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*
Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104; and
Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) are markedly depressed as is expression of IL-12,
a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells
express both the ß1 and ß2 chains of the IL-12 receptor (IL-12R)
and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells
express only the IL-12R ß1 and thus do not tyrosine phosphorylate
STAT4 in response to IL-12. To determine whether SS cells are Th2-like
at the level of IL-12 signal transduction, we analyzed RNA from seven
patients for the presence of message for the IL-12R ß1 and ß2 genes
using RNase protection assays and assessed whether IL-12 induced
tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of
seven SS patients tested, ß2 message was expressed at low to
undetectable levels and its expression could not be stimulated by
either IFN-
or IFN- , which stimulated ß2 expression in control
PBL. The absence of ß2 expression is further supportive evidence for
the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells
also showed severely reduced levels of STAT4, suggesting that they have
a depressed response to any inducer of the STAT4 signal transduction
pathway, including IFN-. This is the first observation linking STAT4
gene expression with a human disease and suggests that dysregulation of
STAT4 expression may be significant to the development and/or
progression of SS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(3, 4, 5, 6). We have also observed that the production of
IL-12, a potent inducer of IFN- and a pivotal cytokine for Th1 cell
differentiation, is depressed in SS (7). Depressed IL-12
levels correlate with an inability to mount an effective Th1 response
as reflected in immune abnormalities seen in these patients. Addition
of exogenous IL-12 to cell cultures of PBL from SS patients can reverse
many of the cytokine and immune abnormalities observed, including
decreased IFN- production and decreased NK cell activity
(8).
Two chains of the IL-12R have been cloned (9, 10, 11, 12), and a
recent report suggests the presence of a third chain (13).
Studies in both humans and mice suggest that the responsiveness to
IL-12 of Th1 cells and the lack of responsiveness of Th2 cells, can be
explained by the types of IL-12R chains expressed on the surface of
these cells. Th1 cells express both the ß1 and ß2 IL-12R subunits,
whereas Th2 cells express only the ß1 subunit (12, 13).
Although both Th1 and Th2 cells can bind IL-12, only Th1 cells are
capable of signaling in response to IL-12, and a functional signal
appears to require binding to a receptor composed of both ß1 and ß2
subunits (9, 11). IL-4 appears to account, at least in
part, for suppressed ß2 expression in Th2 cells, although IFN- can
overcome this suppression under some conditions in the mouse system
(14).
Several studies have shown that signaling by IL-12 occurs through the
Jak/STAT signal transduction pathway with involvement of two members of
the Janus family of tyrosine kinases, Jak2 and Tyk2, and the STAT
proteins 1, 3, and 4 (15, 16). Binding of IL-12 to its
receptor triggers the recruitment and activation of the Janus kinases,
with Jak2 and Tyk2 interacting with the ß2 and ß1 subunits,
respectively, and the subsequent association and tyrosine
phosphorylation (pTyr) of the STAT proteins (17, 18, 19). Mice
with a targeted disruption of the STAT4 gene have decreased
responsiveness to IL-12, an impaired Th1 response, and a Th2 population
that demonstrates markedly enhanced development (20, 21),
characteristics similar to those associated with SS. Furthermore, only
IL-12, the expression of which is depressed in SS patients, and IFN-
(22, 23, 24) have been shown to induce STAT4 phosphorylation
in T cells derived from healthy individuals (17). This
observation raised the possibility that the inability of SS patients to
mount an effective IL-12-directed Th1 response might rest, at least in
part, in their failure to phosphorylate and thus activate STAT
proteins, in particular STAT4, the phosphorylation of which in response
to IL-12 is dependent on the presence of the IL-12R ß2 chain. IFN-
not only activates STAT4 but has also been reported to induce
expression of the IL-12R ß2 chain required for STAT4 phosphorylation
in response to IL-12 in normal T cells (17).
We report here that expression of the IL-12R ß2 gene was low to
undetectable in PBL from six of the seven advanced SS patients tested.
In vitro culture of SS cells with inducers of ß2 expression including
IFN-, IFN-, or IL-12 also failed to stimulate ß2 expression in
these cells. We also show that SS T cells have significantly reduced or
undetectable levels of STAT4 proteins, whereas nonmalignant
CD8+ T cells from patients express normal levels
of STAT4 protein that can be phosphorylated in response to IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- and IFN- were purchased from R&D systems
(Minneapolis, MN), PHA was purchased from Sigma Chemicals (St. Louis,
MO), and rhIL-12 was a gift from Dr. Stanley Wolfe (Genetics Institute,
Amherst, MA).
