0022-1554/90/$3.30
The Journal
of Histochemistry
Copyright
1990
t
by The
and
Vol. 38, No.
Cytochemistry
Histochemical
Society,
Original
Subcellular Localization Human Salivary Glands’ MARGHERITA Dipartimento Received
COSSU,2
for publication
Universita’
October
1, 1989
RIVA,
di Caglthri
and
in revised
form
February
We investigated the subcellular localization of ABH antigem in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monodonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B,
group
substances
glycolipids
with Their
bound
to a protein
activity or lipid
of many Not
secretor
status
the model
all
by
the
independent
mucous
and
as does
all
the
are secreted Szulman of
ABH
and
to ascribe
and
digestive
mucosa,
tissues,
originate
it would
antigens the
in
human
production
apparatus.
tissues
whereas
authors,
MARIA
methods
have
provided
that
in the
mucous
are located glands.
However,
shown the occurrence The demonstration
some
these
of ABH antigens of secretory BGS
and
28,
1990;
of major
mucus-
38:1165-1172, KEY
croscopy;
ABH
by the
della Pubblica 2 Correspondence fologia,
Universita’
Consiglio
Istruzione. to: Prof.
Nazionale Margherita
di Cagliari,
Via
delle
and
Cossu, Porcell
and
Dipartimento 2, 09124
gland;
Immunocytochemistry;
out only in labial
(14,15).
In the
immunoreactive,
latter,
labeled.
resin
as embedding
glands
mucous
whereas
were
Electron
in the
Moreover, to the
mi-
owing
droplets,
These
distribution
sublingual, and minor method on specimens
LR White
ofhuman
which
compound
have
of BGS
to show,
bodies,
considerations
buccal embedded
droplets
failed
filamentous
product
were
mature
et al. (9,10)
secretory
the cytochemical
the
goblet
cisternae
to the use ofthe
the
vestigate
in intestinal Golgi
only
Nakajima
mucous
and and
former
possibly
medium,
(9,10)
droplets
also represent a characteristic mucous glands (13,22,23).
led us to in-
in submandibular,
glands applying an both in LR White
immunogold resin and
in
Epon.
and
Materials
for
the
anesthetized
Cagliari,
the
an-
have
cells. has
Ministero
di CitomorItaly.
Methods
group
removal with
on the
on their
minor
also
and ductal microscopy
Ricerche
(9A1828).
antigens.
carried
cells
sodium
solution
containing
0.1 M cacodylate
fixed
buffer.
solution in graded
After
of 0.2% acetone
resin.
Ultra-thin
sections
Finally,
treated
status
belonged to group for
glutaraldehyde
tetroxide
buccal
glands
with
The clinics of the
to blood AB.
sur-
atropine
patients group
All samples
and
provided but
not
A, four
examined
to had
microscopy.
rinsing in the uranyl and
Darmstadt, FRG). Other mixture, were dehydrated
for immunostaining.
were
group
immediately
1.25%
2 hr in 1% osmium
White
one
by light
were
three
and succinylcholine.
blood
0, and
and
years, who were undergoing
Patients
Six patients
appearance
Specimens
Merck, dehyde
ABO
sublingual,
32-74
pentothal
status.
B, 11 to group
three
aged
of tumors.
serological
secretor
a normal
hydrated Supported
9. 1990
1990)
an aqueous
I
March
Salivary
WORDS:
data
ABH
(1,5,7)
in serous by electron
accepted
and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution ofABH antigens, depending on the type of gland. (J Histochem Cytochem
gery
immunogold that
studies
in
S. LANTINI
Specimens of 18 submandibular, were obtained from patients
distribu-
to the
evidence
endpieces of
BGS
immunofluorescence,
lectin-HRP, further
the
ABH
endoderm,
immunofluorescence BGS
using
it is
Because
expected
ofwater-soluble
Later
(1,6,7,9,10),
by
to
antigens
from
be
the
Se. According
derivatives.
glands
of gly-
secretions,
Substances
Italia.
in addition
form,
ofABH
endodermal
mesodermal
salivary
plasma
glycolipid
gene
in
mixed
to the
their
gene
Se
body sugars
take the form
in
(11), the expression
lectin-gold,
tigens
BGS
on the structural
immunoperoxidase,
salivary
they
have
and
into saliva by secretor subjects only. (20,21) was the first to map the histological
tion
secreting
in the
and
of a few
linked
by Oriol in ecto-
the
tissues
are
chiefly
ofsecretions
depending
is controlled
human
BGS
types,
individuals
H are glycoproteins
in a sequence
molecule.
cell
proposed
in
resides
in the composition
coproteins.
