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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1342-1349
Tissue Inhibitor of Metalloproteinase (TIMP)-1 Induces
Differentiation and an Antiapoptotic Phenotype in Germinal Center B
Cells
By
Liliana Guedez,
Laurel Courtemanch, and
Maryalice Stetler-Stevenson
From the Hematopathology Section, Laboratory of Pathology, National
Cancer Institute, Bethesda, MD.
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ABSTRACT |
Tissue inhibitors of metalloproteinases (TIMPs) have been shown to
be multifunctional factors. Contrasting with their enzyme-inhibitory activity, TIMPs also promote cell growth. Previously, we have reported
an enhanced expression of TIMP-1 by normal reactive B cells and
high-grade lymphomas. In the present study, a series of Burkitt's
lymphoma (BL) cell lines were analyzed for their expression of TIMP-1.
TIMP-1 expression correlates with upregulation of activation and
survival markers. TIMP-1-negative cells express the phenotype
associated with group I BL lines and Epstein-Barr virus (EBV)-negative,
nonendemic BLs (CD10+, CD38+,
sIg+, and CD77+). However,
TIMP-1+ BL lines showed group II/III BL phenotype,
downregulation of the above markers, and upregulation and secretion of
the activation marker CD23. Also, TIMP-1+ cells have high
levels of CD40 expression. To determine whether TIMP-1 is directly
involved in the BL phenotype, an EBV-negative BL line JD38 was infected
with timp-1-expressing retrovirus and analyzed. In the absence
of EBV, upregulation of TIMP-1 is sufficient to induce the same
phenotype seen in TIMP-1+, EBV+ BL lines
(CD10 , CD38, sIg,
CD77, CD23+, CD40 bright). This study not
only suggests a role for TIMP-1 in BLs, but also supports its value as
a prognostic factor.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
THE EXTRACELLULAR matrix (ECM) integrates
cell function in tissues at various levels by regulating cell growth,
differentiation, and cell death.1-3 Likewise, deregulated
cell response to ECM is a determinant of disease processes such as
tumor invasion and metastasis.4 Tissue inhibitors of
metalloproteinases (TIMPs) as components of ECM have been shown to be
important in the control of metastasis by inactivating matrix
metalloproteinases (MMPs).5,6 However, TIMPs have also been
shown to exhibit other functions independent of their MMP inhibitory
activity. TIMP-1 and TIMP-2 have erythroid potentiating activity (EPA)
and promote cell growth in a wide range of cells, including fibroblasts
and hematolymphoid cells.7-10 Moreover, cell lines infected
with human T-cell leukemia virus type 1 (HTLV-1) and
HTLV-2 show upregulated TIMP-1 expression, which is mediated by
transactivator protein (TAX) transactivation of the TIMP-1
gene.11 We have recently reported on the high level of
TIMP-1 expression by high-grade B-cell lymphomas as well as normal,
activated B cells.12 Furthermore, TIMP-1 levels correlate with histological grade in B-cell non-Hodgkin's
lymphomas.13 We have shown that TIMP-1 expression in
Burkitt's lymphoma (BL) cell lines inhibits induction of apoptosis by
Fas-dependent and independent pathways and upregulates
BCL-XL.14 These studies support a role for
TIMP-1 in the growth of hematolymphoid tumors.
BL is a highly malignant B-cell tumor originating from centroblasts in
the germinal center of lymph nodes.15,16 This central area
of the lymph node is characterized by a high rate of apoptosis and the
close interaction with the microenvironment (including ECM) that
promotes the normal process of B-cell maturation.15 Although all Burkitt's lymphomas display chromosomal translocations involving c-myc, they differ in their association with the
Epstein-Barr virus (EBV), with a 95% incidence of infection being
observed in the endemic or African type, whereas only 20% of sporadic
or American cases are associated with EBV.16 Although EBV
infection in BLs is not productive,17 most of the cell
lines established in vitro from BLs show various degrees in the
expression of latent EBV genes, and some of them acquire a
lymphoblastic phenotype.16 Thus, phenotypic changes in
these cell lines are believed to be dependent on latent viral gene
expression and allow classification into groups I to
III.16,17 Group I BL, as well as EBV-negative nonendemic BL
lines, retain the phenotype of the original biopsy (CD10+,
CD38+, sIg+, CD77+) and readily
undergo apoptosis. Group II/III BL lines downregulate these markers,
while acquiring a lymphoblastic phenotype, and are more resistant to
apoptosis.17,18 Unlike group I, these latter lines are
characterized by expression of the full complement of viral
genes.17 Progression toward a lymphoblastic morphology and
resistance to programmed cell death is also obtained in vitro as result
of transformation of normal B cells by some EBV strains.19 These results suggest that EBV gene expression controls not only phenotype in BL but also increases resistance to apoptosis.
