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(Journal of Leukocyte Biology. 2002;72:580-589.)
© 2002 by Society for Leukocyte Biology

IL-13 signal transduction in human monocytes: phosphorylation of receptor components, association with Jaks, and phosphorylation/activation of Stats

Biswajit Roy*, Ashish Bhattacharjee*, Bo Xu*, Dwayne Ford{dagger}, Abby L. Maizel{dagger} and Martha K. Cathcart*

* Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio; and
{dagger} Roger Williams Medical Center, Boston University, School of Medicine, Massachusetts

Correspondence: Dr. Martha K. Cathcart, Department of Cell Biology/NC10, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: cathcam{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-13 regulates monocyte function and is a potent stimulator of 15-lipoxygenase expression. In different cell types, the functional IL-13 receptor complex can be comprised of variable protein components and has not been thoroughly examined in human monocytes. Here, we identify the receptor components and upstream signaling events initiated by IL-13 in primary human blood monocytes. The expression, phosphorylation and associated Jak kinases of the known, variable receptor components, IL-4R{alpha}, IL-2R{gamma}c, IL-13R{alpha}1 and IL-13R{alpha}2, were examined. We determined that IL-4R{alpha} and IL13R{alpha}1 are phosphorylated upon exposure to IL-13. Although IL-2R{gamma}c is also expressed, it is not phosphorylated upon exposure to IL-13. Evaluation of the presence of IL-13R{alpha}2 failed to reveal significant mRNA or protein expression. Earlier, our laboratory showed that IL-13 induced the phosphorylation of Jak2 and Tyk2 in monocytes and that expression of both Jaks was essential for downstream signaling by IL-13. Here, we report that Jak2 is associated with IL-4R{alpha}, and Tyk2 is associated with the IL-13R{alpha}1 component of the IL-13 receptor complex. Additionally, Stat proteins 1{alpha}, 3, 5A, 5B, and 6 are phosphorylated in response to IL-13. Further, the nuclear translocation and DNA binding of each of these Stats were induced by IL-13. These data represent the first complete report of the functional IL-13 receptor complex and early signaling events in human monocytes. This information is critical for understanding the IL-13 response of monocytes in inflammation.

Key Words: human macrophages • cytokine receptors • cytokines • inflammation • monocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-13 is a pleiotropic immune regulatory cytokine that shares structural and biological properties with IL-4 [1 ]. IL-13 is known to promote growth of preactivated B lymphocytes [2 ], induce germ line {varepsilon} transcripts, and direct naive B lymphocytes to switch to immunoglobulin IgE and IgG4 synthesis [3 ]. It also induces expression of the low-affinity receptor for IgE/CD23 and up-regulates class II major histocompatibility complex expression on B lymphocytes and monocytes [4 , 5 ]. In monocytes, IL-13 down-regulates surface expression of the Fc receptor for IgG [5 ] and inhibits synthesis of inflammatory cytokines including tumor necrosis factor-{alpha}, IL-1ß, IL-6, and IL-8 [6 , 7 ]. Moreover, it suppresses synthesis of IL-12, a critical cytokine for differentiation of uncommitted T cells toward the T helper cell type 1 phenotype [5 , 8 ]. Recent reports highlight the central contribution of IL-13 to experimental allergic asthma [9 , 10 ]. The role of this cytokine in inducing monocyte 15-lipoxygenase (15-LO), a lipid-peroxidating enzyme of potential interest in atherosclerosis, asthma, and inflammation in general, is also well documented [11 , 12 ]. We recently reported that two Jak kinases involved in the initial signaling response to IL-13 are required for induction of 15-LO in human monocytes [11 ], yet the monocyte receptor complex components and association of the Jaks with these components remained to be defined.

Receptor complexes for many cytokines have been shown to share components. The composition of the IL-13 receptor complex has been shown to vary between and among differing cell types, but IL-4 and IL-13 receptor complexes can use the IL-4R{alpha} protein (140 kDa). The IL-13 receptor complex has also been reported to putatively share a component that is used by IL-4 as well as several other cytokines, the IL-2R{gamma}c, a finding that remains controversial [13 ]. Two other human IL-13 receptor components have recently been cloned. One of these, referred to here as IL-13R{alpha}1, was cloned by three separate groups [14 15 16 ]. The other, herein referred to as IL-13R{alpha}2, was cloned by Caput et al. [17 ]. Both components are 55–70 kDa and bind IL-13 with different affinities. IL-13R{alpha}1 initially binds IL-13 with subsequent recruitment of the IL-4R{alpha} (140-kDa glycoprotein) to efficiently transduce a signal [14 , 18 ], whereas the IL-13R{alpha}2 can bind IL-13 in the absence of IL-4R{alpha}, but its role in IL-13 signaling is still unclear [17 ]. Earlier, Doucet et al. [19 ] had shown that the pattern of expression of IL-13R{alpha}2 varied from cell line to cell line within the human lung fibroblast cell lineages. Although CCL202 and FPA cell lines expressed IL-13R{alpha}2, the other cell line, ICIG7, had no detectable expression of IL-13R{alpha}2 by reverse transcriptase-polymerase chain reaction (RT-PCR) or on immunoblots. Studies such as these implicate cell lineage-specific variability in the IL-13 receptor constituents.

A series of cytokines and growth factors are known to trigger activation of members of the Jak family of kinases that associate with receptor components. In recently published studies, we found that among the Jaks, only Jak2 and Tyk2 were phosphorylated in response to IL-13 in human monocytes [11 ]. Other studies revealed the inhibition of expression of either of these kinases blocked the IL-13 induction of the 15-LO and were therefore critical components for transducing the cytokine signal. The association of these kinases with IL-13 receptor components was not investigated in these earlier studies; however, Jak1 was shown to associate with IL-4R{alpha} in response to IL-4 in human monocytes [20 ], and another report showed that Jak2 associated with IL-4R{alpha} [21 ].

