INFECTION AND IMMUNITY, Mar. 2010, p. 1012–1021
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 3
High Matrix Metalloproteinase Production Correlates with Immune
Activation and Leukocyte Migration in Leprosy Reactional Lesions?
Rosane M. B. Teles,1Rose B. Teles,1Thais P. Amadeu,1,3Danielle F. Moura,1
Leila Mendonc ¸a-Lima,2Helen Ferreira,1I´talo M. C. F. Santos,1
Jose ´ A. C. Nery,1Euzenir N. Sarno,1* and Elizabeth P. Sampaio1†
Leprosy Laboratory,1and Laboratory for Functional Genomics and Bioinformatics,2Oswaldo Cruz Institute, FIOCRUZ, Rio de
Janeiro, RJ, Brazil, and Department of Histology and Embryology, UERJ, Rio de Janeiro, RJ, Brazil3
Received 7 August 2009/Returned for modification 28 August 2009/Accepted 30 October 2009
Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal
membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear.
In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions
and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The partic-
ipation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy
specimens correlating with the expression of gamma interferon (IFN-?) and tumor necrosis factor alpha
(TNF-?), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii)
increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or
TNF-?. It was observed that IFN-?, TNF-?, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than
lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not
differentially modulated. These data correlated with the detection of the MMP protein evidenced by immuno-
histochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-?,
MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity
(zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae
induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate
an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for
tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy
Matrix metalloproteinases (MMPs) compose a family of
zinc- and calcium-dependent proteolytic enzymes responsible
for extracellular matrix (ECM) remodeling and the regulation
of the trans-ECM migration of leukocytes, an important step in
inflammatory processes as well as infectious diseases. MMPs
are functionally classified according to their relative substrate
specificities but have been shown to overlap. Collagenases
(MMP-1, -8, and -13) degrade type I collagen, whereas gelati-
nases A and B (MMP-2 and -9, respectively) degrade dena-
tured type I (gelatin) and type IV collagens, a major compo-
nent of the basement membrane. Collagenases are produced
by many cell types including lymphocytes, granulocytes, astro-
cytes, and activated macrophages (10, 18).
MMP secretion takes place under tight regulatory mecha-
nisms including transcriptional controls in addition to their
release as proenzymes, requiring activation by specific pro-
teases and cytokines present in the milieu (2, 3, 16). Also, the
postactivation of MMPs is controlled by metalloproteinase tis-
sue inhibitors (tissue inhibitor of MMP [TIMP]), a family of
specific inhibitors, that bind to MMPs in a stoichiometric ratio.
Thus, the matrix-degrading capacity of MMPs depends on the
balance between MMP levels and the availability of extracel-
lular TIMPs (5, 8). Due to their capacity to degrade basement
membrane components, the gelatinases MMP-2 and MMP-9
are key molecules in leukocyte recruitment to inflammatory
sites, which are indispensable for the containment of infection
(27). However, excessive MMP secretion has been related to
tissue damage in many inflammatory disorders (9, 25, 27).
High levels of MMP-9 detected in sera of patients have been
considered to be biomarkers of disease activity (7, 31). Simi-
larly, in tuberculosis (TB) pleurisy, the MMP-9 present in
pleural fluid was associated with the presence of granulomas in
the tissue and with protein staining in the mononuclear cells of
the infiltrate (37). Several studies reported the ability of cyto-
kines to modulate MMP production. In this connection, tumor
necrosis factor alpha (TNF-?) was shown to upregulate
MMP-9 and TNF neutralization for the purpose of abolishing
MMP secretion (33). Gamma interferon (IFN-?) could con-
tribute to the activation of macrophages coexpressing TNF-?,
MMP-2, and MMP-9 (6). Nevertheless, IFN-? has been de-
scribed to be mainly inhibitory (14, 17). In addition, MMP-9
secretion seems to be a common feature of mycobacterial
infection since the induction of these enzymes in response to
Mycobacterium avium, Mycobacterium tuberculosis, and Myco-
bacterium bovis BCG was reported (12, 28). Moreover, the
addition of IFN-? to BCG-infected murine macrophages in-
hibited MMP-9 secretion in vitro (28).
* Corresponding author. Mailing address: Leprosy Laboratory, Os-
waldo Cruz Institute, Oswaldo Cruz Foundation, Av. Brasil 4365,
Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil. Phone and fax: 00
55 21 2270-9997. E-mail: firstname.lastname@example.org.
† Present address: Laboratory of Clinical Infectious Diseases, NI-
AID, NIH, Bethesda, MD 20892.
?Published ahead of print on 14 December 2009.
Leprosy, an infectious disease caused by Mycobacterium lep-
rae, is characterized by clinical patterns related to the immu-
nological status of the patient. At the tuberculoid pole (tuber-
culoid leprosy [TT] and borderline tuberculoid [BT] forms),
skin lesions with epithelioid granulomas are associated with a
TH1 profile and bacterium-killing capacity (paucibacillary [PB]
patients), whereas in lepromatous lesions (lepromatous lep-
rosy [LL] and borderline lepromatous leprosy [BL]), diffuse
macrophage infiltrates loaded with bacteria (multibacillary
[MB] patients) are associated with the absence of a specific
cellular immune response to M. leprae. During the course of
the disease, new skin lesions may occur abruptly, characterized
by dense macrophage and lymphocyte infiltration organized or
not into granulomas typical of a reversal reaction (RR). In the
lepromatous forms, however, the reactional state, referred to
as erythema nodosum leprosum (ENL), has an abundance of
neutrophils and is associated with acute systemic symptoms.
