Proc. Natl. Acad. Sci. USAVol. 92, pp. 7681-7685, August 1995Immunology

Similarities and differences in signal transduction by interleukin4 and interleukin 13: Analysis of Janus kinase activationACHSAH D. KEEGAN*t, JAMES A. JOHNSTONt, P. JUSTIN TORTOLANII, LISA J. MCREYNOLDS*, CAROL KINZER*,JOHN J. O'SHEAt, AND WILLIAM E. PAUL**Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, and *Arthritis and Rheumatism Branch, National Institute of Arthritis,Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892

Contributed by William E. Paul, May 2, 1995

ABSTRACT The cytokines interleukin (IL) 4 and IL-13induce many of the same biological responses, including classswitching to IgE and induction of major histocompatibilitycomplex class II antigens and CD23 on human B cells. It hasrecently been shown that IL-4 induces the tyrosine phosphor-ylation of a 170-kDa protein, a substrate called 4PS, and of theJanus kinase (JAK) family members JAKi and JAK3. Be-cause IL-13 has many functional effects similar to those ofIL-4, we compared the ability of IL-4 and IL-13 to activatethese signaling molecules in the human multifactor-dependentcell line TF-1. In this report we demonstrate that both IL-4 andIL-13 induced the tyrosine phosphorylation of4PS and JAKI.Interestingly, although IL-4 induced the tyrosine phosphor-ylation of JAK3, we did not detect JAK3 phosphorylation inresponse to IL-13. These data suggest that IL-4 and IL-13signal in similar ways via the activation of JAK1 and 4PS.However, our data further indicate that there are significantdifferences because IL-13 does not activate JAK3.

Interleukin (IL) 4 is a type I cytokine produced byT cells, mastcells, and basophils in response to immune recognition recep-tor engagement (1). IL-4 has many biological functions, in-cluding the ability to act as a costimulant for B- and T-cellproliferation, to induce IgE production by activated B cells,and to induce major histocompatibility complex class II mol-ecules on B cells and macrophages (2). Recently it has becomeclear that a closely related cytokine, IL-13, performs many, butnot all, of the biological functions of IL-4 (3-5).The IL-4 receptor complex consists of a high-affinity IL-4-

binding chain (IL-4R) and the common y chain (yc) (6-8).The yc is also used by the IL-2, IL-7, IL-9, and IL-15 receptors(9-13); its expression is required for efficient signal transduc-tion in several murine cell lines (14, 15). Interestingly, a mutantform of human IL-4, with Tyr-124 replaced by an aspartate(mutant Y124D), binds IL-4-responsive cells with high affinitybut is deficient in signaling IL-4-specific biologic responses(16). Mutant Y124D can act as an inhibitor of wild-type IL-4.It has been proposed that the region of the IL-4 moleculearound Tyr-124 may serve as the 'yc interaction site (17).The IL-13 receptor structure remains elusive. It has been

shown that IL-13 can inhibit the binding of 125I-labeled IL-4 tocells that are responsive to both cytokines (18). In addition, theY124D mutant of IL-4 inhibited the IL-4- and IL-13-inducedproliferation of the multifactor dependent line TF-1 (18) andthe production of IgE by activated B cells (19). On the basis ofthese results, it has been proposed that the IL-13 receptorshares a component of the IL-4R that is important for signaltransduction.The signal-transduction pathways activated by IL-4 have

recently been described. IL-4 induces the rapid tyrosine phos-phorylation of the IL-4R (20, 21) and a 170-kDa substrate

termed 4PS (21). Phosphorylated 4PS associates with phos-phatidylinositol 3-kinase as well as other SH2-domain-con-taining proteins and may mediate important proliferativesignals. Interestingly, insulin also induces 4PS phosphorylationin hematopoietic cells (22). 4PS is related to the majorinsulin-induced phosphorylated substrate, insulin receptorsubstrate 1 (22, 23).Recent studies have shown that many cytokines, including

