Human B Lymphocytes Synthesize the 92-kDa Gelatinase, Matrix
Metalloproteinase-9*
Candice
Trocmé
,
Philippe
Gaudin§,
Sylvie
Berthier
,
Claire
Barro
,
Philippe
Zaoui¶, and
Françoise
Morel
From the
UPR ES EA 2019, GREPI, Laboratoire
d'Enzymologie, § Service de Rhumatologie, and
¶ Service de Néphrologie, CHU Albert Michallon, 38043 Grenoble Cedex, France
 |
ABSTRACT |
Matrix metalloproteinases (MMPs) are involved in
the remodeling of connective tissue as well as in disease states
associated with acute and chronic inflammation or tumoral metastatic
processes. Despite detailed and extensive studies of the mechanisms of
lymphocyte extravasation, remarkably little is known about the
expression and regulation of metalloproteinases involved in the
migratory process. By using zymography and reverse
transcription-polymerase chain reaction experiments, we have
demonstrated that Epstein-Barr virus-immortalized B lymphocytes are
able to secrete a 92-kDa metalloproteinase with gelatinolytic activity
which has been purified and identified as being MMP-9. Moreover, the
tissue inhibitor of metalloproteinase was shown to be constitutively
expressed by the B cells. The expression of 92-kDa gelatinase is
mediated by cytokines, growth factors, lipopolysaccharide, concanavalin A, and the tumor promotor phorbol 12-myristate 13-acetate. Time dependence activity increased rapidly up to 24 h of incubation with lipopolysaccharide or concanavalin A stimulation while it requires
a delay and more time to have an optimum effect when cytokines were the
stimulating agents; transforming growth factor-
abolished 92-kDa
gelatinase production. Both staurosporine and wortmannin are inductive
stimuli, and the level of MMP-9 secreted into the media is greater than
that observed with other agents except concanavalin A. Elicitation of
the chemotactic migration of B cells through a model basement membrane
by lipopolysaccharide was shown to be correlated with gelatinase
expression and inhibited by 7 mM captopril. Our study
indicates that Epstein-Barr virus-B lymphocytes express 92-kDa
gelatinase, the production of which can be modified by a variety of
physiological and pharmacological signals which have been shown to
differ according to the cell type.
 |
INTRODUCTION |
Cells of the immune system must invade the surrounding tissue in
order to reach the site of inflammation. High endothelial venules are
specialized postcapillary venules that are found in lymphoïd
tissues which support high levels of lymphocyte extravasation from the
blood (1). The movement of lymphocytes from the circulation into the
tissues requires that cells traverse the capillaries, penetrate the
basement membrane, and migrate into the stroma. The basement membrane
is a major barrier to leukocyte extravasation, which necessitates the
proteolytic cleavage of components, including collagens (predominantly
type IV collagen) and glycoproteins such as laminin (2). Since
metalloproteinases (MMPs)1
are believed to play a critical role in the degradation of the extracellular matrix (3) and to facilitate migration into the surrounding environment (4), we have reasoned that these proteinases may be involved in the movement of human lymphocytes from the circulation into the stroma (5). The matrix metalloproteinases constitute a family of zinc-dependent endopeptidases whose
members have been implicated in such physiological processes as
morphogenesis, angiogenesis (3), and wound repair (6), or the
pathological aspects associated with inflammation (7) and tumor
invasion (4). From the four subclasses of this important protease
family, gelatinase A (72 kDa, type IV collagenase, MMP-2, EC 3.4.24.24) and gelatinase B (92 kDa, type IV collagenase, MMP-9, EC 3.4.24.35) have been reported as being active in the cleavage of all types of
denatured collagens, type IV and type V collagens in their native
forms, elastin, and other matrix proteins (3, 8-10). Gelatinases A and
B are the products of distinct genes and are regulated differently (4).
Their expression can be modulated by soluble mediators such as growth
factors, cytokines, oncogenes, and tumor promotors (3, 11, 12).
Regulation depends on coordinated increases in transcription,
secretion, proteolytic activation or TIMP inactivation, and, in some
instances, association of the activated forms with cell surfaces (7,
13). Neutrophils, eosinophils, macrophages, and T cells all produce and
secrete MMPs with a cell-specific pattern for induction and control of MMP expression, and functional roles in the mediation of immunity and
inflammation (14). Gelatinase A is the most widely distributed MMP,
being produced constitutively by many cell types in culture, particularly fibroblasts (14) and endothelial cells (15). Gelatinase B
can also be secreted by mesenchymal cells in culture, after induction
by cytokines or other agents, but it is a major product of monocytes,
macrophages, T lymphocytes, and tumor cells (14, 16). It is also found
packaged in a granule fraction in polymorphonuclear neutrophils and is
released upon neutrophil stimulation (17, 18). Despite detailed and
extensive studies of the secretion of gelatinase B by infiltrating
neutrophils and much speculation about the mechanisms of their
extravasation (17), little is known about the involvement of MMPs in
the migratory process of immune cells such as lymphocytes. It has been
reported that normal T lymphocytes contain both gelatinase A and
gelatinase B, which may induce basement membrane turnover by a
different regulatory process (5, 19- 22). Here, for the first time, we
describe the expression and regulation of gelatinase B in human B
lymphocytes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Reagents used in this work were obtained from the
following sources: p-aminophenylmercuric acetate (APMA),
phorbol 12-myristate 13-acetate (PMA), EDTA, gelatin agarose,
recombinant (r) interleukin (IL)-10, staurosporine, wortmannin,
lipopolysaccharide (LPS), concanavalin A (ConA) (Sigma);
diisopropylphosphorofluoridate (Fluka, Switzerland); rIL-1
, rIL-2,
rIL-4, rIL-6, rIL-8, rIL-11, rTGF-
, and rTNF-
(Boehringer
Mannheim, France); Superose 12 (Amersham Pharmacia Biotech, Uppsala,
Sweden); rIL-13 (RD System, Abingdon, UK); growth factor reduced
Matrigel® matrix (Becton Dickinson, Bedford, MA); human TIMP-1 ELISA
system and TIMP-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK);
Transwell inserts (Corning Costar Corporation, Cambridge, MA);
Centricon 10 (Amicon, Beverly, MA); TRIzolTM reagent (Life
Technologies, Inc.); captopril
(D-3-mercapto-2-methylpropanoyl-L-proline) was
a generous gift of Bristol Myers-Squibb Company (Paris, France).
Cell Culture--
Lymphocytes from heparinized sterile venous
blood were prepared by Ficoll-Hypaque density gradient centrifugation.
The cells were infected with the B95-8 strain of the Epstein-Barr virus (EBV) as described previously (23). The EBV B lymphocyte cell line was
maintained in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 50 µg/ml kanamycin, and 50 µg/ml streptomycin.
The culture was maintained at 37 °C under a 5% CO2 atmosphere. The medium was changed twice weekly.
Before MMP expression and after extensive washing, the EBV B
lymphocytes were grown in serum-free medium containing 0.2% (w/v) bovine serum albumin and were maintained under the same CO2
atmosphere for 18 h until further use. Cell viability was
monitored by the trypan blue exclusion method; it was of the order of
90%. All the experiments were standardized by using an equal number of cells.