Members of the Jak family of kinases can mediate the phosphorylation of Stat proteins on a single tyrosine leading to Stat translocation and DNA binding [22 23 24 25 26 27 28 ]. So far, six members of the Stat family have been identified and are referred to as Stats1–6. Several of the Stats can be expressed as alternatively spliced isoforms {e.g., 91-kDa form (Stat1{alpha}) and 84-kDa form (Stat1ß); ref [29 ]}.

Stat proteins contain SH2 domains and dimerize after phosphorylation [30 ]. This enables the proteins to be efficiently transported to the nucleus and bind DNA [31 ]. DNA binding may involve interactions with other proteins, such as p48, which forms part of the interferon (IFN)-stimulated gene factor complex binding to the IFN-stimulated response element [32 ]. The ability of individual cytokine receptors to activate overlapping but distinct sets of homo- and heterodimerizing Stat proteins contributes to signal specificity. For example, interferon-{alpha} activates Stats1, 2, and 3 and exerts antiviral- and growth-inhibitory effects in the tumorigenic cell line Daudi [33 ], and prolactin activates Stats1, 3, and 5 in mammary epithelial cells and induces milk protein gene expression [34 , 35 ].

In this study, we report that IL-13 induces the phosphorylation of two receptor components, IL-13R{alpha}1 and IL-4R{alpha}. In contrast, neither IL-13R{alpha}2 nor IL-2R{gamma}c appears to participate in the signaling process. Based on our earlier reports that Jak2 and Tyk2 are phosphorylated in response to IL-13 in these cells, we also investigated the association of Jak2 and Tyk2 with the receptor constituents and found that Jak2 associates with IL-4R{alpha}, and Tyk2 associates with IL-13R{alpha}1. Further, we found that Stat1{alpha}, Stat3, Stat5A/Stat5B, and Stat6 are tyrosine-phosphorylated and activated in response to IL-13. These findings delineate an IL-13-driven, selective signaling pathway from the receptor to the nucleus in human monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Recombinant human IL-13 was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit antisera against Jak1, Jak2, Tyk2, and Jak3 were purchased from Upstate Biotechnology or Santa Cruz Biotechnology (Santa Cruz, CA). Each one of the Jak/Tyk antibodies was essentially noncrossreactive with the other members of the Jak family of kinases and recognized corresponding antigens under native (in 1% Triton X-100 extracts) as well as denaturing conditions.

Antibodies to IL-4R{alpha} were purchased from Santa Cruz Biotechnology and R&D Systems (Minneapolis, MN). Affinity-purified antisera against deduced peptides from the amino-terminal or carboxy-terminal regions of IL-13R{alpha}1 were prepared as previously described [18 ] with the amino-terminal antibody demonstrating higher specificity in Western blotting. Monoclonal rat anti-human IL-2R{gamma}c was purchased from BD Pharmingen (San Diego, CA) and was used for immunoprecipitation. Rabbit anti-human IL-2R{gamma}c (C-20) used for Western blots was obtained from Santa Cruz Biotechnology. Antisera against the IL-13R{alpha}2 were produced in collaboration with Zymed, Inc. (San Francisco, CA). This heterotypic antisera, against the carboxy-terminal amino acids 364–375 of the deduced protein sequence [17 ], was purified by affinity chromatography using the peptide attached to a sulfolink column (Pierce, Rockford, IL) as described [18 ]. The purified antisera against the IL-13R{alpha}2 were capable of recognizing that protein produced subsequent to SuperfectTM (Qiagen, Valencia, CA) mediated transfection of the receptor construct [17 ] in the expression system pcDNA 3.1 (Invitrogen, Carlsbad, CA) into COS-7 cells. In addition, the antisera effectively immunoprecipitated radiolabeled 125I-IL-13 [36 ] subsequent to binding and cross-linking the ligand to cells bearing the receptor.

Rabbit antisera against Stat proteins 1–5 were purchased from Transduction Laboratories (Lexington, KY; Stat1–4) as well as Santa Cruz Biotechnology (Stats2, 4, and 5). Antibodies to Stat5A and Stat5B were obtained from Upstate Biotechnology. Antibodies to Stat6 and phospho-Stat6 were purchased from BD Pharmingen and Cell Signaling Technology Inc. (Beverly, MA), respectively. Antisera raised against two peptides or amino acids 1–178 or 592–731 of the Stat1 protein were also used (Transduction Laboratories). Each of the Stat antibodies used for these studies was essentially noncrossreactive with other Stat molecules and was good for detecting proteins on Western blots as well as immunoprecipitating the respective antigens. Antiphosphotyrosine-Stat antibodies raised against Stat1 (Y701), Stat3 (Y705), and Stat5A/B (Y694,Y699) were purchased from Upstate Biotechnology and were used to detect tyrosine-phosphorylated Stat proteins on Western blots.

For general detection of tyrosine phosphorylated proteins on immunoblots, PY-99 (Transduction Laboratories) or a mixture (1:1) of phosphotyrosine antibodies PY-20 (Santa Cruz Biotechnology) and 4G-10 (Upstate Biotechnology) was used at dilutions of 1:1000.

Isolation of human monocytes
Human peripheral blood monocytes were isolated from heparinized whole blood by sequential centrifugation over a Ficoll-Paque solution and adherence to serum-coated tissue culture flasks as described previously [37 ]. Nonadherent cells were removed from the flasks by subsequent washes using Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% bovine calf serum (BCS; Hyclone, Logan, UT). Adherent cells were released from the flask using 5 mM ethylenediaminetetraacetate and were plated after washing in tissue-culture plates (Costar, Cambridge, MA). The isolated cell preparations had typically more than 95% monocytes, were maintained in DMEM containing 10% BCS at 37°C in the presence of 10% CO2, and were used immediately for experiments.

