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Sjögren’s
QUARTERLY
The Professionals’ Resource on Sjögren’s
Vol. 12, Issue 1 – Winter 2017
SSF Medical and Scientific
Advisory Board
Chair
Nancy L. Carteron, MD, FACR
Members
Esen Akpek, MD
Herbert S. Baraf, MD, MACR
Richard Brasington, MD, FACR
Michael Brennan, DDS, MHS
Steven E. Carsons, MD*
Troy Daniels, DDS, MS*
Denise L. Faustman, MD, PhD
H. Kenneth Fisher, MD, FACP, FCCP
Gary Foulks, MD, FACS
Theresa Lawrence Ford, MD
S. Lance Forstot, MD
Philip C. Fox, DDS*
Robert I. Fox, MD, PhD, FACP*
Tara Mardigan, MS, MPH, RD
Austin Mircheff, PhD
John Daniel Nelson, MD, FACS
Kelly Nichols, OD
Athena Papas, DMD, PhD
Ann Parke, MD
Andres Pinto, DMD
Nelson Rhodus, DMD, MPH
Vidya Sankar, DMD, MHS
Daniel Small, MD, FACP
Neil Stahl, MD
Frederick B. Vivino, MD, FACR
Jeffrey Wilson, MD, FACR
Associate Members
Simon J. Bowman, PhD, FCRP
Janine A. Clayton, MD
Arthur Grayzel, MD, FACR*
Roland Jonsson, DMD, PhD
Stuart S. Kassan, MD, FACP*
Robert Lebovics, MD
Michael Lemp, MD*
Xavier Mariette, MD
Haralampos M. Moutsopoulos, MD*
Manuel Ramos-Casals, MD, PhD
James J. Sciubba, DMD, PhD*
Harry Spiera, MD*
Leo Sreebny, DDS, MS, PhD*
Athanasios G. Tzioufas, MD
Ira J. Udell, MD*
Claudio Vitali, MD
Daniel J. Wallace, MD
Pierre Youinou, MD, Dsc
*Counselor
6707 Democracy Blvd., Ste 325
Bethesda, MD 20817
(301) 530-4420
www.sjogrens.org
©2016 Sjögren’s Syndrome Foundation
The Difficulties of Diagnosing Sjögren’s: Examples of Ro/SSA-Negative
Patients and Previous Erroneous Diagnoses of Patients with Sicca
by Astrid Rasmussen, MD, PhD, Arthritis and Clinical Immunology
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma
Key Messages
The diagnosis of Sjögren’s is difficult and often preceded
by diagnoses of other autoimmune disorders.
l A subset of patients with Sjögren’s present with objective
salivary and lacrimal dysfunction with focal lymphocytic
sialadenitis but without anti-Ro/SSA antibodies.
l
l
Rheumatoid factor and/or ANA increase the risk of misdiagnosing Sjögren’s as rheumatoid arthritis or lupus.
Astrid Rasmussen, MD, PhD
Advances in biomarker and physiopathology discovery
should result in more sensitive and specific tests for Sjögren’s.
Introduction
S
jögren’s is characterized by exocrine gland damage and dysfunction mediated by autoantibodies and lymphocytic infiltrates, resulting in xerostomia
and keratoconjunctivitis sicca. A significant proportion of patients with
Sjögren’s have systemic manifestations including arthritis, fatigue, hematological
abnormalities, pulmonary, renal and peripheral nervous system involvement, and
an increased risk for lymphoma.
Continued on page 2 t
Increased Tear Cathepsin S in Sjögren’s Illustrates
Fundamental Changes in Rab Protein Function in
Lacrimal Gland Acinar Cells
by Maria C. Edman1, Zhen Meng2, and Sarah F. Hamm-Alvarez1,2
1
Department of Ophthalmology, USC Roski Eye Institute and
2
Department of Pharmacology and Pharmaceutical Sciences,
University of Southern California
Maria C. Edman1
Introduction
I
n Sjögren’s, the salivary and lacrimal glands
Zhen Meng2
are infiltrated by lymphocytes. The foci of
lymphocytes are found in between the tearor saliva-producing acinar cells and are often localized
along the major efferent ducts. Another hallmark of
Sjögren’s is the reduction of tear and salivary production
from the acinar cells, a reduction that often is greater
Continued on page 6 t
than what can be explained by the loss of acinar cell
Sarah F. Hamm-Alvarez 1,2
6
Sjögren’s Quarterly
“Tear Biomarker” Continued from page 1 t
An additional 41 patients were recruited from an ophthalmology clinic with a diagnosis of either dry eye (31,
60.9±15.7) or blepharitis (10, 57.4±16.4). Healthy controls
(33, 29.7±8.8) were recruited among staff and students.
