close

Вход

Забыли?

вход по аккаунту

?

Modified aquaporin 5 expression and distribution in submandibular glands from NOD mice displaying autoimmune exocrinopathy.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 56, No. 8, August 2007, pp 2566–2574
DOI 10.1002/art.22826
© 2007, American College of Rheumatology
Modified Aquaporin 5 Expression and Distribution in
Submandibular Glands From NOD Mice Displaying
Autoimmune Exocrinopathy
Muhammad S. Soyfoo,1 Carine De Vriese,1 Huguette Debaix,2 Maria D. Martin-Martinez,3
Chantal Mathieu,4 Olivier Devuyst,2 Serge D. Steinfeld,5 and Christine Delporte1
mice and the 8-week-old NOD mice showed AQP5
primarily at the apical membrane of the salivary
gland acinus. In contrast, in acini from the submandibular glands (but not the parotid glands) from 24week-old NOD mice, AQP5 staining was reduced at the
apical membrane but was increased at the basal membrane. A moderately statistically significant decrease
in pilocarpine-stimulated salivary flow was observed in
24-week-old NOD mice compared with that in agematched BALB/c mice.
Conclusion. Submandibular glands from 24week-old NOD mice displayed inflammatory infiltrates,
increased AQP5 protein expression, and impaired
AQP5 distribution. However, the moderately statistically significant decrease in the salivary flow rate in
these mice did not match the extent of AQP5 misdistribution.
Objective. To investigate the expression and localization of aquaporin 5 (AQP5) in salivary glands and
salivary gland function in the NOD mouse.
Methods. All experiments were performed using
NOD and BALB/c mice (ages 8 weeks and 24 weeks).
Real-time reverse transcription–polymerase chain reaction, Western blotting, and immunohistochemical analysis were used to study the expression and distribution
of AQP5 in salivary glands. In addition, salivary gland
function was determined.
Results. Compared with the levels in BALB/c
mice, relative AQP5 messenger RNA levels were not
significantly modified in the parotid glands from NOD
mice of both ages but were significantly increased in the
submandibular glands from NOD mice of both ages.
Western blot analyses of both salivary gland membranes revealed that the level of AQP5 protein was
increased in 24-week-old NOD mice. Important inflammatory infiltrates were observed in the submandibular
glands, but not in the parotid glands, from 24-week-old
NOD mice. The 8-week-old and 24-week-old BALB/c
Sjögren’s syndrome (SS) is an autoimmune disorder characterized by lymphocyte infiltration of salivary
and lacrimal glands, leading to glandular hypofunction
that often results in characteristic symptoms of dry
mouth and dry eyes (1). Typical salivary gland biopsy
specimens obtained from patients with SS show lymphocytic infiltrates, with an associated reduction (up to
50%) in the number of acinar cells (2). The roles of
reduced numbers of acinar cells and associated glandular dysfunction in the pathogenesis of SS are well
characterized (1). Several factors have been proposed to
contribute to impaired salivary flow, including the proinflammatory cytokines interleukin-1 and tumor necrosis
factor ␣ (which were shown to inhibit both basal and
stimulated secretion [1]), decreased protein kinase C
levels in salivary gland acinar cells from patients with SS
(3), as well as the presence of antibodies against type 3
muscarinic receptor in patients with primary SS (4).
Dr. Soyfoo is recipient of an Erasme Foundation Research
Fellowship. Dr. Devuyst’s work was supported by a grant from the
Fund for National Scientific Research, Belgium and an Arthritis
Research Campaign grant (05/10-328). Dr. Delporte’s work was supported by the Fund for Medical Scientific Research, Belgium (grant
3.4604.05).
1
Muhammad S. Soyfoo, MD, Carine De Vriese, PhD, Christine Delporte, PhD: Université Libre de Bruxelles, Brussels, Belgium;
2
Huguette Debaix, MSc, Olivier Devuyst, PhD: Université Catholique
de Louvain Medical School, Brussels, Belgium; 3Maria D. MartinMartinez, MD: Bordet Institute, Brussels, Belgium; 4Chantal Mathieu,
PhD: Katholieke Universiteit Leuven, Leuven, Belgium; 5Serge D.
Steinfeld, PhD: Erasme University Hospital, Brussels, Belgium.
Address correspondence and reprint requests to Christine
Delporte, PhD, Laboratory of Biochemistry, CP 611, Université Libre
de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium. E-mail:
cdelport@ulb.ac.be.
Submitted for publication November 7, 2006; accepted in
revised form May 11, 2007.
2566
EXPRESSION AND DISTRIBUTION OF AQP5 IN NOD MICE
The discovery of a family of water-specific membrane channel proteins, the aquaporins (AQPs), has
provided insight into the molecular mechanism of membrane water permeability in a variety of tissue (for
review, see refs. 5–8). Among the AQPs identified thus
far, AQP5, AQP1, and AQP8 have been localized to
normal salivary glands, using immunohistochemical
methods (9). In knockout mice, AQP5, which is present
in the apical membrane of acinar cells (5,10–12), was
shown to participate in saliva secretion (13). In contrast,
AQP1, which is present in capillaries and venules adjacent to salivary gland epithelium (14–16), and AQP8,
which is located in myoepithelial cells surrounding the
acini, were not demonstrated, using knockout mice, to
play a physiologic role in saliva secretion (17).
