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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-02-0475.
NEOPLASIA
From the Department of Dermatology, University of
Zürich Medical School, Zürich, Switzerland;
Department of Dermatology, Medical School, J. W. Goethe-University, Frankfurt/Main, Germany; and the
Department of Pathology, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, MA.
Little is known about mechanisms involved in skin-specific homing
of cutaneous T-cell lymphoma (CTCL). Chemokine/chemokine receptor
interactions have been implicated in the homing of lymphoma cells to
various tissue sites. We investigated tissue samples and tumor cell
suspensions of patients with CD30+ CTCL (n = 8) and
CD30 Cutaneous T-cell lymphomas (CTCLs) are part of the
spectrum of extranodal non-Hodgkin lymphomas1 and are
characterized by proliferation of clonally expanded helper T-cells in
skin, but without detectable systemic involvement at least 6 months following diagnosis.2-4 Primary cutaneous
CD30+ large-cell (anaplastic) CTCLs lack the t(2;5)
translocation (NPM/ALK The presence of eosinophils is prominent in certain types of
CTCLs.15,16 Eotaxin/CCR ligand 11 (CCL11)
is one of the most potent eosinophil chemoattractants.17
The question of eotaxin/CCL11 receptor CCR-3 expression in CTCL has
thus far not been addressed.
Here we show that CD30+ large-cell CTCLs express a
functional CCR3 receptor and produce Th-2-like cytokines. These
findings might have implications for the homing of transformed T cells to skin.
Tissue samples and immunohistochemistry
Preparation of tumor cell suspensions
Flow cytometry, intracellular cytokine/chemokine staining All antibody dilutions and washing steps were done in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Sigma). Then, 1 × 106 cells per reaction were stained with the following moAbs: 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated antihuman CD30 (Ki-1) moAb (DAKO) of 10 µg/mL 7B11 antihuman CCR3 moAb. To control for nonspecific staining or Fc-receptor-mediated binding of antibody, the following moAbs were included as negative controls: 1:10 dilution of FITC-conjugated mouse IgG1 (Becton Dickinson, Basel, Switzerland) of 10 µg/mL mouse IgG2a (Ancell). Staining with anti-CCR3 or anti-IgG2a moAb was followed by phycoerythrin (PE)-conjugated goat antimouse F(ab')2 moAb (DAKO) at a 1:20 dilution. All incubations were done at 4°C for 30 minutes. Samples were analyzed by flow cytometry with a FACScalibur System (BD Biosciences) equipped with CellQuest software (BD Biosciences).For intracellular cytokine/chemokine staining, cells were stimulated
with 50 ng/mL PMA and 1 µM ionomycin in RPMI 1640 (Gibco BRL, Basel,
Switzerland) supplemented with 10% FCS (Serotech, Basel, Switzerland)
in 5% CO2, 37°C. After 4 hours, transport inhibitor was added for an additional 2 hours: 0.75 µL/mL GolgiStop (Pharmingen) and 1 µL/mL GolgiPlug (Pharmingen).
