affinities for IL-2 as well as different functional attributes (1,2). Previous studies have demonstrated that both the IL-2Rb andIL-2Rg subunits are required for the formation of functional IL-2Rs and for IL-2 intracellular signaling, whereas the IL-2Ra chainis endowed with IL-2 binding but is devoid of signal-transducingcapabilities (3). Even though none of the IL-2R components pos-sesses any intrinsic catalytic activity (1, 2), one of the earliestbiochemical events observed after T lymphocyte stimulation byIL-2 is the increased tyrosine phosphorylation of several cellularproteins and the subsequent induction of nuclear proto-oncogenescritical for cellular proliferation (1), thus suggesting the activationof cytoplasmic IL-2R-coupled protein tyrosine kinases (PTKs).3 Inthis regard, evidence for the physical and functional association of

PTK activity with the cytoplasmic domains of theb- andg-chainshas been reported in lymphoid cells, and significant advances haverecently been made in identifying the multiple signaling moleculesthat specifically interact with the IL-2R subunits (1, 3, 4). IL-2binding to functional IL-2Rs on T cells leads to the recruitmentand activation of distinct nonreceptor PTKs, such as p56lck (5, 6)of the src family PTKs, Syk PTK of the Syk/ZAP-70 family (7),and JAK1 and JAK3 of the Janus kinase (JAK) family (8–11).Specifically, p56lck, Syk, and JAK1 couple with the cytoplasmicdomain of the IL-2b-chain (5, 7, 8, 11, 12), whereas JAK3 asso-ciates with the cytoplasmic region of the IL-2Rg (8–11).

Although originally identified as a T cell growth factor, IL-2was later shown to exert a wide range of biological effects onseveral other cell types. Functional IL-2Rs have been found on Bcells (13–15), NK cells (3), polymorphonuclear cells (16–18), andmonocytes/macrophages (18–21). We and others have previouslyshown that IL-2 is a powerful activator of human monocytes (19,22). Monocyte stimulation with IL-2 leads to the secretion of sev-eral cytokines (23–27) and growth factors (28–30); to the expres-sion of growth factor receptors (19, 21) and adhesion and costimu-latory molecules (I. Espinoza-Delgado, S. Rottshafer, R. E. Curiel,and M. C. Bosco, manuscript in preparation); and to the enhancedproduction of hydrogen peroxide, superoxide, PGE2, and throm-boxane B2 (20, 31). Furthermore, IL-2 can activate fresh humanmonocytes to exert microbicidal (20) and tumoricidal activities(19, 32) and can potentiate their Ag-presenting ability (I. Espi-noza-Delgado et al., manuscript in preparation). Fresh peripheralblood monocytes constitutively express the IL-2Rb and IL-2Rgchains (19, 33–35), but not the IL-2Ra subunit, which is inducibleby stimulation with IFN-g (18, 20, 21, 36) or LPS (20, 37). IL-2, onthe other hand, can up-regulate the expression of theb-chain (21) andg-chain (34), but is unable to induce thea subunit (20, 21).

Although a large body of information is available on thebiological effects of IL-2 and the expression and regulation ofthe IL-2R components on human monocytes, the biochemical

*Laboratory of Molecular Biology, Giannina Gaslini Institute, Genova Quarto, Italy;Departments of†Medicine and‡Microbiology and Stanley S. Scott Cancer Center,Louisiana State University Medical Center, New Orleans, LA 70112; and Laborato-ries of §Immunoregulation and¶Experimental Immunology, Cytokines MolecularMechanisms Section, National Cancer Institute-Frederick Cancer Research and De-velopment Center, Frederick, MD 21702

Received for publication December 3, 1999. Accepted for publication February24, 2000.

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 This work was supported in part by grants from the Italian Association for CancerResearch and from Telethon Italy (Project A 75).2 Address correspondence and reprint requests to Dr. Igor Espinoza-Delgado, Loui-siana State University Medical Center, 1542 Tulane Avenue, Hematology-Oncology,Suite 604K, New Orleans, LA 70112. E-mail address: iespin@1sumc.edu3 Abbreviations used in this paper: PTK, protein tyrosine kinase; JAK, Janus kinase;HA, herbimycin A; LAK, lymphokine-activated killer cells.

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mechanisms involved in IL-2 signal transduction in these cellshave not yet been elucidated. There is increasing evidence thattyrosine phosphorylation is important in monocyte functions (38),and different members of thesrc family PTKs, such ashck, fgr,and lyn, have been shown to be critical components of the signaltransduction pathway of several monocyte/macrophage-activatingfactors (39–43), thus raising the possibility that PTK activationcould also be involved in mediating monocyte responses to IL-2.However, because the expression of Lck is restricted to T lym-phocytes and NK cells (38) and because cells of the monocyticlineage do not express JAK3 constitutively (39, 44), early IL-2signaling in monocytes should occur via PTKs distinct from thoserequired for regulating T cell functions. The present study wasdesigned to explore the role of PTKs in the activation of humanmonocytes by IL-2 and to investigate whether a monocyte/macro-phage-specific PTK is coupled to the IL-2 signaling pathway inhuman monocytes. We report that PTK activation is required forthe induction of monocyte effector and secretory functions by IL-2,and that IL-2 can specifically affect the expression, phosphoryla-tion, and activation of p59hck kinase in human monocytes.

Materials and MethodsMonocyte isolation and culture conditions

Peripheral blood leukocytes were obtained from normal healthy volunteersby leukapheresis using a Fenwell CS-3000 blood cell separator (Fenwell,Deerfield, IL). Mononuclear cells were separated by density gradient cen-trifugation on lymphocyte separation medium (Organum Teknika,Durham, NC), and then purified in suspension from the unfractionatedmononuclear leukocyte preparation by countercurrent centrifugal elutria-tion in a Beckman JE-6 elutriation chamber and rotor system (Beckman,Palo Alto, CA) as described previously (45). The purity of monocyte prep-arations were 946 3%, as assessed by morphology on Giemsa-stainedcytocentrifuge slide preparations and by flow cytometry using the mono-cyte-specific mAb Leu M3 (Becton Dickinson, Mountain View, CA).Other cells present in the monocyte preparations were as follows: 2–5%basophils, 1–2% lymphocytes, 1% neutrophils, and,1% large granularlymphocytes. Viability, as determined by trypan blue exclusion test, was.99%. Monocytes were cultured in RPMI 1640 (BioWhittaker, Walkers-ville, MD), supplemented with 100 U/ml penicillin, 100 U/ml streptomy-cin, 2 mM glutamine, 20 mM HEPES (Life Technologies, Grand Island,NY), and 10% heat-inactivated FBS (HyClone, Logan, UT).