The reactivation of the immune response and the upregulation
of proinflammatory cytokines have been broadly documented
for both forms of reaction (19–21, 34), and both are implicated
in leprosy morbidity. Immunosuppressors such as corticoste-
roids and thalidomide have been the only treatment options
It was recently reported that MMPs may play a key role in
promoting inflammatory skin damage. These enzymes can be
produced by skin cells such as keratinocytes, Langerhans cells,
and dermal fibroblasts (29, 36). However, no data on the role
of MMPs in leprosy skin lesions currently exist. In order to
investigate the participation of MMPs in the pathogenesis of
leprosy, we analyzed skin and serum samples obtained from
patients at different clinical moments. In addition, the role
played by M. leprae in the induction of MMP production by
blood cells in vitro was investigated. Our data support the
hypothesis that MMPs may be implicated in the local and
systemic responses to M. leprae infection, which may open new
opportunities for therapeutic interventions in leprosy as well as
MATERIALS AND METHODS
Patients. Leprosy patients treated at the Leprosy Out-Patient Unit, Oswaldo
Cruz Foundation, Rio de Janeiro, RJ, Brazil, were diagnosed according to the
Ridley-Jopling classification (30). A total of 32 patients (24 males and 8 females;
mean age ? standard deviation [SD] ? 34.3 ? 14 years) were included in the
study. Patients were classified as having LL (n ? 6), BL leprosy (n ? 16),
borderline lepromatous-borderline tuberculoid (BB) leprosy (n ? 3), or BT
(T-lep) leprosy (n ? 7). All patients were treated with multidrug therapy (MDT)
(rifampin, dapsone, and clofazimine) as recommend by the World Health Or-
ganization. Eighteen out of the 32 patients (4 LL, 11 BL, and 3 BB [L-lep]) who
presented with acute inflammatory reactional episodes (10 ENL and 8 RR
patients) were also evaluated. The study was approved by the Institutional Ethics
Committee of the Oswaldo Cruz Foundation. After written consent, biopsy
specimens and blood were obtained from the patients and processed as described
below. Biopsy specimens of 7 patients were taken before and during reactions (4
ENL and 3 RR patients); 14 patients (2 LL, 5 BL, and 7 BT patients) were
evaluated solely at the moment of leprosy diagnosis and before the initiation
Skin biopsy specimens and immunostaining. Biopsy specimens of skin lesions
were bisected and processed for diagnostic procedures, fixed in formalin, and
stained with hematoxylin and eosin or Wade-Fite stain. Immunostaining for the
detection of MMP-2 and MMP-9 on frozen cryostat sections was performed by
use of the immunoperoxidase method. Sections were acetone fixed, and the
endogenous peroxidase was blocked with 0.3% H2O2. Nonspecific binding was
blocked with goat normal serum for 1 h at room temperature. The sections were
incubated overnight with the primary antibodies (Ab) anti-MMP-9 and anti-
MMP-2 (1:50 dilution; R&D Systems). After washing, the sections were incu-
bated with biotinylated secondary Ab (Dako, Carpinteria, CA) for 1 h at room
temperature. The sections were rinsed, and the avidin-biotin complex (ABC;
Vector Laboratories) was applied for an additional hour, followed by incubation
in ABC reagent and the subsequent addition of substrate (3-amino-9-ethylcar-
bazole [AEC]; Vector Laboratories). Slides were counterstained with Mayer’s
hematoxylin and mounted. After omitting the secondary Ab, control sections
were incubated with nonimmune serum and used as negative controls. The slides
were examined under a Leica DMRB microscope (Leica Microsystems). To
detect the possible colocalization of MMP-9 and CD68 (macrophage marker),
double immunofluorescence was performed. Sections were incubated with
monoclonal antibodies (MAbs) against human MMP-9 (1:100, IgG1; R&D Sys-
tems) and CD68 (1:100, IgG2a; Dako), followed by incubation with isotype-
specific fluorochrome Alexa 488- or Alexa 568-labeled goat anti-mouse Ab (In-
vitrogen Life Technologies). No staining was detected after incubation with
isotype-matched, irrelevant Ab. Images were recorded simultaneously via sepa-
rate optical detectors and superimposed for colocalization analysis. The slides
were then examined with a confocal microscope (TCS-SP MP inverted single-
confocal laser scanning and two-photon laser microscope; Leica, Heidelberg, Ger-
many). The percentage of colocalization between MMP and CD68 staining was
calculated by use of the Andor IQ RGB analysis tool (Andor Technology, Ireland).
PBMC culture. Peripheral blood mononuclear cells (PBMC) were isolated
from the heparinized venous blood of 7 leprosy patients by Ficoll-Hypaque
(Pharmacia Fine Chemicals, Piscataway, NJ) density centrifugation. Cells were
washed in phosphate-buffered saline (PBS) and suspended in RPMI 1640 me-
dium supplemented with 100 U/ml penicillin, 100 ?g/ml streptomycin, 2 mM
L-glutamine, and 10% fetal calf serum (FCS; Gibco BRL, Gaithersburg, MD).
