Research Article
Quantitative Assessment of Basement Membrane in Patients with Sjögren’s Syndrome and Association with Laminin
Mohammad M Pour*, Charles M Cobb and Carole P McArthur
Department of Oral and Craniofacial Sciences, University of Missouri-Kansas City School of Dentistry, and Trauma
Medical Center, Kansas City, USA
*Corresponding author: Mohammad M Pour, Department of Oral and Craniofacial Sciences, University of Missouri-Kansas City School of Dentistry, Kansas City, MO 64108, USA
Published: 18 Jun, 2017
Cite this article as: Pour MM, Charles M Cobb, McArthur
CP. Quantitative Assessment of
Basement Membrane in Patients with
Sjögren’s Syndrome and Association
with Laminin. J Dent Oral Biol. 2017;
2(6): 1049.
Abstract
There is little known about the role of basement membrane and matrix proteins in the pathogenesis
of salivary gland disease associated with Sjögren’s Syndrome (SS). Histologically, at the level of the
light microscope, lymphocytic infiltration is by CD4 T cells.
Our laboratory has studied the basement membrane in salivary gland disease. Preliminary work
indicates abnormalities in the major component of the basement membrane, laminin. This protein
appears to be “trapped” intracellularly in the ductal and acinar epithelial cells as demonstrated
by immunohistochemistry using specific monoclonal antibodies. It remains unclear whether the
laminin dysregulation results from an anabolic or catabolic issue but investigations are ongoing
at several levels. Preliminary transmission electron microscopic (TEM) studies on 6 patients have
revealed variations in the basal lamina are suggestive of a general “narrowing” of this structure.
In contrast, previously published studies report basal lamina “thickening” which is inconsistent
with our observations and published data. The objective of the present study, therefore, is to
attempt to resolve this discrepancy by carrying out sequential measurements on the basal lamina
of ductal and acinar epithelium of diseased salivary glands by TEM from a larger group of patients
(12) and compare them with controls. Results showed that there is a loss of the lamina lucida
portion of the basal lamina, in spite of overall “thickening” that was characterized as structural
disorganization. There was also a marked increase in intracellular acinar cell laminin, shown by
immunohistochemistry and TEM, compared with healthy controls. The potential role of laminin
dysregulation is discussed.
Introduction
Salivary gland dysfunction leading to xerostomia may be seen in various autoimmune diseases,
notably in both primary and secondary Sjögren’s Syndrome (SS) and AIDS, as well as polymyositis/
dermatomyositis and chronic thyroiditis [1,2]. The typical histological picture reported in SS is of
chronic infiltration by mononuclear cells into salivary acini, ducts and regional perivascular areas.
The predominant lymphocytes in each case are of a different phenotype suggesting a different
pathological manifestation. In SS, the lymphocytes are predominantly CD4+ T-helper cells while in
a process of salivary gland disease, as seen in AIDS, for example, they are CD8+. This infiltration is
followed by periductal and perivascular sclerosis and fibrosis, expansion of the lesion and eventual
glandular atrophy or acinar “drop-out”.
Further studies have shown thickening of the ductal basement membrane, which is composed
of laminin, type IV collagen, fibronectin and heparin sulfate proteoglycans [3-5]. Transmission
electron microscopy (TEM) in the areas of irregular thickening demonstrate thickening of the
lamina densa with high electron density [6]. However, this irregular thickening of the lamina
densa is accompanied by the disappearance of the lamina lucida. We have noted in our previous
observations that the same phenomenon of thickening of the lamina densa and the disappearance of
the lamina lucida occurs in minor salivary glands of patients who were infected with HIV.
In a previous study, preliminary TEM studies of minor salivary glands in 6 patients with primary
SS revealed variation in the basement membrane suggestive of a general narrowing of this structure
rather than thickening. Using immunohistochemistry and a monoclonal antibody against the B1
chain of laminin, we have shown that this narrowing of the basement membrane is accompanied
by a marked increase in laminin expression in minor salivary gland ductal epithelial cells in SS.
Although laminin is characteristically an extracellular matrix protein,
light microscopy demonstrates that there is abnormal intracellular
accumulation of laminin in ductal epithelial cells as compared with
fibronectin [7].