In minor salivary glands from patients with SS, a
predominance of AQP5 staining intensity was observed
at the basal membrane of acinar cells (12). This abnormal distribution of AQP5 suggested a defect in AQP5
distribution that likely contributes to a deficiency in fluid
secretion, which is a defining feature of SS. Although a
conflicting report had described normal localization of
AQP5 in 5 patients with SS (18), similar abnormal
localization of AQP5 was reported in lacrimal glands
from patients with SS (19).
The NOD mouse model is a well-described animal model for the study of the autoimmune exocrinopathy that is prevalent in patients with SS (20). The NOD
mouse model allows investigators to study progression of
the disease, which is very difficult to achieve in patients
with SS. In a relatively short period of time, NOD mice
pass from a normal healthy state (at age 8 weeks) to
experiencing a severe form of the disease (at age 24
weeks), characterized by histopathologic modifications
of their salivary glands (inflammatory lymphocytic infiltrates, acinar cells destruction) and impaired saliva
secretion, which also occur in patients with SS (21,22). In
NOD mice, hyposalivation was shown to be preceded by
inflammatory changes in the salivary glands (23). Recently, confocal laser scanning imaging showed abnormal distribution of AQP5 in parotid and submandibular
glands from 20–26-week-old NOD mice compared with
that in glands from 9-week-old BALB/c mice. However,
no data concerning either AQP5 expression or salivary
gland function were provided (24).
To further address the possible involvement of
AQP5 in exocrine salivary gland dysfunction, we studied
the molecular expression and distribution of AQP5 in
salivary glands, linked to measurement of salivary gland
function, using NOD mice of 2 ages (8 weeks old and 24
2567
weeks old; these ages should represent 2 distinct stages
of the disease) and matched control BALB/c mice.
MATERIALS AND METHODS
Animals. NOD mice, originally obtained from Professor Wu (Beijing, China), were housed and inbred in the animal
facility of the Katholieke Universiteit Leuven since 1989. NOD
mice were housed under semi-barrier conditions. All experiments were performed using overnight-starved, 8-week-old or
24-week old female NOD mice or BALB/c mice (purchased
from Harlan, Horst, The Netherlands). Because autoimmune
diabetes also develops spontaneously in NOD mice, mice were
checked for glycosuria, and only nondiabetic mice were included in the study. Maintenance of the animals and all
experimental procedures were performed in accordance with
the guidelines of the ethics committees of the Katholieke
Universiteit Leuven (Leuven, Belgium), which are in accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals.
Semiquantitative real-time reverse transcription–
polymerase chain reaction (RT-PCR). Salivary gland total
RNA was extracted with a NucleoSpin RNA II kit (MachereyNagel, Düren, Germany), treated with DNase, and purified
using the PureLink Micro-to-Midi Total RNA Purification
System (Invitrogen, Merelbeke, Belgium). Prior to reverse
transcription using the SuperScript III kit (Invitrogen), the
quality of the RNA was checked using the Agilent 2100
BioAnalyzer (Agilent, Palo Alto, CA). The primers used for
amplification of mouse AQP5 complementary DNA (cDNA)
were as follows: sense primer 5⬘-TGGAGCAGGCATCCTGTACT-3⬘ and antisense primer 5⬘-CGTGGAGGAGAAGATGCAGA-3⬘ (151-bp amplicon); GAPDH sense primer 5⬘TGCACCACCAACTGCTTAGC-3⬘ and antisense primer
5⬘-GGATGCAGGGATGATGTTCT-3⬘ (176-bp amplicon).
Changes in target gene messenger RNA (mRNA)
levels were determined by semiquantitative real-time RT-PCR
with an iCycler iQ System (Bio-Rad, Hercules, CA), using
SYBR Green I for detection, as described previously (25). All
reactions were performed in duplicate. The PCR conditions
were as follows: 94°C for 3 minutes, followed by 31 cycles of 30
seconds at 95°C, 30 seconds at 61°C, and 1 minute at 72°C. To
exclude amplification by contaminating genomic DNA, samples of RNA that had not been reverse transcribed were run in
a parallel PCR. Standard curves were prepared using serial
4-fold dilutions of cDNA samples for each assay. The efficiency of the reactions was calculated from the slope of the
standard curve (efficiency ⫽ [10⫺1/slope]⫺1). The relative
mRNA expression of AQP5 was investigated in salivary glands
from BALB/c and NOD mice, ages 8 weeks or 24 weeks, after
normalization to GAPDH (ratio ⫽ 2⌬Ct [reporter ⫺ target gene]).
The relative (mean ⫾ SEM) changes in mRNA levels of
AQP5 in salivary glands from NOD mice compared with
BALB/c mice of the same age, after adjustment to
GAPDH (ratio ⫽ [efficiency target gene]⌬Ct [BALB/c – NOD]/
[efficiency GAPDH]⌬Ct [BALB/c – NOD]), were 0.98 ⫾ 0.01 for
AQP5 and 1.04 ⫾ 0.03 for GAPDH.
