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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4287-4295
Generation of Cells With Hodgkin's and Reed-Sternberg Phenotype
Through Downregulation of CD99 (Mic2)
By
Soon Ha Kim,
Eun Young Choi,
Young Kee Shin,
Tae Jin Kim,
Doo Hyun Chung,
Sung Ik Chang,
Noe Kyeong Kim, and
Seong Hoe Park
From the Departments of Pathology and Internal Medicine, Seoul
National University College of Medicine, Seoul, Korea; the Department
of Pathology, Sungkyunkwan University College of Medicine, Suwon,
Korea; and the Department of Anatomy and Medical Genetics, Keimyung
University, School of Medicine, Taegu, Korea.
 |
ABSTRACT |
Despite the fact that Hodgkin's and Reed-Sternberg (H-RS) cells are
morphological hallmarks of Hodgkin's disease (HD), the nature of H-RS
cells still remains to be resolved. Here we report that downregulation
of CD99 (Mic2) leads to the generation of cells with an H-RS
phenotype. IM9 and BJAB B-cell lines that were transfected with an
antisense CD99 expression construct showed the morphological and
immunological characteristics of H-RS cells such as multinuclearity,
expression of CD15, decreased expression of major histocompatibility
complex (MHC) class I and CD45RB, and deregulated secretion of
cytokines. The reduced expression of CD99 was also confirmed in H-RS
cells of patient's lymph nodes and three HD-derived cell lines, L428,
KM-H2, and HDLM-2. Moreover, features characteristic of H-RS cells were
completely abolished by forced expression of CD99 and by a
constitutively active form of Rac, which functions downstream of CD99.
We suggest that CD99 molecules play a crucial role in regulating
functions and morphology of cells through a Rac-Rho signaling pathway
and that the loss of CD99 expression is a significant molecular event
to generate H-RS cells.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
HODGKIN'S DISEASE (HD) is a lymphoid
neoplasm characterized by a low frequency of malignant tumor giant
cells, known as Hodgkin's and Reed-Sternberg (H-RS) cells, in an
abundant background of nonneoplastic inflammatory cells.1,2
The nature and origin of H-RS cells remain controversial even 160 years
after the initial description of HD. Advances in the understanding of
H-RS cell derivation have been made by studies that include
immunophenotyping, genotyping, and cytokine production of H-RS cells in
HD specimens or cell lines from HD tissues.3,4 In HD,
unbalanced cytokine production elicits an abundance of reactive cells
mainly comprising T cells, B cells, macrophages, and so
on.5-9 However, it is intriguing that these infiltrates do
not kill H-RS cells even though many H-RS cells contain Epstein-Barr
virus (EBV) antigens.10,11 Indeed, HD patients show a
severe impairment in their cellular immune responses.12 The
frequent absence of major histocompatibility complex (MHC) class I
expression in H-RS cells provides one explanation for the impaired
CD8+ cytotoxic T-lymphocyte (CTL) response
against EBV protein.13,14
CD99 (Mic2)15-17 is a 32-kD
transmembrane glycoprotein that is involved in cell-cell adhesion
during hematopoietic cell differentiation,18 apoptosis of
immature thymocytes,19 and transport of transmembrane proteins.20 During investigation into the expression
pattern of CD99 in various types of cells, we incidentally found
out that spontaneously occurring H-RS-like multinuclear giant cells
were devoid of CD99 expression. These findings led us to study CD99 expression in lymph nodes and cell lines from HD patients. In all cases
examined, we were not able to see any expression of CD99 in H-RS
cells. Subsequently, we investigated the relationship between
downregulation of CD99 and generation of H-RS-like cells. This
possibility was tested with a use of CD99-deficient IM9 and BJAB B-cell
lines, which individually represent two extremes of B-cell
differentiation. In this study, we were able to confirm that the
enforced downregulation of CD99 generates H-RS-like cells. Because
these cells also had immunological and functional features characteristic of H-RS cells, we suggest that CD99 downregulation is a
significant molecular event to generate H-RS cells.
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MATERIALS AND METHODS |
Tissue and cells.
The lymph nodes were obtained from patients with HD. IM9 (Ig-secreting
lymphoblast) and BJAB (Burkitt's lymphoma) cell lines were obtained
from American Type Culture Collection (ATCC; Rockville, MD). Three
HD-derived cell lines (HDLM-2, L428, and KM-H2) were purchased from
German Collection of Microorganisms and Cell Cultures (DSMZ,
Braunschweig, Germany). All cells were grown in Dulbecco's modified
Eagle's medium (DMEM) or RPMI 1640 media supplemented with 10% or 20% heat-inactivated fetal calf serum (FCS).
Gene constructs and transfection.
A full-length CD99 cDNA was inserted into the HindIII and
Xba I site of mammalian expression vector, RC-CMV, in the sense orientation or antisense orientation. Two constructs of Rac,
constitutively active (L61) or partially active (L61F37A), were kind
gifts of Allan Hall (University College London, London,
UK). For the study of the relationship between Rac and
CD99, CD99 cDNA was cloned at BamHI site in the antisense
orientation and two forms of Rac were inserted at Hpa I site in
the sense orientation of MTIN vector. LTR-driven transcripts for
antisense CD99 and Rac genes are made as a polycistronic mRNA and the
translation of Rac is guided by EMCV IRES sequence. Stable
neomycin-resistant IM9 and BJAB transfectants were established after
lipofectin-mediated (GIBCO-BRL, Gaithersburg, MD) gene
transfer as described.18 Establishment and subcloning of
stable cell lines were accomplished by culturing primary transfectants in the presence of 500 µg/mL of G-418 (GIBCO-BRL) for 1 month. All original and modified cell lines used in this study are
summarized in Table 1.