IL-4, may activate intracellular substrates in a large partthrough a recently reported family of cytoplasmic protein-tyrosine kinases, the Janus (JAK) family (24). To date, fourJAK family kinases have been identified, JAK1, JAK2, JAK3,and TYK2. Biochemical studies in T cells and myeloid cellshave demonstrated that JAK1 and JAK3 are activated afterIL-4 stimulation (25, 26) and, indeed, that JAK3 is coupleddirectly to the yc (12, 27). Because IL-13 induces manybiological functions in common with IL-4, we sought todetermine whether IL-13 also uses JAK1 and JAK3 to mediatesignal transduction. To do this, we analyzed the ability of IL-4and IL-13 to induce JAK1 and JAK3 phosphorylation inseveral cell types that are responsive to both cytokines. Weshow that both IL-4 and IL-13 induce the tyrosine phosphor-ylation of 4PS and JAK1, but unlike IL-4, IL-13 does notinduce the phosphorylation of JAK3. These results suggestthat the signaling events induced by IL-4 and IL-13 are similarin that they both activate the phosphorylation of 4PS andJAK1, but there are also significant differences. The implica-tions of these findings are discussed.

MATERIALS AND METHODSCells and Reagents. The IL-3-dependent myeloid cell line

TF-1 was maintained in RPMI 1640 medium/2 mM glu-tamine/penicillin-streptomycin at 100 ng/ml/10% fetal calfserum/recombinant human IL-3 (10 ng/ml) (R & D Systems).U937 cells were maintained in supplemented RPMI 1640medium. Recombinant human IL-4 expressed in Escherichiacoli was from Alan Levine (Monsanto), whereas human andmouse IL-13 and human colony-stimulating factor 1 wereobtained from R & D Systems. Murine IL-4 was purified frombaculovirus stock as described (28). Bovine insulin was ob-tained from Collaborative Research. Anti-Fc receptor anti-body, anti-MAC-1-fluorescein isothiocyanate (FITC), andanti-I-Ad-FITC were obtained from PharMingen. Anti-serumspecific for the COOH terminus (25) and NH2 terminus (NT)of human JAK3 was prepared. Anti-JAK1, anti-murine JAK3(aa 934-952, JH1 region), and anti-phosphotyrosine (antibody4G10) were obtained from Upstate Biotechnology (Lake Placid,NY).

Abbreviations: IL, interleukin; 4PS, IL-4-induced phosphotyrosine sub-strate; )c, common y chain; IL-4R, IL4 receptor high-affinity IL4-binding chain; NT, NH2 terminus; FITC, fluorescein isothiocyanate.tTo whom reprint requests should be sent at the present address:Immunology Department, Jerome Holland Laboratory, AmericanRed Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Purification of Murine Macrophages. Macrophages werederived from murine bone marrow taken from femurs ofBALB/c mice as described (29). Briefly, bone marrow cellswere incubated at 2 x 106 cells per ml in the presence ofhumancolony-stimulating factor 1 at 10 ng/ml and 37°C for 10 days.The nonadherent cells were aspirated off, and the adherentcells were washed in phosphate-buffered saline (PBS) andgiven fresh medium three times. These cell populations wereroutinely 93-95% macrophages, as determined by cell cytom-etry analysis using anti-MAC-1 antibody. The macrophageswere washed and incubated at 5 x 105 cells per ml in completeRPMI 1640 medium in the presence or absence of murine IL-4(500 units/ml) or murine IL-13 (200 ng/ml) for 24 hr at 37°C.The cells were harvested, washed, and stained with anti-I-Ad_FITC.

Cellular Proliferation Assays. For the study of cellularproliferation, TF-1 cells were incubated at 20,000 cells per wellin a final volume of 0.2 ml of complete RPMI 1640 medium inthe presence or absence of various concentrations of humanIL-4 or human IL-13 for 36 hr at 37°C. The wells were pulsedwith 1 ,uCi of [3H]thymidine per well (1 Ci = 37 GBq) for thefinal 16 hr of culture before harvesting by using a Skatron cellharvestor followed by liquid scintillation counting.

