British Journal of Rheumatology 1997;36:744–747
GELATINASE B IN CHRONIC SYNOVITIS: IMMUNOLOCALIZATION WITH A
MONOCLONAL ANTIBODY
B. GRILLET, J. DEQUEKER, L. PAEMEN,* B. VAN DAMME† and G. OPDENAKKER*
Department of Rheumatology, *Laboratory of Molecular Immunology at the Rega Institute of Medical Research and †Department
of Pathology, University of Leuven, Belgium
SUMMARY
Gelatinase B is a matrix metalloproteinase (MMP-9) involved in the remodelling of extracellular matrices of connective tissues.
With the use of specific monoclonal antibodies against human gelatinase B, the producer cell types were pinpointed in
histopathological sections of a number of arthritic diseases. In cases of acute joint trauma, chondromatosis, villonodular synovitis
and a cyst of a bursa, high numbers of strongly immunopositive neutrophils were observed in additon to weaker staining
macrophages. Activated macrophages with giant cell morphology clearly stained with the gelatinase B-specific monoclonal
antibody in the case of villonodular synovitis and in an epidermoid cyst. However, in the sections from patients with rheumatoid
arthritis, no immunostaining was seen. In other cases of chronic synovitis, however, within the lymphocyte nodular aggregates
a strong gelatinase B expression was observed in morphologically identified dendritic cells. In conclusion, gelatinase B production
in joint disease seems to be predominantly by neutrophils and cell types of the macrophage/antigen-presenting cell lineage.
K : Gelatinase B, Monoclonal antibodies, Immunolocalization.
cytes. It is constitutively present in serum and body
fluids including synovial fluid. Gelatinase B (MMP-9,
mol. wt 292 kDa) is mainly produced by neutrophilic
granulocytes, which release gelatinase B within minutes
after stimulation with the neutrophilic chemokine
interleukin-8 (IL-8) [7]. Gelatinase B is induced in
monocytes by IL-1 and in many other cell types (e.g.
fibroblasts, endothelial cells, keratinocytes) by various
agonists. In terms of protein domain structure,
gelatinase B is so far the most complex MMP. It is the
terminal member of the cascade leading to matrix
degradation and its activity in vitro is modulated at
four levels: de novo gene transcription (e.g. by IL-1),
secretion (e.g. by IL-8), activation (e.g. by stromelysin
and gelatinase A) and specific inhibitors (e.g. tissue
inhibitors of metalloproteinases). Elevated levels of
gelatinase B have been demonstrated in the biological
fluids of patients suffering from various inflammatory
diseases, including RA. Gelatinase B titres vary
considerably in synovial fluid and are correlated with
other parameters of disease activity [8]. In view of the
increased titres of IL-8 and neutrophil cell counts in
synovial fluid of RA patients, and because IL-8
stimulates the rapid release of gelatinase B by
neutrophils, the neutrophil granulocyte is probably the
major source of synovial gelatinase B, although limited
production by monocytes/macrophages is also conceivable. In the synovial membrane of patients with RA,
neutrophil granulocytes are, however, very sparse. In
contrast, mononuclear cells are believed to play an
important role in RA synovial membrane [9]. In
primary cultures of adherent rheumatoid synovial cells,
gelatinase B production was restricted to macrophages
[5]. In order to localize the sites of gelatinase
production in chronic inflammation, we analysed tissue
samples of patients with different chronic inflammatory
disorders of the joints and the surrounding tissue.
C joint inflammation is likely to induce
permanent joint damage. Rheumatoid arthritis (RA),
for instance, is a chronic inflammatory disorder, with
systemic features and joint involvement, resulting in an
erosive synovitis and joint destruction. Many other
disorders are more or less characterized by joint
lesions. Damage to bone, cartilage, tendons and
ligaments is mediated largely by proteinases [1].
Enzymes involved in the breakdown of the matrix
elements, allowing inflammatory cells to migrate into
the site of inflammation, may induce long-term
irreversible damage. Metalloproteinases play an important role in this scenario. They act outside the cell
at neutral pH and are capable of degrading the
molecules of the extracellular matrix [2]. Among these,
collagenase (matrix metalloproteinase-1 or MMP-1)
and its zymogen activators are rate limiting in collagen
degradation [3]. Collagenase activity was previously
detected in the synovial fluid of patients with RA, and
synovial fibroblasts were recognized as the major
source of collagenase [4]. Stromelysin (MMP-3) is
another member of the family of metalloproteinases, its
substrate being non-collagen proteins such as proteoglycans, fibronectin and laminin [2]. Stromelysin is
present in the synovial fluid of patients with RA and
has been detected both in the synovial membrane
(especially in fibroblasts [5]) and in cartilage [6].