Antibodies to AQP5. An antibody resulting from rabbit
immunization using a synthetic peptide corresponding to the
C-terminal 25 amino acids of rat AQP5 (this sequence was
2568
100% identical to the corresponding mouse AQP5 sequence)
was affinity purified, and its specificity was verified by Western
blotting and confocal microscopy (26).
Western blot analysis. Crude plasma membranes were
prepared from mouse salivary glands, as described previously
(27), except that a membrane pellet from 17,000g centrifugation was used, and no ␤-mercaptoethanol was added to the
sample buffer for sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE). The samples were analyzed by
SDS-PAGE using 12% polyacrylamide gels. Proteins were
transferred to polyvinylidene difluoride membranes and immunolabeled using the affinity-purified antibody to AQP5 at a
1:2,000 dilution. The bound antibodies were detected by using
the enhanced chemiluminescence method (Amersham, Piscataway, NJ). Band densities (a measure of “volume”) were
estimated using image analysis apparatus and Bio-Profil
Bio-1D software from Vilbert Lourmat (Marne-la-Vallée,
France). Densitometry in image analysis is based on digitalization of the image in pixels, the intensity of which is coded on
a scale of 256 levels of grey. The measure of volume is the sum
of all pixel intensities comprising the spot and depends on the
number of pixels inside the area of the spot and on the
intensity of these pixels.
Immunohistochemical localization of AQP5. Immediately after being removed, salivary glands were fixed in 4%
buffered formaldehyde, paraffin embedded, and sectioned (5
␮m thick). Some salivary gland sections were stained with
hematoxylin and eosin to visualize the infiltrate. Some sections
were incubated with the primary antibody, affinity-purified
anti-AQP5 (dilution 1:200), overnight at 4°C. The bound
anti-AQP5 antibody was revealed by staining with anti-rabbit
gamma globulin (Nordic, Tilburg, The Netherlands) followed
by peroxidase–antiperoxidase complex (Dako, Glostrup, Denmark). The fixed peroxidase was visualized using diaminobenzidine (Dako). As control, salivary gland sections were incubated either with secondary antibody alone or with anti-AQP5
antibody preadsorbed with the immunizing peptide in 100-fold
excess. The labeling index corresponds to the percentage of the
acinus area that is immunoreactive for rat AQP5 in each region
of the cell. Quantitative analysis of the labeling index of the
immunoreactive acini was performed as previously described
(28,29), using a SAMBA 2005 computer-assisted microscope
system (Unilog, Grenoble, France) equipped with a 20⫻
magnification lens (aperture 0.50 mm).
On each pixel, the tissue-integrated optical density due
to the hematoxylin counterstaining and the brown diaminobenzidine chromogen was computed on 256 densitometric levels in
2-color channels. Immunoreactivity generated by binding of
the histochemical probe was defined by the integrated optical
density values in excess of the mean ⫹2 SD of the corresponding negative control. The computer-assisted microscope and
related quantitative analyses were standardized as follows. A
negative histologic control slide (from which the primary
antibody was omitted) was analyzed for each experimental
condition. The software used on the computer-assisted microscope automatically subtracted the labeling index and mean
optical density values of the negative control sample from the
values of each of the 2 positive samples available for each
experimental condition. Specific software was included in the
computer, to assist the microscope in checking any inherent
shading in the CCD camera–based systems, the glare phenom-
SOYFOO ET AL
enon, and the level of linear precision. The monitoring procedure installed on our computer-assisted microscope showed
that neither shading nor glare nor linearity significantly modified our results (data not shown). Ten acini were analyzed in
each experimental condition.
Salivary flow measurement and protein assay. Mice
were anesthetized with ketamine (60 mg/kg body weight) and
xylazine (8 mg/kg body weight) and injected subcutaneously
with pilocarpine (0.5 mg/kg body weight). Saliva was collected
on a cotton swab for 20 minutes. The weight of the cotton swab
was measured before and after saliva collection. The weight of
the saliva was converted into the volume of saliva, assuming
that 1 ␮g represents 1 ␮l. The amount of saliva was normalized
to microliters of saliva per gram of body weight per 20 minutes.
The protein concentration was determined using the Bradford
method (30).
Statistical analysis. Data are expressed as the mean ⫾
SEM. The Mann-Whitney U test or the unpaired t-test was
used to evaluate statistical differences. All statistical analyses
were carried out using GraphPad InStat version 3.00 for
Windows 95 (GraphPad Software, San Diego, CA).
RESULTS
AQP5 mRNA detection by real-time RT-PCR in
salivary glands from BALB/c and NOD mice. Real-time
RT-PCR analysis of AQP5 mRNA, using GAPDH as
reporter gene, was performed on submandibular and
parotid glands from BALB/c and NOD mice, ages 8
weeks and 24 weeks. In parotid glands from NOD mice
of both ages, as compared with BALB/c mice, the
relative AQP5 mRNA levels were not significantly modified. In submandibular glands from 8-week-old and
24-week-old NOD mice, as compared with those from
age-matched BALB/c mice, the relative AQP5 mRNA
expression levels increased significantly, by 80% and
44%, respectively (Table 1).