Tears were collected from all subjects using an anesthetized Schirmer’s test, and the Schirmer’s strips were placed
in sterile tubes and on ice for a maximum of 4 hours
before analysis. Tears were eluted off the strips and analyzed for Cathepsin S activity using a commercial assay.
Consistent with our hypothesis, we found that the median
tear Cathepsin S activity in patients with Sjögren’s was 4.1fold higher than that in patients with other autoimmune
diseases, 2.1-fold higher than in patients with nonspecific
Cathepsin S is increased in the lacrimal gland
dry eye disease, and 41.1-fold higher than that in healthy
and tears in a mouse model of Sjögren’s.
2
Investigating differences in gene expression between control subjects (Figure 2).
There were no significant differences in tear Cathe lacrimal glands of male NOD mice, a model of
thepsin S activity between patients with primary or
Sjögren’s-like lacrimal gland inflammation, and the
secondary Sjögren’s, indicating that Cathepsin S is a
lacrimal glands of healthy BALB/c mice, we discovered
that, along with pro-inflammatory factors, expression of good marker of Sjögren’s regardless of any concurrent
several members of a family of proteases called Cathep- autoimmune disease. Elevated Cathepsin S activity in
Sjögren’s patient tears was independent of the Schirmsins was increased in NOD mouse lacrimal glands.1
Among these proteins was Cathepsin S, a cysteine prote- er’s test values and of the levels of circulating anti-SSA
ase that is traditionally considered a lysosomal protease. or anti-SSB antibodies. When comparing tear Cathepsin
S levels in patients with other autoimmune diseases to
Cathepsin S has several other intracellular and extrathose with Sjögren’s, values were somewhat elevated
cellular functions in specialized cells that are largely
relative to healthy controls but not to the same extent as
related to inflammation, including 1) participating in
in Sjögren’s patients. Levels were elevated in non-autoactivation of antigen presentation by the major histoimmune dry eye patients as compared to healthy concompatibility complex class II (MHC II); 2) extracellular matrix degradation; and 3) activation of protease- trols. The area under the ROC curve was 0.771, (95%
confidence interval [95% CI 0.703–0.834) for values
activated receptors (PAR). Verification studies of the
initial gene expression analysis in NOD mouse lacrimal in Sjögren’s compared with other autoimmune disease
and 0.686 (95% CI 0.587–0.785) for values in Sjögren’s
glands showed by immunofluorescence that Cathepcompared with dry eye disease. The area under the ROC
sin S content was increased in both the lysosomes and
curve was 0.954 (95% CI 0.893–0.985) for values in
the secretory vesicles of the lacrimal gland acinar cells.
Sjögren’s compared with healthy controls. This study
Furthermore, activity assays showed that its activity
was significantly increased in both lacrimal gland tissue showed that tear Cathepsin S activity may constitute a
novel biomarker for Sjögren’s that can be measured in
lysates and stimulated tears from the male NOD mice
a non-invasive and economical fashion.
relative to control BALB/c mice (Figure 1).
mass due to expansion of lymphocytic foci. In addition to
reduced tear volume, the composition of tear and salivary
proteins changes in Sjögren’s. The precise relationship
between glandular inflammation and alterations in acinar
cell secretory function remains unclear, since the onset
and magnitude of changes in acinar secretion may not
parallel the onset and development of inflammatory foci.
Our work has been focused on elucidating the processes
and cellular machinery that package and transport proteins for secretion in healthy lacrimal gland acinar cells
and studying how this machinery is altered in Sjögren’s.
Tear Cathepsin S activity is a putative
biomarker for Sjögren’s.
Having identified increased tear Cathepsin S activity
as a hallmark of Sjögren’s-like lacrimal gland inflammation using the NOD mouse model, we hypothesized that
tear Cathepsin S activity might serve as a biomarker for
Sjögren’s in patients. To test this hypothesis, we initiated
a clinical study enrolling a total of 278 subjects of which
214 were recruited from a rheumatology clinic and had an
established diagnosis of the following autoimmune diseases: primary Sjögren’s (28 patients, age 47.4±12.4 years);
secondary Sjögren’s (45, 51.4±10.2); Rheumatoid Arthritis (79, 49.9±12.2); Systemic Lupus Erythematosus (40,
40.5±12.3); and other autoimmune disease (12, 48.7±13.1).