Flow cytometric analysis.
For staining, 106 cells were first incubated with relevant
monoclonal antibodies (MoAbs) (1 µg/100 µL) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.1% sodium
azide for 30 minutes at 4°C. The cells were then washed twice with
PBS, stained with fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG antibody, and, after another washing with PBS, analyzed
on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The
antibodies used were purchased from Becton Dickinson (CD15, CD30,
CD45RA, and CD45RB), Pharmingen (CD30 and CD69; San Diego, CA), Serotec
(CD23, CD38, CD40, CD69, and CD80; Oxford, UK), Dako (CD19, CD21, and
CD25; Carpinteria, CA), or obtained from hybridoma culture (MHC class I
[W6/32, ATCC]). The MoAbs to MHC class II (YG18) and CD99 (DN16) were
developed in this laboratory. The secondary antibody used was
FITC-conjugated goat anti-mouse IgG Ab (Dako).
Confocal microscopic analysis of lymph node sections.
Lymph nodes were embedded in Tissue-Tec (Miles Inc, Elkhart,
IN) and frozen on dry ice.
Five-micrometer-thick frozen sections were cut and
mounted onto poly-L-lysine-coated slides, permeabilized, fixed in cold
acetone/methanol (50%/50% vol/vol) for 10 minutes, and blocked in
10% fetal bovine serum (FBS). The slides were incubated overnight with
given MoAbs conjugated to FITC or biotin at a concentration between 10 and 20 µg/mL in PBS/1% BSA/0.02% sodium azide. After washing, they
were incubated for 4 hours with streptoavidin-Texas Red (Caltag,
Burlingame, CA) and mounted with PBS/azide/10% glycerol. The stained sections were examined by immunofluorescence confocal microscopy (BioRad 1024; BioRad Labs, Hercules, CA).
Northern blot analysis.
Cells were dissolved in TRIzol reagent (Life Technologies, Grand
Island, NY) and RNA was extracted following the manufacturer's instructions. Total RNA (20 µg) from each sample was separated by
electrophoresis on a 1.0% agarose/formaldehyde gel and blotted onto
nylon filters (Hybond-N+; Amersham International, Amersham, UK). The
filters were hybridized with [ -32P-dCTP]-labeled cDNA
fragments, then washed under stringent conditions (65°C for 30 minutes in washing buffer composed of 0.1× standard saline
citrate [SSC] and 0.1% sodium dodecyl sulfate
[SDS]), and detected by autoradiography. A full-length CD99 cDNA was
used as a probe. The filters were stripped and rehybridized with a cDNA
probe for  -actin as an internal control.
Cell-cycle analysis.
Asynchronous populations of IM9 transfectants in the log-phase of cell
growth were fixed in 70% ethanol in PBS on ice, pelleted, incubated
with RNase A (0.1 µg/mL) for 30 minutes at 37°C, and then stained
with propidium iodide (40 µg/mL). The cell-cycle profiles (10,000 cells per sample) were analyzed on a FACScan machine.
Cytokine analysis.
IM9 and BJAB transfectants were cultured at 2 × 105/mL in 24-well microplates with either RPMI media alone
or media supplemented with soluble CD40L (supernatant, 1:10 ratio).
After a 48-hour incubation, supernatants were assayed for
interleukin-10 (IL-10) or transforming growth factor 1 (TGF-1)
level by an ELISA kit (R & D, Minneapolis, MN). For the TGF-1 assay,
serum-free medium was used. Culture supernatant of soluble trimeric
CD40L was a gift of Tae Ho Lee (Yonsei University, Seoul, Korea).
| |
RESULTS |
Lack of CD99 expression in H-RS cells from the lymph nodes of HD
patients and in HD-derived cell lines.
In the course of long-term culture of IM9 cells, we noticed that
spontaneously occurring H-RS-like multinucleated giant cells were
consistently devoid of CD99 expression. This finding prompted us to
examine whether CD99 is also downregulated in the H-RS cells from lymph
nodes of HD patients. In 28 cases of HD studied, H-RS cells were
consistently negative for CD99 expression by immunofluorescence and
immunohistochemical analyses. In contrast, activated lymphocytes from
lymph nodes of 15 cases of reactive lymphadenopathy were positive for
CD99 and were taken as controls. The notion that CD99 is upregulated in
activated lymphocytes was based on the flow cytometric finding that
CD69+ cells expressed CD99 in a much higher level than did
CD69 cells in these reactive lymph nodes (data not
shown). Lack of CD99 expression in H-RS cells from HD patients was
again shown with confocal microscopic analysis. Expression of CD99 was
almost completely absent in H-RS cells, whereas a considerable number of surrounding activated lymphocytes showed strong reactivity (Fig 1A). CD99 expression in established
HD-derived cell lines was almost identical to that of H-RS cells from
lymph nodes of HD patients. Two HD-derived cell lines, L428 and KM-H2,
showed no expression of CD99 as detected by Northern blot and flow
cytometric analyses (Fig 2). In the case of
HDLM-2 cell line, expression of CD99 was markedly diminished. These
results strongly suggest that downregulation of CD99 expression is
tightly correlated with the appearance of H-RS cells. For this reason,
we addressed the question of whether forced downregulation of CD99 was
able to produce similar H-RS-like cells.
