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IMMUNOBIOLOGY
From the Flow Cytometry Unit and the Extracellular
Matrix Pathology Section, Laboratory of Pathology, and the
Biostatistics and Data Management Section, Division of Clinical
Sciences, National Cancer Institute, Bethesda, MD.
Tissue inhibitors of metalloproteinases (TIMPs), first described as
specific inhibitors of matrix metalloproteinases, have recently been
shown to exert growth factor activities. It was previously demonstrated
that TIMP-1 inhibits apoptosis in germinal center B cells and induces
further differentiation. Interleukin-10 (IL-10) is reported as a vital
factor for the differentiation and survival of germinal center B cells
and is also a negative prognostic factor in non-Hodgkin lymphoma (NHL).
However, the mechanism of IL-10 activity in B cells and the regulation
of its expression are not well understood. IL-10 has been shown to
up-regulate TIMP-1 in tissue macrophages, monocytes, and prostate
cancer cell lines, but IL-10 modulation of TIMP-1 in B cells and the
effect of TIMP-1 on IL-10 expression has not been previously studied. It was found that TIMP-1 expression regulates IL-10 levels in B cells
and that TIMP-1 mediates specific B-cell differentiation steps. TIMP-1
inhibition of apoptosis is not IL-10 dependent. TIMP-1 expression in
B-cell NHL correlates closely with IL-10 expression and with high
histologic grade. Thus, TIMP-1 regulates IL-10 expression in B-cell NHL
and, through the inhibition of apoptosis, appears responsible for the
negative prognosis associated with IL-10 expression in these tumors.
(Blood. 2001;97:1796-1802) Tissue inhibitors of metalloproteinases (TIMPs) are
a family of closely related proteins that were first
described1-3 as the specific inhibitors of matrix
metalloproteinases (MMP). However, in addition to blocking MMP
activity, TIMPs have also been shown to exert growth factor activities
that are independent of their enzymatic inhibitory
function.4-10 We have previously reported on the
high-level expression of TIMP-1 by normal activated tonsillar B cells
and centroblast-like Burkitt lymphoma cell lines.11 TIMP-1
expression is operative at a specific stage in B-cell development and
is associated with the germinal center phenotype that occurs with the
generation of lymphoblasts.12 TIMP-1 expression by tonsillar germinal center B cells is dependent on the germinal center
milieu, with expression decreasing rapidly on in vitro isolation.11 Induction of endogenous TIMP-1, or treatment
with recombinant TIMP-1 protein, effects further B-cell
differentiation in centroblast-like Burkitt lymphoma cell lines. This
is evidenced by the TIMP-1 modulation of expression of the
differentiation-specific antigens CD10, CD38, surface immunoglobulin
(sIg), CD77, CD40, and CD23.12 We have also previously
shown that TIMP-1 inhibits apoptosis in normal germinal center B cells
and Burkitt lymphoma cell lines.13 In B cells, this is at
least partially attributed to TIMP-1 up-regulation of the antiapoptotic
factor Bcl-XL. However, TIMP-1 may also inhibit apoptosis
by regulating the expression of CD40, CD77, and CD23. Li et
al14 have recently demonstrated that TIMP-1 inhibits
apoptosis in human breast epithelial cells and is induced by the
antiapoptotic factor Bcl-2, indicating this effect is not
tissue specific.
The phenotypic changes induced by TIMP-1 normally occur in the germinal
center during the interaction of B cells and T cells and in proximity
to the TIMP-1-containing germinal center stroma.15-18 CD77 expression is highly restricted to germinal-center B lymphocytes and is a neutral glycolipid expressed by a subset of B lymphocytes that
readily enter programmed cell death.19-21 Apoptosis in
these cells is prevented by CD40 engagement and by soluble
CD23.22 A model of B-cell maturation has been proposed in
which the ligation of CD40 drives differentiating B cells to lose CD77
surface expression and to express membrane and soluble CD23, all
of which are associated with an antiapoptotic state. Thus, TIMP-1 Human interleukin-10 (IL-10) is a cytokine normally produced by
activated T cells, monocytes, B cells, and thymocytes.15 IL-10 contributes to antigen- or mitogen-driven B-cell differentiation and, in non-Hodgkin lymphomas, acts as a growth
factor.23,24 Activation of CD40 ligand induces IL-10
secretion and modulates immunoglobulin production. Furthermore, IL-10
is an important factor for the differentiation and survival of germinal
center B cells.15 Because TIMP-1 expression in Burkitt
lymphoma cell lines induces a more mature phenotype and affects CD40
and immunoglobulin expression,12 a possible effect of
TIMP-1 on IL-10 expression was contemplated. IL-10 has been shown to
up-regulate TIMP-1 in tissue macrophages, peripheral blood monocytes,
and prostate cancer cell lines,25,26 but the IL-10
modulation of TIMP-1 in B cells and the effect of TIMP-1 on IL-10
expression have not been studied. Therefore, we examined the relation
between TIMP-1 and IL-10 expression in a series of cell lines and
non-Hodgkin lymphoma specimens.
