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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4287-4295
By
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.
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.
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.
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 [ 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 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
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.
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).
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 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.
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.
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.
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.
Submitted December 15, 1997;
accepted July 28, 1998.
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.
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Hummel M, Anagnostopoulos I, Dallenbach F, Korbjuhn P, Dimmler C, Stein H:
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Abstract
Introduction
-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.
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 
519.0 cpm) (Fig 