Immunoprecipitation and Immunoblotting. Analysis ofphosphotyrosine-containing proteins was done essentially asdescribed (30). Cells were preincubated in RPMI 1640 mediumwithout serum or cytokines for 2 hr at 37°C. After beingwashed, the cells were resuspended in RPMI 1640 mediumwith 50 ,uM Na3VO4 and incubated in the presence or absenceof human IL-4 or human IL-13 for 5 min at room temperature.The reaction was terminated by 10-fold dilution in ice-coldPBS with 100 ,uM Na3VO4. Cell pellets were lysed in Hepeslysis buffer (50 mM Hepes/150 mM NaCl/0.5% NonidetP-40/1 mM Na3VO4/NaF, pyrophosphate/1 mM phenyl-methylsulfonyl fluoride and protease inhibitor cocktail) andclarified. The soluble fraction (-2 mg) was immunoprecipi-tated with a monoclonal anti-phosphotyrosine antibody, 4G10(Upstate Biotechnology), or anti-JAK3 anti-serum. The pre-cipitates were collected with protein G-agarose beads (Pierce),washed three times in lysis buffer, and eluted with SDS/samplebuffer. The samples were separated on an SDS/7.5% poly-acrylamide gel before transfer to poly(vinylidene difluoride)membranes. The membranes were blocked with PBS/3%bovine serum albumin/0.05% Tween 20. Then the blot wasprobed with anti-phosphotyrosine antibody (1 jig/ml) or anti-JAK3 anti-serum (1:1000). The bound antibody was detectedusing enhanced chemiluminescence (Amersham).

RESULTSThe high degree of functional similarity between IL-4 andIL-13 suggests that they might share signaling pathways. In-deed, L.-M. Wang (personal communication) has recentlyobserved that both IL-4 and IL-13 can induce the tyrosinephosphorylation of 4PS. We analyzed the ability of IL-4 andIL-13 to induce 4PS phosphorylation in two cell types (Fig. 1).As shown by others (L.-M. Wang and J. Pierce, personalcommunication), both IL-4 and IL-13 induced the tyrosinephosphorylation of a 170-kDa protein, termed 4PS (21),detected by anti-phosphotyrosine precipitation followed byanti-phosphotyrosine immunoblotting in the human mono-cytic cell line U937 and the factor-dependent myeloid cell lineTF-1. At the concentrations used, this effect was more pro-nounced in IL-4-treated cells. As anticipated, insulin alsoinduced 4PS phosphorylation in U937 cells. In preliminaryexperiments we found the background levels of JAK phos-phorylation to be quite high in U937 cells (data not shown),and therefore we concentrated our further studies on TF-1cells.

U937

-i ~-J c

200

0

x 97.-

TF-1

CO

C: ,...

C)0-

5.5.

69

FIG. 1. 4PS phosphorylation in response to IL-13. U937 and TF-1cells were deprived of all serum and growth factors for 2 hr beforetreatment with human IL-4 (10 ng/ml), human IL-13 (250 ng/ml), orinsulin (40 ,ug/ml), as indicated. After the reaction was stopped,samples were prepared for immunoprecipitation with anti-phospho-tyrosine followed by immunoblotting with anti-phosphotyrosine asdescribed.

