J Neuropathol Exp Neurol
Copyright Ó 2009 by the American Association of Neuropathologists, Inc.
Vol. 69, No. 1
January 2010
pp. 27Y39
ORIGINAL ARTICLE
Schwann Cells Producing Matrix Metalloproteinases Under
Mycobacterium leprae Stimulation May Play a Role in the
Outcome of Leprous Neuropathy
Ariane Leite Oliveira, MSc, Sérgio Luiz Gomes Antunes, MD, Rosane Magda Teles, PhD,
Ana Caroline Costa da Silva, MSc, Tatiana Pereira da Silva, MSc, Rose Brandão Teles, BSc,
Mildred Ferreira Medeiros, MSc, Constan0a Britto, PhD, Márcia Rodrigues Jardim, MD,
Elizabeth Pereira Sampaio, MD, and Euzenir Nunes Sarno, MD
Abstract
Matrix metalloproteinases (MMPs) mediate demyelination and
breakdown of the blood-nerve barrier in peripheral neuropathies.
Matrix metalloproteinases and tissue inhibitor of metalloproteinase
1 gene expression and secretion were studied in cells of the human
Schwann cell line ST88-14 stimulated with Mycobacterium leprae
and tumor necrosis factor (TNF) and in nerve biopsies from patients
with neural leprosy (n = 21) and nonleprous controls (n = 3).
Mycobacterium leprae and TNF induced upregulation of MMP-2
and MMP-9 and increased secretion of these enzymes in cultured
ST88-14 cells. The effects of TNF and M. leprae were synergistic,
and anti-TNF antibody blockage partially inhibited this synergistic
effect. Nerves with inflammatory infiltrates and fibrosis displayed
higher TNF, MMP-2, and MMP-9 mRNA than controls. Leprous
nerve biopsies with no inflammatory alterations also exhibited higher
MMP-2 and MMP-9; tissue inhibitor of metalloproteinase 1 was significantly higher in biopsies with fibrosis and inflammation. Immunohistochemical double labeling of the nerves demonstrated that
the MMPs were mainly expressed by macrophages and Schwann
cells. The biopsies with endoneurial inflammatory infiltrates and
epithelioid granulomas had the highest levels of MMP-2 and MMP-9
mRNA detected. Together, these results suggest that M. leprae
and TNF may directly induce Schwann cells to upregulate and
secrete MMPs regardless of the extent of inflammation in leprous
neuropathy.
Key Words: Fibrosis, Leprosy, Matrix metalloproteinases, Peripheral neuropathy, Tumor necrosis factor.
INTRODUCTION
Mycobacterium leprae (ML) is the etiologic agent of
leprosy, a chronic infectious disease that predominantly affects
From the Leprosy Laboratory (ALO, SLGA, RMT, ACCdS, TPdS, RBT,
MFM, MRJ, EPS, ENS), and Laboratory of Molecular Biology and
Endemic Diseases (CB), Oswaldo Cruz Institute, Rio de Janeiro, Brazil.
Send correspondence and reprint requests to: Sérgio Luiz Gomes Antunes,
MD, FIOCRUZ-Oswaldo Cruz Institute, Av. Brasil 4365, Manguinhos,
24130-082, Rio de Janeiro, Brazil; E-mail: [email protected]
This study was supported by the Oswaldo Cruz Institute, CNPq, and FAPERJ.
J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
the skin and peripheral nerves. The distinct clinical forms of
the disease (tuberculoid, borderline, and lepromatous) are at
least partially determined by the immune status of the patient.
Peripheral nerve damage is the major determinant of morbidity, that is, the disabilities and physical deformities frequently
found among patients with leprosy (1).
Inflammation is the hallmark of leprosy neuropathy and
is characterized by mononuclear cell infiltration with differentiation to epithelioid cells or acid-fast bacilli (AFB)Yloaded
macrophages in association with a progressive replacement
of myelinated and unmyelinated nerve fibers by proliferating
fibroblasts and collagen deposits (2). If epithelioid granulomas
are formed, there is severe local damage to nerve fibers (3), but
even in the absence of granulomas, insidious and diffuse nerve
destruction gradually evolves into fibrosis. This irreversible
end stage of nerve damage in leprosy needs to be prevented to
avoid permanent disability and physical deformity (4).
Matrix metalloproteinases (MMPs) are a family of zincand calcium-dependent extracellular proteases that include collagenases, gelatinases, stromelysin, and membrane-type
MMPs; their major functions are to remodel the extracellular
matrix (5). Gelatinases A and B, also referred to as MMP-2
and MMP-9, respectively, contribute to human neurological
disorders by degrading neurovascular barriers and facilitating
immune cell migration and demyelination (6Y15). In a neuropathic pain model, MMP-9 has also been related to myelin
basic protein degradation, which results in increased contact
between demyelinated fibers and this specific type of pain (9).
Increased MMP expression has also been detected in human
chronic inflammatory demyelinating polyneuropathy and lupus
erythematosus (10). In addition, we have also recently demonstrated increased MMP-9 mRNA expression that correlates
with increased tumor necrosis factor (TNF) gene expression in
nerve biopsies of pure neural leprosy patients; these findings
suggest the involvement of this enzyme and cytokine in the
pathogenesis of nerve damage in leprosy (11).
Matrix metalloproteinases are thought to be secreted in
physical association with their specific inhibitors, the tissue
inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3,
and TIMP-4) that are known to be responsible for regulating
activities of MMPs. Indeed, TIMP-1 completely blocks the
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
Oliveira et al
proteolytic activity of MMP-9 by forming a 1:1 complex with
this enzyme (13). In general, TIMPs are involved in the downregulation of extracellular matrix degradation and accordingly play a role in the regulation of pathological fibrogenic
processes.
The effects of TNF on MMP-9 expression have been
studied in various cell lines (16, 17), and it has been demonstrated that these effects are promoted by nuclear factorJB, a dimeric transcription factor, and the activator protein 1
binding site (17).