Viability was estimated by trypan blue dye exclusion. A total of 2 ? 106PBMC
were cultured in complete RPMI medium in 24-well plates at 37°C in a humid-
ified CO2incubator and stimulated with M. leprae (10 ?g/ml) or medium alone
(control). After different time periods, culture supernatants were collected and
frozen for quantification by enzyme-linked immunosorbent assay (ELISA), and
the cells were processed for RNA isolation. For the zymography experiments,
PBMC (106cells) were cultured in complete medium with 1% FCS, stimulated
or not with M. leprae. Afterwards, the supernatants were harvested after 24 h and
kept frozen for posterior analysis. For the collection of serum samples, blood was
allowed to clot, and serum aliquots were stored at ?70°C until further use. Blood
for cell isolation and serum samples from 4 healthy donors (blood bank) were
also obtained and assayed in parallel.
RNA extraction and cDNA synthesis. Total RNA from biopsy specimens and
cultured cells was extracted by using Trizol (Invitrogen) according to the man-
ufacturer’s instructions. In the case of biopsy specimens, RNA from the dermis
was processed separately (42), and tissue was homogenized with a Politron
PT-3000 apparatus (Brinkman, Westbury, NY) in 1 ml Trizol. From all samples,
1 ?g of total RNA was used for reverse transcription (33) by using the oligo(dT)
primer (Invitrogen). The resulting cDNA was stored frozen (?20°C) until use.
PCR conditions. Cytokine-specific oligonucleotide primer pair sequences
for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5?-CCACCC AT
GGCAAATTCCATGGCA-3? and 5?-TCTAGACGGCAGGTCAGGTCCA
CC-3?), MMP-2 (5?-GTGCTGAAGGACACACTAAAGAAGA-3? and 5?-TTG
CCATCCTTCTCAAAGTTGTAGG-3?), MMP-9 (5?-CACTGTCCACCCCTC
AGAGC-3? and 5?-GCCACTTGTCGGCGATAAGG-3?), and TIMP-1 (5?-AT
CCTGTTGTTGCTGTGGCTGATAG-3? and 5?-TGCTGGGTGGTAACTCTT
TATTTCA-3?) were obtained from Invitrogen. Samples were amplified by use of
a DNA thermocycler (Perkin-Elmer Cetus, Emeryville, CA), as previously de-
scribed (33), during 25 cycles (94°C for 45 s, 60°C for 45 s, and 72°C for 90 s) for
GAPDH and 30 cycles for MMPs and TIMP-1. PCR products were visualized on
1.7% agarose gels, and the specificity of the amplified bands was validated by
their predicted sizes (GAPDH, 600 bp; MMP-2, 618 bp; MMP-9, 382 bp;
TIMP-1, 630 bp). Densitometry analysis was performed by scanning the images
from the agarose gels (video documenting system; Amersham Pharmacia), and
values were obtained via ImageMaster software (Pharmacia). Each experiment
included a negative control to which no cDNA was added. Serial samples from
a given patient were all assayed in parallel.
Real-time PCR. For quantitative PCR analysis, amplification was carried out
by using 100 ng cDNA added to tubes in triplicate containing TaqMan gene
expression assay mixtures for the detection of interleukin-10 (IL-10), IFN-?,
TNF-?, MMP-9, and TIMP-1 (Applied Biosystems, Foster City, CA) via the ABI
Prism 7000 sequence detection system (Applied Biosystems), as previously de-
scribed (41). The relative expressions of the target messages were continuously
compared during the log phase of PCR in terms of the ?CT(mean threshold
cycle [CT] of 3 replicate experimental sample cDNAs minus the mean CTof 3
replicate GAPDH cDNAs).
VOL. 78, 2010MMP-2 AND MMP-9 EXPRESSION IN LEPROSY REACTION1013
Gelatin zymography. When skin biopsy specimens were used for the zymog-
raphy procedures, the dermis was homogenized in the Politron apparatus in 1 ml
of culture medium (RPMI medium), and the homogenates were stored at ?80°C
until use. Samples to be processed for zymography (culture supernatants and
homogenized biopsy specimens) were recovered, loaded onto a 15% SDS-PAGE
gel containing 1 mg/ml gelatin (Sigma Chemical Co., St. Louis, MO), and pro-
cessed, as described above (28). Briefly, the gels were sequentially treated in
2.5% Triton X-100 for 30 min, followed by an overnight incubation at 37°C in a
gelatinase substrate buffer (50 mM Tris-HCl, 10 mM CaCl2, 0.02% NaN3[pH
8.0]), and stained with 0.5% Coomassie blue. The quantification of gelatinase
levels was achieved by scanning densitometry (Amersham Pharmacia).
In situ zymography. To determine whether proteolytic activity was present in
skin tissue, in situ zymography was performed on unfixed frozen sections using a
gelatin substrate. This technique employed fluorescein isothiocyanate (FITC)-
labeled gelatin (Molecular Probes, Eugene, OR), the proteinase substrate that
loses its fluorescent signal when cleaved. Unfixed frozen sections of tissue were
incubated with reaction buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and
0.2 mM NaN3[pH 7.5]) containing 30 mg/ml gelatin for 48 h. These are optimal
conditions for detecting MMP activity, but activity due to other neutral protein-
ases could also be detected. At the end of the incubation period, fluorescence
was observed by using a light microscope equipped for epifluorescence (Nikon
E400), and images were captured via a digital camera. The same sequential
sections were used as negative controls to which EDTA was added in a reaction
buffer to inhibit gelatinase activity.