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| Fig 1.
Microscopic analysis of H-RS cells from the lymph nodes
of an HD patient and CD99-deficient B-cell lines. (A) Examination on
the distribution of CD30 and CD99 molecules in lymph nodes of an HD
patient by confocal microscopy. The frozen sections of lymph nodes were
double-stained with FITC-conjugated anti-CD30 and biotinylated
anti-CD99 antibodies. CD99 expression was identified by incubation with
Texas Red-conjugated streptoavidin. CD99 molecule is absent in H-RS
cells, whereas a considerable number of surrounding activated
lymphocytes show strong reactivity (original magnification × 200). A CD30+ H-RS cell is shown at higher magnification
(inset; original magnification × 1,000). (B) Localization
of CD15 molecules in AS-TF IM9 cells with confocal microscopic
analysis. AS-TF cells were stained with FITC-conjugated anti-CD15
antibody. Propidium iodide (PI) was included for nuclear
staining. Most of large cells showed intense expression of CD15 in the
Golgi and cytoplamic regions as well as on plasma membrane (original
magnification × 630). Both (C) Vec-TF and (D) AS-TF IM9 cells were
morphologically examined after Wright and Giemsa staining. AS-TF IM9
cells show typical H-RS morphology. (E and F) Restored cell morphology
by exogenous expression of CD99 in Mut-IM9 cells. (E) Spontaneous
CD99-deficient IM9 cells (Mut-IM9) and (F) CD99-TF Mut-IM9 cells
were examined with Wright and Giemsa staining. All slides
(C-F) were processed in parallel and photographed under identical
magnification (40× objective).
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| Fig 2.
Lack of CD99 expression in HD-derived cell lines. (A)
Northern blot analysis for CD99 expression in HD-derived cell lines.
RNA samples were prepared from IM9 (I) and three Hodgkin's cell lines,
HDLM-2 (H), KM-H2 (K), and L-428 (L), and examined for the presence of
CD99 transcripts. As a loading control, the blot was reprobed with
rRNA. (B) Flow cytometric analysis of HD-derived cell lines.
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Production of H-RS-like cells by downregulation of CD99 in B-cell
lines.
To generate CD99-deficient B-cell lines, we transfected IM9 B cells
with antisense CD99 construct as described in Materials and Methods and
obtained stable transfectants. As confirmed by immunoblot, Northern
blot, and flow cytometric analyses (Fig 3), CD99 expression was abolished in the stable IM9 B cell transfectants (defined as AS-TF cells). Three independent stable AS-TF clones showed
a substantial increase in cell size and severe morphological variations
(Fig 1D) compared with control (Fig 1C). About 20% to 30% of the
total cell culture population of AS-TF IM9 cells exhibited a typical
H-RS cell morphology, which has been defined based on the morphologic
criteria: abundant cytoplasm and bilobed or multilobate nuclei with
amphophilic "owl-eyed" nucleoli (Fig 1D). We obtained similar
results in spontaneous CD99-deficient mutant IM9 cells, which had been
subcloned from IM9 cell culture (Mut IM9; Fig 1E), and AS-TF BJAB cells
(data not shown). These results suggest that CD99 downregulation lead
to generation of H-RS-like cells.
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| Fig 3.
Generation of CD99-deficient IM9 cells and
immunophenotyping. (A) Immunoblot and (B) Northern blot analyses of
CD99-deficient IM9 cells. IM9 cells were transfected with either vector
or antisense CD99 expression construct. The Vec-TF (V), AS-TF (A), and
Mut-IM9 cells (M) were examined for CD99 expression. Actin was tested
as an internal control. (C) Immunophenotypic analysis of AS-TF cells.
The cells were first stained with control MoAb or with relevant primary
MoAbs, and then with FITC-conjugated goat anti-mouse IgG antibody.
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Immunological characteristics of H-RS-like cells.
We investigated whether H-RS-like cells generated by downregulation of
CD99 have the immunophenotypic and functional characteristics of H-RS
cells. Immunophenotypic features of AS-TF IM9 cells were compared with
those of vector transfectant (Vec-TF) controls (Fig 3C).
The current consensus is that a significant fraction of
H-RS cells carry a high concentration of CD30 and CD15 in the absence of CD45RB on their surfaces, and this is the immunophenotypic criteria
for the diagnosis of HD.21-27 Flow cytometric analysis showed a fairly constant high-level expression of CD30 in both types of
IM9 cells tested, although AS-TF IM9 cells carried a twofold amount of
CD30 compared with Vec-TF cells. For CD15, IM9 and Vec-TF IM9 cells did
not reveal CD15 expression at all, but more than 20% of AS-TF cells
that had a morphologic phenotype of H-RS cells contained a considerable
amount of CD15 (Fig 3C). Confocal microscopic examination clearly
showed a unique pattern of CD15 localization. As shown in Fig 1B, most
of the large H-RS-like cells showed intense expression of CD15 in the
Golgi and cytoplasmic regions, as well as on plasma membrane. In
contrast, Vec-TF cells were completely negative for CD15 (data not
shown). Regarding CD45RB expression, there was an almost complete
downregulation of this molecule in AS-TF IM9 cells but not in Vec-TF
cells, whereas expression of CD45RA remained high in both cell lines.