To determine biologically equivalent concentrations of IL-4and IL-13, we compared the potency of IL-4 and IL-13 ininducing a proliferative response in TF-1 cells (Fig. 2). Thecells were incubated with various concentrations of IL-4 orIL-13, ranging from 0 to 300 ng/ml. TF-1 cells proliferated inresponse to both cytokines, although the concentration ofIL-13 required for a half-maximal response was =100-foldgreater than that of IL-4.Recent studies have shown that IL-4 induces the tyrosine

phosphorylation of JAK1 and JAK3 (25, 26). We thereforeanalyzed the ability of IL-4 and IL-13 to induce the tyrosinephosphorylation of JAK1 and JAK3 in TF-1 cells (Fig. 3).After factor deprivation, the cells were stimulated with IL-4(10 ng/ml) or IL-13 (250 ng/ml) at concentrations determinedto stimulate maximal proliferative responses. Extracts wereprepared and immunoprecipitated with anti-JAK1 or anti-COOH-terminal JAK3 antibody, followed by immunoblottingwith anti-phosphotyrosine. Both IL-4 and IL-13 induced sim-ilar amounts ofJAK1 tyrosine phosphorylation. However, onlyIL-4, and not IL-13, induced JAK3 tyrosine phosphorylation.Stripping the blot and reprobing with anti-JAK1 or anti-JAK3indicated that equivalent amounts ofJAK protein were loadedin each lane. Neither IL-4 nor IL-13 induced JAK2 or TYK2phosphorylation in these cells (data not shown). In Fig. 4,various concentrations of IL-4 were compared with an optimal

40

30

X 20

v0 IL**'..........13 W... W.0.-

0 .01 .1 1 10 100 1000

Cytokine, ng/ml

FIG. 2. Proliferative response of TF-1 cells to IL-4 and IL-13. TF-1cells were incubated with the indicated doses of human IL-4 andhuman IL-13 for a total of 48 hr. Cells were treated for the last 4 hrof culture with [3H]thymidine.

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Precipitation: a-JAK1

et

0 -J _i

200 -cq

69-

ct-JAK3m

m,

0 J la

Precipitation: a.-NT cc-JAK3

r -r-- I-1 -

- . _wBlot:

200 -t

_ Blot:o,-PY 0

x

-mom

'A M

Blot: ax-JAK1 cx-JAK3

FIG. 3. Activation of JAK kinases by IL-4 and IL-13 in TF-1 cells.TF-1 cells were deprived of serum and IL-3 for 2 hr before treatmentwith human IL-4 (10 ng/ml) or human IL-13 (250 ng/ml). Lysates wereprepared and immunoprecipitated with anti-JAK1 (a-JAK1) or anti-COOH-terminal JAK3 (a-JAK3). Blots were probed with anti-phosphotyrosine (a-PY), then stripped, and reprobed with anti-JAK1or anti-JAK3 as indicated.

concentration of IL-13 (250 ng/ml). The level of 4PS phos-phorylation in cells treated with IL-13 at 250 ng/ml was greaterthan that seen in cells treated with IL-4 at 1 ng/ml and less thanthat treated with IL-4 at 5.0 ng/ml. Tyrosine phosphorylationofJAK3 was induced by as little IL-4 as 0.5 ng/ml. Despite thefact that IL-13 (250 ng/ml) induced more tyrosine phosphor-ylation of 4PS, IL-13 induced no detectable JAK3 phosphor-ylation.To determine whether IL-13 might induce tyrosine phos-

phorylation of JAK3 in another cell type, we analyzed theresponse of murine macrophages to IL-4 or IL-13. Bonemarrow cells cultured in the presence of colony-stimulatingfactor 1 have been reported to be IL-13 responsive (5). BothIL-4 and IL-13 up-regulated major histocompatibility complexclass II expression (I-Ad) on these cells from a mean fluores-cence intensity of 57 to 82 for IL-4 and 86 for IL-13. Macro-phages deprived of serum and colony-stimulating factor 1 weretreated with either IL-4 or IL-13 (Fig. 5). IL-4 induced thestriking tyrosine phosphorylation of JAK3 in these cells,whereas IL-13 failed to do so. Similar results were obtained byusing two different anti-JAK3 anti-sera (anti-NT and anti-mouse JAK3). The results obtained in TF-1 cells and murinemacrophages suggest that JAK3 does not participate in theIL-13 signal-transduction pathway. Although anti-JAK3 anti-sera did not immunoprecipitate tyrosine-phosphorylated JAK3from IL-13-treated cells, we have occasionally observed an