Laminin is the main component of the basement membrane. It is
involved in structural support of the salivary acini and ducts. Laminin
is also involved in maintaining cell polarity. It is hypothesized
that changes in the basement membrane occur as a result of
disorganization brought about by degradation of laminin and type
IV collagen. This process may result from an over expression of
matrix metalloproteases-9 and 3 (MMP-9 & 3) which, in turn, may
result from cytokines released by tissue-infiltrating inflammatory
cells [8]. It has also been demonstrated that the fragmented products
of laminin that are produced due to degradation by MMPs also
promote further MMP expression, possibly in a positive feedback
loop [9]. Thus, it is possible that changes within the basement
membrane, producing irregular areas of thickening or thinning, may
be the result of laminin degradation. There is evidence to support this
weak expression of laminin in the basement membrane of patients
with Sjögren’s Syndrome [10]. Furthermore, the role that MMPs
play in the degradation of laminin and other extracellular matrix
proteins within the basement membrane along with the subsequent
disorganization of the basement membrane that they cause has been
demonstrated [9-11]. Yet, since laminin is an inflammatory mediator,
as well as being involved in maintaining cellular polarity, it may act as
a stimulus for lymphocytic infiltration. The subsequent degradation
of laminin by MMP-9 & 3, following cytokine release, may contribute
to epithelial cell death through apoptosis.
Overall, there is little known about the role of the basal lamina and
matrix proteins in the pathogenesis of salivary gland disease associated
with SS. Furthermore, there has been little explanation regarding the
accumulation of laminin intracellularly following disorganization of
the basal lamina. The objective of this research was to compare the
ductal and acinar basal lamina of minor salivary glands in patients
with SS and healthy individuals by TEM to elucidate pathogenesis and
to confirm whether changes seen in the basal lamina disorganization
in SS, and thereby the basement membrane at the light microscopic
level, are due to an overall narrowing of the basal lamina.
Figure 1
Figure 1
Electron micrograph showing a duct from a normal minor salivary
gland. N: Nucleus; L: Lumen; Large arrows: Basal Lamina; Small arrows:
Mitochondria.
Figure 2
Figure 2
Electron micrograph showing a minor salivary gland from an
individual with Sjögren’s Syndrome. Note the thinning of the basal lamina
compared to that shown in Figure 1. N: Nucleus; L: Lumen; Large arrows:
Basal Lamina; Small arrows: Mitochondria.
Figure 3
Figure 3
Electron micrograph showing a minor salivary gland from an
individual with Sjögren’s Syndrome. Note the degeneration of the epithelial
cell indicated by large arrows. N: Nucleus; L: Lumen.
Figure 4
Figure 4
Electron micrograph showing a duct from a normal minor salivary
gland. N: Nucleus; L: Lumen; Small arrows: Mitochondria.
Figure 5
Figure 5
Electron micrograph showing a minor salivary gland duct from
an individual with Sjögren’s Syndrome. Note the tubal ectasia, indicated by
large arrows and also the presence of increased number of desmosomes. N:
Nucleus; D: Desmosomes; Small arrows: Mitochondria.
Figure 6
Figure 6
Electron micrograph showing a normal minor salivary gland acinar.
N: Nucleus; Large arrows: Secretory granules.
Figure 7
Figure 7
Electron micrograph showing minor salivary gland acinar from
an individual with Sjögren’s Syndrome. Note the formation of mucus island,
pushing the nucleus toward the basal pole indicated by large arrows. N:
Nucleus; Small arrows: Secretory granules.
Figure 8
Figure 8
Frequency of ductal basal lamina thickness between diseased and
non-diseased control specimens.
Materials and Methods
Institutional Review Board (IRB) approval with signed patient
consent was obtained from the UMKC IRB. Minor salivary gland
(MSG) biopsies were obtained from 12 patients (11 Female, 1 Male;
ages 30-78 years) with primary or secondary SS. The non-diseased
control population, also contributing MSG biopsies, consisted of 14
age-matched patients. Criteria for SS were clinical symptoms of dry
eyes and dry mouth, arthritis and a history of at least one positive
serum SS-A (Ro), SS-B (La), anti-nuclear antibody (ANA) or
rheumatoid factor (RF) test. Patients receiving steroid therapy were
excluded from the study.