In our study, CD99-deficient IM9 transfectants showed decreased MHC
class I expression. The absence or reduced expression of MHC class I molecules was also found in three HD-derived cell lines (Fig 2B), again
confirming that these H-RS-like AS-TF cells have immunological features comparable to those of H-RS cells in HD. Both AS-TF and Vec-TF
cells expressed similar levels of other surface molecules such as
CD19, CD21, CD23, CD25, CD40, MHC class II, ICAM-1, LFA-1a, LFA-3, and
CD80 (data not shown). Both types of cells were negative for CD38 (data
not shown).
H-RS cells express high levels of CD30 and CD40, which seem to share
some common biological activities involved in the deregulated secretion
of cytokines.28 The data obtained in the present study showed typical patterns of deregulated secretion of cytokines. Treatment of AS-TF cells with soluble trimeric CD40 ligand induced enhanced release of TGF-1 from cultured AS-TF of both IM9 (1.5-fold) and BJAB cells (1.9-fold), and markedly diminished (9.5-fold) secretion
of IL-10 in AS-TF BJAB cells by comparison with those in
Vec-TF controls (Fig 4A).
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| Fig 4.
Immunologic characteristics of CD99 negative B cells and
rescue from the H-RS phenotype by exogenous expression of CD99. (A)
Deregulation of soluble CD40L-induced cytokine secretion in
CD99-deficient B cells. Cells were cultured in RPMI media alone or in
media supplemented with soluble CD40L (supernatant, 1:10 ratio). (B)
Cell-cycle analysis. Flow cytometric analysis was performed on
asynchronously proliferating Vec-TF (top) or AS-TF IM9 cells (bottom).
The x-axis shows DNA content and the y-axis shows the number of cells.
(C) Growth curve of CD99-deficient IM9 cells. The same number (5 × 104/mL) of cells was plated in 10% FCS-DMEM media at day
0. The total cell numbers were counted at the indicated days. The
result of thymidine uptake is shown in the box. (D) Flow cytometric
analysis of Mut-IM9 and CD99-TF Mut-IM9 cells. (E) Flow cytometric
analysis of Vec-TF and CD99-TF L428 cells. Note the tight relation
between the forward/side scatter profile and the expression of CD99 and
MHC class I.
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Defective cell-cycle progression and chromosome abnormality.
We investigated the growth characteristics of CD99-deficient cells.
AS-TF IM9 cells showed slower kinetics of cell proliferation than did
Vec-TF cells (sixfold decrease at day 5), which was further confirmed
by [3H] thymidine-uptake assays (2981.4  519.0 cpm) (Fig 4C). We also obtained similar results in AS-TF BJAB cells
(data not shown).
To evaluate the cell-cycle distribution and possible arrest or delay
during a certain phase of the cell cycle, the DNA content of
asynchronous cultures of Vec-TF and AS-TF IM9 cells was measured. All
three AS-TF IM9 clones showed a marked accumulation of 4N cells (24.5%
37.5%), a decrease in the number of S phase cells (17.5%
4.0%), and many aneuploidy cells with larger than 4N (0%
to 8%) (Fig 4B) compared with Vec-TF cells. These results indicate that CD99-deficient cells are either arrested at the mitotic
(M) phase of the cell cycle or are defective in cytokinesis, which was
further confirmed with a comparative chromosomal analysis of
CD99-deficient and control IM9 B cells. CD99-deficient cells showed
numerical chromosomal changes (mostly tetrahypoploidy) and frequent
structural abnormalities (data not shown).
Restoration of cell morphology from H-RS cells by forced expression
of CD99.
In the course of long-term culture, we were able to obtain spontaneous
CD99-deficient mutant IM9 cells (Mut-IM9) with an H-RS morphology (Fig
1E), slow growth rate (Fig 4C), and CD15 positivity (data not shown)
like AS-TF IM9 cells. Mut-IM9 cells were shown to be negative for CD99
expression by immunoblot and Northern blot analyses (Fig 3A and B),
suggesting that lack of expression of CD99 on cell surfaces was
attributable to a decrease in the synthesis of CD99 at transcriptional
and translational levels. Because it was essential to test whether
restoration of CD99 expression was able to rescue from the H-RS
phenotype, we transfected the CMV-driven CD99 expression construct into
Mut-IM9 and L428 cells and obtained stable transfectants (CD99-TF).
With a recovery of CD99 expression, the characteristics of H-RS cells
in Mut-IM9 cells were almost completely abolished and they regained a
normal morphology (Fig 1F), growth rate, and surface phenotype (Fig 4C and D). It is notable that CD99-TF Mut-IM9 cells were completely negative for CD15 (data not shown). The CD99-TF L428 cells also acquired a reduced forward and side scatter profile on
fluorescence-activated cell sorting (FACS) analysis, indicating
monomorphism in the size and shape of cells. The frequency of cells
with H-RS morphology was markedly diminished. The intensity of MHC
class I expression was also proportional to that of CD99 expression
(Fig 4E). All these results confirm the presence of a tight correlation
between reduced expression of CD99 and generation of an H-RS phenotype.
Activation of Rho and Rac by engagement of CD99.