MMP-9 and TIMP-1 ELISA. MMP-9 and TIMP-1 levels in serum samples and
supernatants of M. leprae-stimulated PMBC were measured by means of an
enzyme immunoassay with paired antibodies. The MMP-9 and TIMP-1 assays
used reagents from R&D Systems. Plates were coated with mouse anti-MMP-9
and TIMP-1, and the detection antibody from the Quantikine kit (DMP900) was
used. This assay detects total MMP-9, both the proform and the active form.
Statistical analysis. Results are reported as pooled data from an entire series
of experiments and for each group of individuals and are shown as means ?
standard errors of the means (SEM). For data comparisons, the Mann-Whitney,
Kruskal-Wallis, and two-way analysis of variance (ANOVA) tests were used. The
correlation between the level of expression of MMPs and the level of expression
of cytokines was evaluated for each group of patients (MB and PB) via the
Spearman test. The statistical significance level adopted was a P value of ?0.05.
MMP-2 and MMP-9 expression and activity are enhanced
in the dermis of tuberculoid versus lepromatous lesions. An
evaluation of the relative amounts of cytokines and MMP
transcripts was performed by using skin samples from tuber-
culoid leprosy (T-lep group; n ? 6) and lepromatous leprosy
(L-lep group; n ? 6) patients (Fig. 1A). The level of expression
of TNF-? and IFN-? mRNAs in the T-lep dermis was signifi-
cantly higher than that in the L-lep dermis (P ? 0.05). Simi-
larly, levels of expression of the MMP-2 and MMP-9 messages
were also higher (P ? 0.05 and P ? 0.01, respectively) in T-lep
lesions. On the other hand, TIMP-1 and IL-10 message expres-
sion levels did not change significantly under either of the 2
clinical conditions. The association between the expression of
MMPs and the IFN-? and TNF-? cytokines was also evaluated
for each group of patients. In the T-lep (paucibacillary [PB])
group, the correlations between MMP-9 and TNF-? and be-
tween MMP-9 and IFN-? were found to be significant (P ?
0.01). With respect to MMP-2, a correlation was observed only
between this gelatinase and TNF (P ? 0.05). Within the L-lep
group (MB), while all these correlations were possible, none
were statistically significant (P ? 0.05).
MMPs are proteases that must be cleaved to be activated.
The detection of enzyme secretion cannot determine the acti-
vation state of MMPs, and the difference in molecular mass
between pro-MMP-9 and active MMP-9 is only 7 kDa. To
determine gelatinolytic activity in leprosy lesions, zymography
was performed with the tissue homogenates (Fig. 1B). Even
though the characteristic MMP-2 (72-kDa) and MMP-9 (92-
kDa) bands were detected, the lower-molecular-mass bands
with gelatinolytic activity could not be seen. Following densi-
tometry analysis of the gel bands and in accordance with RNA
data, the level of detection of MMP-2 and MMP-9 was found
to be higher in the T-lep dermis than in the L-lep dermis. In
addition, in situ zymography was used to assess skin sections to
demonstrate the localization of MMP activity. As shown in Fig.
1C, gelatinase activity (represented by a focal reduction in
fluorescence) detected in situ in the T-lep lesions was found to
be concentrated in the granuloma structures (Fig. 1C, right),
whereas in the control sections, no change in the fluorescence
pattern was observed (Fig. 1C, left). Furthermore, no gelatin-
ase activity was detected in the L-lep lesions (not shown).
MMP-2 and MMP-9 mRNA activities increase in the dermis
during leprosy reactions. The levels of expression of MMP-2
and MMP-9 and their tissue inhibitor TIMP-1 in the dermis of
18 reactional patients (8 RR and 10 ENL patients were assayed
by reverse transcription-PCR (RT-PCR). MMP-2 and MMP-9
mRNA levels were higher than TIMP-1 levels in both forms of
reaction (Fig. 2A). For ENL patients, differences were signif-
icant between MMP-2 and MMP-9 versus TIMP-1 (P ? 0.01),
whereas for the RR group, significance was noted only be-
tween MMP-9 and TIMP-1 (P ? 0.01) (Fig. 2A). Interestingly,
the extents of expression of MMPs and TIMP-1 detected be-
tween ENL and RR were not different, which is indicative of
their role in triggering tissue damage under both conditions.
It was also of interest to evaluate the expression of MMPs in
sequential biopsy specimens taken from the same patient be-
fore and at the onset of a reaction. A total of 7 patients (3 RR
and 4 ENL patients) were assayed, and real-time PCR was
performed simultaneously for all samples obtained from each
patient. As shown in Fig. 2B, the relative amounts of TNF-?,
MMP-2, and MMP-9 mRNAs were enhanced (P ? 0.05) dur-
ing RR (left) and ENL (right) as opposed to before reaction
(BR). However, TIMP-1 message levels decreased during RR
and varied insignificantly during ENL in comparison to what
their levels were prior to the reaction (P ? 0.05).
To evaluate MMP activation in reactional lesions, zymogra-
phy was performed on extracts from dermis samples of 2 re-
actional patients prior to and during reactions, revealing the
presence of bands corresponding to pro-MMP-2 (72 kDa) and
pro-MMP-9 (92 kDa), which is indicative of dermal gelatinase
production in the skin of leprosy patients (Fig. 2C, left). More
interestingly, the active MMP-2 (65-kDa) and MMP-9 (82-
kDa) forms, absent in BR, were observed in the dermal reac-
tional samples, which may be due to intense MMP activity
during these inflammatory episodes. Densitometry scanning of
the gel confirmed the prevalence of MMP-9 and MMP-2 ac-
tivity detected in the skin lesions prior to and during reactions.
