IL-1
and IL-6 in mouse parotid acinar cells:
characterization of synthesis, storage, and release
Naoko
Tanda1,2,
Hiroe
Ohyama3,
Midori
Yamakawa1,2,
Maria
Ericsson4,
Takanori
Tsuji3,
Jim
McBride3,
Aram
Elovic3,
David T. W.
Wong3, and
Gary R.
Login1,2,3
3 Department of Oral Medicine
and Diagnostic Sciences, Harvard School of Dental Medicine,
1 Department of Pathology and
2 The Charles A. Dana Research
Institute, Beth Israel Deaconess Medical Center and
4 Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02215
 |
ABSTRACT |
Synthesis, storage, and secretion of the
proinflammatory cytokine interleukin-1
(IL-1
) and the
anti-inflammatory cytokine IL-6 have not been established in normal
exocrine gland secretory cells. Parotid glands and isolated acinar
cells prepared from BALB/c mice were homogenized for RNA isolation and
reverse transcription-polymerase chain reaction (RT-PCR). IL-1
and
IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on
supernatants prepared from mouse parotid acinar cell (MPAC)
preparations unstimulated or stimulated between 0 and 10 min with
10
5 M norepinephrine at
37°C. MPACs were fixed in paraformaldehyde, frozen sectioned for
light and electron microscopy, and labeled with antibodies to IL-1
and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in
situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6
(614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant
increase in IL-1
(P < 0.03) and
IL-6 (P < 0.01) release into
supernatants by 10 min that paralleled the time course of amylase
release. In situ hybridization showed the presence of transcripts for
IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1
and
IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows
that MPACs synthesize IL-1
and IL-6 and release these cytokines from
their granules after
- and
-adrenergic stimulation.
reverse transcription-polymerase chain reaction; immunoelectron
microscopy; exocytosis; secretion; parotid gland; cytokines
 |
INTRODUCTION |
RECENTLY, THE EPITHELIAL cells in salivary glands from
patients with Sjögren's syndrome were shown to produce the
proinflammatory cytokine interleukin-1
(IL-1
) and the
anti-inflammatory cytokine IL-6 (4, 15, 21). In fact, parotid saliva
from patients with Sjögren's syndrome contains up to a 40-fold
increase in the concentration of IL-1
and IL-6 compared with
patients without Sjögren's syndrome (15, 21). Furthermore,
IL-1
is a known chemoattractant of leukocytes (5). Taken together,
these data suggest that salivary gland epithelial cells may participate
in the pathogenesis of autoimmune sialadenitis by directing the
accumulation of lymphocytes (i.e.,
CD4+) in diseased glands (15).
However, to understand the pathophysiological responses of IL-1
and
IL-6 in salivary gland pathology, we need first to understand what
role(s) these interleukins have in the saliva of normal individuals.
IL-1
and IL-6 may be physiologically relevant to the maintenance and
function of tissues in the upper gastrointestinal tract. Although no
lethal systemic effects have been observed in mouse knockouts for IL-1,
it is associated with regulation of carbohydrate metabolism in rodents
by a direct physiological effect on beta cells in the endocrine
pancreas (26, 41). For example, chronic administration of IL-1 has been
shown to prevent diabetes in nonobese diabetic mice (25). Studies (27)
on cultured tissues show that IL-1
increases mucus production and
chloride flux in intestinal epithelial cells and increases acute-phase
protein synthesis in hepatocytes.
IL-6 is essential for an optimal acute-phase response after tissue
damage (24, 28) and for an optimal immune response to viruses and
bacteria (28). IL-6 is also essential for mucosal immunoglobulin A
(IgA) production (34). Several lines of converging evidence from recent
work (10, 31) have further established that IL-6 is an essential and
irreplaceable component of the early signaling pathways leading to
liver regeneration. Interestingly, sialoadenectomy in rats decreases
the hepatic regenerative response (31).
Mechanisms of secretion of IL-1
(14) and IL-6 (36) have been
described for hematopoetically derived cells but not for normal
salivary gland epithelial cells or, for that matter, epithelial cells
from other exocrine organs. In this study, we show that normal mouse
parotid acinar cells express the mRNAs for IL-1
and IL-6 and store
these two interleukins in their secretory granules. We further show
that these cytokines are released in response to stimulation by the
,
-adrenergic agonist norepinephrine.
 |
MATERIALS AND METHODS |
Animal care guidelines.
The experiments were approved by the Committee on Animal Research at
Beth Israel Deaconess Medical Center and by the Animal Management
Program at Harvard Medical School. These animal management programs are
accredited by the American Association for the Accreditation of
Laboratory Animal Care, meet National Institutes of Health (NIH)
standards as set forth in the "Guide for the Care and Use of
Laboratory Animals" [DHHS Publication No. (NIH) 86-25, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD
20892], and accept as mandatory the Public Health Service
"Policy on Humane Care and Use of Laboratory Animals by Awardee
Institutions" and NIH "Principles for the Utilization and Care of
Vertebrate Animals Used in Testing, Research and Training." The
animals were housed in the animal care facility at Beth Israel
Deaconess Medical Center.
Saliva collection.
Male mice (18-20 g and 21-28 days old) were fasted overnight
and anesthetized with pentobarbital sodium (50 µg/g body wt). Whole saliva (~1 ml in 30 min) was collected from each mouse
injected subcutaneously with pilocarpine (1.5 µg/mg body wt) (Sigma
Chemical, St. Louis, MO).
Harvest and dispersion of mouse parotid acini.
Male mice were anesthetized as described above and euthanized by
severing the abdominal aorta. Freshly harvested glands from three mice
per experiment were selected for secretion studies, light and electron
microscopy, in situ hybridization, or RNA analysis. Acinar cell
isolation was done using a standard collagenase digestion method as previously described (33). Cell preparations consisted of
5-30 acinar cells per aggregate.
