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Enhanced degradation of proteins of the basal lamina and stroma by matrix metalloproteinases from the salivary glands of Sjgren's syndrome patientsCorrelation with reduced structural integrity of acini and ducts.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 9, September 2003, pp 2573–2584
DOI 10.1002/art.11178
© 2003, American College of Rheumatology
Enhanced Degradation of Proteins of the Basal Lamina and
Stroma by Matrix Metalloproteinases From the
Salivary Glands of Sjögren’s Syndrome Patients
Correlation With Reduced Structural Integrity of Acini and Ducts
Eduardo Goicovich, Claudio Molina, Paola Pérez, Sergio Aguilera, Juan Fernández, Nancy Olea,
Cecilia Alliende, Cecilia Leyton, Rafael Romo, Lisette Leyton, and Marı́a-Julieta González
the acinar and ductal basal lamina revealed abnormalities ranging from disorganization to disappearance of
this ECM structure. These changes were paralleled by
an important loss of microvilli on the apical surface, as
well as decreased unstimulated salivary flow. Interestingly, the results were similar in LSGs from all SS
patients, regardless of the proximity of infiltrating
mononuclear cell foci.
Conclusion. Our observation that the proteolytic
action of MMPs toward ECM macromolecules is increased in SS patients provides a rationale for understanding the dramatic changes in the structural organization observed in the basal lamina and apical surface
of acini in these patients. The results provide new
evidence that acinar and ductal cells from the LSGs of
SS patients display a molecular potential, with increased capacity to markedly disorganize their ECM
environment and, thus, damage their architecture and
functionality.
Objective. To determine the effect of matrix metalloproteinase (MMP) activity from the labial salivary
glands (LSGs) of Sjögren’s syndrome (SS) patients on
proteins of the extracellular matrix (ECM) that form
the basal lamina and stroma, and to compare this effect
with the structural integrity of acini and ducts as well as
the functionality of the LSGs.
Methods. Gelatinase activity was determined by
zymography. The digestion pattern of extracellular matrix (ECM) macromolecules was detected by gel electrophoresis and quantified by densitometry. The structural
integrity of acini and ducts was evaluated by light and
electron microscopy. Secretory function was evaluated
by measuring unstimulated salivary flow and by scintigraphy.
Results. LSG extracts showed increased levels of
proteolytic activity toward purified proteins of the basal
lamina (laminin and type IV collagen) and stroma
(types I and III collagen and fibronectin). Enhanced
degradation was most evident for fibronectin, laminin,
and type IV collagen. Analysis of the ultrastructure of
Important changes in acinar and ductal morphology and function, together with pronounced extracellular matrix (ECM) remodeling, are detectable in the
labial salivary glands (LSGs) of Sjögren’s syndrome (SS)
patients (1–3). The classic symptoms of this disease,
xerostomia and keratoconjunctivitis sicca, have been
related to the presence of autoantibodies and cytokines
and to progressive denervation of the LSGs (4–7).
Physiologic remodeling of ECM components depends on a balanced expression of proteolytic enzymes
and their inhibitors, such as matrix metalloproteinases
(MMPs) and tissue inhibitors of metalloproteinases
(TIMPs), respectively (8,9). Some pathologic processes
are associated with excessive or insufficient ECM degra-
Dr. González’ work was supported by a grant from the
Fondecyt-Chile (grant 1020755). Dr. Pérez’ work was supported by
postdoctoral fellowships from Conicyt, Mecesup-Postgraduate University of Chile (grant 99-03) and from the Laboratorio Tecno-FarmaChile.
Eduardo Goicovich, Biochemist, Claudio Molina, DDS, MSc,
Paola Pérez, Biochemist, Sergio Aguilera, MD, Juan Fernández, PhD,
Nancy Olea, BSc, Cecilia Alliende, BSc, Cecilia Leyton, BSc, Rafael
Romo, DDS, Lisette Leyton, PhD, Marı́a-Julieta González, MSc:
University of Chile, Santiago, Chile.
Address correspondence and reprint requests to Marı́aJulieta González, MSc, Institute of Biomedical Sciences, Faculty of
Medicine, University of Chile, Casilla 70061, Santiago 7, Chile. E-mail:
jgonzale@machi.med.uchile.cl.
Submitted for publication January 21, 2003; accepted in
revised form May 1, 2003.
2573
2574
dation (10–12), thereby highlighting the importance of
the ECM for the maintenance of epithelial architecture
and function (13,14). The basal lamina plays an important role in this maintenance function, since it lies at the
interface between epithelial cell plasma membranes and
the connective tissue. The ECM is also responsible for
transmitting signals to cells via cell surface receptors,
which include integrins and proteoglycans (3,5,15–20).
