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Archives of Oral Biology (2006) 51, 967973

www.intl.elsevierhealth.com/journals/arob

Salivary histatins in human deep posterior lingual


glands (of von Ebner)
Marco Piludu a,*, Maria Serenella Lantini a, Margherita Cossu a,
Monica Piras a, Frank G. Oppenheim b, Eva J. Helmerhorst b,
Walter Siqueira b, Arthur R. Hand c
a

` Degli Studi di Cagliari, Cagliari, Italy


Dipartimento di Citomorfologia, Universita
Department of Periodontology and Oral Biology, Goldman School of Dental Medicine,
Boston University, Boston, MA, United States
c
Department of Craniofacial Sciences, School of Dental Medicine,
University of Connecticut, Farmington, CT, United States
b

Accepted 24 May 2006

KEYWORDS
von Ebners glands;
Secretory granules;
Antimicrobial proteins;
Electron microscopy

Summary
Objective: Human saliva contains a family of low molecular weight histidine-rich
proteins, named histatins, characterised by bactericidal and fungicidal activities in
vitro against several microbial pathogens, such as Streptococcus mutans and Candida
albicans. They represent a major component of an innate host non-immune defense
system.
In an earlier study we described the distribution of histatins in the glandular
parenchyma of human major salivary glands, confirming that all human major salivary
glands are involved in the secretion of histatins into saliva. In the present study we
determined the expression and localisation of histatins in human posterior deep
lingual glands (von Ebners glands) by means of immunoelectron microscopy.
Design: Thin sections of normal human salivary glands, embedded in Epon resin, were
incubated with rabbit polyclonal antibodies specific for human histatins and successively with a gold conjugated goat anti-rabbit IgG used as secondary antibody.
Sections incubated with medium devoid of primary antibody or containing nonimmune serum were used as controls.
Results: The serous secreting cells represented the main source of histatins in the
glandular parenchyma of von Ebners glands. At the electron microscopic level,
labeling was associated with rough endoplasmic reticulum, Golgi complex and
secretory granules that represented the main cytoplasmic site of histatin localisation.

* Corresponding author at: Dipartimento di Citomorfologia, Cittadella Universitaria, Monserrato, Cagliari I-09042, Italy.
Tel.: +39 070 675 4058; fax: +39 070 675 4003.
E-mail address: mpiludu@unica.it (M. Piludu).
00039969/$ see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2006.05.011

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M. Piludu et al.
However, variability in the intensity of labeling was observed among adjacent cells.
Conclusions: The present results show for the first time that human von Ebners
glands produce and represent a significant source of histatins, supporting the hypothesis of their important role in preventing microbial assaults on the tissues in the
posterior region of the tongue and in the circumvallate papillae.
# 2006 Elsevier Ltd. All rights reserved.

Introduction
Salivary histatins contribute with other salivary
peptides and proteins, such as peroxidase, lactoferrin and lysozyme,1 to provide an important antimicrobial defense mechanism in the oral cavity since
they have been demonstrated to possess antimicrobial activities against several oral microrganisms.24
They represent significant constituents of a nonimmune innate host defense system that is believed
to play an important role in protecting oral tissues,
especially in childhood when efficacious immune
responses are not yet developed.
Although histatins have antibacterial activities,
they possess mainly fungicidal activities in vitro and
have been proposed as potential therapeutic agents
against opportunistic fungal human pathogens like
Candida albicans.5 C. albicans is one of the most
commonly encountered human pathogens, responsible for opportunistic mucosal and systemic infections in AIDS patients6,7 and in immunocompromised patients undergoing anticancer therapy or organ transplantations.
During the last 20 years histatins have been well
characterised biochemically and functionally.2,8
They are represented by a family of low molecular
weight histidine-rich proteins whose major members histatin 1 and 3 have been isolated from human
saliva and represent the products of two encoded
genes, HIS1 and HIS2, respectively.9 Although the
antifungal activity of histatins is well documented,
the mechanism by which they kill or inhibit the
growth of fungi is still not clear. It has been reported
that incubation with histatin 5 causes disruption of
the cell membrane and cell wall of C. albicans and
non-lytic loss of adenosine triphosphate.10 Recently,
the Trk1 potassium transporter was identified as the
pathway for ATP loss.11 Helmerhorst et al.12,13 have
shown that, after its internalisation, histatin 5 is
specifically targeted to the yeast mitochondria,
causing a loss of mitochondrial transmembrane
potential and inducing the generation of reactive
oxygen species (ROS).13,14
The studies cited above have established the
properties of histatins in vivo and in vitro, and their
physiological concentrations in human salivary samples collected from parotid, submandibular and
sublingual glands, highlighting the contribution of