Immunoprecipitation and Western blotting
Freshly isolated monocytes were counted and plated in six-well or 10-cm plates and were allowed to adhere for 2 h. The cells were then pretreated for 15 min with addition of sodium orthovanadate solution (100 µM) followed by treatment with or without IL-13 (250–500 pM) or IL-4 (670 pM) for 10, 15, or 30 min (as indicated). Orthovanadate does not enter the cells [38 , 39 ]. Extensive studies in our laboratory have shown that treatment of human monocytes with 100 µM sodium orthovanadate does not induce the Jak or Stat pathway as is observed with cell-permeable pervanadate and instead preserves phosphorylation signals of the phosphoproteins upon Western analysis, likely by reducing phosphatase activity immediately upon lysis. In these experiments, this treatment did not change the results qualitatively but merely improved the preservation of the phosphorylation. For receptor studies, postnuclear lysates were prepared using a lysis buffer of 1% Triton X-100, 150 mM NaCl, 50 mM NaF, 30 mM ß glycerophosphate, 0.5 mM phosphoserine, 0.5 mM phosphotyrosine, 1.0 mM phosphothreonine, 1.5 mM p-nitrophenylphosphate, 50 mM Tris, pH 7.4, 1 mM sodium orthovanadate, 500 µM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). The cells were kept on ice for 30 min and centrifuged at 9300 g for 15 min at 4°C, the supernatant was collected, and protein concentration was determined. The lysates were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes as previously described [11 ]. For Stat experiments, whole cell extracts and nuclear extracts were used as noted and prepared according to previously published protocols [40 , 41 ] with the exception of 0.5 M salt, which was used in the whole cell lysis buffer. The blocker used for direct Westerns with antiphospho Stat antibodies was 5% milk in phosphate-buffered saline (PBS) with 0.1% Tween 20; otherwise, it was 5% bovine serum albumin in PBS/Tween 20.

For immunoprecipitation experiments, the lysates (0.5–1.0 mg/ml, usually 0.5–2.0 mg total lysates) were incubated with immunoprecipitating antibodies (4–6 µg/ml as noted) for 1–2 h at 4°C with constant rotation. Immune complexes were collected using prewashed Sepharose-protein A or Sepharose-protein G beads (20–50 µl packed bead vol per ml extract). The beads were washed three times with lysis buffer. The immune complexes were released by boiling the beads in SDS sample buffer and then analyzed by SDS-PAGE followed by electrophoretic transfer to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked and subsequently probed with specific antibodies and developed using enhanced chemiluminescence (ECL; Pierce). For immunoprecipitation of IL-13R{alpha}1, the antisera against the aminoterminal peptide were used. Sodium pyrophosphate, p-nitrophenyl phosphate, phosphoserine, phosphotyrosine, phosphothreonine, and ß-glycerophosphate were not included in the lysis buffer for phosphoprotein immunoprecipitation experiments.

In numerous experiments, immunoblots were stripped and reprobed to assess equal loading and/or equal immunoprecipitation. In these instances, the blots were incubated at 50°C for 30 min in a stripping buffer containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM ß-mercaptoethanol. The blots were then washed three to four times for 15 min each with PBS-Tween 20 (0.1% v/v) and were then reprobed with a different primary antibody and developed using ECL.

Electrophoretic mobility shift assay (EMSA)
To assess the DNA binding activity of Stat proteins upon IL-13 treatment, EMSA was performed using nuclear extracts from human monocytes and specific Stat probes. Briefly, nuclear proteins from monocytes with or without IL-13 treatment were extracted by the method described above. The double-stranded Stat1-, Stat3-, and Stat5-specific probes were obtained from Santa Cruz, and the SBE1 probe (5'-GCTCTTCTTCCCAGGAACTCAATG-3') was used for Stat6. The Stat probes were labeled with 32P using T4-polynucleotide kinase (Promega, Madison, WI) and were incubated with 3 µg nuclear proteins at room temperature for 20 min. The protein-DNA complexes were resolved on 5% denaturing polyacrylamide gels. After drying, the gels were exposed to X-ray films at -80°C.

Determination of mRNA for IL-13R{alpha}2
The presence of mRNA for the IL-13R{alpha}2 was performed as previously described using a nested protocol [18 ]. The first-stage reaction results in a 1088-bp product, and the internal primers for the second reaction results in amplification of a 437-bp product.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of receptor components in IL-13-treated human monocytes
To determine whether IL-4R{alpha} was phosphorylated on tyrosine in response to IL-13, its phosphorylation status was analyzed using antiphosphotyrosine immunoprecipitation (using PY-99) followed by detection with an IL-4R{alpha} antibody on a Western blot (Fig. 1 A ). PY-99-immunoprecipitated IL-4R{alpha} was detected in the immunoprecipitate from untreated monocyte cell lysates. This result was confirmed by performing experiments using the antibody to IL4-R{alpha} for immunoprecipitation and then probing with the antibody to phosphotyrosine. IL-13 caused a substantial increase in tyrosine phosphorylation of IL4-R{alpha} in these experiments (Fig. 1B , upper panel). After stripping, this blot was reprobed with antibody to IL4-R{alpha}, and equal loading was evident in the two lanes (Fig. 1B , lower panel). A similar result was obtained by probing a blot of the PY99 immunoprecipitate with antibody to IL4-R{alpha} derived from another source (data not shown).