Loss of adhesive interactions between epithelial cells
and their microenvironment modifies gene expression
and may lead to cell death (1,21).
We recently demonstrated that LSGs from control subjects and SS patients showed gelatinolytic activity
for MMP-2 and MMP-9 (3). Activation studies revealed
that both enzymes were predominantly present in their
latent form. The highest MMP-9 activity was detected in
patients with severe, active primary SS. No differences in
MMP-2 activity were observed between patients and
controls. MMPs, as detected by immunolocalization,
were found only in acinar and ductal cells and were
homogeneously distributed throughout the LSGs. The
expression of MMP-2 and MMP-9 paralleled their gelatinolytic activity. MMP-3, which was detectable only by
immunologic methods, was absent in control subjects,
but was abundantly expressed in SS patients. Moreover,
levels of MMP protein in acinar and ductal cells were
unrelated to the proximity of infiltrating mononuclear
cells (3).
Other investigators have reported the production
of MMPs by mononuclear cells (22). However, our
results suggested that the increased gelatinolytic activity
of the LSGs from SS patients was due to the expression
of some MMPs in acinar and ductal cells (3), and was
not a consequence of the increased presence of mononuclear cells. Such increased MMP activity is directly
related to greater damage of parenchymal glands, but is
most likely not associated with the presence of infiltrating cells (3).
The expression of MMPs is induced by cytokines,
such as interleukin-1␣, interleukin-6, tumor necrosis
factor ␣, and interferon-␥ (6,23). Studies of LSGs from
patients with primary SS showed that these cytokines are
expressed by infiltrating mononuclear cells and by acinar
and ductal cells (24,25). Thus, MMPs could be induced
in acinar and ductal cells by autocrine and paracrine
pathways. Considering the mechanisms of induction and
activation described for these enzymes and the cell types
in which they are expressed (3,26,27), we hypothesized
that MMPs could be secreted locally and degrade the
basal lamina proteins of acini and ducts, as well as the
stromal microenvironment.
GOICOVICH ET AL
Gelatin and casein are substrates that are frequently used to test the enzymatic activity of tissue
MMPs in vitro. However, proteolysis of these substrates
is not sufficient evidence to predict that these proteolytic
enzymes would also degrade proteins of the ECM in vivo
(28,29). Thus, a specific objective of the present study
was to analyze the proteolytic activity of MMPs from
extracts of LSGs obtained from SS patients toward
purified proteins of the basal lamina (laminin and type
IV collagen) and stroma (type I collagen, type III
collagen, and fibronectin) and to compare this activity
with that exerted toward in vivo substrates. We also
evaluated the structural integrity of acini and ductal
basal lamina, as well as the secretory properties of the
LSGs.
Analysis by gel electrophoresis and scanning densitometry revealed that extracts of LSGs from SS patients clearly contained increased proteolytic activity
toward the proteins of interest. Degradation was most
pronounced for fibronectin, laminin, and type IV collagen. Consistent with the findings of our biochemical
analysis, the ultrastructure of acinar and ductal basal
lamina showed abnormalities ranging from disorganization to disappearance of this ECM structure. Such
changes were paralleled by a loss of microvilli on the
apical surface, which suggests that inappropriate cytoskeletal reorganization could disfavor exocytosis of
secretory granules. These results demonstrate that uncontrolled ECM remodeling of both the basal lamina
and stroma due to excessive MMP activity may disrupt
the architecture of parenchymal glands and thereby
promote a loss of function.
PATIENTS AND METHODS
SS patients and control subjects. For this study, 15
patients with primary SS were selected and diagnosed according to the American–European Consensus Group criteria (30).
Only patients with all the following signs and symptoms were
included: keratoconjunctivitis sicca, xerostomia, low unstimulated whole salivary flow (ⱕ1.5 ml/15 minutes), abnormal
scintigraphy curve (median or flat), presence of both anti-Ro
and anti-La autoantibodies, and positive findings on LSG
biopsy. Group A patients (n ⫽ 9) had an age range of 28–52
years (mean 42 years). Their focus scores ranged from 1 to 2,
with 80–90% of remnant parenchyma present, and without
fibrous or adipose tissue. LSGs from this group possessed
MMP-9 gelatinolytic activity ranging from 1.4 to 2.9 (mean
2.4), expressed as follows: (兺 pixels area2) ⫻ 10–3. Group B
patients (n ⫽ 6) had an age range of 51–70 years (mean 64
years). Their focus scores were ⬎2, with 20–60% of remnant
parenchyma present, and with fibrous and/or adipose tissue.