Purification of MMP-9 from Human Neutrophils and Production of
Antisera--
Secreted gelatinase B was purified from 0.8 nM PMA-stimulated human neutrophils as described previously
(24). Briefly, released proteins were fractionated by DEAE-Sephacel
anion exchange chromatography and affinity chromatography on gelatin
agarose. Fractions containing gelatinase activity were pooled and
processed using preparative SDS-polyacrylamide gel electrophoresis
(PAGE) (5-15% gel). A 92-kDa band corresponding to the native
neutrophil gelatinase was electroeluted and injected into rabbits for
antibody production. Purified gelatinase (50 µg/0.5 ml of
phosphate-buffered saline) was mixed with 0.5 ml of Freund's complete
adjuvant and injected into rabbits. Booster injections using Freund's
incomplete adjuvant were started on day 15 and repeated every 2nd week
for 2 months. Blood was collected 2 weeks after the last booster and
allowed to clot. Antisera were separated by centrifugation and stored
at
20 °C. Serum immunoglobulins were pelleted down twice with
33-40% (w/v) (NH4)2SO4, and
filtered through a DEAE-cellulose matrix to eliminate plasma
2-macroglobulin. Immunoglobulins G (IgG) were further
isolated onto protein A-Sepharose. The specificity of antisera and
immunoglobulins with respect to binding and inhibition has been
reported previously (24).
Purification of a Metalloproteinase with Gelatinolytic Activity
from EBV B Lymphocytes in Culture--
A metalloproteinase with
gelatinolytic activity, from crude EBV B lymphocyte culture medium
supernatant, was purified using substrate affinity chromatography on
gelatin agarose, and gel filtration on FPLC Superose 12. Approximately 40 ml of serum-free, conditioned medium (108
EBV B lymphocytes) was dialyzed against 0.05 M Tris-HCl, pH
7.6, buffer containing 0.005 M CaCl2, 0.02%
(w/v) NaN3, 0.5 M NaCl, 0.05% (v/v) Brij 35. The dialyzed medium was applied to a gelatin agarose column that had
been equilibrated in the same buffer. After extensive washing with
equilibration buffer containing 1 M NaCl, the bound enzyme
was eluted with 10% (v/v) dimethyl sulfoxide, which was added to the
latter buffer. Fractions with zymographically determined gelatinolytic
activity were pooled, concentrated by ultrafiltration through Centricon
10, and applied to a Superose 12 column prepared in 0.05 M
Tris-HCl, pH 8, buffer containing 0.3 M NaCl and 0.05%
(v/v) Brij 35. The fractions were collected by FPLC, at a flow rate of
0.25 ml/min at a pressure of 1 MPa. Eluates showing gelatinolytic
activity were pooled and frozen until further use.
Migration Assay--
Cell migration was quantified using
Transwell inserts as described previously (5). 106 EBV B
lymphocytes in 0.5 ml of serum-free culture medium containing 0.2%
(w/v) BSA were added to the 12-mm diameter inserts of Transwell chambers over 12-µm pore polycarbonate filters with a continuous even
coating of 100 µl of growth factor reduced Matrigel® matrix, which
separated the cells from 1.5 ml of the same medium in the lower
compartment. In this experiment, EBV B cells were preincubated with 10 ng/ml LPS at 37 °C for 18 h in the 0.2% BSA serum-free medium
and then added to the upper compartment of the insert. The Transwell
chambers were incubated in a 5% CO2 environment at
37 °C for 24 h. In some experiments, different concentrations of captopril were added to the two compartments. Cells in the lower
compartment were detached by shaking and collected for counting.
SDS-PAGE and Immunoblotting--
Proteins were separated in
parallel with appropriate controls and molecular weight markers using
SDS-PAGE (25) in 10% (w/v) acrylamide gel with a 5% (w/v) stacking
gel, and stained with Coomassie Brilliant Blue R-250. Nitrocellulose
transfer of proteins separated by SDS-PAGE was performed according to
the method described by Towbin et al. (26). After this, the
blotting membranes were incubated with specific antiserum raised
against neutrophil-purified gelatinase (1:200 dilution) in 0.05 M Tris-HCl, 0.2 M NaCl, 0.05% (v/v) Tween 20, pH 7.5, followed by goat anti-rabbit IgG alkaline phosphatase conjugate
(1:1000 dilution in the same buffer), and stained with nitro blue
tetrazolium 5-bromo-4-chloro-3-indolyl phosphate reagent, according to
the manufacturer's instructions.
Gelatin Zymography--
Zymographic analysis was carried out in
10% (w/v) SDS-polyacrylamide gels containing gelatin (0.5 mg/ml), as
described previously (15, 16). The proteins collected from conditioned
medium or chromatography eluates were concentrated using Centricon 10. They were applied to the gel in a sample buffer containing 2.3% (w/v) SDS but lacking
-mercaptoethanol and were not boiled prior to loading. The gels were washed twice for 15 min in 0.05 M
Tris-HCl, pH 7.6, containing 0.005 M CaCl2,
0.001 mM ZnCl2, and 2.5% (w/v) Triton X-100,
in order to remove SDS, followed by 5-min washes in buffer devoid of
Triton X-100. After 3.5 h of incubation at 37 °C with 0.001 M APMA in the same buffer, containing 1% (w/v) Triton
X-100, the gels were stained with Coomassie Brilliant Blue R-250 and
destained as described previously (27). Zones of enzymatic activity
were shown by negative staining and quantitated by scanning densitometry at 600 nm (CD 60, Desaga, Sarstedt Gruppe). Enzyme activity was expressed in arbitrary units from a standard curve corresponding to the gelatin zymography of increasing concentrations of
latent purified gelatinase B (27).
Protein content was estimated using the micro BCA method (28). TIMP-1
was measured by using a two-site ELISA "sandwich" format (ELISA
system, code RPN 2611, Amersham Life Science, Inc.). Standards
(purified TIMP-1, Amersham Life Science, Inc.) and samples (flow-through from gelatin agarose matrix and culture medium) were
incubated with anti-TIMP-1 antibody. The TIMP-1 was detected by a
conjugate (peroxidase-labeled antibody to TIMP-1); the reaction was
then quantitated as indicated by the manufacturer's instruction. The
concentration of TIMP-1 in a sample was determined by interpolation from a standard curve.
Analysis of the Isolated 92-kDa Gelatinase and TIMP-1 mRNAs
by Reverse Transcription (RT)-Polymerase Chain Reaction
(PCR)--
Total RNA was isolated (29) from about 108 EBV
B lymphocytes, using a modification of the single-step method described
by Chomczynski and Sacchi (29), involving 5-min incubation of the total
cell pellets with 1 ml of TRIzolTM reagent. The cell
pellets were disrupted by repetitive push-pull through a 1-ml Pipetman
tip. Cell lysates were transferred to RNase-free sterile Eppendorf
tubes, and RNA was extracted over 0.2 ml of chloroform by
centrifugation (10,000 × g, 15 min, 4 °C). RNA was
precipitated from the supernatant phase with 0.5 ml of isopropyl
alcohol and washed in 70% (v/v) ethanol. The optical density of RNA
resuspended in sterile water was recorded
(A260 nm/280 nm ratio >1.8-2.0). The yield
from this procedure varied between 50 and 200 µg of total RNA.