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  		Figure 1. IL-13 induces tyrosine phosphorylation of IL-4R{alpha}. Human blood monocytes were treated with IL-13 (250 pM) for 10 min or left untreated as indicated. (A) The cells were lysed; the cleared cell lysates were used for immunoprecipitation with the phosphotyrosine antibody, PY-99. Immune complexes were collected using protein A beads and analyzed by immunoblotting with antibody to IL-4R{alpha}. (B) Cell lysates were immunoprecipitated with antibody to IL-4R{alpha}, and immune complexes were analyzed by Western blotting with antibody to phosphotyrosine. Arrows indicate the position of IL-4R{alpha} as calculated from molecular weight markers in adjacent lanes. The lower panel displays the results of reprobing the stripped blot from the upper panel with IL-4R{alpha} antibody to assess equal immunoprecipitation and loading of protein in each lane.

 
The phosphorylation status of IL-13R{alpha}1 and IL-13R{alpha}2 receptor components was investigated next. Two different antibodies against N-terminal or C-terminal fragments of IL-13R{alpha}1 were used in our studies, as described in Materials and Methods [18 ]. Blots of SDS-PAGE gels of IL-13R{alpha}1 immunoprecipitates, derived from 1 mg each of untreated and IL-13-treated monocyte cell lysates, were probed with antiphosphotyrosine (PY-99). The results indicate increased (~3.4-fold) phosphorylation of IL-13R{alpha}1 in IL-13-treated monocytes with a low but detectable basal level of phosphorylated protein in the untreated cells (Fig. 2 ). The blot was stripped and reprobed with antibody to IL-13R{alpha}1 to evaluate the loading in the two lanes, and equal loading was observed (Fig. 2A , bottom panel). This result was confirmed by detecting phosphorylated IL-13R{alpha}1 by probing a blot of an SDS gel of the PY-99 immunoprecipitate with the antibody raised against the N-terminal sequence of the receptor component (Fig. 2B) . The results thus indicate that IL-13R{alpha}1 is an active constituent of the IL-13 signaling complex.



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  		Figure 2. IL-13 induces tyrosine phosphorylation of IL-13R{alpha}1. Freshly isolated monocytes were treated with IL-13 (250 pM) for 10 min or left untreated and lysed, and the lysate was immunoprecipitated with (A) a mixture of two antibodies (1:1) raised against the C-terminal and N-terminal peptide sequences of the protein or (B) antibody to phosphotyrosine (PY-99). The immunoprecipitated proteins were collected using protein A beads and analyzed on a 7.5% SDS-PAGE and were blotted with (A) PY-99 or (B) IL-13R{alpha}1 antibody. (A) The bottom panel displays the results of reprobing the stripped blot from the upper panel with IL-13R{alpha}1 antibody (N-terminal) to assess the equal loading of proteins in each lane. Arrows indicate calculated positions of the molecules based on migration of molecular weight markers in adjacent lanes.

 
The presence of specific mRNA for IL-13R{alpha}2 was first evaluated by RT-PCR [18 ] using RNA derived from unstimulated, purified monocytes. The predicted product of 1088 bp from the first reaction of the nested procedure was barely visible or undetectable (data not shown). The second reaction of the nested procedure produced the predicted band of 437 bp consistently (data not shown). The results indicate that only a marginal amount of mRNA for IL-13R{alpha}2 was produced, a finding consistent with previously published work on cells of the monocytic lineage. Furthermore, using antibodies to IL-13R{alpha}2, Western blots of unfractionated, freshly isolated monocyte-cell lysates revealed a nonspecific banding pattern (data not shown). These results are also consistent with recently completed radiolabeled 125I-IL-13-binding studies that selectively detect the presence of IL-13R{alpha}2 [42 ]. Freshly isolated monocytes demonstrated equivocal binding, while subsequent to dendritic cell differentiation; a definitive receptor presence could be demonstrated (D. Ford et al. manuscript in preparation).

Although the IL-2R{gamma}c protein was detected in untreated and IL-13-treated monocyte lysates (Fig. 3 A , bottom panel), immunoprecipitation experiments using a rat monoclonal antibody, clone TUGH4, to human IL-2R{gamma}c, revealed no detectable signal when probed with the antibody to phosphotyrosine (Fig. 3A) . We also used antiphosphotyrosine antibodies to immunoprecipitate the monocyte lysates, and immunoprecipitates from untreated or IL-13-treated cells showed no detectable IL-2R{gamma}c (~68 kDa) when probed with the {gamma}c antibody (Fig. 3B) . To confirm that our approaches could detect the phosphorylation of this receptor, we performed an identical experiment but used IL-4 to stimulate the monocytes. Data for the IL-2R{gamma}c immunoprecipitation and probe with antiphosphotyrosine are shown in Figure 3C . In contrast to IL-13, results from this study indicate that IL-4 induces phosphorylation of IL-2R{gamma}c. A reprobe of this blot indicated equal immunoprecipitation and loading of the IL-2R{gamma}c (Fig. 3C) . The reverse antibody experiment gave essentially identical results, indicating phosphorylation of IL-2R{gamma}c in response to IL-4 (data not shown).



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  		Figure 3. IL-2R{gamma}c is not phosphorylated in response to IL-13. Monocytes were treated with IL-13 (500 pM) for 10 min or left untreated and lysed, and the lysate was immunoprecipitated with (A) a purified rat monoclonal anti-human IL-2R{gamma}c antibody or (B) antibody to phosphotyrosine (PY-99). The immunoprecipitated proteins were collected using protein G beads. The immunoprecipitated proteins were analyzed on a 10% SDS-PAGE gel and blotted with (A) PY-99 or (B) a rabbit polyclonal antibody to human IL-2R{gamma}c. (A) The bottom panel displays the results of reprobing the stripped blot from the upper panel with the anti-IL-2R{gamma}c polyclonal antibody. Arrows indicate the migration position of molecular weight markers that were run in adjacent lanes and the calculated position of IL-2R{gamma}c. The arrowhead marks the migration of the heavy chain of IgG. (C) Similar experiments to those conducted in A and B were also performed, but monocytes were activated with IL-4 (670 pM) instead of IL-13. The results of a Western blot showing the reactivity of anti-PY-99 with IL-2R{gamma}c immunoprecipitates from lysates of IL-4-treated and -untreated monocytes are presented in the upper panel. The lower panel represents a reprobing of the stripped blot with antibody to IL-2R{gamma}c.

