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[Cancer Research 64, 3271-3275, May 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Soluble Interleukin-13R{alpha}2 Decoy Receptor Inhibits Hodgkin’s Lymphoma Growth in Vitro and in Vivo

Young Trieu1, Xiao-Yan Wen1, Brian F. Skinnider2, Mark R. Bray2, Zhihua Li1, Jaime O. Claudio1, Esther Masih-Khan1, Yuan-Xiao Zhu1, Suzanne Trudel1, J. Andrea McCart1, Tak W. Mak2 and A. Keith Stewart1

1 Division of Experimental Therapeutics, Toronto General Research Institute, McLaughlin Centre for Molecular Medicine, and 2 Advanced Medical Discovery Institute, Departments of Immunology and Medical Biophysics, University Health Network, University of Toronto, Toronto, Ontario, Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have demonstrated that the malignant Reed-Sternberg cells of Hodgkin’s lymphoma (HL) secrete and are responsive to interleukin (IL)-13. We hypothesized that overexpression of a soluble IL-13 decoy receptor (sIL-13R{alpha}2) via adenoviral-mediated gene transfer would inhibit IL-13-induced Reed-Sternberg cell proliferation. Western blot and ELISA analysis verified expression of sIL-13R{alpha}2 in cell lysates and supernatants of AdsIL-13R{alpha}2-transduced COS-7 cells. Treatment of two IL-13-responsive HL-derived cell lines, HDLM-2 and L-1236, with AdsIL-13R{alpha}2-conditioned medium, resulted in the inhibition of cell proliferation, and down-regulated the phosphorylation of signal transducer and activator of transcription 6 (STAT6), an important mediator of IL-13 signaling. i.v. delivery of AdsIL-13R{alpha}2 in NOD/SCID mice with s.c. implanted HDLM-2 cells delayed tumor onset and growth while enhancing survival compared with control mice. Intratumoral administration of AdsIL-13R{alpha}2 led to the regression or stabilization of established tumors and was associated with diminished STAT6 phosphorylation. Our data demonstrate that AdsIL-13R{alpha}2 can suppress HL growth in vitro and in vivo.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hodgkin’s lymphoma (HL) is a lymphoproliferative disorder characterized by the unusually rare malignant Reed-Sternberg (RS) cells surrounded by a background of reactive infiltrates (1) . The unique histology and eosinophilia in this malignancy, as well as the constitutional symptoms observed in patients, such as fever, weight loss, and night sweats, are thought to be due to an abnormal pattern of cytokine secretion (2) . Indeed, cDNA microarray analysis revealed that interleukin (IL)-13 expression was up-regulated in HL-derived cell lines (3) . In situ hybridization demonstrated the coexpression of IL-13 and the IL-13-specific receptor chain, IL-13R{alpha}1, in HL cell lines as well as in primary HL tumors. Furthermore, antibody-mediated IL-13 neutralization resulted in a dose-dependent inhibition of proliferation of two RS cell lines (3, 4, 5) . Together, these results suggest that RS cells not only secrete IL-13 but also use the cytokine as a growth factor, possibly by an autocrine or paracrine mechanism (3, 4, 5, 6, 7) . As such, modulation of the IL-13 signaling pathway is a logical therapeutic target for HL.

