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NEOPLASIA
From the Department of Internal Medicine I, Division of
Hematology & Hemostaseology, Institute of Immunology, Institute of
Pathophysiology, Department of Obstetrics and Gynecology, Division of
Gynecological Endocrinology and Reproductive Medicine, University of
Vienna, Austria; Axxima Pharmaceuticals, Martinsried, Germany;
Servicio de Hematologia, Mast Cell Unit, Hospital Ramón y Cajal,
University of Alcalá de Henares, Madrid, Spain; and St Anna
Children's Hospital, Vienna, Austria.
Recent data suggest that mast cells (MCs) in patients with systemic
mastocytosis or mast cell leukemia express a CD2-reactive antigen.
To explore the biochemical nature and function of this antigen, primary
MCs as well as the MC line HMC-1 derived from a patient with mast cell
leukemia were examined. Northern blot experiments revealed
expression of CD2 messenger RNA in HMC-1, whereas primary nonneoplastic
MCs did not express transcripts for CD2. In cell surface staining
experiments, bone marrow (BM) MCs in systemic mastocytosis (n = 12)
as well as HMC-1 cells (30%-80%) were found to express the T11-1 and
T11-2 (but not T11-3) epitopes of CD2. By contrast, BM MCs in
myelodysplastic syndromes and nonhematologic disorders (bronchiogenic
carcinoma, foreskin phimosis, uterine myeomata ) were consistently
CD2 Mastocytosis is a term collectively used for a
heterogeneous group of disorders characterized by abnormal
proliferation and accumulation of mast cells (MCs) in one or multiple
organs.1-5 Cutaneous and systemic variants of the disease
have been described.1-8 Cutaneous mastocytosis is usually
detected in childhood and shows a benign course with spontaneous
regression in many cases.8 Systemic mastocytosis (SM) may
develop at any age throughout lifetime and is characterized by
persistent disease and multiorgan involvement.1-7 The
formation of multifocal dense aggregates of MCs in the bone marrow (BM)
is the pathologic hallmark of SM.9 In a small group of
patients, a malignant course or even mast cell leukemia (MCL) develops.1-5,9-12 Little is known, however, about the
mechanisms that contribute to abnormal growth and accumulation of MCs
or disease progression in patients with mastocytosis.
Recent data suggest that distinct gain-of-function mutations in
C-KIT, a gene that encodes the tyrosine kinase
receptor for stem cell factor (SCF, a major growth factor for MCs), are
involved in the pathophysiology of mastocytosis. Such point mutations
are associated with ligand-independent receptor activity and autonomous growth of mast cells.13,14 One of these mutations,
Asp816Val (C-KIT), has been detected in most patients with
SM and MCL15-17 as well as in the human MCL-derived cell
line HMC-1.13 The emerging hypothesis is that the
mutation-induced activation of the SCF receptor (KIT) is directly
involved in the abnormal growth of MCs in patients with SM. The notion
that patients with SM frequently exhibit C-KIT mutations
also supports the concept that SM is a clonal neoplastic
disease.15-17
A number of attempts have been made in the past to establish the
surface receptor profile of MCs in patients with mastocytosis. In
common with normal MCs, MCs in patients with SM or MCL express substantial amounts of KIT.12,18-23 Other surface
receptors are also coexpressed on normal and neoplastic
MCs.18-23 However, recent data suggest that several
surface molecules are differentially expressed on neoplastic versus
normal MCs.20-23 The observation that MCs in patients with
SM or MCL react with antibodies against leukocyte function-associated
antigen-2 (LFA-2, or CD2) is a remarkable finding. In fact, LFA-2 is
expressed physiologically on thymocytes, activated T cells, and a
subset of natural killer cells,24-32 whereas normal MCs
and other normal myeloid cells apparently are CD2 The CD2 antigen (LFA-2) was originally characterized as a T-cell
molecule that mediates sheep erythrocyte rosette
formation.24 However, the CD2 molecule on T lymphocytes
contains several distinct epitopes with specific functional
properties.24-32 Moreover, these epitopes are variably
expressed on T cells. Thus, the T11-1 and T11-2 epitopes of CD2 are
expressed on both resting and activated T cells, whereas the T11-3
epitope is only expressed when T cells are activated by antigen or
other stimuli.24-32 The T11-1 epitope on T cells is
primarily involved in adhesive cell functions (including sheep
erythrocyte binding), whereas the T11-3 epitope appears to be
associated with cell activation and related biochemical processes.24-32
The aims of the present study were to analyze expression of CD2 in
normal and neoplastic human MCs at the messenger RNA (mRNA) and protein
level, to study the epitope composition of CD2 on MCs, and to examine
the regulation and functional role of CD2 epitopes on MCs.