Aliquots of 5 µg of total RNA were reverse-transcribed in 20 µl of
RT buffer, using oligo(dT) primers and a cDNA synthesis kit used
according to the manufacturer's instructions. cDNA (2.5 µl per
test) was immediately amplified by PCR, using 2.5 units of
Taq polymerase in 100 µl (final volume) of Taq
buffer containing 0.2 mM dNTP and 0.25 µM
sense and antisense oligonucleotides. The oligonucleotides used as
primers were synthesized from the timp-1, mmp-9 gene
sequences of the EMBL cDNA library as follows. MMP-9 (92-kDa
gelatinase) primers, sense 5'-116-136 bp/antisense 5'-392-372 bp were
designed to amplify a 277-bp cDNA fragment; TIMP-1 primers: sense
5'-1-18 bp/antisense 5'-769-746 bp (769-bp complete cDNA) (Table
I). Thirty-five cycles (denaturation, 1 min at 94 °C; annealing, 1 min at 57 °C for MMP-9, at 60 °C for TIMP-1; extension, 2 min at 72 °C) followed by a 7-min
elongation period were performed with a M J Research PTC 150 thermocycler with Peltier effect. Commercial actin primers were run in
parallel PCR tests as a control for PCR and RNA extraction efficiency. Aliquots of 10 µl of PCR products in bromphenol blue solution were
run together with a scale of DNA ladders (markers VI, Boehringer Mannheim, France) on 1.5% (w/v) agarose gels containing 1 µg/ml ethidium bromide. The bands were photographed using Polaroid film and
UV transillumination.
View this table:
[in this window]
[in a new window]
|
Table I
Composition of oligonucleotide primers
Positions of the 5'-ends of the primers are numbered from the ATG
inhibition codon of the MMP-9 or TIMP-1 gene. The MMP-9 gelatinase
primers and TIMP-1 primers correspond to cDNA fragments of 277 and
769 bases, respectively.
|
|
Sequencing of the PCR Products--
PCR products were
gel-purified and automatically sequenced by Genome Express Ltd.,
Grenoble, France, with forward and backward primers for TIMP-1 and
MMP-9.
Northern Blot Analysis--
24 h after addition of 0.5 nM PMA, 1 µg/ml concanavalin A, or 10 ng/ml LPS, total
RNA was extracted from EBV B lymphocytes by the TRIzolTM
method as described previously. Poly(A)+ RNA was isolated
onto an oligo(dT) cellulose matrix by rocking total RNA with oligo(dT)
cellulose in a high salt buffer (400 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.2% (w/v) SDS, pH 7.4)
for 2-3 h at room temperature and elution of mRNA with a buffer
with no salt (5 mM Tris, 1 mM EDTA, 0.2% (w/v)
SDS, pH 7.4). After denaturation at 65 °C for 15 min,
poly(A)+ RNA from control or test samples was
size-fractionated on a 1% (w/v) agarose-formaldehyde gel in 1× MOPS
buffer, blotted onto a positive nylon membrane in 10× SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7) by
capillary action and immobilized by UV cross-linking using a UV
Stratalinker 1800 (Stratagene, La Jolla, CA). The 277-bp mmp-9 PCR product was labeled with digoxigenin-11-dUTP
(Boehringer Mannheim, France) by PCR with the same oligonucleotide
primers as those used before and hybridized to the filter overnight at 42 °C in a high SDS buffer (7% (w/v) SDS, 50% (v/v) formamide, 5×
SSC, 2% (w/v) blocking reagent, 50 mM sodium phosphate, pH 7, 0.1% (w/v) N-lauroylsarcosine). The membrane was washed
twice in 2× SSC with 0.1% (w/v) SDS at room temperature for 5 min and twice in 0.1× SSC with 0.1% (w/v) SDS at 50 °C for 15 min. The detection was performed using anti-digoxigenin (Fab) fragments conjugated to alkaline phosphatase followed by a chemiluminescent reaction using the CDP-Star system (Boehringer Mannheim, France), according to the manufacturer's instructions. Chemiluminescent signals
were detected by exposing the blot onto Hyperfilm MP (Amersham Life
Science, Inc.) for 10 min. The integrity and equal gel loading of
mRNA were assessed by visualizing the remaining 28 and 18 S ribosomal RNA bands under UV light after staining with ethidium bromide
and by a second hybridization of the membrane with a
digoxigenin-labeled probe of the housekeeping g3pdh gene.
Quantitation (ratio mRNA for gelatinase B/mRNA for G3PDH) was
performed by scanning densitometry at 400 nm of the bands of MMP-9 and
G3PDH seen on x-ray films (CD 60, Desaga, Sarstedt Gruppe).
Statistical Methods--
The variations are expressed as
mean ± S.E.; p values were calculated by Student's
paired t test.
 |
RESULTS |
Production of a Metalloproteinase with Gelatinolytic Activity by
Epstein-Barr Virus-immortalized B Lymphocytes--
Human B cells were
isolated from peripheral blood, transformed by Epstein-Barr virus, and
cultured in vitro as described under "Experimental
Procedures." We analyzed EBV B lymphocytes for metalloproteinase activity secreted into the culture-conditioned medium. Because of low
levels of expression, the isolation of a metalloproteinase with
gelatinolytic activity was carried out onto a gelatin agarose matrix
(Fig. 1A). Analysis of the
eluates by gelatin zymography (Fig. 1A, inset)
revealed that proenzyme was present in eluates 11-15, which were
recovered from the gelatin affinity chromatography once dimethyl
sulfoxide was added to the NaCl washing buffer; maximum gelatinolytic
activity occurred in fractions 12 and 13 in both the control experiment
and after treatment of the cells with 0.5 nM PMA. Treatment
with PMA induced a high level of secretion of the gelatinolytic enzyme
compared with the control. Similar results were observed with
untransformed B lymphocytes (not shown). Metalloproteinases are known
to be secreted as latent precursors of higher molecular weight than the
mature enzyme. 1.8 mM APMA induced the conversion of the
proenzyme to active forms (Fig. 1B, lane 1 (latent) and
lane 2 (active)), yielding gelatinolytically active products
at 78 and 71 kDa molecular masses. 10 mM EDTA completely
inhibited the activity of all the gelatinases (Fig. 1B,
lane 3 (latent) and lane 4 (active)). The
inhibition of gelatinolytic activity by EDTA indicated that the enzyme
displays the characteristics of a metalloproteinase. Gelatinase-free
TIMP-1 was recovered in the flow-through of the gelatin agarose
chromatography during the isolation of 92-kDa gelatinase, as shown in
Fig. 1A. TIMP-1 was quantitated using ELISA, giving a 94%
recovery of TIMP-1 (flow-through versus culture medium). The
92-kDa gelatinase isolated on gelatin agarose was purified by FPLC gel
filtration chromatography (not shown). The eluates which show
gelatinolytic activity were pooled and processed using SDS-PAGE and
Western blotting (Fig. 1, C and D). About 10 ng
of purified gelatinase were isolated from 196 ml of serum-free
conditioned medium of 8 × 108 EBV B lymphocyte
culture in a purification process which yielded a 471-fold purified
protein. The final material ran as a major 90-kDa band on reducing
SDS-polyacrylamide gel electrophoresis (Fig. 1C, lane
2). Western blot analysis of the purified protein with a
polyclonal antibody specific for neutrophil MMP-9 labeled a protein of
molecular mass in the range of 90 to 94 kDa (Fig. 1D). This
corresponds to the 92-kDa protein with gelatinolytic activity detected
by zymography (Fig. 1A, inset). Additional
confirmation that the enzyme was indeed gelatinase B (MMP-9) was
obtained by detection of specific mRNA by PCR. Total RNA was
extracted from EBV B lymphocytes (108 cells) and 5 µg of
total RNA were subjected to RT and amplification by PCR, as described
under "Experimental Procedures." As shown in Fig.
