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NEOPLASIA
From the Amgen Institute and Department of Oncologic
Pathology, Ontario Cancer Institute, Toronto, Canada; the Departments
of Medical Biophysics and Immunology, University of Toronto, Canada;
the Department of Pathology, British Columbia Cancer Agency, Vancouver,
Canada; the Department of Hematology/Oncology, Georg August University,
Göttingen, Germany; and the Department of Hematology/Oncology,
University Medical Center, Freiburg, Germany.
The unique clinicopathologic features of Hodgkin lymphoma (HL) are
due to the multiple cytokines produced by its neoplastic cells, the
Hodgkin and Reed-Sternberg (HRS) cells. Cytokine signaling is mediated
through the signal transducer and activator of transcription (STAT)
family of transcription factors. Immunoblotting and
immunohistochemistry were used to examine cell lines and tissue
sections derived from patients with HL and non-Hodgkin lymphoma (NHL)
for expression of activated STAT proteins. Constitutive phosphorylation
of STAT6 and STAT3 was common in HL. STAT6 was constitutively
phosphorylated in 5 of 5 HL cell lines and in HRS cells from 25 of 32 (78%) classical HL cases. STAT3 was constitutively phosphorylated in 4 of 5 HL cell lines and in HRS cells from 27 of 31 (87%) classical HL
cases. Only 4 of 24 NHL cases demonstrated constitutive STAT6
activation, whereas STAT3 activation was observed in 6 of 13 (46%)
cases of B-cell NHL and 8 of 11 (73%) cases of T-cell NHL.
Constitutive STAT5 phosphorylation was not a common feature of HL or
NHL. STAT6 mediates signaling by interleukin 13 (IL-13), a cytokine
frequently expressed by HRS cells. Antibody-mediated neutralization of
IL-13 resulted in significant decreases in both cellular proliferation and levels of phosphorylated STAT6 of HL cell lines. In conclusion, constitutive STAT6 phosphorylation is a common and distinctive feature
of HRS cells in classical HL, whereas STAT3 activation was regularly
present in both HL and NHL. These results suggest that IL-13 signaling
is largely responsible for the constitutive STAT6 activation observed
in HRS cells and further implicate IL-13 as an important growth factor
in classical HL.
(Blood. 2002;99:618-626) Hodgkin lymphoma (HL) has distinct pathologic and
clinical features that distinguish it from non-Hodgkin lymphoma (NHL).
In HL, the neoplastic Hodgkin and Reed-Sternberg (HRS) cells make up
only a small proportion of the clinically detectable mass, the bulk of
the tumor being composed of a reactive infiltrate of lymphocytes,
eosinophils, plasma cells, histiocytes, and fibroblasts.1 Patients with HL also often present with fever, night sweats, and
weight loss, consistent with an abnormal pattern of cytokine expression. Studies of HL-derived cell lines and tissues involved by HL
have shown that HRS cells produce multiple cytokines, including interleukin-5 (IL-5), IL-6, IL-7, IL-9, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin The interaction of cytokines with their specific cell surface receptors
triggers the activation of intracellular signaling cascades that
ultimately have effects on multiple cellular functions. The signal
transducer and activator of transcription (STAT) family of proteins
plays a central role in cytokine signaling.7 STAT proteins
are latent transcription factors located in the cytoplasm that become
activated by phosphorylation on a single tyrosine residue in response
to cytokine receptor stimulation. This tyrosine phosphorylation leads
to STAT dimerization and translocation to the nucleus, followed by
activation of transcription resulting in changes to gene
expression.7 The 7 members of the STAT family can be
broadly divided into 2 groups based on their activation patterns in
vivo. STAT1, STAT3, STAT5a, and STAT5b are activated by a wide variety
of extracellular and intracellular stimuli, whereas STAT2, STAT4, and
STAT6 show a more restricted pattern of activation and are triggered by
only 1 or 2 extracellular factors.8 For example, STAT3 is
activated by IL-2, IL-6, IL-7, IL-9, IL-10, and IL-15,8 as
well as by intracellular tyrosine kinases such as src and
abl.9,10 Similarly, STAT5 is activated in response to
IL-2, IL-3, IL-5, IL-7, IL-9, and GM-CSF.8 In contrast, STAT6 is primarily activated by IL-4 and IL-13.11
Importantly, constitutive activation of STATs, particularly STAT3 and
STAT5, has been associated with oncogenic
transformation.12
We previously identified IL-13 and IL-13R expression as common features
of HRS cells in HL.3,4 IL-13 and IL-4 have similar biologic functions,13 and both cytokines activate
STAT611,14; however, IL-4 is not typically expressed by HRS
cells.15 We have therefore hypothesized that IL-13
expression by HRS cells and their autocrine response to this cytokine
may be linked to HL pathogenesis. To pursue this line of investigation,
we examined the activation of several STAT proteins in a series of
lymphomas from patients with HL and NHL. Our results further implicate
IL-13 signaling via STAT6 as a major factor in the pathogenesis of HL.