Patients
Venous blood samples were collected from seven SS patients
before commencing treatment with IFN- and during or soon after
IFN- treatment as indicated (Table I
). Circulating T cells of
patients 1–6 were characterized as 80–99% SS cells as measured by
1-µm section analysis of peripheral blood buffy coats and by FACS
analysis. Patient 7 had 35% SS and 15% CD8+
cells before IFN- and 77% SS cells and 5%
CD8+ cells 1 year later during IFN-
treatment.
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PBL were prepared as described (21). Briefly, venous blood was drawn from SS patients and normal healthy donors (all with informed consent) and collected into heparinized tubes. Blood was diluted 2-fold with Dulbecco’s PBS, layered over Ficoll-Hypaque (Pharmacia, Piscataway, NJ), and centrifuged for 30 min at 500 x g. The interface containing the mononuclear cell fraction was washed with Dulbecco’s PBS by centrifugation. Monocytes were depleted by adherence to gelatin-coated tissue culture flasks, and the PBL resuspended in complete medium at the indicated cell concentrations. PBL that were skewed to either Th1 or Th2 phenotypes were generated by culturing in RPMI 1640,10% FCS plus 5 µg/ml PHA in the presence of either IL-12 (1 ng/ml), and anti-IL-4 (1/5000 dilution of 4F2 ascites) for the Th1 cells or IL-4 (50 ng/ml) and anti-IL-12 (1/5000 of CB6 ascites) for the Th2 cells (25). Under these conditions, cultures are routinely two-thirds CD4+ and one-third CD8+, with virtually no contaminating monocytes, NK or B cells.
In certain experiments, CD8+ T cells were removed using BioMag immunobeads (PerSeptive Diagnostics, Cambridge, MA) according to the manufacturer’s protocol. Briefly, PBL were incubated with anti-CD8+ mAb (Immunotech, Westbrook, ME) for 30 min at 4°C. Cells were then washed to remove unbound Ab and combined with immunomagnetic beads coated with goat anti-mouse Fc-specific Abs (PerSeptive Diagnostics) at a bead-cell ratio of 25:1. Positively selected CD8+ T cells or clonal malignant T cells were recovered in a magnetic field (with a purity of >98%). The selection process was repeated on the cells not bound to the beads in this first round of separation to maximize yields. The remaining unbound cells typically contained <1% CD8+. Cells were either used immediately after purification or stored in liquid nitrogen in 95% FBS/5% DMSO until use.
Ribonuclease protection assay (RPA)
Total RNA was isolated from PBL with TRIzol reagent (Life Technologies, Grand Island, NY), resuspended in diethylpyrocarbonate-treated water, and stored at -20°C. RPA was conducted as described (26). Briefly, antisense RNA probes were prepared with the used of the T7 promoter in Bluescript (Stratagene, La Jolla, CA) or Sp6 promoter in PCRII (Invitrogen, San Diego, CA) and labeled with 400–800 Ci/mmol [32P]UTP or [32P]CTP. Probes were purified by extraction from a 6% acrylamide sequencing gel. Depending on availability, 2–10 µg of total RNA were hybridized with antisense probe (5 x 105 cpm) in 80% formamide buffer at 60°C for 16 h. The IL-12R ß1 and ß2 probes, generated from human cDNA clones, cover nucleotides 1752–2102 and 2069–2449, respectively (11). A human cyclophilin antisense probe (Ambion, Austin, TX) was used as an internal control for standardization of expression levels between samples. Samples were processed as described (26) and fractionated on a 6% sequencing gel. The dried gels were exposed to a PhosphorImager screen for 1–4 days, and the relative signals were quantitated with Image Quant (Molecular Dynamics, Sunnyvale, CA). Where comparisons were made between the individual messages, the values were standardized for C or U content of the protected fragments.
Determination of STAT protein and phosphorylation levels
Cells depleted of monocytes and/or CD8+
cells were cultured for 3 days in RPMI 1640/10% FBS containing (PHA, 2
µg/ml), washed twice with medium, starved for 20 h in RPMI 1640
containing 2% FBS, and stimulated with IL-12 (22 ng/ml), IFN- (1000
U/ml), or IFN- (1000 U/ml) for 15 min at 37°C. After lysis in ice
cold buffer (5 mM Na2EDTA, 50 mM NaCl, 30 mM
Na4P2O7,
50 mM NaF, 0.1 mM Na3VO4,
20 mM Tris (pH 7.6), 1% Triton X-100, 0.5 µg/ml leupeptin, 0.7
µg/ml pepstatin A, 50 µg/ml PMSF), cell lysates were cleared by
centrifugation and incubated with anti-STAT4 or anti STAT1 (C-20 or
E23, respectively, Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C
for 60 min. Immune complexes were collected by incubation with protein
A-agarose (60 min, 4°C), washed three times with lysis buffer, and
heated for 4 min in a boiling water bath in sample buffer for SDS-PAGE.