It is well established that the small GTP-binding proteins Rac and Rho
participate in the control of cell morphology, cell aggregation, and
cytokinesis.29,30 The findings that engagement of CD99
induces the aggregation of various types of lymphoid
cells15,18 and diminished CD99 expression leads to
defective cytokinesis led us to investigate whether stimulation of CD99
has an effect on the activities of Rac and Rho. Anti-CD99 MoAb
triggered homotypic aggregation of IM9 cells within 1 hour
(Fig 5A, a). This CD99-induced aggregation
was blocked by pretreatment of IM9 cells with C3 transferase, a
bacterial enzyme known to inactivate Rho through ADP
ribosylation31 (Fig 5A, b). We also observed that phorbol
myristate acetate (PMA)-induced aggregation was blocked
by C3 pretreatment (positive control experiment, data not shown). To
investigate whether Rac also participates in this particular signaling
pathway, we generated two kinds of IM9 transfectants that contained
either a constitutively active form of Rac (L61) or a mutant form of
Rac (L61F37A) that is defective in the morphology signaling
pathway.31 Despite the similar expression level of CD99 in
both transfectants, only the L61F37A-TF cells showed reduced
CD99-induced aggregation (Fig 5A, c and d). Expression of the
transfected Rac genes was confirmed by the immunoblot analysis (data
not shown). In this assay, we used isotype-matched MoAb as negative
control. This control antibody induced very weak aggregation (data not
shown). These results indicated that Rac and Rho were in the downstream
of CD99 with respect to cell aggregation signaling pathway.
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| Fig 5.
Function of Rac and Rho in CD99 signaling pathway. (A)
Blocking of CD99 MoAb-induced aggregation of IM9 cells by C3 exoenzyme
or L61F37A Rac mutant. Normal IM9 cells pretreated with (b) or without
(a) the inhibitor of C3 transferase (20 µg/mL; UBI, Lake Placid, NY)
for 36 hours were dispersed into single cells by repeated pipetting,
incubated with control antibody or anti-CD99 MoAb for 1 hour, and
assayed for cell aggregation. L61-TF (c) or L61F37A-TF (d) IM9
transfectants were stimulated with anti-CD99 MoAb for 1 hour and
assayed for aggregation. (B) Gene constructs that contain L61 or
L61F37A Rac in sense orientation and CD99 in antisense orientation. (C)
Immunophenotypic analysis. All transfectants were examined for CD99
expression by flow cytometric analysis. L61 Rac rescues cell morphology
from the H-RS phenotype, with a concomitant decrease in forward and
side scatter and increased MHC class I expression, in CD99-deficient
IM9 cells.
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Rescue from the H-RS phenotype in CD99-deficient IM9 cells by
expression of a constitutively active Rac.
Previous observation suggests that a constitutively active form of Rac
might rescue from the H-RS phenotype in CD99-deficient IM9 B cells if
basal activities of Rac and Rho are maintained via CD99. To address
this issue, LTR-driven expression constructs were designed that contain
CD99 gene in the antisense orientation alone (AS-TF) or with either L61
or L61F37A Rac gene in the sense orientation (AS-L61-TF or AS-F37A-TF)
(Fig 5B). AS-TF cells acquired H-RS morphology, with a concomitant
increase in forward and side scatter. In contrast, AS-L61-TF IM9 cells
that express both L61 Rac and antisense CD99 showed normal morphology
of IM9 cells, which was accompanied by the acquisition of proper MHC
class I expression (Fig 5C), indicating that increased Rac activity
rescued from the H-RS phenotype of CD99-deficient IM9 cells. In
contrast, L61F37A-TF cells retained H-RS morphology.
| |
DISCUSSION |
In this study we have shown that downregulation of CD99 is tightly
linked to generation of cells with the H-RS phenotype. Evidence
supporting the presence of a relationship between reduced expression of
CD99 and H-RS-like cell generation are presented here. In all 28 cases
of HD examined, CD30+ H-RS cells in lymph nodes were
negative for CD99 expression, while surrounding reactive lymphocytes
from HD and activated lymphocytes from benign lymphadenopathy expressed
a high level of CD99 molecule on their cell surfaces. In addition,
established HD-derived cell lines showed markedly reduced or no
expression of CD99 molecules. More convincing evidence was obtained
from transfection studies. Downexpression of CD99 in B-cell lines led
to the generation of H-RS-like cells irrespective of differentiation
stages of cells. AS-TF cells exhibited most of the features
characteristic of H-RS cells: (1) the typical H-RS cell morphology,
characterized by abundant cytoplasm and bilobed or multilobated nuclei
with amphophilic "owl-eyed" nucleoli, (2) the markedly diminished
expression of MHC class I and CD45RB; (3) expression of CD15 and its
unique localization in the Golgi region; (4) the typical pattern of
deregulated cytokine secretion; and (5) defective cell-cycle
progression and chromosomal abnormalities. Finally, cell morphology and
proliferation activity were restored to normal by the forced expression
of CD99 in Mut-IM9 and L428 cells.
The constitutive expression of CD30 and CD15 and the absence of CD45RB
have been currently used as immunophenotypic criteria for diagnosis of
HD.21-27 In the present study, even though high-level expression of CD30 was already detected in Vec-TF, CD30 expression was
upregulated by twofold with downregulation of CD99. This result is in
agreement with a previous report that CD30 can also be expressed in
transformed or activated lymphocytes, rather than being H-RS cell-specific antigens.32 As for the CD15 molecule, its
expression was confined to cells with H-RS morphology, which represent
20% of total AS-TF IM9 cells. This was further confirmed by confocal microscopic analysis. A large amount of CD15 was expressed in the
cytoplasm of H-RS-like AS-TF cells with a less intense expression on
their surfaces. Interestingly, this finding was in sharp contrast with
that of Vec-TF cells and CD99-TF Mut IM9 cells where expression of CD15
was not visible. It is well known that expression of CD15 with its
unique intracellular localization is one of the most important points
for the differential diagnosis between HD and large cell lymphoma of
anaplastic type.33-35 Therefore, based on present data
regarding CD15 expression, it seems to be reasonable to say that AS-TF
cells are more likely to be H-RS cells than other types of neoplastic
lymphoid cells.