Cell lines, tissues, and reagents
Formalin-fixed paraffin-embedded biopsy specimens representing the
following diseases were investigated: 32 cases of classical HL,
including 21 cases of nodular sclerosis HL (NSHL), 8 cases of mixed
cellularity HL (MCHL), and 3 cases of lymphocyte-depletion HL (LDHL); 4 cases of nodular lymphocyte predominance HL (NLPHL); 5 cases of
T-cell-rich B-cell lymphoma (TCRBCL); 8 cases of DLBCL; 6 cases of
ALCL with a T/null cell phenotype; and 5 cases of peripheral T-cell
lymphoma, unspecified (PTCL). All cases were diagnosed according to the
Revised European-American Lymphoma Classification.16 All
patients were negative for the human immunodeficiency virus. By
immunohistochemistry, the Reed-Sternberg cells in classical HL cases
were CD15+ and CD30+, and the lymphocytic and
histiocytic cells in NLPHL cases were CD20+,
CD15 Phosphospecific polyclonal antibodies that detect the corresponding
STAT protein only when it is phosphorylated at the indicated tyrosine
residue were used as follows: Phospho(Tyr705)-STAT3, Phospho(Tyr694)-STAT5, and Phospho(Tyr641)-STAT6 (Cell Signaling Technology, Beverly, MA). For immunoblotting, antibodies were used at a
1:1000 dilution. For immunohistochemistry, anti-P-STAT5 and
anti-P-STAT6 were used at 1:100, and anti-P-STAT3 was used at 1:200.
Antibodies detecting total STAT5 and STAT6 were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA), and the antibody detecting total
STAT3 was from Cell Signaling Technology. The antibody detecting EBV
latent membrane protein-1 (LMP-1) was purchased from Dako (Carpinteria, CA).
Immunoblotting
Immunohistochemistry Phosphorylated STAT proteins were detected in formalin-fixed paraffin-embedded tissue sections using standard immunohistochemical techniques. Briefly, tissue slides were immersed in 10 mM sodium citrate buffer, pH 6.0, and heated to maximal pressure in a microwave pressure cooker (Dako). Slides were allowed to cook for an additional 2 minutes to complete heat-induced antigen retrieval. Slides were then incubated with the primary antibody overnight at room temperature. Antibody binding was detected using a biotinylated antirabbit IgG, Vectastain avidin-biotinylated enzyme complex kit, and 3,3'-diaminobenzidine (Vector Labs, Burlingame, CA). Cases were considered positive if 10% or greater of the malignant cell population demonstrated nuclear staining.In some experiments, the specificity of immunohistochemical staining was confirmed by preabsorbing the phosphospecific antibody preparation for 1 hour at room temperature with a 10-fold excess (by weight) of the tyrosine-phosphorylated peptides against which the antibodies were raised (kindly provided by Cell Signaling Technology). The absorbed antibody preparation was then used for immunohistochemical staining as above. In situ hybridization Interleukin 4 messenger RNA (mRNA) in formalin-fixed paraffin-embedded tissue and IL-13 mRNA in cytospin preparations of cell lines was detected by in situ hybridization as previously described.4 Cytospins were prepared according to the manufacturer's directions (Shandon, Pittsburgh, PA), air-dried, and fixed in 50% methanol/50% acetone at room temperature for 6 minutes. The IL-4 probe was a 278-base pair (bp) EcoRI/EcoRV fragment of pcD-hIL-4 (ATCC) subcloned into pBluescript