Western blot quantification of AQP5 expression
in salivary glands from BALB/c and NOD mice. Protein
immunoblots of membranes from salivary glands of
8-week-old and 24-week-old BALB/c and NOD mice
were probed with an affinity-purified anti-AQP5 antibody and an anti–␤-actin antibody. Western blot analysis
revealed the presence of an ⬃27-kd band corresponding
to AQP5. The band density, also called the measure of
volume, is the sum of all pixel intensities composing the
spot and depends on both the number of pixels inside
the area of the spot and the intensity of these pixels.
Linear relationships were determined by plotting separately the values for AQP5 and ␤-actin band volumes
versus increasing amounts (0.050–20 ␮g) of salivary
gland membrane proteins in BALB/c and NOD mice
(data not shown). All further Western blot analyses were
EXPRESSION AND DISTRIBUTION OF AQP5 IN NOD MICE
Table 1. Relative expression of aquaporin 5 messenger RNA in
salivary glands from BALB/c and NOD mice*
Age group; salivary gland,
mouse
Age 8 weeks
Parotid gland
BALB/c (n ⫽ 4)
NOD (n ⫽ 6)
Submandibular gland
BALB/c (n ⫽ 5)
NOD (n ⫽ 6)
Age 24 weeks
Parotid gland
BALB/c (n ⫽ 5)
NOD (n ⫽ 5)
Submandibular gland
BALB/c (n ⫽ 6)
NOD (n ⫽ 6)
Relative
expression, %
100 ⫾ 22
88 ⫾ 13
100 ⫾ 6
180 ⫾ 26†
100 ⫾ 8
116 ⫾ 29
100 ⫾ 6
144 ⫾ 13†
* Values are the mean ⫾ SEM. RNA extraction and reverse
transcription–polymerase chain reaction were performed as described
in Materials and Methods. Messenger RNA expression levels were
compared after adjustment to GAPDH as reporter gene. Similar
results were obtained when using different reporter genes, including
hypoxanthine guanine phosphoribosyltransferase, cyclophilin A, and 18S.
† P ⬍ 0.05 versus age-matched BALB/c mice, by unpaired t-test.
2569
performed using 2.5 ␮g of salivary gland membrane
protein.
AQP5 expression in parotid and submandibular
gland membranes from 8-week-old NOD mice was not
significantly different from that in membranes from
age-matched BALB/c mice (Figures 1A and B). In
parotid and submandibular glands from 24-week-old
NOD mice, AQP5 expression was significantly increased, by 3.7-fold and 1.7-fold, respectively, compared
with that in salivary glands from age-matched BALB/c
mice (Figures 1C and D).
Histopathologic findings in salivary glands from
BALB/c and NOD mice. Parotid and submandibular
glands (Figures 2A–C) from 8-week-old and 24-week old
BALB/c mice and those from 8-week-old NOD mice
were normal. Parotid glands from 24-week-old NOD
mice were normal and devoid of any lymphocytic infiltrates (results not shown). Submandibular glands from
24-week-old mice displayed numerous large lymphocytic
infiltrates (Figure 2D).
Figure 1. Western blot quantification of aquaporin 5 (AQP5) expression in salivary
glands from BALB/c and NOD mice. Western blot analysis was performed using
anti-AQP5 antibody (1:2,000 dilution) and 2.5 ␮g of membrane (pellet from 17,000g
centrifugation) from the parotid (A and C) or submandibular (B and D) glands of
8-week-old (A and B) and 24-week-old (C and D) BALB/c and NOD mice. Bars show
the mean and SEM results from 6 mice per group. ⴱ ⫽ P ⬍ 0.05 versus age-matched
BALB/c mice, by Mann-Whitney U test.
2570
SOYFOO ET AL
Figure 2. Localization of aquaporin 5 (AQP5) in submandibular glands from BALB/c mice at ages 8 weeks and 24 weeks (A
and C) and NOD mice at ages 8 weeks and 24 weeks (B and D). AQP5 was localized on submandibular gland sections from
BALB/c and NOD mice, as described in Materials and Methods. Images are representative of immunohistochemical staining
performed on salivary gland sections from 3 mice per group. Negative control staining was performed in the absence of
anti-AQP5 antibody or with anti-AQP5 antibody previously incubated with the immunizing peptide (results not shown). Insets show
higher-magnification views. (Original magnification ⫻ 40.)
Immunohistochemical localization of AQP5 and
quantitative analysis of AQP5-immunoreactive acini in
salivary glands from BALB/c and NOD mice. Immunohistochemical analysis revealed AQP5 labeling at both
the apical and basolateral membranes of salivary gland
acini from BALB/c and NOD mice, at age 8 weeks and
age 24 weeks (Figure 3). Computer-assisted microscopy
was performed to quantitatively evaluate AQP5 distribution in the immunopositive acini of parotid and
submandibular glands from both BALB/c and NOD
mice, at age 8 weeks and 24 weeks. A labeling index
representing the percent of the acinus area stained with
anti-AQP5 antibody was determined for both the apical
and basolateral membranes.