Cytokines and reagents

Highly purified rIL-2 from Escherichia coli(sp. act., 183 106 IU/mg; 1Chiron unit corresponds to 6 IU; LPS content,,0.6 pg/ml) was providedby Chiron (Emeryville, CA). Human rIFN-g (sp. act., 2.023 107 IU/mg)was provided by Dr. Michael Shepard (Genentech, San Francisco, CA).The PTK inhibitor herbimycin A (HA) was purchased from Life Technol-ogies and/or was a gift from Dr. Satoshi Omura (Kitaato Mimato-Ku, To-kyo, Japan); it was prepared as a 1.75-mM stock solution in DMSO (FisherScientific, Pittsburgh, PA). ATP disodium salt was purchased from Sigma(St. Louis, MO). Special care was taken to ensure endotoxin-free condi-tions in all the experiments, and all reagents were demonstrated to beendotoxin free by theLimulusamebocyte lysate test (M. A. Bioproducts,Walkersville, MD; sensitivity, 0.06 IU/ml).

Northern blot analysis

Monocytes were cultured for the indicated time points in 15-cm Lux plates(Miles Scientific, Wapersville, CA) at 23 106 cells/ml in the presence ofIL-2 or HA, alone or in combination. Cells were then lysed in Trizol (LifeTechnologies), and total RNA was purified according to the manufacturer’sinstructions. Twenty micrograms of total RNA from each sample was elec-trophoresed under denaturing conditions on a 1.2% agarose gel containing2.2 M formaldehyde, blotted onto Nytran membranes (Schleicher &Schuell, Keene, NH), and cross-linked by UV irradiation. Membranes wereprehybridized at 42°C in Hybrisol solution (Oncor, Gaithersburg, MD) andhybridized overnight with 23 106 cpm/ml of ana-32P-labeled probe.Membranes were then washed three times at room temperature for 10 mineach time in 23SSC-0.1% SDS, and twice at 60°C for 15 min each timein 0.23 SSC-0.1% SDS before being autoradiographed using KodakXAR-5 films (Eastman Kodak, Rochester, NY) and intensifying screens at280°C. Probes were labeled by random priming reaction using a commer-

cial kit (Roche, Indianapolis, IN) and [a-32P]dCTP (3000 Ci/mmol; Am-ersham, Arlington Heights, IL). The sp. act. was always.109 cpm/mg. Thefollowing cDNAs were used as probes and were provided by each of therespective researchers listed below: human IL-8 full-length cDNA by Dr.K. Matsushima (Kanazawa University Cancer Institute, Kanazawa, Japan),human IL-1b full-length cDNA by Dr. D. Carter (Upjohn Pharmacia,Kalamazoo, MI), the 900-bpPstI fragment of the human IL-6 cDNA (46),human TNF-a cDNA by Dr. S. A. Nedospasov (Institute of MolecularBiology, Academy of Sciences of Russia, Moscow, Russia), andhckcDNAby Dr. Zack Howard (Laboratory of Molecular Immunoregulation, Divi-sion of Basic Sciences, National Cancer Institute, National Institutes ofHealth). The human GAPDH probe was purchased from Clontech (PaloAlto, CA).

Detection of cytokine release

Monocytes were cultured in 15-cm Lux plates at 23 106 cells/ml and werestimulated for 18 h with the indicated factors. At the end of the incubationperiod, cell-free supernatants were harvested and assayed for IL-1b,TNF-a, IL-6, and IL-8 activity, using an IL-6-specific ELISA from Bio-Source (Camarillo, CA) and IL-1b-, TNF-a-, and IL-8-specific ELISAsfrom R&D System (Minneapolis, MN), according to the manufacturer’sinstructions.

Cytotoxicity assay

The cytotoxicity assay was performed as previously described (19).Briefly, monocytes were cultured for 18 h in 96-well round-bottom plates(Dynatech, Alexandria, VA) at 23 105 cells/well in medium alone or inmedium containing optimal concentrations of IL-2, IFN-g, or various dosesof HA, alone or in combination. The plates were then extensively washedbefore the addition of labeled tumor target cells. Cytolytic activity wasmeasured in a 48-h111In release assay against the human colon carcinomacell line HT29 (American Type Culture Collection, Manassas, VA). Targetcells were labeled by incubating 53 106 tumor cells with 40mCi of 111In(Amersham) for 20 min at room temperature. Effector cells were incubatedwith 5 3 103 111In-labeled target cells at an E:T cell ratio of 20:1 at 37°Cfor 48 h. Plates were then centrifuged at 3503 g, 75ml of the supernatantwas harvested, and the radioactivity was measured. The results are ex-pressed as the percentage of111In released, calculated from the meancounts per minute of triplicate determinations as [(experimental counts perminute2 spontaneous counts per minute)/(total counts per minute2 spon-taneous counts per minute)]3 100. The SEMs were consistently,10% ofthe means. The spontaneous release of111In from target cells cultured alonewas between 8 and 10% of the total radioactivity incorporated.

Western blot analysis

For analysis of hck protein levels, monocytes were cultured in 15-cm Luxplates at 23 106 cells/ml with IL-2 for the indicated times, washed inice-cold PBS, and solubilized in lysis buffer (10 mmol/L Tris, 50 mmol/LNaCl, 5 mmol/L EDTA, and 1% Triton X-100, pH 7.6) containing 10mg/ml of the protease inhibitors aprotinin, leupeptin, water-soluble PMSF,and pepstatin A (Roche, Mannheim, Germany) by end-over-end rotationfor 20 min at 4°C. Insoluble material was removed by centrifugation, andthe protein content was determined using a protein assay kit (Bio-Rad,Richmond, CA). Equal amounts of protein from each sample were dena-tured by boiling for 5 min after the addition of 1 vol of 23 sample buffer(125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 10% 2-ME, and 20% glycerol),electrophoresed under reducing conditions on 10% SDS-PAGE, and trans-ferred to Immobilon nitrocellulose membranes (Millipore, Bedford, MA)using a semidry transfer apparatus (Pharmacia LKB, Piscataway, NJ).Membranes were blocked for 2 h at room temperature in blocking buffer(5% dry milk, 0.1% Tween 20, and 13PBS) and subsequently probed inblocking buffer for 1 h with affinity-purified rabbit polyclonal Ab specificfor human p59hck, Lyn, c-Fgr, c-Yes,fyn, JAK3 (Santa Cruz Biotechnol-ogy, Santa Cruz, CA) or anti-JAK1 mAb (Transduction Laboratories, Lex-ington, KY). The blots were then washed three times for 10 min each timein wash buffer (2.5% powdered milk, 0.1% Tween 20, and 13PBS), in-cubated for 30 min in blocking buffer containing 200mg/ml of HRP-linkedaffinity purified goat anti-rabbit antiserum (Kirkegaard & Perry Laborato-ries, Gaithersburg, MD), and extensively washed. Bound Ab was detectedby the enhanced chemiluminescence Western blotting detection kit (Am-ersham, Aylesbury, U.K.), and the membranes were autoradiographed us-ing Kodak XAR-5 films.