SK (Stratagene, La Jolla, CA). The probe for IL-13 has been previously described.3Cytokine production Supernatants of cell cultures (5 × 105/mL) were recovered 48 hours after medium exchange and assayed by enzyme-linked immunosorbent assay (ELISA) for IL-13 (Biosource International, Camarillo, CA) and IL-4 (R & D Systems, Minneapolis, MN). The sensitivities of IL-13 and IL-4 detection were 12 pg/mL and 10 pg/mL, respectively.Analyses of cell proliferation and apoptosis Cells were cultured in 96-well flat-bottomed plates at 3 × 104 cells/well for 24, 48, or 72 hours in the presence of varying concentrations of anti-IL-13 or isotype control antibodies (BD Pharmingen, San Diego, CA). Cells were also treated with varying concentrations of IL-13 (R & D Systems). [3H]-thymidine (1µCi [0.037 MBq]/well; Amersham Pharmacia Biotech, Piscataway, NJ) was added to each well for the last 16 hours of incubation. The cells were harvested on filters and the incorporation of [3H]-thymidine into cellular DNA was measured as previously described.17 Apoptosis was determined by FACS analysis using the TACS annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (R & D Systems) following the manufacturer's instructions.
HL-derived cell lines demonstrate variable STAT activation patterns To investigate the activation status of STAT proteins in HL-derived cell lines, L-428, KM-H2, HDLM-2, L-1236, and L-540 cells were analyzed for phosphorylated STAT proteins (P-STAT) by immunoblotting. All 5 HL cell lines stained positively for P-STAT6, whereas the 2 DLBCL, 1 ALCL, and 2 B-lymphoblastoid cell lines examined were all negative (Figure 1). Four of 5 HL cell lines showed comparable levels of P-STAT6, but significantly lower levels were present in L-540 cells. Phosphorylation of STAT3 and STAT5 in HL cell lines was more variable (Figure 1). P-STAT3 was detected in 4 of 5 HL cell lines, with high levels in HDLM-2 and L-540 but significantly lower levels in L-428 and KM-H2. P-STAT5 was detected in 3 of 5 HL cell lines, with the highest levels seen in HDLM-2, and lower levels in L-540 and KM-H2. P-STAT3 and P-STAT5 were also detected in KARPAS-299, a cell line derived from an ALCL with t(2;5). The DLBCL and lymphoblastoid cell lines did not show any evidence of STAT3 or STAT5 tyrosine phosphorylation.
HRS cells in HL-involved tissue also demonstrate variable STAT activation patterns The utility of the anti-P-STAT antibodies in fixed cellular material was first tested in cytospin preparations of HDLM-2 (positive for P-STAT3, P-STAT5, and P-STAT6 by immunoblotting) and LCL-GK (negative for P-STAT3, P-STAT5, and P-STAT6 by immunoblotting). HDLM-2 showed nuclear staining for all 3 P-STAT antibodies, whereas LCL-GK cells were negative (data not shown). To investigate the activation status of STAT proteins in lymphomas, immunohistochemical analysis of phosphorylated STAT proteins was performed on primary biopsy material from patients with HL and NHL and on normal lymphoid tissue. In a section of a benign reactive human tonsil, P-STAT6 (Figure 2A) and P-STAT5 (Figure 2C) were present in only a few nuclei of scattered lymphocytes outside of the germinal center. Basal STAT3 activation in the normal tonsil was significantly higher, with numerous P-STAT3+ cells within and outside the germinal center (Figure 2B).