Cell handling and stimulation.
Cell viability was >90% determined by trypan blue dye (Sigma
Chemical) exclusion. The acinar cell aggregates were diluted in minimal
essential medium (MEM) with Earle's balanced salts and 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) buffer (Life Technologies, Gaithersburg, MD) to a
final hematocrit reading of 2% [cell suspension aliquoted in
75-mm capillary tubes (Drummond Scientific, Broomall, PA) and
centrifuged for 3 min in a hematocrit centrifuge (Clay Adams, New York,
NY)], corresponding to
~106 cells/ml. Cell
suspensions were maintained at 37°C (i.e., up to 10 min) until
stimulation. Cells were stimulated with
10
5 M norepinephrine (Sigma
Chemical) in MEM containing
10
7 M ascorbic acid at
37°C in an Erlenmeyer flask in a gyrorotary shaker (New Brunswick
Scientific, Edison, NJ). At the precise time point (0, 2, 5, and 10 min
after stimulation), 1.2-ml aliquots of acinar cells in suspension were
added to a 12-ml syringe (Monoject, St. Louis, MO) that was connected
to a 3.0-µm filter (Millipore, Bedford, MA). We collected 0.9-ml
aliquots of supernatant either for IL-1
or IL-6 and 0.2-ml aliquots
of supernatant for amylase in separate tubes and immediately froze them
at
70°C. At each time point, 100-µl aliquots of cells in
suspension were collected to determine total amylase. Unstimulated
acinar cells from the same mouse harvests were handled identically and
in parallel to stimulated aliquots at each time point.
RNA isolation and reverse transcription-polymerase chain reaction
confirmation by specific restriction enzyme digestion of
IL-1
and IL-6.
Total RNA was isolated from two whole, mouse parotid gland preparations
(300 µg RNA/gland) and one dispersed mouse parotid acinar cell
preparation (300 µg RNA/from 6 glands) using the guanidine isothiocyanate method (12). Then the RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the detection of
IL-1
and IL-6 mRNAs using rat-specific primers for IL-1
and IL-6.
RNA from freshly excised mouse parotid gland or dispersed parotid acini
was also isolated. The RT-PCR reaction was performed as previously
described (30). The efficiency of rat primer homology to mouse was
determined by oligo primer analysis software (National Biosciences,
Plymouth, MN). Rat IL-1
primers included the 22-nucleotide sense
primer 5'-AAA TGC CTC GTG CTG TCT GAC C-3' (nucleotides 474-495) and the 24-nucleotide antisense primer 5'-CTG CTT
GAG AGG TGC TGA TGT ACC-3' (nucleotides 787-810). Rat
IL-1
primers used to detect mouse IL-1
signals in the mouse
parotid gland by RT-PCR are 91% (20/22) and 96% (23/24) homologous to
that of the mouse for 5' primer and 3' primer, respectively
(19). Rat IL-6 primers included the 22-nucleotide sense
primer 5'-CAA GAG ACT TCC AGC CAG TTG C-3' (nucleotides
1-22) and the 25-nucleotide antisense primer 5'-TGG CCG AGT
AGA CCT CAT AGT GAC C-3' (nucleotides 1-25). Rat IL-6
primers used to detect mouse IL-6 signals in mouse parotid gland by
RT-PCR are 95% (21/22) and 88% (22/25) homologous to that of the
mouse for 5' primer and 3' primer, respectively (20). The
amplified products were visualized on an ethidium bromide-stained 2%
agarose gel. Confirmation of product identity was accomplished by
specific restriction enzyme digestion of the PCR products using
Pvu II (New England Biolaboratories,
Beverly, MA) for IL-1
and Bsr I
(New England Biolaboratories) for IL-6.
In situ hybridization.
In situ hybridization on 5-µm frozen sections (
20°C)
(CM1800, Leica, Wein, Austria) was performed using
35S-labeled sense and antisense
mouse specific cytokine riboprobes under conditions previously
established in our laboratory (30). IL-1
plasmid DNA was donated by
Dr. Patrick Gray (ICOS, Bothell, WA), and IL-6 plasmid DNA was donated
by Dr. Stephen Galli (Beth Israel Deaconess Medical Center). All slides
were processed for autoradiography (Kodak, Rochester, NY) and
counterstained with Giemsa stain. The use of ribonuclease A and T1 in
posthybridization washing was important for eliminating nonspecific
binding. Sense riboprobes were used as controls for interpretation of
the in situ hybridization results.
Enzyme-linked immunosorbent assay.
IL-1
and IL-6 were measured by immunoreactivity in a double-sandwich
enzyme-linked immunosorbent assay (ELISA) format using commercially
available kits for mouse cytokines (Endogen, Cambridge, MA). Briefly,
acinar cell supernatants and mouse saliva were used immediately after
thawing from
70°C. Using a Centricon 3 filter system
(Amicon, Beverly, MA), we concentrated 0.9-ml aliquots of acinar cell
supernatants or saliva 10- to 15-fold. Aliquots were placed into
IL-1
or IL-6 antibody-coated, 96-well microtiter plates (Endogen).
After incubation and washing, a secondary biotinylated antibody was
added for IL-1
detection, followed by a conjugated poly-horseradish
peroxidase (HRP)-80 streptavidin (Research Diagnostics, Flanders, NJ)
and substrate (3,3',5,5'-tetramethylbenzidine;
Endogen). The IL-6 kit used a rat anti-mouse HRP conjugate
in place of the biotinylated antibody. Three to six aliquots, each from
independent acinar cell harvests for IL-1
and IL-6 (unknowns and
standards), were run in duplicate. Colorimetric reactions were read on
a Thermomax microplate reader (Molecular Devices, Menlo Park, CA).