Monoclonal antibodies
Cytokines, buffers, and other reagents
Isolation and purification of primary MCs Primary MCs were isolated from BM, lung, foreskin, and uterus. In each case, informed consent was obtained from patients or parents (circumcision of juvenile foreskin) before surgery or BM biopsy. BM was obtained from the iliac crest of 13 patients with indolent SM and 8 with myelodysplastic syndromes (MDSs; subtypes: refractory anemia [RA], n = 2; RA with ringed sideroblasts [RARS], n = 1; RA with excess of blasts [RAEB], n = 2; RAEB in transformation [RAEB-T], n = 3). Diagnoses were established according to published criteria.5,33 None of the MDS patients exhibited focal dense infiltrates of BM MCs excluding a coexisting SM. BM was also obtained from 4 donors with reactive MC hyperplasia and 1 with normal BM. Aspirated BM was collected in preservative-free heparin, diluted 1:1 (vol/vol) in phosphate-buffered saline, and prepared for flow cytometry as described.20,21 BM mononuclear cells (MNCs) were isolated using Ficoll. Surgical specimens of lung (lobectomy; 15 patients with bronchiogenic carcinoma), uterus (uterine myomata, n = 5), and juvenile foreskin (n = 5) were collected and subjected to MC isolation as described.34 In brief, tissue was chopped, washed in Mg2+/Ca++-free Tyrode buffer, and then incubated in collagenase type II (2 mg/mL) at 37°C for 1 to 3 hours. Dispersed cells were recovered by filtration through Nytex cloth and washed. Cell suspensions were cultured at 37°C in RPMI 1640 medium with 10% FCS and antibiotics for at least 24 hours before analysis. In stimulation experiments, lung and uterus MCs were incubated in rhSCF (100 ng/mL) or control medium for up to 48 hours. In 7 donors, lung cells were further enriched for MC by elutriation as described.34 In brief, cells were loaded at a flow rate of 12 mL/min into a Beckman elutriator equipped with a JE-6B rotor (Beckman Instruments, Palo Alto, CA). Fractions were recovered at increasing flow rates (14, 18, 20, 30 mL/min). A selection was performed based on the content of MCs. In one donor, 2 fractions contained more than 90% MCs (total cell number 6 × 107). These MCs were cultured overnight (37°C, 5% CO2), washed, split, exposed to either rhSCF (100 ng/mL) or control medium for 2 hours (37°C), and then subjected to RNA isolation. In the other 6 donors, elutriated MCs were further enriched by cell sorting using anti-KIT mAb YB5.B8.34 MCs were sorted as KITbright cells and were more than 98% pure after isolation. The 6 lung MC preparations were pooled (total cell number 7 × 106) and used for Northern blot experiments.Culture of HMC-1 cells and sorting of CD2+ and
CD2  namely, a CD2+ and a CD2
subpopulationsorting experiments were conducted to separate these 2 cell subsets using the PE-conjugated mAb RPA-2.10 (CD2) and a FACStar
Plus (Becton Dickinson). In stimulation experiments, HMC-1 cells
(subpopulations and unseparated) were exposed to CD2 antibodies
(39C1.5, 6F10.3, VIT13; 3-6 µg/mL), rh cytokines (SCF, 10-1000 ng/mL;
other cytokines, 100 ng/mL), or control medium for 1 to 48 hours
(37°C).
Culture of human MCs from cord blood cell-derived progenitors To analyze expression of CD2 on immature "nonneoplastic" MCs, we cultured MCs from their CD34+ cord blood progenitors. Cord blood was obtained from full-term deliveries (n = 11) after informed consent was given by mothers. MNCs were obtained using Ficoll and enriched for CD34+ cells by mAb QBEND/10 and magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) as described.36 After MACS, CD34+ cells were further enriched by fluorescence-activated cell sorting (FACS) using mAb 8G12 (CD34) and a FACS Vantage SE (Becton Dickinson). The resulting purity of CD34+ cord blood MNCs was more than 98%. The MC differentiation assay was performed according to published techniques.37-39 In brief, cells were cultured in RPMI 1640 medium containing 10% FCS, L-glutamine, antibiotics, and rhSCF (100 ng/mL) for up to 63 days (37°C, 5% CO2). During the first 7 days, SCF was used as the single growth factor. Thereafter, cells were divided: One part was maintained in rhSCF (100 ng/mL) and rhIL-6 (100 ng/mL) and the other part in rhSCF (100 ng/mL), rhIL-4 (10 U/mL), rhIL-6 (100 ng/mL), and rhIL-10 (10 ng/mL). Cultures were fed in 2-week intervals. On days 21, 42, and 63, cultured cells were examined for viability, cell number, morphology, and expression of CD2 and other antigens.Cell surface staining techniques HMC-1 and cultured cord blood MNC-derived MCs were analyzed by single-color flow cytometry. Surface marker expression on lung and uterine MCs was analyzed by 2-color staining using unconjugated "first-step" mAb, FITC-conjugated "second-step" antibody, and PE-labeled CD117 mAb as described.40 Expression of surface antigens on BM MCs was determined by 3- or 4-color staining using conjugated mAb (FITC: CD2, CD25; PE: CD2, CD117; peridinin chlorophyll protein: CD45; APC: CD117) as reported.20-23,41 In brief, 1.5 × 106 cells were incubated with combinations of mAb (CD2/CD117/CD45 or CD25/CD117/CD45) for 10 minutes. Erythrocytes were then lysed in FACS lysing solution (Becton Dickinson). After washing, cells were examined by flow cytometry on a FACSCalibur or FACScan (Becton Dickinson). Staining reactions were controlled using isotype-matched mAb. To analyze surface marker expression on single metachromatic MCs (lung, n = 7; foreskin, n = 5; uterus, n = 4; mastocytosis-BM, n = 3; cultured MCs), a toluidine blue/immunofluorescence (TB/IF) double-staining technique was applied.42,43 Briefly, cells were incubated with mAb for 30 minutes (4°C), washed, and exposed to FITC-conjugated