IL-13 is an immunoregulatory cytokine secreted predominantly by activated T cells that exerts its effects mainly on B cells and monocytes (8 , 9) . In B cells, IL-13 enhances survival and proliferation, and stimulates immunoglobulin heavy chain class switching to IgE and IgG4. IL-13 is a key mediator of allergic inflammation and has been implicated in the pathogenesis of asthma (10, 11, 12, 13) . IL-13 and IL-4 have similar biological properties, which are due in part to the sharing of a receptor complex. Two distinct chains of the IL-13 receptor (IL-13R) have been cloned, IL-13R{alpha}1 and IL-13R{alpha}2 (14, 15, 16) . Binding studies show that IL-13R{alpha}1 binds IL-13 weakly on its own. However, when coupled with the IL-4R{alpha} chain the resulting heterodimer complex binds IL-13 with a greater affinity, and mediates signaling via the Janus-activated kinase-signal transducers and activators of transcription (STAT) pathway (17 , 18) . In contrast, IL-13R{alpha}2 can bind IL-13 avidly on its own, but does not appear to signal. One possible explanation for this observation is that unlike IL-13R{alpha}1, the intracellular domain of IL-13R{alpha}2 is shorter and is devoid of an obvious signaling motif or the Janus-activated kinase-STAT binding sequence (19) . This has led to speculation that IL-13R{alpha}2 is a dominant-negative inhibitor or decoy receptor (20, 21, 22) . This decoy characteristic is supported by the findings that IL-13R{alpha}2 can mediate the internalization of IL-13 independent of IL-13 signaling (23) . Furthermore, this receptor has been found as a secretable form in mouse serum and urine (24) . Interestingly, a soluble IL-13R{alpha}2-Fc (sIL-13R{alpha}2-Fc) fusion protein has been engineered to specifically block the detrimental effects induced by IL-13, including asthma (11 , 12) and Schistosoma mansoni-induced hepatic fibrosis (25) . Wood et al. (26) and Chiaramonte et al. (27) recently generated IL-13R{alpha}2-deficient mice and showed that IL-13R{alpha}2 serves to limit IL-13-mediated responses in vivo. Therefore, we have generated a recombinant adenovirus that expresses a soluble IL-13 decoy receptor (sIL-13R{alpha}2) to target Hodgkin tumor cells and have examined the biological consequences of sIL-13R{alpha}2-mediated blockade of IL-13 in vitro and in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Recombinant AdsIL-13R{alpha}2.
Recombinant AdsIL-13R{alpha}2 was generated using the Admax Cre/loxP system. Detailed methods for the construction and propagation of human adenovirus vectors were described by Hitt et al. (28) . Briefly, the sIL-13R{alpha}2 gene (provided by M. R. B.) that was engineered with a stop codon at amino acid position 346 to encode the extracellular domain of the full-length human IL-13R{alpha}2 was cloned into the HindIII and SacI sites of the pDC316 shuttle plasmid by blunt end ligation. Twenty µg of the resulting pDC316-sIL-13R{alpha}2 vector was mixed with 20 µg of the pBHGlox{Delta}E1,3Cre adenoviral vector and cotransfected into 293Cre4 cells by calcium phosphate precipitation. The plaque formed from the rescued virus was picked, amplified in 293Cre4 cells, and purified by cesium chloride gradient ultracentrifugation. The collected virus band was dialyzed against three changes of 10 mM Tris-HCl (pH 8.0) and stored at –70°C in small aliquots containing 10% glycerol.

Cell Lines and Reagents.
All of the cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. 293Cre4 cells were grown in complete MEM containing 2 mM glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 µg/ml Fungizone plus 10% FCS. COS-7 cells were grown in DMEM containing Fungizone and 10% FCS. HL-derived cell lines, HDLM-2 and L-1236, and the lymphoblastoid cell line LCL-GK were grown in RPMI 1640 supplemented with Fungizone and 10% FCS.

The monoclonal mouse-antihuman IL-13R{alpha}2 and polyclonal goat-antihuman IL-13R{alpha}2 antibodies, and recombinant human IL-13R{alpha}2-Fc protein were all purchased from R&D Systems (Minneapolis, MN). Phospho(Tyr641)-STAT6 antibody (Cell Signaling Technology, Beverly, MA) was used for Western blot and immunohistochemical analyses at 1:1000 and 1:300 dilutions, respectively. The monoclonal STAT6 antibody (BD Transduction Laboratories, Lexington, KY) was used to detect total STAT6.