Cells
Consecutive high-grade (n = 10) and low-grade (n = 21) B-cell
non-Hodgkin lymphomas were retrieved from the Hematopathology Section
frozen-tissue bank at the National Cancer Institute (National Institutes of Health, Bethesda, MD). Hematoxylin-eosin-stained sections from the original case were reviewed, and complete
immunophenotyping was performed to confirm the diagnosis.
Immunophenotyping was performed on frozen sections and paraffin
sections by the immunoperoxidase technique and on cell suspensions by
flow cytometry. High-grade cases included 8 diffuse large B-cell
lymphomas and 2 Burkitt lymphomas. Low-grade cases included 8 small
lymphocytic lymphomas and 13 follicle-center lymphoma (all follicular,
nondiffuse, and grade I or II).
cDNA production
Reverse transcription-polymerase chain reaction TIMP-1 and IL-10 expression was determined by semiquantitative reverse transcription (RT)-PCR. Multiple quantities of cDNA and cycle numbers were used to optimize the range for PCR determinations. Equal quantities of cDNA were then used in PCR amplification. The following primers were used for TIMP-1 RT-PCR: 5'-ATAGTCGACATGGCCCCCTTTGAG CCCCTG-3' and 5'GGAATTCCTC AGGCTATCTGGGACCGCAGGGA-3'. The following primers (specific for human IL-10; do not amplify viral IL-10) were used for IL-10: 5'-ACCAAGACCCAGACATCAAG-3' and 5'-GAGGTACAATAAGGTTTCTCAAG-3'. -Actin primers were as follows:
5'-AAGAGAGGCATCCCTCACCT-3' and 5'-TACATGGCTGGGGTGTTGAA-3'. Thirty-one
B-cell non-Hodgkin lymphomas (10 high grade, 21 low grade) were
examined for TIMP-1 expression. Twenty-seven B-cell non-Hodgkin
lymphomas (10 high grade, 17 low grade) were examined for IL-10
expression because of the availability of adequate cDNA.
Amplified products were electrophoresed on a 4% low melting point
agarose gel, and bands were visualized with 2% ethidium bromide and
transferred onto a nylon membrane (Dupont-NEN Research, Boston, MA) by
Southern blot analysis. Blots were hybridized using standard conditions
with TIMP-1, IL-10, and Enzyme-linked immunosorbent assay determinations Secreted TIMP-1 was quantitated by using a TIMP-1 enzyme-linked immunosorbent assay (ELISA; Oncogene Research, Cambridge, MA) kit that detects free TIMP-1 and MMP-bound TIMP-1. Secreted IL-10 was quantitated using an Immunotech (Westbrook, ME) ELISA kit that detects human IL-10 and the Epstein-Barr virus (EBV) IL-10 viral homolog. Equal numbers of cells (5 × 105/mL) were cultured in fresh media. After 48 hours, cells were centrifuged and supernatants were tested for TIMP-1 or IL-10 following the manufacturer's instructions. All ELISA determinations were performed 3 times, with triplicate samples within each determination.Reverse zymography Metalloproteinase inhibitory activity was assayed by electrophoresis in polyacrylamide gels containing gelatin as matrix metalloproteinase substrate and HT1080 fibroblast culture conditioned media as a source of matrix metalloproteinase, as previously described.27 Briefly, 12% or 15% polyacrylamide gels were prepared with Tris-HCl containing 0.1% sodium dodecyl sulfate (SDS), 2.5 mg/mL gelatin, and 10% (vol) HT1080 fibroblast culture-conditioned media. Aliquots of conditioned media (15 µL) were suspended in sample buffer (50 mol/L Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, pH 6.8) and applied to gels followed by electrophoresis at 20 mA/gel. Gels were incubated in 2.5% (vol/vol) Triton X-100 for 60 minutes to remove SDS and then