STAT activation was then studied in biopsies of malignant lymphomas.
P-STAT6 was detected in nuclei of HRS cells in 25 of 32 (78%) of
classical HL cases (Table 1 and Figure
3A). Six cases showed fainter
cytoplasmic staining in addition to nuclear staining. In contrast to
classical HL, the lymphocytic and histiocytic cells in all 4 cases of
NLPHL were P-STAT6
Among the classical HL cases, P-STAT6 was differentially expressed
according to subtype, with NSHL cases demonstrating a significantly higher rate of P-STAT6 expression compared to MCHL or LDHL (Table 2). Of the NSHL cases, 20 of 21 (95%)
contained P-STAT6+ HRS cells, whereas only 4 of 8 (50%) of
MCHL cases and 1 of 3 (33%) of LDHL cases contained
P-STAT6+ HRS cells (P = .003). The majority of
cells expressing P-STAT6 demonstrated the morphology of HRS cells,
whereas few cells in the reactive infiltrate were P-STAT6+.
The same cases of classical HL were previously analyzed for IL-13
expression by in situ hybridization,4 and STAT6 activation was significantly associated with IL-13 expression. P-STAT6 was expressed in 24 of 27 (89%) of IL-13+ cases, but in only 1 of 5 (20%) of IL-13
The activation status of other STATs was determined in HL tissue samples. Nuclear P-STAT3 was detected in HRS cells in 27 of 31 (87%) of classical HL cases (Figure 3F), with no difference in P-STAT3 expression among subtypes of classical HL. Five cases showed fainter cytoplasmic staining in addition to nuclear staining. P-STAT3 was detected in a significant proportion of cells in the reactive infiltrate, including lymphocytes, macrophages, fibroblasts, endothelial cells, and neutrophils, as well as in HRS cells. STAT3 activation was not specific for classical HL, because it was also present in lymphocytic and histiocytic cells in 2 of 4 cases of NLPHL (Figure 3G), and in the malignant cell populations in 5 of 6 cases of ALCL (Figure 3H), 3 of 8 cases of DLBCL (Figure 3I), 3 of 5 cases of TCRBCL, and 3 of 5 cases of PTCL. P-STAT5 was detected in HRS cells in only 8 of 31 (26%) of classical HL cases (Figure 3J,K), and was absent in the lymphocytic and histiocytic cell population of NLPHL (Figure 3L). Among NHLs, P-STAT5 was present only in the malignant cells of 3 of 6 cases of ALCL (Figure 3M-O). Hodgkin lymphoma is associated with EBV in 30% to 50% of cases, and EBV LMP-1 is known to activate STAT proteins, particularly, STAT1 and STAT3.18 We therefore studied the relationship between LMP-1 expression and STAT protein phosphorylation in classical HL. LMP-1 was expressed by HRS cells in 11 of 29 (38%) cases of classical HL (Table 2). No correlation was found between LMP-1 expression and phosphorylation of STAT3, STAT5, or STAT6. To confirm the specificity of the phosphospecific antibodies used for
immunohistochemical analysis, the antibodies were preincubated with the
phosphorylated peptides against which the antibodies were raised. HRS
cells from a case of NSHL stained positively for P-STAT6 when antibody
alone was used (Figure 4A); however, preincubation of the antibody with P-STAT6 peptide abrogated this staining (Figure 4B). Specific P-STAT6 staining was maintained when the
anti-P-STAT6 antibody was preincubated with P-STAT3 peptide (Figure
4C) or P-STAT5 peptide (Figure 4D). Similarly, preincubation of
anti-P-STAT3 antibody with P-STAT3 peptide and preincubation of
anti-P-STAT5 antibody with P-STAT5 peptide blocked specific staining
with each antibody (not shown).