Immunoblotting.
COS-7 cells were transduced with AdsIL-13R{alpha}2 or Addl70.3 at a multiplicity of infection (moi) of 100. Whole cell lysates and conditioned media were collected at 72 h after infection. The conditioned media were concentrated to about one-fiftieth of its original volume by centrifugal filtration through the Mr 10,000 cutoff membrane (Millipore, Bedford, MA). Western blot analysis was performed using the mouse-antihuman IL-13R{alpha}2 antibody (R&D) to detect expression of sIL-13R{alpha}2 in cell lysates. Protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) conjugated to the goat-antihuman IL-13R{alpha}2 antibody (R&D) was used to immunoprecipitate sIL-13R{alpha}2 in conditioned media. The mouse-antihuman IL-13R{alpha}2 antibody (R&D) was then applied to probe for sIL-13R{alpha}2 by Western.

ELISA.
An ELISA kit for the quantitative determination of sIL-13R{alpha}2 in mouse serum or in conditioned media was developed in conjunction with the YES-Biotech Laboratories Ltd. (ANOGEN, Toronto, Ontario, Canada). Quantification of sIL-13R{alpha}2 expression was done according to the manufacturer’s instructions. Briefly, the mouse-antihuman IL-13R{alpha}2 antibody (R&D) was precoated onto a microplate. Standards and samples were then added to the wells with biotin-conjugated goat-antihuman IL-13R{alpha}2 antibody (R&D). Recombinant human IL-13R{alpha}2-Fc protein (R&D) was used as standard controls. The sensitivity of sIL-13R{alpha}2 detection was 5 ng/ml.

Immunohistochemistry.
Phosphorylated STAT6 was detected in formalin-fixed, paraffin-embedded tumor tissue sections using standard immunohistochemical techniques. Briefly, paraffin sections were dewaxed and microwave-heated in 10 mM of sodium citrate buffer (pH 6.0) and then incubated with antiphosphorylated STAT6 antibody overnight at room temperature. Staining was done with biotinylated goat-antirabbit IgG (Vector Labs, Burlingame, CA) and horseradish peroxidase-conjugated Ultra Streptavidin (Signet Labs, Dedham, MA). Color development was done with NovaRed solution (Vector Labs).

Analyses of Cell Proliferation and Apoptosis.
HDLM-2, L-1236, and LCL-GK cells were cultured in 96-well flat-bottomed plates in triplicate at 3 x 104 cells/well in the presence of various dilutions of AdsIL-13R{alpha}2 or Addl70.3 conditioned media for 72 h. Cell proliferation was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s protocol. To confirm the infectivity of adenovirus on HDLM-2, L-1236, and LCK-GK cells, an Ad-yellow fluorescent protein (29) vector was used to infect cells at a moi of 100. At 72 h after infection, the cells were analyzed for expression of yellow fluorescent protein by flow cytometry on a FACScan (BD Biosciences, San Jose, CA) using the CellQuest software following the manufacturer’s instructions. In a separate study the cell lines were infected with either AdsIL-13R{alpha}2 or Addl70.3 at a moi of 100 for 2 h, washed, and then seeded at the same density as before. Cell growth and apoptosis at 72 h after infection were evaluated by MTT and Annexin V assays, respectively. Apoptosis was determined by fluorescence-activated cell sorter analysis using the TACS Annexin V-FITC apoptosis detection kit (R&D) according to the manufacturer’s instructions. All of the experiments were done in triplicate.