LSGs from this group displayed variable levels of MMP-9
gelatinolytic activity (0.6–2.4; mean 1.2).
PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS
The control group was composed of 5 subjects who had
consulted their physicians because of oral and ocular dryness,
but who did not fulfill the criteria for SS. Their age range
(40–50 years; mean 48 years) was similar to that of the SS
group. Findings of serology and scintigraphy were normal, and
unstimulated whole salivary flow was normal. Biopsied LSGs
were normal, with scarce and scattered distribution of mononuclear cells, well-preserved parenchyma, and without fibrous
or adipose tissue. MMP-9 gelatinolytic activity in these individuals was in the range of 0.5–0.7 (mean 0.6).
LSGs were obtained in the morning (after at least 2
hours of fasting), using the technique described by Daniels
(31). All patients gave their informed consent before undergoing LSG biopsy. The study protocol was approved by the
Ethics Committee of the Faculty of Medicine, University of
Chile.
Preparation of LSG extracts. LSGs were homogenized
in extraction buffer for ECM components (0.5M Tris HCl, pH
7.5, 1% Triton X-100, 10 mM CaCl2, 200 mM NaCl) at a ratio
of 1:5 (weight/volume) at 4°C with a glass/glass conical homogenizer. The homogenate was then subjected to three 5-minute
freeze–thaw cycles and centrifuged at 13,000g for 30 minutes at
4°C. The detergent-soluble supernatants were recovered and
stored at –70°C for further analysis. All steps of the procedure
were done in the presence of a protease inhibitor cocktail (4
mM phenylmethylsulfonyl fluoride, 100 ␮g/ml of leupeptin, 2
mM benzamidine in DMSO). Protein concentrations in the
extracts were determined as described elsewhere (32).
Evaluation of gelatinolytic activity of MMPs in LSG
extracts by zymography. Detergent extracts of glands from the
study subjects were analyzed by zymography to determine the
enzymatic activity of MMPs. Zymography was performed
using the protocol previously described by our group (3). In
order to activate proMMPs, the extracts were preincubated
with 1 mM APMA in the presence of protease inhibitors for 2
hours at 37°C. Unlike other proteases, all members of the
MMP family contain Zn2⫹ at the catalytic site (33); in addition,
they require Ca2⫹ for stability and activity (34). To demonstrate that the observed proteolytic activity was due to the
presence of MMPs, 4 mM EDTA was added to the incubation
medium of the zymographs for 30 minutes at 37°C.
Preparation of ECM macromolecules. Types I, III, and
IV collagen (Sigma, St. Louis, MO) were dissolved at 2 mg/ml
in 0.1M acetic acid and then dialyzed against 2 liters of MMP
test buffer (150 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM
CaCl2, 0.02% NaN3) for 48 hours at 4°C, with changes of
dialysis buffer every 12 hours. Each collagen preparation was
stored in aliquots of 1 mg/ml at –70°C. Fibronectin (500 ␮g;
Sigma) was reconstituted in MMP test buffer to a final
concentration of 1 mg/ml. A commercial solution of laminin
(Sigma) was diluted to 1 mg/ml. These solutions were also
stored as aliquots at –70°C.
Digestion of ECM macromolecules. Aliquots (5 ␮g) of
proteins from each LSG extract were activated by 1 mM
APMA for 2 hours at 37°C. Protein aliquots were incubated
separately with 20 ␮g of type I, III, or IV collagen or with 10
␮g of laminin or fibronectin, in 30-␮l total volume with an
MMP test buffer containing a protease inhibitor cocktail. The
samples were incubated at 37°C for 0–48 hours, and samples
were taken every 12 hours. At time points shorter than 12
hours, no obvious changes were detectable. Purified proteins
2575
incubated without LSG extract at time 0 and at 24 hours or
with LSG extract in the presence of EDTA for 24 hours served
as controls. The reaction was stopped by the addition of 4 mM
EDTA at specific time points. Samples were subsequently
analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (35), and
gels were stained with Coomassie blue R250.
Densitometric analysis. Gels were scanned using Corel
Photo-Paint (Corel, Ottawa, Ontario, Canada) on a gray scale
at a resolution of 1,200 interpolated dots per inch. The
intensity of the bands was analyzed using UN-SCAN-IT gel
software, version 4.1 for Windows (Silk Scientific, Orem, UT).
The intensity was expressed as follows: (兺 pixels area2) ⫻ 10–3.
Morphologic studies. LSGs were processed for morphologic evaluation by either light microscopy or transmission
electron microscopy (TEM). For light microscopy, samples
were fixed with alcoholic Bouin’s fixative and embedded in
Paraplast. Samples for TEM were fixed in Karnovsky’s fixative,
postfixed in OsO4, and embedded in Epon (36). The samples
were coded to ensure unbiased collection of data.