By use of in situ zymography (Fig. 2D), gelatinolytic activity
was located inside the inflammatory infiltrate in RR lesions, as
demonstrated by the absence of fluorescent gelatin degrada-
tion (Fig. 2D, right), similar to what was observed for the T-lep
lesions (Fig. 1C).
MMP-2 and MMP-9 are differentially modulated in leprosy
skin lesions. The levels of expression of MMP-2 and MMP-9
were then evaluated by immunohistochemistry and confocal
microscopy (Fig. 3 and 4, respectively). In T-lep lesions (n ?
1014 TELES ET AL.INFECT. IMMUN.
4), positive staining was concentrated in the granuloma cell
components, although focal epidermal positivity was also ob-
served (Fig. 3A). The positivity of MMP-9 seen in L-lep skin
samples (n ? 2) was mildly dispersed in the dermis (Fig. 3B),
whereas immunostaining for TIMP-1 showed slight and diffuse
positivity, making a descriptive analysis difficult (not shown).
Confocal microscopy performed on 3 patient samples (T-lep
lesions) (Fig. 4A to C) showed a colocalization of MMP-9 with
CD68, confirming that macrophages were the main source of
MMP-9 production in these lesions (Fig. 4C). As for the L-lep
lesions (n ? 2), it cannot be ruled out that dermal fibroblasts
and endothelial cells (Fig. 4D to F) were positive since no
FIG. 1. Expression of MMPs and cytokines and MMP activity in leprosy skin. Biopsy specimens were taken from lepromatous (L-lep) (n ?
6) and tuberculoid (T-lep) (n ? 6) lesions. Real-time PCR was performed to evaluate the levels of expression of TNF-?, IFN-?, IL-10,
MMP-9, MMP-2, and TIMP-1 mRNAs. Results are the relative expression levels of the target message presented as means ? SEM for each
group of patients tested. The levels of expression of MMPs and the cytokines but not TIMP-1 were significantly enhanced in the T-lep group
compared to the L-lep group. (B) Gelatin zymography was performed on dermis samples from 3 L-lep and 3 T-lep patients. Bands shown
in the gel (representative of 2 patients in each group) correspond to the characteristic 72-kDa MMP-2 and 92-kDa MMP-9. Densitometry
analysis was performed, and values in the graph are pooled data for all patients tested. (C) MMP gelatinolytic activity in leprosy skin lesion
samples was assayed by in situ zymography. For T-lep patients, gelatinolytic activity was identified as the dark areas (arrows) in the
inflammatory infiltrate (b) compared to the negative control sections to which EDTA was added (a), and no protein activity was observed.
Scale bar, 50 ?m.*, P ? 0.05;**, P ? 0.01.
VOL. 78, 2010MMP-2 AND MMP-9 EXPRESSION IN LEPROSY REACTION 1015
FIG. 2. Detection of MMP mRNA and protein activity in leprosy reactions. MMP and TIMP-1 mRNA levels in reactional skin lesions were assayed
by RT-PCR. (A) MMP-2 and MMP-9 mRNA levels were higher than those of TIMP-1 in both forms of reaction (ENL, n ? 10; RR, n ? 8). No
differences were detected between ENL and RR, but significant differences were seen between MMP-2 and MMP-9 in comparison with TIMP-1 in ENL
and between MMP-9 and TIMP-1 in the RR group (**, P ? 0.01). (B) Evaluation of TNF-?, MMP, and TIMP-1 mRNAs in skin lesions obtained from
the same patient and assayed before reaction (BR) and during reaction (3 RR and 4 ENL patients). The mRNA level was significantly enhanced
during reaction in the same patient compared to that before reaction, except for TIMP-1, the level of which decreased during RR (*, P ? 0.05).
(C) Zymography evaluation of MMP-9 protein activity in reactional lesions (RR, 2 patients) showed bands that corresponded to pro-MMP-2 and
pro-MMP-9 in the dermis before and during reactions. Data in the graph represent data from densitometry analyses of the gel. (D) In situ
zymography identified gelatinolytic activity in RR skin lesions in the dark areas (arrows) of the tissue inflammatory infiltrate (b), which was absent
in the negative control sections (a). Scale bar, 50 ?m.
1016 TELES ET AL.INFECT. IMMUN.
predominant colocalization with CD68 was observed (Fig. 4F).
The MMP-9 and CD68 colocalization percentage rate ob-
served for T-lep skin was 19%, and that for L-lep skin was 3%.
Immunohistochemistry analysis of MMPs assayed in reac-
tional lesions demonstrated strong positivity, corresponding to
the inflammatory infiltrate in both RR and ENL (Fig. 3C to F).
The presence of focal MMP positivity (MMP-2 [Fig. 3C]
andMMP-9 [Fig. 3E]) was detected in the epidermis of RR
lesions, in contrast to the absence of positivity in the ENL
epidermis (MMP-2 [Fig. 3D] and MMP-9 [Fig. 3F]). More-
over, while in RR (Fig. 3C and E), MMP staining in the skin
was found to be concentrated in the granuloma (similarly to
T-lep lesions), in ENL, the lesions showed intense positivity,
completely covering the inflammatory infiltrate (Fig. 3D and
F). In these lesions, not only macrophages but also other cell
types, including granulocytes, could be MMP producers.
