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Peptide-containing nerves in labial salivary glands in sjgren's syndrome.

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Objective. The presence and spatial distribution
of peptide-containing nerves in labial salivary glands
from 10 Sjogren’s syndrome patients were compared
with those in salivary glands from 7 healthy controls.
Methods. Immunoperoxidase staining was used
to demonstrate vasoactive intestinal peptide (VIP)immunoreactive (IR) fibers, postganglionic sympathetic
fibers containing the C-flanking peptide of neuropeptide
Y (CPON), and sensory fibers containing calcitonin
gene-related peptide (CGRP)and substance P.
From the Institute of Molecular Immunology, Hospital for
Joint Diseases, New York University Medical School, New York,
New York; the Department of Histochemistry, Royal Postgraduate
Medical School, London, United Kingdom; and the Department of
Anatomy and the Institute of Dentistry, Helsinki University, Helsinki, Finland.
Supported in part by grants from the Finnish Academy, the
Finska Lakaresallskapet, and the Perklen Foundation.
Y j o T. Konttinen, MD, PhD: Visiting Professor, Institute
of Molecular Immunology, Hospital for Joint Diseases, New York
University Medical School; Mika Hukkanen, BSc: PhD Student,
Department of Histochemistry, Royal Postgraduate Medical School;
Pertti Kemppinen, MSc: Visiting Researcher, Institute of Molecular
Immunology, Hospital for Joint Diseases, New York University
Medical School; Margaretha Segerberg, MD, PhD: Visiting Senior
Investigator, Institute of Molecular Immunology, Hospital for Joint
Diseases, New York University Medical School; Timo Sorsa, DDS:
Acting Consultant, Institute of Dentistry, Helsinki University;
Maria Malmstrom, DDS: Associate Professor, Institute of Dentistry, Helsinki University; Sam Rose, PhD: Senior Investigator,
Institute of Molecular Immunology, Hospital for Joint Diseases,
New York University Medical School; Silviu Itescu, MD: Senior
Investigator, Institute of Molecular Immunology, Hospital for Joint
Diseases, New York University Medical School; Julia M. Polak,
MD, DSc, FRCPath: Professor, Department of Histochemistry,
Royal Postgraduate Medical School.
Address reprint requests to Y j o T. Konttinen, MD, PhD,
Molecular Biology Laboratory, Department of Anatomy, University
of Helsinki, Siltavuorenpenger 20 A, SF-00170 Helsinki, Finland.
Submitted for publication April 16, 1991; accepted in revised form February 16, 1992.
Arthritis and Rheumatism, Vol. 35, No. 7 (July 1992)
Results. Acini, intralobular ducts, small arteries,
and postcapillary veins were richly innervated by VIP-IR
fibers, whereas CPON-, CGRP-, and substance P-IR
fibers were restricted to blood vessels. Peptide-containing
nerves were found surrounding, but not in the middle of,
the highly inflamed mononuclear cell areas.
Conclusion. This topologic distribution suggests
involvement of VIP-IR fibers in vascular, motor, and
secretory components of the reflex salivary secretion,
whereas the distribution and the vasoactive actions of
CPON, CGRP, and substance P suggest a role in the
regulation of the salivary gland circulation, and thus of
transcapillary flow. Excessive release may contribute to
a neurogenic inflammation. Local depletion and absence
of trophic neuropeptide stimuli may contribute to
acinar atrophy.
Aside from Sjogren’s syndrome, an autoimmune disease of unknown etiology characterized by
keratoconjunctivitis sicca, xerostomia, and focal adenitis, psychogenic and iatrogenic factors are also
common causes of “dry mouth.” These factors relate,
respectively, to inhibitory influences of higher central
nervous system control on salivary nuclei and to peripheral anticholinergic effects, and thus illustrate the role of
the autonomic nervous system in the control of reflex
salivary flow. The sialopenia of Sjogren’s syndrome,
however, has been generally attributed to autoimmune
destruction of the glandular parenchyma, the flow of
saliva being roughly proportional to the mass of the
secretory end-piece elements. More recently, it has
become evident that in addition to the integrated vascular, motor, and secretory control of reflex salivary flow,
the autonomic nervous system delivers trophic stimuli to
parenchymal tissue ( 1 4 ) .