were incubated overnight in developing buffer (50 M Tris-HCl, 0.2 M NaCl, 5 mmol/L CaCl2, 0.02% (wt/vol) Brij-35, adjusted to pH 7.6). This restores the matrix metalloproteinase activity, resulting in gelatin degradation. Gels were stained for 3 hours in 30% methanol, 10% glacial acetic acid, 0.5% Coomassie blue G-250 (Bio-Rad, Richmond, CA), destained for 1.5 to 2 hours in 30% methanol and 10% glacial acetic acid, and dried overnight. TIMPs were visualized as nondegraded gelatin staining positive with Coomassie blue.TIMP-1-IL-10 incubations JD38 cells (3 × 105 cells/mL) were incubated in serum-free media with both native recombinant TIMP-1 and reduced and alkylated TIMP-1 (0-500 ng/mL) for 24 and 48 hours. JD38 cells (3 × 105 cells/mL) were incubated in serum-free media with 0 to 25 ng/mL recombinant IL-10 (Genzyme, Cambridge, MA). TIMP-1-expressing clones were incubated with 1 µg/mL neutralizing anti-TIMP-1 monoclonal antibody (Oncogene Research Products). IL-10 was inactivated by incubation with neutralizing antibodies to IL-10 (0.5 µg/mL) and with 50 pg/mL soluble IL-10 receptor (R & D Systems, Minneapolis, MN). Incubation with an equivalent quantity of isotype control antibody protein was performed in each experiment. Conditioned media were collected after 24- and 48-hour incubation in serum-free RPMI or AIM V medium (Gibco BRL). Supernatants were cleared of cells by centrifugation and, in some experiments, whole cell lysates prepared using RIPA buffer (150 nM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris-HCL, pH 8.0) containing protease inhibitors. Protein concentration of supernatants and whole cell lysates was determined by BCA protein assay from Pierce (Rockville, IL).Flow cytometric immunophenotyping Cells (1 × 106) were washed twice in phosphate-buffered saline containing 1% bovine serum albumin before staining, as described,12 with fluorochrome-conjugated monoclonal antibodies against the following antigens: fluorescein isothiocyanate (FITC)-CD38, FITC-CD20, phycoerythrin (PE)-CD5, FITC-CD45, PE-CD14, FITC-mouse IgG1, and PE IgG2 (DAKO, Carpinteria, CA); PE- CD38 (Becton Dickinson, San Jose, CA); PE-CD40 and unconjugated CD77 (Immunotech, Miami, FL); and FITC-CD10 (Coulter, Miami, FL). Labeled cells were analyzed in a Facscan (Becton Dickinson, San Jose, CA) with Cellquest software to determine the percentage of positive cells and fluorescence intensity (ie, quantitation of antibody-staining intensity). After data acquisition, fluorescent calibrated beads (Flow Cytometry Standards, San Juan, Puerto Rico) were used to standardize fluorescence intensity of different antigens and data were expressed as molecule equivalent of surface fluorochrome units.Cell viability-proliferation assays All cells were reviewed microscopically for morphologic evidence of apoptosis such as blebbing and nuclear condensation. Viability was determined by cellular exclusion of trypan blue. Apoptosis was measured by light scatter as previously described28 and by staining with Annexin V-FITC from Caltag (Burlington, CA) according to the manufacturer's protocol. To measure proliferation, cells were incubated with bromodeoxyuridine (BrdU) for 30 minutes at 37°C. S-phase and BrdU incorporation were simultaneously detected by flow cytometric analysis of staining with the DNA-binding fluorescent dye propidium iodide- and FITC-labeled anti-BrdU (Becton Dickinson, San Jose, CA), according to manufacturer's guidelines.