Increased MMP-9 serum levels were detected in sera from
reactional patients. To verify the systemic release of MMP-9
and TIMP-1 into peripheral blood, serum samples from 12
unreactional patients (L-lep, n ? 7; T-lep, n ? 5), 11 reactional
patients (ENL, n ? 6; RR, n ? 5), and 4 healthy controls were
collected and measured by ELISA. Figure 5 shows that
MMP-9 levels were significantly higher in both L-lep (169 ?
29.3 ng/ml; P ? 0.001) and T-lep (205 ? 29 ng/ml; P ? 0.01)
reactional and unreactional patients. In the ENL group, an
elevated serum level of MMP-9 (563 ? 52.2 ng/ml), but not
TIMP-1, was observed (P ? 0.05). The highest MMP-9 serum
levels detected in ENL were higher than the values found for
the control group (389 ? 84 ng/ml; P ? 0.05). While not
significant, the same profile was noted for RR patients. In
contrast, among L-lep patients, TIMP-1 values (407.2 ? 82
FIG. 3. MMP-2 and MMP-9 expression levels in leprosy skin le-
sions. Immunostaining of MMPs was performed in cryostat sections,
visualized by the immunoperoxidase method, and counterstained with
hematoxylin. (A) In T-lep lesions, MMP-9 positivity was concentrated
inside the granuloma and was also detected in the focal areas in
keratinocytes. (B) In L-lep skin, MMP-9 was only mildly expressed,
demonstrating a disperse distribution in the inflammatory infiltrate. (C
and E) In RR, positive MMP-2 (C) and MMP-9 (E) staining was
limited to the inflammatory infiltrate. The epidermal basal layer is
almost continuously positive. (D and F) In ENL, dense MMP-2 stain-
ing (D) and, on the other hand, diffuse MMP-9 (F) are superimposed
onto the inflammatory infiltrate occupying the whole dermis. MMP-2
was also detected in the epidermis, while MMP-9 was not. Images are
representative of each group of patients tested (BT ? 4; LL ? 2; RR ?
3; ENL ? 2). Scale bars, 50 ?m.
FIG. 4. Coexpression of MMP-9 and CD68 detected in leprosy skin
samples. (A to C) Confocal microscopy was performed with patient
samples (T-lep ? 3; L-lep ? 2), and immunostaining for MMP-9
(A) and CD68 (B) in samples from T-lep patients showed MMP-9
colocalizing with inflammatory macrophages (CD68?cells) in the
granuloma (C); the colocalization of MMP-9 (green; Alexa 488) and
CD68 (red; Alexa 568) is indicated by orange staining. (D to F)
MMP-9 (D) and CD68 (E) staining in the L-lep lesions did not show
any colocalization (F). DAPI blue staining indicates the nuclei, and the
dotted line indicates the dermal-epidermal limit. Images were visual-
ized and captured by use of a confocal microscope (Leica) and are
representative of each group of patients tested. Scale bar, 40 ?m.
FIG. 5. MMP-9 and TIMP-1 detected in patient sera. Sera were
obtained from 12 unreactional (7 T-lep and 5 L-lep) and 11 reactional
(5 RR and 6 ENL) patients in addition to 4 healthy donors (controls),
all evaluated by ELISA. Results represent means ? SEM. The
Kruskal-Wallis test was used to compare the different groups (**, P ?
0.01;***, P ? 0.001;*, P ? 0.05 [compared to the control group]).
Two-way ANOVA was used to compare MMP-9 and TIMP-1 levels
within the same group (#, P ? 0.05).
VOL. 78, 2010MMP-2 AND MMP-9 EXPRESSION IN LEPROSY REACTION1017
ng/ml) were higher than those of MMP-9 (169 ? 29.3 ng/ml;
P ? 0.05).
M. leprae induces MMP-9 expression and activity in PBMC
of leprosy patients. Analysis of the clinical specimens de-
scribed so far indicates that MMP-2 and MMP-9 are being
produced in situ during the course of the disease. To determine
whether M. leprae is able to induce MMP-9 production, as was
previously reported for other mycobacteria (12, 28), PBMC
from leprosy patients (n ? 7) were stimulated in vitro with M.
leprae after different culture periods, and mRNA and protein
expression levels were evaluated as described above. Following
M. leprae stimulation (10 ?g/ml), the induction of the TNF-?
message was already highly upregulated after 1 and 3 h of
culture (P ? 0.01) (Fig. 6A). TNF-? secretion in the culture
FIG. 6. M. leprae induces MMP-9 expression in PBMC in vitro. PBMC obtained from L-lep patients, both unstimulated (control [C]) and
stimulated with M. leprae (ML) (10 ?g/ml), were assayed after different culture periods. (A) TNF-? mRNA was evaluated by real-time PCR (left),
and protein levels were assessed by ELISA (right). The relative quantification (RQ) of TNF-? message induced by M. leprae already showed
enhanced expression after 1 h of culture. (B) Evaluation of MMP-9 mRNA (left) and protein (right) levels indicated a peak of the response at 6
and 20 h, respectively. (C) MMP-9 activity measured by zymography showed representative bands in the supernatants of the PBMC cultures
corresponding to pro-MMP-9 (92 kDa) and active MMP-9 (88 kDa). Densitometry scanning of the gel confirmed the predominance of MMP-9
induced by M. leprae versus the control cells. Results are means (?SEM) for data from 7 independent experiments (*, P ? 0.05;**, P ? 0.01).