2, MMP-9 and TIMP-1 PCR products were
clearly observed. RT-PCR yielded low but significant levels of MMP-9
products (Fig. 2, lane 1) while the amount of TIMP-1
transcript seemed to be higher (Fig. 2, lane 2); the
amplification products have the expected size and 99.3 and 99.5%
homology, respectively, with TIMP-1 and MMP-9 cDNA published
sequences.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Isolation of a metalloproteinase with
gelatinolytic activity from serum- free conditioned medium of cultured
EBV B lymphocytes. A, serum-free conditioned medium of
resting (control) or 0.5 nM PMA-stimulated 108
EBV B lymphocytes cultured at 37 °C for 72 h was filtered
through a gelatin agarose matrix as described under "Experimental
Procedures." A 92-kDa metalloproteinase with gelatinolytic activity
was eluted from the matrix by using 10% Me2SO and 1 M NaCl added to the equilibrium buffer. Fractions
11-15 of the control ( ) or PMA assay ( ) were concentrated
onto Centricon 10 (Amicon) and submitted to gelatin zymography
(inset). Following Coomassie staining, white
bands represent zones of lysis of the gelatin substrate.
TIMP-1 ( ) was measured using ELISA, as reported under
"Experimental Procedures" and expressed as
nanograms/107 cells. Results are representative of 20 experiments. B, fractions of the previous gelatin
agarose chromatography performed with the conditioned medium from
PMA-stimulated EBV B lymphocytes were pooled and subjected to gelatin
zymography, after activation of the proteinase using 1.8 mM
APMA (lanes 2 and 4) at 37 °C for 45 min in
the absence (lanes 1 and 2), or the presence
(lanes 3 and 4) of 10 mM EDTA;
lanes 1 and 3, control experiment without APMA
activation. C, the pooled fraction from B
was subjected to FPLC Superose 12 gel filtration for purification as
described under "Experimental Procedures." The purified
gelatinase-enriched pooled fraction was submitted to SDS-PAGE.
Lane 1, gelatin agarose, 35 µg; lane 2, FPLC
Superose, 20 µg. Results are representative of three experiments. D, Western blotting of the
purified protein. Specific antibodies were polyclonal antibody raised
against neutrophil MMP-9. Staining was performed with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. kDa
represents the molecular mass standards. Results are representative of
three experiments.
|
|

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 2.
RT-PCR of actin, MMP-9 and TIMP-1 of RNA from
unstimulated EBV B lymphocytes. The experiment was performed with
5 µg of RNA for each specimen and 35 cycles for the PCR as
described under "Experimental Procedures." Samples without RT
showed no PCR products. Results presented illustrate 1 of 10 experiments.
|
|
Taken together, these results conclusively indicate that EBV B
lymphocytes secrete a 92-kDa gelatinolytic enzyme which can be
identified as MMP-9 according to the following criteria: molecular weight, specific inhibition by EDTA, the pattern of activation products
generated by APMA, binding to gelatin agarose matrix and cleavage of
gelatin, identification of specific mRNA, and immunoreactivity.
Regulation of Expression of the Metalloproteinase with
Gelatinolytic Activity Secreted from Epstein-Barr Virus-immortalized B
Lymphocytes--
Under the conditions used for short term cultures,
EBV B lymphocytes secreted a slight constitutive gelatinolytic
proteinase of approximately 92-kDa molecular mass, which has been
identified as being MMP-9. Treatment of EBV B lymphocytes with PMA
resulted in an enhanced production of the 92-kDa gelatinase (Fig.
1A, inset). To determine the optimal PMA
concentration for enzyme induction, the cells were incubated in medium
containing increasing concentrations of PMA, for 18 h. Maximum
induction of gelatinase activity was observed in the range of 0.1 to
0.5 nM and was correlated with good cell viability. The
kinetics of enzyme induction were then examined. The cells were grown
in medium containing 0.5 nM PMA, and at defined time
periods, medium samples were withdrawn. Gelatinase was then isolated
onto a gelatin agarose matrix and analyzed for gelatinolytic activity.
Increased levels of secreted proteinase activity were visible 10 h
post-treatment (Fig. 3). As PMA is an
inducer of the membrane-associated protein kinase C and in order to
determine whether it was inducing gelatinase activity via activation of
protein kinase C in EBV B lymphocytes, the effect of staurosporine, a
protein kinase C inhibitor, was investigated. For these studies, EBV B
lymphocytes were cultivated in the absence or the presence of
increasing concentrations of staurosporine up to 20 nM
(Fig. 4A). Staurosporine
increased gelatinase expression and at the concentrations used, had no
effect on cell survival as determined by trypan blue exclusion; in all
treatment groups, the viability of the cells was 90% (data not shown).
A similar stimulation was observed after incubation of EBV B
lymphocytes for 18 h with wortmannin, suggesting the involvement
of phosphatidyl inositiol 3-kinase in signal transduction pathways.
Basal expression of gelatinase was optimum with 20 nM
staurosporine and 200 nM wortmannin (Fig. 4, A
and B), and four times more than that measured when PMA was
the stimulating agent. EBV B lymphocytes were then exposed to a variety
of physiological and pharmacologic modulators. In order to determine
the best conditions for enzyme induction, the cells were incubated in
medium containing increasing concentrations of stimulating agents. The
metalloproteinase from the medium was isolated onto a gelatin agarose
matrix and analyzed by zymography as reported previously for PMA. As
shown in Table II and Figs. 3 and
5, basal expression of gelatinase
activity was affected by exposure of the cells to different cytokines,
TNF-
, TGF-
, LPS, ConA, or PMA. Increased levels of secreted
gelatinolytic enzyme were visible at 10 and 8 h post-treatment of
the cells with PMA (Fig. 3), and LPS or ConA (Fig. 5B),
respectively, but only at 24 h when IL-13 was the stimulating
agent (Fig. 5A). Stimulation of EBV B lymphocytes showed
variations in gelatinase secretion according to the stimulus (Table
II). The results obtained at 18 + 72 h of culture showed a
significant 2-fold increase in IL-1
-induced gelatinolytic activity,
compared with the control. Interleukins, IL-2, IL-4, IL-6, IL-10,
IL-11, and TNF-
failed to up- or down-regulate significantly
gelatinase biosynthesis, while IL-13 required more time to have an
optimum effect (Fig. 5A); 1 ng/ml TGF-
completely abolished gelatinase synthesis and secretion. The production of gelatinase was strongly stimulated by LPS in a
dose-dependent manner, up to 20 ng/ml (not shown). It was
optimum with 10 ng/ml LPS and 1 µg/ml ConA at 24-h incubation.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of gelatinase B (92 kDa)
induction in EBV B lymphocytes stimulated by 0.5 nM
PMA. 2-5 × 107 EBV B lymphocytes were
stimulated by 0.5 nM PMA ( ) or not (control, ) and
cultivated at 37 °C (+5% CO2) at different incubation
times. The supernatant was next collected and assayed for gelatinase B
production using gelatin zymography. Gelatinase activity was
quantitated by scanning densitometry and expressed from arbitrary units
extrapolated to a concentration of MMP-9 obtained from a standard curve
drawn up after gelatin zymography of latent purified gelatinase B (24,
27). Results are mean ± S.E. and representative of four
experiments (each in triplicate). Inset, dose-response
effect of PMA on gelatinase B production. 2-5 × 107
EBV B lymphocytes were stimulated by increasing PMA concentrations up
to 0.5 nM and cultivated for 18 h at 37 °C.