Figure 1. Protein gene product 9.5 (PGP9.5) and synaptophysin (SYN)staining of labial salivary gland (LSG) from a healthy control subject
(A and B) demonstrate rich innervation, whereas LSG tissue from a patient with Sjogren's syndrome (C and D)demonstrates only a few nerve
fibers in the areas of inflammation. Immunoreaction with vasoactive intestinal peptide in LSG tissue from a patient with Sjogren's syndrome
reveals axon terminals and pretenninal varicosities over the entire salivary gland tissue (E), but few in the areas of inflammation (F).
(Avidin-biotin-peroxidase complex techniques.)
It has been shown that parasympathetic denervation or reduced reflex activation induced by a liquid
diet, but not prolonged blockade of muscarinic cholin-
ergic receptors by atropine, rapidly causes acinar
atrophy, which can be prevented by treatment with
vasoactive intestinal peptide (VIP) (2,3,7-10). Neu-
8 17
ropeptides have also been implicated in the regulation
of several other resident tissue cells and immigrant
inflammatory cells and are able to induce neurogenic
inflammation. Because of the possible role of the
peripheral nerves in the physiology and pathology of
salivary gland tissues, we sought to characterize various classes of nerves in labial salivary glands (LSG) in
patients with Sjogren's syndrome by using neuropeptides as markers for parasympathetic, sympathetic,
and sensory fibers.
Biopsies. We examined labial salivary glands obtained from 8 patients with primary Sjogren's syndrome and
2 patients with Sjogren's syndrome secondary to rheumatoid
arthritis or systemic lupus erythematosus. Biopsy samples of
normal LSG were available from 7 subjects who did not have
focal sialadenitis; these were used as controls.
From each study subject 5-8 LSGs were removed
and fixed in Zamboni's fluid for 6 hours. After washes in 15%
sucrose in 0.1M phosphate buffered saline, pH 7.4, the LSG
were embedded in Tissue-Tek OCT compound (Lab-Tek
Products, Elkhart, IN) and stored at -20°C until analyzed.
Antisera and immunostaining. To visualize total innervation of the labial salivary glands, 2 neuronal markers,
namely, protein gene product 9.5 and synaptophysin, were
used as described in detail elsewhere ( I f ) . Antisera to
synthetic VIP, the C-flanking peptide of neuropeptide Y
(CPON), tyrosine hydroxylase, calcitonin generelated peptide (CGRP), and substance P were raised in rabbits. The
specificity of these neuropeptide antisera has been confirmed
and reported previously (12-14). Substance P antiserum also
reacts with substance K and neuropeptide K, and therefore
detects tachykinin-like immunoreactivity. The specificity of
the peptide antisera was further assessed by incubation of
control sections with antiserum preabsorbed with an excess
(1 nmoleslml of diluted antiserum) of the corresponding
antigen. The VIP, CPON, CGRP, and substance P antigens
were all purchased from Cambridge Research Biochemicals
(Cambridge, UK).
To control for method specificity, we included the
following for comparison: omission of the primary antiserum, use of normal rabbit serum (diluted 1 :1,OOO), use of 2
irrelevant antisera (rabbit antigastrin, diluted 1:2,OOO, and
rabbit antipancreastatin, diluted 1:4,OOO),or omission of one
of the subsequent steps in the avidin-biotin-peroxidase complex (ABC) method. Immunoperoxidase staining was performed as described in detail elsewhere (1 1).