goat antimouse antibody. After washing, cells were fixed in glutaraldehyde (0.025%) for 1 minute, washed, and stained with TB (0.0125%) for 8 minutes. Then, cells were again washed and examined under a fluorescence microscope.In situ staining experiments To confirm expression of CD2 in neoplastic MCs (focal BM infiltrates in SM), in situ staining was performed on BM sections (SM, n = 8; RAEB, n = 2; MC hyperplasia, n = 3). BM biopsy specimens were fixed in formalin, decalcified in ethylenediaminetetraacetic acid, and embedded in paraffin. Immunohistochemistry was performed on serial BM sections (2 µm) by avidin-biotin-immunoperoxidase staining technique.44-46 Endogenous peroxidase was blocked with 0.3% H2O2. The mAbs AB75 (CD2) and G3 (antitryptase) were diluted (AB75: 1:20; G3: 1:5000) in TBS with 1% bovine serum albumin (Behring, Marburg, Germany) and applied for 60 to 90 minutes. For CD2 staining, sections were heated in an autoclave prior to antibody exposure. Isotype-matched mAbs were used as negative control. After exposure to first-step mAbs, slides were washed and incubated with biotinylated horse antimouse immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA) for 30 minutes. After washing, slides were exposed to avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) for 30 minutes. DAB (CD2) or AEC (antitryptase) was used as chromogen. Slides were counterstained in Mayer hemalum and mounted. Double immunohistochemistry was also performed. For this purpose, CD2-stained slides were photographed, remounted, washed, and then stained with antitryptase mAb G3 (1:5000). In a separate set of experiments, serial sections of lesional skin obtained from 3 patients with cutaneous mastocytosis and 3 with SM were examined. Immunocytochemistry was performed on HMC-1, lung MCs, and cultured MCs. HMC-1 and lung MCs were exposed to control medium or rhSCF (100 ng/mL) for 1 to 48 hours. After incubation, cells were spun on cytospin slides, fixed in acetone, washed, and incubated with CD2 mAb 6F10.3 (1:20) or antitryptase mAb G3 (1:5000) for 60 minutes. Slides were then washed and incubated with biotinylated horse antimouse IgG. Slides were again washed and exposed to streptavidin-biotin-alkaline phosphatase complex (Dako, Glostrup, Denmark) for 30 minutes. Antibody reactivity was made visible with vector red alkaline phosphatase substrate.Northern blot analysis Total RNA was extracted from untreated and SCF-treated lung MCs and HMC-1 cells by the guanidinium isothiocyanate/cesium chloride extraction technique.47 Northern blot analysis was performed as described.48,49 In brief, 10 µg RNA was size-fractionated on 1.2% agarose gels, transferred to synthetic membranes (Hybond N, Amersham, Little Chalfont, United Kingdom) with 20 × SSC (1 × SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0) overnight, and cross-linked to membranes by UV irradiation (UV Stratalinker 1800, Stratagene, CA). RNA was prehybridized at 65°C for 4 hours in 5 × SSC, 7% sodium dodecyl sulfate (SDS), 10 × Denhardt solution (1 × Denhardt solution = 0.02% bovine serum albumin, 0.02% polyvinyl pyrolidone, and 0.02% Ficoll), 10% dextran sulfate, 20 mM sodium phosphate (pH 7.0), sonicated salmon sperm DNA (100 µg/mL), and poly(A)+ (100 µg/mL). Hybridization was performed with a 32P-labeled synthetic oligonucleotide probe specific for CD2 (29-mer: 5'-GCTGACAGGCTCGACACTGGATTCCTTGC-3', gene position: base pairs 601-629).50 After stripping the blot, a probe specific for GAPDH (29-mer: 5'-CCATGGTGGTGAAGACGCCAGTGGACTCC-3')51 was applied. All probes were from MWG Biotech (Ebersberg, Germany). Blots were washed once in 3 × SSC, 5% SDS, 10 × Denhardt solution, and 20 mM sodium phosphate, pH 7.0, for 30 minutes at 65°C, and once in 1 × SSC and 1% SDS for 30 minutes at 65°C. Bound radioactivity was visualized by exposure to XAR-5 films at 70°C using intensifying screens (Eastman Kodak, Rochester, NY).
Aggregation assay and sheep erythrocyte rosette formation To demonstrate functional significance of CD2 expression, a sheep erythrocyte rosette formation assay was applied. For this purpose, primary BM MCs (SM, n = 2; reactive BM, n = 1) were highly purified (> 98% purity) by cell sorting using a CD117 mAb as described.34 In addition, HMC-1 subsets (CD2+ and CD2) were examined. Cells were incubated with sheep
erythrocytes (Behring) at 37°C for 15 minutes. Cells were then
centrifuged at 200g and incubated at 4°C for 18 hours. In
blocking experiments, CD2+ HMC-1 cells were preincubated
with the T11-1 antibody 39C1.5 (100 µg/mL), isotype-matched control
antibody, or control medium for 30 minutes (4°C) and then washed
prior to incubation with sheep erythrocytes. Sheep erythrocyte rosette
formation and formation of aggregates was analyzed using an inverted
microscope (Olympus, Vienna, Austria).
Histamine release experiments Histamine release experiments were conducted using BM MNCs (SM, n = 2; reactive BM, n = 2), unseparated HMC-1, as well as sorted HMC-1 subsets (CD2+ vs CD2 subset). Histamine
release was performed essentially as described.52 Cells
were resuspended in histamine release buffer (Immunotech) and adjusted
to a final cell concentration of 0.5 × 106/mL. Cells
were incubated with mAb 39C1.5 (T11-1) (3 µg/mL), 6F10.3 (T11-2) (3 µg/mL), or VIT13 (T11-3) (3 µg/mL), a combination of mAbs (3 or 6 µg/mL of each mAb), or control buffer for 3 hours at 37°C. After
incubation, cells were centrifuged and the cell-free supernatants
recovered. Liberated histamine was measured by radioimmunoassay (Immunotech). Total histamine was quantified in cell lysates. Histamine
release was expressed as percentage of total histamine.