Gelatinase activity was quantitated as described previously. The
results are mean ± S.E. and representative of four experiments
(each in triplicate).
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Dose-response effect of staurosporine
(A) and wortmannin (B) on constitutive
gelatinase B and TIMP-1 production. 2-5 × 107
EBV B lymphocytes were treated with increasing concentrations of
staurosporine (up to 20 nM) (A) and wortmannin
(up to 200 nM) (B) and cultivated for 18 h
at 37 °C (+5% CO2). Gelatinase production was measured
using gelatin zymography and densitometry, as described under
"Experimental Procedures." Results are the mean ± S.E. from
three experiments (each in triplicate).
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Induction of gelatinolytic activity in EBV B lymphocytes stimulated
with cytokines, and growth factor, TGF-
Conditioned medium from stimulated EBV B lymphocytes was withdrawn
after 18 h and 18 h + 72 h of culture and filtered
through a gelatin agarose matrix. The eluted fractions were analyzed
for gelatinase activity by zymography. Zones of enzymatic activity were
shown by negative staining and quantitated by scanning densitometry at
600 nm (CD60, Desaga, Sarstedt Gruppe). The arbitrary units that were
obtained were extrapolated to a concentration of MMP-9 from a range of
increasing concentrations of purified gelatinase B of neutrophils (27).
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of gelatinase induction by
cytokines, LPS, or ConA in EBV B lymphocytes. A,
2-5 × 107 EBV B lymphocytes were either stimulated
or not stimulated (control) ( ) by 1 ng/ml IL-1 ( ),
or 50 ng/ml IL-13 ( ) at different incubation times. Serum-free
conditioned medium containing 0.2% (w/v) BSA was withdrawn, and 92-kDa
metalloproteinase with gelatinolytic activity was isolated onto a
gelatin agarose affinity matrix and quantitated by zymography as
described under "Experimental Procedures." B, same
experiment as in A, using 10 ng/ml LPS ( ), 1 µg/ml ConA
( ) as the stimulating agent. The results in A and in
B are representative of two experiments, except for the
control, the results of which are mean ± S.E. (three
experiments). C, Northern blot analysis of mmp-9
transcription of stimulated EBV B lymphocytes. 3 × 108 EBV B lymphocytes were cultivated with 0.5 nM PMA, 1 µg/ml ConA, or 10 ng/ml LPS for 24 h at
37 °C (+5% CO2). Total RNA was extracted and oligo(dT)
cellulose purified poly(A)+ RNA was analyzed by Northern
blot using a digoxigenin-labeling MMP-9 DNA probe as described under
"Experimental Procedures." The filter was reprobed with a specific G3PDH DNA probe; mRNA for gelatinase
was quantitated by normalizing to mRNA for G3PDH. Ribosomal RNA
bands (28 and 18 S) visualized under ultraviolet light after staining
with ethidium bromide show the amounts and quality of the RNA
(bottom panel). The results are representative of two
experiments.
|
|
92-kDa Gelatinase mRNA expression by EBV B lymphocytes was
evidenced by using a reverse transcription-polymerase chain reaction and Northern blot. We were successful in performing RT-PCR for 92-kDa
gelatinase and TIMP-1 from resting EBV B lymphocytes (Fig. 2). When EBV
B lymphocytes were cultured in the presence of cytokines or growth
factors, there was no increase in the levels of the enzyme and TIMP-1
inhibitor mRNAs (not shown); these findings differ from the slight
but significant increase in both proteins secreted after stimulation of
the cells by IL-1
, IL-8, and IL-13. On the contrary when PMA, LPS,
and ConA were the stimulating agents the MMP-9 mRNA message was
enhanced while there was no significant differences versus
control for TIMP-1 (not shown). Northern blot analysis of
poly(A)+ RNA prepared from EBV B lymphocytes was carried
out with a cDNA probe specific for human MMP-9 to determine if the
changes in secreted gelatinolytic activity were reflective of
significantly increasing amounts of mRNA present (Fig.
5C). Poly(A)+ mRNA from resting cells
hybridized as a single band which corresponded to the MMP-9 transcript
(30). Treatment of EBV B lymphocytes with PMA, ConA, or LPS increased
significantly the expression of MMP-9 mRNA of 1.7-, 2.7-, and
3.5-fold, respectively, as shown by densitometric analysis (ratio of
mRNA for gelatinase B/mRNA for G3PDH).
EBV B Lymphocyte Migration through a Basement Membrane
Equivalent--
The recruitment of blood B cells to tissue sites of
immune responses and chronic inflammation involves their adhesion to
and movement between endothelial cells, and migration through the vascular basement membrane and into tissues. The spontaneous and LPS-
or ConA-enhanced migration of human EBV-transformed B cells across a
layer of growth factor-reduced Matrigel® which consists of basement
membrane matrix constituents was carried out in order to assess the
role of secreted 92-kDa gelatinase in the transmigration process.
106 EBV B cells were layered onto a polycarbonate micropore
filter coated with Matrigel® (Fig. 6).
Migration assays were performed using unstimulated and
LPS-prestimulated B lymphocytes. The specificity of gelatinase-mediated
migration was demonstrated by using captopril, a recently reported
inhibitor of zinc MMPs and angiogenesis (31, 32). As expected,
prestimulation of the EBV B lymphocytes with 10 ng/ml LPS induced a
large migration increase (Fig. 6) which was inhibited by 10 mM EDTA (not shown). Captopril was able to inhibit
LPS-induced migration at concentrations ranging from 1 to 15 mM. The dose-response curve illustrated first a sharp rise in inhibition of migration up to a 7 mM captopril
concentration followed by a plateau in inhibitory activity at captopril
concentrations from 7 to 15 mM (Fig. 6, inset).