IL-4

oo -u ' Ppt:4PS -0- £ US. oc-PY

Blot:a-PY

ccs.i.( -PYP.: w

97

69

x-JAK3

FIG. 5. IL-13 fails to activate JAK3 in murine macrophages.Biological responses of IL-4 and IL-13 were measured by I-Adinduction on murine bone-marrow-derived macrophages. The meanfluorescence intensity of anti-I-Ad-FITC staining was 57, 82, and 86 foruntreated, IL-4-treated, or IL-13-treated macrophages, respectively.For the phosphorylation studies, macrophages were deprived ofcolony-stimulating factor 1 and serum and treated with IL-4 (10ng/ml) or IL-13 (250 ng/ml), as indicated. Samples were precipitatedwith anti-NT JAK3 (a-NT) or anti-mouse JAK3 specific for the JH1region and probed with anti-phosphotyrosine (a-PY) or anti-mouseJAK3 (a-JAK3) as indicated.

increase in tyrosine phosphorylation of a 125-kDa proteinafter IL-4 and IL-13 treatment of TF-1 cells (Fig. 6). Inaddition, the anti-COOH-terminal JAK3 anti-sera weaklyimmunoblotted a 125-kDa protein from anti-phosphotyrosineimmunoprecipitates, suggesting the possibility that IL-13 in-duces the phosphorylation of an unidentified protein that isweakly recognized by anti-JAK3 in an immunoblot. Thus bothIL-4 and IL-13 induce 4PS and JAKi phosphorylation, butIL-13 does not induce JAK3 phosphorylation.

DISCUSSIONReceptors for interferons and the type I cytokines utilizemembers of the Janus family of tyrosine kinases to mediatesignal transmission (24). For example, growth hormone, IL-3,granulocyte/macrophage colony-stimulating factor, and gran-ulocyte colony-stimulating factor all have been shown toactivate JAK2; interferon a activates JAKi and TYK2; inter-

Precipitation: cc-PY cc-PYBlot: a-PY cx-JAK3

o cI200 c

200 -

Co

0

xJAK3 - - °r''- a-JAK3 a-PY

JAK3 -- at-J A K3 c-JAK3

FIG. 4. IL-4 dose-response. TF-1 cells were treated as described in

Fig. 3, except that the various doses of IL-4 (0.5-10 ng/ml) were used,

and a single dose (250 ng/ml) of human IL-13 was used. Lysates were

prepared and immunoprecipitated (Ppt) with anti-phosphotyrosine

(a-PY) or anti-NT JAK3. Blots were probed with anti-phosphoty-

rosine or anti-NT JAK3, as indicated.

00 -

97 -4 4

MI - m . z

FIG. 6. IL-13 induces the tyrosine phosphorylation of a 125-kDaprotein (arrow). TF-1 cells were deprived of serum and IL-3 for 2 hrbefore treatment with human IL-4 (10 ng/ml) or human IL-13 (250ng/ml). Lysates were prepared and immunoprecipitated with anti-phosphotyrosine. The blots were probed with anti-phosphotyrosineand anti-COOH terminal JAK3 as indicated.