The present study demonstrates that ML induces MMP-9
upregulation and secretion in a Schwann cell (SC) line in
vitro and that this effect is enhanced in response by the addition of TNF to the cultures. This key cytokine acts in synergy with ML to cause maximal MMP-9 upregulation. These
in vitro data parallel the findings of upregulation of MMP-2
and MMP-9 in leprosy nerve biopsies and suggest a novel
mechanism by which ML interactions with nerve components
result in nerve damage.
MATERIALS AND METHODS
Patient Nerve Biopsies
The peripheral nerve biopsies of 24 patients (5 women
and 19 men) with a mean age of 45.5 T 14.1 years and with
clinical evidence of peripheral neuropathy were studied. The
biopsies had been performed to confirm the diagnosis of leprosy. In 21 of these patients with neuropathy, there were no
verifiable skin lesions and negative skin smears; the patients
were diagnosed with neural leprosy. The diagnosis of neural
leprosy was based on the histopathologic findings and the
detection of AFB or ML-DNA detected by polymerase chain
reaction (PCR) in the nerve biopsies and/or antiYphenolic
glycolipid 1 positivity in the sera (18). Accordingly, 3 patients
who showed no evidence of leprosy as the etiology of their
peripheral neuropathy and whose nerve biopsies showed a
normal histological appearance were designated as the control group (G1).
Nerve biopsies were collected at the Leprosy Outpatient
Clinic of the Leprosy Laboratory, Oswaldo Cruz Foundation,
Rio de Janeiro, Brazil. All patients received the treatment recommended for paucibacillary patients described in the Brazilian Ministry of Health guidelines. Patients with clinical and
laboratory evidence of diseases such as diabetes mellitus, alcoholism, hepatitis B or C, human immunodeficiency virus or
human T-lymphotropic virus I infection, rheumatoidal diseases, or toxic, drug-induced, or hereditary neuropathies were
excluded from the leprosy neuropathy groups. Patients in the
control group had diabetic, alcoholic, or vasculitic neuropathy
based on clinical, laboratory, and electroneuromyographic
data. This study was approved by the Ethics Committee on
Human Research of the Oswaldo Cruz Foundation.
ML and Reagents
Irradiated armadillo-derived ML was provided by
Dr P. Brennan (Department of Microbiology, Colorado State
University, Fort Collins, CO). The ML was tested for purity and
the absence of endotoxin. According to the limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, MD),
28
all stimuli used for in vitro cultures were shown to contain less
than 0.1 U/mL endotoxin. In a previous publication, Marques
et al (19) demonstrated that no armadillo protein was present in
the dead ML sample used in this study. Recombinant human
TNF prepared in Escherichia coli was purchased from R&D
Systems (Minneapolis, MN).
SC Culture
The ST88-14 tumor cell line established from malignant
schwannoma (neurofibrosarcoma) of patients with type 1 neurofibromatosis (20) was generously donated by J.A. Fletcher
(Dana Farber Cancer Institute, Boston, MA). ST88-14 cells
have been tested in the Leprosy Laboratory and validated as
a model of ML-SC interaction (20, 21). Although they are
transformed cells, they exhibit neural markers of differentiation such as myelin basic protein, glial fibrillary acidic protein, S100 protein, and laminin (21). Responses obtained in
experiments using human primary SCs are very similar to
those involving ST88-14 cells (data not shown).
Cells were grown in RPMI-1640 medium (Gibco BRL,
Gaithersburg, MD) supplemented with 100 U of penicillin/mL,
100 Kg of streptomycin/mL, 2 mmol/L L-glutamine, and 15%
fetal calf serum (Gibco BRL) in a humidified carbon dioxide
incubator at 37-C. For the experimental assays, the attached
ST88-14 cells were released by using trypsin/EDTA (0.25%,
1 mmol/L) for 1 minute, washed in RPMI-FBS, suspended
in complete medium, and further cultured (7 104 cells/well)
onto 24-well plates and (5 105 cell/well) 6-well plates
(Falcon, Franklin Lakes, NJ) for in vitro stimulation.
In Vitro Experiments
For morphological evaluation, cells were seeded onto
glass coverslips and stimulated with ML and TNF separately
and in combination for 24 hours. The coverslips were then
removed from the medium, and the cells were fixed in 4%
paraformaldehyde for 10 minutes at room temperature (RT),
washed in 0.01 mol/L PBS, and immunolabeled with antiY
MMP-9 for confocal microscopy. For ELISA and zymography
analysis, the SCs were cultured onto 24-well plates (Falcon) for
24 hours at 37-C in the presence or absence of one of the
following stimuli: ML at a multiplicity of infection of 50:1, or
TNF at a final concentration of 25 ng/mL, or a combination of
both. For gene expression analysis, the cells were cultured onto
6-well plates (Falcon) in the presence or absence of stimulus
for 1 to 24 hours at 37-C. The TNF dosage and incubation
times were determined in previous kinetic and dose-response
experiments. The time points tested were 1, 3, and 6 hours after
adding TNF to the cultures. Six hours was considered the optimal time response. Concentrations were based on a previous
report (22).
RNA Isolation and cDNA Synthesis
Nerve fragments stored in liquid nitrogen were recovered and homogenized using a Politron PT-3000 in 1 mL of
Trizol (Invitrogen, Carlsbad, CA). All samples were processed at the same time. Schwann cells were cultured onto
6-well plates in the presence or absence of stimulus for 1 to
24 hours at 37-C and suspended in 1 mL Trizol. Total RNA
was extracted according to the manufacturer’s instructions.
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
RNA purity was verified by the OD260/280 absorption ratio
of 1.9 to 2.0. cDNA was synthesized using the Superscript
III first-strand RT-PCR Kit (Invitrogen).