A, B, and
distribution
antigenic
membranes
Se
(BGS)
a wide
fluids.
whereas
and
been
Introduction Blood
Group
Caglthri
09124
1165-1172, 1990 Printed in USA.
Article
of Blood
ALESSANDRO
di Citomorfologia,
8, pp.
Inc.
3 hr at room and
in buffer, same
buffer
acetate.
embedded
specimens, in graded were
collected
they were stained
temperature
in a
1% paraformaldehyde
The
they
were
and
placed
for
overnight
samples
in Epon
in
post-fixed were
(Glycide
then ether
in de100;
after fixation in the same alacetone and embedded in LR on nickel
grids
with uranyl
and
acetate
processed
and bis1165
Downloaded from jhc.sagepub.com at FLORIDA INTL UNIV on June 14, 2015
1166
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..
(
;
.,
1
LOCALIZATION
muth
OF
subnitrate
ABH
(12).
ANTIGENS
The
grids
were
IN
SALIVARY
examined
1167
GLANDS
in aJEOL
electron
1005
Finally,
microscope.
types,
Immunostaining. munogold
For
staining
clonal
technique,
antibodies
as secondary particles
specific
ing was performed
ment, other
results
and
gold-conjugated
control
sections
control
of the
anti-mouse
were
applied
antiserum
the
mouse
Santa
Barbara,
CA)
with
colloidal
Belgium).
The
immunostain-
to the schedule
the incubation
anti-ABH
serum
at a dilution
incubated
omitting
sera
of 1:30. the
serum
The
antibodies
In each
in the
experi-
antibody.
was used
ervation
In
antigens
were
revealed
to
the
relationship ment and density
was
lumina.
intracellular
sometimes
3), whereas
and
membranes
mens
their
were
we found
particle
within
Golgi and
unreactive
(Figure
number
ofunreactive
by their
morphological
type
dc-
i.e. , A antigen
donor,
AB
subjects.
of A and B antigens
cells
subjects
and
Otherwise,
well as from tribution
A, B, and
with
the
rized
as follows.
donor’s
from
tribution type
entirely
showed
cells
cently
demonstrated H antigen
donors,
with
Such
variations
an identical 2 and
the
and
distribution
labeled
the mucous
4).
within
In sublingual
droplets
from AB 0
as
disand
type
can
be summa-
buccal
glands
Nakajirna
Therefore,
our
antigenic and
quite
similar A and
(Figure
to
that
of
in
were
weakly
6).
submandibular (Inset) A labeled
and
Nakajima
et al.
folds
reof
(9)
have
ofmucous
cells
glands labeled
and
Epon
isolo-
from or
that
and
sites.
that
leave
It should
secretory
are reachable
embedding
et al. (9,10) by BGS
intact
be noted,
BGS
maintain
by antibodies
without
etching
even
after
or deplasticiz-
have
in the
late
suggested
that
phases
of granule
on labial glands In our specimens,
the antigenic
activity
maturation,
be-
only the mature mucous no relation was observed
between the maturative phases of the mucous droplets, deduced from their number, size, and arrangement, and the distribution pattern oflabeling. label deposition observations maturation
In addition, the frequent in Golgi cisternae indicates,
made phases.
by Roth
An unexpected negligible
glands
we believe
and Roth et al. (15) used nonresins as embedding media.
acrylic
demonstrate
cause in their experiment granules were labeled.
result
amounts
that
although
ing
BGS
H,
show
buccal
mucous
BGS
in their
these
type
labeled
One
the
capacity
in early
B donors
H antigen
opposite
possible
glands
of some with the
are present
A and
B, whereas
cells ofall
differ
in type
glands.
observation in keeping
that
abundant
of A and
and they
et al. (14), was that
glands
the sublingual
the but
was true
of
interpretation
are capable
to convert
is
of produc-
it into
A or
B
antigen. However, such considerations are not borne out by the literature, except for the demonstration that 70% of salivary BGS A comes from sublingual and minor mucous glands (8). Another possibility
gland Golgi
membranes
antigens
et al. (9)
and
results
is that
in all
Figure 1. Submandibular gland from a type 0 subject, embedded in LA White resin and treated with anti-H tural features of the cells are not well preserved. Original magnification x 8000. Bar = 1 pm. Figure 2. Epon-embedded arrows, Golgi apparatus.
an-
et al. (15) have
plasma
binding
activity
submandibular
droplets
abundant
described
B antigens
find-
group
treatments.