In 8-week-old mice, the apical and basolateral
labeling indices of the parotid glands from NOD mice
were not significantly different from those of the parotid
glands from BALB/c mice (apical [n ⫽ 6], 35.15 ⫾ 2.01
versus 35.06 ⫾ 1.44% [P ⫽ 0.589]; basolateral [n ⫽ 6],
18.87 ⫾ 1.77 versus 17.42 ⫾ 0.91% [P ⫽ 0.699]) (Figure
3A). In 24-week-old mice, the apical and basolateral
labeling indices of the parotid glands from NOD mice
were not significantly different from those of the parotid
glands from BALB/c mice (apical [n ⫽ 6], 25.09 ⫾ 4.54
versus 14.07 ⫾ 1.60% [P ⫽ 0.065]; basolateral [n ⫽ 6],
6.57 ⫾ 1.56 versus 4.95 ⫾ 0.61% [P ⫽ 0.310]) (Figure
3B). In 8-week-old mice, the apical and basolateral
labeling indices of the submandibular glands from NOD
mice were not significantly different from those of the
submandibular glands from BALB/c mice (apical [n ⫽
6], 4.03 ⫾ 0.45 versus 3.82 ⫾ 0.43% [P ⫽ 0.699];
basolateral [n ⫽ 6], 0.91 ⫾ 0.14 versus 0.70 ⫾ 0.05%
[P ⫽ 0.485]) (Figure 3C). In the submandibular glands
from 24-week-old NOD mice, the apical labeling index
decreased significantly, by 75% (1.04 ⫾ 0.14 versus
4.13 ⫾ 0.35% in BALB/c mice [n ⫽ 6]; P ⫽ 0.002), while
EXPRESSION AND DISTRIBUTION OF AQP5 IN NOD MICE
2571
and 24 weeks of age (Figure 4). At 8 weeks of age, the
salivary flow rates in NOD mice (n ⫽ 6) and BALB/c
mice (n ⫽ 6) were not statistically significantly different
(7.38 ⫾ 0.92 versus 8.90 ⫾ 0.75 ␮l of saliva/gm of body
weight/20 minutes; P ⫽ 0.310), while at 24 weeks of age,
salivary flow in NOD mice (n ⫽ 6) decreased significantly, by 29%, compared with that in BALB/c mice
(n ⫽ 6) (6.95 ⫾ 0.48 versus 9.73 ⫾ 0.88; P ⫽ 0.041).
DISCUSSION
Figure 3. Quantitative analysis of aquaporin 5 (AQP5)–immunoreactive acini in salivary glands from BALB/c and NOD mice. Quantitative analysis of the labeling index of the labeled acini was performed as described in Materials and Methods. The labeling index was
determined for parotid (A and B) and submandibular (C and D) glands
from 8-week old (A and C) or 24-week-old (B and D) BALB/c mice
(open bars) or NOD mice (solid bars). Bars show the mean and SEM
results from 6 mice per group (10 acini for each animal). ⴱ ⫽ P ⬍ 0.05
versus age-matched BALB/c mice, by Mann-Whitney U test.
the basolateral labeling index increased significantly, by
⬃50% (2.45 ⫾ 0.11 versus 1.66 ⫾ 0.06% in BALB/c mice
[n ⫽ 6]; P ⫽ 0.002) (Figure 3D).
Measurement of the salivary flow rate in BALB/c
and NOD mice. Pilocarpine-stimulated salivary flow was
measured in BALB/c and NOD mice at 8 weeks of age
Figure 4. Measurement of the salivary flow rate in BALB/c and NOD
mice. Salivary flow was measured in 8-week-old and 24-week-old
BALB/c mice (open bars) and NOD mice (solid bars), as described in
Materials and Methods. Bars show the mean and SEM results from 6
mice per group. ⴱ ⫽ P ⬍ 0.05 versus age-matched BALB/c mice, by
Mann-Whitney U test.
In normal salivary glands, localization of AQP5 is
restricted to the apical membrane of acinar cells (5,10–
12). The mechanism that leads to saliva secretion involves secretion of a primary isotonic fluid by the acini,
from which considerable NaCl is resorbed during passage through the ducts. Ductal cells possess a sodium
channel (31,32), a chloride channel (10,31), and a
sodium/proton exchanger (10,31) that are likely involved
in NaCl reabsorption, as well as a potassium/proton
exchanger that may mediate potassium and bicarbonate
secretion (31,33). The involvement of AQP5 in saliva
secretion was clearly demonstrated using AQP5knockout mice that displayed remarkably reduced salivary flow as well as hypertonic and hyperosmolar saliva
(13).
Although the biologic mechanisms leading to
hyposalivation and hypolacrimation symptoms in SS
remain poorly understood, it is increasingly evident that
dryness of the eyes and mouth does not result solely
from gland destruction. Recent studies have shown that
AQP5, a water channel protein, could play a role in the
pathogenesis of SS. Indeed, the contribution of impaired
AQP5 distribution to the deficiency of fluid secretion, a
defining feature of SS, was supported by the abnormal
distribution of AQP5 in minor salivary glands (12) and
lacrimal glands (19) from patients with SS. Conflicting
data describing the normal localization of AQP5 in
minor salivary glands from 5 patients with SS (18) could
result from the use of distinct antibodies and/or methodologies (12).