Real-Time PCR
Matrix metalloproteinase 2, MMP-9, TNF, and TIMP-1
mRNA expressions were determined in nerve tissue and/or
SC cultures using a TaqMan real-time PCR kit (Applied
Biosystems, Foster City, CA) by the ABI Prism 7000 sequence detection system (Applied Biosystems). Polymerase
chain reaction amplification was carried out using 100 ng
cDNA added to triplicate tubes with 12.5 KL of the master mix
and 1.25 KL of the probe (FAM-MGB) containing forward
and reverse primers for glyceraldehyde-3-phosphate dehydrogenase, MMP-2, MMP-9, and TIMP-1 (Applied Biosystems).
The amplification parameters consisted of a hold cycle at
50-C for 5 minutes to activate the uracil N9-glycosylase,
followed by a hold cycle at 95-C for 10 minutes to inactivate
N9-glycosylase and release the activity of the DNA polymerase, 50 cycles in 2 steps at 95-C for 15 seconds to separate the
strands, and 60-C for 1 minute to anneal primers and probes
and extend the new strand. Glyceraldehyde-3-phosphate dehydrogenase was used as a normalizer. Its expression in the nerves
was confirmed to be stable and consistent with our previous
publications and it may be superior to A-actin as a housekeeping control (23Y25). Relative mRNA expression was analyzed during the log phase of PCR using the comparative
cycle threshold (CT) method (i.e. mean CT of a target gene
minus mean CT of glyceraldehyde-3-phosphate dehydrogenase)
(26). Samples were considered negative when the signal did not
attain threshold levels of up to 45 cycles. No template controls
for each gene showed any contaminating DNA.
MMP-2, MMP-9, and TIMP-1 ELISA
Matrix metalloproteinase 2, MMP-9, and the TIMP-1
protein secreted from ST88-14 cells were assayed by ELISA
(R&D Systems). Supernatants from stimulated and unstimulated cells were used in a 1:10 dilution. The lowest detection
limit of the assay was 0.08 ng/mL.
Zymography
Gelatin zymography was performed to confirm MMP-2
and MMP-9 enzyme activities, as previously published (27).
Briefly, supernatants from stimulated and unstimulated SC
cultures containing 20 Kg of total proteins were quantified by
the Bio-Rad Protein Assay kit and mixed with an equal volume of the sample buffer (80 mmol/L Tris-HCl [pH 6.8] containing 4% sodium dodecyl sulfate and 10% glycerol (0.01%
bromphenol blue).
For electrophoresis, 10% sodium dodecyl sulfate polyacrylamide resolving gels containing 1% gelatin were overlaid
with 5% stacking gels and samples were loaded. After running
the gel at 25 mA for 2 hours, it was washed 3 times in 150 mL
of 2.5% Triton X-100 solution (15 minutes each) and incubated in 250 mL of 50 mmol/L Tris-HCl (pH 7.5) containing
10 mmol/L CaCl2 and 0.02% NaN3 at 37-C for 18 hours.
After incubation, the gels were stained in 150 mL of 0.1%
Coomassie blue in acetic acid, methanol, and distilled water at
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M. leprae Stimulates Schwann Cell MMPs
a volume ratio of 1:3:6, respectively, for 60 minutes. The gels
were then destained in a solution containing 25% acetic acid
and 50% methanol. After destaining, the gels were immersed
in distilled water for 20 minutes and immediately scanned.
Gelatinolytic activity was demonstrated as clear bands against
a blue background. The intensities of the gelatinolytic bands
corresponding to MMP-2 and MMP-9 were measured by
computer-assisted image analysis. All experimental samples
were run in parallel with 10 pg/well of recombinant proY
MMP-9, a prestained standard (Invitrogen); an aliquot of culture medium was used as an internal control. Because of the
possible background that could occur because of the presence
of serum in the samples, the positivity of the control aliquots
were densitometrically subtracted from the values obtained
for each of the samples (control, ML, TNF, and ML + TNFY
stimulated samples).
Immunolabeling in SCs
Schwann cells were cultured onto 24-well plates containing glass coverslips covered with 4% silane (Sigma Chemical
Co, St Louis, MO). The samples were washed with PBS and
fixed in 1% paraformaldehyde, blocked and permeabilized in
0.5% Triton X-100/10% goat serum and 10% fetal calf serum
in PBS, and then incubated with the antiYMMP-9 antibody.
After rinsing with 0.01 mol/L PBS, fluorescein isothiocyanate
goat anti-rabbit IgG secondary antibody was added and incubated for 30 minutes at RT. Cellular nuclei were stained in
blue with bisbenzimide and Evans blue was used to stain cytoplasm in red; coverslips were mounted with Vectashield
mounting medium (Vector Laboratories, Burlingame, CA). The
specific positive signal was green. Images were obtained using
LSM510 META-ZEISS laser scanning.
Histopathologic and Immunohistochemistry
Methods on Nerve Biopsies
Nerve biopsies were divided into 3 fragments: one of
the fragments was submitted for routine diagnostic procedures (hematoxylin and eosin, Gomori’s trichrome, and Wade
method for AFB); another was frozen and stored in liquid N2 for
immunohistochemistry; the third was processed for Epon embedment for obtaining 0.5-Km-thick toluidine blueYstained
and ultrathin sections for transmission electron microscopy
analysis (28).
Immunolabeling was conducted in paraffin-embedded
sections for single immunoperoxidase labeling as previously
described (11) and in frozen sections for double labeling and
observation with a confocal microscopy (Zeiss LSM 510
META-Germany). In brief, for immunoperoxidase, inhibition
of endogenous peroxidase was performed with 3% H2O2.
