Elucidating the pathogenesis of Sjogren's syndrome: Analysis of lower lipbiopsies

Sjögren*s syndrome (SS) is a systemic autoimmune disorder of unknown etiology,
characterized by mononuclear cell infiltration in exocrine glands, principally
the lacrimal and salivary glands. Other organ systems are frequently involved
and 5% of SS patients develop lymphoma. The inflammation of exocrine glands is
most prominent, resulting in dry eyes (keratoconjunctivitis sicca) and dry
mouth (xerostomia). The salivary gland dysfunction with associated difficulty
in eating and swallowing is the major complaint of patients. SS may occur
isolated, termed primary SS, or in association with another defined autoimmune
disease, such as rheumatoid arthritis, termed secondary SS. Primary SS is one
of the most common autoimmune diseases affecting 0.6% of the total population
in the Netherlands with women being nine times more likely to be affected than
men. Generally there are two age-peaks seen in primary SS patients, the first
between 20-30 years and the second around 50-60 years of age.
The exact pathogenesis of SS is unknown, but it is clearly multifactorial. The
presence of multiple autoantibodies in the peripheral blood of patients with SS
and the signs of chronic inflammation in the target organ, indicate an
autoimmune response against the exocrine glands. This is supported by several
findings at the histological level, such as the presence of large persistent
mononuclear foci, consisting of both T lymphocytes (~80%) and B lymphocytes
(~20%) in salivary and lacrimal glands in patients with SS. Evidence confirms
that lymphocytic disturbances, including formation of ectopic germinal centers
and aberrations of cellular signaling play a significant role, but still the
exact function has to be elucidated. Variable degrees of acinar cell atrophy
and progressing fibrosis can also be observed. Currently, there are different
hypothetic models for the SS pathogenesis. The *classical* model to explain
glandular hypofunction is tissue loss secondary to immune attack mediated by a
combination apoptosis and cytotoxic cell death. In the *non-apoptotic* model
glandular hypofunction follows immune-mediated inhibition of acinar secretory
function.
While there is effective palliative therapy for lacrimal manifestations, no
effective treatment exists for the salivary gland dysfunction. Current
palliative treatment for salivary gland dysfunction includes artificial saliva,
frequent dental prophylaxis, and/or stimulation by muscarinic agonists, such as
pilocarpine and cevimeline, and the use of steroids and other immunomodulatory
agents. However, systemic immunosuppressive and immunomodulatory therapies,
including classical immunosuppressives and TNF-alpha inhibitors, are largely
ineffective.
Since SS primarily involves the salivary and lacrimal glands, tissue analysis
of the salivary gland is essential to gain more insight into the pathogenesis
of the disease process. Therefore, descriptive studies of SS salivary glands
may provide a better understanding of the events that take place in vivo and
complement experimental animal studies and in vitro studies. Moreover,
knowledge about the pathogenesis can be used for the identification of new
targets and development of innovative therapies for SS.

To evaluate if the salivary gland infiltrate and structural changes are
specific for SS, it is important to compare SS salivary gland tissue to that of
other (aspecific) inflammatory and non-inflammatory salivary glands.

To gain more insight into the pathogenetic processes involved in SS by
analyzing the infiltrate and structural changes in salivary glands of SS
patients and compare these findings to those in other (aspecific) inflammatory
and non-inflammatory salivary gland diseases.

1.1 Patients:
Inclusion criteria:
Patients undergoing lower lip biopsies for diagnostic purposes.

1.2 Clinical evaluation and demographic measurements:
Individual patient information will be reported in a form (Case Report From
(CRF). Recording of:
- Demographic data (date of birth, sex, and race)
- Medical history (sicca symptoms, arthralgia, medication)
- Diagnosis
- Disease duration
- Presence of autoantibodies in serum (anti-ENA, anti-SSA and anti-SSB)
- Histological evaluation of the salivary glands (focus score)

1.3 Collection of material:
Salivary gland (lower lip):
In all patients undergoing diagnostic lower lip biopsies, salivary gland tissue
will be collected and processed using a standard protocol for formalin fixation
to allow standard histological evaluation for diagnostic purposes. Samples will
also be frozen in Tissue-Tek OCT compound for immunohistochemistry (IHC) or
snap frozen for PCR, micro-array, T Cell Receptor (TCR) and B Cell Receptor
(BCR) analysis, and protein expression analysis.

Serum and Peripheral Blood Mononuclear Cells (PBMC):
From all patients, 54 ml blood will be collected (6 heparine tubes of 9 ml
each). Serum will be isolated for detection of immunoglobulins (Ig*s) and
cytokine levels. Also, PBMC*s (± 54x10exp6) will be isolated and
ribonucleicacid (RNA) will be extracted from these cells. PBMC RNA will be
used in TCR and BCR analysis and compared to RNA samples from salivary glands.
This will give us the opportunity to compare in parallel systemic versus tissue
levels.

1-time blood withdrawal (54 ml).
1-time lower lipbiopsy: 1 extra lipbiopsy in patients having already a
diagnostic biopsy in daily patient care.