Amylase and lactate dehydrogenase biochemistry.
Amylase was determined using a
4,6-ethylidene-(G7)-p-nitrophenyl-(G1)-
,D-maltoheptaoside
substrate (Sigma Chemical), and lactate dehydrogenase (LDH) was
determined using a pyruvate substrate (Sigma Chemical) in a centrifugal
autoanalyzer (COBAS-BIO, Roche Diagnostic, Nutley, NJ) according to the
method of Crouse et al. (11).
Frozen ultrathin sectioning for electron microscopy.
Cells in suspension were fixed in 8% formaldehyde in 200 mM HEPES at
room temperature for 1 h and pelleted. Pellets were cryoprotected by
immersion in 2.3 M sucrose overnight at 4°C. Fifty-nanometer ultrathin sections were cut at
125°C (Reichert Ultracut S,
Leica) on a diamond knife (Diatome US, Fort Washington, PA). Sections were removed from the knife with 2.3 M sucrose droplets, placed on
Formvar-coated nickel grids (Electron Microscopy Sciences, Fort
Washington, PA), and floated section side down on phosphate-buffered saline (PBS) in a humidor for ~1 h at 20°C before immunolabeling.
Protein A-gold and immunogold labeling of frozen ultrathin
sections.
Gold labeling on sections on nickel grids at 20°C was done as
previously described (30), modified for frozen ultrathin sections (37,
38). Grids were floated section side down on 10-µl drops of reagents
at 20°C. Reagents were used in the following sequence: 1) 0.5% fish skin gelatin (FSG;
Fisher Scientific, Pittsburgh, PA) and 0.02 M glycine for 10 min;
2) primary goat IgG anti-mouse IL-1
(R & D Systems, Minneapolis, MN) or primary rabbit IgG
anti-mouse IL-6 (R & D) were used undiluted or diluted 1:2, 1:5, 1:10,
1:25, or 1:100 in 5% FSG in PBS (PBS-FSG) for 30 min at 20°C;
3) PBS-FSG, for four 15-min washes;
4) secondary rabbit anti-goat 10-nm
gold (Sigma Chemical) diluted 1:80 in PBS-FSG or 10-nm protein A-gold (Dr. G. Posthuma, Utrecht, The Netherlands) diluted 1:30 in PBS-FSG for
30 min at 20°C; 5) PBS-FSG, for
four 15-min washes; 6) distilled water for four 15-min washes; 7)
contrasted with 0.3% uranyl acetate (Electron Microscopy Sciences) in
2% methyl cellulose (Sigma Chemical) for 10 min at 4°C; and
8) excess liquid was removed with
filter paper and sections were air dried.
Controls included omission of the primary antibody, substitution of the
specific primary antibody with nonimmune IgG to determine nonspecific
binding [i.e., normal goat IgG (Sigma Chemical) in place of goat
anti-mouse IL-1
and normal rabbit IgG (Sigma Chemical) in place of
rabbit anti-mouse IL-6] or with an irrelevant antibody [i.e., rabbit IgG anti-histamine-bovine serum albumin (Accurate Chemical and Scientific Company, Westbury, CT) diluted 1:10 or 1:100 in
place of rabbit anti-mouse IL-6]. An absorption control was done
for IL-1
by preincubating the primary antibody [diluted 1:5,
1:10, or 1:25 in Hanks' balanced salt solution (Life
Technologies)] with murine IL-1
peptide (5 µg/ml; R & D
Systems) for 30 min at 20°C before using the mixture for
immunolabeling.
Quantitative analysis of gold-labeled compartments.
Thin sections were examined systematically in a Philips 300 electron
microscope (Philips, Mahwah, NJ) in the same order, beginning at the
top left corner of the grid and proceeding to the lower right corner of
the grid, until at least 10 acinar cells were photographed. Electron
micrographs of gold-labeled sections were printed at ×41,250, and
8 to 10 prints containing a combined total of at least 104 granules
from each experimental and control group were used for stereological
analysis. Cell images included the nucleus, plasma membrane, multiple
cytoplasmic granules, and mitochondria.
Area determinations were done by a point counting method using a square
lattice overlay (40) with a density of 1 point per 4 cm2 on each print to measure the
area of the nucleus and the cytoplasm (i.e., area of the cell excluding
the nucleus) and with a density of 1 point per 1 cm2 to measure the area of the
mitochondria and granules (29). Labeling density was expressed as the
number of gold particles per square micrometer of the cellular
compartment of interest. Nonspecific IL-1
and IL-6 labeling
densities for nuclear and cytoplasm compartments were calculated as the
number of gold particles per square micrometer. Gold particles were
counted as independent when they were separated from one another by at
least one gold particle diameter (i.e., 10 nm).
Statistical analysis for ELISA and gold-labeling studies.
IL-1
and IL-6 readings were normalized to 1 pg/ml at 0 min, and the
percent increase in IL-1
and IL-6 levels in the supernatant was
calculated for the 2-, 5-, and 10-min time points. Differences between
the percent increase in IL-1
and IL-6 in the supernatants of
unstimulated and stimulated acinar cells were examined for statistical
significance using the one-tailed two-sample
t-test.
Differences among gold-labeling conditions were examined for
statistical significance using one-way, unblocked analysis of variance.
Pairwise differences were evaluated by the Newman-Keuls multiple-sample
comparisons test. P < 0.05 was
considered a significant difference. Data organization and analysis
were performed on the PROPHET system (Bolt, Barenek, and Newman,
Cambridge, MA), a national computer resource, sponsored by The National
Center for Research Resources.
 |
RESULTS |
RT-PCR analysis of IL-1
and IL-6 in whole parotid
gland and acinar cell preparations.
RT-PCR results showed the expected size of 336 bp for the mouse IL-1
PCR product (19) (Fig.