Nakajima
of H anti-
buccal
previous
the
rather,
mucus samples
ing
gland
and
displayed
and
mu-
However,
of blood
Roth
membrane-bound
both
the
the relative
pattern
the
labeling.
membrane
applied;
tissue
which
from
or alter
that
osmication
from
the secretory
cells,
in the basolateral
osmicated their
faint or in some cases no reactivity was seen 5). In submandibular glands from A, B, and cells appeared rich in H antigen, with a dis-
whereas
unreactive
However,
on
located
experiments,
results
method,
absorptive
techniques
remove
in fact,
2
B and
glands
our
data
are chiefly
localization
A antigen and
In our
between
a post-embedding
goblet
speci-
in glands from
in
sublingual,
strongly
whereas (Figure mucous
pattern 0
Using
histochemical
antigens
site of antigen
cytological
the glycoproteinaceous
on the blood
only those
greatly
group.
(Figures
in
present
donors.
varied
serological
A and B donors
gous antigen, for H antigen AB subjects,
was
AB type
antigens
appeared
ofmucous
only
Submandibular,
donors
which
was present
B antigen
H antigen
of ABH
was dependent
to the
ABH
glands.
specific
appear
regard
tigens.
that no
organdIes
features
ofsalivary
the
discrepancies with
is acquired
expression
of the
A and
0 type
cells
were
previous
that
our failure
3). In most mucous
confirm
to reveal any membrane labeling may be ascribed to some preparation steps, such as osmium post-fixation and Epon embedding,
(Figures cell
8).
histochemical
appeared
cisternae
7 and
several
deposition;
all other
within
(Figures
Epon.
cells.
The
gen,
of gold
droplets
granules
ABH imdiscrepanimmuno-
to the mucous
mucous
bodies
always
a variable
confined
in
demilunes,
were confined
of labial glands. By contrast, we have never observed munoreactivity associated with plasma membranes. Such cies probably are not attributable to the different
after
embedded
results demonstrating
mucous
found
with
their size, number, and arrangeIn addition, a moderate labeling
filamentous
not distinguishable
reactive
site
detected
the
examined,
1). Therefore,
The
was observed between the label distribution.
and
glands
routinely
was usually
2) and
specific
(Figure
samples
immunoreactivity (Figure
were
use
in all
secretory
of all blood
acini,
the gold particles
of their
droplets
some
in place
five subjects who we presume are nonwere negative in all experiments. in LR White resin showed poor pres-
features
decided
The to be
cous ings
of cytological we
ments
present
intestinal group
trials
component
(3,6,10,20,21)
antibody.
the exception of those from secretors. Control sections The specimens embedded
where
specimens
ofserous
Discussion the
Results Blood
ducts,
in cells
gold
at a dilution
primary
(Dako)
the clear
gland
was seen
and
with
with primary
using
non-immune
supplied
H labeling
and intercalated
im-
mono-
1gM coated
Beerse,
obtained
a mouse
we
(Dako;
according
were
sections
primary
antigens
but extending
best
BGS
as primary
Uanssen;
essentially
antiserum,
to 24 hr. The of 1:20
a goat
diameter)
of
using
for ABH
antiserum (15-nm
J anssen
demonstration
in a few submandibular
some
salivary
antibody.
from a type 0 subject, treated with anti-H antibody. Only mucous apparatus. Original magnification x 8000; Inset x 16,000. Bars
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glands
the
negative
Immunolabeling
cells are intensely = 1 pm.
cells,
is evident
labeled.
butthe
the
H-rich
ultrastruc-
SC, serous
cell;
.
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Figure
4. Epon-embedded
.
sublingual gland from a type B subject, Original magnification x 16,000. Bar buccal
gland
from
a type 0 subject,
1
y
4 Figure points
J
r
=
treated with anti-B 1 pm.
treated
with
anti-H
-
antibody.
antibody.