1018TELES ET AL.INFECT. IMMUN.
supernatants reached a peak after 6 h (P ? 0.01). The level of
expression of MMP-9 mRNA was seen 3 h after the addition of
the mycobacteria, and it was even higher after 6 h (P ? 0.01)
and 12 h (P ? 0.05) (Fig. 6B). The ability of M. leprae to induce
MMP was also observed for the PBMC obtained from healthy
donors (not shown). The induction of MMP-9 was enhanced
after 6 h, and levels were higher still after 20 h (P ? 0.05) (Fig.
6B). For the same cultures, no modification of TIMP-1 or
MMP-2 occurred during the experiments (not shown).
To determine whether both pro-MMP-9 and active MMP-9
can be detected in M. leprae-stimulated cultures, zymography
was performed on the supernatants of PBMC stimulated or not
with the mycobacteria (24-h cultures). The visualization of the
bands in the gel in Fig. 6C corresponds to both pro-MMP-9 (92
kDa) and active MMP-9 (88 kDa) following M. leprae activa-
tion. Pooled densitometry analysis from the different experi-
ments demonstrated a significant induction of MMP-9 (P ?
0.02) in comparison to the control supernatants (Fig. 6C).
Overall, the data confirm the ability of M. leprae to induce the
expression of the active forms of MMP-9.
TNF synergizes with M. leprae in the induction of MMP-9 in
PBMC cultures. Since TNF-? was previously shown to pro-
mote MMP secretion (32, 35), we then investigated whether M.
leprae and TNF-? could drive MMP production in vitro. Cells
from 5 L-lep patients were stimulated with mycobacteria (10
?g/ml), TNF-? (10 ng/ml), or M. leprae and TNF-? for 6 and
24 h, when MMP-9 and TIMP-1 production was evaluated, as
described above. As shown in Fig. 7A (top), the MMP-9
mRNA level increased in the presence of TNF-? (P ? 0.05)
and M. leprae (P ? 0.05) in addition to M. leprae and TNF-?
(P ? 0.001) in comparison with the control cells. Moreover,
increased levels of MMP-9 mRNA were detected when M.
leprae- and TNF-?-stimulated cells were compared with M.
leprae- or TNF-?-stimulated cultures (P ? 0.05). With regard
to protein release, the MMP-9 levels induced by M. leprae and
TNF in culture supernatants (64.5 ? 7.3 ng/ml) were higher
than those induced by M. leprae alone (45 ? 8 ng/ml) but not
significantly different from the levels induced by TNF alone
(56 ? 10.6 ng/ml) (Fig. 7A, bottom). For TIMP-1 (Fig. 7B),
although its levels were slightly elevated in comparison to
those of M. leprae alone, the variation in mRNA expression
induced by M. leprae plus TNF was insignificant (P ? 0.05)
(bottom). Moreover, protein levels were not altered by either
M. leprae or TNF (Fig. 7B, bottom).
Reactional episodes are very intriguing events occurring during
the course of leprosy, an infectious disease. Although both innate
immunity and adaptive immunity against the pathogen are in-
volved in the specific mechanisms leading to skin and nerve dam-
age in both RR and ENL, the precise components are not com-
pletely understood. In the present study, higher MMP-2 mRNA,
MMP-9 mRNA, and protein levels were detected together
with enhanced TNF-? and IFN-? expression levels in the le-
sions of tuberculoid patients and in those undergoing a reac-
tion. MMP proteolytic activity depends not only on the number
of transcripts but also on the degree of molecular cleavage in
the membrane itself, together with the action of specific inhib-
itor molecules present in the tissue environment. In summary,
our data evidenced a positive MMP/TIMP ratio induced by M.
leprae infection during the inflammatory process both in vivo
FIG. 7. M. leprae and TNF-? induce MMP-9 production in vitro. Shown are data for PBMC from L-lep patients, both unstimulated (control
[C]) and stimulated with M. leprae (ML) (10 ?g/ml), TNF-? (10 ng/ml), and M. leprae plus TNF-? for 6 h (mRNA) and 24 h (protein release).
MMP-9 (A) and TIMP-1 (B) mRNA (top) and protein (bottom) levels were assayed by real-time PCR and ELISA, respectively. The values
represent means ? SEM (n ? 5). Statistical differences were evaluated by the Mann-Whitney test (*, P ? 0.05;**, P ? 0.01;***, P ? 0.001).
VOL. 78, 2010MMP-2 AND MMP-9 EXPRESSION IN LEPROSY REACTION 1019
and in vitro. By use of in situ zymography, local MMP activity
in both tuberculoid and reactional skin lesions associated with
the granuloma infiltrates was confirmed.
During a reaction, patients suddenly present with an inflam-
matory infiltrate that invades the skin abruptly as a result of
TH1 cytokine release. Due to immune cell migration, IFN-?
and TNF-? are upregulated, and an enhanced expression of
MMPs was evidenced in situ. In this scenario, the heteroge-
neous clinical and histopathological presentations ranging
from localized to extensive and/or necrotic lesions suggested
that both the tissue environment and remodeling play a crucial
role in the clinical presentation of skin damage.