1A,
lanes
1, 3,
and 5) and of 614 bp for the mouse
IL-6 product (20) (Fig. 1B,
lanes
1, 3,
and 5). Confirmation of product
identity was accomplished by specific restriction enzyme digestion of
the respective PCR products. Pvu II
made one cut in the IL-1
PCR product, yielding two daughter bands at
197 and 139 bp (Fig. 1A,
lanes
2, 4,
and 6).
Bsr I made two cuts in the IL-6 PCR
product, yielding three daughter bands at 321, 278, and 15 bp (Fig.
1B,
lanes
2, 4,
and 6).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
Reverse transcription-polymerase chain reaction (RT-PCR) and
restriction enzyme characterization of interleukin-1 (IL-1 )
(A) and IL-6
(B) RT-PCR products visualized on
2% agarose gels. M, 100-bp markers.
Lane 1, undigested RT-PCR products from a
dispersed acinar cell preparation;
lane 2, digested RT-PCR products from a
dispersed acinar cell preparation;
lane 3, undigested RT-PCR products from a
homogenized parotid gland; lane 4, digested RT-PCR products from the
corresponding homogenized parotid gland;
lane 5, undigested RT-PCR products from a
second homogenized parotid gland; and
lane 6, digested RT-PCR products
corresponding to the second homogenized parotid gland. The 336-bp
IL-1 RT-PCR products (A,
lanes 1, 3,
and 5) were subjected to
Pvu II restriction enzyme digestion
giving rise to the expected 197-bp and 139-bp fragments
(A,
lanes 2, 4,
and 6). The 139-bp fragment is faint
but present in lanes 4 and
6 in
A. The 614-bp IL-6 RT-PCR products
(B,
lanes 1, 3,
and 5) were subjected to
Bsr I
digestion giving rise to the expected 321-, 278-, and 15-bp fragments
(B,
lanes 2, 4,
and 6); only the 321- and 278-bp
fragments are seen on the gel.
|
|
In situ hybridization of IL-1
and IL-6 mRNA in
parotid gland.
To determine the cellular localization of IL-
and IL-6 mRNAs in
slices of mouse parotid gland, in situ hybridization was performed
using mouse-specific riboprobes. IL-1
mRNA (Fig.
2, A and
B) and IL-6 mRNA (Fig.
3, A and
B) were detected in the glandular
epithelial cells. The control samples using IL-1
(Fig. 2,
C and
D) and IL-6 (Fig. 3,
C and
D) sense cDNA strands showed no
specific labeling of any cell type. Inspection of tissue sections of
the gland at high magnification in the light microscope showed minimal
label in the interstitium, similar to the sense strand control (data
not shown).

View larger version (168K):
[in this window]
[in a new window]
|
Fig. 2.
In situ hybridization of IL-1 . Frozen sections of normal mouse
parotid gland fixed in 8% paraformaldehyde and 0.2 M HEPES buffer
(A-D)
lightly stained with Giemsa. A and
C: bright-field examination.
B and
D: dark-field microscopy to highlight
autoradiographic grains of sections in A and C,
respectively. A and
C: sections show well-preserved
lobular architecture containing striated ducts (* in lumen) and
secretory epithelial cells. A and
B: tissue sections were hybridized
with a 35S-labeled mouse IL-1
antisense strand riboprobe. C and
D: sections were hybridized with the
mouse IL-1 sense strand control riboprobe. Autoradiographic grains
are seen in greatest density over the glandular epithelial cells, and
they highlight the lobular architecture within the tissue section
(B).
D: fewer autoradiographic background
grains are seen using the IL-1 sense strand control probe.
A-D:
magnification, ×100. Bar, 250 µm.
|
|

View larger version (K):
[in this window]
[in a new window]
|
Fig. 3.
In situ hybridization of IL-6. Frozen sections of normal mouse parotid
gland fixed in 8% paraformaldehyde and 0.2 M HEPES buffer
(A-D)
lightly stained with Giemsa. A and
C: bright-field examination.
B and
D: dark-field microscopy to highlight
autoradiographic grains of sections in A and C,
respectively. A and
C: sections show well-preserved
lobular architecture containing striated ducts (* in lumen) and
secretory epithelial cells. A and
B: tissue sections were hybridized
with a 35S-labeled mouse IL-6
antisense strand riboprobe. C and
D: sections were hybridized with the
mouse IL-6 sense strand control riboprobe. Autoradiographic grains are
seen in greatest density over the glandular epithelial cells, and they
highlight the lobular architecture within the tissue section
(B).
D: fewer autoradiographic background
grains are seen using the IL-6 sense strand control probe.
A-D:
magnification, ×100. Bar, 250 µm.
|
|
Release of IL-1
, IL-6, and amylase from mouse
parotid acinar cells.
Enriched preparations of dispersed acinar cells stimulated with
10
5 M norepinephrine
released significantly more IL-1
(P < 0.03) (Fig.
4A) and
IL-6 (P < 0.01) (Fig.
5A) into
the supernatant by 10 min than unstimulated preparations. Although the
absolute amounts of IL-1
and IL-6 in supernatants varied between
experiments, the percent increase between time points was reproducible.
Stimulated whole mouse saliva contained 2.8 pg IL-1
/ml and 41.3 pg
IL-6/ml.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Normalized %increase of IL-1 in supernatants containing enriched
populations of dispersed mouse parotid acinar cells unstimulated
(hatched bars) and stimulated (filled bars) with
10 5 M norepinephrine at 0, 2, 5, and 10 min determined by enzyme-linked immunosorbent assay
(ELISA) (A). %Total release of
amylase ( , stimulated; , unstimulated) and lactate dehydrogenase
(LDH) ( , stimulated; , unstimulated) from the same populations of
cells used for ELISA (B).