BL cell lines have been widely used as a model to study both B-cell
malignant transformation and normal mechanisms of the B-cell
physiology.15,20 We have previously shown TIMP-1 expression by centroblast-like BL cell lines but not by follicular lymphoma cell
lines, indicating that TIMP-1 may be operative at a specific stage in
the germinal center.12 As phenotype reflects germinal center stage, as well as EBV latency group, we undertook the study of
the effect of TIMP-1 expression in BL. In the present study, we have
determined that TIMP-1 is not expressed by EBV-negative and group I BL
lines. Furthermore, greater levels of TIMP-1 are shown by lymphoblastic
BL lines. The data indicate that TIMP-1 expression is correlated with a
blast phenotype. To determine whether TIMP-1 was involved in the
phenotypic changes in the BLs, an EBV-negative Burkitt's line
transfected with human timp-1 was also analyzed. Results
strongly suggest that TIMP-1 upregulation is sufficient to induce a
more mature, activated phenotype in Burkitt's lines even in the
absence of EBV. This report further supports the importance of TIMP-1
expression in BLs and suggests that similar studies should be
undertaken for other B-cell malignancies with the hope of determining
the value of TIMP-1 as a prognostic factor and for formulating novel
modalities of treatment.
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MATERIALS AND METHODS |
Cell cultures and treatments.
The BL cell lines JD38, DW6, AG876, ST846, and PA682 were kindly
provided by Dr Ian Magrath21-23 from the Lymphoma Biology Section, National Cancer Institute; Jijyoe and Daudi were obtained from
ATCC, Rockville, MD. The LXSN retroviral transduction was used to
induce TIMP-1 expression in the TIMP-1-negative BL cell line JD38 as
described.14 Briefly, TIMP-1 cDNA was obtained by
polymerase chain reaction and subcloned into LXSN by using DNA
recombinant techniques. Empty LXSN or TIMP-1-LXSN
constructs were transfected into packaging cell lines. Nonadherent JD38
cells were cocultured with adherent LXSN or LXSN-TIMP-1 packaging
cells (GP+envAM12) for 48 hours. LXSN and TIMP-1-LXSN infected JD38 cells were then selected by using 2,500 mg/mL G418 (GIBCO-BRL, Grand
Island, NY) for 10 days. Individual clones were selected by repeated
limiting dilution. Clonality was confirmed by restriction enzyme
digestion with Sma I and Southern blot analysis, with unique sites of integration detected in the JD38 cell clones used in this
study. JD38 cell clones have been stable for TIMP-1 expression and
phenotype for over 2 years. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin G, 100 mg/mL streptomycin sulfate, and L-glutamine (GIBCO BRL, Rockville, MD) and incubated in 5% CO2, 95% humidity at
37°C. TIMP-1-JD38 (clone 24) cells (3 × 105
cells/mL) were also incubated in RPMI 1640 media containing 1% FBS and
5 µg/mL of anti-TIMP-1 neutralizing antibody (Oncogene Sciences, La
Jolla, CA) or isotype control (Jackson ImmunoResearch, West Grove, PA).
After 48 hours, cells were checked for phenotypic changes by
immunostaining and flow cytometry. Three independent experiments were
performed.
TIMP-1 enzyme-linked immunosorbent assay (ELISA) determination.