A third group within the family of MMPs is formed
by the gelatinases. These enzymes degrade denatured
collagen, gelatin and some other extracellular matrix
components. Two proteins have been isolated: gelatinase A (MMP-2, mol. wt 272 kDa) is produced by
monocytes, fibroblasts, endothelial cells and keratinoSubmitted 5 August 1996; revised version accepted 10 December
1996.
Correspondence to: B. Grillet, U.Z. Pellenberg-Reumatologie,
Weligerveld 1, B-3212 Pellenberg, Belgium.
= 1997 British Society for Rheumatology
744
GRILLET ET AL.: GELATINASE B IN CHRONIC SYNOVITIS
Immunohistochemistry was performed using a highly
specific monoclonal antibody against gelatinase B [10].
MATERIALS AND METHODS
Tissues
Synovial tissue from a range of joint inflammations
was obtained from biopsies and surgical interventions
carried out for other medical reasons. Thirteen samples
were analysed: 11 from the synovial membrane of the
joint, two from cysts.
The tissues were fixed in formaldehyde and routinely
processed through paraffin.
Monoclonal gelatinase B antibody and Western blot
analysis
Gelatinase B-specific monoclonal antibodies were
developed recently [10]. One of these, REGA-2D9, is
an IgG1 monoclonal with a dissociation constant of
2.9 × 10−9  towards pure human gelatinase. B. The
corresponding hybridoma was grown in pristaneprimed mice, the ascites collected and purified on
protein A columns, and used for immunohistochemical
localization of gelatinase B. In Western blot analysis of
reduced crude synovial fluid samples, the monoclonal
REGA-2D9 reacted with only one protein band which
was gelatinase B specific.
Immunolocalization
Studies were performed using standard immunohistochemical techniques. To inactivate endogenous
peroxidase, the slides were pre-treated with 0.3%
hydrogen peroxide in methanol. The samples were
incubated for 30 min at room temperature with 50 ml
aliquots of the REGA-2D9 monoclonal antibody.
After three washes with phosphate-buffered saline
(PBS), the slides were incubated with biotin-linked
rabbit anti-mouse IgG1 (Dako) for 30 min. Finally,
after three washes with PBS, the slides were incubated
with peroxidase-labelled avidin–biotin complex (Dako)
for 30 min. The peroxidase reaction was performed
with 0.6 mg/ml 3-3'-diamino-azo-benzidine in 0.01%
hydrogen peroxide in PBS. Control slides were treated
in an identical manner, except that an irrelevant IgG1
mouse mAb was used in the first step. All sections were
counterstained with haematoxylin.
RESULTS
Clinical picture
Tissue samples were analysed from the 13 patients,
who were aged between 14 and 65 yr (mean 45 yr);
seven were males, six were females. They all suffered
from a joint inflammation for more than 6 weeks.
RA, in agreement with the criteria of the American
College of Rheumatology (ACR) [11], was present in
two patients. In two other patients, a chronic synovitis
indicative for RA was present, albeit without satisfying
the criteria for RA.
One patient suffered from pes planus resulting in
mechanical damage to the joint. In two other patients,
the synovitis occurred after an acute mechanical insult
to the joint. Osteochondromatosis was diagnosed in
745
two patients. A villonodular synovitis was seen in two
others.
In two patients the biopsy was from a cyst: an
epidermoid cyst in one patient and a cyst of the bursa
synovialis of the popliteal muscle in the other patient.
Histopathology
Considering the 11 samples of joint tissue, hyperplasia of the synovial lining was seen in five patients
(two with RA, two with synovitis following an acute
mechanical insult and one patient with chondromatosis).
In eight cases, the inflammation was characterized by
a lymphocytic infiltrate organized in nodular aggregates.
In a patient with chondromatosis, granulocytes were
abundantly present.
In the cases of villonodular synovitis and in the cysts,
the infiltrate contained predominantly macrophages,
frequently accompanied by giant cells.
Immunohistology
Immunoreactive gelatinase B was present in seven of
the 13 samples.
Neutrophil granulocytes were abundantly present in
the synovial tissue of one patient with chondromatosis
(Fig. 1A). They were seen in three other samples as
well. Staining of macrophages was seen in all seven
positive specimens (Fig. 1B). These cells had the
histological aspect of dendritic cells in two samples: one
was from a patient with a chronic synovitis not
fulfilling the ACR criteria of RA (Fig. 1C), the other
was from a patient with chondromatosis. In the five
other samples, the macrophages were the predominant
positive cells of the inflammatory infiltrate (two
samples of villonodular synovitis and two cysts). Giant
cells were positive as well (Fig. 1D).