 
Association of the receptor components with Jak/Tyk kinases
To evaluate the possible association of Jak/Tyk kinases with IL-4R{alpha} and IL-13R{alpha}1, we immunoprecipitated Jak/Tyk kinases individually, from untreated or IL-13-treated monocyte cell lysates and analyzed the SDS-PAGE blots with antibodies to the receptor components. Results of these experiments are presented in Figures 4 and 5. Antibodies to Jak1 and Jak2 immunoprecipitated IL-4R{alpha}, and Jak3 and Tyk2 did not (Fig. 4A) . The data indicate that the recruitment of Jak1 to IL-4R{alpha} is IL-13-dependent, Jak2 is endogenously associated with the receptor, and association increases upon treatment with IL-13. Reversing the antibodies in immunoprecipitator and Western confirmed the coassociation of Jak2 with IL-4R{alpha} (Fig. 4B) . Reprobing the blots with antibodies corresponding to those used for immunoprecipitation confirmed that equal amounts of specific proteins were immunoprecipitated from the lysates (Fig. 4A and 4B , lower panels).



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  		Figure 4. Jak1 and Jak2 associate with IL-4R{alpha}. Monocytes (20 million cells per group) were treated with IL-13 or left untreated. (A) Both groups were lysed and immunoprecipitated with antibodies specific for Jak1, Jak2, Tyk2, or Jak3. The immunoprecipitated proteins were absorbed to protein A beads, extracted, and analyzed on a 7.5% SDS-PAGE blot and probed with an antibody to IL-4R{alpha}. The arrow indicates the estimated position of the IL-4R{alpha} protein. The bottom panel displays the blot from subsequent reprobing, after stripping, of the individual two-lane strips, using antibodies against corresponding Jak/Tyk kinases. Near-equal immunoprecipitation of proteins was observed. (B) Monocytes were treated as above, but antibody to IL-4R{alpha} was used for immunoprecipitation, and the blot of the SDS-PAGE was then probed with antibody to Jak2. The bottom panel shows the results of reprobing this stripped immunoblot with antibody to IL-4R{alpha} to evaluate equal immunoprecipitation and loading.

 
Similar experiments, where Jak/Tyk kinases were immunoprecipitated from untreated or IL-13-treated monocyte cell lysates and were blotted with antibody to IL-13R{alpha}1, showed that predominantly Tyk2 and not other Jaks distinctly coimmunoprecipitated IL-13R{alpha}1 (Fig. 5A) . Similar findings were demonstrated when the IP and Western antibodies were reversed as shown in Figure 5B . Results indicated that the association between Tyk2 and IL-13R{alpha}1 existed in untreated cells and was modestly enhanced (25–40%, n=3) by IL-13 treatment. Reprobing the same blots with the antibodies used for immunoprecipitation confirmed equal loading of the immunoprecipitated proteins (Fig. 5A and 5B , lower panels).



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  		Figure 5. Tyk2 associates with IL-13R{alpha}1. (A) Monocytes were treated and immunoprecipitated as described in the legend to Figure 4 . The blot was probed with an antibody against the N-terminal peptide sequence of the IL-13R{alpha}1 protein. The arrow indicates the calculated, expected position of IL-13R{alpha}1. The bottom panel shows the results of reprobing, after stripping, the individual strips with corresponding Jak/Tyk antibodies to assess equal immunoprecipitation and loading of proteins in each lane. (B) IL-13R{alpha}1 was immunoprecipitated from monocyte lysates (20 million per group), untreated or treated with IL-13, and analyzed on a 7.5% SDS-PAGE. The blot was probed with antibody to Tyk2, and the arrow indicates the calculated, predicted position of Tyk2. The bottom panel shows the same blot that was stripped and reprobed with IL-13R{alpha}1 antibody to assess equal immunoprecipitation and loading of proteins in each lane.

 
Earlier reports from our lab [11 ] showed that both Jak2 and Tyk2, but not Jak1 and Jak3, were phosphorylated on tyrosine in IL-13-treated human blood monocytes. The association experiments above thus show that although Jak2 associates with IL-4R{alpha}, Tyk2 associates with the other constituent of the IL-13 receptor complex, IL-13R{alpha}1.

Tyrosine phosphorylation and activation of Stats
Because the Stat proteins are early and important substrates of phosphorylated and activated Jak/Tyk kinases, we next studied which of the six known Stats were involved in the IL-13 signaling pathway. Previously, Stat6 has been reported as being phosphorylated on tyrosine in response to IL-13. We conducted experiments to test this in our monocyte culture system and found that Stat6 does indeed become phosphorylated. Data from a representative experiment are shown in Figure 6 where Stat6 was detected in PY-99 immunoprecipitates in IL-13-treated monocyte-nuclear lysates (Fig. 6A) or in whole cell lysates when directly probed with antiphospho-Stat6 antibody (Fig. 6B) . Equal loading of protein is observed in the reprobe of the upper blot in Figure 6B with antibody to Stat6.