Animal Studies.
Six to 8-week-old female NOD/SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). For Addl70.3 and AdsIL-13R{alpha}2 dose-finding studies, three different viral doses of 1.2 x 108, 6 x 108, or 3 x 109 plaque-forming units (pfu) were i.v. delivered via the tail vein. Serum samples from mice receiving a dose of 6 x 108 pfu were collected on days 2, 7, 14, 21, and 28, and used to quantify for sIL-13R{alpha}2 expression by ELISA. To generate a HL tumor model, 1 x 107 HDLM-2 cells in 0.2 ml of PBS were s.c. injected into the flanks of mice. Two days after tumor challenge, 6 x 108 pfu of AdsIL-13R{alpha}2 or Addl70.3 control vector was i.v. injected. A second i.v. injection of the viruses was given 3 weeks after the first viral dose. In a separate study, 1 x 109 pfu of either AdsIL-13R{alpha}2 or Addl70.3 was injected intratumorally in mice with established tumor volumes of approximately 65–70 mm3, and these mice were subsequently followed for tumor size. In a parallel study, tumor samples were excised from mice sacrificed 3 days after treatment, and formalin-fixed, paraffin-embedded tissue sections were prepared for immunohistochemistry. Caliper measurements of the longest perpendicular tumor diameters were done twice weekly to estimate tumor volume using the formula: length x width2 x 0.5. Mice were sacrificed when their tumors reached 15 mm or became ulcerated. The statistical significance of tumor regression was calculated by Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of sIL-13R{alpha}2 in Cell Lysates and Conditioned Media of AdsIL-13R{alpha}2-Infected Cells.
We have generated a recombinant adenovirus that expresses the soluble IL-13 decoy receptor, AdsIL-13R{alpha}2. To confirm expression of sIL-13R{alpha}2 at the protein level, COS-7 cells were transduced with either AdsIL-13R{alpha}2 or Addl70.3 at a moi of 100. Three days after viral infection, cell lysates and conditioned media were prepared from the transduced cells, and expression of sIL-13R{alpha}2 was verified by Western blot analysis (Fig. 1)Citation . ELISA analysis was used to quantify the amount of sIL-13R{alpha}2 in unconcentrated and concentrated conditioned media of AdsIL-13R{alpha}2 infected COS-7 cells, which measured 1.5 and 85 µg/ml, respectively.



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  		Fig. 1. Expression of soluble interleukin (sIL)-13R{alpha}2 in cell lysates and conditioned media of AdsIL-13R{alpha}2 transduced COS-7 cells. Recombinant AdsIL-13R{alpha}2 or Addl70.3 control virus was used to infect COS-7 cells at a multiplicity of infection of 100. Whole cell lysates and conditioned media were collected at 72 h after infection. Cell lysates and conditioned media were then subjected to SDS-PAGE, followed by Western blot analysis.

 
sIL-13R{alpha}2 Inhibits Proliferation and Down-Regulates STAT6 Phosphorylation in HL-Derived Cell Lines.
To determine the biological activity of the expressed soluble decoy receptor in culture, an MTT assay was used to evaluate the proliferation status of two IL-13-responsive HL-derived cell lines, HDLM-2 and L-1236, and the control lymphoblastoid LCL-GK treated with sIL-13R{alpha}2. The cell lines were cultured with various dilutions of conditioned media derived from either Addl70.3 or AdsIL-13R{alpha}2-infected cells. Treatment of the three cell lines with Addl70.3 conditioned media did not affect their rate of cell growth (Fig. 2A)Citation . Conversely, AdsIL-13R{alpha}2 supernatants inhibited the proliferation of HDLM-2 and L-1236 in a dose-dependent manner, whereas the LCL-GK cell line was unaffected by this treatment (Fig. 2B)Citation . Maximal inhibition of cell growth in both HL cell lines was observed at a 1:4-fold dilution that corresponds to a concentration of ~22.5 µg/ml of sIL-13R{alpha}2.



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  		Fig. 2. Adenovirus-derived soluble interleukin (sIL)-13R{alpha}2 inhibits proliferation of cultured HL-derived cells. The IL-13 responsive HL cell lines, HDLM-2 and L-1236, and control LCL-GK cells were cultured in the presence of 1:8 and 1:4 fold dilutions of Addl70.3 (A) or AdsIL-13R{alpha}2 (B) conditioned media for 72 h. AdsIL-13R{alpha}2 conditioned media dilutions of 1:8 and 1:4 correspond to 11 and 22.5 µg/ml of sIL-13R{alpha}2, respectively. Cell proliferation was assessed by measuring MTT dye absorbance cells. Absorbance (O.D.) values are normalized relative to the growth of untreated controls at 72 h. Values are the mean from experiments done in triplicate; bars, ±SD.