Statistical analysis To determine the levels of significance, appropriate statistical tests including the paired Student t test and ANOVA were applied. Results were considered to be statistically significant at P < .05.
Evaluation of CD2 expression on BM MCs Three different staining techniques were applied to investigate CD2 expression in BM MCs in patients with SM, MDSs, and normal or reactive BM. As assessed by multicolor flow cytometry, the KIT+ BM MCs in patients with SM (n = 12) stained positive with CD2 mAb S5.2 and CD25 mAb 2A3 (Figure 1 and Table 1). Moreover, BM MCs in SM were found to react with the epitope-specific CD2 mAbs 39C1.5 (T11-1) and 6F10.3 (T11-2) but did not react with the T11-3-specific mAb VIT13. BM MCs in MDSs (n = 6) or normal or reactive BM (n = 3) were not recognized by any of the CD2 mAbs applied. Expression of CD2 on BM MCs in SM was confirmed by combined TB/IF staining. In these experiments, TB+ BM MCs were recognized by mAbs against KIT, CD25, and CD58 as well as by the CD2 mAbs 39C1.5, 6F10.3, S5.2, and T11 but not by mAb VIT13 (T11-3). These data show that BM MCs in SM express T11-1 and T11-2 but not T11-3 epitopes of CD2. In a next step, CD2 expression in BM MCs was analyzed by double immunohistochemistry. These experiments revealed expression of CD2 in spindle-shaped neoplastic tryptase-positive BM MCs (Figure 2). Serial section staining confirmed expression of CD2 in BM MCs in SM. In MDSs and normal or reactive BM, MCs did not stain positive for CD2. These results suggest that CD2 (T11-1/T11-2) is selectively expressed in BM MCs in patients with SM. A summary of staining results is shown in Table 1.
Evaluation of CD2 expression on primary MCs obtained from extramedullary organs The phenotype of MCs may depend on the organ/tissue environment. We therefore asked if CD2 could be a marker of organ-specific phenotypic heterogeneity of MCs. To address this question, we analyzed expression of CD2 in primary nonneoplastic MCs in extramedullary organs. MCs were isolated from lung, uterine tissue, and juvenile foreskin. As assessed by multicolor flow cytometry, KIT+ MCs in various organs expressed CD58 but did not express detectable amounts of CD2 (Table 2). Identical results were obtained by combined TB/IF staining. In fact, MCs from lung, uterus, and foreskin reacted with mAbs against KIT and CD58 but did not react with CD2 mAbs (Table 2). In a separate set of experiments, CD2 expression in MC in lesional skin in patients with SM (n = 3) or cutaneous mastocytosis (n = 3) was analyzed by immunohistochemistry. In all patients, skin MCs stained positive for tryptase. In SM, skin MCs were also found to react with CD2 antibody AB75, whereas skin MCs in cutaneous mastocytosis were found to be CD2. Thus, expression of CD2 in MC seems to be a
disease-related phenomenon but not determined by a specific
environment.
Evaluation of CD2 expression on immature cultured MCs We next asked whether CD2 is expressed at an early stage of MC development. To address this question, cultured immature (nonneoplastic) MCs derived from their CD34+ cord blood progenitors were examined. MC progenitors were cultured in the presence of SCF+IL-6 or SCF+IL-6+IL-4+IL-10 and analyzed on days 21, 42, and 63. As assessed by flow cytometry, cultured MCs were found to react with mAb against KIT and CD58 (Table 3). However, independent of the culture condition applied, MCs failed to react with CD2 mAbs (Table 3). Cultured MCs were also found to be negative for CD25 (IL-2R ) and CD123 (IL-3R).
Detection of CD2 mRNA in neoplastic MCs To study CD2 expression in normal and neoplastic MC at the mRNA level, Northern blot experiments were performed using a CD2-specific oligonucleotide probe and RNA of unstimulated or SCF-stimulated lung MCs and unstimulated or SCF-stimulated HMC-1 cells. HMC-1 cells were found to express CD2 mRNA in a constitutive manner (Figure 3). By contrast, lung MCs did not express detectable transcripts for CD2 (Figure 3). Incubation of HMC-1 cells with rhSCF (100 ng/mL, 2 hours) did not result in a change of expression of CD2 mRNA. SCF also failed to induce expression of CD2 mRNA in lung MCs.
Detection of CD2 epitopes on HMC-1 cells Six different CD2 mAbs were applied to detect the CD2 antigen on HMC-1 cells. As assessed by flow cytometry, unstimulated HMC-1 cells were recognized by all CD2 mAbs applied except VIT13 (T11-3) (Table 2). These data suggest that HMC-1 expresses T11-1 and T11-2 epitopes of CD2 but does not express T11-3. Interestingly, 2 distinct subpopulations of HMC-1 were detected by flow cytometrynamely, a distinctively
CD2+ and a distinctively CD2 cell fraction.
These 2 populations were seen with all CD2 mAbs applied (except VIT13)
(Figure 4). The CD2+ fraction
accounted for 30% to 80% of all HMC-1 cells.