After cell-surface antigen staining with 10 µg/mL mouse IgG1
antihuman CCR3 moAb or by purified mouse IgG1 (BD Biosciences) followed
by PE-cyanin 5 (PE-Cy5)-conjugated goat antimouse antibody
(DAKO) at a 1:10 dilution, intracellular cytokine staining was
performed with 1:100 PE-conjugated anti-IL-4 moAb and 1:100
FITC-conjugated anti-IFN- Internalization experiments Recombinant chemokines eotaxin/CCL11, RANTES/CCL5, and MCP-3/CCL7 were obtained from R&D Systems. Cell suspensions from lesional skin were obtained as described in "Preparation of tumor cell suspensions" and were cultured for 2 days in RPMI 1640 supplemented with 10% FCS. Short-term cultures were incubated with 200 nM CCR3 ligands eotaxin/CCL11, RANTES/CCL5, MCP-3/CCL7, or untreated medium, respectively, for 40 minutes at 37°C in a 5% CO2 atmosphere. After chemokine incubation, cells were washed extensively in PBS; the final washing step was performed in acidic glycine buffer, pH 3.0, to remove bound chemokine located on noninternalized receptor for 1 minute at 37°C. To determine the amount of surface-expressed CCR3, cells were stained with antihuman CCR3 (10 µg/mL) and PE-conjugated goat antimouse F(ab')2 antibody (DAKO) at a dilution of 1:20; each Ab was incubated for 30 minutes at 4°C.Actin polymerization assay Actin polymerization was tested as previously described.19 Briefly, cells derived from a CD30+ cutaneous lymphoma line (Mac-1) (1.25 × 106/mL)20 were resuspended in RPMI-1640 medium containing 0.5% bovine serum albumin (BSA) at 37°C and incubated with 100 ng/mL eotaxin (R&D Systems) for varying amounts of time. At the indicated time points, 400 µL cell suspension was added to 100 µL solution containing 4 × 107 M FITC-labeled phalloidin, 0.5 mg/mL 1- -lysophosphatidylcholine, and 18% formaldehyde (all from Sigma)
in phosphate-buffered saline (PBS). After incubation at 37°C
for 10 minutes in the dark, fixed cells were centrifuged at
400g for 5 minutes at room temperature and subsequently
resuspended in PBS containing 0.5% BSA. Cells were analyzed by flow
cytometry on a FACSCalibur (Becton Dickinson), and all time points are
plotted relative to the mean relative fluorescence of the sample before
addition of eotaxin.
Migration assay The number of CD30+ lymphoma cells migrating in response to recombinant eotaxin (R&D Systems) across 5-µm pore size polycarbonate filters (6.5-mm diameter) was assessed in 24-well Transwell chambers (Costar Corning, NY). First, 600 µL warm (37°C) assay medium (RPMI containing 0.5% BSA) containing various concentrations of eotaxin were added to the lower wells. Lymphoma cells were suspended at 2 × 106 cells per milliliter in warm assay medium (37°C), and 100 µL cell suspension was added to the upper chamber of each Transwell chamber. The plates were incubated for 3 hours at 37°C in 10% CO2. The migrated cells in the lower chambers were collected, and the number of migrated cells was counted by acquisition for 60 seconds with a flow cytometer.
Chemokine receptor CCR3 is expressed in CD30+
large-cell CTCL, but not in CD30 Twenty CTCLs, consisting of 8 CD30+ large-cell
lymphoma, 6 mycosis fungoides, and 6 Sézary syndrome patient
samples, were analyzed by immunohistochemistry. CD30 was expressed in
all large-cell CTCLs (Table 1). Variation
in the admixture of inflammatory cells accounted for the difference in
numbers of CD30+ cells in the infiltrate. Staining for the
chemokine receptor CCR3 demonstrated expression of CCR3 in 7 of 8 CD30+ CTCLs investigated (Table 1; Figure
1). No epidermotropism of CCR-3+ cells was observed. There was faint staining of
keratinocytes, which is explained by the recently described CCR3
expression on these cells.21 Mycosis fungoides (n = 6)
and Sézary syndrome (n = 6) sections were CD30
CCR3 and CD30 coexpression in freshly isolated tumor cell suspensions from CD30+ CTCL To confirm our immunohistochemistry data on a single-cell level and to assess expression levels of CCR3 on CD30+ lymphoma cells, we performed flow cytometric analysis of tumor cell suspensions. Tumor cells suspensions were derived from fresh biopsy material, which was available in 3 patients with CD30+ large-cell CTCL (nos. 1, 7, and 8). Cells were double-stained for CCR3 and CD30 and analyzed by flow cytometry. Compared with isotype control, a strong coexpression of the chemokine receptor CCR3 and CD30 was detected (Figure 2; patient no. 7). Strong staining in immunohistochemistry (Figure 1) translated into a high level of CCR3 expression on CD30+ tumor cells in flow cytometric analysis (Figure 2; patient no. 7). The majority of CD30+ cells were CCR3+. Expression levels were comparable to those seen in eosinophils (data not shown).