Before immunoprecipitation, aliquots were taken for protein assay
(bicinchoninic acid assay, Pierce, Rockford, IL) and for Coomassie blue
staining. Immunoprecipitates from extracts containing the same amount
of protein were analyzed by Western blotting with Abs against pTyr
(4G10 (Upstate Biotechnology, Lake Placid, NY) and PY20 (Transduction
Labs, Lexington, KY)), STAT4 (L-18 (Santa Cruz Biotechnology)) or STAT1
(E23 (Santa Cruz Biotechnology)) by chemiluminescence (Renaissance
(NEN-DuPont, Boston, MA)) with HRP-conjugated anti-Ig (Figs. 4 and 5). The blots were always analyzed for pTyr first and then stripped by
incubation for 30 min at 50°C in stripping medium (2% SDS, 0.05 M
Tris (pH 6.8), 0.1 mM ß-mercaptoethanol). Stripping was confirmed
before reanalysis with anti-STAT4 or anti-STAT1. Western
blotting shown in Fig. 6 was conducted with alkaline
phosphatase/5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium
for visualization. In this case identical samples were Western blotted
and developed with either anti-pTyr or anti-STAT4.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Levels of IL-12R ß1 and ß2 subunit mRNA in PBL from 7 SS
patients and four normal controls were determined by RPA. Samples for
RNA were obtained either before or during IFN- therapy, which
involved three weekly injections for extended periods of time (Table I
). Six of the seven patients had little
or no detectable ß2 message (Fig. 1).
ß2 message levels were markedly lower than in RNA extracted from
freshly isolated control PBL which typically express low levels of ß2
and were even lower than in the Th2-skewed normal control PBL. By
contrast, ß1 message levels were 3- to 5-fold higher in the SS
samples than in normal PBL but were still significantly lower than in
the Th1-skewed normal controls. Unlike ß2 message levels which
remained essentially unchanged before or during IFN- therapy, ß1
message levels increased from 30 to 50% in samples taken from several
patients during IFN- treatment, suggesting that some patient cells
might be responding to IFN- at the level of receptor gene expression
(Fig. 1).
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and IFN- induce expression of IL-12R
ß2 in normal PBL but not in SS cells
To further compare the effects of biological response modifiers on
IL-12R gene expression in normal controls and SS patients, freshly
isolated PBL were cultured in the presence of either IFN-, which has
been reported to induce human ß2 expression (27), or
IFN-, which has been reported to induce ß1 and ß2 expression in
humans (28) and mice (29). Freshly isolated
normal control PBL had low but relatively similar levels of ß1 and
ß2 mRNA, whereas no ß2 message was detected in the RNA isolated
from SS patient 1 (Fig. 2). Both ß1 and
ß2 message levels increased 2- to 3-fold in control cells cultured in
the presence of IFN- for 24 h, but there was no significant
increase in either ß1 or ß2 message levels in the SS cells even
after 56 h.
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on ß1 and ß2 message levels in control PBL
was even greater than that of IFN-, with 5- and 7-fold increases
respectively (Fig. 2). These results are consistent with previous
reports in mice and humans that IFN- enhances IL-12 responsiveness
(29, 30). In contrast, ß2 expression in SS cells was
unaffected by IFN- and remained undetectable, although ß1 message
levels increased more than 4-fold by 18 h and remained elevated
56 h after addition of IFN-. Because the increases in the
levels of ß1 message were similar in both the control and patient
RNA, ß1 expression was likely induced in the patient’s SS cells in
addition to the normal cells because 99% of the T cells of patient 1
were identified as SS cells by FACS analysis of clonal Vß expression,
and NK cell numbers were also very low. The inability to detect any
increase in ß2 message suggests that ß2 transcription may be
suppressed in the SS cells, although we cannot eliminate the
possibility that increased message turnover is also involved. The
extremely low number of normal T cells in this patient precluded
determination of induced ß2 expression in the nonmalignant T cells.
The finding that IFN- and IFN- induce both receptor genes in
normal PBL supports the previous report that IFN- up-regulates IL-12
receptors and IL-12 responsiveness in freshly isolated human PBL
(29), although no effect of IFN- on either ß1 or ß2
expression was detected in similar studies using human Th cell lines
(27).