Separation and properties of CD2+ and CD2 HMC-1 cells were separated from each other by cell
sorting and cultured separately for 140 days. In all 3 experiments,
both cell populations showed comparable survival and growth
characteristics as well as comparable morphologic features. After
sorting, most of the surface CD2+ HMC-1 cells appeared to
be CD2+ by immunocytochemistry, whereas the surface
CD2 cells were all CD2 in their cytoplasm
(Figure 2C,D). During the entire culture period (140 days), HMC-1
sorted for CD2 remained all CD2+, and the sorted
CD2 cells all remained CD2 (Figure
5).
Sheep erythrocyte rosette formation of CD2+ MCs The CD2 antigen was originally described as sheep erythrocyte receptor. In the present study, primary BM MCs and HMC-1 cells were examined for their capacity to bind sheep erythrocytes in a specific manner. For this purpose, BM MCs of patients with SM (n = 2) and reactive marrow (n = 1) as well as HMC-1 cells (CD2+ versus CD2 subset) were examined. In both patients with
SM, BM MCs (50% and 70%, respectively) were found to form rosettes
with sheep erythrocytes (Figure 6A),
whereas no rosette formation was seen on MCs in the reactive marrow
(< 10% of MC formed rosettes) (Figure 6B). Corresponding results
were obtained with HMC-1 cells. In particular, when cultured in the
presence of sheep erythrocytes, CD2+ HMC-1 cells were found
to form significantly more rosettes than the CD2 subset
(P < .05) (Figure 6C,D and Table
4). In addition, the CD2+
HMC-1 cells showed an increase in spontaneous formation of aggregates compared with CD2 cells (10%-30% increase). The
increased sheep erythrocyte rosette and aggregate formation of
CD2+ HMC-1 cells was inhibited by preincubation with mAb
39C1.5 directed against the T11-1 epitope of CD2, whereas no inhibition
was seen with antibodies against T11-2 or T11-3.
Modulation of expression of CD2 epitopes on MCs In line with previous observations,53 incubation of HMC-1 with rhSCF (100 ng/mL; 1-48 hours) resulted in decreased expression of KIT. In contrast, no difference in binding of CD2 mAbs to HMC-1 cells was seen after SCF incubation. The other cytokines tested (IL-1 through IL-18, GM-CSF, G-CSF, M-CSF) also failed to modulate expression of CD2 on HMC-1. We next asked whether expression of CD2 on tissue MCs is inducible by cytokines such as SCF. However, incubation of primary lung or uterine MCs with rhSCF was not followed by CD2 expression. In a next step, HMC-1 subsets were stimulated with epitope-specific mAbs. In these experiments exposure of CD2+ HMC-1 cells to mAb against T11-1 (39C1.5) or T11-2 (6F10.3) resulted in detectable expression of the T11-3 epitope (Figure 7).
Effects of CD2 antibodies on histamine release Activation of T cells through CD2 can be initiated by 2-epitope stimulation (T11-2, T11-3) using a mixture of specific mAbs. To examine whether activation of neoplastic MCs via CD2 is associated with cell activation, histamine release experiments were performed using BM MCs (SM, n = 2; control cases, n = 2) and HMC-1 subsets (CD2+ versus CD2). Three different CD2 mAbs
were applied. In 2 SM patients, exposure of BM MCs to a combination of
CD2 mAbs against T11-3 and T11-1 or against T11-3 and T11-2 resulted in
a substantial release of histamine above control (Figure
8). A slight increase in release was also
obtained by using only one CD2 mAb. By contrast, in the control cases
(reactive BM), neither the mAb combinations nor the single mAbs applied
were found to induced histamine release above control (not shown).
Corresponding results were obtained with HMC-1 cells. In fact,
incubation of CD2+ HMC-1 cells with a mixture of mAbs
specific for T11-2 and T11-3 resulted in a significant increase in
spontaneous histamine release (121% ± 32% of control, n = 6,
P < .05), whereas no increase in histamine release in
response to CD2 mAbs (alone or in combination) was seen when
CD2 HMC-1 cells were examined (T11-2 plus T11-3:
89% ± 6% of control, n = 3, P > .05).
Recent data suggest that MCs in patients with SM express a CD2-reactive surface antigen.20-23 In the present study, we have examined the biochemical nature and function of this antigen using primary MCs as well as the MC line HMC-1. The results of our study show that MCs in patients with SM as well as HMC-1 express T11-1 and T11-2 epitopes of CD2. Expression of CD2 was confirmed at the mRNA and protein level as well as by functional studies. In fact, primary neoplastic MCs as well as HMC-1 formed rosettes with sheep erythrocytes. Moreover, incubation of neoplastic MCs with T11-1 or T11-2 antibodies resulted in exposure of the T11-3 epitope, and costimulation through T11-3 resulted in substantial release of histamine. These data point to a functional role of CD2 epitopes on neoplastic MCs. To provide evidence for specific expression of CD2 on MCs in systemic MC disease, we analyzed patients with SM, MDS, as well as normal or reactive BM. However, the CD2 antigen was only detectable on BM MCs in SM but not in other disease states. Selective expression of CD2 in SM was also confirmed by immunohistochemistry. All in all, CD2 expression appears to be a specific marker of systemic MC disorders. The phenotype of MCs may depend on diverse factors, including the maturation stage of MCs and the tissue environment.18,42,43 We therefore examined CD2 expression on MCs in various organs as well as in cultured immature MC progenitors. In a first step, MCs in various organs were analyzed. However, CD2 was not detected on lung MCs, uterine MCs, or foreskin MCs in patients without mastocytosis. By contrast, in patients with SM and cutaneous involvement, skin MCs were found to be CD2+. These data suggest that CD2 expression in MCs is determined by the disease rather than by the tissue environment. In a next step we asked if CD2 expression occurs in an early phase of MC development. To address this question, immature MCs cultured from their CD34+ progenitor cells were examined. However, at all stages of differentiation analyzed, MC progenitors failed to express CD2. These data further confirmed that CD2 expression on MCs is a disease-specific abnormality. The HMC-1 cell line is derived from a patient suffering from
MCL.35 Like primary neoplastic MCs in SM, HMC-1 cells
contain the C-KIT mutation
Asp816Val.13 HMC-1 cells are also known to stain positive for CD2.54 In the present study, the HMC-1
cell line was applied as a useful tool for the investigation of
expression and function of CD2 epitopes on neoplastic MCs. Like primary
MCs in patients with SM, resting HMC-1 cells were found to react with antibodies against T11-1 and T11-2 epitopes of CD2 but did not react
with T11-3 antibody. A remarkable finding was that HMC-1 is composed of
2 distinct subsets The typical formation of MC aggregates in the BM of patients with
SM is the histological hallmark of the disease.1-5,9 Because normal and neoplastic MCs consistently express CD58, the natural ligand of CD2, we were interested to know whether the CD2
antigen on neoplastic MCs would fulfill adhesive functions. For this
purpose, we analyzed the formation of sheep erythrocyte rosettes as
well as aggregate formation in sorted HMC-1 subsets. In these
experiments, the CD2+ HMC-1 cell subset formed sheep
erythrocyte rosettes and aggregates, whereas only few, if any, rosettes
were detectable in CD2 Little is known about the regulation of expression of CD2 in MCs.