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  		Figure 6. IL-13 induces tyrosine phosphorylation of Stat6 protein in monocytes. (A) Monocytes (20 million per group) were untreated or treated with IL-13 (500 pM) or IL-4 (670 pM) for 30 min and were lysed, and the nuclear extracts were immunoprecipitated with the phosphotyrosine antibody PY-99. The immunoprecipitated proteins were collected using protein G beads and were run on 8% SDS-PAGE gel and immunoblotted with Stat6 antibody. Jurkat cell extract was run in an adjacent lane of the same gel as a positive control, and the immunoreactive band migrated just above 97 kDa, identically with the monocyte band (data not shown). (B) Human blood monocytes (5x106) were untreated or treated with IL-13 (500 pM) and IL-4 (670 pM) for 30 min. The cells were lysed, and the whole cell extracts (75 µg/lane) were loaded on an 8% SDS-PAGE gel and immunoblotted with antiphospho Stat6 (Tyr 641) antibody. The bottom panel shows the same blot that was reprobed with Stat6 antibody to evaluate equal loading of proteins.

 
Phosphorylation of other Stat family members in monocytes has remained unexplored. We next conducted experiments to evaluate whether any other Stat proteins were phosphorylated in monocytes as a result of IL-13 stimulation. The remaining Stat proteins (Stats1–5) were individually immunoprecipitated from untreated and IL-13-treated monocyte lysates. The immunoprecipitates were then evaluated in Western blots. The blot was probed with phosphotyrosine antibody (PY-99) to detect IL-13-induced changes in the phosphotyrosine content of the immunoprecipitated Stat proteins. We observed that although Stat1, Stat3, Stat5A, and Stat5B were phosphorylated on tyrosine in response to IL-13, other Stats, Stat2 and Stat4, were not detected by the PY-99 antibody (Fig. 7 A ). In each case, the blots were individually reprobed with corresponding Stat antibodies to ensure adequate and equal immunoprecipitation and loading of each Stat protein. The antibody to Stat1, in Figure 7A , was against the C-terminus of the protein and typically detects mostly Stat1{alpha} (91 kDa).



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  		Figure 7. IL-13 induces tyrosine phosphorylation of Stat proteins in monocytes. (A) Monocytes (30 million per group) were untreated or treated with IL-13 for 15 min, lysed, and immunoprecipitated with antibodies specific for Stat1, Stat2, Stat3, Stat4, Stat5A, and Stat5B. The immunoprecipitated proteins were collected on protein A beads and separated on a 7.5% SDS-PAGE, transferred onto a PVDF membrane, and probed with PY-99. The bottom panel shows the stripped and reprobed individual sections of the membrane blot with corresponding antibodies, demonstrating nearly equal immunoprecipitation and loading of proteins in each lane. (B) Monocytes (20 million per group) were untreated or treated with IL-13 (500 pM) or IL-4 (670 pM) for 30 min and were lysed, and the nuclear extracts were immunoprecipitated with the phosphotyrosine antibody PY-99. The immunoprecipitated proteins were collected using protein G beads. Three different blots were prepared by running the samples on 8% SDS-PAGE gels, and the blots were probed individually with antibodies to Stat1, Stat3, and Stat5. Arrows indicate predicted positions of Stat proteins, based on the migration of molecular weight markers. In the Stat3 blot, lysate from A431 cells was run in an adjacent lane of the same gel as a positive control and as an indicator of the migration of Stat3 (data not shown). (C) Monocyte lysates were prepared from untreated or IL-13-treated cells, and protein concentrations were determined. Lysate protein (50 µg) from treated or untreated monocytes was loaded in adjacent lanes and probed separately using anti-phospho-Stat antibodies against Stat1, Stat3, and Stat5. The positions of the markers loaded in adjacent lanes are indicated.

 
To confirm these data, we also used antiphosphotyrosine (PY-99) to immunoprecipitate tyrosine-phosphorylated proteins from whole cell lysates of untreated and IL-13-treated monocytes followed by blotting with antibody against each of the three Stats previously shown to be phosphorylated, Stat1, Stat3, and Stat5. The results, shown in Figure 7B , confirmed the above studies in that PY-99 immunoprecipitated each of the three Stats found to be phosphorylated in the prior experiments. The Stat1 antibody used in these experiments was prepared against the common N-terminus and is capable of detecting Stat1{alpha} and Stat1ß proteins. It appears that both were phosphorylated on tyrosine in response to IL-13. Stat3 was detected in the IL-13-treated cell lysates, and the Stat5 blot, using an antibody against the common N-terminus, indicated increased levels in the IL-13-treated cell lysates as compared with low but detectable levels in PY-99 immunoprecipitates of unstimulated cell lysates. Additional evidence that Stats2 and 4 are not phosphorylated on tyrosine was derived from a reprobe of the blot shown in Figure 6A . This blot, which was positive for Stat6 phosphorylation, gave no detectable signal when reprobed with antibodies to Stat2 or Stat4.

The results displayed in Figure 7B indicate that IL-4 also induces the phosphorylation of Stats1, 3, and 5 in addition to Stat6. IL-4, in contrast to IL-13, induced lower levels of phosphorylation of Stats1 and 5 and a more robust phosphorylation of Stat3.

The tyrosine phosphorylation status of Stat1, Stat3, and Stat5 was additionally examined using antiphospho-Stat antibodies on Western blots. For detecting phospho-Stats, 50 µg monocyte whole cell lysates from untreated and IL-13-treated monocytes was run on SDS-PAGE, transferred onto a PVDF membrane, blocked with milk, and blotted separately with antiphospho-Stat antibodies raised against Stat1, Stat3, and Stat5. Phosphotyrosine-Stat1 antibody detected Stat1{alpha} and a much weaker, barely detectable band for Stat1ß only in lanes where IL-13-treated cell lysates were loaded. Phosphorylated Stat3 was only detected in lysates from IL-13-treated monocytes, and Stat5A and Stat5B were phosphorylated in the IL-13-treated cells (Fig. 7C) . In the blot shown for Figure 7B , Stat5A and Stat5B were not resolved, but in this blot, Stat5A and 5B were resolved as a result of running the gel for a longer time, and both bands were reactive with the antiphospho-Stat5 antibody. Thus, it appears from results in Figure 7A and 7C , that Stat5A and 5B are phosphorylated. Taken together, our data indicate that multiple but selective Stats are activated in monocytes in response to IL-13, including Stat1{alpha}, 3, 5A, 5B, and 6.