Features of normal labial salivary glands. Normal LSGs were innervated throughout, as revealed by
the 2 neuronal markers synaptophysin and protein
gene product 9.5 (Figure 1). Although the 2 markers
can demonstrate rich innervation of salivary gland
Figure 2. Vasoactive intestinal peptide-immunoreactive (VIP-IR)
axon terminals and preterminal varicosities (arrows) in a labial
salivary gland from a patient with systemic lupus erythematosus and
secondary Sjogren's syndrome. VIP-IR nerves are seen close to the
end-piece cells in the acini (a). (Avidin-biotin-peroxidase complex
tissue, they do not reveal the neuropeptide content of
the local nerves. Among all neuropeptides we studied,
VIP-immunoreactive (VIP-IR) nerves were the most
abundant and most widely distributed. They were seen
throughout the glandular parenchyma, often surrounding the end-piece cells in the LSG acini (Figure 2).
Thus, they were in the close vicinity of secretory
mucous cells and myoepithelial cells. In addition,
striated ducts and the more sparsely distributed intercalated ducts contained VIP-IR nerves in the periductal connective tissue.
In contrast to VIP, CPON-IR nerves were
much more sparsely distributed and were only found
around blood vessels. In the muscular wall of resistance blood vessels, CPON-IR fibers were seen to
accompany the vessels, forming a plexus. No
CPON-IR fibers were seen in relation to various
components of the tubuloalveolar glands themselves.
This does not exclude a role for the sympathetic
nervous system in reflex salivary secretion, because
staining for tyrosine hydroxylase, the rate-limiting
enzyme in catecholamine synthesis, and therefore a
useful marker for postganglionic sympathetic nerves,
was found to be more widely distributed and often
close to salivary ducts or acini.
CGRP-IR and substance P-IR fibers were
mainly seen in association with blood vessels, covering the same segments innervated by VIP-IR, CPONIR, and tyrosine hydroxylase-IR fibers. More rarely,
CGRP- and substance P-IR free nerve endings were
Figure 3. Vasoactive intestinal peptide-irnmunoreactive (VIP-IR)
varicosities are seen close to the salivary acini and ducts in labial
salivary gland tissue from the same patient as in Figure 2. There is
a focus (fJof inflammatory mononuclear cells in the center of this
field. where no salivary acini or VIP-IR nerves are seen, although
the focus contains VIP-IR fibers in the peripheral zones (pz).
(Avidin-biotin-peroxidase complex techniques.)
seen in various parts of the gland, including the
peritubular and periacinar tissue and in the periphery
of some inflammatory cell areas.
Features of labial salivary glands from Sjogren’s
syndrome patients. Neuroanatomy and peptide immunohistochemistry of the noninflamed areas of LSGs
from patients with Sjogren’s syndrome were similar to
those in normal LSGs. However, Sjogren’s syndrome
is characterized histopathologically by focal accumulation of inflammatory mononuclear cells, so-called
focal sialadenitis. Peptide-containing nerves were seen
in small infiltrates and in the peripheral zones of the
large infiltrates, but were absent from the central areas
of more extensive accumulations of inflammatory
cells. In such intensively inflamed areas, glandular
atrophy or absence of secretory parenchyma was also
evident (Figures 1 and 3). The specificity of the immunostaining was demonstrated by comparison with the
control sections.
Labial salivary glands are richly innervated, as
has been recently demonstrated by confocal laser
scanning microscopy of sections of normal tissue
stained with the 2 neuronal markers protein gene
product 9.5, a major noncytoskeletal, cytoplasmic
neuronal protein with ubiquitin C-terminal hydrolase
activity, and synaptophysin, a 38-kd calcium-binding
integral membrane protein of synaptic vesicles (1 1).