Secreted TIMP-1 was quantitated by using a Biotrak TIMP-1 ELISA kit
(Amersham, Arlington Heights, IL) that detects free TIMP-1, as well as
MMP-bound TIMP-1. Equal numbers of cells (5 × 105/mL)
were cultured in fresh medium. After 48 hours, cells were centrifuged
and supernatants were tested for TIMP-1 following the manufacturer's
instructions. All ELISA determinations were performed three times, with
triplicate samples within each determination.
Western blot analysis.
BL cell lines as well as TIMP-1-transfected and LXSN-control JD38 cell
clones were cultured (5 × 105 cells/mL) for 48 hours
in serum-free RPMI media. Conditioned media was collected after
centrifugation, and 20 to 40 µL was electrophoresed in a 4% to 20%
(wt/vol), polyacrylamide/sodium dodecyl sulfate (SDS) gel at 100 V for
60 minutes at room temperature. Proteins were electroblotted onto
polyvinylidine difluoride membrane (Novex, San Diego, CA). After
blocking with Tris-buffered saline (TBS) containing 5% nonfat dry
milk, membrane was washed and blotted with monoclonal anti-TIMP-1
antibody (Oncogene Sciences) with 1:1000 dilution. After repeated
washes in TBS, the blots were developed with a horse radish
peroxidase-conjugated goat antimouse antibody diluted 1:10000 (Pierce,
Rockville, IL) and a chemiluminescence kit (DuPont, NEN, Boston, MA).
Blots were exposed to luminescence detection film (Amersham, Arlington
Heights, IL). Three independent analyses were performed.
TIMP-1 functional analysis.
Two independent TIMP-1-JD38 cell lines, clones 20 and 24, as well as
control LXSN-JD38 cells (5 × 105 cells/mL) were
incubated in serum-free conditions for 24 hours. Supernatants (25 mL)
were cleared from cells by centrifugation and concentrated 10 times by
ultrafiltration. Equal amounts of total protein were separated and
analyzed by reverse zymography as previously reported.24
Briefly, electrophoresis was performed in a 15% acrylamide gel
containing 2.25 mg/mL porcine gelatin (Sigma, St Louis, MO), 0.125%
SDS and 160 ng/mL of progelatinase (gift from Dr William
Stetler-Stevenson, the Extracellular Matrix Pathology Section,
Laboratory of Pathology, National Cancer Institute) at 120 V for 1.5 hours. The reverse zymogram was removed and washed in 2.5% Triton-X
for 3 hours and constant shaking. After incubation with activating
enzyme buffer (50 nmol/L Tris, pH 7.5, 200 nmol/L NaCl, 5 mmol/L
CaCl2, 0.02% Brij-35) at 37°C for 15 hours, zymogram was fixed in methanol/acetic acid and stained with 0.5% Coomassie blue. TIMP-1 was visualized as dark blue band of 28-kD mass by indication of the inhibition of gelatin digestion. Supernatant from
HT1080 cells was used as positive control for the detection of TIMPs.
Immunostaining.
After washing twice in phosphate-buffered saline (PBS) containing 1%
bovine serum albumin (BSA), 1 × 106 cells were
incubated with fluorochrome-conjugated monoclonal antibodies (MoAbs)
against the following B-cell differentiation antigens: fluorescein
isothiocyanate (FITC)-CD38, FITC-CD20, phycoerythrin (PE)-CD23, PE-CD5,
FITC-CD45/PE-CD14, FITC-mouse IgG1, and Isotype controls
FITC-IgG/PE-IgG2 (Dako, Carpenteria, CA); PE-CD19 and PE-CD38 (Becton-Dickinson, San Jose CA); FITC-antihuman
IgG1, IgM, IgA, IgD (Tago, Burlingame, CA); PE-CD40 and
unconjugated CD77 (Immunotech, Miami, FL); and FITC and PE-CD10
(Coulter, Miami, FL). After each incubation at 4°C in the dark,
cells were rinsed with PBS and secondary antibody FITC goat-antimouse
(Caltag, San Francisco, CA) was added, when necessary, for 30 minutes
followed by washing twice with PBS. Five independent immunostaining
analyses were performed.
Flow cytometry analysis.