TIMP-1 up-regulation of IL-10 expression IL-10 expression was studied in a series of Burkitt lymphoma cell lines with known TIMP-1 expression.12,13 Figure 1A shows that IL-10 is expressed and secreted by all TIMP-1+ Burkitt lymphoma lines studied (AG876, PA682, Jijyoe, DW6). In contrast, TIMP-1 Burkitt
cell lines do not secrete IL-10 (Daudi, ST846, JD38). Furthermore,
IL-10 expression is proportional to levels of TIMP-1 secretion. To
determine whether IL-10 expression was dependent on TIMP-1 expression,
we analyzed the TIMP-1 Burkitt lymphoma cell line JD38
transfected by retroviral infection with the human TIMP-1 gene. ELISA
used for the IL-10 determination also detected viral IL-10. The
EBV Burkitt cell line JD38 offered another advantage: we
could examine the effects of TIMP-1 and IL-10 in the absence of viral
IL-10. Figure 1B shows the effects of TIMP-1 expression, mediated by retroviral vector, on human IL-10 expression by JD38 cells.
Up-regulation of TIMP-1 induces the secretion of IL-10, as shown by 3 independent TIMP-1 clones (clones 8, 20, 24). TIMP-1-induced IL-10
levels are similar to those seen in TIMP-1-expressing,
IL-10+ Burkitt cell lines. In contrast,
TIMP-1 JD38 cells carrying empty LXSN vector and parental
cells do not secrete IL-10.
To further determine the role of TIMP-1 in controlling IL-10
expression, JD38 cells were cultured in the presence of increasing concentrations of human recombinant TIMP-1 and analyzed for IL-10 secretion. Figure 2A shows that when
cells are cultured in serum-free conditions, exogenous TIMP-1 not only
maintains high viability (as shown previously) but also induces JD38
cells to secrete IL-10. Furthermore, inhibition of secreted TIMP-1 in
the TIMP-1-expressing JD38 clone 24 by a neutralizing anti-TIMP-1
antibody results in the decreased expression of IL-10 (data not shown).
This demonstrates that TIMP-1 can regulate the production of IL-10 by
Burkitt lymphomas. This activity of TIMP-1 on IL-10 induction is
independent of its enzymatic inhibitory function. Figure 2B
demonstrates that reduced and alkylated TIMP-1 (a form devoid of all
MMP inhibitory function but retaining growth factor
activity13,29) can still induce IL-10 secretion in a
concentration-dependent manner. Induction of IL-10 secretion by TIMP-1
is evidence supportive of an autocrine loop. We have previously
demonstrated saturable cell surface binding of TIMP-1 to JD38 cells
that is consistent with a TIMP-1 receptor.13 This suggests
that TIMP-1 binding to a cell surface receptor mediates IL-10
expression.
Biologic activity of IL-10 versus TIMP-1 in B cells In previous studies we have shown that TIMP-1 is a survival and differentiation factor for B cells.12,13 These effects could be directly mediated by TIMP-1 activity or TIMP-1 induction of IL-10. We therefore studied the effect of recombinant IL-10 on B-cell differentiation and inhibition of apoptosis. JD38 cells are highly serum dependent and rapidly undergo apoptosis in serum-free media. Although rTIMP-1 prevents the induction of programmed cell death in JD38 cells under serum-free conditions,13 it also induces IL-10 expression. The number of viable cells under serum deprivation was dose dependent in the JD38 cells incubated with 0 to 25 ng/mL rIL-10 (Figure 3A). However, treatment with rIL-10 at concentrations observed in TIMP-1+ cell lines did not prevent apoptosis of the TIMP-1 JD38 cells
in serum-free media (Figure 3B). Il-10 increased proliferation in the
cells, as measured by BrdU incorporation (Figure 3B-C). Although the
differences in BrdU incorporation are modest, they are significant and
only represent a 30-minute incubation. Differences in the number of
viable cells were observed after 48 hours, a much more sensitive
indicator of the action of IL-10. Thus, the increased number of viable
cells was attributed to increased cell growth in the presence of
identical levels of apoptosis. Although IL-10 has been reported to
prevent germinal center B-cell apoptosis,30 this effect is
differentiation dependent IL-10 also induces B-cell apoptosis in
specific B-cell populations.31 It is apparent that it is
TIMP-1, not IL-10, that prevents apoptosis in the current model;
rTIMP-1 prevents apoptosis and IL-10 has been shown to induce TIMP-1
expression in monocytes and prostate cancer cells.25,26 To