Eotaxin/CCL11 induces CCR3 down-regulation on CD30+ cutaneous tumor cells Detection of CCR3 protein on CD30+ tumor cells does not necessarily imply the existence of a functional receptor. Freshly isolated cell suspensions did not provide sufficient material to perform migration assays. A different approach to demonstrate the existence of a functional chemokine receptor is receptor internalization. Binding of ligand to its cognate receptor leads to receptor down-regulation, which can be assessed by flow cytometry.22 After incubation with eotaxin/CCL11, RANTES/CCL5, MCP-3/CCL7, or medium alone, surface expression of CCR3 on short-term cultured tumor cells was analyzed by flow cytometry with the use of anti-CCR3 moAb. Receptor-bound chemokine might interfere with antibody binding; therefore, cells were washed in acidic glycine buffer, as described, to dissociate the chemokine ligand from its receptor.22 As shown in Figure 3A, the CCR3 ligand eotaxin/CCL11, but not medium alone, is able to induce a 1.9-fold decrease of CCR3 expression owing to down-regulation of CCR3. In contrast, CCR3 ligands RANTES/CCL5 and MCP-3/CCL7, which possess a lower affinity to CCR3 than eotaxin/CCL11, did not induce an internalization of CCR3 (data not shown).
Functional expression of CCR3 on a CD30+ cutaneous lymphoma cell line Investigation of receptor functionality in fresh tumor cell suspensions is limited by both the low number and the heterogeneity of cells. To further explore functional CCR3 expression in CD30+ lymphoma cells, we investigated the expression of CCR3 in CD30 lymphoma cell lines. The cell line Mac-1 coexpressed CD30 and CCR3 (data not shown).20 As a first step, actin polymerization was investigated. Actin polymerization, which controls a number of processes regulating cell migration and reorganization of the actin cytoskeleton, has been shown to be an early event in the migratory response to chemokines.23 To examine whether eotaxin induces reorganization of the cytoskeleton in CD30+ lymphoma cells, filamentous actin was measured in the CD30+CCR3+ lymphoma cell line20 with the use of fluorescein phalloidin. Stimulation with 100 ng/mL eotaxin induced a transient 85% increase in intracellular F-actin in CD30+ lymphoma cells within 75 seconds (Figure 3B), indicating the transduction of a migration signal to the cytoskeleton after binding of eotaxin to CCR3 on the CD30+ lymphoma cells.Next we assessed whether engagement of CCR3 by its ligand eotaxin was leading to cell migration. Migration assays through 5-µm pore size polycarbonate membranes during 3 hours were performed with the use of recombinant human eotaxin as chemoattractant (Figure 3C). Data show the typical curve of chemokine-induced migration, with a peak cell migration induced at 100 ng/mL. CCR3 ligand eotaxin/CCL11 is expressed by CD30+ CTCL We investigated the expression pattern of CCR3 ligands in CD30+ and CD30 CTCL. Immunohistochemical
staining of lesional skin for eotaxin/CCL11, MCP-3/CCL7, and
RANTES/CCL5 was performed. Positive dermal immunoreactivity for
eotaxin/CCL11 occurred in 17 of 18 CTCLs (Table 1). In
CD30+ CTCL, eotaxin/CCL11 protein was detected in tumor
cell aggregates demonstrated as being CD30+ in serial
sections (Figure 4B). Eotaxin/CCL11
protein was also detected in connective tissue cells surrounding tumor
cells. Each of 8 CD30+ CTCLs showed an immunopositivity for
eotaxin/CCL11 associated with tumor cell aggregate, and in 5 of 8 there
was expression by connective tissue cells (Figure 4B; Table 1). In
contrast, positive staining for eotaxin/CCL11 in CD30
CTCL was confined to connective tissue cells, while
infiltrating lymphoma cells were negative (Figure 4A; Table 1). In one
case of CD30 CTCL, eotaxin/CCL11 was also present in
infiltrating lymphoma cells. Eosinophil infiltration was found in each
of 5 CD30+ large-cell CTCLs and in 3 of 12 CD30 CTCLs (Table 1). Staining for RANTES/CCL5 was
positive in 4 CD30+ CTCLs, while MCP-3/CCL7 was expressed
in only 1 case (Table 1).