Around 3-5 labial salivary glands were obtained from each
patient. A single gland from each patient was bisected and onehalf
was processed for light microscopy (fixation in 10% buffered
formalin solution) and Greenspan scoring. The remaining one-half
was fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer at pH
7.0 for examination by TEM. The remaining glands (2 to 4) from each
patient were processed and evaluated by light microscopy to provide
a biopsy Grade for severity of SS.
Basal laminae adjacent to both ductal and acinar cells were
measured at multiple points from photographs over a light-box. With
respect to acinar cells, between 35 and 439 (mean 160) measurements
were from each patient’s biopsy specimen. Similarly, 7 to 111 (mean
31) measurements were made of the basal lamina associated with
ductal epithelial cells. Measurements could not be obtained on ductal
epithelium from 3 SS patients as there were no ducts present in these
specific specimens. Ducts also could not be located in 1 control
specimen. Analysis of frequency of distributions of thickness in
both acinar and ductal basal lamina in SS and control patients was
performed using the students T-test.
Figure 9
Figure 9
(A) Electron micrograph of minor salivary gland acinar from an
individual with Sjögren’s Syndrome. As shown with large arrows, there is
significant staining of laminin with monoclonal antibody intracellularly.
(B) Electron micrograph of minor salivary gland acinar from a healthy
control. Note the absence of staining by immunohistochemistry for laminin
intracellularly.
Results
Figures 1 to 7 are electron micrographs comparing minor salivary
glands from SS and control patients. A comparison of a normal
salivary gland (Figure 1) with that from a patient with SS (Figure 2)
demonstrates the typical irregularities found within the basal lamina.
Table 1 is a list of patients showing the number of measurements of
ductal and acinar basal laminae from each patient and the histologic
grade of each biopsied gland. The mean thickness of acinar basal
laminae in patients with SS compared to control specimens was a mean
of 190 nm ± 31.3 nm vs. a mean of 178 nm ± 40.3 nm, respectively.
The mean thickness of ductal basal laminae for SS patients was 147
nm ± 11.5 nm vs. and a mean thickness of 189 nm ± 81.0 nm in non-diseased
controls.
The bar graph (Figure 8) demonstrates the frequency of ductal
basal lamina thickness between diseased and non-diseased control
specimens. Both SS and control patients had the same frequency of
thickness when the basal laminae of the two groups were 0.24 μm
or less in thickness. However, basal laminae that were greater than
0.24 μm proved to be thicker in healthy patients more frequently
than that of SS patients. Tables 2 and 3 provide the frequency of
basal lamina thickness between SS and non-diseased patients in more
detail. As noted in the bar graph, ductal cell basal laminae in healthy
controls were generally thicker on average compared to SS patients,
when basal lamina 0.24 μm. In acini, however, the basal laminae were
thicker consistently for SS patient specimens vs. that of control group
specimens, regardless of any parameter of thickness. Overall, this
data reveals irregularities in basal laminae thickness when comparing
acinar and ductal basal laminae in the two patient groups. Despite
our observations, based on trends in the measurements, the present
study was unable to show a statistically significant difference in the
thickness of the basal laminae between SS vs. control patient biopsy
specimens.
Figures 3 to 7 detail some of the structural changes that
characteristically occur in SS patients. Figure 3 shows a degenerating
epithelial cell, indicating a pathologic process that may be related to a
dysfunctional disorganization within the basal lamina. Figures 4 and
5 show minor salivary gland ductal cells from SS and control patients.
As can be seen in Figure 5, there is tubal ectasia and an increase in
the number of desmosomes. Figures 6 and 7 allow comparison
of acinar cells in SS and normal salivary glands, with emphasis on
the fusion of granules forming “mucus islands”, which displace the
nucleus in the SS specimen. Figure 9A and 9B present the results of
the immunohistochemical staining of laminin in minor salivary gland
acini using a monoclonal antibody. Figure 9B shows a significant
intracellular accumulation of laminin (dark staining) compared to
that of the control specimen.