It has been reported that all HD cases examined showed a near-complete
lack of expression of MHC class I molecules on the surfaces of H-RS
cells.13,14 In this study, downregulation of CD99 was
always accompanied by decreased expression of MHC class I molecule.
Because the intensity of CD99 was proportional to the extent of MHC
class I expression, we suggest that downregulation of MHC class I
molecule may be a secondary event to the loss of CD99. Indeed, we have
recently showed that CD99 ligation led to the marked upregulation of
MHC class I and class II molecules on the cell surfaces.20
The results obtained here are considered to have a biological relevance
in vivo in the following aspects. The downregulation of MHC class I
molecule can render cells resistant to CTLs and provide a selective
growth advantage for H-RS cells in vivo. In this way, H-RS cells may
escape from host immune surveillance and survive. However, the
mechanism leading to this downregulation of MHC class I molecule has
yet to be unraveled. With our previous study, it appeared that MHC
class I and II molecules were regulated at the level of vesicular
transport.20
H-RS cells express a high level of CD30 and CD40, which are known to be
involved in the deregulated secretion of cytokines.28 This
might provide a favorable condition for growth of H-RS cells and their
surrounding reactive bystander cells. Typical deregulated secretion of
cytokines was shown in AS-TF cells. We presume that increased TGF-1
production by H-RS cells might cause decreased immune response in vivo
by the suppression of the surrounding reactive bystander cells. Based
on these findings, we suggest that CD99 molecules play a crucial role
in controlling the interaction between H-RS cells and T cells in HD
through a network of interactive signals generated by cytokine-mediated
(CD40-CD40L) events.
CD99-deficient B cells exhibited slower kinetics of cell proliferation
due to markedly decreased S phase with accumulation at mitotic (M)
phase, a defect in cytokinesis. Furthermore, numerical chromosomal
changes and frequent structural abnormalities were also found. These
results are in support of the recent proposal that multinuclear RS
cells are derived from originally mononuclear Hodgkin's cells through
nuclear division with disturbance of cytokinesis.36 We
interpreted that cell-cycle blockage at M phase by CD99 downregulation is incomplete and typical H-RS cells are continuously provided by
faster growing mononuclear variants, which may comprise a big proportion of bona fide tumor cells in the peripheral blood of HD
patients.37 However, we thought that these CD99-deficient H-RS-like cells had been in cell cycle beyond restriction point, which
might be an explanation for Ki67 positivity in H-RS cells from
patients' lymph nodes.
Experiments performed in this study support the idea that molecular
signals generated by CD99 engagement are associated with the
organization of the cytoskeleton. First, cell cycle and cytokinesis were disrupted by reduced expression of CD99. Second, the treatment of
C3 exoenzyme completely abolished CD99-induced cell aggregation. Third,
overexpression of a mutant form of Rac rendered cells resistant to
aggregate-formation after CD99 engagement. Fourth, cell shape and MHC
class I expression were restored to a normal state by increased Rac
activity despite the absence of CD99 expression. Until recently,
members of the Rho subfamily were believed to be involved in the
regulation of cytoskeletal organization in response to extracellular
signals, influencing the actin- and microtubule-based
system.38,39 On the basis of these facts, it can be
suggested that the activity of CD99 may be linked to the organization
of the cytoskeleton or activation of cytoskeletal components.
Our findings suggest that the progression of HD may occur when a
genetic alteration of H-RS precursor cells leads to the downregulation of CD99. At this point, the mechanism by which CD99 expression is
downregulated is unknown. We suggest several possibilities for the
diminished expression of CD99. One possibility is the transcriptional
downregulation of CD99, because CD99 mRNA was undetectable in some of
the HD-derived cell lines and in spontaneously occurring Mut-IM9 cells.
Another possible causative event is the mutation of the CD99-encoding
gene during tumorigenesis. According to the clinical studies, HD is
frequently associated with immunosuppressive states such as viral
infection40 that may allow the expansion of a lymphoid
precursor of H-RS cells. Therefore, various viral gene products
including LMP-1 might be candidates for the downregulation of CD99
expression.
In this report it is suggested that CD99 may play a major role in the
maintenance of cell shape, cell integrity, and cell division through
Rac-Rho signaling pathway. We suggest that the loss of CD99 expression
is a significant molecular event for the generation of H-RS cells. This
idea might be an adequate explanation for the actual presence of both
EBV+ and EBV HD cases.41
| |
ACKNOWLEDGMENT |
We are grateful to Dr A. Hall (Department of Biochemistry, University
College London, London, UK) for providing us with L61Rac and L61F37A
Rac genes, to Peter N. Goodfellow (SmithKline Beecham, Essex, UK) for
helpful criticism and discussion, and to Sean Bong Lee (MGH Cancer
Center, Boston, MA) for helpful comments on the manuscript.
| |
FOOTNOTES |
Submitted December 15, 1997;
accepted July 28, 1998.
S.H.K. and E.Y.C. contributed equally to this work.