There was no toxic effect, as measured by trypan blue exclusion, of
captopril to the cell in culture up to 12 mM. In the
absence of stimulus, 10 mM captopril had no effect on basal
migration corroborating that at this concentration it was not toxic to
the B lymphocytes.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Transwell migration. Resting or 10 ng/ml
LPS-prestimulated EBV B lymphocytes (106 cells) were added
to the upper chamber of a Transwell over a continuous coating of growth
factor reduced Matrigel® matrix and incubated in 0.5 ml of 0.2% (w/v)
BSA serum-free culture medium supplemented (or not, control) with 10 mM captopril added to the two chambers. After 24-h
incubation at 37 °C in a 5% CO2 atmosphere, the B cells
in the lower compartment were collected by shaking and numbered by
trypan blue exclusion. LPS preincubation of the B cells was performed
at 37 °C for 18 h. The results are representative of four
experiments, each in quadriplicate. Inset, dose-response
effect of captopril on cell migration. 106 resting EBV B
lymphocytes in 0.2% (w/v) BSA serum-free culture medium were layered
onto the upper Transwell chamber and incubated at 37 °C in a 5%
CO2 atmosphere for 24 h. Increasing concentrations of
captopril (up to 15 mM) were added to the two chambers. At
the end of incubation, the B cells which migrated in the lower chamber
were detached and numbered. The results are representative of three
experiments each in duplicate.
|
|
 |
DISCUSSION |
One of the effector functions of activated MMPs expressed in
immune cells is to promote transbasement membrane migration of lymphocytes. Here, we have shown that EBV-immortalized B lymphocytes constitutively express gelatinase B and that expression can be modulated by cytokines, growth factors, and tumor promotors; there was
no evidence of phenotypic expression of gelatinase A. The characteristics of the 92-kDa gelatinase isolated from EBV-immortalized B lymphocytes and demonstrated in this study are similar to those reported for neutrophils, monocytes, and tumor cells (19, 33-35). It
is a metalloproteinase that is dependent on zinc and calcium ions, with
degradative activity against gelatin. Our data indicate that the
isolated 92-kDa form is a proenzyme that can be activated in
vitro by organomercurials and in vivo after secretion
as all metalloproteinases except membrane-type MMPs and MMP-11.
Disruption of the endothelial basal lamina is a prerequisite for
migration of immunoactive cells through post-capillary venules. It has
been reported that normal human T cells express two matrix
metalloproteinases, gelatinase A and B, both being detectable in their
inactive proenzyme forms as well as in their active forms (5). However,
the enzymatic events that underlie this capability in B cells are
unknown. Here, we demonstrate the selective and constitutive expression
in EBV B lymphocytes of progelatinase B, which can be modulated by
different mediators. Among the proinflammatory cytokines, only IL-1
and IL-8 enhanced gelatinase synthesis, while IL-6 and TNF-
had no significant effect. The expression of MMP with gelatinolytic activity was suppressed by TGF-
, providing an interesting contrast with the
MMP-enhancing expression observed in T lymphocytes, which suggests the
existence of a differential regulation of the MMP gene in the two
lymphocyte cell types (19, 22). IL-4, another anti-inflammatory
cytokine, has no effect, and in contrast, IL-13 induces significant
gelatinase expression but as IL-1
and IL-8 requires a delay for the
response. There is no possible contamination of the medium by other
sources of gelatinase; this was ensured by the use of serum-free media
and a highly purified EBV-immortalized B cell population.
As observed with lymphocytes and with various cell lines,
metalloproteinase expression and regulation is cell type-specific. In
uterine cervical fibroblasts, the synthesis of pro-MMP-9 was recently
shown to be regulated by PMA, IL-1, and TNF-
, but the combined
effect of PMA or TNF-
and then of IL-1 resulted in distinct responses suggesting a different regulation process of production (36).
Differential signaling pathways mediated by TGF-
were also evidenced
in dermal fibroblasts and epidermal keratinocytes (37). Finally the
expression and suppression of matrix metalloproteinase biosynthesis was
differently reported. While IL-4 and IL-10 inhibited 92-kDa gelatinase,
matrilysin, and collagenase in mononuclear phagocytes (38-40), they
had little or no effect upon fibroblast (40) or EBV B lymphocyte (this
work) MMP expression. In contrast, TGF-
suppressed gelatinase
production in B cells, while it had no effect upon the mononuclear
phagocyte production of matrilysin, interstitial collagenase, or 92-kDa
gelatinase (38). TGF-
is known to achieve its anti-inflammatory
properties by the inhibition of IL-8 gene expression (41). These
results argue in favor of a great complexity of regulation mechanisms
of MMPs produced by the inflammatory cells and a strict dependence on
cell specificity.
T and B lymphocytes, neutrophils, and macrophages attracted to damage
sites by various chemotactic factors are often in close spatial
proximity and may interact with each other or with resident tissue
cells. The consequences of the interaction have been demonstrated recently by the induction of MMP expression when neutrophils adhere to
the endothelium (15) or by a direct contact between T cells and
monocytes (42, 43). The present results suggest that gelatinase B
expression in B lymphocytes might depend on an imbalance between pro-
and anti-inflammatory stimuli, through which the cytokines could play a
central role. Our results therefore have shown that inflammation will
depend not only on the presence of proinflammatory mediators such as
IL-1, IL-8, and IL-13 but also on the absence of negative regulatory
factors such as TGF-
(41). Like other MMPs, gelatinase B is secreted
from cells as an inactive zymogen (pro-MMP-9) and the recent finding
that pro-MMP-9 forms a specific complex with the MMP inhibitor TIMP-1
(16, 44) has introduced another level of complexity into the regulatory
mechanisms of MMP activity. In the present work, during the 92-kDa
gelatinase purification process, we did not collect any
gelatinase-TIMP-1-associated complex, while the ratio of TIMP-1
versus progelatinase B in the culture medium recovered after
72 h of culture time was in favor of the inhibitor
(TIMP-1/MMP-9 = 1000). The local concentration of these molecules
(mediators and inhibitors) will thus be critical in infiltration of
inflammatory cells and regulation.
An interesting finding of our study is that the constitutive expression
of latent 92-kDa progelatinase by EBV B lymphocytes is greatly enhanced
when they are stimulated by LPS, ConA, and PMA. In many inflammatory
cell types, including macrophages, MMP genes encoding gelatinase B but
not gelatinase A respond in a similar fashion to LPS and PMA
stimulation (45) and contain cis-acting elements such as AP-1 and
NF-
B-binding sites in their promoter region (45). The gelatinase
content of EBV B lymphocytes was investigated after homogenization of
the cells and found to be in the same range as that measured in the
culture medium at resting states (0.17 ng/107 cells inside,
versus 0.11-0.18 ng/107 cells outside,
respectively). Secretion of gelatinase in the medium increased upon PMA
stimulation without any modification of its content inside the cells
(not shown). The results suggest that, contrary to neutrophils which
release outside of the cells their storage granule content upon
stimulation (18, 46), there was no accumulation of gelatinase inside
the B lymphocytes before secretion. Moreover, a 20-fold increase of
92-kDa gelatinase production was recently demonstrated in bronchial
epithelial cells (47) upon LPS stimulation with a minimal change of
mRNA level, suggesting post-transcriptional modifications. RT-PCR
and Northern blot experiments carried out with EBV B lymphocytes
indicated that message was increased upon PMA, LPS, and ConA
stimulation, while it was unchanged when cytokines or growth factors
were the ligand. These latter aspects are now under work, particularly
referring to the half-life of 92-kDa gelatinase mRNA according to
the stimulation conditions.