Immunofluorescent-labeled cells were analyzed in a FACSCAN
(Becton-Dickinson, San Jose, CA) with CellQuest software
(Becton-Dickinson) to determine the percentage of positive cells.
Fluorescence intensity was also calculated. After data acquisition,
fluorescent calibrated beads (Flow Cytometry Standards, San Juan, PR)
were used to standardize fluorescence intensity of different antigens
and expressed as molecule equivalent of surface fluorochrome (MESF)
units.
Determination of total IgM.
In addition to cell surface IgM, parental JD38 cells, as well as
LXSN-JD38 and TIMP-1-JD38 cells, were also analyzed
for their production of intracellular and secreted IgM. Equal number of cells (106 cells/mL) were incubated in fresh culture media.
After 48 hours, supernatants were cleared by centrifugation. Cell
pellets were rinsed with PBS and proteins extracted with
RIPA buffer (150 mmol/L NaCl, 1% nonidet
P-40 [NP-40], 0.1% SDS, 50 mmol/L Tris-HCL, pH 8.0) containing the
protease inhibitors 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.23 U/mL aprotinin, and 10 mmol/L leupeptin. After 30 minutes incubation at
4°C, cell lysates were centrifuged in a microcentrifuge and
postnuclear fractions were collected. IgM was measured in protein
fractions as well as in tissue culture supernatants by a
double-antibody ELISA. Plates for the IgM ELISA were prepared by the
Diagnostic and Clinical Research Division of PharMingen (San Diego,
CA). Determination of total IgM was performed in triplicate for three
experiments.
Quantitation of secreted CD23.
Cells (1 × 106) were cultured in 2 mL of media. After
48 hours, supernatants were cleared of cells by centrifugation.
Concentration of secreted CD23 was determined by a double-antibody
sandwich ELISA (Biosource, Camarillo, CA) and read at 450 nm optical
density. Quantitation of secreted CD23 was performed in triplicate for three experiments.
Correlation analysis.
Association of TIMP-1 production and expression intensity of cell
surface markers was analyzed by using Spearman rank correlation coefficients (Biostatistics and Data Management Section, National Cancer Institute).
| |
RESULTS |
BL phenotype and TIMP-1 expression.
Both EBV-negative, as well as EBV-positive BL lines with differential
viral latency expression were assessed for TIMP-1 secretion. Figure 1 shows production of TIMP-1 by the
seven lines studied. Lines DW6, PA682, Jijyoe, and AG876 secrete
TIMP-1. These cell lines also show advanced (ie, group II/III) EBV
latency.21-23 Lack of TIMP-1 expression is seen by the
group I line Daudi, as well as by the EBV-negative lines JD38 and ST846
(this confirms our previous results showing lack of TIMP-1 RNA
expression in these cell lines).14 Phenotypic analysis of
these cell lines shows changes in the cell surface markers typically
seen in germinal center tumors. Figure 2
shows representative flow cytometric analyses for two (JD38 and Daudi)
of the three TIMP-1-negative tumors analyzed as well as for four
(PA682, AG876, Jijyoe, and DW6) TIMP-1-positive lines.
TIMP-1-negative BL lines express CD10, CD77, and cell surface IgM,
whereas TIMP-1-positive BL lines show downregulation of these markers
accompanied by upregulation of the activation marker CD23. These
results not only indicate that TIMP-1 expression correlates with EBV
latency group in BL cell lines but is also associated in these tumors
with changes in the germinal center phenotype that occurs with the
generation of lymphoblasts. All cell lines were CD5 negative and
expressed the same levels of CD20, CD19, and CD45 (data not shown).
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| Fig 1.
TIMP-1 production by BLs. Cell lines were assayed
for TIMP-1 secretion by ELISA analysis of conditioned media. Number
below cell lines indicate negative or EBV latency grade. The X-axis shows BL cell lines studied. The Y-axis shows TIMP-1 in ng/mL. Data
represent triplicate determinations ± standard deviation (SD) of
three experiments.
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| Fig 2.