We have addressed this issue by incubating primary normal MCs and HMC-1
cells with a number of cytokines and CD2 antibodies. So far, we have
not been able to identify a cytokine that would induce expression of
CD2 in normal MCs, MC progenitors, or CD2 In T cells, CD2 has been characterized as a critical functional
surface antigen that can mediate major activation
signals.24-32 In previous years, the activation signals
delivered by CD2 were thought to be restricted to T cells. In later
years, however, it was found that CD2 is also expressed in other cells.
In 1991, Arulanandam et al described that CD2-transfected mouse MCs
release histamine in response to T11-2 plus T11-3
antibody.55 We therefore asked whether MCs in SM could be
a potential target of CD2-dependent triggering and could release
histamine in response to CD2 mAbs. For this purpose, we analyzed
histamine release in resting and epitope-stimulated HMC-1 cells as well
as primary BM MCs in patients with SM. Interestingly, in SM, incubation
of isolated BM MCs with T11-2 plus T11-3 mAbs or T11-1 plus T11-3 mAbs
resulted in release of histamine, whereas no release was obtained with
MCs enriched from reactive BM. Corresponding results were obtained with
HMC-1 cells. In fact, when CD2+ HMC-1 cells were exposed to
T11-2 and T11-3 mAbs, a significant increase in spontaneous histamine
release was observed, whereas no increase in histamine release was seen
in CD2 In conclusion, we show that neoplastic MCs in patients with SM express functionally active LFA-2 (CD2) epitopes on their surface. The notion that MCs in SM specifically express functional CD2 may have pathophysiologic and clinical implications.
We thank Yasamin Majlesi and Hans Semper for skillful technical assistance.
Submitted December 6, 2000; accepted August 13, 2001.
Supported by Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich (FWF) grant no. P-12517, Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 01/0413), and Fundación Oftalmológica J. Cortés. R.N. is a recipient of a grant FPI from Comunidad de Madrid.
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: P. Valent, Dept of Internal Medicine I, Div of Hematology & Hemostaseology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria; e-mail: peter.valent{at}akh-wien.ac.at.
1. Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: a review. Histopathology. 1979;3:349-365[Medline] [Order article via Infotrieve]. 2. Parwaresch MR, Horny H-P, Lennert K. Tissue mast cells in health and disease. Pathol Res Pract. 1985;179:439-461[Medline] [Order article via Infotrieve]. 3. Metcalfe DD. Classification and diagnosis of mastocytosis: current status. J Invest Dermatol. 1991;96:2S-4S[CrossRef][Medline] [Order article via Infotrieve]. 4. Valent P. Biology, classification and treatment of human mastocytosis. Wien Klin Wochenschr. 1996;108:385-397[Medline] [Order article via Infotrieve]. 5. Valent P, Horny H-P, Escribano L, et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res. 2001;25:603-625[CrossRef][Medline] [Order article via Infotrieve]. 6. Travis WD, Li CY, Bergstralh EJ, Yam LT, Swee RG. Systemic mast cell disease: analysis of 58 cases and literature review. Medicine. 1988;67:345-368[Medline] [Order article via Infotrieve]. 7. Horan RF, Austen KF. Systemic mastocytosis: retrospective review of a decade's clinical experience at the Brigham and Women's Hospital. J Invest Dermatol. 1991;96:5S-13S[CrossRef][Medline] [Order article via Infotrieve]. 8. Caplan RM. The natural course of urticaria pigmentosa. Arch Dermatol. 1963;87:146-153. 9. Horny H-P, Parwaresch MR, Lennert K. Bone marrow findings in systemic mastocytosis. Hum Pathol. 1985;16:808-814[Medline] [Order article via Infotrieve]. 10. Travis WD, Li CY, Hoagland HC, Travis LB, Banks PM. Mast cell leukemia: report of a case and review of literature. Mayo Clin Proc. 1986;61:957-966[Medline] [Order article via Infotrieve]. 11. Dalton R, Chan L, Batten E, Eridani S. Mast cell leukemia: evidence for bone marrow origin of the pathological clone. Br J Haematol. 1986;64:397-406[Medline] [Order article via Infotrieve]. 12. Baghestanian M, Bankl HC, Sillaber C, et al. A case of malignant mastocytosis with circulating mast cell precursors: biologic and phenotype characterization of the malignant clone. Leukemia. 1996;10:159-166[Medline] [Order article via Infotrieve]. 13. Furitsu T, Tsujimura T, Tono T, et al. Identification of mutations in the coding sequence of the protooncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of the c-kit product. J Clin Invest. 1993;92:1736-1744.