Immunoprecipitation and tyrosine phosphorylation analysis

Monocytes were stimulated in 50-ml conical polypropylene tubes (Falcon,Becton Dickinson Labware, Lincoln Park, NJ) at 53 106 cells/ml of warm

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RPMI with 1000 U/ml of IL-2 for brief periods of time, washed in ice-coldPBS, and solubilized in lysis buffer containing protease and phosphataseinhibitors (10 mM sodium tetrapyrophosphate, 50 mM sodium fluoride,and 5 mM sodium orthovanadate). Depending on the experiment, clarifiedcell lysates were incubated rotating end-over-end overnight at 4°C with 3mg/ml of anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, LakePlacid, NY), anti-JAK3 rabbit polyclonal antiserum, anti-JAK1 mAb, anti-hck rabbit polyclonal antiserum, or normal rabbit serum that had beenprebound to protein A/G Plus-agarose beads (Santa Cruz Biotechnology).The beads were extensively washed with buffer containing 0.1% TritonX-100, and precipitated material was eluted by boiling in SDS samplebuffer for 5 min, run on 10% SDS-PAGE, and transferred to Immobilonmembranes. For immunoblotting, anti-phosphotyrosine and anti-hck Abswere used at a concentration of 1mg/ml in blocking buffer, and Westernblot analysis was performed as described above.

Tyrosine kinase assay

hck immune complex tyrosine kinase assays were conducted by incubatingthe immunoprecipitatedhck tyrosine kinase from lysates of unstimulatedand IL-2-stimulated cells in the presence or the absence of ATP and visu-alizing incorporated phosphate on tyrosines by immunoblotting. Immobi-lized proteins were washed three times with lysis buffer followed by asingle wash with kinase buffer containing 25 mM HEPES (pH 7.3), 0.1%Triton X-100, 100 mM NaCl, 10 mM MgCl2, 3 mM MnCl2, and 200mMsodium orthovanadate. Isotope-free tyrosine kinase reactions were initiatedby the addition of 15mM unlabeled ATP and allowed to incubate at 37°Cfor 15 min. The reactions were quenched by washing the protein A/GPlus-agarose beads with lysis buffer and eluting bound material by boilingin SDS-sample buffer for 4 min. The material in each lane represents im-munoprecipitates from;1 3 108 cells.

Densitometry analysis

The intensities of the bands were quantitated from the autoradiographsgenerated from every experiment using ana Imager 2000 (Innotech, SanLeandro, CA). Whenever applicable, the results were normalized to thehousekeeping gene GAPDH.

ResultsEffects of HA on IL-2-induced monocyte tumoricidal activity

To study the requirement for PTKs in human monocyte activationby IL-2, experiments were performed to analyze the effects of thePTK inhibitor HA on IL-2-induced monocyte tumoricidal activity.Previous studies have indicated that this inhibitor is effectiveagainst most tyrosine kinases, but does not significantly affectPKC, phosphorylase kinase, phospholipase C, or cyclic nucleotide-dependent protein kinases (47, 48). Monocytes were stimulatedwith an optimal dose of IL-2 for 18 h in the presence or the ab-sence of increasing concentrations of HA and then assayed forcytotoxic activity against the human colon carcinoma cell line HT-29. Fig. 1 shows the results of one representative experiment ofthree performed with monocytes from different donors. IL-2-treated monocytes exerted high levels of tumoricidal activity(49%), which was markedly decreased in a dose-dependent man-ner by treatment with HA. Interestingly, this function of mono-cytes was exquisitely sensitive to the drug, because a concentrationof HA as low as 0.01mM was sufficient to cause a 54% reductionand a concentration of 0.5mM was able to completely suppressIL-2-induced monocyte-mediated cytotoxicity. As depicted in Fig.1 and previously reported (19), a similar activation of humanmonocytes to a tumoricidal stage can be achieved by cell stimu-lation with IFN-g; however, IFN-g-dependent monocyte cytotox-icity was relatively more resistant to the effects of HA at all dosestested than IL-2-induced monocyte cytotoxicity. In fact, concen-trations of HA that inhibited the monocyte response to IL-2 (0.01–0.1 mM) did not affect IFN-g-induced effects, and a concentrationas high as 1mM was required for a 51% reduction of IFN-g-mediated cytolysis (Fig. 1). Similar results were obtained using thePTK inhibitor genistein, which at a dose of 20mM inhibited IL-2-induced monocyte cytotoxicity by 48%, but only decreased IFN-

g-induced monocyte cytotoxicity by 6% (data not shown). At theconcentrations used, HA did not affect monocyte viability, as de-termined by the trypan blue dye exclusion test. The vehicle forHA, DMSO, alone at the equivalent concentrations used did notinhibit IL-2-induced monocyte cytotoxicity. These results demon-strate that the activation of monocyte effector functions by IL-2 isextremely sensitive to PTK inhibition, suggesting a role for PTK inmonocyte response to IL-2.