Protein kinase C signaling pathways have been involved in the
expression of metalloproteinase genes; surprisingly staurosporine, a
broad spectrum protein kinase inhibitor, not only failed to block
response but itself stimulated expression of gelatinase B (this work)
or of collagenase (48). This observation is of particular interest in
view of the finding that staurosporine possesses tumor-promoting
activity in mouse skin keratinocytes (49). The discordant effects of
protein kinase C inhibitors on gelatinase expression raise questions
with respect to their therapeutic use in the treatment of cancer since
recent reports suggest that gelatinase B may play a role in metastasis
development (45).
Lymphocytes continuously circulate from the blood through lymphoid and
other tissues, and back through the lymphatics to the blood (1), the
first critical step in lymphocyte migration being adhesion to the
vascular endothelium. Gelatinases A and B were recently shown to
mediate the invasion of the basement membrane by cytotrophoblasts and
tumor cells in vitro (4); inhibition of T cell homing by
interference with gelatinase function was proposed to represent a
useful approach to the treatment of T cell-mediated autoimmune disease
(5, 50). Degradation of the basement membrane by MMP-9 was reported to
play an important role in transmigration of human eosinophils (51) as
of lymphocytes (5, 22) or neutrophils (52). The data presented above or previously reported (32) clearly identify captopril as being a general
inhibitor of the migratory behavior of cells. The demonstrated ability
of captopril to inhibit in vitro gelatinase B (31) may partly explain its antiangiogenic activity. The study presented here
argues in favor of the expression of gelatinase B by B lymphocytes as
tools for migration through high endothelial venules, and for induction
of the proteolysis process at the cell surface. The latter aspect has
yet to be demonstrated and is currently under study.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. L. Matrisian for
critical reading of the manuscript and helpful suggestions. We also
thank Dr. G. Klein for his assistance and stimulating comments, M. Berthe and L. Laval for expert secretary assistance, Dr. R. Griffin for
reviewing the manuscript, and Dr. M. Willison for linguistic
corrections.
 |
FOOTNOTES |
*
This work was supported in part by grants from the
Delegation Regionale à la Recherche Clinique, CHU de Grenoble,
the Faculté de Médecine-Université J. Fourier,
Grenoble, the Fondation pour la Recherche Médicale, Paris, the
Direction de la Recherche et des Etudes Doctorales, Paris, and the
Bristol Myers Squibb Company.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratoire
d'Enzymologie, CHU Albert Michallon-BP 217, 38043 Grenoble Cedex 9, France. Tel.: (33) 76 76 54 83; Fax: (33) 76 76 56 08.
The abbreviations used are:
APMA, p-aminophenylmercuric acetatebp, base pair(s)BSA, bovine
serum albuminConA, concanavalin AEBV, Epstein-Barr virusIL, interleukinLPS, lipopolysaccharideMMP, matrix metalloproteinasePMA, phorbol 12-myristate 13-acetateRT, reverse transcriptionPCR, polymerase chain reactionPAGE, polyacrylamide gel electrophoresisTIMP tissue inhibitor of metalloproteinase, TGF, transforming growth
factorTNF, tumor necrosis factorFPLC, fast protein liquid
chromatographyELISA, enzyme-linked immunosorbent assayMOPS, 4-morpholinepropanesulfonic acidG3PDH, glyceraldehyde-3-phosphate
dehydrogenase.
 |
REFERENCES |
-
Girard, J. P.,
and Springer, T. A.
(1995)
Immunol. Today
16,
449-457[CrossRef][Medline]
[Order article via Infotrieve]
-
Aumailley, M.,
and Verrando, P.
(1993)
Médecine/Sciences
9,
926-933
-
Matrisian, L. M.
(1992)
Bioessays
14,
455-463[Medline]
[Order article via Infotrieve]
-
Powel, W. C.,
and Matrisian, L. M.
(1996)
in
Attempts to Understand Metastasis Formation (Gunthert, U., Shlag, P. M., and Birchmeier, W., eds), pp. 1-21, Springer Verlag, New York
-
Leppert, D.,
Waubant, E.,
Galardy, R.,
Bunnett, N. W.,
and Hanser, S. L.
(1995)
J. Immunol.
154,
4379-4389[Abstract/Free Full Text]
-
Buisson, A. C.,
Zahm, J. M.,
Polette, M.,
Pierrot, D.,
Bellon, G.,
Puchelle, E.,
Birembaut, P.,
and Tournier, J. M.
(1996)
J. Cell. Physiol.
166,
413-426[CrossRef][Medline]
[Order article via Infotrieve]
-
Owen, C. A.,
and Campbell, E. J.
(1995)
Semin. Cell Biol.
6,
367-376[Medline]
[Order article via Infotrieve]
-
Woessner, J. F.
(1991)
FASEB J.
5,
2145-2154[Abstract/Free Full Text]
-
Werb, Z.,
and Alexander, C. M.
(1989)
in
Textbook of Rheumatology (Kelley, W. N., Harris, E. D., Ruddy, S., and Sledge, C. B., eds), pp. 248-268, W. B. Sanders, Philadelphia
-
Birkedal-Hansen, H.
(1995)
Curr. Opin. Cell Biol.
7,
728-735[CrossRef][Medline]
[Order article via Infotrieve]
-
Mauviel, A.
(1993)
J. Cell. Biochem.
53,
288-295[Medline]
[Order article via Infotrieve]
-
Ries, C.,
and Petrides, P. E.
(1995)
Biol. Chem. Hoppe-Seyler
376,
345-355[Medline]
[Order article via Infotrieve]
-
Chen, W. T.
(1992)
Curr. Opin. Cell Biol.
4,
802-809[Medline]
[Order article via Infotrieve]
-
Goetzl, E. J.,
Banda, M. J.,
and Leppert, D.
(1996)
J. Immunol.
156,
1-4[Abstract]
-
Zaoui, P.,
Barro, C.,
and Morel, F.
(1996)
Biochim. Biophys. Acta
1290,
101-112[Medline]
[Order article via Infotrieve]
-
Murphy, G.,
and Crabbe, T.
(1995)
Methods Enzymol.
248,
470-484[Medline]
[Order article via Infotrieve]
-
Mainardi, C. L.,
Hasty, K. A.,
and Hibbs, M. S.
(1988)
in
The Control of Tissue Damage (Glauert, A. H., ed), pp. 139-147, Elsevier Science, New York
-
Morel, F.,
Dewald, B.,
Berthier, S.,
Zaoui, P.,
Dianoux, A. C.,
Vignais, P. V.,
and Baggiolini, M.
(1994)
Biochim. Biophys. Acta
1201,
373-380[Medline]
[Order article via Infotrieve]
-
Zhou, H.,
Bernhard, E. J.,
Fox, F. E.,
and Billings, P. C.