determine whether IL-10 induces TIMP-1 in B cells, we analyzed the
supernatant of IL-10-treated cells for TIMP-1 protein by both ELISA
and reverse zymography (data not shown). In addition, RT-PCR for TIMP-1
cDNA was performed on the IL-10-treated cells. IL-10 induction of
TIMP-1 was not observed by any of the methods used. To further prove
that TIMP-1-mediated inhibition of apoptosis occurs independently of
IL-10, JD38 clones 20 and 24 (positive for both TIMP-1 and IL-10
expression) were incubated in serum-free conditions in the presence of
both IL-10 neutralizing antibody and IL-10 receptor blocking
(nonactivating) antibody (ie, IL-10 activity simultaneously blocked by
2 methods). After the addition of both neutralizing antibodies, the
JD38 clones 20 and 24 did not undergo apoptosis (TIMP-1 is still
active). However, the addition of TIMP-1-neutralizing antibody did
induce apoptosis (Figure 3C). Isotype controls for anti-IL-10,
anti-IL-10 receptor, and anti-TIMP-1 antibodies did not induce
apoptosis. In summary, apoptosis of these Burkitt lymphoma cells is
prevented by the presence of TIMP-1 alone, whereas IL-10 stimulates
cell proliferation. Neutralization of IL-10 in the presence of TIMP-1
did not result in programmed cell death.
TIMP-1 effects further B-cell differentiation, and TIMP-1-expressing
lines are mature, activated B cells in a pre-plasma cell stage.12 Induction of TIMP-1 (Figure
4A-B) or addition of recombinant TIMP-1
protein down-regulates CD10, CD38, CD77, and surface immunoglobulins while up-regulating CD23 and CD40 in JD38 cells. Neutralization of
TIMP-1 by anti-TIMP-1 antibody reverses this effect (Figure 4C). The
induction of a more mature phenotype may be a direct action of TIMP-1
or secondary to IL-10 expression. Therefore, we incubated JD38 cells
with rIL-10 and analyzed for the changes in immunophenotype. We did not
observe any changes in levels of expression of CD40, CD10, or CD77
after the addition of IL-10 to TIMP-1
Expression of TIMP-1 and IL-10 in B-cell non-Hodgkin lymphoma The in vitro effects of TIMP-1 on the survival and induction of IL-10 by B cells could have important implications in the clinical behavior and response to therapy of B-cell malignancies. IL-10 expression has been associated with a poor prognosis in non-Hodgkin lymphoma and may be important in the development of B-cell neoplasia. These findings prompted us to determine the relation between TIMP-1 and IL-10 expression in a series of B-cell non-Hodgkin lymphoma of either high grade (n = 10) or low grade (n = 21) using semiquantitative RT-PCR normalized to actin expression. A statistically significant
difference (P < .00001) in the expression of TIMP-1 is
observed between high- and low-grade tumors (Figure 5A), as determined by the Wilcoxon rank
sum test. In high-grade B-cell lymphomas, 100% had high level TIMP-1
(greater than 0.7 relative density units). All low-grade tumors were
either negative for TIMP-1 (n = 2 of 21) or had low level expression
(19 of 21; 90%), as determined by less than 0.65 relative density.
These data clearly establish a direct correlation between TIMP-1
expression and the histologic grade of B-cell lymphomas and further
support our hypothesis that TIMP-1 may play a role in the pathogenesis of B-cell lymphomas.
The same lymphoma cases were examined for human IL-10 mRNA by semiquantitative RT-PCR using primers that specifically detect human IL-10 mRNA, and not viral IL-10. IL-10 levels are strongly associated with tumor grade (P < .00001) by the Wilcoxon rank sum test. As demonstrated in Figure 5B, all high-grade lymphomas expressed IL-10, whereas only 4 of 17 (23.5%) low-grade lymphomas were positive. There was a high correlation between TIMP-1 and IL-10 expression in these tumors (P = .00001; Fisher exact test). Data from our in vitro studies demonstrate that IL-10 expression correlates with and is dependent on TIMP-1 expression. Clinical data on B cell lymphomas further demonstrate a strong correlation between TIMP-1 expression, IL-10 expression, and histologic grade. Together these data suggest that TIMP-1 controls the expression of IL-10 in B-cell tumors. In view of TIMP-1 antiapoptosis activity in B cells,13 TIMP-1 may affect prognosis by determining the expression of factors involved in the survival and growth of B-cell tumors.