HA inhibits proinflammatory cytokine production by IL-2-treatedmonocytes

Human monocytes can be stimulated by IL-2 to release severalproinflammatory cytokines (49). If signaling via IL-2R is mediatedby PTK activation, responses of monocytes to IL-2 other than cy-totoxicity would also be susceptible to inhibition by HA. To in-vestigate this possibility, HA was tested for its effects on IL-2-induced monokine production. Supernatants from monocytesstimulated for 18 h with IL-2, alone or in combination with HA(0.1 mM), were assayed for the presence of IL-1b, TNF-a, IL-6,and IL-8 (Fig. 2). Secretion of all four cytokines was induced byIL-2, as previously reported (22, 33, 50), although the absolutelevels detected varied somewhat from donor to donor (33) (datanot shown). HA almost completely abrogated the effects of IL-2 onTNF-a, IL-1b, and IL-6 secretion and decreased monocyte releaseof IL-8 relative to that in IL-2-treated cells in all the experimentsperformed. The specificity of this inhibition was demonstrated bythe inability of HA to block in a meaningful manner IL-1b induc-tion of IL-6 and IL-8. The HA diluent DMSO had no effect onIL-2-induced monokine protein expression (Fig. 2). To investigatewhether the effects of HA were exerted at the level of gene ex-pression, total RNA was extracted from monocytes stimulated for6 h with optimal doses of IL-2 or 0.1mM HA, alone or in com-bination, and Northern blot analysis was performed (Fig. 3A). Thistime point was shown to be optimal for mRNA induction by IL-2(22). As depicted in Fig. 3A, control monocytes expressed unde-tectable or very low constitutive levels of IL-6, IL-8, IL-1b, and

FIGURE 1. Dose-dependent inhibition of IL-2-induced monocyte tu-moricidal activity by HA. Monocytes were cultured for 18 h in mediumalone or in medium supplemented with IL-2 (1000 U/ml) or IFN-g (500U/ml), in the presence or the absence of increasing concentrations of HAand then assayed for tumoricidal activity as described inMaterials andMethods. Results from one representative experiment are plotted as themeans of triplicate determinations (SEM,,10% of the mean).

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TNF-a mRNAs that were markedly up-regulated by stimulationwith IL-2. Addition of HA at the onset of the culture potentlysuppressed IL-1b, IL-6, and IL-8 and completely inhibited TNF-amRNA expression in IL-2-stimulated monocytes. HA alone didnot induce expression of the message for any of the cytokinestested and did not affect the mRNA levels of the housekeepinggene GAPDH.

Dose-response experiments (Fig. 3B) demonstrated that as littleas 0.01mM HA was sufficient to cause a detectable reduction inIL-2-induced TNF-a mRNA expression, although higher doses ofthe drug were required to inhibit the expression of the other cyto-kine mRNAs. A major suppression of all cytokine mRNA levelswas reached at 0.1mM, and only slight further reductions of IL-6and IL-8 mRNA was detectable at 1mM (Fig. 3B). DMSO alonedid not affect IL-2-induced monokine expression at either themRNA (data not shown) or the protein (Fig. 2) level. Similar re-sults were obtained in three independent determinations, althoughdifferent degrees of mRNA levels were evident, representing do-nor-to-donor variations. Based on these findings, we can concludethat tyrosine kinase activation is required for IL-2-induced secre-tory functions of human monocytes.

IL-2 induces protein tyrosine phosphorylation in humanmonocytes

Activation of the IL-2R in T lymphocytes rapidly induces tyrosinephosphorylation of a variety of substrates (51). Because tyrosinekinase activation appeared to be involved in regulating the re-sponses of monocytes to IL-2, we next examined the effects ofIL-2 on the phosphorylation of cellular proteins on tyrosine resi-dues. Fresh human monocytes were stimulated with 1000 U/ml ofIL-2 for the indicated times and subjected to detergent lysis andimmunoprecipitation with anti-phosphotyrosine mAb. The protein

samples were separated by SDS-PAGE and analyzed further byimmunoblotting with a chemiluminescence detection system. Asshown in Fig. 4,upper panel, IL-2 induced a marked increase in

FIGURE 2. HA suppresses monocyte secretion of proinflammatory cy-tokines. Monocytes were cultured in medium alone or in medium supple-mented with 1000 U/ml of IL-2 or 10 ng/ml of IL-1b, alone or in combi-nation with 0.1 mM HA. Supernatants were harvested after 18 h andassayed for the indicated cytokines by specific ELISA. Results from onerepresentative experiment of three performed, expressed as picograms permilliliter per 2 3 106 cells/ml, are shown.

FIGURE 3. HA inhibits cytokine mRNA expression in IL-2-treatedmonocytes.A, Total RNA was extracted from monocytes stimulated for 6 hwith IL-2 (1000 U/ml) or HA (0.1mM), alone or in combination, andanalyzed by Northern blotting.B, Monocytes were cultured for 6 h withIL-2 (1000 U/ml) in the presence or the absence of increasing concentra-tions of HA,and Northern blot analysis was performed on total RNA. Theblots were sequentially hybridized with the cDNAs for the indicated cy-tokines. GAPDH levels were determined to ensure that equal amounts ofRNA were loaded in each lane.

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the phosphorylation on tyrosine residues of several cellular pro-teins, ranging from 46–180 kDa (indicated by the arrows), withinthe first 5 min of incubation. IL-2-dependent tyrosine phosphory-lation was further augmented after 15 min of stimulation, reacheda plateau at 30 min, and remained steady for at least 60 min duringcontinuous incubation with IL-2. Among the cellular substratesthat underwent phosphorylation in response to IL-2 were proteinsmigrating with apparent molecular masses from 35 kDa (after 30min of treatment) to 46 kDa (after 5 min of treatment), which weredetected upon overexposure of the film (Fig. 4,lower panel). Thisexperiment as well as two other similar experiments gave compa-rable results, strongly suggesting that IL-2 is able to directly stim-ulate phosphorylation of proteins on tyrosine residues and providesfurther evidence that PTK activation is induced in response totreatment with IL-2 in fresh human monocytes.