We next conducted experiments to assess IL-13-induced Stat functional activation by examining nuclear translocation and the acquisition of DNA-binding ability. The results of these studies are shown in Figure 8 . Our data indicate that each of the Stats that was phosphorylated in response to exposure to IL-13 was induced to translocate to the nuclear fraction (Fig. 8A) to detectable levels by 15 min. Stat functional activation was also assessed by the IL-13 induction of DNA-binding activity (Fig. 8B) . These data are representative of at least three independent experiments with similar results. In each case, cold competitor oligonucleotides (50-fold) caused nearly complete inhibition of the signal (data not shown). Each of the Stats, previously shown to be phosphorylated in response to IL-13, was shown to acquire DNA-binding activity.



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  		Figure 8. Nuclear translocation and DNA binding activity of Stat proteins are induced in monocytes by IL-13. (A) Human blood monocytes (10 million per group) were untreated or treated for different time intervals with IL-13 (500 pM). The cells were lysed, and the nuclear extracts (10 µg/lane) were loaded on 8% SDS-PAGE gels and probed with antibodies to Stat1, Stat3, Stat5, and Stat6. The arrows indicate the predicted positions of each Stat based on the migration of molecular weight markers loaded in adjacent lanes. (B) Five million monocytes were untreated or treated with 500 pM IL-13 for 30 min. Nuclear proteins were extracted as described previously, and 3 µg nuclear proteins was subjected to EMSA analysis using the 32P-labeled-specific Stat probes. Arrows indicate the positions of Stat protein and DNA complexes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In general, interleukins are believed to exert their effects on target cells through receptors according to the model described by Schindler and Darnell Jr. [43 ]. This model indicates that cytokine ligands induced heterodimerization of two cell surface receptor molecules. The heterodimerized, nontyrosine kinase receptor components are known to recruit specific Jak/Tyk kinases, and these receptor-associated kinases phosphorylate each other and then phosphorylate the receptor molecules. The phosphorylated receptors can then recruit Stat proteins that are phosphorylated and detach from the receptors, form dimers and translocate to the nucleus. The phosphorylated Stats are known to bind specific DNA sequences and thus regulate transcription of certain genes.

Among the receptor components previously identified to contribute to the IL-4/IL-13 cytokine receptor complexes [18 , 36 , 44 , 45 ], our studies show that only IL-4R{alpha} and IL-13R{alpha}1 are phosphorylated on tyrosine in response to IL-13 in human blood monocytes. No phosphorylation of IL-13R{alpha}2 was observed. IL-13, in contrast to IL-4, did not induce the phosphorylation of IL-2R{gamma}c. The lack of IL-2R{gamma}c involvement in the IL-13 response is consistent with previous studies showing involvement of this receptor component in IL-4 responses but not in IL-13 responses of a mast cell line [46 ], B lymphocytes [47 ], or lymphohematopoietic cells [48 ]. This finding might also explain why Jak3 is involved in the IL-4 response in monocytes but is not involved in the IL-13 signaling pathway, as Jak3 is known to associate with IL-2R{gamma}c [49 , 50 ]. IL-4 has also been shown to be able to stimulate certain cells in the absence as well as the presence of IL-2R{gamma}c. In contrast to signaling through Jak3/Stat6, in the absence of IL-2R{gamma}c, IL-4 activation can still proceed but instead involves a Jak1/Stat6 pathway [51 ].

Four models of IL-13 receptor complex composition have been proposed by Murata et al. [44 ] to represent the differing components between and among cell types and cell lines. Although Model I for the IL-13R involves IL-13R{alpha}1 and IL-13R{alpha}2 subunits, Model II includes IL-13R{alpha}1 and IL-4R{alpha}. Both of the other two models, III and IV, contain the IL-2R{gamma}c chain of the IL-2 receptor along with IL-13R{alpha}1 and IL-4R{alpha}. Therefore, we believe that Model II best represents the IL-13 receptor complex in monocytes.

So far, mostly nontyrosine kinase receptors have been reported to transduce signals involving the Jak/Tyk kinases; the epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor tyrosine kinase receptors are exceptions in that they can use Jak kinases for signal transduction [27 ]. Physical association of Jak/Tyk kinases with profoundly different nontyrosine kinase receptors has been shown, and association and phosphorylation of Jak/Tyk kinases on a particular receptor molecule vary between cells. Interactions between Jak kinases and receptor components can be constitutive as well as inducible, as reported in numerous studies including those presented here.

Our studies demonstrate that Jak1 and Jak2 associated with IL-4R{alpha}. Cross-immunoprecipitation studies confirmed this finding. Significantly, although the association of Jak1 to IL-4R{alpha} was induced in response to ligand binding, Jak2 was bound to IL-4R{alpha}, irrespective of the cytokine treatment. Further, we showed that Tyk2, another kinase phosphorylated in response to IL-13, bound to IL-13R{alpha}1 prior to exposure to IL-13, and association was modestly induced in response to the ligand. Reports by others have indicated that upon exposure of T lymphocytes to IL-4, IL-4R{alpha} can bind and phosphorylate Jak1 [52 ]. At present, we cannot explain the IL-13-responsive binding of Jak1 to IL-4R{alpha} and noninvolvement of Jak1 phosphorylation in the IL-13 response. This appears to be another important difference between the IL-4 and IL-13 activation pathways. One might speculate that if only one Jak associates with each molecule of IL-4R{alpha}, then endogenous levels of Jak1, differing in various cell types, may alter the downstream signaling by IL-13. Further evidence that Jak1 is not involved in the IL-13 response of human monocyte, at least as it relates to 15-LO expression, is derived from our recently published studies showing that inhibition of Jak1 expression did not interfere with IL-13-mediated 15-LO expression [11 ]. Others have reported that IL-4 and IL-13 induce Jak2 phosphorylation [21 , 53 ] and that Jak2 is constitutively associated with IL-4R{alpha} in human colon carcinoma cells [21 ]. This finding is consistent with the IL-13 signaling in monocytes as reported here. Similarly, IL-13 induction of Tyk2 phosphorylation was also observed in human colon carcinoma cells, similar to our results in human monocytes [11 , 21 ]. Thus, some aspects of Jak/Tyk association and phosphorylation are similar, and some are different than observations in other cell types. One common finding from numerous labs in a variety of cell types, and consistent with our findings, is that Jak3 is not phosphorylated in response to IL-13 [21 , 48 , 54 55 56 ].