BL phenotype and TIMP-1 expression. Representative flow
cytometric analysis of cell lines with differential TIMP-1 secretion as
shown in Fig 1. JD38 and Daudi are negative for TIMP-1 whereas DW6,
PA682, AG876, and Jijyoe express variable levels. TIMP-1-negative BL
lines express high-level CD10, surface IgM, and CD77 (similar results
obtained with all three TIMP-1-negative lines studied). TIMP-1+ BL lines show downregulation of the follicular
center markers CD10, surface IgM, and CD77 along with upregulation of
activation marker CD23 seen with all four TIMP-1-positive BL lines
studied. The X-axis shows log fluorescence intensity. The
Y-axis shows the number of cells. Horizontal bars in histograms
indicate nonantibody binding as determined by irrelevant isotype
control antibodies. Data are representative of five independent
experiments.
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TIMP-1 levels and B-cell differentiation.
We next determined whether TIMP-1 expression was related to
differentiation state in BL cell lines. Immunoglobulin (sIg) and CD38
antigen expression both vary during B-cell development and can be used
to determine B-cell differentiation.25 Downregulation of
surface IgM in the TIMP-1+ cell lines indicates that these
tumors may be more mature (Fig 2). During B-cell maturation, the level
of CD38 expression decreases from the early B cell, which is a high
expressing cell, to the mature B cell (low expressing), and then levels
are again elevated on terminally differentiated plasma cells.
Figure 3A shows intensity of fluorescence
of CD38 by various BL lines in relation to their TIMP-1 expression. The
TIMP-1-negative cell lines (ST846, JD38, and Daudi) and low level
TIMP-1 secreting line DW6 express CD38 in the range of 1 × 106 to 2 × 106 fluorescence (MESF) units,
whereas cell lines expressing levels higher than 2 ng/mL TIMP-1 are
either negative for CD38 or show one log decrease in fluorescence
intensity. These results, in conjunction with the other phenotypic
findings, are consistent with TIMP-1-negative BL lines representing
earlier stages of B-cell differentiation than high TIMP-1-expressing
cells. Although the highest TIMP-1-positive cell lines, AG876 and
Jijyoe, show no sIgM (Fig 2) and, therefore, are more mature cells,
these tumors express lower CD38 levels than those usually seen in
plasmacytoid cells. Association of TIMP-1 and CD38 intensity of
expression shows a moderate inverse correlation (r =  .70, P2 = .08). Therefore, these
lines can be described as mature, activated B cells in a preplasma
stage.
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| Fig 3.
TIMP-1 levels are associated with intensity of CD40 and
CD38 expression. (A) CD38 expression in BL lines shows a moderate inverse correlation (r = .70, P2 = .08) with TIMP-1 production, BL lines expressing higher TIMP-1 levels
(<2 ng/mL) are either CD38 negative or show significantly lower
CD38(dim) intensity. The X-axis shows secreted TIMP-1 as determined by
ELISA. The Y-axis shows CD38 expression as determined by flow cytometry
and expressed in MESF as explained in the Materials and Methods. (B) A
direct strong correlation (r = .88, P2
= .02) between TIMP-1 production and intensity of CD40 is observed
among BL cell lines. The X-axis shows secreted TIMP-1. The Y-axis shows
CD40 fluorescence units expressed as MESF as explained in the Materials
and Methods. Data represent duplicate MESF determinations of three
experiments. ELISA detection of TIMP-1 levels were performed in
triplicates for three experiments.
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Apoptosis plays a central role during B-cell development. We have
previously shown that expression of TIMP-1 in BL cell lines inhibits
apoptosis induced by various treatments including fas activation, serum
starvation, and gamma radiation.14 TIMP-1 upregulates
BCL-XL and the inhibitor of NF-kB, IkB, but does not affect
BCL-2.14 CD40 has been shown to regulate apoptosis in B
cells. Ligation of CD40 with MoAb causes inhibition of cell death of
both germinal center B cells and immature B cells.26 Also,
cytokines such as interleukin-13 (IL-13) have been shown to affect cell
survival through upregulation of CD40 in peripheral B
cells.27 Expression of CD40 in BLs also shows a strong
correlation (r = .88, P2 = .02) with TIMP-1
levels (Fig 3B). Unlike TIMP-1-negative cell lines, DW6, PA682, and
Jijyoe express up to 2 logs higher fluorescence units of CD40.