B-cell differentiation follows a complex series of steps and is tightly regulated. Naive B cells are produced in the bone marrow and travel to T-cell-rich extrafollicular areas, where they may be stimulated to proliferate by T cells and antigen-presenting interfollicular dendritic cells.32,33 Stimulated B cells invade germinal centers to either undergo apoptosis or to differentiate into highly proliferative CD77-expressing centroblasts. The factors controlling the process of B-cell differentiation versus apoptosis in the germinal center are not completely understood. Stimulated T cells play an important role in B-cell stimulation. Germinal center stromal cells, such as follicular dendritic cells, are also essential for germinal center B-cell survival and differentiation. Proliferation within the germinal center is dependent on CD40 activation and on IL-2 and IL-4. CD40 activation is important in the early survival of these cells because CD40 engagement drives B cells to lose CD77 (a neutral glycolipid associated with susceptibility to apoptosis).19-21 Activation of CD40 by its ligand is also reported to induce IL-10 secretion and to modulate immunoglobulin production. On stimulation with IL-10 in conjunction with CD40 activation, centroblasts differentiate into nonproliferating centrocytes, with down-regulation of CD77 and up-regulation of CD40. Because TIMP-1 alone affects all these changes, CD40-induced B-cell differentiation may be dependent on TIMP-1. Centrocytes can differentiate into either memory cells or plasma cells, and IL-10 and CD40 are both important in this process.34-37 TIMP-1 expression by B cells is dependent on their environment and on their state of differentiation. We have demonstrated11 that TIMP-1 is expressed by isolated germinal center B cells and that expression decreases rapidly, within 24 hours, under conventional culture conditions. Cell death on the culturing of isolated germinal center B cells is coincident with decreased TIMP-1 expression by these B cells.15 Addition of exogenous TIMP-1 prevents the germinal center B-cell apoptosis that normally occurs in vitro. Therefore, germinal center B cells express TIMP-1 in the appropriate environment, and TIMP-1 inhibits programmed cell death in these cells, indicating that TIMP-1 plays an important role in germinal center B-cell survival.13 Culturing of the isolated B cells with germinal center stromal cells also prolongs their survival. This observation is interesting because TIMP-1 is expressed by germinal center stromal cells and macrophages.41,25 Thus, germinal center stromal cells may promote B-cell survival directly by secreting TIMP-1 or possibly by stimulating germinal center B cells to produce TIMP-1. In addition to promoting survival, TIMP-1 affects germinal center B-cell differentiation by up-regulating CD40 and CD23 and down-regulating CD77.12 Our current study demonstrates that TIMP-1 induces IL-10 expression, an important step in B-cell differentiation. Furthermore, it is TIMP-1, not IL-10, that regulates the expression of CD40, CD77, and CD10. IL-10 cannot substitute for TIMP-1 in its actions. This is consistent with previous studies indicating that CD77+ germinal center B cells do not express IL-10.38 Our data indicate that exogenous IL-10 promotes proliferation of the centrocytes in the absence of TIMP-1 but that TIMP-1 alone is able to inhibit apoptosis and to induce further differentiation in these cells. Based on these data, it appears that TIMP-1 plays an important role in B-cell differentiation. Previous reports of the effects of IL-10 and CD40 activation in normal germinal center B cells were likely biased by the presence of contaminating TIMP-1 from fetal calf serum, which contains high levels of TIMP-1, or germinal center stromal cells, which expresses TIMP-1. Monocytes or tissue macrophages, which express TIMP-1 on IL-10 stimulation,25 might also have been present. These studies should be interpreted with caution because observed effects may, in part, result from TIMP-1 in the growth media from exogenous (serum) or endogenous (IL-10-stimulated macrophage) sources. We hypothesize that TIMP-1 expression is induced in centroblasts
by the appropriate germinal center environment and that TIMP-1 not only
inhibits apoptosis but also induces differentiation to the centrocyte