Nonspecific binding was blocked with 10% normal serum
(from horse for MMP-2, goat for MMP-9, and mouse for
TIMP-1) for 1 hour at RT, after which the normal serum was
removed by tipping it out of the slides. This was followed by
the immediate application of the following primary antibodies
diluted in 0.01 mol/L PBS: mouse anti-human MMP-2 (1:50;
R&D Systems), polyclonal rabbit anti-human MMP-9 (1:500;
Chemicon), and mouse anti-human TIMP-1 (1:50; R&D
Systems). Each slide was then rinsed 3 times with 0.01 mol/L
PBS for 5 minutes followed by the application of the second
29
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Oliveira et al
antibody (the ready-to-use Envision plus Dual Link System peroxidase DAKO K4061) for 30 minutes at RT in a humid chamber, rinsed with PBS, developed with 3¶3-diaminobenzidine
(Vector), counterstained with Harris hematoxylin, dehydrated,
and finally, mounted with Entellan medium-2 (Merck, Darmstadt, Germany).
Control sections were run according to the following
procedures: the primary antibody was omitted on one of the
nerve sections, and only diaminobenzidine (Sigma) was applied
on another. The slides were observed via a Nikon microscope
(Nikon Eclipse 400, Japan). The images were captured via a
Cool Snap Digital Camera (Media Cybernetics, Bethesda, MD)
and analyzed by way of ImagePro image analysis software
(Media Cybernetics).
J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
for 1 hour at RT, rinsed 3 times for 5 minutes each in PBS, and
then mounted with coverslip and Vectashield mounting media
(Vector).
Statistical Analysis
Results are reported as mean T SD for each group of
experiments. Multiple comparisons were evaluated by the
Kruskal-Wallis test using the Graph Prism (GraphPad, San
Diego, CA). Statistical comparisons among the groups were
performed by 1-way analysis of variance with a post hoc test
using Graph-PAD Prism software (GraphPad); values of
p G 0.05 were considered significant.
RESULTS
Double Labeling of MMPs, CD68 Macrophages,
and S100 Protein SC Markers
ML Increased MMP Gene Expression and
Secretion in SCs
The frozen sections were fixed for 10 minutes in cold
absolute acetone, allowed to dry at RT, and immersed in
0.01 mol/L PBS. Blocking solution composed of 2% normal
goat serum at RT was used for 30 minutes. The drops of
blocking solution were removed by turning the slides, followed
by the application of the antiYMMP-2 (1:50), antiYMMP-9
(1:500), and antiYTIMP-1 (1:50) first primary antibodies to each
section for 1 hour at RT, then by rinsing the slides 3 times with
0.01 mol/L PBS for 5 minutes each. The sections were again
incubated with the blocking solution (2% normal goat serum
diluted with 0.01 mol/L PBS), which was removed by turning
the slides, followed by the application of the goat anti-mouse
first second antibody IgG1 568 Alexa Fluor (1:1000; Invitrogen) for MMP-2, and for TIMP-1, goat anti-rabbit IgG 568
Alexa Fluor (1:1000; Invitrogen), for MMP-9 for 1 hour at RT,
and finally, rinsed thrice with 0.01 mol/L PBS, 5 minutes each.
The second primary antibodies, mouse anti-human CD68
(1:100; Dako, Carpinteria, CA) and rabbit anti-cow S100 protein (1:100; Dako) diluted with 0.01 mol/L PBS + 2% normal
goat serum, were applied to the sections for 1 hour at RT and
rinsed 3 times for 5 minutes each in PBS. The 1:1000 PBS + 2%
normal goat serum diluted goat anti-mouse 488 Alexa Fluor
(1:1000; Invitrogen) for the anti-CD68 and the goat anti-rabbit
488 Alexa Fluor (1:1000; Invitrogen) second secondary antibodies for the anti-S100 protein were dropped on the sections
Initial investigations focused on the effect of ML on
MMP-2, MMP-9, and TIMP-1 mRNA expression in SCs with
real-time RT-PCR. Matrix metalloproteinase 2, MMP-9, and
TIMP-1 mRNA were detected in the unstimulated control cells.
After 6 hours of ML infection, the cultures showed significant
upregulation of mRNA encoding MMP-2 (Fig. 1A), MMP-9
(Fig. 1B), but not of TIMP-1 (Fig. 1C). Because MMP-9 activity in the extracellular milieu is blocked by TIMP-1, which
binds to its catalytic domain (29), the MMP-9/TIMP-1 tissue
ratio corresponds to the balance between these 2 opposing
activities. The MMP-9/TIMP-1 ratio also indicated a predominance of MMP-9 over TIMP-1 expression (data not shown).
To determine whether the effect of ML on mRNA expression was reflected in SC secretion patterns, MMP-2,
MMP-9, and TIMP-1 protein levels were measured by ELISA
in the supernatants of ML-stimulated SCs after the cells
were stimulated for 24 hours. Matrix metalloproteinase 2
and MMP-9 were constitutively expressed in the SCs. The
presence of ML increased MMP-2 secretion after 24 hours
compared with control cells (Fig. 2A). Similarly, MMP-9 secretion was significantly upregulated by ML after 12 hours
(Fig. 2B). On the other hand, TIMP-1 secretion was not modified by ML in the observation period (Fig. 2C); increased
secretion of TIMP-1 despite low mRNA expression could be
explained by the secretion of stored protein.
FIGURE 1. Mycobacterium leprae (ML) induces matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase 1
(TIMP-1) gene expression in ST88-14 cells in vitro. Matrix metalloproteinase 2 (A), MMP-9 (B), and TIMP-1 (C) mRNA in
response to ML was assessed for 24 hours after stimulation (n = 4). *p G 0.05, **p G 0.01 when compared with control untreated
cells (C). Results are expressed as mean T SD of 4 separate experiments.
30
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
M. leprae Stimulates Schwann Cell MMPs
FIGURE 2. Mycobacterium leprae (ML) induces matrix metalloproteinase (MMP) secretion but not tissue inhibitor of metalloproteinase 1 (TIMP-1) by ST88-14 cells. Matrix metalloproteinase 2 (A), MMP-9 (B), and TIMP-1 (C) were assayed in cultured
ST88-14 cells for 24 hours after stimulation with ML with a multiplicity of infection of 2.5. Results represent mean picograms per
milliliter T SD of 4 experiments; *p G 0.05, **p G 0.01 versus control cells (C).