The NOD mouse, a recognized appropriate
model for studying the autoimmune exocrinopathy that
is prevalent in patients with SS, was used to investigate
the possible involvement of AQP5 in exocrine gland
salivary dysfunction. Compared with the parotid and
submandibular glands from control BALB/c mice, those
from 8-week-old NOD mice had no inflammatory infiltrates, no significant modification in AQP5 protein
levels, no significant modification of AQP5 apical and
basolateral labeling indices, and no significant salivary
2572
flow decrease. However, the submandibular, but not the
parotid, glands demonstrated a significant increase in
relative AQP5 mRNA levels. Although a correlation
seems to exist between relative AQP5 mRNA levels and
AQP5 protein levels in parotid glands from both
BALB/c mice and NOD mice, an apparent discrepancy
between AQP5 mRNA levels (increased) and protein
levels (unchanged) was observed in the submandibular
glands and could be attributable to higher protein
turnover. Because, from the physiologic point of view,
protein levels are more relevant than mRNA levels, it is
important to note that in the salivary glands from
8-week-old NOD mice presenting no autoimmune exocrinopathy, AQP5 protein levels and AQP5 localization
in the acini were not modified.
In contrast, the submandibular glands from 24week-old NOD mice displayed substantial inflammatory
infiltrates throughout the glands, a significant increase in
AQP5 protein levels, and a significant reduction in the
apical labeling index associated with significant augmentation in the basolateral labeling index. In addition,
24-week-old NOD mice displayed a moderately statistically significant decrease in salivary flow (P ⫽ 0.041),
which appeared to be marginal, because there was no
significant difference between saliva flow rates from
8-week-old and 24-week-old mice. However, although a
correlation seems to exist between the increase in the
relative AQP5 mRNA levels and the AQP5 protein
levels observed in the submandibular glands, the apparent discrepancy between AQP5 mRNA levels (unchanged) and protein levels (increased) in the parotid
glands could be attributable to low turnover of protein.
In the submandibular acini from 24-week-old NOD
mice, impaired AQP5 localization was observed. The
24-week-old NOD mice used in this study displayed
submandibular gland lymphocytic infiltrates and a moderately reduced salivary flow rate, which is consistent
with results of other studies (22–24).
A recent study demonstrated abnormal AQP5
distribution in parotid and submandibular glands from
20–26-week-old NOD mice (using 9-week-old BALB/c
mice as controls) (24). In the current study, additional
data concerning the distribution of AQP5 in salivary
glands from NOD mice of 2 ages were provided by
performing quantitative analysis of the labeling index of
AQP5-labeled acini. Moreover, at the molecular level,
the expression of AQP5 (at both the mRNA and protein
levels) was evaluated. At the physiologic level, salivary
gland function was determined by measuring saliva flow.
The data collected from NOD mice of 2 ages (8 weeks
old and 24 weeks old, representing 2 distinct stages of
SOYFOO ET AL
the disease) were then compared with those collected
from matched control BALB/c mice. The unexpected
increase in AQP5 protein expression observed in the
submandibular glands from 24-week-old NOD mice
might reflect a compensatory mechanism, because
AQP5 does not distribute properly in acinar cells and is
therefore unlikely to play its normal physiologic role.
The absence of inflammatory infiltrates in the
parotid glands from 24-week-old NOD mice was quite
unexpected. Parotid glands from 24-week-old NOD
mice showed no increase in mRNA but did have an
increase in the AQP5 protein level, which could not
reflect a compensatory mechanism, because AQP5 distributes normally in acinar cells and is therefore likely to
play its normal physiologic role. Because hyposalivation
has been shown to be preceded by inflammatory changes
in salivary glands from NOD mice (23), submandibular
glands, rather than parotid glands, are likely to contribute to the moderate saliva flow dysfunction observed in
our study. A link seems to exist between inflammation
and abnormal AQP5 distribution. This hypothesis is
based on observations that the parotid glands from
24-week-old NOD mice displayed no inflammatory infiltrates and no changes in AQP5 localization, while the
submandibular glands displayed inflammatory infiltrates
and abnormal AQP5 distribution. Further studies will be
required to understand the mechanisms leading to the
increased level of AQP5 protein in parotid glands from
24-week-old NOD mice, and to characterize the possible
link existing between inflammatory changes and abnormal AQP5 distribution.
In 24-week-old mice, several explanations might
account for the discrepancy between the moderate reduction in the salivary flow rate and the extent of AQP5
misdistribution. First, whole saliva (containing saliva
from the parotid and submandibular glands) was collected, although parotid glands predominantly contribute to the salivary flow rate (34). Second, increased
AQP5 protein expression in the submandibular glands
might compensate for AQP5 misdistribution. Finally,
the ability of water to permeate properly localized
AQP5 might be reduced.
Abnormal expression and distribution of AQP5
in acinar cells from submandibular glands from NOD
mice and minor salivary glands from patients with SS
(12) suggest a defect in AQP5 distribution in autocrine
exocrinopathy. Similar observations of altered protein
trafficking in patients with SS have been shown for the
Na ⫹ -independent chloride–bicarbonate anion exchanger (35). In SS, autoantibodies against AQP5 are
unlikely to account for its abnormal distribution, be-
EXPRESSION AND DISTRIBUTION OF AQP5 IN NOD MICE
cause no antibodies to human AQP5 were detected in
sera from patients with SS (12). AQP5 down-regulation
and possibly altered distribution are also likely involved
in the symptom of dry mouth following radiation therapy
for head and neck cancer (36). Consequently, water
channel gene transfer has been considered as a potential
therapeutic approach in patients with SS, as well as in
patients with impaired saliva production resulting from
radiation therapy for head and neck cancer (37). Since
the discovery that increased fluid secretion could be
observed after adenovirus-mediated gene transfer of
AQP1 cDNA to irradiated rat salivary glands (14),
transfer of human AQP1 cDNA for the correction of
radiation-induced hypofunction has been extensively
documented in several species (37).