14.
Kitayama H, Tsujimura T, Matsumura I, et al.
Neoplastic transformation of normal hematopoietic cells by constitutively activating mutations of c-kit receptor tyrosine kinase.
Blood.
1996;88:995-1004
15.
Nagata H, Worobec AS, Oh CK, et al.
Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.
Proc Natl Acad Sci U S A.
1995;92:10560-10564 16. Longley BJ, Tyrrell L, Lu SZ, et al. Somatic c-kit activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat Gen. 1996;12:312-314[CrossRef][Medline] [Order article via Infotrieve].
17.
Longley BJ, Metcalfe DD, Tharp M, et al.
Activating and dominant inactivating c-kit catalytic domain mutations in distinct forms of human mastocytosis.
Proc Natl Acad Sci U S A.
1999;96:1609-1614 18. Valent P. Mast cell differentiation antigens: expression in normal and malignant cells and use for diagnostic purposes. Eur J Clin Invest. 1995;25:715-720[Medline] [Order article via Infotrieve]. 19. Castells MC, Friend DS, Bunnell CA, et al. The presence of membrane-bound stem cell factor on highly immature nonmetachromatic mast cells in the peripheral blood of a patient with aggressive systemic mastocytosis. J Allergy Clin Immunol. 1996;98:831-840[CrossRef][Medline] [Order article via Infotrieve]. 20. Escribano L, Orfao A, Villarrubia J, et al. Expression of lymphoid-associated antigens in mast cells: report of a case of systemic mast cell disease. Br J Haematol. 1995;91:941-943[Medline] [Order article via Infotrieve]. 21. Escribano L, Orfao A, Villarrubia J, et al. Sequential immunophenotypic analysis of mast cells in a case of systemic mast cell disease evolving to a mast cell leukemia. Cytometry. 1997;30:98-102[CrossRef][Medline] [Order article via Infotrieve].
22.
Escribano L, Orfao A, Diaz-Agustin B, et al.
Indolent systemic mast cell disease in adults: immunophenotypic characterization of bone marrow mast cells and its diagnostic implications.
Blood.
1998;91:2731-2736 23. Escribano L, Orfao A, Villarrubia J, et al. Immunophenotypic characterization of human bone marrow mast cells: a flow cytometric study of normal and pathologic bone marrow samples. Anal Cell Pathol. 1998;16:151-159[Medline] [Order article via Infotrieve]. 24. Meuer SC, Hussey RE, Fabbi M, et al. An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein. Cell. 1984;36:897-906[CrossRef][Medline] [Order article via Infotrieve]. 25. Krensky AM, Sanchez-Madrid F, Robbins E, Nagy JA, Springer TA, Burakoff SJ. The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions. J Immunol. 1983;131:611-616[Abstract]. 26. Selvaraj P, Plunkett ML, Dustin M, Sanders ME, Shaw S, Springer TA. The T lymphocyte glycoprotein CD2 binds cell surface ligand LFA-3. Nature. 1987;326:400-403[CrossRef][Medline] [Order article via Infotrieve]. 27. Peterson A, Seed B. Monoclonal antibody and ligand binding sites of the T cell erythrocyte receptor (CD2). Nature. 1987;329:842-846[CrossRef][Medline] [Order article via Infotrieve]. 28. Sayre PH, Reinherz EL. Structure and function of the erythrocyte receptor CD2 on human T lymphocytes: a review. Scand J Rheumatol. 1988;76:131-144. 29. Bierer BE, Sleckman BP, Ratnofsky SE, Burakoff SJ. The biologic roles of CD2, CD4, and CD8 in T-cell activation. Ann Rev Immunol. 1989;7:579-599[CrossRef][Medline] [Order article via Infotrieve]. 30. Moingeon P, Chang HC, Sayre PH, et al. The structural biology of CD2. Immunol Rev. 1989;111:111-144[CrossRef][Medline] [Order article via Infotrieve]. 31. Bell GM, Imboden JB. CD2 and the regulation of T-cell anergy. J Immunol. 1995;155:2805-2807[Medline] [Order article via Infotrieve]. 32. Holter W, Schwarz M, Cerwenka A, Knapp W. The role of CD2 as a regulator of human T-cell cytokine production. Immunol Rev. 1996;153:107-122[CrossRef][Medline] [Order article via Infotrieve]. 33. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol. 1982;51:189-199[Medline] [Order article via Infotrieve]. 34. Willheim M, Agis H, Sperr WR, et al. Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/C-kit. J Immunol Methods. 1995;182:115-129[CrossRef][Medline] [Order article via Infotrieve]. 35. Butterfield JH, Weiler D, Dewald G, Gleich GJ. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res. 1988;12:345-355[CrossRef][Medline] [Order article via Infotrieve]. 36. De Wynter EA, Coutinho LH, Pei X, et al. Comparison of purity and enrichment of CD34+ from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems. Stem Cells. 1995;13:524-532[Abstract].
37.