Supported by a grant (HMP-96-M-2-1073) of the '96 Good Health R&D
Project, Ministry of Health & Welfare, Republic of Korea.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Seong Hoe Park, MD, Department
of Pathology, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110-799, Korea; e-mail:
pshoe{at}plaza.snu.ac.kr.
| |
REFERENCES |
1.
Drexler HG:
Recent results on the biology of Hodgkin and Reed-Sternberg cells. I. Biopsy material.
Leuk Lymphoma
8:283, 1992[Medline]
[Order article via Infotrieve]
2.
Kadin ME:
Pathology of Hodgkin's disease.
Curr Opin Oncol
6:456, 1994[Medline]
[Order article via Infotrieve]
3.
Gruss HJ, Pinto A, Duyster J, Poppema S, Herrmann F:
Hodgkin's disease: A tumor with disturbed immunological pathways.
Immunol Today
18:156, 1997[Medline]
[Order article via Infotrieve]
4.
Haluska FG, Brufsky AM, Canellos GP:
The cellular biology of Reed-Sternberg cell.
Blood
84:1005, 1994[Free Full Text]
5.
Gruss HJ, Brach MA, Drexler HG, Bonifer R, Mertelsmann RH, Herrmann F:
Expression of cytokine genes, cytokine receptor genes, and transcription factors in cultured Hodgkin and Reed-Sternberg cells.
Cancer Res
52:3353, 1992[Abstract/Free Full Text]
6.
Hsu SM, Krupen K, Lachman LB:
Heterogeneity of interleukin 1 production in cultured Reed-Sternberg cell lines HDLM-1, HDLM-1d, and KM-H2.
Am J Pathol
135:33, 1989[Abstract]
7.
Merz H, Fliender A, Orscheschek K, Binder T, Sebald W, Muller-Hermelink HK, Feller AC:
Cytokine expression in T-cell lymphoma and Hodgkin's disease. Its possible implication in autocrine or paracrine production as a potential basis for neoplastic growth.
Am J Pathol
139:1173, 1991[Abstract]
8.
Newcom SR, Ansari AA, Gu L:
Interleukin-4 is an autocrine growth factor secreted by the L-428 Reed-Sternberg cell.
Blood
79:191, 1992[Abstract/Free Full Text]
9.
Jucker M, Abts H, Li W, Schindler R, Merz A, Gunter A, von Kalle C, Schaadt M, Diamantstein T, Feller AC:
Expression of interleukin-6 and interleukin-6 receptor in Hodgkin's disease.
Blood
77:2413, 1991[Abstract/Free Full Text]
10.
Muller N, Evans A, Harris NL, Comstock GW, Jellum E, Magnus K, Orentreich N, Polk BF, Vogelman J:
Hodgkin's disease and Epstein-Barr virus. Altered antibody pattern before diagnosis.
N Engl J Med
320:689, 1989[Abstract]
11.
Weiss LM, Movahed LA, Warnke RA, Sklar J:
Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease.
N Engl J Med
320:502, 1989[Abstract]
12.
Frisan T, Sjoberg J, Dolcetti R, Boiocchi M, De Re V, Carbone A, Brautbar C, Battat S, Biberfeld P, Eckman M, Ost A, Christensson B, Sundstrom C, Bjorkholm M, Pisa P, Masucci M:
Local suppression of Epstein-Barr virus (EBV)-specific cytotoxicity in biopsies of EBV-positive Hodgkin's disease.
Blood
86:1493, 1995[Abstract/Free Full Text]
13.
Poppema S, Visser L:
Absence of HLA class I expression by Reed-Sternberg cells.
Am J Pathol
145:37, 1994[Abstract]
14.
Oudejans JJ, Jiwa NM, Kummer JA, Horstman A, Vos W, Baak JP, Kluin PM, van der Valk P, Walboomers JM, Meijer CJ:
Analysis of major histocompatibility complex class I expression on Reed-Sternberg cells in relation to the cytotoxic T-cell response in Epstein-Barr virus-positive and -negative Hodgkin's disease.
Blood
87:3844, 1996[Abstract/Free Full Text]
15.
Gelin C, Aubrit F, Phalipon A, Raynal B, Cole S, Kaczorek M, Bernard A:
The E2 antigen, 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.
EMBO J
8:3253, 1989[Medline]
[Order article via Infotrieve]
16.
Dworzak MN, Fritsch G, Buchinger P, Fleischer C, Printz D, Zellner A, Schollhammer A, Steiner G, Ambros PF, Gadner H:
Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood.
Blood
83:415, 1994[Abstract/Free Full Text]
17.
Goodfellow PJ, Darling SM, Thomas NS, Goodfellow PN:
A pseudoautosomal gene in man.
Science
234:740, 1986[Abstract/Free Full Text]
18.
Hahn JH, Kim MK, Choi EY, Kim SH, Sohn HW, Ham DI, Chung DH, Kim TJ, Lee WJ, Park CK, Ree HJ, Park SH:
CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.
J Immunol
159:2250, 1997[Abstract/Free Full Text]
19.
Bernard G, Breittmayer JP, de Matteis M, Trampont P, Hofman P, Senik A, Bernard A:
Apoptosis of immature thymocytes mediated by E2/CD99.
J Immunol
158:2543, 1997[Abstract]
20.
Choi EY, Park WS, Jung KC, Kim SH, Kim YY, Lee WJ, Park SH:
Engagement of CD99 induces upregulation of TCR and MHC class I and II molecules on the surface of human thymocytes.