Effects of TNF and ML on MMP and TIMP-1
Gene Expression and Secretion
Schwann cells were stimulated with 10 ng/mL TNF or
ML separately and in combination for 6 hours. Tumor necrosis factor alone slightly increased MMP-2 and MMP-9
mRNA levels, but the combination of TNF and ML caused
higher levels of MMP gene expression than either of the individual stimulus (Figs. 3A and, B). The same profile was
observed with respect to TIMP-1 expression (Fig. 3C), but
the induction of TIMP-1 was less than that of MMP-9.
To confirm that there was increased MMP production
by ML + TNF, MMP and TIMP-1 secretions were measured
in ST88-14 culture supernatants after 24 hours of stimulation.
Mycobacterium leprae did not significantly increase MMP-2
secretion nor did TNF or ML + TNF have any effect on the
secretion of this enzyme. By contrast, MMP-9 secretion increased in SCs stimulated with ML alone, TNF alone, and
with both in combination, thereby confirming the gene expression results. Tissue inhibitor of metalloproteinase 1 secretion was not modulated after 24 hours of culture by either
of the stimuli (Fig. 4A). The higher production of MMP-9 in
SCs stimulated by ML + TNF was confirmed by immunocytochemical labeling of the enzyme and observation by confocal microscopy (Fig. 4B).
The variation in the MMP-2 and MMP-9 expression
levels in response to ML in the experiment shown in Figure 2,
as opposed to the one in Figure 4, was attributable to the use
of 2 different vials of dead ML in each experiment, that is,
one was recently produced, whereas the other had been stored
at j70-C for several months. It should be kept in mind,
however, that aleatory variations in the responses assessed by
ELISA are to be expected.
Effects of ML and TNF on ProYMMP-9 and
ProYMMP-2 Secretion
Because MMP secretion by ELISA was highest 24 hours
after stimulus, this time point was chosen for determining
MMP-2 and MMP-9 activity. Gelatin zymography showed
gelatinolytic bands migrating at 92 and 72 kd and corresponding to proYgelatinase B and proYgelatinase A, respectively. No
FIGURE 3. Tumor necrosis factor (TNF) acts in synergy with Mycobacterium leprae (ML) in increasing matrix metalloproteinase 2
(MMP-2 [A]), MMP-9 (B), and tissue inhibitor of metalloproteinase 1 (TIMP-1 [C]) mRNA expression by ST88-14 Schwann cells.
Schwann cells were stimulated with TNF 10 ng/mL and ML (multiplicity of infection, 50:1) either alone or in combination.
Expression was measured at 6 hours and analyzed by real-time RT-PCR. *p G 0.05, **p G 0.01. Results represent mean T SD of
4 experiments performed. C, control untreated cells.
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Oliveira et al
J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
FIGURE 4. Tumor necrosis factor (TNF) acts in synergy with Mycobacterium leprae (ML) to increase matrix metalloproteinase 9
(MMP-9) but not MMP-2 or tissue inhibitor of metalloproteinase 1 (TIMP-1) secretion in ST88-14 Schwann cell (SC) culture
supernatants. (A) Schwann cells were stimulated with TNF alone (10 ng/mL), with ML alone (multiplicity of infection, 50:1), or
with both stimuli in combination. Matrix metalloproteinase 9 secretion at 24 hours was analyzed by ELISA. *p G 0.05, **p G 0.01;
and (B) MMP-9 immunocytochemical labeling of ST88-14 SCs increased with ML, TNF, and ML + TNF stimuli in comparison to
controls. Cell nuclei were stained with bisbenzimide (blue); the cytoplasm was metachromatically stained with Evans blue (red);
and the MMP-9Yspecific signal is green. Note that the ML- and TNF-stimulated cultures show higher numbers of green MMP9Ypositive cells and that TNF + ML applied in association potentiated this effect.
bands corresponding to the active form of either enzyme could
be identified (Fig. 5A). Densitometric analysis of the bands
showed that all stimuli upregulated proYMMP-9 compared
with control, whereas MMP-2 was modulated albeit not significantly by ML alone (Fig. 5B). In similar experiments using
primary human SCs, both stimuli induced the MMP-9 active
form (86 kd band); this effect was comparable to that observed
with the controls. These data suggest that detection of active
MMP-9 may have resulted from the usual addition of
heregulin to the primary SC culture medium because no active
form of MMP-9 was observed in the absence of this component (data not shown).
FIGURE 5. Tumor necrosis factor (TNF) acts in synergy with Mycobacterium leprae (ML) to increase the matrix metalloproteinase 9
(MMP-9) proactive enzyme in ST88-14 Schwann cell supernatants. Schwann cells were stimulated with TNF alone (10 ng/mL),
with ML alone (multiplicity of infection, 50:1), or with both stimuli in combination. Matrix metalloproteinase 9 activity at 24 hours
was analyzed via zymography (A) and by band densitometry (B). Results represent mean picograms per milliliter T SD of
4 experiments and are shown as a fold induction relative to untreated control.
32
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
M. leprae Stimulates Schwann Cell MMPs
ML alone tended toward a reduction in MMP-9 gene expression after the addition of the anti-TNF antibody, but this
was not statistically significant. As expected, the culture stimulated with TNF alone showed a decrease in MMP-9 gene
expression levels. Interestingly, in cultures to which ML +
TNF were added, TNF blockage partially inhibited MMP-9
expression, reducing it to levels similar although slightly
lower than those obtained in ML onlyYstimulated cultures.
Anti-TNF was unable to completely block ML + TNF stimuli, suggesting that ML alone can induce MMP upregulation
(Fig. 6), but it was not possible to rule out the possibility that
this effect was mediated by ML-induced TNF expression.