Statistical analysis. The results of degradation analyses were expressed as the mean ⫾ SEM intensity. Differences
in intensities were analyzed using the Mann-Whitney U test. P
values less than 0.05 were considered significant.
RESULTS
Digestion of basal lamina molecules. To confirm
the presence of MMPs, all LSG extracts were previously
checked by zymography, as shown in Figure 1. The
zymogram shows representative samples from a control
subject and an SS patient. The 92-kd and 84-kd bands
corresponded to proMMP-9 and active MMP-9, respec-
Figure 1. Zymographic analysis of the gelatinolytic activity of matrix
metalloproteinases (MMPs) from extracts of labial salivary glands
(LSGs) obtained from Sjögren’s syndrome (SS) patients and controls.
Shown are representative samples from an SS patient (lanes 4–6) and
a control subject (lanes 1–3). Each lane was loaded with 30 ␮g of
protein derived from the LSG extracts and then subjected to zymography. Lanes 1 and 4, Untreated LSG extracts; lanes 2 and 5, LSG
extracts treated with 1 mM APMA; lanes 3 and 6, LSG extracts treated
with 4 mM EDTA. Results are representative of 3 independent
experiments.
2576
GOICOVICH ET AL
Figure 2. Analysis of laminin degradation by extracts of labial salivary glands (LSGs) from
Sjögren’s syndrome (SS) patients and controls. A, Purified laminin (10 ␮g) was incubated
with LSG extracts (5 ␮g) that had been preactivated with 1 mM APMA. Digestion time 1 ⫽
12 hours; digestion time 2 ⫽ 24 hours. Lane 1, Purified laminin incubated for 24 hours
without gland extract, showing 2 bands (␤/␥ and ␣) with apparent Mr of 220,000 and 400,000,
respectively. All incubations were done in the presence of a protease inhibitor cocktail (see
Patients and Methods). Proteins were separated on a 7% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis gel and stained with Coomassie blue R250. Results are
representative of those obtained for 5 SS patients and 5 controls, each of which was analyzed
in duplicate to confirm the reproducibility of the results. Lane St, Prestained molecular
weight standards: myosin (212 kd) and myelin basic protein/␤-galactosidase (158 kd). B,
Densitometric analysis of the gel shown in A. LM ⫽ laminin; E ⫽ LSG extract; t1 ⫽ 12
hours; t2 ⫽ 24 hours.
tively. The 72-kd and 64-kd bands corresponded to
proMMP-2 and active MMP-2, respectively. When extracts were incubated with APMA, lower Mr MMPs were
detected; however, gelatinolytic activity was higher in
patients than in controls. When the extracts were incubated in presence of EDTA, these bands were no longer
observed.
Laminin and type IV collagen are the main
components of the basal lamina. The ability of MMPs
from the LSGs of SS patients to degrade these physiologic substrates was examined and compared with that of
control subjects. MMPs were preactivated with APMA,
and the reaction was stopped by EDTA. The degradation products obtained after different incubation times
were analyzed by SDS-PAGE and densitometry. The
LSG extract loaded in each lane (5 ␮g in each case) was
carefully titrated to avoid any overlap of electrophoretic
pattern between the bands in the glandular extracts and
those of the ECM substrates.
Using laminin, 2 different bands of apparent Mr
200,000 and Mr 400,000, corresponding to the laminin
␤/␥ and ␣ subunits, respectively, were detected (Figure
2A). Upon treatment with gland extracts, a decrease in
intensity of these bands was observed. This effect was
more evident in the patients than in the controls (Figures 2A and B). Purified laminin incubated in the
absence of extract for 24 hours is also shown in Figures
2A and B for comparison. Laminin degradation by
PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS
2577
Table 1. Comparison between laminin and fibronectin degradation at 24 hours and salivary function in SS patients and controls*
Laminin,
mean ⫾ SEM
Control (n ⫽ 5)
SS patients
Group A (n ⫽ 9)
Group B (n ⫽ 6)
Laminin ⫹ LSG extract,
mean ⫾ SEM
Fibronectin ⫹
LSG extract,
mean ⫾ SEM
USF,
mean (range)
␣
␤␥
␣
␤␥
Fibronectin,
mean ⫾ SEM
0.4 ⫾ 0.08
0.6 ⫾ 0.09
0.29 ⫾ 0.05
0.45 ⫾ 0.06
4.9 ⫾ 0.6
4.6 ⫾ 0.3
3.5 (2.6–4.6)
0.5 ⫾ 0.09
0.4 ⫾ 0.06
0.7 ⫾ 0.10
0.6 ⫾ 0.08
0.09 ⫾ 0.04†
0.17 ⫾ 0.07†
0.12 ⫾ 0.01†
0.28 ⫾ 0.14†
4.7 ⫾ 0.4
5.0 ⫾ 0.5
2.0 ⫾ 0.4†
2.9 ⫾ 0.7†
1.05 (0.8–1.3)
0.7 (0.5–1.1)
Scintigraphy
curve
Normal
Median
Median (n ⫽ 3);
flat (n ⫽ 3)
* Degradation was evaluated as the decreased intensity of electrophoretic bands of laminin and fibronectin. Intensity was measured as (兺 pixels
area2) ⫻ 10⫺3. Unstimulated salivary flow (USF) is expressed in milliliters per 15 minutes. Sjögren’s syndrome (SS) patient groups were classified
as described in Patients and Methods.