Neutralization of IL-4 in SS cell cultures fails to result in the
induction of IL-12R ß2 expression by either IFN- or IL-12
SS is characterized by production of Th2 cytokines including IL-4,
which has been shown to suppress ß2 expression (14, 30, 31). However, neutralization of IL-4 activity in SS cultures
with anti-IL-4 Abs (anti-IL-4) did not result in any detectable
ß2 message in the presence or absence of IFN- (Fig. 3A). By contrast, the
induction of both ß1 and ß2 expression by IFN- in normal control
PBL was enhanced by the presence of anti-IL-4, with the greatest
effect on ß2 expression (Fig. 3B).
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A). However,
anti-IL-4 had no detectable effect on ß2 expression in the SS
cells even when IL-12 was added. STAT4 protein levels are undetectable in PBL from SS patients with high levels of SS cells
The biological effects of the IFNs and IL-12 on T cells are
mediated through the Jak/STAT signal transduction pathways. In
particular, signal transduction by both IFN- and IL-12 proceeds in
part, through the activation of STAT4 (17, 18, 32).
Because activation of STAT4 is important for development of an
effective Th1 response, we compared the ability of SS cells and PBL
from normal healthy individuals to activate the STAT4 signal
transduction pathway, as measured by tyrosine phosphorylation of STAT4,
in response to IL-12 or IFN- (Fig. 4).
Extracts for immunoprecipitations were prepared from cells of SS
patients in whom >85% of the lymphocytes represented a single
clonotypic TCR Vß rearrangement and were thus derived from the
identical transformed clone. STAT4 protein was essentially undetectable
in the SS cell extracts from patient 2 (Fig. 4) or patient 1 (data not
shown). In some cases, low levels of STAT4 protein could be detected if
a 10-fold greater number of SS cells (2 x 108)
were used to prepare the extracts (data not shown). We attribute the
detection of STAT4 protein under these conditions to the small
proportion of normal T cells present.
To determine whether the expression of other STAT proteins was also
repressed in SS cells we examined protein extracts prepared from PBL
from patients 1 and 2 for the expression of STAT1. STAT1 is required
for signaling by both IFN- and IFN- (33) and has
also been reported to be activated by IL-12 in human T cells
(28). Fig. 5 shows the
results of the immunoblotting experiments, which clearly detect STAT1
protein and pTyr phosphorylation at similar levels in both patients 1
and 2 and the normal control.
The normal CD8+ T cells from SS patients with moderate levels of SS cells express STAT4 and phosphorylate it in response to IL-12
To determine whether the patient’s normal T cells also have
reduced levels of STAT4, we prepared protein extracts from PBL of
patient 7, in whom 
15% of the T cells were normal
CD8+ cells and 35% of the T cells were SS
cells at the time of sampling. Immunoblotting revealed reduced levels
of STAT4 protein as compared with the normal controls and greatly
decreased, but none the less detectable levels of tyrosine
phosphorylation of STAT4 in response to IL-12 (Fig. 5A). To
determine whether the reduced levels of STAT4 phosphorylation
correlated with the lack of ß2 expression on the SS cells, the
analysis was repeated using extracts prepared from the patient PBL
depleted of the normal CD8+ T cells. Indeed,
STAT4 protein levels were greatly reduced by removal of the
CD8+ T cells and tyrosine phosphorylation of
STAT4 was no longer detectable under these conditions (Fig. 5B), suggesting that most of the STAT4 protein and all of
the STAT4 phosphorylation detected in these experiments could be
attributed to the CD8+ T cells. To ensure that
equivalent amounts of the normal and CD8+
depleted SS immunoprecipitates were Western blotted, the precipitated
extracts were analyzed on an SDS-acrylamide gel by Coomassie blue
staining (Fig. 5C) to confirm the protein concentrations
determined with bicinchoninic acid reagent (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, that have potential
therapeutic utility in the treatment of CTCL.