 
Previous studies have demonstrated that the transcription factor STAT6 acts as an important mediator of IL-13 signaling, and it has been shown to be constitutively phosphorylated in HL-derived cell lines as well as in RS cells of primary HL tumors due to the autocrine secretion of IL-13 (5 , 30) . Therefore, we examined whether sIL-13R{alpha}2-mediated blockade of IL-13 could down-regulate expression of phosphorylated STAT6 in HL cell lines. HDLM-2 and L-1236 cells were cultured in medium alone (untreated) or treated with conditioned medium derived from AdsIL-13R{alpha}2 or Addl70.3 control virus for 20 h, and expression of phosphorylated STAT6 was then analyzed by immunoblotting. Compared with controls, conditioned medium containing sIL-13R{alpha}2 dramatically decreased the basal levels of STAT6 phosphorylation in both cell lines (Fig. 3)Citation . Taken together, our data additionally support a possible connection between IL-13 and the activation of its downstream signaling target STAT6 with the proliferation of HL-derived cells.



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  		Fig. 3. Down-regulation of constitutive signal transducer and activator of transcription 6 (STAT6) phosphorylation in HL cell lines after treatment with soluble interleukin (sIL)-13R{alpha}2. HDLM-2 and L-1236 cells were cultured for 20 h in medium alone (untreated), in the presence of AdsIL-13R{alpha}2 or Addl70.3 conditioned media. Equal amounts of whole cell lysates were then subjected to SDS-PAGE, followed by Western blot analysis with antiphosho (Tyr641)-STAT6 antibody (top row). The membrane was stripped and reblotted with an antibody against total STAT6 to confirm equal loading (bottom row).

 
AdsIL-13R{alpha}2 Infection of RS Cells Decreases Proliferation and Enhances Apoptosis.
To confirm the infectivity of RS cells by adenovirus, HDLM-2, L-1236, and LCL-GK cell lines were infected with an Ad-yellow fluorescent protein (29) vector at a moi of 100, which resulted in 67%, 31%, and 41% of the cells expressing the yellow fluorescent protein by flow cytometry analysis, respectively (data not shown). The same panel of cell lines was then transduced with AdsIL-13R{alpha}2 or Addl70.3 control vector at a moi of 100. Cell proliferation and apoptosis were assessed at 72 h after viral infection by MTT and annexin V assays, respectively. By MTT, a decrease in proliferation (as measured by the absorbance ratio) in HDLM-2 (to 62% of control) and L-1236 (to 76% of control) was determined (Fig. 4A)Citation . Compared with cells infected with Addl70.3 versus AdsIL-13R{alpha}2, a corresponding increase in the proportion of annexin V-positive cells from 12% ± 2% to 28 ± 2.5% (P < 0.05) in HDLM-2 and from 17% ± 2% to 24% ± 2% (P < 0.05) in L-1236 cells was observed (Fig. 4B)Citation . In contrast, the rate of cell proliferation and cell death in the control LCL-GK cell line remained virtually unchanged.



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  		Fig. 4. AdsIL-13R{alpha}2 infection decreases proliferation and enhances apoptosis in HL cell lines. HDLM-2, L-1236, and control LCL-GK cell lines were infected with either AdsIL-13R{alpha}2 or Addl70.3 control vector at a multiplicity of infection of 100. Analyses of cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (A) and apoptosis by Annexin V staining (B) were performed at 72 h postinfection. In B, a representative plot of one experiment is shown.