A: there is a significantly
(P < 0.03) higher % of IL-1 /ml
in supernatant from stimulated cells than from unstimulated cells at 10 min poststimulation. B: by 10 min,
average amylase release is 36% from norepinephrine-stimulated cells
and 5% from unstimulated cells. LDH release by 10 min is 6% from
norepinephrine-stimulated cells and 7% from unstimulated cells.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Normalized %increase of IL-6 in supernatants containing enriched
populations of dispersed mouse parotid acinar cells unstimulated
(hatched bars) and stimulated (filled bars) with
10 5 M norepinephrine at 0, 2, 5, and 10 min determined by ELISA
(A). %Total release of amylase
( , stimulated; , unstimulated) and LDH ( , stimulated; ,
unstimulated) from the same populations of cells used for ELISA
(B).
A: there is a significantly
(P < 0.01) higher % of IL-6/ml in
supernatant from stimulated cells than from unstimulated cells at 10 min poststimulation. B: by 10 min,
average amylase release is 42% from norepinephrine-stimulated cells
and 7% from unstimulated cells. LDH release by 10 min is 8% from
norepinephrine-stimulated cells and 4% from unstimulated cells.
|
|
Amylase determinations from the same mouse acinar cell preparations
that were used for ELISA showed maximal release by 10 min in stimulated
populations (31% and 35% above unstimulated release, Figs.
4B and
5B, respectively). Spontaneous amylase
release by 10 min was 5% and 7% of total amylase release in
unstimulated acinar cell preparations (Figs.
4B and
5B, respectively). Importantly, LDH
determinations also from the same acinar cell preparations showed that
cell death was <8% in stimulated and in unstimulated groups (Figs.
4B and
5B, respectively) at the 10-min time
point. Taken together, these findings show that mouse parotid acinar cells used for ELISA determinations of IL-1
and IL-6 responded to
norepinephrine stimulation and that the release of stored contents was
not the result of cell death. These data further show that the release
of IL-1
and IL-6 paralleled the time course of amylase release.
Ultrastructural localization of IL-1
and IL-6.
Dispersed mouse parotid acinar cells were labeled with gold particles,
indicating IL-1
(Fig.
6A) and
IL-6 (Fig. 6C) in the matrix of
storage granules. IL-1
labeling density was significantly (P < 0.01) higher in granules (21 ± 11 gold particles/µm2)
than in mitochondria (12 ± 4 gold
particles/µm2), nucleus (5 ± 2 gold particles/µm2),
and cytoplasm (2 ± 0.5 gold
particles/µm2) (Table
1). IL-6 labeling density showed no
significant difference between granules (11 ± 4 gold
particles/µm2) and
mitochondria (15 ± 9 gold
particles/µm2), but background
labeling was significantly lower (P < 0.01) in the nucleus (4 ± 3 gold
particles/µm2) and the
cytoplasm (1 ± 0.4 gold
particles/µm2) (Table
2). Interestingly, IL-1
and IL-6
labeling of mitochondria were more focal, often associated with the
outer mitochondrial membrane, and not all mitochondria were labeled
(data not shown). Occasionally, labeling for IL-1
was seen in Golgi
structures. Gold particle labeling was rarely seen in the nucleus,
cytoplasm, or along the luminal or basolateral plasma membranes (data
not shown).

View larger version (147K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoelectron microscopy of IL-1
(A and
B) and IL-6
(C and
D) on frozen ultrathin sections of
mouse parotid acinar cells. A:
sections were labeled with primary goat anti-mouse IL-1 (diluted
1:5) and secondary rabbit anti-goat 10 nm immunogold (diluted 1:80).
B: nonimmune goat immunoglobulin G
(IgG) was substituted for the primary antibody to IL-1 .
C: sections were labeled with a
primary rabbit anti-mouse IL-6 (diluted 1:100) and secondary 10 nm
protein A-gold (diluted 1:30). D:
nonimmune rabbit IgG was substituted for the primary antibody to IL-6.
Gold label for IL-1 (A) and for
IL-6 (C) is seen on the matrices of
the cytoplasmic storage granules. No. of gold particles on granules is
reduced in nonimmune controls (B and
D).
A-D:
magnification, ×41,250. Bar, 0.25 µm.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Protein A-gold labeling density of IL-6 in various cell compartments of
mouse parotid gland acinar cells
|
|
Controls for IL-1
included the omission of primary antiserum to
IL-1
or substitution of nonimmune goat IgG for the primary antiserum
(Fig. 6B). An additional control,
preabsorption of the primary goat anti-mouse IL-1
antiserum with its
peptide in solution, reduced granule and mitochondrial labeling (Table
1, experiment 2). Controls for IL-6 included
substitution of nonimmune rabbit IgG for the primary antiserum to IL-6
(Fig. 6D), omission of the primary
antiserum, or substitution of an irrelevant rabbit antihistamine BSA
for the primary antiserum to IL-6. Omission, substitution, and
irrelevant antibody controls showed significantly
(P < 0.01) lower labeling compared
with specific labeling for IL-1
(Table 1) and IL-6 (Table 2).
 |
DISCUSSION |
This study provides new information on the presence of inflammatory
cytokines in normal exocrine cells. Using RT-PCR, we showed that
healthy mouse parotid gland and dispersed parotid acinar cells contain
IL-1
and IL-6 mRNA. We further showed using protein A and immunogold
electron microscopy that these interleukins are localized in the
granules of acinar cells. Finally, by ELISA and biochemical methods, we
showed that IL-1
and IL-6 release from norepinephrine-stimulated
acinar cells paralleled the time course of amylase secretion.