each major salivary gland in the secretion of histatins in the oral cavity. Recent immunohistochemical
studies on histatin distribution in human major
salivary glands have confirmed that serous components of the glandular parenchyma represent the
main source of salivary histatins.15 However, relatively few data exist on the nature of secretion
products in human minor salivary glands, especially
concerning production and secretion of histatins.16
The small volume of saliva produced by individual
minor salivary glands, and the inaccessibility of
some glands to saliva collecting procedures, have
limited studies of the function and secretions of
minor salivary glands. In 1990 Azen et al.17 highlighted in rhesus macaque, as well as in mice, the
presence of mRNAs for several antimicrobial peptides, histatins included, pointing out the additional
role of von Ebner secretions in defence mechanism.
Although Spielman and coworkers18 developed a
technique for collection of secreted saliva from
human von Ebner glands and analysed some of the
proteins present, few additional studies have been
reported.
In order to investigate the involvement of minor
salivary glands in histatin secretion and to further
define histatin localisation, in this study we investigated their presence in human posterior deep
lingual glands (of von Ebner) by means of a postembedding immunogold staining method.

Materials and methods


Samples of normal von Ebners glands were obtained
from four consenting patients, three men and one
woman, aged 4660 years undergoing surgery at the
Otorhinolaryngology Clinic of the University of
Cagliari. All procedures were approved by the
human experimentation committee.
For light microscopic studies small pieces of the
samples were cut and fixed in a solution of 4%
formaldehyde in 0.1 M sodium cacodylate buffer,
pH 7.4. The tissue samples were rinsed with the
same buffer, dehydrated in ethanol and successively
embedded in Epon resin (Glycide Ether 100, Merck,
Darmstadt). One micrometer sections were collected on Super Frost slides and rinsed with phosphate-buffered saline (PBS) containing 1% bovine

Immunolocalization of histatins in von Ebner s glands


serum albumin (BSA) and 5% normal goat serum
(NGS) to block non-specific binding. Incubation with
rabbit polyclonal antibody to human histatin,
diluted 1:500 or 1:1000 in 1% bovine serum albumin
(BSA), was performed at room temperature for
90 min. ELISA characterisation studies of the affinity
purified
anti-histatin
antibody
preparation
employed in our studies have shown that it reacts
with histatin 3 and 5 and to a lesser extent with
histatin 1, but not with other purified salivary proteins such as PRPs and amylase, or with whole saliva,
which is devoid of intact histatins (data not shown).
After rinsing in PBS and distilled water, immunocomplexes were revealed by a silver enhancement
procedure. The sections were stained with Azure IIToluidine Blue, observed and photographed in a
Leica DMR light microscope.
For electron microscopy, small pieces of the same
specimens were fixed in a mixture of 3% paraformaldehyde0.1% glutaraldehyde in 0.1 M cacodylate buffer, rinsed in the same buffer, dehydrated in
ethanol and embedded in Epon resin.
Ultrathin sections collected on nickel grids were
treated with 1% BSA5% NGS in PBS or in high salt
buffer (HSB) to block non specific binding. The
sections were incubated overnight at 4 8C with
the same antibody described above, diluted 1:100
or 1:200 in 1% BSA5% NGS in PBS or in HSB. In
control sections the primary antibodies were
omitted or substituted with rabbit normal serum.
Furthermore, in order to assess the specificity of the

969
antibody used, some grids were treated with the
primary antibodies preincubated overnight at 4 8C
with histatin. After rinsing, the grids were incubated
for 1 h at room temperature with the secondary
antibody, a goat anti-rabbit IgG labeled with
15 nm diameter gold particles (Auroprobe EM G15,
Amersham International plc, Little Chalfont, England), diluted 1:50 in 1% BSA-PBS. The grids were
washed with PBS and then with distilled water,
stained with uranyl acetate and bismuth subnitrate,
and finally observed and photographed in a JEOL
100S transmission electron microscope.