Up-regulation ofhck-PTK mRNA and protein expression byIL-2 in human monocytes

Several members of thesrc family PTKs are constitutively ex-pressed in mononuclear phagocytes and have been shown to beimportant components of the signal transduction pathway of sev-

eral monocyte/macrophage-activating factors (39). To gain in-sights into the biochemical mechanisms leading to monocyte ac-tivation by IL-2, experiments were designed to investigate whetherIL-2 specifically affected the expression and activation of a specificmember of thesrc family PTKs in human monocytes. Cell lysatesfrom monocytes cultured for 6 and 12 h with IL-2 were subjectedto Western blot analysis using specific Abs against the varioussrcPTKs. In agreement with previous reports (38, 52), we detectedconstitutive levels of expression offyn, Lyn, c-Fgr, andyes thatwere not up-regulated in response to IL-2 (Fig. 5). Interestingly, ofthe constitutively expressedsrc PTKs, only the steady-state levelsof hckmRNA dramatically increased in a time-dependent mannerupon exposure of resting monocytes to IL-2. As shown by North-ern blot analysis (Fig. 6A), increased amounts of the 2.2-kbhcktranscripts were observed as early as 3 h after IL-2 stimulation,while maximal mRNA accumulation (as determined by densito-metric analysis of the intensities of the bands and normalizationwith GAPDH) occurred within 6 h and remained stable until 18 hafter the onset of the culture. The accumulation of mRNA wasparalleled by enhanced expression of p59hck that reached a plateauafter 12 h of culture, as assessed by Western blot (Fig. 6B). Boththe 56- and 59-kDa isoforms ofhck were equally up-regulated inIL-2-treated monocytes. Three independent experiments yieldedcomparable results, although slight fluctuations in the degree ofinduction were detectable due to donor variability. To determinewhether the increased expression ofhck in response to IL-2 wasthe general consequence of the activated phenotype on humanmonocytes, cells were treated with IFN-g, a well-known monocyteactivator. As shown in Fig. 7, IL-2 induced a 2.5-fold increase inhck expression over that in medium-treated cells. On the otherhand, IFN-g failed to affect the expression ofhck in a meaningful

FIGURE 4. Kinetics of IL-2-dependent induction of protein tyrosinephosphorylation in human monocytes. Monocytes were stimulated withIL-2 (1000 U/ml) for the indicated time points. Cell lysates were immu-noprecipitated with 4G10 anti-phosphotyrosine mAb prebound to proteinA/G Plus-agarose, run on a 10% SDS-polyacrylamide gel, and immuno-blotted with the 4G10 mAb. Two different autoradiograms of the same blotfrom one of three separate experiments are shown. Molecular mass stan-dards (kilodaltons) and Ig heavy chains (IgG) are indicated. The arrowsdenote those proteins whose level of tyrosine phosphorylation is increasedby IL-2.

FIGURE 5. Thesrc PTKs are constitutively expressed in human mono-cytes. Monocytes were incubated in medium alone or in medium supple-mented with 1000 U/ml of IL-2 for the indicated times. The cells were thenlysed, and the amount of protein was equalized in each sample. The pro-teins were separated on a 10% SDS-PAGE and analyzed by immunoblot-ting as described inMaterials and Methodsusing the indicated Abs. Ananti-GAPDH immunoblot was performed to control that comparableamounts of protein were loaded into each lane.

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way. These results provide the first evidence that the expression ofhck PTK can be markedly induced by IL-2 in human monocytes.

IL-2 induceshck tyrosine phosphorylation in human monocytes

Having established that IL-2-activated monocytes express in-creased levels of p59hck, we asked whether this PTK could beinvolved in the IL-2 signal transduction pathway in monocytes.Monocytes were stimulated with 1000 U/ml of IL-2 for the indi-cated periods of time, and tyrosine phosphorylation was assessedon immunoprecipitated p59hck by immunoblotting with an anti-phosphotyrosine Ab. As shown in Fig. 8,upper panel, a certaindegree of basal tyrosine phosphorylation of p59hck was present inunstimulated monocytes. IL-2 treatment resulted in a rapid, time-dependent augmentation ofhck phosphorylation that started asearly as 1 min after IL-2 addition, peaked at 5 min after stimula-tion, and declined thereafter. A second peak of phosphorylationwas evident after 60 min of culture. Both the p56 and p59 isoformsof hckwere equally tyrosine phosphorylated. Stripping of the blotfollowed by direct immunoblotting forhck demonstrated that atthese early time points IL-2 treatment did not affect the amounts ofhck immunoprecipitated (Fig. 8,bottom panel). Similar resultswere obtained in three independent experiments. These resultsdemonstrate that thehckprotein becomes tyrosine phosphorylatedin response to IL-2 and suggest that p59hck may be involved inIL-2 signal transduction in human monocytes.

p59hck is activated by IL-2 stimulation in human monocytes

To better assess the importance ofhck in regulation of the mono-cyte response to IL-2, we next examined the catalytic activity ofhck PTK following IL-2 stimulation. Human monocytes were ex-

posed to 1000 U/ml of IL-2 for 2 min, and cell lysates were sub-jected to immunoprecipitation by a specific anti-hck or a controlantiserum. Immunoprecipitated proteins were then subjected to thein vitro tyrosine kinase assay. As shown in Fig. 9, although acertain degree of basalhckkinase activity was present in unstimu-lated monocytes (lane 2), a significant (11-fold) increase in phos-phate incorporation on tyrosine residues was detectable whenhckimmunoprecipitates from IL-2-stimulated cells were incubatedwith ATP in vitro (lane 4). Both the 56- and 59-kDa isoforms ofhck were equally phosphorylated upon cell exposure to IL-2. By

FIGURE 6. IL-2 up-regulates the constitutive expression ofhckmRNAand protein in human monocytes. Monocytes were cultured for the indi-cated time points in medium alone (2) or in medium supplemented with1000 U/ml of IL-2 (1).A, Total RNA was isolated and analyzed by North-ern blotting forhck mRNA expression (upper panel). The blot was thenrehybridized with the GAPDH probe as a control for RNA loading (lowerpanel).B, Protein lysates were prepared and analyzed by Western blottingusing a rabbit polyclonal anti-hckantiserum. The two 56- and 59-kDaisoforms ofhck are indicated.

FIGURE 7. IL-2, but not IFN-g, up-regulates the expression of p59hck.Cells were cultivated for the indicated lengths of time in the presence or theabsence of 1000 U/ml of IL-2. Monocytes were then lysed, and the amountof protein was equalized in each sample. The proteins were separated on a10% SDS-PAGE and analyzed by immunoblotting as described inMate-rials and Methodsusing rabbit anti-hckAb. An anti-GAPDH immunoblotwas performed to control that comparable amounts of protein were loadedinto each lane.

FIGURE 8. Tyrosine phosphorylation ofhckin response to IL-2. Mono-cytes were stimulated with 1000 U/ml of IL-2 for the indicated time pointsand then lysed. Cell lysates were incubated overnight with anti-hckpoly-clonal rabbit antiserum prebound to protein A/G Plus-agarose. Immuno-precipitatedhck was run on a 10% SDS-PAGE and immunoblotted with4G10 anti-phosphotyrosine mAb (a-PY; top). An anti-hckimmunoblot(bottom) was performed to control that comparable amounts of p59hck hadbeen immunoprecipitated.