In six of our patients, the SS cells can be classified as Th2 T cells
based on the relative absence of message for the IL-12R ß2 chain,
supporting previous studies that classify SS cells as Th2 cells based
on cytokine secretion profiles (3, 4, 34, 35). ß2
expression appears to be strongly suppressed in these cells because in
vitro exposure to IFN- and IFN- both of which induced ß2
message in control PBL, was ineffective on SS cells. SS cells do
express the GATA-3 protein (data not shown), which has been suggested
to suppress Th1 differentiation (36, 37). However,
IFN-, which was previously reported to partially overcome the
suppression of ß2 expression in normal mouse Th2 cells
(14), did induce significant levels of ß1 message in the
SS cells but not ß2, suggesting that factors in addition to GATA-3
may be involved. The induction of ß1 in the SS cells by IFN-
further suggests that signal transduction pathways for induction of
IL-12R ß1 and ß2 expression by IFN- are independent at some
level. Indeed, the synergy demonstrated between IFN- and IL-12 in
activating cytotoxic activity of the normal CD8+
T cells against autologous SS cells demonstrated by Seo et al.
(38) might reflect, in part, the induction of IL-12R
expression in the CD8+ T cells by IFN-,
consequently enhancing their responsiveness to IL-12.
The apparent lack of ß2 chain expression by most SS cells is consistent with their characterization as Th2 cells. However, normal Th1 and Th2 cells, in both humans (27) and murine models (19, 31) have similar levels of STAT4 protein, although we find that STAT4 levels in the Th2-like SS cells are essentially undetectable as compared with their normal counterparts. By contrast, STAT1 protein levels appear to be normal in SS cells; therefore, the lack of STAT4 that we detect is not symptomatic of a more general effect on all STAT proteins. These results suggest that SS cells may have the capacity to signal through the unaffected STAT pathways and raise the question as to whether some SS cells could be susceptible to the growth-inhibiting effects of the IFNs. Understanding at what point in the progression of SS the STAT4 expression is lost, whether a similar loss of STAT4 is associated with other forms of CTCL, and how the loss of STAT4 correlates with the loss of ß2 expression may be an important element in understanding the development and progression of this disease and in designing treatments for its control.
The studies described here also clearly show that the normal patient
CD8+ cells retain the capacity to respond to
IL-12 by phosphorylating STAT4 and thus express functional IL-12
receptors. The phosphorylation of STAT4 detected in extracts of SS
cultures with significant numbers of normal CD8+
T cells supports our previous report that IL-12 can partially reverse
the Th2 cytokine profile of PBL isolated from CTCL patients. The
increases in IFN- production and NK cell activity detected in these
studies (7) was likely due to the effects of IL-12 on the
normal T cells present in these preparations. These hypotheses are
further supported by our in vitro data (39) as well as
those of others (40) derived from clinical trials
utilizing recombinant IL-12 which suggest that
CD8+ cytotoxic T cells are activated in patients
receiving IL-12 therapy. Thus, IL-12 can mediate immune-augmenting
effects on the nonmalignant cells of SS patients, and this may lead to
a profound clinical benefit.
The cells of patient 5 expressed significantly higher levels of ß2
message than those of the other six patients, suggesting that
manifestation of the Th2 phenotype may not absolutely depend on
silenced ß2 expression. On the other hand, the ratio of ß1 to ß2
message in this patient was 6:1, comparable to with ratios we find in
PBL cultures skewed to the Th2 phenotype by growth in the presence of
IL-4 and anti-IL-12, and unlike the 1:1 to 2:1 ratios in cultures
skewed to the Th1 phenotype (see Fig. 1). Using enhanced
chemiluminescence, we could detect some STAT4 expression in this
patient but not phosphorylation of STAT4 (data not shown), suggesting
that signal transduction by IL-12 through STAT4 is blocked. Perhaps the
levels of ß2 expression as compared with that of ß1 are too low to
assemble significant numbers of functional receptors. Whether ß2
expression is a unique characteristic of this particular patient or
whether it is a marker that may be significant for classification of a
SS subtype will require the identification and characterization of
additional ß2-expressing SS patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Louise C. Showe, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: SS, Sézary syndrome; CTCL, cutaneous T cell lymphoma; RPA, RNase protection assay; pTyr, phosphotyrosine.
Received for publication December 28, 1998. Accepted for publication July 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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zary syndrome and mycosis fungoides. J. Clin. Invest. 67:523.
receptor ß-chain). Blood 88:1383.: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781.[Abstract]
2a and etretinate. Br. J. Dermatol. 125:456.[Medline]
and IFN- in IL-12-induced T helper cell-1 development. J. Immunol. 156:1442.[Abstract]
and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. Eur. J. Immunol. 27:647.[Medline]
zary syndrome expresses a Th2-type profile [see comments]. J. Invest. Dermatol. 103:29.[Medline]
zary syndrome: its enhancement by interferon- (IFN-) and IL-12 and fluctuations in association with disease activity. Clin. Exp. Immunol. 112:403.[Medline]
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