Furthermore, induction of TIMP-1 expression in JD38 cells upregulates
CD40 expression (Fig 5B). These results indicate an association between
TIMP-1 levels and CD40 expression intensity and provide an additional
mechanism by which TIMP-1 may inhibit apoptosis.
Effects of TIMP-1 transfection on the phenotype of JD38 BL cells.
The pattern of gene expression we observe in TIMP-1+
Burkitt's cell lines has been previously reported as controlled by EBV proteins. For instance, CD23 is increased by nuclear antigen-2 (EBNA-2)
and latent membrane protein-1 (LMP-1) in Burkitt's cell lines with
type-III latency.28 To determine whether TIMP-1 expression was also dependent on EBV state of latency, we analyzed the
EBV-negative BL cell line JD38 transfected via retroviral infection
with the human timp-1. TIMP-1 secretion by two independent
TIMP-1-JD38 cell clones (20 and 24) was similar to the TIMP-1
expression by EBV-positive BL lines, whereas control LXSN-JD38 and
parent JD38 cells are negative for TIMP-1 as analyzed by Western blot
(Fig 4A and B). Functional MMP inhibitory
activity of TIMP-1 secreted by transfected JD38 cells was confirmed by
reverse zymography (Fig 4C).
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| Fig 4.
Detection and functional analysis of TIMP-1. (A) Western
blot analysis of unconcentrated TIMP-1 in conditioned media of various BL lines: lane 1, rTIMP-1 (300 ng) positive control; lane 2, JD38; lane
3, ST846; lane 4, Daudi; lane 5, AG876; lane 6, PA682; lane 7, DW6.
JD38 cells were transfected with human timp-1 or empty LXSN vector as described in the Materials and Methods. (B)
Western blot analysis of TIMP-1 by TIMP-1-JD38 cell clones: lane 1, rTIMP-1 (300 ng) positive control; lane 2, parental JD38 cells; lane 3, TIMP-1-JD38 cell clone 20; lane 4, TIMP-1-JD38 cell clone 24; lane 5, LXSN-JD38 cells. (C) Reverse zymogram of TIMP-1 in the concentrated
conditioned media of transfected JD38 cells: lane 1, LXSN-JD38 cells;
lane 2, TIMP-1-JD38 cell clone 20; lane 3, TIMP-1-JD38 cell clone 24;
lane 4, positive control HT1080 cells. Representative results of three
independent analyses.
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Figure 5A shows TIMP-1 production by
TIMP-1-JD38 cell clones (20 and 24) but not by those cells carrying
empty vector LXSN or parental JD38 cells. Flow cytometry
analysis of these cell lines shows downregulation of CD10, CD38, CD77,
and sIgM in the TIMP-1-JD38 cells. Upregulation of TIMP-1 expression
also induces CD23 expression and increased CD40 as shown by an enhanced
fluorescence intensity signal when compared with parental JD38 cells
and LXSN-JD38 cells (Fig 5B). Incubation of TIMP-1-JD38 clone 24 with
anti-TIMP-1 antibody inhibited the observed phenotypic changes. Unlike
treatment with control antibody, treatment with anti-TIMP-1 antibody
decreases the percentage of cells expressing CD23 while increasing the
percentage of cells expressing CD10 and CD77
(Fig 6). These results indicate that the
observed phenotypic changes in the TIMP-1-JD38 clone are secondary to
the secreted TIMP-1 protein. This is consistent with our previous
observations that secreted TIMP-1 inhibits apoptosis in BL cell
lines.14
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| Fig 5.
Effects of induced TIMP-1 in the phenotype of the
EBV-negative JD38 cells. (A) TIMP-1 secretion shown by two independent
TIMP-1-JD38 cell clones (20 and 24) compared with JD38 cells
transfected with vector alone (LXSN) and JD38 parental cells (parent).