To verify eotaxin/CCL11 expression in infiltrating cells on a
single-cell level, we performed intracellular eotaxin/CCL11 staining of
cells isolated from freshly obtained biopsy specimens of patients with
CD30+ CTCL. As shown in Figure
5, eotaxin/CCL11 protein was detected in
cells with high forward- and side-scatter properties corresponding to
infiltrating tumor cells.
CCR3+ tumor cells express IL-4 protein It has been previously shown that CD30+ CTCL lesions contain mRNA related to Th-2 cytokine differentiation such as IL-4.24 To analyze the functional differentiation state of CCR3-bearing tumor cells, we performed 3-color staining of tumor cell suspensions obtained from lesional CD30+ CTCL skin. Staining of tumor cell suspensions with IFN- , IL-4, and CCR3 showed
a clear predominance of IL-4+ cells within the
CCR3+ cell population (Figure
6; patient no. 8; representative of 3 experiments). Ratio of IL-4- to IFN--expressing cells was 24 (12.3% versus 0.5%). This indicates a skewing of CCR3+
cells toward Th-2 cytokine production. Analysis of CCR3
lesional cells did not demonstrate a predominance of IFN- or IL-4
protein (data not shown).
We found selective expression of the chemokine receptor CCR3 in
CD30+ large-cell CTCL. No CCR3 expression was seen in
CD30 CTCLs are a group of lymphoproliferative disorders with clonal
expansion of transformed T cells in skin.25 Surface
expression of CD30 distinguishes a subtype of large-cell CTCL with slow
progression, indolent behavior, and favorable
prognosis.26-29 CD30 is a member of the tumor necrosis
factor receptor family, which was originally described as Ki-1 antigen
on Hodgkin and Reed-Sternberg cells in Hodgkin
disease.30,31 The favorable prognosis of CTCL may be
related in part to the fact that dissemination to other body compartments occurs only late during disease development. This might be
suggestive of chemotactic forces that keep lymphoma cells confined to
the skin. The insight into the field of chemokine/chemokine receptor
interactions has been developing rapidly.32 Recently, chemokine receptor expression has been correlated with differential recruitment of polarized Th-1 or Th-2 T cells as well as secretion of
Th-1 or Th-2 cytokines.33-37 It has been shown
that CCR3 is preferentially expressed in vitro by Th-2
cells.33,35 In this context, the presence of eosinophils
as well as expression of Th-2 cytokine mRNA in lesions of
CD30+ cutaneous lymphomas is of interest.24 We
reasoned that recruitment of CD30+ Th-2-like lymphoma
cells might be mediated by the presence of CCR3 ligands in skin in
association with constitutive expression of CCR3 on lymphoma cells. The
availability of a monoclonal antibody to CCR3 allowed us to perform the
present study.38 Seven of 8 CD30+ CTCLs
demonstrated expression of CCR3 on lesional tumor cells (Table 1;
Figure 1). This finding was confirmed by flow cytometric staining of
tumor cell suspensions (Figure 2). Interestingly, there was high
expression of CCR3 on lymphoma cells, comparable to the expression on
eosinophils, which was also reflected by strong staining intensity for
CCR3 with the use of immunohistochemistry (Figure 1).