Results
Morphology
The structure of the human von Ebners glands
observed in the present study was similar to that
reported in previous investigations.19,20 Their glandular parenchyma consisted mainly of tubulo-alveolar serous units whose ducts opened into the sulci of
circumvallate papillae.
Serous secretory cells exhibited spherical and
dense secretory granules, representing the most
prominent cytoplasmic feature, and a well developed rough endoplasmic reticulum (rER) at the cell
base and Golgi complex in the perinuclear region.
The duct cells varied from cuboidal to columnar
displaying desmosomes on their lateral surfaces and

Figure 1 Immunogold silver stain of 1-mm Epon sections of human deep posterior lingual gland showing histatin
localisation. Labeling (black silver deposits) is present in the secretory cells (arrows). Note that some acinar cells exhibit
variable labeling intensity or are devoid of reactivity (asterisks). The apical cytoplasm of some duct cells (D) also is
labelled (arrows). (Inset) Details of labelled secretory acinus. Bars = 20 mm.

970
hemidesmosomes on their basal surfaces. The rER
and Golgi complex were well developed, and mitochondria were numerous and randomly distributed
in the cytoplasm. There were no apparent differences in the structure of the glands from male and
female patients.

Immunocytochemistry
Specific histatin reactivity was observed at both the
light and electron microscopic levels at each dilution of the anti-histatin antibody used.
Light microscopic immunogold silver staining
revealed histatin reactivity in the secretory elements of the glandular parenchyma, however, significant variability of the histatin expression was
observed among serous secretory cells (Fig. 1). Most
of the acinar cells showed strong reactivity mainly in

Figure 2 EM immunogold labelling of thin sections of


human deep posterior lingual gland. Portion of acinar
secretory unit. Secretory cells display strong histatin
reactivity within their secretory granules (SG) that represent the main cytoplasmic feature. (Inset) Labeled secretory granules. Gold particles are uniformly distributed
within secretory granules. The acinar cell nucleus (N)
and mitochondria are unlabeled. L, lumen. Bars = 1 mm.

M. Piludu et al.
their secretory granules, whereas a smaller number
of acinar cells displayed weaker labeling or were
devoid of reactivity. Specific labeling also was
observed in the apical portion of some ductal cells.
No reactivity was detected in the control sections
when the primary antibody was omitted from the
labeling sequence or non-immune rabbit serum was
substituted for the primary antibody or when the
primary antibodies were preincubated overnight
with histatin before incubation with sections of
von Ebner tissues.
Electron microscopic immunogold labeling confirmed and extended the light microscopic investigations. Intense histatin labeling was observed
within the acinar secretory granules (Fig. 2). These
granules appeared to be the main source of histatin
reactivity in the glandular parenchyma. However,
differences in histatin expression were detected
among the secretory cells; in some cases, acinar
cells were devoid of labeling or exhibited reactivity
in only a few secretory granules (Fig. 3A and B). In
most cases, the rER and Golgi complex were labeled
(Fig. 4A and B), whereas the nucleus, mitochondria
and other cellular organelles were unlabelled.

Figure 3 EM immunogold labelling of thin sections of


human deep posterior lingual gland. (A and B) Some acinar
secretory cells contain heavily labeled secretory granules
(LSG) whereas adjacent cells contain poorly or unlabeled
granules (UG). L, lumen; N, nucleus. Bars = 1 mm.

Immunolocalization of histatins in von Ebner s glands

Figure 4 EM immunogold labelling of thin sections of


human deep posterior lingual gland. (A) Small regions of
acinar cell cytoplasm are shown. Labeling is associated
with Golgi-like structures (A, arrows) and rER-like structures (B, arrows). Bars = 0.2 mm.