 
Systemic Delivery of AdsIL-13R{alpha}2 Delays Tumor Growth and Enhances Survival.
As a prelude to antitumor efficacy studies, we conducted a dose-finding study using the Addl70.3 control vector and AdsIL-13R{alpha}2 by systemic delivery of the viruses in NOD/SCID mice. At 3 x 109 pfu both vectors were toxic, as mice died within 1–3 days of injection. However, mice were able to tolerate a dose of 6 x 108 pfu of either vector, and subsequently serum samples were collected on multiple days and used to quantify for expression of sIL-13R{alpha}2 by ELISA. Whereas serum levels of sIL-13R{alpha}2 were not detectable in control mice, mice receiving AdsIL-13R{alpha}2 could be detected for expression of the soluble receptor, which peaked at 7.7 µg/ml on day 2, declined sharply to 0.15 µg/ml on day 21, and was no longer detectable by day 28 (data not shown).

To assess the efficacy of AdsIL-13R{alpha}2, NOD/SCID mice were s.c. implanted with the HL-derived cell line, HDLM-2. Two days after tumor challenge, 6 x 108 pfu of AdsIL-13R{alpha}2 (n = 6) or the Addl70.3 (n = 7) control virus was systemically delivered via tail vein injections. A third group of tumor challenged mice was left untreated (n = 6) and served as an additional control. Compared with the untreated and Addl70.3 group, mice in the AdsIL-13R{alpha}2 group demonstrated a delay in tumor onset, and as a result developed significantly smaller tumors (Fig. 5A)Citation . The delay in tumor progression correlated with serum levels of sIL-13R{alpha}2, which peaked at 11.3 µg/ml at 2 days after viral administration, and decreased sharply by the end of the third week (Fig. 5B)Citation . At 23 days after tumor challenge, the mean tumor volume from two independent experiments in AdsIL-13R{alpha}2-treated mice was 48 ± 33 mm3 versus 131 ± 35 mm3 in the control group (P < 0.05).



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  		Fig. 5. i.v. delivery of AsIL-13R{alpha}2 delays tumor growth and enhances survival. NOD/SCID mice were implanted s.c. with HDLM-2 cells on day 0. Two days after tumor challenge, 6 x 108 plaque forming units of either AdsIL-13R{alpha}2 (n = 6) or Addl70.3 (n = 7) control virus was given via tail vein injections. A second injection of the viruses at the same viral dose was delivered 3 weeks after the first viral administration. A third group of tumor challenged mice was left untreated (n = 6) and served as an additional control over the same study period. A, tumor volume (mean mm3; bars, ±SD) plotted against days after tumor challenge. B, serum levels of soluble interleukin (sIL)-13R{alpha}2 were quantified by ELISA after each administration of AdsIL-13R{alpha}2. C, a survival curve was plotted based on mice sacrificed when their tumors reached 15 mm or became ulcerated. Arrows indicate the day of injections.

 
In an attempt to prolong and elevate the expression of serum sIL-13R{alpha}2 in AdsIL-13R{alpha}2-treated mice, a second i.v. injection of the virus was delivered 3 weeks after the first viral dose. A repeat injection of AdsIL-13R{alpha}2 restored the serum levels of sIL-13R{alpha}2; however, the peak expression of the soluble decoy receptor was significantly lower than that of the first viral delivery (Fig. 5B)Citation . Expression of sIL-13R{alpha}2 in mice persisted for an additional 2 weeks and resulted in a parallel delay in tumor progression (Fig. 5A)Citation . Consistent with the tumor growth rate, an improvement in survival was observed in mice treated with AdsIL-13R{alpha}2. The median survival in the control group was 45 days but had not been reached by day 68 in the treatment group (Fig. 5C)Citation .