Histopathologic Grouping of Leprous
Nerve Biopsies
FIGURE 6. Tumor necrosis factor (TNF) blockade with antiTNF antibody suppresses the induced matrix metalloproteinase 9 (MMP-9) expression in ST88-14 Schwann cell cultures
stimulated with Mycobacterium leprae (ML) + TNF. Schwann
cells preincubated with anti-TNF antibody for 30 minutes were
stimulated with ML, TNF, or ML + TNF. Results represent the
mean T SD of 3 experiments.
To correlate MMP expression and histopathologic nerve
patterns, the biopsies were divided into 5 groups, G1 to G5
(Table; Fig. 7). G1 biopsy specimens (n = 3) were nonleprosy
with normal histological appearances and considered the
control nonleprous group; G2 (n = 4) were neural leprosy
without an inflammatory infiltrate but with loss of myelinated nerve fibers (Fig. 7a), which was confirmed in semithin
preparations. Although there was no evidence of fibrosis in
the endoneurium, focal expansion of the extracellular matrix
was observed in G2. G3 (n = 4) and G4 (n = 10) biopsy
specimens showed typical leprous endoneurial inflammatory
infiltrates; some had epithelioid granulomas (5 biopsies) or
Inhibition of TNF Blocks MMP-9 Gene
Upregulation in ML-Stimulated SC Cultures
Inhibition of TNF activity upon the addition of antiTNF antibody reduced MMP-2 and TIMP-1 gene expression
levels in SC cultures as a result of all the stimuli but in a nonsignificant way (data not shown). Cultures stimulated with
TABLE. Histopathologic Features of Nerve Biopsies
Groups
G1
G2
G3
G4
G5
Nerve Biopsied
Mononuclear Cell Infiltrates
Epithelioid Granulomas
AFB+ Macrophages
Loss of Fibers
AFB Detected by
Wade Method
R DCB ulnar
L DCB ulnar
L Sural
L Sup Fib
L DCB ulnar
R Sural
R Sural
R Sural
R DCB ulnar
R Sural
R Sural
R Sural
L Sural
L Sural
L Sup Fib
R DCB ulnar
R DCB ulnar
L DCB ulnar
R DCB ulnar
L Sural
L Sural
L DCB ulnar
L Sural
L DCB ulnar
j
j
j
j
j
j
j
+
+
+
+
+
+
+
+
+
+
+
+
+
+
j
j
j
j
j
j
j
j
j
j
j
j
j
+
j
+
j
j
j
j
+
+
j
+
j
j
j
j
j
j
j
j
j
j
+
j
+
+
j
+
+
j
j
j
j
j
j
j
j
j
j
j
j
j
j
+
+
j
+
j
+
+
+
+
+
+
j
+
+
+
+
+
+
+
+
j
j
j
j
j
j
+
+
j
+
+
j
+
+
j
j
j
j
j
+
j
j
j
j
AFB, acid-fast bacilli; DCB ulnar, dorsal cutaneous branch of ulnar nerve; G, group; L, left; R, right; Sup Fib, superficial fibular nerve.
+, finding present; j, finding absent.
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Oliveira et al
34
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
AFB-loaded macrophages (n = 5) or SCs (n = 2). Mononuclear infiltrates were found in all G3 and G4 biopsies. G5
biopsy specimens had only sparse residual inflammatory cells
(Table). The degree of fibrosis distinguished the G3, G4, and
G5 groups. G3 biopsy specimens had focal inflammatory
infiltrates but no fibrosis (Fig. 7b); G4 had larger fibrotic
endoneurial regions amid inflammatory infiltrates that partially replaced nerve fibers (Fig. 7c); in the G5 group (n = 3),
nerve compartments showed dense fibrous tissue (Fig. 7d).
Remyelinated fibers and clusters of axonal regeneration were
seen in semithin sections of G3 and G4 (Fig. 7e); mononuclear cells closely apposed to fibroblasts were observed in
nerves with endoneurial fibrosis (Fig. 7f ).
MMP-2 and MMP-9 Are Increased In Leprous
Nerve Biopsies With Inflammatory Infiltrate
and Fibrosis
The 5 groups of biopsies expressed different MMP and
TIMP-1 levels. G4 and G2 showed significantly higher
MMP-2 message expression compared with G1 (Fig. 8A).
Matrix metalloproteinase 9 was also highly expressed in G2,
G3, and G4 compared with G1 (Fig. 8B). In contrast, MMP-2
and MMP-9 were not as strongly expressed in G5 (Figs. 8A
and B). Tissue inhibitor of metalloproteinase 1 expression in
G3 and G4 showed a tendency toward upregulation, but this
was not statistically significant (Fig. 8C). The resulting TNF
expression profile was found to be similar to MMP-9 and
MMP-2 profiles (Fig. 8E).
In summary, G3 and G4 displayed higher TNF, MMP2, and MMP-9 mRNA expression than G1 and G2. Interestingly, with the exception of TNF, G2 also exhibited higher
MMP-2 and MMP-9 message levels than G1.
MMP-2 and MMP-9 Are Expressed in SCs and
Macrophages In Situ
Matrix metalloproteinase 2 was expressed in both the
SCs and axons of the control group in addition to the
remaining nerve fibers in G3 and G4. Both of these groups
also exhibited a moderate-to-severe decrease in numbers of
myelinated fibers depending on the degree of inflammation
and fibrosis. CD68-positive macrophages were costained
with antiYMMP-2 (Figs. 9aYd), but G5 did not show any
MMP-2 immunoreactivity.
Matrix metalloproteinase 9 immunoreactivity was
observed in nerve fibers (both SCs and axons) of G1 and in
the remaining fibers of both G3 (Fig. 9e) and G4, colocalizing with S100-positive SCs (Figs. 9fYh). Most of the S100+
SCs expressed MMP-9; only a few SCs in the merged images
were not stained yellow.
M. leprae Stimulates Schwann Cell MMPs
Matrix metalloproteinase 9 and CD68 double-positive
macrophages were also detected in the endoneurial compartments of G3 and G4 nerves. In G5, MMP-9 positivity was
observed in flat elongated cells that morphologically corresponded to fibroblasts.