Investigating the trafficking machinery
delivering Cathepsin S to the tears
Initially, we hypothesized that the principal source of
increased Cathepsin S in the lacrimal glands of the male
NOD mice might be due to the increased number of infiltrating macrophages which are abundant in this protein
and which also accompany lymphocytic infiltration of
the gland. Later, we determined that, using laser capture
microdissection to allow PCR analysis of gene expression
within specific cell populations, the increase in Cathepsin
S expression occurs primarily in the acinar cells themselves. Cathepsin S that is normally produced in healthy
acinar cells is delivered primarily to the endo-lysosomal
compartments, so we were interested in mapping the ma-
Sjögren’s Quarterly
7
Figure 1: Cathepsin S (CTSS) activity in lacrimal gland
lysates and tear fluid from NOD and BALB/c mice.
Twelve-week-old male mice were anesthetized and tear fluid
collected after stimulation with carbachol. Lacrimal gland
(LG) lysates were prepared from the stimulated glands after
tear collection. Activity is expressed as relative fluorescence
units (RFU)/10 µg of lysate or RFU/10 µL of tears, n=7.
***P < 0.001. Adapted from (1) © The Association for
Research in Vision and Ophthalmology.
CTSS Activity (RFU)
14000
***
12000
10000
8000
***
6000
4000
BALB/c
NOD
2000
chinery responsible for delivering Cathepsin S to the tears
in acinar cells exhibiting a Sjögren’s-like phenotype.3
Exocrine secretion is regulated by Rab proteins, small
GTPases which serve as sorting codes for vesicular trafficking in the cells. Rab3 and Rab27 subfamilies are involved in
the process of regulated exocytosis in many secretory cells
and play roles in the regulated release of secretory proteins
from lacrimal acinar secretory cells. Rab3D, Rab27a and
Rab27b are all expressed in lacrimal gland acinar cells.
Utilizing Rab3D, Rab27a, Rab27b and Rab27a/b deficient
mice, we were able to show that the secretion of Cathepsin S activity into tears was highly affected by changes in
the relative Rab3D and Rab27 isoform expression relative
to other tear proteins. Specifically, Cathepsin S activity
in tears of mice lacking Rab27 isoforms was significantly
reduced, but Cathepsin S activity in tears of mice lacking
Rab3D was significantly elevated (Figure 3).
Figure 3: Changes in Cathepsin S (CTSS) secretion in Rabdeficient mice.
The Cathepsin S activities were measured as relative fluorescence units per microgram of tear protein. Values are
0
presented as relative values relative to those from C57BL/6
LG
Tear
(C57) mice, which were arbitrarily set as 100. Cathepsin S
secretion was significantly higher in Rab3D knockout (3DKO)
Figure 2: Cathepsin S (CTSS) activity in tears from patients mice (N=10) but lower in mice deficient in both Rab27 a
with autoimmune diseases (Sjögren’s n=73, RA n=79, SLE
and b isoforms (27KO) (N=12), Rab27a mutant (ash) (N=6)
n=40, and others n=12), patients with blepharitis n=10, pa- and Rab27b knockout (27bKO) (N=8) mice compared with
tients with dry eye n=31, and healthy control subjects n=33. healthy C57BL/c mice (N=29). *, significantly increased; #,
The Cathepsin S activities were measured as relative fluores- significantly decreased. Adapted from publication in Am J
cence units (RFU) per milligram of tear protein. Each patient Physiol Cell Physiol (3).
contributed 2 Schirmer’s test strips; thus, the number of sam250
ples in each group is twice the number of patients recruited
to each group. The shaded bars represent the 50th to 90th
200
percentile, the solid bars represent the 10th to 50th percentile, and the junction represents the median. P vs Sjögren’s
150
after adjustment for age, per group; RA <0.0001, SLE
<0.0001, Other autoimmune, 0.0041, Blepharitis, < 0.0001,
100
Dry eye, 0.0005, Healthy controls, <0.0001. Reprinted with
50
permission from Arthritis Rheumatol (2).
CTSS (% of C57)
*
0
18000
CTSS Activity (RFU/Protein)
#
#
#
C57 3DKO 27KO ash 27bKO
16000
14000
12000
10000
8000
6000
4000
2000
0
SS
RA
SLE
Blepharitis
Dry
Eye
Others
Healthy
Control
To further confirm the reliance of Cathepsin S secretion on Rab27 activity, we conducted different in vitro
studies in cultured lacrimal gland acinar cells, confirming that newly synthesized Cathepsin S was secreted
from Rab27-enriched secretory vesicles and, further,
that expression of dominant negative Rab27b reduced
the stimulated secretion of Cathepsin S. By using highresolution 3D-structured illumination microscopy of
Continued on page 8 t
8
“Tear Biomarker” Continued from page 7 t
mouse lacrimal gland acinar cells, we found that Rab3D
and Rab27 isoforms reside in different proportions on
microdomains localized on acinar secretory vesicles,
suggesting that their relative association with secretory
vesicles may tailor the vesicle contents and thus the
composition of the tear film. Since recent studies have
demonstrated that Rab3D expression and localization
are altered in the acinar cells of Sjögren’s patients, we
hypothesize that a loss of Rab3D function may underlie the altered profile of tear Cathepsin S secretion in
Sjögren’s-model mice and in patients.4,5 This model is
supported by our findings of lower gene expression and
altered Rab3D localization in male NOD mouse lacrimal gland acinar cells and by the findings of elevated
Cathepsin S secretion in the Rab3D-deficient mice.