The filamentous
Original
bodies
magnification
1168
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(FB) are always
x 8000.
Bar
=
devoid
1 pm.
of labeling.
Arrow
LOCALIZATION
OF
ABH
ANTIGENS
IN
SALIVARY
GLANDS
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Figure 5. (a) Epon-embedded sublingual gland from a type A subject, Original magnifications: a x 8000; b x 8500. Bars = 1 pm.
treated
with anti-A
antibody.
L, lumen.
(b) The same
gland
treated
with
anti-H
antibody.
1169
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COSSU,
1170
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.
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Figure 6. (a) Epon-embedded magnifications: a x 16,000;
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1
J
4 treated
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(b) The same
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gland
treated
with anti-H
antibody.
Original
LOCALIZATION
OF
ABH
ANTIGENS
IN
SAUVARY
GLANDS
1171
..
Figure 7. Epon-embedded submandibular gland from the type AB subject, treated with anti-H antibody. Seromucous cells. Original magnification x 16,500. Bar = I pm.
.:,.
..4 .
,-.,-
.-
.
4..’.
:
.
-:
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Figure 8. Epon-embedded submandibular gland from a type B subject, treated with anti-H antibody. Intercalated duct. Original magnification x 8000. Bar 1 pm.
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..,
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1172
COSSU,
cells,
and
early, theory
intermediate, has been
the
of ABH
A- or B-rich and proposed
antigens
it appears
described
unsuitable
we consider
Another structures
have
the
large
that
they
soluble
BGS.
examined.
are endowed
with
neutral
However,
al. (5) have visualized membranes of serous
sali-
for acid
mucins
the mucous
more
as oligosaccharide presence of BGS
recently
Hamper
and,
unlike
Our
results
(2).
granules
in that
Y, Ishitani
antigens
and glycosidase
Acta
M, Okamura
digestion.
J Histochem
peroxidase
9.
their
to the classical with the wateret al. (1) and
conjugates.
10.
contributions ofwhole saliva
M, Ito N, Nishi K, Okamura of blood group substances complexes. J Histochem
elements
and
is not
intercalated
we have found
surprising,
duct
gold
ductal
particles
because
cells,
In some
within
it is well
cells of human
salivary
but
submansecre-
known
glands
that
dowed with mucins that share some histochemical features with those of mucous cells (2,17,18). It is interesting to note that the distribution pattern of BGS in our specimens parallels that of the sulfomucins as identified in histochemical experiments (2). It is conceivable that salivary BGS belong content
to this class of substances, has been demonstrated
because in salivary
a considerable BGS (19).
andMrA.
Cadaufortheir
va/u.
K, CaselitzJ, Seifert G, Seitz R, Poschmann A: The occurrence ofblood group substances (A, B, H, Le-a, Le-b) in salivary glands and salivary gland tumors. An immunohistochemical investigation. J Oral Pathol 15:334, 1986
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Auger DW, Paterson KL, Rowley PSA: Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy. Histochem J 19:555, 1987 K,
Hirota
T: Localization
formalin-fixed, paraffin-embedded creas with lectin-HRP conjugates. 1985
related
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Biochem
Soc Trans
the of intestinal
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human Acta
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Histochem
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and pan-
Cytochem
18: 644,
20.
trans-tubular goblet cells.
observations
con-
19:105,
on human
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P, Watkins WM: group A gene.
Subcellular Biochem
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Spicer 5: Light microscopic histochemical detection of sugar residues in secretory glycoproteins of rodent and human tracheal glands with lectin-horseradish peroxidase conjugates and the galactose oxidase-Schiff sequence. J Histochem Cytochem 31:391, 1983 Wilborn salivary
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Structural and histochemical Ala J Med Sci 5:180, 1968
Sirigu P. Parodo G, Pilato D: Rilievi istochimici maggiori dell’uomo. Rass Med Sarda 76:655,
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AE:
The
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P. Watkins WM: Immunolocalization 1,3N-acetyl-galactosaminyltransferase
RothJ, Greenwell ucts of the blood
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AE: The
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Cited
Nishi
16.
18.
1. Hamper
N,
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Greenwell
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We are gratefulto Ms S. Bernardini-Foddis able technical assistance.
3. Ito
15.
sulfate
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