Although the presence of IL-1
has been established in many
hematopoetically derived cells (14), in normal human epidermis (23),
and in normal human minor salivary gland acinar and ductal cells (7),
its mechanism(s) of release from any cell is not known (14). Because
biochemical [e.g., subcellular fractionation of activated
monocytes (1)] and morphological studies [e.g., immunocytochemistry (13)] have not detected IL-1
in Golgi or endoplasmic reticulum in these cell types, novel secretory mechanisms have been proposed (35). For example, in blood monocytes, pro-IL-1
mRNA is translated on polysomes associated with microtubules in the
cytosol (35) and is subsequently myristoylated to facilitate localization to the cell membrane (14). The
IL-1
-converting enzyme on the inner surface of the cell membrane
subsequently cleaves a 14-kDa amino-terminal portion of the molecule,
releasing mature IL-1
(17 kDa) (14). The mature form is transported
extracellularly through a putative membrane channel (14).
In view of the current thinking regarding the intracellular processing
of IL-1
, how can we explain the granule label we observed in parotid
acinar cells? First, pro-IL-1
can be secreted by macrophages in the
absence of the IL-1
-converting enzyme (3), indicating that IL-1 can
be released by more than one mechanism. In such cases,
pro-IL-1
has been found in intracellular vesicles (protected from
protease digestion) and was presumably released by exocytosis (35).
Second, immunoelectron microscopy data show colocalization of IL-1
with lamellar bodies in normal human epidermal cells (i.e., stratum
granulosum), thereby implicating exocytosis as the primary process of
IL-1
release in these cells (13). Therefore, it is possible that
epithelial cells process IL-1
differently than hematopoetically
derived cells.
In contrast to IL-1
, IL-6 is a classic secretory protein with a
signal sequence (36). Accordingly, it has been identified by a variety
of methods in the Golgi and in the endoplasmic reticulum in peripheral
blood mononuclear cells (36) and in normal keratinocytes (reviewed in
Ref. 32). Immunohistochemical studies of salivary glands show IL-6
localized in striated and intercalated duct cells (but not in acinar
cells) within the normal margins of human salivary gland tumors (17)
and in mononuclear cells (but not in acinar or ductal cells) of MRL/lpr
mouse salivary glands (22). An immunocytochemical study of cultured
human parotid pleomorphic adenoma cells shows IL-6 in Golgi and
cytoplasmic granules, but pharmacological inhibition of exocytosis was
necessary for IL-6 accumulation in these cells (16). Our study is the
first to show the subcellular localization of IL-6 in normal parotid
acinar cells from healthy mice.
Mitochondrial labeling of IL-1
and IL-6 in our acinar cell
preparations was unexpected. However, Beesely et al. (2) also identified pro-IL-1
by immunoelectron microscopy in mitochondria of
normal and lipopolysaccharide-stimulated peripheral blood monocytes and
within the double membrane in mitochondrial-enriched fractions. IL-1
has also been identified by immunocytochemistry and immunoprecipitation in mitochondria of normal peripheral blood monocytes (2).
Although IL-1
is not synthesized by mitochondrial DNA, the first 21 amino acids of the mature IL-1
do correspond to
mitochondrial-targeting signals (2). Evidence for this targeting
phenomenon was reported by Tomoda et al. (39), who showed that IL-1
binding to mitochondrial membranes decreased the severity of
acetylsalicylate-induced mitochondrial damage. Association of IL-1
with mitochondrial membranes is supported indirectly by biochemical
data showing that IL-1
-converting enzyme can complex with Bcl-2 (9),
an integral membrane protein mainly located on the outer membrane of
mitochondria (42).
We considered the possibility that IL-1
and IL-6 mRNA expression in
isolated acinar cells may be induced secondarily to the acinar cell
dispersion procedure. Therefore, we used established cell dispersion
methods (33) appropriately modified to reduce upregulation of IL-1
and IL-6. For example, our media and dispersion solutions did not
contain known inducers (e.g., endotoxin or serum factors) of IL-1 and
IL-6 genes (6, 14). Collagenase and hyaluronidase have not been shown
to induce IL-1
and IL-6 (14). Because potent inducers of IL-1
can
increase IL-1
mRNA levels in some cell types within 15 min (14), we
verified the presence of IL-1
mRNA in unstimulated parotid glands
harvested within seconds from normal mice.
Elucidation of IL-1
and IL-6 trafficking in parotid acinar cells may
help to clarify the role(s) for these cytokines in health and in the
progression of autoimmune diseases. Recent data report upregulation of
IL-1
and IL-6 mRNA in salivary glands of mouse models of
sialadenitis (8, 22) and in human Sjögren's syndrome (15, 21);
however, the cellular sources of these cytokines have not been well
defined. Such upregulation of IL-1
in another common autoimmune
disease, Hashimoto's thyroiditis, has been shown to increase Fas
expression in glandular epithelial cells, resulting in gradual
destruction of the tissue in the absence of immune effector cells (18).
Because our findings show that healthy mouse parotid acinar cells
produce and store the proinflammatory cytokine IL-1
and the
anti-inflammatory cytokine IL-6, it will be of interest to determine if
intracellular alterations in the concentrations of these cytokines in
animal models genetically predisposed to autoimmune sialadenitis can
also lead to secretory cell destruction in the absence of a lymphocytic
infiltrate.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Dental Research
Grants DE-10059 (G. R. Login), DE-10335 (D. T. W. Wong) and DE-08680
(D. T. W. Wong).
 |
FOOTNOTES |
Address for reprint requests: G. R. Login, Dept. of Pathology, Beth
Israel Deaconess Medical Center, East Campus, 330 Brookline Ave.,
Boston, MA 02215.
Received 6 August 1997; accepted in final form 8 October 1997.
 |
REFERENCES |
1.
Bakouche, O.,
D. C. Brown,
and
L. B. Lachman.
Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release.