Because the NOD mice used in this study demonstrated moderately decreased salivary flow, it would
be of particular interest to address in further studies
whether impaired localization of AQP5 is also observed
in other NOD mouse models demonstrating either no or
a very significant salivary flow decrease (24,38). Additional studies investigating the basic mechanisms of
AQP5 trafficking may also provide valuable insight into
the regulation of AQP5 distribution in salivary glands
from both control and NOD mice.
In conclusion, submandibular glands from 24week-old NOD mice displayed inflammatory infiltrates,
increased AQP5 protein expression, and impaired
AQP5 distribution. In 24-week-old NOD mice, however,
the moderately statistically significant decrease in the
salivary flow rate did not match the extent of AQP5
misdistribution.
ACKNOWLEDGMENTS
We thank M. Stiévenart for secretarial assistance and
Jos Laureys for technical assistance.
AUTHOR CONTRIBUTIONS
Dr. Delporte had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Soyfoo, De Vriese, Devuyst, Steinfeld, Delporte.
Acquisition of data. Soyfoo, De Vriese, Debaix, Mathieu, Devuyst,
Delporte.
Analysis and interpretation of data. Soyfoo, De Vriese, Debaix,
Martin-Martinez, Devuyst, Steinfeld, Delporte.
Manuscript preparation. Soyfoo, De Vriese, Debaix, MartinMartinez, Mathieu, Devuyst, Steinfeld, Delporte.
Statistical analysis. Soyfoo, Debaix, Delporte.
2573
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
REFERENCES
1. Fox RI. Sjögren’s syndrome [review]. Lancet 2005;366:321–31.
2. Andoh Y, Shimura S, Sawai T, Sasaki H, Takishima T, Shirato K.
23.
Morphometric analysis of airways in Sjögren’s syndrome. Am Rev
Respir Dis 1993;148:1358–62.
Tornwall J, Konttinen Y, Tuomen R, Tornwall M. Salivary gland
acinar epithelial cells are deficient in their protein kinase C
expression in Sjögren’s syndrome. Lancet 1997;349:1914–5.
Bacman S, Sterin-Borda L, Camusso JJ, Arana R, Hubscher O,
Borda E. Circulating antibodies against rat parotid gland M3
muscarinic receptors in primary Sjögren’s syndrome. Clin Exp
Immunol 1996;104:454–9.
Agre P, Brown D, Nielsen S. Aquaporin water channels: unanswered questions and unresolved controversies [review]. Curr
Opin Cell Biol 1995;7:472–83.
Borgnia M, Nielsen S, Engel A, Agre P. Cellular and molecular
biology of the aquaporin water channels [review]. Annu Rev
Biochem 1999;68:425–58.
King LS, Agre P. Pathophysiology of the aquaporin water channels
[review]. Annu Rev Physiol 1996;58:619–48.
Verkman AS, van Hoek AN, Ma T, Frigeri A, Skach WR, Mitra A,
et al. Water transport across mammalian cell membranes [review].
Am J Physiol 1996;270(1 Pt 1):C12–30.
Delporte C, Steinfeld S. Distribution and roles of aquaporins in
salivary glands [review]. Biochim Biophys Acta 2006;1758:
1061–70.
He X, Tse CM, Donowitz M, Alper SL, Gabriel SE, Baum BJ.
Polarized distribution of key membrane transport proteins in the
rat submandibular gland. Pflugers Arch 1997;433:260–8.
Nielsen S, King LS, Christensen BM, Agre P. Aquaporins in
complex tissues. II. Subcellular distribution in respiratory and
glandular tissues of rat. Am J Physiol 1997;273:C1549–61.
Steinfeld S, Cogan E, King LS, Agre P, Kiss R, Delporte C.
Abnormal distribution of aquaporin-5 water channel protein in
salivary glands from Sjögren’s syndrome patients. Lab Invest
2001;81:143–8.
Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS.
Defective secretion of saliva in transgenic mice lacking aquaporin-5 channels. J Biol Chem 1999;274:20071–4.
Delporte C, O’Connell BC, He X, Lancaster H, O’Connell A,
Agre P, et al. Increased fluid secretion following transfer of the
aquaporin-1 cDNA to irradiated rat salivary glands. Proc Natl
Acad Sci U S A 1997;94:3268–73.
Li J, Nielsen S, Dai Y, Lazowski KW, Christensen EI, Tabak LA,
et al. Examination of rat salivary glands for the presence of the
aquaporin CHIP. Pflugers Arch 1992;428:455–60.
Nielsen S, Smith BL, Christensen EI, Agre P. Distribution of the
aquaporin CHIP in secretory and resorptive epithelia and capillary
endothelia. Proc Natl Acad Sci U S A 1993;90:7275–9.