† P ⬍ 0.05.
MMPs was not significantly enhanced by increasing the
incubation time beyond 24 hours (Figure 2A, lanes 4 and
7, and Table 1).
In patients (Figure 3B), protein bands characteristic of type IV collagen, with apparent Mr of 110,500
and 91,000 (lane 8) showed increased or decreased
intensity, respectively (compare lanes 9 and 10 in Figure
3B with those in Figure 3A [controls]). Degradation
products of type IV collagen with apparent Mr of 79,500
and 52,300 were observed. The former band was of
comparable intensity in both patients and controls,
whereas the latter band was detectable only in the
patients (Figures 3A and B, lanes 9 and 10). Slight
differences in type IV collagen digestion were apparent,
depending on the incubation time (Figure 3C).
Digestion of stroma molecules. Types I collagen,
type III collagen, and fibronectin are important components of the gland stroma. We compared the MMP
activity in extracts of LSGs from patients and control
subjects with the use of these substrates.
For types I and III collagen, a group of proteolytic fragments with apparent Mr ranging from 100,000
to 70,000 were detectable both in the control subjects
(Figures 3A and B, lanes 3 and 4) and in the SS patients
(Figures 3A and B, lanes 6 and 7). However, the
intensity of these bands was higher in the SS patients. A
second group of fragments (Mr range 60,000–45,000)
was detected in the patients only when type I collagen
was used as a substrate. The intensity of these bands was
dependent on the incubation time.
A third fragment that was observed when type III
collagen was used as a substrate (Mr 54,000) was detected only in the SS patients (Figure 3B, lanes 6 and 7,
bottom arrowhead). Two bands with apparent Mr of
212,000 and 142,000 showed decreased intensity (Figure
3B, lane 7, upper arrowheads) compared with the intensity of purified type III collagen (Figure 3B, lane 5).
In samples from control subjects, fibronectin was
the only protein that did not undergo important changes
when exposed to gland extracts for different incubation
times (Figure 4A, lanes 3 and 4). In the SS patients,
however, the susceptibility of fibronectin to MMPmediated degradation was dependent on the incubation
time (Figure 4A, lanes 6 and 7). An increase in the
electrophoretic mobility of fibronectin was observed
upon incubation with gland extracts from either group of
subjects (Figure 4B).
Analysis by gel electrophoresis and scanning densitometry revealed that extracts of LSGs from SS patients clearly contained increased proteolytic activity
of MMPs toward the basal lamina and stromal substrates studied. Proteolytic activities were higher and less
variable in group A patients than in group B patients
(Table 1).
Structural changes in acini and ducts. This study
focused on alterations found in acini and ducts, changes
that have previously been related to mononuclear cell
infiltrates (31). In a histologic analysis of LSGs from
control subjects, acinar and ductal cells displayed typical
basal–apical polarity, whereby nuclei were basally located and secretion granules were visible at the apical
pole. While mucous cells normally form a very tight
acinar lumen (Figure 5A), a variety of modifications
were observed in LSGs obtained from SS patients. These
included dilated acinus lumen, loss of nuclear polarity
(Figure 5B), detachment of acinar cells from the basal
lamina (Figure 5C), and cellular debris in the lumen
duct (Figure 5D).