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contrast, cell lysates subjected to immunoprecipitation by normalrabbit serum (control) were negative (Fig. 9,lanes 5–8), and rep-robing of the immunoblots withhckantiserum verified equal pro-tein loading (data not shown). To further substantiate the potentialrole of hck in the activation process of monocytes by IL-2, exper-iments were performed to investigate whether HA inhibited IL-2-inducedhck catalytic activity. Monocytes were preincubated withmedium in the presence or the absence of increasing concentra-tions of HA. After 8 h of culture, 1000 U/ml of IL-2 was added for2 min, lysates were prepared, andhckcatalytic activity was deter-mined as described above. As depicted in Fig. 10, HA decreasedp59hckcatalytic activity in a dose-dependent manner. These resultsdemonstrate that the specific activity ofhck PTK is increased byIL-2 stimulation of monocytes and strongly suggest thathck is anintegral component of the signaling pathway involved in IL-2-induced monocyte activation.

Expression and functional status of JAK1 and JAK3 in restingand activated monocytes

To determine whether JAK1 and/or JAK3 are involved in the earlystages of monocyte activation by IL-2, we investigated their ex-

pression on resting and activated monocytes. Northern blot anal-ysis revealed very low basal levels of JAK1 mRNA in medium-treated cells (Fig. 11,upper panel). Incubation of monocytes witheither 1000 U/ml of IL-2 or 500 U/ml of IFN-g for up to 6 h didnot affect JAK1 mRNA expression. Similar results were obtainedin four independent experiments. To determine whether JAK1 pro-tein paralleled the expression of JAK1 mRNA, Western blot anal-ysis was performed. Monocytes cultured for 3 h in medium aloneexpressed very low levels of JAK1. The expression of JAK1 in-creased with culture, reaching a maximum at 9 h, the latest pointtested. Neither IL-2 nor IFN-g meaningfully affected JAK1 ex-pression (Fig. 11,lower panel). The same lysates were then usedto investigated the expression of JAK3 in monocytes. Western blotanalysis revealed that JAK3 was almost undetectable in medium-treated monocytes. On the other hand, both IL-2 and IFN-ginduced the expression of JAK3 in a time-dependent manner(Fig. 12).

Having established that IL-2-treated monocytes express in-creased levels of JAK3, we investigated whether this kinase couldbe involved in the early stages of IL-2 activation of human mono-cytes. Fresh monocytes were treated with 1000 U/ml of IL-2, andtyrosine phosphorylation was determined on immunoprecipitatedJAK3 by immunoblotting with an antiphosphotyrosine Ab. Freshresting monocytes were treated with IL-2 for 5, 10, 15, 30, and 60min. However, only cells treated for 5 min are shown, because theresults of the time course were unequivocally identical, indepen-dent of the length of treatment. As shown in Fig. 13,upper panel,

FIGURE 9. IL-2-induced p59hck catalytic activity analyzed by in vitrotyrosine kinase assay. Monocytes were incubated with medium (2) or1000 U/ml of IL-2 (1) for 2 min at 37°C. Lysates were prepared andsubjected to immunoprecipitation with rabbit polyclonal anti-hck (a-hck;lanes 1–4) or preimmune rabbit serum (CTRL;lanes 5–8). Immunopre-cipitates were assayed for in vitro kinase activity in the absence (2) or thepresence (1) of 15mM unlabeled ATP. Reaction products were then re-solved by SDS-PAGE, followed by anti-phosphotyrosine immunoblotting,as described inMaterials and Methods.

FIGURE 10. HA inhibits IL-2-induced p59hck catalytic activity. Mono-cytes were incubated in medium alone or in medium supplemented with0.05mM HA ( lanes 3and7) or 0.1mM HA ( lanes 4and8). After 8 h ofincubation, 1000 U/ml of IL-2 was added for 2 min, lysates were preparedand subjected to immunoprecipitation as described in Fig. 9, and in vitrokinase activity was determined as described inMaterials and Methods.

FIGURE 11. Expression of JAK1 in human monocytes.Upper panel,Monocytes were cultured for the indicated lengths of time in medium aloneor in medium supplemented with either 1000 U/ml of IL-2 or 500 U/ml ofIFN-g. Total RNA was isolated and analyzed by Northern blot for JAK1mRNA expression. The blot was then rehybridized with GAPDH probe asa control for RNA loading.Lower panel, Cells were cultivated for theindicated lengths of time in the presence or the absence of 1000 U/ml ofIL-2. Monocytes were then lysed, and the amount of protein was equalizedin each sample. The proteins were separated on a 10% SDS-PAGE andanalyzed by immunoblotting as described inMaterials and MethodsusingJAK1 mAb. An anti-GAPDH immunoblot was performed to control thatcomparable amounts of protein were loaded into each lane.

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1000 U/ml of IL-2 failed to induce tyrosine phosphorylation ofJAK3 in fresh resting monocytes. On the other hand, monocytesthat have been preactivated for 18 h with 500 U/ml of IFN-g andthen treated with IL-2 for 10 min displayed a clear increase intyrosine phosphorylation of JAK3. YT cells treated with IL-2 for5 min displayed a major tyrosine phosphorylation of JAK3. Im-munoblotting of the membrane with JAK3 confirmed that freshmonocytes had low or undetectable levels of JAK3 protein, whichmight explain the lack of tyrosine phosphorylation in resting non-preactivated monocytes. On the other hand, monocytes preacti-vated for 18 h with IFN-g had high levels of JAK3, almost com-parable to those in YT cells. Similar results were obtained in twoindependent experiments. To determine the tyrosine phosphoryla-tion status of JAK1 on fresh and preactivated monocytes, lysates

obtained from the previous experiment were immunoprecipitatedwith JAK1 mAb. In sharp contrast with JAK3, the levels of ty-rosine phosphorylation for JAK1 were almost completely unde-tectable in preactivated monocytes (only seen in overexposedfilms, data not shown) and were definitely undetectable in freshresting monocytes. YT cells displayed a moderate tyrosine phos-phorylation of JAK1. Western blot with JAK1 confirmed that lowlevels of JAK1 protein were present in resting monocytes. On thecontrary, preactivated monocytes and YT cells had high levelsof JAK1.