Yang B, Zhao D, Solenov E, Verkman AS. Evidence from
knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport. Am J Physiol Cell Physiol 2006;291:
C417–23.
Beroukas D, Hiscock J, Jonsson R, Waterman SA, Gordon TP.
Subcellular distribution of aquaporin 5 in salivary glands in
primary Sjögren’s syndrome. Lancet 2001;358:1875–6.
Tsubota K, Hirai S, King LS, Agre P, Ishida N. Defective cellular
trafficking of lacrimal gland aquaporin-5 in Sjögren’s syndrome.
Lancet 2001;357:688–9.
Van Blokland SC, Versnel MA. Pathogenesis of Sjögren’s syndrome: characteristics of different mouse models for autoimmune
exocrinopathy [review]. Clin Immunol 2002;103:111–24.
Humphreys-Beher MG. Animal models for autoimmune diseaseassociated xerostomia and xerophthalmia. Adv Dent Res 1996;10:
73–5.
Humphreys-Beher MG, Peck AB. New concepts for the development of autoimmune exocrinopathy derived from studies with the
NOD mouse model. Arch Oral Biol 1999;44 Suppl 1:S21–5.
Jonsson MV, Delaleu N, Brokstad KA, Berggreen E, Skarstein K.
Impaired salivary gland function in NOD mice: association with
2574
24.
25.
26.
27.
28.
29.
30.
changes in cytokine profile but not with histopathologic changes in
the salivary gland. Arthritis Rheum 2006;54:2300–5.
Konttinen YT, Tensing EK, Laine M, Porola P, Tornwall J,
Hukkanen M. Abnormal distribution of aquaporin-5 in salivary
glands in the NOD mouse model for Sjögren’s syndrome. J Rheumatol 2005;32:1071–5.
Ni J, Cnops Y, McLoughlin RM, Topley N, Devuyst O. Inhibition
of nitric oxide synthase reverses permeability changes in a mouse
model of acute peritonitis. Perit Dial Int 2005;25 Suppl 3:S11–4.
Delporte C, O’Connell BC, He X, Ambudkar IS, Agre P, Baum
BJ. Adenovirus-mediated expression of aquaporin-5 in epithelial
cells. J Biol Chem 1996;271:22070–5.
Dai YS, Ambudkar IS, Horn VJ, Yeh CK, Kousvelari EE, Wall SJ,
et al. Evidence that M3 muscarinic receptors in rat parotid gland
couple to two second messenger systems. Am J Physiol 1991;261(6
Pt 2):C1063–73.
Camby I, Nagy N, Lopes MB, Schafer BW, Maurage CA, Ruchoux
MM, et al. Supratentorial pilocytic astrocytomas, astrocytomas,
anaplastic astrocytomas and glioblastomas are characterized by a
differential expression of S100 proteins. Brain Pathol 1999;9:1–19.
Steinfeld S, Penaloza A, Ribai P, Decasetecker C, Danguy A,
Gabius HJ, et al. D-mannose and N-acetylglucosamine moieties
and their respective binding sites in salivary glands of Sjögren’s
syndrome. J Rheumatol 1999;26:833–41.
Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 1976;72:248–54.
SOYFOO ET AL
31. Cook DI, Van Lennep EW, Roberts ML, Young JA. Secretion by
the major salivary glands. In: Johnson LR, editor. Physiology of
the gastrointestinal tract. New York: Raven; 1994. p. 1061–117.
32. Moran A, Davis VH, Turner RJ. Na⫹ channels in membrane
vesicles from intralobular salivary ducts. Am J Physiol 1995;268:
C350–5.
33. Knauf H, Lubcke R, Kreutz W, Sachs G. Interrelationships of ion
transport in rat submaxillary duct epithelium. Am J Physiol
1982;242:F132–9.
34. Levy S, Nagler A, Okon S, Marmary Y. Parotid salivary gland
dysfunction in chronic graft-versus-host disease (cGVHD): a longitudinal study in a mouse model. Bone Marrow Transplant
2000;25:1073–8.
35. Vazquez JJ, Vazquez M, Idoate MA, Montuenga L, MartinezAnso E, Castillo JE, et al. Anion exchanger immunoreactivity in
human salivary glands in health and Sjögren’s syndrome. Am J
Pathol 1995;146:1422–32.
36. Li Z, Zhao D, Gong B, Xu Y, Sun H, Yang B, et al. Decreased
saliva secretion and down-regulation of AQP5 in submandibular
gland in irradiated rats. Radiat Res 2006;165:678–87.
37. Baum BJ, Zheng C, Cotrim AP, Goldsmith CM, Atkinson JC,
Brahim JS, et al. Transfer of the AQP1 cDNA for the correction
of radiation-induced salivary hypofunction. Biochim Biophys Acta
2006;1758:1071–7.
38. Lodde BM, Mineshiba F, Kok MR, Wang J, Zheng C, Schmidt M,
et al. NOD mouse model for Sjögren’s syndrome: lack of longitudinal stability. Oral Dis 2006;12:566–72.
Документ
Категория
Без категории
Просмотров
2
Размер файла
229 Кб
Теги
expressions, submandibular, distributions, exocrinopathy, mice, gland, modified, autoimmune, displaying, nod, aquaporin
1/--страниц
Пожаловаться на содержимое документа