The cytokines associated with CD4?T-cell subpopulations
may influence MMP production in a different manner. TH2
cytokines like IL-4 and IL-10 were previously shown to pre-
dominate in lepromatous leprosy lesions (19, 22) and to down-
regulate MMP-9 (4). However, the effects of TH1 cytokines
are not as clear. IL-15 and IL-18 are able to upregulate MMP-9
in macrophages (1), while, conversely, the effects of IFN-? are
predominantly inhibitory (14, 17). In experimental models of
mycobacterial infection, the addition of IFN-? inhibits MMP-9
production by peritoneal macrophages (28). In contrast, IFN-?
was shown previously to synergize with IL-1? to upregulate
MMP-9 activity in M. tuberculosis-infected monocytes (11).
In tuberculosis (TB), the upregulation of MMP-9 was
correlated with the widespread tissue destruction that is a
hallmark of this disease (37). In leprosy, however, tissue
destruction occurs only during a reaction. It is noteworthy
that in tuberculoid leprosy lesions, they do not promote
tissue proteolysis in the skin even when granulomas are
abundant, in contrast to what occurs with TB, suggesting a
tight balance between proteolytic enzymes and their regu-
latory systems. Moreover, in TB granuloma, MMP-9 positivity
is localized around the necrotic area (37). In leprosy (both in
tuberculoid and RR lesions), MMPs are localized in the cen-
tral area of the granuloma, in which macrophages and epithe-
lioid cells are predominant. Necrosis is rarely seen in T-lep
lesions even though caseous abscesses are a frequent finding in
nerve lesions, suggesting that in the skin, MMP regulatory
mechanisms are more efficient at balancing their proteolytic
To our knowledge, this is the first time that the ability of M.
leprae to induce MMP expression in blood mononuclear cells
has been demonstrated. The induction of MMP was found to
be preceded by TNF-? transcription and release, suggesting
that, in vitro, the production of MMP-9 could be mediated by
TNF-?. The addition of TNF to the cultures increased the
levels of MMP production, reinforcing this hypothesis. TNF-?
induces MMP-9 expression in many systems (24, 38), while
MMP-9 has the ability to activate TNF-? release from its mem-
brane precursor (43). Endogenous TNF-? stimulates MMP-9
gene transcription in monocytes through NF-?B activation (3),
and p38 mitogen-activated protein kinase (MAPK) inhibitors
were able to downregulate MMP-9 mRNA and protein expres-
sion (24). Therefore, inhibitors of MAPK pathways could benefit
from the damage caused by excessive inflammation.
A serum MMP-9 and MMP-9/TIMP-1 imbalance may be a
risk factor for triggering inflammatory processes. The detec-
tion of systemic MMPs has been used broadly for correlations
with disease severity for many inflammatory conditions such as
rheumatoid arthritis, type 2 diabetes, multiple sclerosis, lupus
erythematosus, as well as cancer (7, 26, 31) and may have great
value in disease diagnosis and prognosis. However, compar-
ative research should be carried out with caution since the
results often seem to be overly influenced by the collection
and processing techniques adopted (15). TIMPs inhibit the
active MMP forms by specifically binding with the catalytic
site, and they were also reported to sometimes inhibit the
latent forms as well. Therefore, any disturbance in this bal-
ance may lead to tissue damage. TIMP-1 is more capable of
binding with the active forms, especially that of MMP-9, but
can also bind to active MMP-2 (13, 23, 39, 40).
In this study, the MMP-9 levels detected in patient sera
correlated with the increased level of expression of this protein
in reactional lesions. The low TIMP-1 levels seem to become
imbalanced with MMP expression when disseminated leuko-
cyte migration is in course during a reaction. Similarly, in
lepromatous patients, lower MMP-9 levels in sera correlated
with the low cell turnover reported for these lesions and could
be related to sustained TIMP-1 levels. Unexpectedly, the sys-
temic MMP-9 levels in tuberculoid patients were similar to
those found for the lepromatous group. Nevertheless, TIMP-1
levels were significantly more elevated in the latter group.
The systemic response observed herein seems to correlate
with the imbalance in proteolytic activity at the lesion site in
the T-lep and reactional groups. Moreover, the highest serum
levels of MMP-9 detected in ENL correlated with the clinical
and histopathological findings whenever disseminated in-
flammatory lesions, characterized by intense macrophage
and granulocytic infiltrates, appeared all over the body. The
intense positivity seen via immunohistochemistry confirmed
these data. Conversely, some studies have shown that aug-
mented synthesis and MMP-9 secretion are induced via cyto-
kines, mainly TNF-?, secreted by activated macrophages
present in the inflammatory infiltrates (35).
In this study, a large number of clinical specimens taken
from stable, untreated leprosy lesions during a reaction con-
firmed participation of MMPs in the disease. Higher mRNA
levels in skin biopsy specimens correlated with the expression
of the proinflammatory cytokines TNF-? and IFN-?. On the
other hand, the detection of protein in the inflammatory infil-
trate correlated with tissue damage and increased levels of
MMPs in the sera during the reaction, which was especially
true for ENL. Furthermore, the induction of MMP-9 by M.
leprae alone seemed to be reinforced by the addition of TNF-?.
Overall, these results pave the way for new therapeutic inter-
ventions during leprosy reactions.
We are indebted to A. L. Oliveira and L. M. B. Pinheiro for their
technical support and J. Grevan for revising the text in English.
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Editor: A. J. Ba ¨umler
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