IL-1beta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1beta (IL-1beta ) 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-1beta 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-1beta 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-1beta (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-1beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1beta and IL-6 and release these cytokines from their granules after alpha - and beta -adrenergic stimulation.

reverse transcription-polymerase chain reaction; immunoelectron microscopy; exocytosis; secretion; parotid gland; cytokines

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RECENTLY, THE EPITHELIAL cells in salivary glands from patients with Sjögren's syndrome were shown to produce the proinflammatory cytokine interleukin-1beta (IL-1beta ) 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-1beta and IL-6 compared with patients without Sjögren's syndrome (15, 21). Furthermore, IL-1beta 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-1beta 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-1beta 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-1beta 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-1beta (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-1beta 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 alpha ,beta -adrenergic agonist norepinephrine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-1beta 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-1beta 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-1beta and IL-6 mRNAs using rat-specific primers for IL-1beta 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-1beta 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-1beta primers used to detect mouse IL-1beta 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-1beta 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-1beta 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-1beta 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-1beta or IL-6 antibody-coated, 96-well microtiter plates (Endogen). After incubation and washing, a secondary biotinylated antibody was added for IL-1beta 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-1beta 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)-alpha ,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-1beta (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-1beta 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-1beta by preincubating the primary antibody [diluted 1:5, 1:10, or 1:25 in Hanks' balanced salt solution (Life Technologies)] with murine IL-1beta 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-1beta 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-1beta and IL-6 readings were normalized to 1 pg/ml at 0 min, and the percent increase in IL-1beta 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-1beta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RT-PCR analysis of IL-1beta 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-1beta 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-1beta 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).


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Fig. 1.   Reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme characterization of interleukin-1beta (IL-1beta ) (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-1beta 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-1beta and IL-6 mRNA in parotid gland. To determine the cellular localization of IL-beta and IL-6 mRNAs in slices of mouse parotid gland, in situ hybridization was performed using mouse-specific riboprobes. IL-1beta 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-1beta (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).


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Fig. 2.   In situ hybridization of IL-1beta . 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-1beta antisense strand riboprobe. C and D: sections were hybridized with the mouse IL-1beta 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-1beta sense strand control probe. A-D: magnification, ×100. Bar, 250 µm.


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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-1beta , 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-1beta (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-1beta 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-1beta /ml and 41.3 pg IL-6/ml.


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Fig. 4.   Normalized %increase of IL-1beta 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 (square , stimulated; star , unstimulated) and lactate dehydrogenase (LDH) (open circle , stimulated; triangle , unstimulated) from the same populations of cells used for ELISA (B). A: there is a significantly (P < 0.03) higher % of IL-1beta /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.


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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 (square , stimulated; star , unstimulated) and LDH (open circle , stimulated; triangle , 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-1beta 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-1beta and IL-6 paralleled the time course of amylase release.

Ultrastructural localization of IL-1beta and IL-6. Dispersed mouse parotid acinar cells were labeled with gold particles, indicating IL-1beta (Fig. 6A) and IL-6 (Fig. 6C) in the matrix of storage granules. IL-1beta 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-1beta 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-1beta 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).


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Fig. 6.   Immunoelectron microscopy of IL-1beta (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-1beta (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-1beta . 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-1beta (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.

                              
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Table 1.   Immunogold labeling density of IL-1beta in various cell compartments of mouse parotid gland acinar cells

                              
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Table 2.   Protein A-gold labeling density of IL-6 in various cell compartments of mouse parotid gland acinar cells

Controls for IL-1beta included the omission of primary antiserum to IL-1beta or substitution of nonimmune goat IgG for the primary antiserum (Fig. 6B). An additional control, preabsorption of the primary goat anti-mouse IL-1beta 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-1beta (Table 1) and IL-6 (Table 2).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-1beta 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-1beta and IL-6 release from norepinephrine-stimulated acinar cells paralleled the time course of amylase secretion.

Although the presence of IL-1beta 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-1beta in Golgi or endoplasmic reticulum in these cell types, novel secretory mechanisms have been proposed (35). For example, in blood monocytes, pro-IL-1beta 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-1beta -converting enzyme on the inner surface of the cell membrane subsequently cleaves a 14-kDa amino-terminal portion of the molecule, releasing mature IL-1beta (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-1beta , how can we explain the granule label we observed in parotid acinar cells? First, pro-IL-1beta can be secreted by macrophages in the absence of the IL-1beta -converting enzyme (3), indicating that IL-1 can be released by more than one mechanism. In such cases, pro-IL-1beta 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-1beta with lamellar bodies in normal human epidermal cells (i.e., stratum granulosum), thereby implicating exocytosis as the primary process of IL-1beta release in these cells (13). Therefore, it is possible that epithelial cells process IL-1beta differently than hematopoetically derived cells.

In contrast to IL-1beta , 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-1beta and IL-6 in our acinar cell preparations was unexpected. However, Beesely et al. (2) also identified pro-IL-1beta by immunoelectron microscopy in mitochondria of normal and lipopolysaccharide-stimulated peripheral blood monocytes and within the double membrane in mitochondrial-enriched fractions. IL-1beta has also been identified by immunocytochemistry and immunoprecipitation in mitochondria of normal peripheral blood monocytes (2).

Although IL-1beta is not synthesized by mitochondrial DNA, the first 21 amino acids of the mature IL-1beta 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-1beta with mitochondrial membranes is supported indirectly by biochemical data showing that IL-1beta -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-1beta 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-1beta 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-1beta and IL-6 (14). Because potent inducers of IL-1beta can increase IL-1beta mRNA levels in some cell types within 15 min (14), we verified the presence of IL-1beta mRNA in unstimulated parotid glands harvested within seconds from normal mice.

Elucidation of IL-1beta 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-1beta 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-1beta 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-1beta 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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