Our study shows that the major neuropeptide in such
local peripheral nerves is vasoactive intestinal peptide,
an octacosapeptide originally isolated from the porcine
small intestine and characterized by a potent vasodilatory action (15). VIP is present in neurons rich in
acetylcholinesterase and choline acetyltransferase
(16). Our results suggest that VIP-IR nerves in human
LSG innervate all components of the gland, including
the secretory end-piece cells, myoepithelial cells, salivary ductal cells, and the blood vessels. The topologic
distribution of VIP suggests an important role for this
neuropeptide in an autonomously controlled, integrated, reflex salivary flow. This is composed of
that the
and Secretory responses act in concert to produce
adequate site- and time-specific salivation.
Comparison of the LSGs from the 10 Sjogren’s
syndrome patients with the LSGs from the 7 normal
control subjects without focal sialadenitis revealed a
clear and unusual pattern of innervation in the patients
with Sjogren’s syndrome: VIP and other neuropeptide-IR nerves were absent from the areas of intense
inflammation, which are also the areas of the so-called
acinar atrophy. This structural change, rather than any
neurally mediated functional change(s), is regarded as
a cause of decreased salivary flow in Sjogren’s syndrome. It is therefore of interest that attention has
more recently focused not on the immediate effects of
neuropeptides on reflex salivary flow, but on the
longer-lasting influences of trophic neuropeptides.
Parasympathetic denervation by severing nerves or by
ligating ducts leads to acinar atrophy, which according
to preliminary observations, can be prevented by
administering VIP (2,3,7-10). Prolonged treatment
with the anticholinergic muscarinic receptor blocker
atropine does not lead to this type of atrophy, which
suggests that this effect is mediated by nonclassic
neuropeptide mediators that are probably derived
from the parasympathetic nerves (2,3). Neuroimmunohistochemical localization of VIP-IR nerves would
allow direct delivery of trophic peptides to acinar cells
in normal and mildly inflamed glands. However, glandular atrophy could result from the absence of such
influence, as deduced from the mapping studies, in the
intensely inflamed areas.
Neuropeptide tyrosine (NPY) is a 36-amino
acid polypeptide initially isolated from pig brain. It is
known for its potent vasoconstrictor activity. NPY has
been localized in neurons that contain tyrosine hydroxylase (a cytoplasmic enzyme) and dopamine-p
hydroxylase (an intravesicular enzyme) and, not surprisingly, in norepinephrine-containinggranules (17).
Minth and coworkers, in 1984, noticed a C-flanking
peptide in preproNPY (1 8). The deduced amino acid
sequence of the precursor suggested that there were 2
sites of proteolytic processing, which would generate 3
peptides having 28 (signal peptide), 36 (NPY), and 30
(COOH-terminal peptide or CPON) amino acid residues (18). In contrast to VIP-IR fibers, CPON-IR
nerves and “afferent” substance P- and CGRP-IR in
human LSG seem only to innervate blood vessels.
This topologic distribution suggests that these neuropeptides may be mainly involved with the vascular
component of the reflex salivary flow. Interestingly,
tyrosine hydroxylase-IR nerves were more widely
distributed than were those showing CPON immunoreactivity, which may suggest that only a portion of
the postganglionic sympathetic fibers in human LSG
contain both CPON and norepinephrine.
Due to multiple effects of neuropeptides on
cellular immune and inflammation processes, it can be
speculated that neuropeptides might contribute t o the
focal sialadenitis. Such speculations have been presented for other diseases, such as rheumatoid arthritis
(19). Interactions between neuropeptides and inflammatory cells might occur at the periphery of focal
mononuclear cell infiltrates. On the other hand, the
absence of neuropeptides in the central areas might
deplete normal salivary gland tissue of important
trophic stimuli. Neuropeptides might thus contribute
to the pathogenesis of Sjogren’s syndrome in two
ways. In addition to their role in the regulation of the
reflex salivary flow, they may impart a neurogenic
component to the focal adenitis and they may contribute to the acinar atrophy.
We (YTK, PK, MS) are grateful to Dr. Robert J.
Winchester for giving us an opportunity to spend a research
sabbatical at his molecular immunology laboratory.
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containing, syndrome, nerve, labial, sjgren, gland, salivary, peptide
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