The Y-axis shows secreted TIMP-1 as determined by ELISA of conditioned
media. Data represent triplicate determinations ± SD of three
experiments. (B) Flow cytometry analysis of TIMP-1-transfected JD38
cell clones 20 and 24 shows downregulation of follicular markers and
upregulation of CD23 and CD40 compared with parental JD38 and LXSN-JD38
control cells. The X-axis shows log fluorescence intensity. The Y-axis shows the number of cells. Every plot shows staining with irrelevant isotype control antibodies (empty histograms). Data represent five
independent flow cytometric determinations.
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| Fig 6.
Flow cytometric analysis of TIMP-1-JD38 cells treated
with TIMP-1 neutralizing antibody. TIMP-1-JD38 clone 24 cells were
treated with an isotype control (A) or anti-TIMP-1 antibody (B) as
explained in the Materials and Methods. Two-color flow cytometry shows
anti-TIMP-1 treatment decreases percentage of CD23 positive cells from
89.7% (A) to 27.4% (B), and increases both the percentage of cells
expressing CD10 from 0.55% (A) to 17.8% (B), and CD77 from 1.2% (A)
to 13.6% (B), respectively. Isotype control plots (IgG Ctrl) are also
shown. Data are representative results of three independent
determinations.
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To further determine TIMP-1 effect on immunoglobulin production,
parental and both TIMP-1-JD38 cell clones were analyzed for their
intracellular as well as secreted IgM expression.
Table 1 shows a significant decrease in the
total IgM production by the two TIMP-1 overexpressing clones (20 and
24) compared with control JD38 cells. In contrast to parental and
LXSN-JD38 cells, TIMP-1-JD38 cells downregulate cell surface
immunoglobulin (Fig 5B). Thus, the downregulation in the surface IgM by
TIMP-1-JD38 cell clones does not result in intracellular accumulation
or augmented IgM secretion (Table 1). On the contrary, TIMP-1
upregulation resulted in a 50% reduction in the intracellular IgM
levels and an 80% decrease of secreted IgM. These results clearly show
that in the absence of EBV, induction of TIMP-1 in BLs is sufficient to
modulate immunoglobulin expression.
The CD23, also known as low affinity receptor for IgE (FceRII),
is a multifunctional 45-kD membrane glycoprotein that is cleaved into a
biologically active 37-kD soluble fragment (sCD23).29 In
activated B cells, upregulation of membrane CD23 is usually accompanied
by CD23 secretion.30 TIMP-1 transfected JD38 cells were
also tested by ELISA for their expression of secreted CD23 and compared
with JD38 control cells as well as with TIMP-1+ and
TIMP-1 BL lines. Table 2
shows percentage of positive cells for membrane-CD23 as determined by
flow cytometry and units/mL of secreted CD23 as assayed by ELISA. The
TIMP-1-negative BL cell lines fail to express CD23 whereas the
TIMP-1-positive lines show secretion and cell surface expression of
CD23. Induction of TIMP-1 expression in JD38 cells resulted in
upregulation of CD23 expression and secretion compared with parental
and LXSN-JD38 controls. In addition, in all TIMP-1-positive BL cell
lines studied, a higher level of CD23 secretion correlated inversely
with surface CD23, indicating a higher CD23 cleavage by these.
| |
DISCUSSION |
The extracellular matrix has been shown to regulate various functions
in hematolymphoid cells.30 Soluble components
such as metalloproteinases and their inhibitors have been reported to
be involved in proteolytic cleavage of receptors and ligands important
for the survival and/or growth of hematolymphoid
cells.31-33 Additionally, we have recently reported on the
expression of matrix metalloproteinases and TIMPs by human normal and
neoplastic lymphoid cells.12 In this study, TIMP-1
expression in B-cell lymphomas did not correlate with expression of
matrix metalloproteinases. Furthermore, TIMP-1 was expressed by the
centroblast-like BL cell lines but not by the follicular lymphoma lines
studied. The observation of discordant TIMP-1 production in B cells and
germinal-center-specific expression prompted us to investigate the
role of TIMP-1 expression in BL cell lines. Previously, we have shown
that TIMP-1 upregulation in Burkitt's cell lines inhibits apoptosis
and induces BCL-XL.14 In the present study,
TIMP-1 was secreted by BL lines previously described as group II/III,
which corresponds to advanced stage in EBV latency. Unlike the latter
cell lines, BL cells retaining the original biopsy phenotype
(CD10+, CD38+, CD77+, and
sIg+) did not secrete TIMP-1. Additionally, TIMP-1 was
associated with upregulated expression intensity of the survival
antigen CD40 as well as expression and secretion of the activation
marker CD23.