J Immunol
161:749, 1998[Abstract/Free Full Text]
21.
Kanzler H, Kuppers R, Hansmann ML, Rajewsky K:
Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.
J Exp Med
184:1495, 1996[Abstract/Free Full Text]
22.
Poppema S, De Jong B, Atmosoerodjo J, Idenburg V, Visser L, De Ley L:
Morphologic, immunologic, enzymehistochemical and chromosomal analysis of a cell line derived from Hodgkin's disease. Evidence for a B-cell origin of Sternberg-Reed cells.
Cancer
55:683, 1985[Medline]
[Order article via Infotrieve]
23.
Schmid C, Pan L, Diss T, Isaacson PG:
Expression of B-cell antigens by Hodgkin's and Reed-Sternberg cells.
Am J Pathol
139:701, 1991[Abstract]
24.
Hummel M, Ziemann K, Lammert H, Pileri S, Sabattini E, Stein H:
Hodgkin's disease with monoclonal and polyclonal populations of Reed-Sternberg cells.
N Engl J Med
333:901, 1995[Abstract/Free Full Text]
25.
Benharroch D, Meguerian-Bodoyan Z, Lamant L, Amin C, Brugieres L, Terrier-Lacombe M-J, Haralambieva E, Pulford K, Pileri S, Morris SW, Mason DY, Delsol G:
ALK-positive lymphoma: A single disease with a broad spectrum of morphology.
Blood
91:2076, 1998[Abstract/Free Full Text]
26.
Kuppers R, Rajewsky K, Zhao M, Simons G, Laumann R, Fischer R, Hansmann ML:
Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.
Proc Natl Acad Sci USA
91:10962, 1994[Abstract/Free Full Text]
27.
Kanzler H, Hansmann ML, Kapp U, Wolf J, Diehl V, Rajewsky K, Kuppers R:
Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed-Sternberg cells of a Hodgkin's lymphoma patient.
Blood
87:3429, 1996[Abstract/Free Full Text]
28.
Gruss HJ, Ulrich D, Braddy S, Armitage RJ, Dower SK:
Recombinant CD30 ligand and CD40 ligand share common biological activities on Hodgkin and Reed-Sternberg cells.
Eur J Immunol
25:2083, 1995[Medline]
[Order article via Infotrieve]
29.
Ridley AJ, Paterson HF, Jonston CL, Diekmann D, Hall A:
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:401, 1992[Medline]
[Order article via Infotrieve]
30.
Nobes CD, Hall A:
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:53, 1995[Medline]
[Order article via Infotrieve]
31.
Lamarche N, Tapon N, Stowers L, Burbelo PD, Aspenstrom P, Bridges T, Chant J, Hall A:
Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and JNK/SAPK MAP kinase cascade.
Cell
87:519, 1996[Medline]
[Order article via Infotrieve]
32.
Stein H, Mason D, Gerdes J, O'Connor N, Wainscoat J, Pallesen G, Gatter K, Falini B, Delsol G, Lemke H, Schwarting R, Lennert K:
The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: Evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.
Blood
66:848, 1985[Abstract/Free Full Text]
33.
Grogan TM:
Hodgkin's disease, in
Jaffe ES
(ed):
Surgical Pathology of the Lymph Nodes and Related Organs, vol 16. Philadelphia, PA, Saunders, 1995, p 133.
34.
Agnarsson BA, Kadin ME:
The immunophenotype of Reed-Sternberg cells. A study of 50 cases of Hodgkin's disease using fixed frozen tissues.
Cancer
63:2083, 1989[Medline]
[Order article via Infotrieve]
35.
Agnarsson BA, Kadin ME:
Ki-1 positive large cell lymphoma.
Am J Surg Pathol
12:264, 1988[Medline]
[Order article via Infotrieve]
36.
Knecht H, McQuain C, Martin J, Rothenburger S, Drexler HG, Berger C, Bachmann E, Kittler ELW, Odermatt BF, Quesenberry PJ:
Expression of the LMP1 oncoprotein in the EBV negative Hodgkin's disease cell line L-428 is associated with Reed-Sternberg cell morphology.
Oncogene
13:947, 1996[Medline]
[Order article via Infotrieve]
37.
Wolf J, Kapp U, Bohlen H, Kornacker M, Schoch C, Stahl B, Mucke S, von Kalle C, Fonatsch C, Schaefer HE, Hansmann ML, Diehl V:
Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin-Reed Sternberg cells.
Blood
87:3418, 1996[Abstract/Free Full Text]
38.
Van Aelst L, D'Souza-Schorey C:
Rho GTPases and signaling networks.
Genes Dev
11:2295, 1997[Free Full Text]
39.
Tapon N, Hall A:
Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton.
Curr Opin Cell Biol
9:86, 1997[Medline]
[Order article via Infotrieve]
40.
Herndier BG, Sanchez HC, Chang KL, Chen YY, Weiss LM:
High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of HIV-associated Hodgkin's disease.
Am J Pathol
142:1073, 1993[Abstract]
41.
Hummel M, Anagnostopoulos I, Dallenbach F, Korbjuhn P, Dimmler C, Stein H:
EBV infection patterns in Hodgkin's disease and normal lymphoid tissue: expression and cellular localization of EBV gene products.
Br J Haematol
82:689, 1992[Medline]
[Order article via Infotrieve]
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Abstract
Introduction