Intratumoral Delivery of AdsIL-13R{alpha}2 Demonstrates Antitumor Efficacy and Inhibits STAT6 Phosphorylation.
We next investigated whether local delivery of AdsIL-13R{alpha}2 can suppress or decrease the rate of tumor progression in mice with established tumors. Tumor-challenged mice were allowed to reach a tumor volume of approximately 65–70 mm3. These mice were then randomly divided into two groups and were administered intratumorally with 1 x 109 pfu of either AdsIL-13R{alpha}2 (n = 7) or Addl70.3 (n = 6). Within 5 days of viral injection, 6 of the 7 mice from the AdsIL-13R{alpha}2 group exhibited tumor regression or stabilization, whereas tumor progression in mice that received the Addl70.3 control vector continued at the expected rate (P < 0.05; Fig. 6ACitation ).



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  		Fig. 6. Regression of established Hodgkin’s lymphoma tumors with decreased signal transducer and activator of transcription 6 phosphorylation after intratumoral delivery of AdsIL-13R{alpha}2. NOD/SCID mice with established s.c. tumor volumes of approximately 65–70 mm3 were injected intratumorally (indicated by arrow) with 1 x 109 plaque-forming units of either AdsIL-13R{alpha}2 (n = 7) or Addl70.3 (n = 6) control virus. A, tumor volume (mean mm3; bars, ±SD) plotted against days after viral delivery. B, expression of phosphorylated-signal transducer and activator of transcription 6 was examined by immunohistochemistry in formalin-fixed, paraffin-embedded sections prepared from untreated, Addl70.3 treated, and AdsIL-13R{alpha}2 treated tumors at 3 days after viral administration. Original magnifications, x400.

 
In a parallel study, mice with established tumors were also given an equivalent dose of either AdsIL-13R{alpha}2 or Addl70.3 intratumorally. These mice were sacrificed 3 days after treatment, and tumor samples were removed to examine for expression of phosphorylated STAT6 by immunohistochemical analysis. Compared with untreated and Addl70.3-treated tumors, a decrease in the phosphorylation and nuclear localization of STAT6 was observed in tumors treated with AdsIL-13R{alpha}2 (Fig. 6B)Citation . Together, these results additionally implicate a role for IL-13 and STAT6 in mediating their effects on RS cell proliferation in vitro and in vivo.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-13 was postulated recently to be an autocrine growth factor for the neoplastic RS cells of HL (3, 4, 5, 6, 7) . Accordingly, modulation of the IL-13 signaling pathway may serve as a potential pharmacological target in HL. To validate this target we have used a gene therapy strategy using a recombinant adenovirus, AdsIL-13R{alpha}2, that expresses sIL-13R{alpha}2. Conditioned medium containing sIL-13R{alpha}2 inhibited proliferation in two IL-13-responsive HL-derived cell lines, HDLM-2 and L-1236, but had no effect on the control lymphoblastoid LCL-GK cell line. Furthermore, direct infection of HDLM-2 and L-1236 with AdsIL-13R{alpha}2 led to a decrease in cell growth and a corresponding increase in apoptosis in both cell lines.

STAT6 has been shown to be a key mediator of IL-13 signaling (30 , 31) . More importantly, Skinnider et al. (5) demonstrated that constitutive STAT6 phosphorylation in HL cell lines and in RS cells of primary tumors was associated with the autocrine secretion of IL-13. In our studies, treatment of HDLM-2 and L-1236 cells with sIL-13R{alpha}2 resulted in a decrease in the phosphorylation of STAT6 in both cell lines. These results are in keeping with data presented by Skinnider et al. (5) and others (3 , 6 , 7) , and add credence to the proposed IL-13-induced autocrine growth mechanism of RS cells. Significantly, our data provide a strong correlation among sIL-13R{alpha}2-mediated blockade of IL-13, decreased STAT6 phosphorylation, and inhibition of HL cell proliferation.