(1993)
Biochim. Biophys. Acta
1177,
174-178[CrossRef][Medline]
[Order article via Infotrieve]
-
Weeks, B. S.,
Schnaper, H. W.,
Handy, M.,
Holloway, E.,
and Kleinman, H. K.
(1993)
J. Cell. Physiol.
157,
644-649[Medline]
[Order article via Infotrieve]
-
Montgomery, A. M. P.,
Sabzevari, H.,
and Reisfeld, R. A.
(1993)
Biochim. Biophys. Acta
1176,
265-268[CrossRef][Medline]
[Order article via Infotrieve]
-
Xia, M.,
Leppert, D.,
Hauser, S. L.,
Sreedharan, S. P.,
Nelson, P. J.,
Krensky, A. M.,
and Goetzl, E. J.
(1996)
J. Immunol.
156,
160-167[Abstract]
-
Cohen-Tanugi, L.,
Morel, F.,
Pilloud-Dagher, M. C.,
Seigneurin, J. M.,
François, P.,
Bost, M.,
and Vignais, P. V.
(1991)
Eur. J. Biochem.
202,
649-655[Abstract]
-
Morel, F.,
Berthier, S.,
Guillot, M.,
Zaoui, P.,
Massoubre, C.,
Didier, F.,
and Vignais, P. V.
(1993)
Biochem. Biophys. Res. Commun.
191,
269-274[CrossRef][Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.,
and Favre, M.
(1973)
J. Mol. Biol.
80,
575-599[Medline]
[Order article via Infotrieve]
-
Towbin, U. K.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354[Abstract]
-
Woessner, J. F.
(1995)
Methods Enzymol.
248,
510-528[Medline]
[Order article via Infotrieve]
-
Smith, P. K.,
Krohn, R. I.,
Hermanson, G. T.,
Mallia, A. K.,
Gartner, F. H.,
Provenzano, M. D.,
Fujimoto, E. K.,
Goeke, N. M.,
Olson, B. J.,
and Klenk, C.
(1985)
Anal. Biochem.
150,
76-85[Medline]
[Order article via Infotrieve]
-
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Wilhem, S. M.,
Collier, I. E.,
Marmer, B. L.,
Eisen, A. Z.,
Grant, G. A.,
and Goldberg, G. I.
(1989)
J. Biol. Chem.
264,
17213-17221[Abstract/Free Full Text]
-
Sorbi, S.,
Fadly, M.,
Hicks, R.,
Alexander, S.,
and Arbeit, L.
(1993)
Kidney Int.
44,
1266-1272[Medline]
[Order article via Infotrieve]
-
Volpert, O. V.,
Ward, W. F.,
Lingen, M. W.,
Chesler, L.,
Solt, D. B.,
Johnson, M. D.,
Molteni, A.,
Polverini, P. J.,
and Bouck, N. P.
(1996)
J. Clin. Invest.
98,
671-679[Abstract/Free Full Text]
-
Masure, S.,
Proost, P.,
Van Damme, J.,
and Opdenakker, G.
(1991)
Eur. J. Biochem.
198,
391-398[Abstract]
-
Pourmotabbed, T.,
Solomon, T. L.,
Hasty, K. A.,
and Mainardi, C. L.
(1994)
Biochim. Biophys. Acta
1204,
97-107[Medline]
[Order article via Infotrieve]
-
Winwood, P. J.,
Schuppan, D.,
Iredale, J. P.,
Kawser, C. A.,
Docherty, A. J. P.,
and Arthur, M. J. P.
(1995)
Hepatology
22,
304-315[Medline]
[Order article via Infotrieve]
-
Sato, T.,
Ito, A.,
Ogata, Y.,
Nagase, H.,
and Mori, Y.
(1996)
FEBS Lett.
392,
175-178[CrossRef][Medline]
[Order article via Infotrieve]
-
Mauviel, A.,
Chung, K.,
Agarwal, A.,
Tamai, K.,
and Uitto, J.
(1996)
J. Biol. Chem.
271,
10917-10923[Abstract/Free Full Text]
-
Busiek, D. F.,
Baragi, V.,
Nehring, L. C.,
Parks, W. C.,
and Welgus, H. G.
(1995)
J. Immunol.
154,
6484-6491[Abstract/Free Full Text]
-
Mertz, P. M.,
DeWitt, D. L.,
Stetler-Stevenson, W. G.,
and Wahl, L. M.
(1994)
J. Biol. Chem.
269,
21322-21329[Abstract/Free Full Text]
-
Lacraz, S.,
Nicod, L. P.,
Chicheporiche, R.,
Welgus, H. G.,
and Dayer, J. M.
(1995)
J. Clin. Invest.
96,
2304-2310[Medline]
[Order article via Infotrieve]
-
Smith, W. B.,
Noack, L.,
Khew-Goodall, Y.,
Isenmann, S.,
Vadas, M. A.,
and Gamble, J. R.
(1996)
J. Immunol.
157,
360-368[Abstract]
-
Lacraz, S.,
Isler, P.,
Vey, E.,
Welgus, H. G.,
and Dayer, J. M.
(1994)
J. Biol. Chem.
269,
22027-22033[Abstract/Free Full Text]
-
Romanic, A. M.,
and Madri, J. A.
(1994)
J. Cell Biol.
125,
1165-1178[Abstract]
-
Itoh, Y.,
and Nagase, H.
(1995)
J. Biol. Chem.
270,
16518-16521[Abstract/Free Full Text]
-
Houde, M.,
Tremblay, P.,
Masure, S.,
Opdenaker, G.,
Oth, D.,
and Mandeville, R.
(1996)
Biochim. Biophys. Acta
1310,
193-200[Medline]
[Order article via Infotrieve]
-
Borregaard, N.,
and Cowland, J. B.
(1997)
Blood
89,
3503-3521[Free Full Text]
-
Yao, P. M.,
Buhler, J.-M.,
d'Ortho, M. P.,
Lebargy, F.,
Delclaux, C.,
Harf, A.,
and Lafuma, C.
(1996)
J. Biol. Chem.
271,
15580-15589[Abstract/Free Full Text]
-
Shoshan, M. C.,
and Linder, S.
(1992)
J. Cell. Biochem.
55,
496-502
-
Lyons, J. G.,
Birkedal-Hansen, B.,
Pierson, M. C.,
Whitelock, J. M.,
and Birkedal- Hansen, H.
(1993)
J. Biol. Chem.
268,
19143-19151[Abstract/Free Full Text]
-
Fabry, Z.,
Topham, D. J.,
Fee, D.,
Herlein, J,
Carlino, J., A.,
Hart, M. N.,
and Sriram, S.
(1995)
J. Immunol.
155,
325-332[Abstract]
-
Okada, S.,
Kita, H.,
George, T. J.,
Gleich, G. J.,
and Leiferman, K. M.
(1997)
Am. J. Respir. Cell Mol. Biol.
17,
519-528[Abstract/Free Full Text]
-
Delclaux, C.,
Delacourt, C.,
d'Ortho, M. P.,
Boyer, V.,
Lafuma, C.,
and Harf, A.
(1996)
Am. J. Respir. Cell Mol. Biol.
14,
288-295[Abstract]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.