CD30 Investigation of receptor functionality in fresh tumor cell suspensions is limited by both the low number and the heterogeneity of cells. To further address functionality of the CCR3 receptor, we used the CD30-expressing cutaneous lymphoma cell line Mac-1. Engagement of the CCR3 receptor by its ligand eotaxin led to a signal toward the cytoskeleton involved in cell migration: actin polymerization (Figure 3B). Furthermore, directed migration of CD30+ lymphoma cells toward an eotaxin gradient was observed (Figure 3C). To establish a relationship between CCR3 expression and Th-2 cytokine
production, we assessed the cytokine expression profile of
CCR3+ tumor cells. Earlier reports have demonstrated Th-2
cytokine mRNA in lesions of CD30+ CTCL.24 On
the protein level, we observed strong expression of IL-4 by the
majority of CCR3+ cells, while few cells expressed IFN- The CCR3 ligand eotaxin/CCL11 is known to be produced by human dermal
fibroblasts.44,45 Eotaxin/CCL11 not only has agonistic functions but is also a natural antagonist for
CCR2.46 Our data demonstrate the presence of eotaxin/CCL11
protein in skin of CTCL lesions (Table 1; Figure 4). In
CD30+ as well as in CD30 In summary, we have shown functional CCR3 expression in CD30+ large-cell CTCL as well as expression of its ligand eotaxin in skin. Expression of CCR3 on CD30+ T cells may provide a link between the recruitment of lymphoma cells and their functional state as Th-2 cells. As suggested for breast cancer,48 pharmacological modulation of chemokine receptors may open the way to new treatment modalities.
We are especially grateful to LeukoSite for providing the 7B11 antibody. We thank B. Fruet, C. Dudli, and B. Mueller for technical assistance with immunohistochemical staining.
Submitted February 12, 2002; accepted August 27, 2002.
Prepublished online as Blood First Edition Paper, September 26, 2002; DOI 10.1182/blood-2002-02-0475.
Supported by grants from the Swiss and French Cancer Leagues (to F.O.N.).
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: Frank O. Nestle, Department of Dermatology, University of Zürich Medical School, Gloriastrasse 31, 8091 Zürich, Switzerland; e-mail: nestle{at}derm.unizh.ch.
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Abstract
Introduction
80°C. Representative serial 5- to 7-µm cryostat sections were mounted on polylysine-coated slides and fixed in 100% acetone for 10 minutes. After washing in
phosphate-buffered saline (PBS) without Ca or Mg (Biochrom, Berlin,
Germany), nonspecific antibody binding sites were blocked by
means of normal rabbit serum (DAKO, Copenhagen, Denmark), for 15 minutes at room temperature. For chemokine receptor staining, the
following antibodies were used: 5 µg/mL mouse immunoglobulin G2a (IgG2a) antihuman CCR3 (monoclonal antibody
[moAb] 7B11 [kindly provided by LeukoSite, Cambridge, MA]
as well as monoclonal rat IgG2A [R&D Systems, Abingdon, United
Kingdom]); CCR4 (rabbit polyclonal IgG [Santa Cruz Biotechnology,
CA); and CCR8 (goat polyclonal IgG [Alexis, Lausen, Switzerland]).
For chemokine staining, the following antibodies were used: 1:100 ratio
of antihuman eotaxin to CCL11 moAb (R&D Systems); 1:30 ratio
of antihuman monocyte chemoattractant protein 3 (MCP-3) to CCL7
(Pharmingen/BD Biosciences, Basel, Switzerland); and 1:30
ratio of antihuman RANTES (regulated on activation, normal T expressed
and secreted) to CCL5 (Pharmingen/BD Biosciences). For
chemokine receptor staining, negative controls with antibody diluent
(DAKO) instead of primary antibodies were included. For chemokine
staining, the following isotype controls were used: 10 µg/mL mouse
IgG1 (Ancell, Bayport, MN); mouse IgG2a (Ancell); and 1:300 dilution of
normal goat serum (DAKO). Incubation time was 1 hour at room
temperature for chemokine receptor staining; chemokine Abs and
corresponding isotype controls were incubated overnight at room
temperature. As detection system, the alkaline phosphatase/anti-alkaline phosphatase system followed by New Fuchsin (DAKO) as substrate, containing levamisole for blocking of endogenous alkaline phosphatase, was used as described.