DiscussionActivation of cytoplasmic tyrosine kinases is an important aspectof signal transduction mediated by IL-2. Several distinct nonre-ceptor PTKs are known to be physically and functionally coupledto the IL-2R complex in T lymphocytes, and the concerted actionof these molecules is required for triggering the full-scale activa-tion of downstream signaling pathways leading to cellular re-sponses (3). Most of these kinases, however, have a limited patternof expression, restricted to T and NK cells, and are undetectable inother IL-2-responsive cells, such as those of the monocyte/macro-phage lineage (38, 52). Despite our increasing knowledge on thebiological effects of IL-2 on human monocytes, there is currentlylittle information regarding the biochemical mechanisms throughwhich these responses are mediated. A recent report from Mussoet al. (39) demonstrated that JAK-3 PTK is involved in IL-2-signaltransduction in monocytes preactivated with IFN-g, but not infresh peripheral blood monocytes. Therefore, one question of in-terest was to establish whether PTKs played any role in IL-2 sig-naling in resting human monocytes and to identify the kinasesinvolved. In the present study we clearly demonstrate for the firsttime that PTK activation is required for IL-2 triggering of botheffector and secretory functions of fresh human monocytes. More-over, we show that stimulation with IL-2 results in both tyrosinephosphorylation and catalytic activation of p59hck, an srcfamilyPTK specifically expressed in cells of the myeloid lineage (38, 52),strongly suggesting thathckkinase is an integral component of thesignaling pathway elicited by IL-2 in monocytes. Specific phar-macologic inhibitors represent powerful tools in exploring the roleof PTKs in receptor signal transduction. HA has been shown to beone such PTK inhibitor; it is able not only to block the enzymaticfunction of these enzymes but also to specifically degrade them,although sparing serine/threonine kinases (53, 54). Earlier studieshave reported the ability of HA to exert inhibitory effects on IL-2-dependent regulation of T and NK cell gene expression and pro-liferation (55, 56). However, the range of effective concentrationsvaried depending on the cell lineage and the biological functiontested. Nanomolar concentrations of HA (100 nM) were sufficientto maximally block IFN-g secretion by NK cells as well as NK andLAK cell tumoricidal activity (56), whereas higher amounts (1–2mM) were required for the inhibition of IL-2-induced proliferationof T lymphocytes, tyrosine phosphorylation, and activation ofp21ras (57). Here we demonstrated that the biological effects ofIL-2 on monocytes were extremely sensitive to HA inhibition, withas little as 0.01mM HA sufficient to cause a 54% reduction ofIL-2-induced monocyte tumoricidal activity and a concentration of0.5 mM being able to almost completely abrogate cytotoxicity.Thus, monocytes, compared with NK and LAK cells, appear to besimilarly sensitive to PTK inhibition by HA (56). However, cellpreincubation with the drug was required for the suppression ofIL-2-induced LAK and NK killing (56), whereas the addition ofHA at the onset of the culture was sufficient to exert a profoundinhibition of both IL-2-induced cytotoxic activity and cytokine

FIGURE 12. JAK3 expression is induced by either IL-2 or IFN-g. Hu-man monocytes were cultivated for the indicated lengths of time in thepresence or the absence of 1000 U/ml of IL-2 or 500 U/ml of IFN-g. Cellswere then lysed, and the amount of protein was equalized in each sample.The proteins were separated on a 10% SDS-PAGE and analyzed by im-munoblotting as described inMaterials and Methodsusing rabbit anti-JAK1 Ab. An anti-GAPDH immunoblot was performed to control thatcomparable amounts of protein were loaded into each lane.

FIGURE 13. The tyrosine phosphorylation status of JAK3 and JAK1 inresting and preactivated monocytes in response to IL-2.Upper panel, Freshresting monocytes were stimulated with 1000 U/ml of IL-2 for 5 min (lane1) or with 500 U/ml of IFN-g for 18 h followed by IL-2 for 5 and 10 min(lanes 2and 3, respectively). YT cells were treated for 5 min with IL-2(lane 4). Cells were then lysed and incubated overnight with anti-JAK3polyclonal rabbit antiserum prebound to protein A/G Plus-agarose. Immu-noprecipitated JAK3 was run on a 10% SDS-PAGE and immunoblottedwith 4G10 anti-phosphotyrosine mAb (a-PY; top). An anti-JAK3 immu-noblot (bottom) was performed to control for immunoprecipitation.Lowerpanel, The same cellular lysates and experimental procedures used in theupper panelexperiment were used to determine JAK1 tyrosine phosphor-ylation status.

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production by monocytes. IFN-g and IL-2 stimulate similar levelsof tumoricidal activity in human monocytes (19); however, thesensitivity of IL-2-dependent effects to HA greatly exceeded thatof IFN-g-mediated responses. Induction of monocyte cytotoxicityby IFN-g was, in fact, relatively resistant to the inhibitory activityof HA at all doses tested. A concentration of HA as high as 1mMwas required for a 51% suppression of killing, thereby suggestingthat the two cytokines act on monocytes through different signalingpathways.