Discussion
The morphological features of the glandular parenchyma of von Ebners glands have been investigated previously in humans, as well as in other
mammalian species.16,1924 Compared to other
minor salivary glands, von Ebners gland is unique
in that its secretions are serous in nature and enter
the oral cavity in association with the main sites of
taste receptors. In humans, von Ebners glands consist mainly of serous tubular alveolar secretory units
whose granules, located in the apical portion of the
cytoplasm, represent the most prominent feature.
The ducts of the glands open into the sulci of the
circumvallate and foliate papillae. Immunocytochemical and biochemical studies have revealed
the presence of several different secretory proteins
in the secretions of von Ebners glands in various
species. Some proteins, such as amylase,16,25 peroxidase,26 lysozyme and lactoferrin,27 PRPs,
statherin and histatins,17 are also secreted by other
salivary glands. In contrast, other secretory proteins, such as lingual lipase,2831 von Ebners gland
protein (VEGP)18,32 and Ebnerin33 are produced only
by von Ebners glands.

971
Our results show, for the first time in humans, the
presence and distribution of histatins in von Ebners
glands. This study makes a significant contribution
to further defining the nature of the secretory
products and assessing the serous nature of the
glandular parenchyma in human von Ebners glands.
Secretory granules, rER and Golgi complex display
histatin reactivity, suggesting for histatins a similar
secretion pattern to that observed previously in
major salivary glands.15 The presence of intense
histatin labeling within the secretory granules coincides with our previous findings on histatin localisation in major salivary glands, where the serous
components of the glandular parenchyma represented the main source of histatin production.
Our previous findings15 showed variability in the
intragranular localisation of histatin in the submandibular and sublingual serous cells, where the dense
cores of their secretory granules exhibited a greater
labeling density than the pale areas of the granule
content. In von Ebners glands, however, histatins
were found to be distributed uniformly within the
granules. This pattern was similar to that observed
in the parotid gland, suggesting that parotid and von
Ebners glands may share similar mechanisms and
pathways for processing, packaging and secretion of
histatins. However, variability of histatin expression
was observed in the parenchyma of von Ebners
gland, where some secretory cells were unlabeled
or contained secretory granules with different
levels of labeling.
It is well known that histatins originate from two
genes, HIS1 and HIS2, whose primary products
undergo proteolytic processing.9,34 One possible
explanation for the cell-to-cell variability in expression could be found in differential activation of the
two genes that code for the histatins. Although the
molecular mechanisms that lead to histatin gene
activation are not known, previous studies have
reported variations in salivary histatin levels in
different conditions. In normal healthy individuals
donor age seems to affect the concentration of
histatins in saliva.35 Daily variations in histatin level
in whole saliva were observed by Castagnola et al.,36
and differential levels of histatins were reported in
HIV-positive patients37 and in patients affected by
oral candidiasis.38
Due to their postulated role in the chemical sense
of taste, von Ebners glands have been the most
frequently studied minor salivary gland, especially
in animal models, where the nature of their secretory products have been extensively investigated.
Because VEGP is a member of the lipocalin protein
family, and has homology with odorant binding proteins, it has been suggested to function in the
regulation of taste sensation.32 Ebnerin, a much

972
larger protein than VEGP, is present the bottom of
the trenches surrounding circumvallate papillae and
also was suggested to function in taste phenomena.33 Moreover, recent immunohistochemical studies have demonstrated that von Ebners glands
secrete carbonic anhydrase VI, whose function has
been suggested to be the protection of taste receptor cells.39
Antimicrobial activity also appears to be an
important function of the secretions of von Ebners
glands. Earlier studies highlighted the presence of
several antimicrobial proteins and peptides in rhesus macaque and mouse taste-bud tissues, pointing
out the additional antibacterial and antifungal properties of von Ebners gland secretions.17,26,27 The
strong histatin reactivity found in these glands support the hypothesis of their important role in preventing microbial assaults on the tissues of the
posterior region of the tongue and especially on
the taste-bud tissue located in the sulci of circumvallate papillae.
In conclusion, the involvement of human von
Ebners glands in the production of histatins has
been demonstrated. The role of these glands in
the secretion of several antimicrobial peptides for
the protection of taste receptors and the posterior
region of the oral cavity seems clearly established.
Further work is required, however, in order to completely characterise the products and functions of
these interesting glands.

M. Piludu et al.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Acknowledgements
The authors wish to thank Mrs. S. Bernardini and Mr.
A. Cadau for their technical assistance. This investigation was supported by the Ministero dellIstruzione, dellUniversita
` e della Ricerca, I.Z.S. Sassari,
NIH/NIDCR grants DE05672, DE07652 and DE14950.

15.

16.
17.

18.

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