These results together with the observations of CD38 expression and IgM
production suggest that higher TIMP-1 levels are associated with a
mature, activated B cell phenotype in EBV+ BLs. However,
the present report also shows that inducing TIMP-1 expression in the
EBV-negative JD38 was sufficient to generate the same phenotypic
changes as those seen in EBV+ lymphomas. That this is a
TIMP-1-specific effect is indicated by the observed reversal of these
phenotypic changes by TIMP-1 neutralizing antibody. These results
suggest that TIMP-1 expression may be an important factor in the
maturational or activation state of B cells. The TIMP-1 upregulated
expression of the B-cell survival factor, CD40, may also contribute to
the inhibition of apoptosis. Our data suggest that TIMP-1 expression
could convey a poor prognosis in these high-grade lymphomas.
Considering that the germinal center is the origin of BL and that
important steps of B-cell differentiation also occur in the germinal
center, numerous studies have been directed to describe B-cell
subpopulations in this region of the lymph node.34,35 CD77
expression is highly restricted to germinal-center B
lymphocytes.36 This antigen is a neutral glycolipid
expressed by a subset of B lymphocytes that readily enter programmed
cell death.37,38 Apoptosis in these cells is prevented by
CD40 engagement and by soluble CD23.39 Rescue from
apoptosis by CD40 is mediated by a Bcl-2-independent
mechanism.40 Based on these previous studies, a model of
B-cell maturation has been proposed in which ligation of CD40 drives
cells to lose CD77 and express membrane and soluble CD23, which in
turns acts as an autocrine factor. Unlike these studies in which normal
B lymphocytes were infected with an EBV strain, the present report
shows that in the absence of EBV, upregulation of TIMP-1 induces the
same changes, probably through similar mechanisms used by EBV. Thus,
TIMP-1 may play a role in the normal development of the B cells in that
it may provide an ECM signal to prevent programmed cell death. The
phenotypic changes observed here normally occur in the germinal center
during the interaction of B cells and T cells and in close proximity to
the extracellular matrix in the germinal center
stroma.39,41,42 Moreover, TIMP-1 expression has been
observed in this stroma.43 The outcome of this interaction depends on the affinity of B-cell receptors for antigens, either resulting in death by apoptosis of B cells expressing low affinity receptors or survival of memory B cells. TIMP-1 by upregulating expression of CD40 and decreasing CD77 could be implicated in the
survival of both differentiating normal and neoplastic B cells. This is
supported by previous results from our laboratory showing that TIMP-1
expression in B cells confers resistant to apoptosis.14 TIMP-1-mediated rescue of differentiated cells from apoptosis and
restoration of normal development of the mammary gland has been
previously reported in a transgenic huTIMP-1 mouse model.44 The present study further supports a role for TIMP-1 as survival factor
in differentiated B cells. Future studies are indicated to determine
not only the prognostic value of TIMP-1 in B-cell neoplasias, but also
its clinical significance in other malignancies.45,46 Gaining insights into the mechanism of TIMP-1 action may show new
therapeutic approaches to the treatment of lymphomas as well as solid
tumors.
| |
FOOTNOTES |
Submitted January 9, 1998;
accepted April 19, 1998.
Address reprint requests to Maryalice Stetler-Stevenson, MD, PhD,
Building 10, Rm 2A33, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Susan Wormsley (PharMingen Inc) for her valuable help
determining IgM levels by ELISA, Dr Seth M. Steinberg (Head,
Biostatistics and Data Management, National Cancer Institute) for
statistical analysis, Chris Benton (Amersham) for providing the TIMP-1
ELISA kit. The authors also thank Drs William G. Stetler-Stevenson, Susan Hoegie, and Megan Lim for their valuable comments.
| |
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Abstract
Introduction
Abstract