J. Immunol.
138:
4249-4255,
1987[Abstract/Free Full Text].
2.
Beesley, J. E.,
R. Bomford,
and
J. A. Schmidt.
Ultrastructural localization of interleukin 1 in human peripheral blood monocytes; evidence for IL-1
in mitochondria.
Histochem. J.
22:
234-244,
1990[Medline].
3.
Beuscher, H. U.,
C. Gunther,
and
M. Rollinghoff.
IL-1
is secreted by activated murine macrophages as biologically inactive precursor.
J. Immunol.
144:
2179-2183,
1990[Abstract/Free Full Text].
4.
Boumba, D.,
F. N. Skopouli,
and
H. M. Moutsopoulos.
Cytokine mRNA expression in the labial salivary gland tissues from patients with primary Sjögren's syndrome.
Br. J. Rheumatol.
34:
326-333,
1995[Medline].
5.
Camp, R. D., N. J. Fincham, J. S. Ross, K. B. Bacon, and A. J. Gearing.
Leukocyte chemoattractant cytokines of the epidermis.
J. Invest. Dermatol. 95, Suppl.: 108S-110S, 1990.
6.
Campbell, I. L.,
M. V. Hobbs,
J. Dockter,
M. B. Oldstone,
and
J. Allison.
Islet inflammation and hyperplasia induced by the pancreatic islet- specific overexpression of interleukin-6 in transgenic mice.
Am. J. Pathol.
145:
157-166,
1994[Abstract].
7.
Cauli, A.,
G. Yanni,
C. Pitzalis,
S. Challacombe,
and
G. S. Panayi.
Cytokine and adhesion molecule expression in the minor salivary glands of patients with Sjögren's syndrome and chronic sialoadenitis.
Ann. Rheum. Dis.
54:
209-215,
1995[Abstract].
8.
Chandrasekar, B.,
H. S. McGuff,
T. B. Aufdermorte,
D. A. Troyer,
N. Talal,
and
G. Fernandes.
Effects of calorie restriction on transforming growth factor
1 and proinflammatory cytokines in murine Sjögren's syndrome.
Clin. Immunol. Immunopathol.
76:
291-296,
1995[Medline].
9.
Chinnaiyan, A. M.,
K. O'Rourke,
B. R. Lane,
and
V. M. Dixit.
Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death.
Science
275:
1122-1126,
1997[Abstract/Free Full Text].
10.
Cressman, D. E.,
L. E. Greenbaum,
R. A. DeAngelis,
G. Ciliberto,
E. E. Furth,
V. Poli,
and
R. Taub.
Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.
Science
274:
1379-1383,
1996[Abstract/Free Full Text].
11.
Crouse, S. P.,
R. E. Cross,
N. C. Parker,
and
J. Savory.
Rapid enzymatic determination of amylase in serum and urine using a centrifugal analyzer.
Ann. Clin. Lab. Sci.
9:
420-424,
1979[Abstract].
12.
Davis, L. G.,
M. D. Dibner,
and
J. F. Battey.
Guanidine isothiocyanate preparation of total RNA.
In: Basic Methods in Molecular Biology, edited by L. G. Davis,
M. D. Dibner,
and J. F. Battey. New York: Elsevier Science, 1986, p. 130-136.
13.
Didierjean, L.,
D. Salomon,
Y. Merot,
G. Siegenthaler,
A. Shaw,
J. M. Dayer,
and
J. H. Saurat.
Localization and characterization of the interleukin 1 immunoreactive pool (IL-1
and
forms) in normal human epidermis.
J. Invest. Dermatol.
92:
809-816,
1989[Abstract].
14.
Dinarello, C. A.
Biologic basis for interleukin-1 in disease.
Blood
87:
2095-2147,
1996[Abstract/Free Full Text].
15.
Fox, R. I.,
H. I. Kang,
D. Ando,
J. Abrams,
and
E. Pisa.
Cytokine mRNA expression in salivary gland biopsies of Sjögren's syndrome.
J. Immunol.
152:
5532-5539,
1994[Abstract/Free Full Text].
16.
Gallo, O.,
D. Bani,
G. Toccafondi,
F. Almerigogna,
and
O. F. Storchi.
Characterization of a novel cell line from pleomorphic adenoma of the parotid gland with myoepithelial phenotype and producing interleukin-6 as an autocrine growth factor.
Cancer
70:
559-568,
1992[Medline].
17.
Gandour-Edwards, R.,
S. B. Kapadia,
P. H. Gumerlock,
and
L. Barnes.
Immunolocalization of interleukin-6 in salivary gland tumors.
Hum. Pathol.
26:
501-503,
1995[Medline].
18.
Giordano, C.,
G. Stassi,
R. De Maria,
M. Todaro,
P. Richiusa,
G. Papoff,
G. Ruberti,
M. Bagnasco,
R. Testi,
and
A. Galluzo.
Potential involvement of FAS and its ligand in the pathogenesis of Hashimoto's thyroiditis.
Science
275:
960-963,
1997[Abstract/Free Full Text].
19.
Gray, P. W.,
D. Glaister,
E. Chen,
D. V. Goeddel,
and
D. Pennica.
Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1
.
J. Immunol.
137:
3644-3648,
1986[Abstract/Free Full Text].
20.
Grenett, H. E.,
N. L. Fuentes,
and
G. M. Fuller.
Cloning and sequence analysis of the cDNA for murine interleukin-6.
Nucleic Acids Res.
18:
6455,
1990[Medline].
21.
Grisius, M. M.,
D. K. Bermudez,
and
P. C. Fox.
Salivary and serum interleukin 6 in primary Sjögren's syndrome.
J. Rheumatol.
24:
1089-1091,
1997[Medline].
22.