Morphologic changes observed both in the basal
lamina and the apical pole of acinar cells were similar in
both groups of SS patients, although the small amount of
gland tissue present in patients of group B made the
evaluation of these changes difficult in regions distant
from mononuclear cell foci (data not shown). The acinar
2578
GOICOVICH ET AL
Figure 3. Analysis of the degradation of types I, III, and IV collagen by extracts of labial salivary glands (LSGs) from A, a control subject and B,
a patient with Sjögren’s syndrome (SS). Purified collagen (20 ␮g) was incubated with LSG extracts (5 ␮g) that had been preactivated with 1 mM
APMA. Digestion time 1 ⫽ 12 hours; digestion time 2 ⫽ 24 hours. Proteins were separated on a 7% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis gel and stained with Coomassie blue R250. Lane 1, LSG extract incubated for 24 hours. Lane St, Prestained molecular weight
standards. Arrowheads at lane 7 indicate bands of apparent Mr of 212,000, 142,000, and 54,000. Braces at lanes 4 and 7 indicate groups of polypeptide
fragments. Electrophoretic patterns are representative of all patients and controls. C, Densitometric analysis of the bands that showed intensity
differences or new fragments in A and B, for both the control subject (open bars) and the SS patient (crosshatched bars). Values are the mean and
SEM relative intensity.
PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS
2579
Figure 4. Analysis of fibronectin degradation by extracts of labial salivary glands
(LSGs) from Sjögren’s syndrome (SS) patients and controls. A, Purified fibronectin
(10 ␮g) was incubated with LSG extracts (5 ␮g) that had been preactivated with 1
mM APMA. Digestion time 1 ⫽ 12 hours; digestion time 2 ⫽ 24 hours. Lane 1,
Purified fibronectin without gland extract incubated for 24 hours, showing a band
with an apparent Mr of 400,000. All incubations were done in the presence of a
protease inhibitor cocktail (see Patients and Methods). Proteins were separated on
a 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and stained with
Coomassie blue R250. Lane St, Prestained molecular weight standards. Electrophoretic patterns are representative of all SS patients and controls. B, Densitometric
analysis of the gel shown in A. FN ⫽ fibronectin; E ⫽ LSG extract; t1 ⫽ 12 hours;
t2 ⫽ 24 hours.
and ductal cell images shown in this article were captured from gland regions distant from mononuclear cell
foci.
Ultrastructural changes in basal lamina acini
and ducts. In control subjects, the acini and ducts of the
basal lamina showed normal structural features (Figure
6A). However, in the SS patient, major modifications
were observed, such as disorganization of the ultrastructural components of the basal lamina of acini and ducts
and a massive infiltration of bundles of stromal collagen
fibers (Figure 6B). The basal lamina was often found to
be absent, and in this case, the collagen fibers were in
direct contact with the basolateral plasma membrane
(Figure 6D). Other changes observed in acinar cells
included swollen cisternae of both the endoplasmic
reticulum and mitochondria, nuclear vesiculation, and
increased endosomal vesicles (Figures 6C and D).
Ultrastructural changes in the apical region of
acini. In control subjects, acini had a narrow lumen, with
structural characteristics of normal gland epithelia, such
as microvilli and caveolae. Secretion granules with low
electron density and a well-defined structure were observed in the apical cytoplasm of acinar cells from the
controls (Figure 7A). In SS patients, however, the acini
showed a strongly dilated lumen, loss of microvilli, and
loss of normal structure of the remaining microvilli
(Figures 7B and C). In the lumen, abundant cellular
debris and, unexpectedly, bundles of stromal collagen
fibers were found (Figure 7D). The electron density of
secretion granules and other cytoplasmic components
2580
GOICOVICH ET AL
Figure 5. Morphologic changes in acini and ducts in labial salivary glands (LSGs) from patients
with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject,
showing normal architecture of acini. B, Acinus in an LSG section from an SS patient, showing 2
acinar cells that have lost nuclear polarity (arrow) and have a dilated lumen. C, Acinus in an LSG
section from an SS patient, showing total detachment of acinar cells. Outlined area marks the limit
where the basal lamina should be seen; however, this has been replaced by stromal fibers. D, Duct
in an LSG section from an SS patient, showing cellular debris in the lumen (arrow). Images of acini
and ducts represent regions distant from mononuclear cell foci. (Original magnification ⫻ 390 in
A; ⫻ 900 in B, C, and D.)
was higher in SS patients than in control subjects.
Moreover, these granules were fused within the cells.
Structures with the appearance of secretion granules
were observed in the acinar lumen (Figures 7B and C).
Consistent with the findings of our biochemical
analysis, the ultrastructure of acinar and ductal basal
lamina showed abnormalities ranging from disorganization to disappearance of this ECM structure. Such
changes were paralleled by a loss of microvilli on the
apical surface, suggesting that inappropriate cytoskeletal
reorganization may impair exocytosis of secretory granules.