All 3 cases in which the HRS cells were negative for P-STAT3, P-STAT5, and P-STAT6 showed positive staining of cells within the reactive infiltrate for P-STAT3, excluding the possibility of false-negative results due to degradation of phosphorylated proteins before fixation or during tissue processing. L-1236 is an IL-13-responsive HL-derived cell line We previously studied the proliferative response of HL-derived cell lines to antibody-mediated neutralization of IL-13. The proliferation of HDLM-2 cells was found to be IL-13 dependent, whereas that of L-428 and KM-H2 cells could not be inhibited by the addition of anti-IL-13 antibody.3 We have extended these analyses to the HL-derived cell lines L-1236 and L-540. IL-13 expression was evaluated by ELISA and by in situ hybridization. IL-13 expression in L-1236 cells was detected by in situ hybridization with an IL-13-specific antisense probe (Figure 5A); no signal was observed when a control sense probe was used (Figure 5B). Cytospin preparations of L428 (IL-13+ by Northern analysis3) and LCL-GK (IL-13 by Northern analysis3) served as
positive and negative controls, respectively. ELISA (sensitivity limit
12 pg/mL) was not sensitive enough to detect IL-13 expression in L-1236
cells. IL-13 expression by L-540 cells could not be demonstrated by
either method.
To investigate the effects of IL-13 on L-1236 cells,
proliferation in the presence of neutralizing anti-IL-13 antibody was measured by determining [3H]-thymidine incorporation.
After 72 hours, the proliferation of L-1236 cells was suppressed to
41% of that of untreated L-1236 cells (Figure 5C), whereas an isotype
control antibody had no effect on proliferation (Figure 5D). In
contrast, anti-IL-13 antibody had no effect on the proliferation of
either L-540 cells or the lymphoblastoid cell line LCL-GK. Treatment of
L-1236 cells with increasing concentrations of anti-IL-13 antibody
revealed that the inhibition of proliferation was dependent on the dose
(Figure 5E). The decreased proliferation induced by IL-13
neutralization was due to an increase in the apoptotic rate, as
determined by annexin V staining. Treatment of L-1236 cells with 20 µg/mL anti-IL-13 for 48 hours resulted in a reduction in viable,
nonapoptotic (annexin-V STAT6 phosphorylation in HL-derived cell lines is largely IL-13 dependent STAT6 is activated primarily in response to IL-4 and IL-13. However, previous reports have shown that IL-4 is rarely expressed in HL.15 In the present study, IL-4 was undetectable in ELISAs of culture supernatants from the HL-derived cell lines L-428, KM-H2, HDLM-2, L-1236, and L-540. Biopsy samples from tissues involved by HL were examined for IL-4 mRNA expression by in situ hybridization. HRS cells in all 16 cases examined were negative for IL-4 (data not shown). Two cases showed IL-4 signal in scattered lymphocytes within the reactive infiltrate. A case of peripheral T-cell lymphoma that showed specific signal with an IL-4 antisense probe and no signal with an IL-4 sense probe was used as a control.We next explored whether IL-13 was responsible for the constitutive
STAT6 activation seen in HL cell lines. The IL-13-responsive HL cell
lines L-1236 (Figure 6A) and HDLM-2
(Figure 6B) were incubated for 18 hours in medium alone, medium
containing anti-IL-13 antibody, or medium containing the isotype
control. Expression of P-STAT6 was then analyzed by immunoblotting.
Treatment with neutralizing antibody to IL-13 resulted in the
inhibition of basal STAT6 phosphorylation in both cell lines (Figure
6A,B; lanes 1-5). To confirm the specificity of this effect, L-1236
cells were incubated in medium alone for 18 hours, followed by a
15-minute incubation with either medium alone, medium plus 5 ng/mL
IL-13, or medium plus 5 ng/mL IL-13 that had been preincubated with 20 µg/mL anti-IL-13 antibody for 60 minutes. Exogenous IL-13 alone
resulted in an increase in STAT6 phosphorylation above basal levels,
whereas there was no increase when the exogenous IL-13 was neutralized
with anti-IL-13 antibody (Figure 6A, lanes 6-8). Taken together, our
data demonstrate a strong association of IL-13 expression and
activation of its downstream signaling mediator STAT6 with the
proliferation of HL-derived cells.