Tissue inhibitor of metalloproteinase 1 was not identified in the control group but was coexpressed in a few CD68positive cells and cytoplasmic granules of mast cells in G4
mast cells (Figs. 9iYl). Fibroblast-like cells expressed TIMP-1
in G5.
Correlations of MMP Expression and
Histopathologic Findings
The biopsies with inflammatory infiltrates (particularly
G3 and G4) expressed markedly higher TNF, MMP-2, and
MMP-9 levels (p G 0.06, p G 0.003, and p G 0.001 vs
controls, respectively). Likewise, the biopsies with epithelioid granulomas displayed significantly higher levels of
MMP-9 compared with those without this finding (p G 0.03).
Finally, there was higher MMP-9 (p G 0.02) and MMP-2
(p G 0.03) expression in G2, that is, the group with noninflammatory histological alterations but with decreased
numbers of myelinated fibers and a slight accumulation of
endoneurial extracellular matrix.
DISCUSSION
The present results suggest that ML + TNF not only
modulate MMP-2, MMP-9, and TIMP-1 expression, but that
MMPs, cytokines, and ML also seemed to be involved in the
final configuration of leprous nerve damage. Moreover, the in
vitro data suggest that as a result of their interactions with
ML, SCs (either alone or in association with TNF) can induce
MMP-9 upregulation production and are, therefore, one of
the probable cellular mediators of leprosy nerve damage.
Mycobacterium leprae upregulates MMP-2 and MMP9 mRNA expression and protein production in SC cultures
at several time intervals subsequent to stimuli, in the absence,
however, of increased TIMP-1 expression. It is worth remarking that the in vitro experiments were conducted with
dead ML and that any actively secreted bacterial product
might change the expression and secretion profile obtained
if live ML were used. Mycobacterium leprae did not induce
SC proliferation during the time points tested. Significant induction of SC proliferation was only observed after
48 hours of stimuli (data not shown), thus ruling out the
possibility that MMP upregulation was caused by increased
SC proliferation.
The upregulation of the MMP-2 and MMP-9 detected
in the G2 biopsies with nonspecific noninflammatory
FIGURE 7. Examples of each group (G) of nerve biopsies: (a) G2, nerve with loss of myelinated fibers. Endon, endoneurium. (b)
G3, nerve with focal perivascular infiltrate (arrow) in the endoneurium without fibrosis. (c) G4, nerve with endoneurial and
perineurial inflammatory infiltrate (endo inf) and fibrosis amid inflammatory cells. (d) G5, nerve with severe endoneurial fibrosis
(end fibrosis); there are scant residual inflammatory cells in the endoneurium. (e) G2, nerve with loss of myelinated fibers, few
remyelinated fibers (short arrow), and a cluster of sprouting regenerating axons (long arrow). Toluidine blueYstained semithin
section. (f) G4, electron photomicrograph of a nerve fascicle showing endoneurial fibrosis (end fibrosis) and contact between a
mononuclear cell (arrowhead) and a fibroblast (long arrow). A small cluster of ML bacilli can be seen in the cytoplasm of a
Schwann cell (short arrow). (aYd) Gomori trichrome stain.
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35
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Oliveira et al
J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
FIGURE 8. Matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tumor necrosis factor (TNF)
mRNA expression determined by real-time polymerase chain reaction in nerve biopsies of controls (G1) and neural leprosy biopsy
groups (G2YG5). (A) Matrix metalloproteinase 2 expression was higher in G2 and G4. (B) Matrix metalloproteinase 9 was higher
in G2, G3, and G4. (C) Tissue inhibitor of metalloproteinase 1 was not significantly increased in any of the groups compared with
the controls, but the MMP-9/TIMP-1 ratio was significantly higher in G4 and in G2 compared with G1 (D). (E) Tumor necrosis
factor was increased in G4, coinciding with the higher expression of MMP-9 (D). Results represent mean T SD of 3 or more
biopsies for each group. *p G 0.05, **p G 0.01.
histopathologic alterations suggests that increased expression does not depend exclusively on inflammation; this is
consistent with our results in relation to SCs in in vitro cultures. The data also suggest that ML alone can increase both
the mRNA and secretion of MMPs by SCs irrespective of
the inflammatory process. Because leprosy affects the peripheral nervous system in an asymmetrical and segmental pattern
(which is not always accurately detected by standard biopsy
procedures), the existence of lesions in the portions of the
nerve proximal to the biopsied segments cannot be excluded.
It is known that TNF is involved in the mediation of
MMP-9 expression under diverse conditions (30Y33). Inversely, MMP-9 can activate the release of TNF from its
transmembrane precursor (34) and inactivate TNF-mediated
signaling by sequestering the TNF receptors, TNFRI (p55)
and TNFRII (p75) (35). Studies in the rat sciatic nerve have
indicated that endoneurial MMP-9 expression follows TNF
expression temporally and spatially (3, 14, 15). We emphasized TNF over other proinflammatory cytokines in this study
because of its upstream position in MMP-9 activation, and
corresponding results in a previous study were also confirmed herein. Furthermore, TNF-induced MMP-9 activity
could trigger the breakdown of the blood-nerve barrier,
36
recruiting macrophages to the endoneurium in a manner similar to that in Shubayev’s model (24). Other cytokines not
studied in this investigation (e.g. interleukins 2, 12, and 8,
and interferon-F) also likely participate in the inflammatory
response in leprosy nerves. More experimental models are
needed to test their roles.
We emphasize the importance of ML as an inducer of
MMP-2 and MMP-9 production in SCs and provide evidence
that TNF is implicated in this message upregulation and
increased protein secretion (36). Synergy between TNF and
other pathogens in increased MMP-9 expression and secretion has also been reported in other infections (37, 38).