Hamano, H.,
I. Saito,
N. Haneji,
Y. Mitsuhashi,
N. Miyasaka,
and
Y. Hayashi.
Expressions of cytokine genes during development of autoimmune sialadenitis in MRL/lpr mice.
Eur. J. Immunol.
23:
2387-2391,
1993[Medline].
23.
Hauser, C.,
J. H. Saurat,
A. Schmitt,
F. Jaunin,
and
J. M. Dayer.
Interleukin 1 is present in normal human epidermis.
J. Immunol.
136:
3317-3323,
1986[Abstract/Free Full Text].
24.
Heinrich, P. C.,
L. Graeve,
S. Rose-John,
J. Schneider-Mergener,
E. Dittrich,
A. Erren,
C. Gerhartz,
U. Hemann,
C. Lutticken,
U. Wegenka,
O. Weiergräber,
and
F. Horn.
Membrane-bound and soluble interleukin-6 receptor: studies on structure, regulation of expression, and signal transduction.
Ann. NY Acad. Sci.
762:
222-236,
1995[Medline].
25.
Jacob, C. O.,
S. Aiso,
S. A. Michie,
H. O. McDevitt,
and
H. Acha-Orbea.
Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor (TNF): similarities between TNF-
and interleukin 1.
Proc. Natl. Acad. Sci. USA
87:
968-972,
1990[Abstract].
26.
Jafarian-Tehrani, M.,
A. Amrani,
F. Homo-Delarche,
C. Marquette,
M. Dardenne,
and
F. Haour.
Localization and characterization of interleukin-1 receptors in the islets of Langerhans from control and nonobese diabetic mice.
Endocrinology
136:
609-613,
1995[Abstract].
27.
Jarry, A.,
G. Vallette,
J. E. Branka,
and
C. Laboisse.
Direct secretory effect of interleukin-1 via type I receptors in human colonic mucous epithelial cells (HT29-C1.16E).
Gut
38:
240-242,
1996[Abstract].
28.
Kopf, M.,
H. Baumann,
G. Freer,
M. Freudenberg,
M. Lamers,
T. Kishimoto,
R. Zinkernagel,
H. Bluethmann,
and
G. Kohler.
Impaired immune and acute-phase responses in interleukin-6-deficient mice.
Nature
368:
339-342,
1994[Medline].
29.
Login, G. R.,
and
A. M. Dvorak.
Microwave energy fixation for electron microscopy.
Am. J. Pathol.
120:
230-243,
1985[Abstract].
30.
Login, G. R.,
J. T. Yang,
K. P. Bryan,
E. C. Digenis,
J. McBride,
A. Elovic,
D. O. Quissell,
A. M. Dvorak,
and
D. T. W. Wong.
Characterization of the synthesis and storage of TGF-
in rat parotid acinar and intercalated duct cells.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G553-G562,
1997[Abstract/Free Full Text].
31.
Michalopoulos, G. K.,
and
M. C. DeFranes.
Liver regeneration.
Science
276:
60-66,
1997[Abstract/Free Full Text].
32.
Paquet, P.,
and
G. E. Pierard.
Interleukin-6 and the skin.
Int. Arch. Allergy Immunol.
109:
308-317,
1996[Medline].
33.
Quissell, D. O.,
and
R. S. Redman.
Functional characteristics of dispersed rat submandibular cells.
Proc. Natl. Acad. Sci. USA
76:
2789-2793,
1979[Abstract].
34.
Ramsay, A. J.,
A. J. Husband,
I. A. Ramshaw,
S. Bao,
K. I. Matthaei,
G. Koehler,
and
M. Kopf.
The role of interleukin-6 in mucosal IgA antibody responses in vivo.
Science
264:
561-563,
1994[Medline] .
35.
Rubartelli, A.,
F. Cozzolino,
M. Talio,
and
R. Sitia.
A novel secretory pathway for interleukin-1
, a protein lacking a signal sequence.
EMBO J.
9:
1503-1510,
1990[Abstract].
36.
Schindler, R.,
J. Mancilla,
S. Endres,
R. Ghorbani,
S. C. Clark,
and
C. A. Dinarello.
Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF.
Blood
75:
40-47,
1990[Abstract].
37.
Slot, J. W.,
and
H. J. Geuze.
Gold markers for single and double immunolabelling of ultrathin cryosections.
In: Immunolabelling for Electron Microscopy, edited by J. M. Pola,
and I. M. Varndell. New York: Elsevier Science, 1984, p. 129-142.
38.
Slot, J. W.,
and
H. J. Geuze.
A new method of preparing gold probes for multiple-labeling cytochemistry.
Eur. J. Cell Biol.
38:
87-93,
1985[Medline].
39.
Tomoda, T.,
K. Takeda,
T. Kurashige,
H. Enzan,
and
M. Miyahara.
Experimental study on Reye's syndrome: inhibitory effect of interferon
on acetylsalicylate-induced injury to rat liver mitochondria.
Metabolism
41:
887-892,
1992[Medline].
40.
Williams, M. A.
Stereological Techniques, Quantitative Methods in Biology. New York: Elsevier Science, 1977.
41.
Wogensen, L. D.,
V. Kolb-Bachofen,
P. Christensen,
C. A. Dinarello,
T. Mandrup-Poulsen,
S. Martin,
and
J. Nerup.
Functional and morphological effects of interleukin-1
on the perfused rat pancreas.
Diabetologia
33:
15-23,
1990[Medline].
42.
Yang, J.,
X. Liu,
K. Bhalla,
C. N. Kim,
A. M. Ibrado,
J. Cai,
T.-I. Peng,
D. P. Jones,
and
X. Wang.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275:
1129-1132,
1997[Abstract/Free Full Text].
AJP Gastroint Liver Physiol 274(1):G147-G156
0193-1857/98 $5.00
Copyright © 1998 the American Physiological Society