The multiple cytokines produced by HRS cells in HL are thought to promote HRS cell growth and survival and to recruit the characteristic reactive infiltrate. To investigate cytokine signaling in HRS cells in vivo, we have examined the activation of STAT proteins, transcription factors that mediate cytokine signaling. STAT activation requires phosphorylation at specific tyrosine residues and translocation from the cytoplasm to the nucleus.7 Activation of STAT3, STAT5, and STAT6 was assessed in HL and NHL cell lines by immunoblotting, using antibodies that specifically recognize the tyrosine-phosphorylated form of a given STAT protein. The same antibodies were used in immunohistochemical analyses of various lymphoma and normal tissues, revealing both the phosphorylation and nuclear localization of an activated STAT protein. In addition to nuclear staining, a minority of cases demonstrated weaker staining in the cytoplasm, the site of STAT protein phosphorylation. STAT6 was activated in all 5 HL-derived cell lines examined and in a variable proportion of HRS cells in 78% of classical HL cases. Strikingly, HRS cells were P-STAT6+ in 95% of cases of the nodular sclerosis subtype of HL. STAT6 is activated primarily in response to the Th2 cytokines IL-4 and IL-13.14 Mice deficient for STAT6 have defects in both IL-4- and IL-13-mediated functions, including IL-4-mediated B- and T-cell proliferation, Th2 cytokine expression, immunoglobulin class switching, and up-regulation of CD23 and major histocompatibility complex class II molecules.19-21 These data demonstrate the critical role of this transcription factor in the signaling pathway of these cytokines. We have previously shown that IL-13 and its receptor are commonly expressed by HRS cells,3,4 whereas HRS cells and cells within the reactive infiltrate express IL-4 at a significantly lower level compared to IL-13 (present study, Herbst et al,15 and Dukers et al22). We therefore hypothesized that STAT6 activation in HL is primarily due to expression of IL-13. Both the in vitro and in vivo data in the present study support this hypothesis. Antibody-mediated neutralization of IL-13 in 2 IL-13-responsive HL cell lines resulted in a significant decrease in P-STAT6. Furthermore, there was a correlation between antibody-mediated neutralization of IL-13, decreased STAT6 phosphorylation, and reduced HL cell proliferation. However, complete abrogation of STAT6 activity was not observed in the antibody neutralization experiments, and low levels of P-STAT6 were present in L-540 cells that do not express detectable levels of IL-13 or IL-4. These findings suggest that signals other than IL-4 and IL-13 may contribute to STAT6 activation in HL. In vivo, STAT6 activation correlated positively with IL-13 expression in HL-derived tissues. Our results indicate that IL-13 signaling is active in a majority of HL cases, and that this cytokine plays an important role in HRS cell growth. It is possible that IL-13 is also acting in a paracrine manner contributing to the inflammatory infiltrate in HL.4 This may be through the IL-13-stimulated release of chemokines by HRS cells, or through direct effects of IL-13 on the reactive cells that express IL-13R, particularly B cells and macrophages. However, we did not detect significantly increased P-STAT6 levels within the reactive infiltrate compared to normal tonsillar lymphoid tissue. In contrast to the persistent STAT6 activation in HRS cells, STAT6 activation is a transient event under normal physiologic conditions. Immunohistochemistry, which provides only a snapshot of the activation pattern, may not be sensitive enough to detect subtle differences in STAT6 activation in normal cells. Therefore, our data do not permit us to determine the extent of the role of IL-13 in the pathogenesis of the reactive infiltrate. In contrast to its frequent activation in HRS cells in classical HL
cases, STAT6 was rarely activated in normal lymphoid tissue and in
cases of NHL. Normal tonsillar tissue showed only a few P-STAT6+ cells, and a large majority of NHL
tissue sections were P-STAT6 STAT3 is activated by several extracellular and intracellular stimuli,
including IL-10, the IL-6 family of cytokines, IL-2, and other