Substrate degradation and secretory functionality. Table 1 shows the unstimulated salivary flow and
scintigraphy data. The mean unstimulated salivary flow
was 1.05 ml/15 minutes (range 0.8–1.3) in group A
patients and 0.7 ml/15 minutes (range 0.5–1.1) in group
B patients. Both these values are lower than the minimum accepted value for classification as SS (30). Scintigraphy curves were all in the median category for
group A patients, while in group B patients, the curves
were flat or median. Both parameters were normal in
control subjects. Thus, low unstimulated salivary flow
and abnormal scintigraphy curves correlated with higher
substrate degradation as well as loss of ultrastructural
integrity and functionality of the LSG. However, low
proteolytic activity was observed in 3 of 6 group B
patients, with low levels of residual gland tissue and high
levels of mononuclear cells and fibrous tissue (Table 1).
DISCUSSION
In this study, we present the first evidence that
MMP activity in LSGs from SS patients can degrade
specific components of both the basal lamina and stroma
with different intensities, laminin ⬎ fibronectin ⬎ type
IV collagen ⬎ type III collagen ⬎ type I collagen
(Figures 1–4). Moreover, we observed dramatic changes
in the architecture of these structures, which suggests
that they might indeed be tissue-specific targets of these
enzymes (Figures 5–7). This proteolytic activity was also
correlated with the secretory function of the glands
PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS
2581
Figure 6. Ultrastructural changes in the basal domain of acini in labial salivary glands (LSGs) from
patients with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject,
showing a normal appearance of the basal lamina (arrowheads). Note also the normal-appearing
mitochondria (m). B, LSG section from an SS patient, showing a thin, but disorganized, basal lamina
(arrowheads), with a swollen mitochondrion in the cytoplasm. C, LSG section from an SS patient, showing
the relationship between acinar and myoepithelial cells (myo). There is a disorganized basal lamina, with
a cotton-like appearance (arrowhead). Swollen mitochondria and dilated rough endoplasmic reticulum
(rer) are visible in the acinar cell. D, LSG section from an SS patient, showing the absence of basal lamina.
There are bundles of collagen fibers (col) in the intercellular space (arrow). Note the dilated rough
endoplasmic reticulum and polymorphic nuclei (n) in the acinar cell. Dⴕ, Higher magnification view of
boxed area in D, showing a myoepithelial cell process in tight contact with collagen fibers. Images of acini
and ducts represent regions distant from mononuclear cell foci. Bars ⫽ 1 ␮m.
(Table 1). Group B patients had a higher content of
inflammatory cells, lower amounts of parenchymal tissue, and lower levels of proteolytic activity, which were
detected mainly in patients whose LSGs were enriched
in fibrous/adipose tissue. The nature of this group
explains the heterogeneity of results. However, despite
these variations, findings in group B support our hypothesis that epithelial cells play a crucial role in the events
that ultimately lead to gland destruction. Our previous
findings provided evidence that expression of MMPs in
acini and ducts was increased in SS patients (3), supporting the idea tested in the present study that MMPs may
act locally on their basal lamina and thereby lead to the
progressive gland destruction that is characteristic of SS.
In this respect, it is important to note that the basal
lamina architecture of other structures (e.g., blood vessels) did not show any apparent changes (data not
shown).
Lower molecular weight forms of MMPs were
detected when extracts from patients and controls were
treated with APMA. However, the gelatinolytic activity
was more pronounced in patients (4-fold higher than in
the controls) (Figure 1). High levels of TIMP-1 have
been found in LSGs from all study subjects, patients as
well as controls (Gonzalez J: unpublished observations),
and as previously described (37), activation of some
MMPs with trypsin or activation of MMP–TIMP-1 complexes with APMA or trypsin generates lower molecular
2582
GOICOVICH ET AL
Figure 7. Ultrastructural changes in the apical domain of acini in labial salivary glands (LSGs) from
patients with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject,
showing the normal appearance of the apical domain. Note the normal-appearing of microvilli (mi),
secretion granules (sg), and a narrow lumen (L). B, LSG section from an SS patient, showing a very dilated
lumen with a large amount of cellular debris. C, High magnification view of the apical pole of an acinar
cell in an LSG section from an SS patient, showing few and abnormal microvilli. There is coalescence of
secretion granules and abundant cellular debris in the lumen. D, LSG section from an SS patient, showing
a lumen with bundles of collagen fibers (col; arrow). Note the differences in electron density and shape
among secretion granules in B, C, and D, compared with those showed in A. Images of acini and ducts
represent regions distant from mononuclear cell foci. Bars ⫽ 1 ␮m.
weight MMPs with decreased activity. This might explain the small effect observed with APMA in LSGs
from control subjects (Figure 1, lane 2). However, if
MMP–TIMP-1 complexes interact with other MMPs
(such as MMP-3) before activation, the lower molecular
weight MMP that is generated has a higher activity (38).