The ability of HA to suppress the monocyte response to IL-2was not restricted to cytotoxicity, but was also evident on IL-2-induced expression of IL-1b, TNF-a, IL-8, and IL-6 mRNA, andaccordingly, cytokine secretion was reduced to baseline levels inthe presence of the drug. This inhibition was specific, because HAfailed to inhibit IL-1b-induced IL-6 and IL-8. Inhibition was dosedependent and occurred at the same doses as those that were ef-fective in blocking cytotoxicity. No effect on cell viability or onGAPDH mRNA levels was observed upon treatment with the drug,demonstrating that the decrease in monocyte responses to IL-2could not be accounted for by toxicity of the treatment and ex-cluding the possibility of a general and nonspecific inhibition ofmRNA synthesis. A causal relationship between inhibition of PTKactivity and suppression of IL-2-induced monocyte functions wasalso supported by the observation that 20mM genistein, anothertyrosine kinase inhibitor structurally unrelated to HA and known tohave different nonspecific activities (58), markedly decreasedmonocyte tumoricidal activation by IL-2 (data not shown). Thesimplest interpretation of our results is that IL-2R engagement cantrigger the activation of one or more PTKs and protein tyrosinephosphorylation events critical for IL-2 intracellular signal trans-duction, and that HA (and genistein) inhibited PTK activation byIL-2, thereby preventing monocyte responses. Indeed, we demon-strated that IL-2, at a concentration optimal for inducing monocytebiological activities, was able in resting monocytes to directlystimulate rapid tyrosine phosphorylation of several proteins, rang-ing from 35 to 180 kDa. This increase was detectable within thefirst 5 min of treatment, reached a maximum by 30 min, and re-mained steady for at least 60 min during continuous incubationwith IL-2. This time course of tyrosine phosphorylation is similarto that observed by Saltzman et al. (59) in the cytotoxic T cell lineCTLL and peripheral blood T lymphocytes. The recruitment andactivation of Lck, JAK3, JAK1, and Syk PTKs following IL-2binding to its receptor on T cells are well documented (1, 3, 4, 51).However, the expression of Lck PTK is restricted to T lympho-cytes and NK cells, and we demonstrated that resting monocytesexpress low levels of JAK1. Furthermore, JAK1 was not tyrosinephosphorylated on resting or preactivated monocytes despite therelatively high levels of JAK1 protein on the later cells. In contrast,Syk PTK, in agreement with previous observations (7), was con-stitutively expressed, but did not undergo phosphorylation in re-sponse to IL-2 (data not shown). In agreement with Musso et al.(39), we demonstrated that JAK3 was present in preactivated, butnot in resting, monocytes. Furthermore, Villa et al. (44) recentlyreported that monocytes from a JAK3-SCID patient exhibited nor-mal response to IL-2 in terms of cytokine production, thus dem-onstrating that the stimulatory activity of IL-2 on monocytes wasunaffected by the lack of JAK3. These observations indicate thatthese kinases, although involved in T lymphocyte activation byIL-2, are not absolutely required for early IL-2 signaling in freshlyisolated monocytes and suggest the participation of other PTKs inthis pathway. Several members of thesrc family PTKs are con-stitutively expressed in mononuclear phagocytes and have beenshown to be important components of the signal transduction path-way of different monocyte/macrophage-activating factors

(39–43). Because HA at 0.1mM has been reported to have a directblocking effect onsrc PTKs (47), and consistent increases in thetyrosine phosphorylation of proteins in the 50–60 kDa range,which is the size of thesrckinases (52), were observed in responseto IL-2, we hypothesized that a myeloid-specific member of thesrcfamily PTK could be involved in the early signal transductionevents activated by IL-2 in monocytes. Herein, we provide the firstevidence that IL-2 induced increased expression, tyrosine phos-phorylation, and rapid activation of p59hcksrc family PTK. Mono-cyte treatment with IL-2 resulted in a significant up-regulation ofthe expression ofhck mRNA relative to that in control cells; thiswas paralleled by p59hck protein accumulation.hckmRNA induc-tion occurred very rapidly, within 3 h of stimulation, suggesting adirect response to IL-2, and was likely to be IL-2 specific, becauseno changes inhck transcript levels were observed in IFN-g-treatedmonocytes (42) (data not shown). The finding that p59hck under-went tyrosine phosphorylation within 5 min of stimulation withIL-2 and the fact that this phosphorylation is associated with anincrease in p59hck kinase activity strongly indicate that one of theearliest intracellular events triggered by IL-2 in monocytes is theactivation ofhck PTK. The functional relevance ofhck in freshmonocytes is further suggested by the fact that HA inhibits IL-2-inducedhck catalytic activity. These findings clearly suggest acritical role for p59hck kinase in IL-2-induced protein tyrosinephosphorylation and the IL-2 signaling pathway in monocytes. Theeffects of IL-2 onhckwere selective, because we did not detect anychange in the expression of the othersrc family PTKs constitu-tively expressed in monocytes, such asfgr, lyn, fyn, andyes. How-ever, we cannot completely rule out the involvement of otherssrcfamily PTK in IL-2 signaling in freshly isolated monocytes. Stud-ies are currently ongoing in our laboratory to investigate the effectsof IL-2 on the catalytic activity of the otherssrc PTK members.The effect of IL-2 onhck expression was specific and was notassociated with the activated phenotype of monocytes, becauseIFN-g, a potent activator of monocytes, failed to enhance the ex-pression ofhck.

Despite the close structural relationship among thesrc familymembers, it is likely that they probably subserve different func-tions in cells of the monocyte/macrophage lineage. Moreover,these results, together with earlier findings by others (60, 61)showing that IL-2 could specifically regulate the activities offyn,lyn, andblk src family members in B cell lines not expressingp56lck, indicate that some flexibility exists in the ability of differentsrc-like PTKs to couple to the IL-2R and participate in IL-2 signaltransduction. In addition, it raises the possibility that cell lineage-specific responses to IL-2 may be determined, at least in part, bythe repertoire ofsrc family PTKs expressed in the cell. Extensivestudies in T cells have demonstrated the importance of the coop-eration among distinct PTKs in IL-2 signaling (1, 3). It is likelythat more than one class of signal transduction molecule contrib-utes to the cascade of intracellular events that results in IL-2-in-duced monocyte activation. IL-2 has been shown to induce theexpression of JAK3 mRNA and protein in IFN-g-pretreated mono-cytes and to trigger subsequent kinase phosphorylation, thus sug-gesting that JAK3 is a component of the IL-2 signal transductionpathway in activated monocytes (Ref. 39 and the present study).Our results indicate that in fresh resting monocytes thehck geneproduct is involved at an early step in IL-2 signal transductioncascade, and that JAK3 phosphorylation occurs at a later pointfrom the src PTK, although additional experiments will be re-quired to establish a causal role for the two kinases in IL-2 sig-naling. We propose a model in which IL-2 binding to its receptorin monocytic cells elicits the rapid activation ofhckkinase and thetriggering of downstream events resulting in monocyte responses,

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including up-regulation of IL-2Rb/g-chains (21, 34, 35), increasesin p59hckexpression (present study), and induction as well as latteractivation of JAK3 (39), thereby augmenting the number of func-tional IL-2Rs on the cell surface and the expression of intracellularsignaling molecules, leading to a more efficient cell response toIL-2. Studies are currently in progress to determine whetherhck isconstitutively associated with the IL-2R complex in freshly iso-lated monocytes or whether it is recruited upon IL-2 stimulation;furthermore, the studies will attempt to elucidate the contributionsof other PTKs to signal transduction events activated by IL-2. Inconclusion, the current observations provide important initial in-sights into IL-2 signal transduction processes in monocytic cellsand emphasize a key role for specific tyrosine phosphorylationevents.

AcknowledgmentsWe thank Dr. Carmen S. Garcia and Dr. James M. Mwatibo for theircritical review of this manuscript.

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