cytokines sharing the IL-2R Like STAT3, STAT5 is activated by several stimuli, including engagement
of cytokine receptors sharing the IL-2R Mounting evidence indicates that STAT proteins, particularly STAT3 and
STAT5, are involved in oncogenesis.12 Constitutive STAT3
activation has been identified in a wide variety of
malignancies,12 and expression of a constitutively
activated STAT3 molecule in immortalized fibroblasts leads to cellular
transformation.29 STAT5 has been shown to be essential for
transformation by bcr-abl in chronic myelogenous
leukemia.30,31 Activation of STAT3 and STAT5 is thought to
contribute to oncogenesis by preventing apoptosis through
bcl-xl up-regulation,32,33 and by stimulating
proliferation via cyclin D1 up-regulation.29 The
activation of STAT6 in malignancies is less well-studied. Constitutive
activation of STAT6 has been demonstrated in human T-cell leukemia
virus- associated T-cell leukemia/lymphoma,34 as well as
in leukemia associated with the p190bcr-abl.35
A role for STAT6 in IL-4-mediated T-cell proliferation has also been
reported,19,20,36,37 mediated through up-regulation of the
IL-4R In conclusion, we have examined activation of STAT proteins in HL to study cytokine activity in this disease. Neither STAT3 nor STAT5 activation was specifically associated with HL, whereas STAT6 activation was a common and distinctive feature of HRS cells. We also showed that STAT6 activation in these cells is due in part to IL-13 signaling. Our previous work implicated IL-13 as an autocrine growth factor for HRS cells.3,4 Given the role of STAT6 in the proliferation of components of the normal immune system,19,20 it is likely that IL-13 is mediating its growth-promoting effects on HRS cells via STAT6. Constitutive IL-13 activity and STAT6 phosphorylation may also underlie other aspects of HL such as the recruitment of the reactive infiltrate. For example, IL-13 signaling through STAT6 is involved in the release of chemokines such as macrophage-derived chemokine,39,40 a molecule involved in Th2 cell recruitment that is frequently expressed by HRS cells in classical HL.41 Our results strongly imply that neutralization of IL-13 signaling by targeting either IL-13R or STAT6 activation may positively affect the course of disease in a majority of patients with HL. As previously discussed,4 inhibition of IL-13 signaling for therapeutic purposes may not be seriously detrimental to the immune system of patients, because it should not compromise a Th2 response mediated through IL-4, allowing the patient some measure of immune protection.
The authors thank J. Ho for excellent technical support, M. Bray for critical comments, and M. Saunders for scientific editing.
Submitted May 7, 2001; accepted July 15, 2001.
Supported in part by grants from the Canadian Institute of Health Research and the National Cancer Institute of Canada.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Tak W. Mak, Ontario Cancer Institute, Amgen Institute, 620 University Ave, Suite 706, Toronto, ON, Canada M5G 2C1; e-mail: tmak{at}oci.utoronto.ca.
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Abstract
Introduction
, and transforming growth factor 
(TGF-
) and L-540 (
) and
control LCL-GK (
) cells were treated with either 20 µg/mL
anti-IL-13 antibody (C) or 20 µg/mL of an isotype control (D).
Proliferation was measured at 24, 48, and 72 hours by
[3H]-thymidine incorporation. The y-axis shows the
proliferation of the treated cells as a percentage of the corresponding
untreated control, calculated as the mean of triplicate samples.
(E) L-1236 cells were treated with various doses of
anti-IL-13 antibody and [3H]-thymidine incorporation was
measured at 72 hours. The value of 56 446 cpm (untreated control at 72 hours) was taken as 100% proliferation. (F) L-1236 cells were treated
with various doses of exogenous IL-13 and [3H]-thymidine
incorporation was measured at 48 hours. The value of 34 688 cpm
(untreated control at 48 hours) was taken as 100% proliferation. These
experiments were repeated twice with similar results.
chain (including IL-7, IL-9, and IL-15),
and oncogenic tyrosine kinases such as src and abl.
harboring tumor cells of Hodgkin's disease.
Blood.
1996;87:2918-2929