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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RIVA,

LANTINI

#{149}i’_ .. ..

.

_; f1)

.

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.

.

..L

.

-

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.4

.

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t-&.

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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

Downloaded from jhc.sagepub.com at FLORIDA INTL UNIV on June 14, 2015

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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45

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...

.

.

.

.

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........

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-

.

I

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. .

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I

I

#{149} . #{149}

:

y

14

IL:

-p

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r

#{149}A ? :

AT 3. Epon-embedded to Golgi apparatus.

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

.F

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-

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‘

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,

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,;

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-,

I

I

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

‘

RIVA,

LANTINI

-

.

.

..4

..4..

.i

‘j”

d4

..

.

:

.

..,

.

..,

...

. ...,.

.

-.

-. .

.

..:.1

-..

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.

.

.

.,I.

.

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I

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I

.

.

j .

.

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,....,

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?

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F,

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rr

%

: !:

Figure 6. (a) Epon-embedded magnifications: a x 16,000;

-.

T

.::

#{149}

.

!

4j

IL.

.-

(

:

:;

iyIr

)*

-

r

.:

c:

S

#{149}1I1 submandibular gland from a type A subject, b x 17,000. Bars = 1 pm.

1

J

4 treated

with anti-A

antibody.

(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..’.

:

.

-:

;

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.

I

::.

.#{149}:

ft

.,.

.

,

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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

2. HarrisonJD,

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

antigens.

Biochem

Soc Trans

the of intestinal

of blood

human Acta

salivary

Histochem

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antigens

in

glands

and pan-

Cytochem

18: 644,

20.

trans-tubular goblet cells.

observations

con-

19:105,

on human

ofbbood group and blood group

network of the Golgi apparatus Eur J Cell Biol 46:105, 1988

P, Watkins WM: group A gene.

Subcellular Biochem

distribution

Soc Trans

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ofthe

prod-

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BA,

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

WH: glands.

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,

Szulman

AE:

The

histological

stances in man as disclosed and its relation to A and 21.

15:596,

rapid staining method for enhancing the treated with uranyl acetate.J Microscopic

P. Watkins WM: Immunolocalization 1,3N-acetyl-galactosaminyltransferase

RothJ, Greenwell ucts of the blood

Schulte

tissue

sulle

diversity

ghiandole

Szulman

AE: The

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of the

by immunofluorescence. B antigens. J Exp Med

histological

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B, Riva A: Salivary glands. man oral embryology and histology.

salivari

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19. Sonju T, Rolla G: Further studies of the chemistry of a human blood-group group substance. Acta Pathol Microbiol Scand 83:215, 1975

Cited

Nishi

16.

18.

1. Hamper

N,

and

Greenwell

17. ShacklefordJM, in mammalian

We are gratefulto Ms S. Bernardini-Foddis able technical assistance.

3. Ito

15.

sulfate

Acknowledgments

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