stage. TIMP-1 induces IL-10 expression (Figure 6) and up-regulates CD40, which makes the
cells responsive to CD40 activation. In CD40-activated B cells, IL-10
is known to induce cell proliferation and to stimulate the further
differentiation of B cells into plasma cells. The phenotypic changes
induced by TIMP-1 normally occur in the germinal center during the
interaction of B cells and T cells and in proximity to the
TIMP-1-containing germinal center stroma.15-18
Therefore, a view of B-cell differentiation evolves in which germinal
center environment induces TIMP-1 expression in differentiating B
cells. This effect could be initiated or augmented by germinal center
stromal cell TIMP-1. TIMP-1 then inhibits apoptosis and directly
induces further B-cell differentiation. TIMP-1 also induces IL-10
expression, which, in the presence of CD40 ligand activation, results
in further differentiation to the plasma cell stage. T cells and
monocytes can affect B-cell differentiation by the secretion of IL-10,
which not only stimulates B-cell differentiation but also induces
TIMP-1 expression by macrophages and monocytes. Thus, TIMP-1 may play a
pivotal role in humoral-mediated immunity by controlling B-cell
differentiation from the centroblast to the plasma cell stage.
TIMP-1-deficient mice display altered immunity.42
However, B-cell function has not been studied in these mice.
IL-10 is a strong B-cell activator and is proposed to act as an autocrine growth factor in certain non-Hodgkin lymphomas.23,24,39 It has been proposed that IL-10 can contribute to B-cell malignant disorders, in particular those associated with EBV infection, through B-cell activation or immunosuppression.39 TIMP-1 is important in the growth and survival of B cells, and, in the current study, we have shown that TIMP-1 can regulate IL-10 expression in B cells. To examine the clinical relevance of this correlation, we studied the relation between TIMP-1, IL-10, and tumor grade in a series of high-grade and low-grade B-cell lymphomas. We found a highly significant correlation between TIMP-1 expression and histologic grade. This is consistent with previous findings40 and indicates that TIMP-1 may be a negative prognostic indicator in non-Hodgkin lymphoma. Furthermore, TIMP-1 expression in this series of B-cell non-Hodgkin lymphomas correlates with human (ie, not EBV) IL-10 expression. TIMP-1 appears to play a role in B-cell neoplasia and may up-regulate IL-10 in these tumors. Because TIMP-1 is known to inhibit apoptosis, the reported association of IL-10 with a negative prognosis may be a function of the association of IL-10 with TIMP-1 expression in addition to IL-10 activity as a proliferation factor. The mechanism(s) of IL-10 action in B-cell tumors and in normal B-cell maturation is poorly understood. Because these mechanisms are crucial to understanding B-cell differentiation and because they are potential therapeutic targets for B-cell lymphomas, they are actively studied by a number of investigators. In the current study, we have shown that TIMP-1 regulates IL-10 production in B cells and that the induction of TIMP-1 expression may be a key effector in the regulation of IL-10 in B-cell malignancies. In addition, there is a strong correlation of high histologic grade with TIMP-1 and IL-10 expression. Furthermore, IL-10 expression directly correlates with TIMP-1 expression in these tumor samples. These observations confirm previous reports of IL-10 as a negative prognostic indicator in lymphoma. However, our data indicate that TIMP-1 expression controls IL-10 expression and is responsible for the poor prognosis in these tumors. Additional studies are needed to determine the specific role TIMP-1 plays in the development of B-cell malignancies.
Submitted April 21, 2000; accepted November 10, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maryalice Stetler-Stevenson, Flow Cytometry Unit, Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bldg 10, Rm 2N-108, Bethesda, MD 20892; e-mail: stetler{at}box-s.nih.gov.
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Abstract
Introduction
) and IL-10 (
). All ELISA determinations were
performed 3 times, with triplicate samples within each
determination. (B) TIMP-1 induction of IL-10. LXSN retroviral
transduction was used to induce TIMP-1 expression in the
TIMP-1
) and proliferation (