Unlike MMP-9, MMP-2 is constitutively and widely
expressed and usually forms a molecular complex with
TIMP-2. In the present study, MMP-2 secretion was not
significantly different in SC cultures stimulated with ML,
TNF, or ML + TNF, or even in the absence of stimulus (data
not shown). In contrast, MMP-2 message expression increased significantly as a result of all types of stimuli. The
discrepancy between expression and secretion might be
explained by the fact that MMP release is regulated by
intracellular and extracellular mechanisms in addition to
transcriptional ones.
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
M. leprae Stimulates Schwann Cell MMPs
FIGURE 9. Representative immunohistochemical staining for matrix metalloproteinases (MMP) and tissue inhibitor of
metalloproteinase 1 (TIMP-1) in leprous nerve biopsy groups. (aYd) Matrix metalloproteinase 2Yimmunoreactive mononuclear
cells in a perineurial infiltrate of a G4 leprous nerve (a); colocalization of CD68 ([b] green); MMP-2 immunoreactivity ([c] red);
and merge (d). (eYh) Matrix metalloproteinase 9Yimmunoreactive Schwann cells (SCs) of large and small myelinated nerve fibers
(outer positive collar) in a G3 leprous nerve (e); colocalization of S100 protein ([f] green); and MMP-9 immunoreactivity ([g]
red); merge (h). Not all S100-immunolabeled SCs are stained in yellow. (iYl) Tissue inhibitor of metalloproteinase
1Yimmunoreactive mononuclear cells in an endoneurial infiltrate of a G4 biopsy (i); colocalization of some CD68 ([j] green);
TIMP-1 ([k] red); and merge (l).
Using gelatin zymography, it was possible to detect proY
MMP-2 and proYMMP-9 enzymatic activity. Montgomery
et al (39) demonstrated that B cells in culture secrete only
proYMMP-9 but not the active form. Active MMP-9 was
only detected when the cultures were pretreated with aminophenylmercuric acetate. Similarly in this study, proYMMP-2
and proYMMP-9, but not active MMPs, were only detected in
the supernatants of SC cultures. These data indicate that both
ML and TNF upregulated MMP-9 protein production but had
no effect on MMP activity in SC cultures. Nonetheless, this
conclusion cannot be extended to an in situ analysis that
detected the presence of both enzymes in the biopsies of
leprous cutaneous lesions, in contrast to the peripheral blood
mononuclear cell cultures from leprous patients, in which this
form was undetectable (manuscript in preparation).
The higher MMP-2 and MMP-9 mRNA expression in
G3 and G4 nerve biopsies is consistent with MMP
pathogenic activity, that is, these are the groups that showed
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active inflammatory infiltrates in the nerves with variable
(moderate-to-severe) loss of myelinated fibers. The higher
TIMP-1 expression in G4 and in G3 can be attributed to the
delayed resolution of the inflammatory infiltrate caused by
the persistence of ML or its components. Chronic and
prolonged inflammation elicits a persistent increase in
MMP-9 expression with concomitant TIMP-1 upregulation,
accounting for the higher G4 MMP-9/TIMP-1 ratio compared
with the ratio in the G3 group. Thus, TIMP-1 elevation
concomitant to persistent MMP-9 activity (in reality, 2
opposing activities) renders the nerve vulnerable to proteolytic and fibrogenic activities, resulting in the replacement of
nerve fibers and assembly of excessive fibrous tissue. High
levels of nerve damage and fibrosis were associated with the
highest MMP-9 and TIMP-1 expression.
The correlations of MMP and TIMP-1 immunohistochemical expression with marked pathological alterations in
leprosy nerves suggest that the infiltrating inflammatory cells
37
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J Neuropathol Exp Neurol Volume 69, Number 1, January 2010
Oliveira et al
are responsible for the upregulation of these enzymes; the
SCs and axons were reduced in number because of the partial
or total inflammatory replacement of myelinated nerve fibers
in the G3 and G4 groups. Matrix metalloproteinase 9Y and
MMP-2Yimmunoreactive cells were identified as macrophages in G3 and G4 biopsies on the basis of their
coexpression of CD68 and their concomitant S100 proteinY
negative labeling. Matrix metalloproteinaseYimmunoreactive
macrophages were scarce in G5 as the fibrosis observed in
this group engulfed residual inflammatory cells. Therefore, it
can be assumed that the increased message expression of the
enzymes studied almost certainly was caused by the
inflammatory proteolytic activity of infiltrating macrophages
and repairing fibroblasts in leprosy nerves.
Matrix metalloproteinase 2 and MMP-9 expression in
nerve biopsies were associated with the presence of inflammatory infiltrates and, in particular, with the presence of
epithelioid granulomas. Biopsies with mature granulomatous
infiltrates usually show a severe reduction in the number of
both myelinated and nonmyelinated nerve fibers. This highly
destructive profile might be a consequence of the higher TNF
and MMP-9 expression found in this group. These results are
consistent with those of a previous publication (11), in which
biopsies with higher levels of immune response also exhibited higher MMP expression.
In conclusion, this investigation suggests that on 1
hand, MMP-2, MMP-9, and TIMP-1 contribute individually
and/or jointly to the final configuration of the nerve damage
and fibrosis in leprosy neuropathy, and on the other hand,
that ML-stimulated SCs mediate MMP-9 upregulation preceding inflammatory infiltrate. Despite these results, it
remains questionable as to whether MMP-9 upregulation is
the most critical event in the breakdown of the blood-nerve
barrier. It is, therefore, imperative that experimental studies
using animal models be conducted to test this hypothesis.
Once inflammation has been established and the nerves have
been partially or totally damaged, the cells that assume the
sustained production of these enzymes are infiltrating macrophages. Future studies could focus on the role of MMP
inhibitors because their use, particularly of MMP-9, in
patients with leprosy neuropathy might prevent the initial
recruitment of macrophages and halt nerve damage, thereby
preventing irreversible nerve fibrosis in the more advanced
stages of the disease.
ACKNOWLEDGMENTS
The authors thank Helen Ferreira and Vânia Valentim
for their expert technical assistance and Judy Grevan for editing the text in English.
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