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feron y activates JAK1 and JAK2; and IL-4 and IL-2 activateJAK1 and JAK3, respectively. Stimulation of the type Icytokine receptors leads to the activation of these JAK-familykinases and to the tyrosine phosphorylation, activation, andtranslocation of factors involved in gene transcription, termedsignal transducers and activators of transcription (STAT).Recent work has demonstrated that STATs are also involvedin the signaling pathway from the IL-4 and IL-13 receptors(31-35).The cytokines IL-4 and IL-13 have many overlapping bio-

logic functions and most likely share a receptor component(18, 19). It has been shown that both cytokines induced thetyrosine phosphorylation of 4PS (L.-M. Wang and J. Pierce,personal communication). Recently a sequence motif (14Rmotif) was identified in the cytoplasmic domain of the IL-4Rthat is important for signaling phosphorylation of insulinreceptor substrate 1/4PS (30). The observation that IL-13signals 4PS phosphorylation suggests that one of the chains ofthe IL-13 receptor also contains an I4R motif.

Interestingly, although IL-4 clearly induced the tyrosinephosphorylation of JAK3, we did not detect IL-13-inducedphosphorylation of JAK3. Similar results were obtained byusing three different anti-JAK3 antibodies (compare Figs.3-5) and two different cell types. Thus, IL-13 can activate thephosphorylation of 4PS and JAK1 without the participation ofJAK3. However, IL-13 occasionally induced the phosphory-lation of a 125-kDa protein that was weakly immunoblotted bythe anti-JAK3 antisera. It is possible that this immunoreactivespecies represents another JAK kinase used by the IL-13receptor that is weakly recognized by anti-JAK3.These results are partially consistent with previous hypoth-

eses of IL-4 and IL-13 signaling (5, 8). IL-4 signaling most likelybegins with the heterodimerization of the IL-4R and the yc,followed by activation of their associated kinases. On the basisof the ability of the mutant form of human IL-4, mutantY124D, to block IL-13-mediated biological responses, it wassuggested that the IL-13 receptor also used the yc (17).Because it has been shown that JAK3 predominantly associ-ates with the yc (12, 27), we initially anticipated that IL-13would activate JAK3. However, recent results suggest that theIL-13 receptor may not use the yc but the IL-4R in combina-tion with an alternative chain termed y' (35). The data that wepresent here would support this hypothesis.Tony et al. (17) and Lin et al. (35) have demonstrated that

antibodies to the IL-4R that block IL-4 action also blockIL-13 action, and Smerz-Bertling and Duschl (36) haverecently shown that IL-4 and IL-13 induce the tyrosinephosphorylation of the IL-4R. In addition, it was found thatboth IL-4 and IL-13 induce similar STAT activity in COS-7cells that do not express yc or JAK3 (35) and that they bothcan induce a proliferative response in the B9 plasmacytomathat lacks yc expression (37). Moreover, both IL-4 and IL-13induced CD23 expression and IgE production in B cellslacking -yc derived from patients with X chromosome-linkedsevere combined immunodeficiency (38). These results sug-gest that the component shared between the IL-4 and IL-13receptors is most likely the IL-4R and not the -yc and that theIL-4R and IL-13 receptor can either signal independently oruse an alternative signaling chain (-y').The ability of IL-13 to use the IL-4R to signal would explain

its ability to stimulate the phosphorylation of JAK1 and 4PSand to activate similar STATs. Recent studies have shown thatJAK3 interacts with the yc and that JAK1 interacts with theIL-2R 13 chain. Additionally, it was shown that JAK1 caninteract with the IL-4R (39) and, therefore, would act as afunctional component of both the IL-4 and IL-13 receptorsignaling complexes. In addition, the IL-13 receptor complexwould contain the I4R motif to recruit the 4PS molecule (30)and several critical downstream tyrosine residues shown tointeract with IL-4 STAT (34). Several important issues remain

to be clarified, such as the identity of the IL-13-binding chain,the alternative -y chain (y'), and the identity of the tyrosinekinase, potentially another JAK kinase, associated with the y'receptor.

We acknowledge Drs. Jackie Pierce and Warren Leonard for helpfuldiscussions and sharing unpublished work, Helen Wang and JaneHu-Li for excellent technical assistance, and Carol Butler for editorialassistance. P.J.T. is a Howard Hughes Medical Institute-NationalInstitutes of Health Research Scholar.

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