To examine whether sIL-13R{alpha}2-mediated IL-13 neutralization had similar effects in vivo we assessed the efficacy of AdsIL-13R{alpha}2 in a HL xenograft model. Consistent with our in vitro data, systemic delivery of AdsIL-13R{alpha}2 in tumor-challenged mice delayed tumor progression and growth as compared with untreated or Addl70.3-treated mice. Moreover, an overall survival advantage was observed in mice receiving AdsIL-13R{alpha}2. Interestingly, the delayed tumor onset correlated with serum levels of sIL-13R{alpha}2, which persisted for ~3 weeks after viral delivery. A repeat injection of AdsIL-13R{alpha}2 extended the duration of serum sIL-13R{alpha}2 expression and additionally impeded the rate of tumor growth. In a separate study, intratumoral delivery of AdsIL-13R{alpha}2 or Addl70.3 was given to mice with established tumors. Within 5 days of treatment, mice from the AdsIL-13R{alpha}2 group exhibited tumor regression or stabilization. Immunohistochemical examination of AdsIL-13R{alpha}2-treated tumors revealed a reduction in the phosphorylation and nuclear localization of STAT6.

In addition to its proliferative role in HL, IL-13 has been shown to be a critical mediator for the exaggeration of asthmatic symptoms that is independent of IL-4 (11 , 12) . Indeed, targeting IL-13 with a soluble IL-13R{alpha}2-Fc fusion protein has demonstrated remarkable efficacy in a mouse asthma model (11 , 12) . However, this approach requires repeated injections of the therapeutic protein. Furthermore, the protein needs to be expressed appropriately and be highly purified, a process that may result in the loss of biological activity of soluble IL-13R{alpha}2. For this reason, a gene therapy strategy could offer an alternative approach. Zavorotinskaya et al. (32) recently generated a recombinant adeno-associated viral vector that expresses the IL-4 receptor antagonist to target airway hyper-responsiveness induced by IL-13 and showed sustained protein expression in vivo. In the present study, systemic delivery of AdsIL-13R{alpha}2 resulted in transient serum expression of sIL-13R{alpha}2. Compared with a first-generation adenovirus, an adeno-associated viral or a helper-dependent adenoviral vector is theoretically superior due to a reduction in host immune response against the vector, and, thus, allowing for prolonged and stable transgene expression (33, 34, 35, 36) . As it may prove to be more clinically relevant if our adenoviral vector were to be modified into an adeno-associated viral or helper-dependent adenoviral system, future gene therapy strategies against HL will involve the development of an adeno-associated viral or helper-dependent adenoviral vector expressing sIL-13R{alpha}2. Several other factors will affect whether this will be a clinically relevant treatment for patients with HL. Although only 10% of newly diagnosed HL patients have detectable serum IL-13 levels (37) , the remaining patients may have locally elevated IL-13 levels in their tumors. Serum and tumor levels of naturally occurring soluble IL-13R{alpha}2 may also play a role in the effectiveness of such treatment. Our experiments show that tumor growth is not completely inhibited when treated with AdsIL-13R{alpha}2, suggesting that there is a population of cells that may be IL-13-independent. Future studies will address the ability of Hodgkin cells to develop inherent and acquired resistance to IL-13 blockade. Whereas this may be the case in patients, such a treatment would be given in conjunction with other therapeutic agents, including conventional chemotherapeutic drugs or radiation.

In conclusion, we demonstrate the proof of principle that a recombinant adenovirus expressing the soluble IL-13 decoy receptor, AdsIL-13R{alpha}2, can inhibit IL-13-induced proliferation of RS cells in vitro and in vivo. Our data additionally reinforce the notion that blocking the IL-13 autocrine loop offers a paradigm that can be used to treat Hodgkin’s lymphoma.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: A. Keith Stewart, McLaughlin Centre for Molecular Medicine, 620 University Avenue, Suite 8–202, Toronto, Ontario, Canada M5G 2C1. Phone: (416) 946-4566; Fax: (416) 946-2087; E-mail: kstewart{at}uhnres.utoronto.ca

Received 12/ 4/03. Revised 1/28/04. Accepted 2/19/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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