Table 1
Discussion
Previous unpublished studies in our laboratory revealed a
statistically significant narrowing of the basal lamina in acinar and
ductal cells of SS patients vs. that of non-diseased controls. However,
results of the current study do not support previous findings and,
interestingly, are in contrast toother reports, which generally describe
“thickening” of basal laminae in SS. Although our cases of SS consisted
of 1 male (30 years) and 11 females (40-77 years) all with welldefined
primary disease, it is pertinent that in none of the previous
publications concerning the nature of the basal lamina were actual
measurements of the basal lamina carried out as was in the present
Observations in previous reports are of a distinctly qualitative
nature with statements describing basal laminae as “irregularly
thickened”. Such statements are open to a variety of interpretations
and unsupported by actual measurements.
The lack of statistically significant narrowing in our present
study suggests that it is possible that differences between our data
and others are attributable to sampling differences or clinical criteria
for diagnosis. The differences in thickness that have been attributed
to this study may be coincidental, but we suggest that it reinforces
the fact that the basal laminae are irregular in contour. We have
concluded that the finding of a “narrow” basal lamina is consistent
with the abnormal intracellular accumulation of laminin-1 isoform
which we have recently demonstrated in SS [7]. This isoform appears
to be “trapped” intracellularly in ductal and acinar cells and may
prevent the transportation and incorporation of laminin into the
basal lamina, thereby providing an explanation for the narrowing we
found in some patients.
It has been suggested by Konttinen et al. [12] and Royce et al.
[13], that laminin-1 is responsible for the maintenance of healthy
acini and is weakly expressed in acini of patients with SS, resulting
in acinar atrophy. The postulation is that the absence of laminin-1
from damaged or disorganized basal laminae in SS affects the normal
migration and differentiation of acinar progenitor cells which depend
on signaling from laminin for maturation and development. This
reinforces our theory that laminin degradation coincides with basal
lamina changes, which according to the current study, is a sign of
irregularity of the basal lamina. As such, given the results from our
previous study, intracellular accumulation of laminin may accompany
its degradation in the basal lamina. It remains unclear whether any
of these processes are attributable to a catabolic or anabolic process
taking place in the ductal or acinar cells.
Acinar atrophy seen histologically in patients with salivary gland
disease attributable to SS or other diseases, including AIDS, tend to
coincide with scattered ductal cell hyperplasia within lymphoid tissue.
Hyperplastic ducts have most often been observed in association with
TEM studies that reveal islands of epithelial cell aggregates (epimyoepithelial
islands) extending from the ductal epithelium [6,14].
Other changes seen by TEM include dilation of intercellular spaces
in acini and microvascular changes in periacinar microcirculation
that occur at the time of pathogenesis of SS in patients presenting
with xerostomia [3]. As the study by Perminova [3], it may be the
disturbances in the periacinar microcirculation that are important in
contributing to the on-set of Sjögren’s Syndrome, thus, suggesting a
persistent incomplete understanding of the sequence of steps in the
pathogenesis of salivary gland disease in SS.
At this point, there may be other variables to consider, but what
has been ascertained by our lab is the development of irregularities
in the basal laminae of patients with SS due to changes that are
concomitant with intracellular laminin accumulation. We have
been able to show previously that high levels of laminin mRNA
exist in labial salivary glands in Sjögren’s Syndrome, and that
this intracellular substance is laminin and not some laminin like
substance [7]. As discussed earlier in this paper, the mechanism of
laminin degradation in the basal lamina may occur as a result of the
activation of MMPs by infiltrating lymphocytes into salivary gland
tissues. Therefore, it may still be difficult to describe the changes in the
basement membrane as just narrowing of the basal lamina, but rather
a process of disorganization due to laminin degradation accompanied
by intracellular accumulation leading to overall irregularities that are
not consistently narrow or thick.
Table 2
Table 2
Frequency distributions of total ductal basal lamina thickness in 14 controls and 6 representative Sjögren’s patients.
Table 3
Table 3
Frequency distributions of total acinar basal lamina thicknesses in 14 control and 6 representative Sjögren’s patients.
Acknowledgement
The research was supported by grants to CM from the Arthritis Foundation and an RO-3 from the National Institute of Dental and Craniofacial Research.
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