Valent P, Spanblöchl E, Sperr WR, et al.
Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture.
Blood.
1992;80:2237-2245 38. Saito H, Ebisawa M, Tachimoto H, et al. Selective growth of human mast cells induced by steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol. 1996;157:343-350[Abstract].
39.
Ochi H, Hirani WM, Yuan Q, Friend DS, Austen KF, Boyce JA.
T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro.
J Exp Med.
1999;190:267-280 40. Wimazal F, Ghannadan M, Müller MR, et al. Expression of homing receptors and related molecules on human mast cells and basophils: a comparative analysis using multi-color flow cytometry and toluidine blue/immunofluorescence staining technique. Tissue Antigens. 1999;54:499-507[CrossRef][Medline] [Order article via Infotrieve]. 41. Orfao A, Escribano L, Villarubia J, et al. Flow cytometric identification and enumeration of mast cells from both normal and pathological human bone marrow samples. Am J Pathol. 1996;149:1493-1499[Abstract].
42.
Valent P, Ashman LK, Hinterberger W, et al.
Mast cell typing: demonstration of a distinct hemopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes.
Blood.
1989;73:1778-1785 43. Ghannadan M, Baghestanian M, Wimazal F, et al. Phenotypic characterization of human skin mast cells by combined staining with toluidine blue and CD antibodies. J Invest Dermatol. 1998;111:689-695[CrossRef][Medline] [Order article via Infotrieve]. 44. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577-580[Abstract].
45.
Irani AA, Schechter NM, Craig SS, De Blois G, Schwartz LB.
Two types of human mast cells that chave distinct neutral protease compositions.
Proc Natl Acad Sci U S A.
1986;83:4464-4468 46. Horny HP, Sillaber C, Menke D, Kaiserling E, Wehrmann M, Stehberger B. Diagnostic value of immunostaining for tryptase in patients with mastocytosis. Am J Surg Pathol. 1998;22:1132-1140[CrossRef][Medline] [Order article via Infotrieve]. 47. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299[CrossRef][Medline] [Order article via Infotrieve].
48.
Valent P, Bevec D, Maurer D, et al.
Interleukin 4 promotes expression of mast cell ICAM-1 antigen.
Proc Natl Acad Sci U S A.
1991;88:3339-3342
49.
Baghestanian M, Hofbauer R, Kiener H-P, et al.
The c-kit ligand stem cell factor and anti-IgE promote expression of monocyte chemoattractant protein 1 (MCP-1) in human lung mast cells.
Blood.
1997;90:4438-4449
50.
Sayre PH, Chang HC, Hussey RE, et al.
Molecular cloning and expression of T11 cDNAs reveal a receptor-like structure on human T lymphocytes.
Proc Natl Acad Sci U S A.
1987;84:2941-2945
51.
Ercolani L, Florence B, Denaro M, Alexander M.
Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene.
J Biol Chem.
1988;263:15335-15341
52.
Valent P, Besemer J, Muhm M, Majdic M, Lechner K, Bettelheim P.
Interleukin-3 activates human blood basophils via high affinity binding sites.
Proc Natl Acad Sci U S A.
1989;86:5542-5546 53. Baghestanian M, Agis H, Bevec D, et al. Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells. Exp Hematol. 1996;24:1377-1386[Medline] [Order article via Infotrieve]. 54. Valent P, Bettelheim P. Cell surface structures on human basophils and mast cells: biochemical and functional characterization. Adv Immunol. 1992;52:333-423[Medline] [Order article via Infotrieve].
55.
Arulanandam AR, Koyasu S, Reinherz EL.
T cell receptor-independent CD2 signal transduction in FcR+ cells.
J Exp Med.
1991;173:859-868
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  H Agis, M-T Krauth, I Mosberger, L Mullauer, I Simonitsch-Klupp, L B Schwartz, D Printz, A Bohm, G Fritsch, H-P Horny, et al. Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7 J. Clin. Pathol., April 1, 2006; 59(4): 396 - 402. [Abstract] [Full Text] [PDF]
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 W. R. Sperr, J. Drach, A. W. Hauswirth, J. Ackermann, M. Mitterbauer, G. Mitterbauer, M. Foedinger, C. Fonatsch, I. Simonitsch-Klupp, P. Kalhs, et al. Myelomastocytic Leukemia: Evidence for the Origin of Mast Cells from the Leukemic Clone and Eradication by Allogeneic Stem Cell Transplantation Clin. Cancer Res., October 1, 2005; 11(19): 6787 - 6792. [Abstract] [Full Text] [PDF]
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 S. Florian, M. Ghannadan, M. Mayerhofer, K. J. Aichberger, A. W. Hauswirth, G.-H. Schernthaner, D. Printz, G. Fritsch, A. Bohm, K. Sonneck, et al. Evaluation of normal and neoplastic human mast cells for expression of CD172a (SIRP{alpha}), CD47, and SHP-1 J. Leukoc. Biol., June 1, 2005; 77(6): 984 - 992. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
was purchased from Pharma Biotechnology
(Hannover, Germany); rhIL-16, rhIL-17, and rhIL-18 from Antigenix
America (New York, NY); and rhGM-CSF was provided by Sandoz Vienna
(Vienna, Austria). Collagenase type II was purchased from Sebak (Suben,
Austria), RPMI 1640 medium and fetal calf serum (FCS) from PAA
Laboratories (Linz, Austria), and Iscoves modified Dulbecco medium
(IMDM), L-glutamine, penicillin, and streptomycin from
Gibco Life Technologies (Gaithersburg, MD).