Stat proteins are activated in response to the binding of a number of cytokines and growth factors to their specific receptors. The Stats are activated by phosphorylation of one particular tyrosine residue located in the C-terminus [27 ]. Tyrosine phosphorylation allows Stat proteins to dimerize via binding of the SH2 domain of one Stat molecule to the phosphotyrosine of another activated Stat molecule. The dimerized Stat complex is transported to the nucleus where it binds DNA and thus regulates transcription of target genes [57 ]. Our observations indicate that Stat1{alpha}, Stat3, Stat5A, Stat5B, and Stat6 are tyrosine-phosphorylated in IL-13-treated primary human monocytes. A recent report has also observed the phosphorylation of Stats1 and 3 in response to IL-13 in human normal and tumor lung fibroblasts [58 ]. In normal B cells, studies by Izuhara et al. [47 ] have shown that IL-13-induced phosphorylation of Stat6, in contrast to that induced by IL-4, is independent of Jak3 and IL-2R{gamma}c. This finding is consistent with the results presented here with primary human monocytes.

The serine phosphorylation of Stats is known to be essential for optimal promotion of transcription but not for dimer formation or nuclear translocation of the Stat complex [59 , 60 ]. Recent reports suggest that there is serine phosphorylation of Stats, Stat1{alpha} (Ser-727), Stat3 (Ser-727), Stat5A (Ser-725), and Stat5B (Ser-730), although predictably, this phosphorylation is mediated by different kinases [61 , 62 ]. We have recently shown that Stat1 and Stat3 are phosphoryated on Serine 727 in response to IL-13 (B. Roy, A. Bhattacharjee, B. Xu, and M. K. Cathcart, manuscript in preparation). Further studies are required to understand exactly how tyrosine and serine-phosphorylated Stat1 and Stat3 participate in the IL-13-mediated regulation of gene transcription.

The formation of homodimers and heterodimers between tyrosine-phosphorylated Stats as well as serine phosphorylation appears to be an important mechanism for providing specificity in gene induction. These additional, regulatory events potentially enable a limited group of transcription factors to orchestrate cell type-specific or cell stage-specific effects observed in response to many cytokines and growth factors. Recently, in Stat6-transfected, transformed epithelial cells, Stat-6 has been reported to regulate the IL-4-mediated induction of 15-LO [63 ]. Prior studies also suggested Stat6 regulation of IL-4-induced 15-LO enzymatic activity in murine macrophages [64 ]. Our studies indicate Stat6 is not the only Stat with potential to mediate the IL-13 induction of 15-LO expression in primary human monocytes. Further, our results suggest quantitatively differential Stat activation induced by IL-4 and IL-13. This may explain the differing cellular responses to these cytokines.

In summary, we report here the immediate, early components of the IL-13 signaling pathway in human monocytes. Our results, schematically presented in Figure 9 , indicate that IL-13 signals through IL-13R{alpha}1 and IL-4R{alpha}, two receptor components characterized earlier in different cells types. Both of these receptor components were phosphorylated upon exposure of monocytes to IL-13. Each was associated with members of the Jak/Tyk kinase family, which are known to be phosphorylated, activated, and linked to expression of 15-LO expression in response to IL-13 in these cells; i.e., Jak2 and Tyk2 were shown to be associated with IL-4R{alpha} and IL-13R{alpha}1, respectively. Jak1 also associated with IL-4R{alpha} in response to IL-13 exposure, but Jak1 was not phosphorylated. We have therefore depicted Jak1 or Jak2 interacting with this component of the IL-13 receptor. Tanner et al. have reported that the peptide sequence of PXXPXP is an absolutely required receptor sequence for binding to Jaks [65 ]. It is interesting to note that IL-4R{alpha} has two of these sequences, 42 amino acids apart. It is therefore theoretically possible that IL-4R{alpha} could bind more than one Jak; however, there are no data to support this. Our studies have also revealed the selective tyrosine phosphorylation and activation of Stat proteins 1{alpha}, 3, 5A, 5B, and 6 in response to IL-13 in this system. The identification of the IL-13-induced signal transduction cascade in monocytes suggests novel regulation of IL-13 responses in these cells. Differences between IL-4 and IL-13 signal transduction in monocytes were identified, as well as differences between IL-13 signal transduction in monocytes as compared with other cell types. Further studies are needed to evaluate the roles of these pathway components in regulating gene expression in response to IL-13. Identification of the IL-13-induced signaling pathways may provide novel targets for therapeutic intervention in the IL-13-driven, inflammatory processes that appear to continue to be of critical importance in allergic asthma and atherogenesis.



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  		Figure 9. Model of the predicted IL-13 receptor complex and upstream signal-transducing components.

 


   ACKNOWLEDGEMENTS
 
B. R. and A. B. contributed equally to this work and are both considered first authors. This work was supported by funding to M. K. C. from NIH-HL51068.

Received March 6, 2002; revised April 8, 2002; accepted May 1, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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