Our working model, shown in Figure 4, proposes that
a loss of Rab3D from secretory vesicles in lacrimal gland
Figure 4: Working model for functions of Rab3D and Rab27
in exocytosis of tear proteins in healthy and Sjögren’s lacrimal gland acinar cells.
Rab3D and Rab27b are located to secretory vesicles but
with different abundances. We speculate that the recruitment
of low amounts of cathepsin S to secretory vesicles normally
occurs from endo-lysosomal compartments in a process
driven by one or both Rab27 isoforms, which then remain
associated with secretory vesicles enriched largely in Rab3D
beneath the apical plasma membrane. In Sjögren’s, Rab3D
gene and protein expression are decreased and the distribution of some cellular Rab3D protein is shifted to accumulation with large basolateral organelles. The loss of Rab3D on
secretory vesicles may result in decreased recruitment of
some tear proteins to secretory vesicles, and also permits
and/or promotes the increased flow of Cathepsin S through
Rab27-mediated processes to generate altered secretory
vesicles with a relative enrichment in Rab27 isoforms containing increased Cathepsin S. Adapted from publication in
Am J Physiol Cell Physiol (3).
Sjögren’s Quarterly
acinar cells affected by Sjögren’s contributes to an imbalance in Rab27-to-Rab3D activity and is a driver of the
enhanced release of Cathepsin S activity into tears. Currently, we are further investigating the complex interplay
between these Rab proteins of the regulated secretory
pathway and their responses to changes in the signaling
milieu including cytokine mediators of inflammation.
We are also investigating the origin of the increased
Cathepsin S expression in lacrimal gland acinar cells in
Sjögren’s, which likely also contributes to the relative
upregulation of the Rab27-mediated pathway that promotes increased Cathepsin S secretion to tears. n
References
1. Li X, Wu K, Edman M, Schenke-Layland K, MacVeigh-Aloni M, Janga
SR, Shulz B, Hamm-Alvarez SF. Increased expression of cathepsins and
obesity-induced proinflammatory cytokines in lacrimal glands of male
NOD mouse. Invest Ophthalmol Vis Sci. 2010 Oct; 51(10):5019-29.
2. Hamm-Alvarez SF, Janga SR, Edman MC, Madrigal S, Shah M, Frousiakis
SE, Renduchintala K, Zhu J, Bricel S, Silka K, Bach D, Heur M, Christianakis S, Arkfeld DG, Irvine J, Mack WJ, Stohl W. Tear cathepsin S as a
candidate biomarker for Sjogren’s syndrome. Arthritis Rheumatol. 2014
Jul;66(7):1872-81.
3. Meng Z, Edman MC, Hsueh PY, Chen CY, Klinngam W, Tolmachova
T, Okamoto CT, Hamm-Alvarez SF. Imbalanced Rab3D versus Rab27
increases cathepsin S secretion from lacrimal acini in a mouse model of
Sjogren’s Syndrome. Am J Physiol Cell Physiol. 2016 Jun;310(11):C942-54.
4. Bahamondes V, Albornoz A, Aguilera S, Alliende C, Molina C, Castro I, Urzua U, Quest AF, Barrera MJ, Gonzalez S, Sanchez M, Hartel S, Hermoso M,
Leyton C, Gonzalez MJ. Changes in Rab3D expression and distribution in
the acini of Sjogren’s syndrome patients are associated with loss of cell polarity and secretory dysfunction. Arthritis Rheum. 2011 Oct;63(10):3126-35.
5. Kamoi M, Ogawa Y, Nakamura S, Dogru M, Nagai T, Obata H, Ito M, Kaido
M, Kawakita T, Okada Y, Kawakami Y, Shimmura S, Tsubota K. Accumulation of secretory vesicles in the lacrimal gland epithelia is related
to non-Sjogren’s type dry eye in visual display terminal users. PloS One.
2012;7(9):e43688.
Editor’s Note
Dr. Edman received an SSF research grant in 2015/2016
and 2016/2017 for her project entitled “Tear fluid and
serum levels of Cathepsin S and its endogenous inhibitor
Cystatin C as biomarkers for Sjogren’s.”
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