Overall positivity of the mononuclear cells was
significant, but weaker than that of neutrophil
granulocytes. Control immunohistochemistry with an
irrelevant mouse IgG1 was negative in all cases
(Fig. 1F).
CONCLUSION AND DISCUSSION
Gelatinase B was localized by immunohistochemistry with a specific monoclonal antibody in the
synovial tissue of patients with joint diseases.
Neutrophil granulocytes are a major source of
gelatinase B in the synovial fluid, but are rarely present
in the synovial membrane. Neutrophils were present in
three samples of synovial membrane and in one cyst.
These neutrophils yielded the strongest immunoreactivity observed. This is in accordance with previous
biochemical data: neutrophils contain gelatinase B
granules which are promptly released under the
influence of IL-8, whereas other cells, e.g. monocytes,
synthesize de novo smaller amounts of gelatinase B
when triggered by IL-1 [7, 8].
Cells of the monocyte/macrophage lineage stained
for gelatinase B in 7/13 samples. As macrophages are
dependent on cytokine stimulation to produce gelatin-
746
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 7
F. 1.—Legend opposite.
GRILLET ET AL.: GELATINASE B IN CHRONIC SYNOVITIS
747
F. 1.—Immunohistochemistry of gelatinase B in chronic synovial diseases. Gelatinase B was detected by means of a specific mouse monoclonal
antibody in formalin sections of joint tissues. The second antibody was a biotin-linked rabbit anti-mouse IgG1. Staining was performed by
peroxidase-labelled avidin–biotin complex. Immunopositivity is indicated by red–brown colouring. (A) Strong gelatinase B staining of
granulocytes in a patient with chondromatosis (original magnification ×300). (B) Gelatinase B immunoreactivity of granulocytes and
macrophages in a patient with a synovitis secondary to an acute trauma (original magnification ×640). (C) A lymphocytic nodular aggregate
in a patient with a chronic synovitis. Immunohistochemical staining for gelatinase B in mononuclear cells with dendritic morphology (original
magnification ×600). (D) Staining of gelatinase B in macrophages and giant cells of a patient with a villonodular synovitis (original magnification
×400). (E) Immunohistochemical staining for gelatinase B in mononuclear cells with dendritic morphology in a patient with chronic synovitis
(original magnification ×400). (F) The same sample as in (E), using an irrelevant IgG1 mouse monoclonal antibody in the first step as a control,
demonstrating the lack of staining (original magnification ×400).
ase B [8], the present data are in agreement with the
stimulated state of macrophages in the samples of
synovitis tissue. On several occasions, macrophages
organized into giant cells were seen in the samples of
villonodular synovitis and in an epidermoid cyst. These
giant cells stained well with the anti-gelatinase B
monoclonal antibody.
In a patient with a chronic synovitis with
lymphocytic infiltration and in a patient with
chondromatosis, morphologically identified dendritic
cells embedded in aggregates of lymphocytes stained
for gelatinase B.
Besides their potential antigen-presenting role,
macrophages are believed to play an important role in
driving or regulating the chronic inflammatory process
by the production of cytokines such as IL-1, tumour
necrosis factor-a (TNF-a) and IL-6. In addition,
monocytes are involved in the secretion of gelatinase B
in vitro [3, 8]. Here we document the specific cellular
production of gelatinase B molecules in disease states.
Functionally, the gelatinase can be used by the
macrophage in several ways: to clear the way for its
own migration, but also in the proteolytic conversion
of extracellular matrix components into remnant
epitopes generating autoimmunity (REGA-model)
[12].
In contrast to the other metalloproteinases collagenase and stromelysin that are produced by the synovial
lining [4], especially the fibroblasts [5], gelatinase B is
produced by the infiltrating cells of monocyte/macrophage lineage.
Gelatinase may be responsible for the destruction of
cartilage and bone, but the abundance of gelatinase B
in synovial fluid compared to synovial tissue [8]
emphasizes the role of synovial fluid as a potential
source of gelatinase B in tissue damage. Further
analysis will be needed to determine to what extent
immunotyping with gelatinase B can be used as a
marker for a particular disorder or for a more
damaging joint disease.
A
The present study was supported by the ‘Geconcerteerde Onderzoeksacties’ of the Flemish Government,
by the General Saving and Retirement Fund (ASLK),
and by the National Fund for Scientific Research
(NFWO). LP holds a fellowship from the Charcot
Foundation, Belgium. The help of Erik Martens in
monoclonal antibody production and purification is
appreciated.
R
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