In view of these observations, it is possible that the
differences in APMA activation observed in samples
from patients and controls (Figure 1, lanes 2 and 5)
could be explained by the finding that MMP-3 is highly
expressed in SS patients but not in controls (3).
The disorganization observed in the basal lamina
was accompanied by important changes in organelle
morphology, particularly at the apical surface of acinar
and ductal cells and in the lumen. Normally, adhesion
molecules (e.g., integrins) connect components of the
basal lamina with the inside of these cells by providing
attachment sites for the cytoskeleton (3,18,39–42). This
dynamic framework regulates cytoskeletal organization
and, as a consequence, the location of organelles (e.g.,
nuclear polarity), cellular volume, structure, and organization of the apical microvilli (13,43,44). In SS patients, different degrees of detachment were observed
between the basal lamina and either acini or ducts,
which correlated with a loss of cytoskeletal organization.
Thus, both a complete lack of cellular architecture and
structural disorganization of microvilli at the apical
plasma membrane were detected (Figures 5–7).
In acinar cells, maintenance of the integrity of the
exocytotic machinery is essential to ensure that secretory
granules fuse with the apical plasma membrane and
release their content to the lumen (45,46). The alter-
PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS
ations observed in our studies concerning the morphology of the apical compartment, including loss of microvilli, fusion, and accumulation of secretion granules,
suggest that acinar secretion in LSGs is perturbed in SS
patients (Figures 7). The alterations in the exocytic
machinery caused by these structural changes and the
disruption of the cytoskeleton could account for the
reduction in unstimulated salivary flow and the modifications in the scintigraphy curves (Table 1).
Aquaporin 5, a channel protein that regulates the
movement of water across the apical membrane of
salivary gland acinar cells, has been found by some
researchers to accumulate in the cytoplasm of cells from
SS patients (47,48), although other investigators have
not identified such localization (49). Thus, based on our
findings, the accumulation of aquaporin 5 in these
patients may be attributed to a deficiency in vesicle
sorting from the trans Golgi network to the plasma
membrane. An important point here is that both the
fusion of secretory granules with the plasma membrane
and the sorting of molecules from the trans Golgi
network to the apical surface are processes that are
highly dependent on the integrity of cytoskeletal elements (3,50).
Furthermore, other studies have demonstrated
the presence of various alterations caused by a loss of
interaction between the basal lamina and epithelial cells,
such as changes in the movement of cell electrolytes and
disruption of tight junctions between neighboring cells
(51,52). The presence of bundles of collagen fibers in the
acinar lumen of SS patients (Figure 7D) is indicative of
a loss of tight interactions between cells. The disruption
of these structural and functional barriers could allow
components in the stroma and lumen that are normally
separated to interact.
Increased electron density in the cytoplasm of
acinar cells and loss of cellular volume have been used as
an indicator of changes in cell osmolarity and apoptosis
(53). Thus, the modifications observed in the LSGs from
SS patients might be the consequence of cell apoptosis.
MMPs degrade ECM molecules, and it has been
demonstrated that the resulting fragments of fibronectin
and laminin induce the expression of MMP (54). Therefore, it is possible that a positive feedback loop is
established that promotes gland damage. Stromal collagen is also a good inducer of MMPs (54). Interestingly,
severe gland damage was found only in some cases,
indicating the possible existence of a compensatory
mechanism for replacing dying cells. In this respect,
higher levels of cell proliferation using appropriate
markers have been found in acinar and ductal cells of SS
patients (Alliende C, et al: unpublished observations).
2583
So far, xerostomia has been mainly explained by
imbalances in cytokine levels (6), production of autoantibodies against the M3 muscarinic receptor (7), desensitization to secretagogues (55), lack of sorting of aquaporin 5 (47–49), and mononuclear cell infiltration (1,2).
Our study is the first to indicate that increased MMP
activity may lead to both the basal and apical changes
detected in acinar and ductal cells of LSGs from SS
patients. Such changes could explain xerostomia at a
molecular level, since the lack of communication between these cells and their microenvironment may lead
to disorganization of the cytoskeleton and, as a consequence, deregulation of normal vesicle trafficking.
ACKNOWLEDGMENTS
We would like to dedicate this article to the late
Michael Humphreys-Beher, MD (Department of Oral Biology,
University of Florida, Gainesville) for his outstanding contribution to the current understanding of autoimmune exocrinopathy with the use of murine models. We thank Drs. Andrew
Quest and Remigio López (Facultad de Medicina, ICBM,
Universidad de Chile, Santiago, Chile) for their support,
discussions, and critical review of the manuscript.
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