ARTICLE |
Correspondence to: Gary R. Login, Dept. of Oral Pathology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115. E-mail: glogin@caregroup.harvard.edu
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Summary |
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IL-1ß, TNF-, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3
, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1ß and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1ß and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:10331041,2000)
Key Words:
immunohistochemistry, lymphocytes, CD3, macrophage, F4/80, CD20, ELISA, cytokines, Sjögren's syndrome, mouse
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Introduction |
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The progressive development of Sjögren's disease was recently shown to coincide with expression of IL-1ß, IL-6, and TNF- in human (
The histopathology of salivary glands affected by autoimmune disease shows clones of CD4+ T-cells (
A new paradigm for autoimmune exocrinopathy suggests that cytokine-induced, programmed cell death ( enhance the secretion of acinar cell (but not duct cell)-sythesized matrix metalloproteinase-2 (MMP-2) expression (e.g., a 72-kD collagenase) (
may cause degradation of the basement membrane and disruption of acinar cell architecture (
-fodrin antigen (
, and IL-6 in parotid acinar cells preceding lymphocyte infiltration of salivary glands in aging NOD mice.
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Materials and Methods |
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Animal Care Guidelines
The experiments were approved by the Beth Israel-Deaconess Committee on Animal Research and by the Harvard Medical School's Animal Management Program. These animal management programs are accredited by the American Association for the Accreditation of Laboratory Animal Care, meet National Institutes of Health standards as set forth in the "Guide for the care and use of laboratory animals" [DHHS publication (NIH) 86-25, revised 1996], and accept as mandatory the PHS "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 Beth Israel-Deaconess animal care facility.
Harvest and Dispersion of Mouse Parotid Acini
NOD and BALB/c (control strain) male mice ages 45 weeks and 1012 weeks were used in these studies. Mice were fasted overnight, anesthetized with sodium pentobarbital (Veterinary Laboratories; Lenexa, KS) (50 µg/g), and sacrificed by severing the abdominal aorta. Freshly harvested glands from three mice were pooled per experiment and used for secretion studies, light microscopy, RNA isolation, RT-PCR, and enzyme assays. All experiments were done in triplicate.
Acinar cell isolation was done using a standard collagenase digestion method (
Cell Handling and Stimulation
At precise time points (0, 5, 10, and 20 min after stimulation), 2.5-ml aliquots of acinar cells in suspension were added to a 12-ml syringe (Monoject; St Louis, MO) that was connected to a 3-µm filter (Millipore; Bedford, MA). Aliquots (1.8-ml) of supernatant for either IL-1ß, TNF-, or IL-6 ELISA and 0.6-ml aliquots of supernatant for amylase and lactate dehydrogenase (LDH) release were collected in separate tubes. and immediately frozen at -70C. The filter was immersed in 1 ml of distilled water for determination of total cellular IL-1ß, TNF-
, or IL-6 by ELISA. Aliquots (100-µl) of cells in suspension were collected at each time point to determine total amylase, LDH, and DNA. Unstimulated acinar cells from the same mouse harvests were handled identically and in parallel to stimulated aliquots at each time point to determine basal secretion. The entire time required to sample a single time point was less than 20 sec.
Histology
Parotid glands and spleens were fixed in 10% neutral buffered formalin for 2 hr at 20C and processed routinely on a Tissue-Tek VIP processor (Miles; Naperville, IL), beginning with ethanol dehydration (
Immunohistochemical Detection of CD3 and F4/80
Using a Vectastain Elite ABC kit (Vector Laboratories; Burlingame, CA), 4-µm paraffin sections of mouse parotid gland were stained for a cytoplasmic T-cell-specific antigen, CD3, with rabbit anti-human CD3 IgG (1:30, 1 hr, 20C) (Dako; Carpinteria, CA), secondary biotinylated swine anti-rabbit IgG (1:200, 30 min, 20C) (Dako), and avidinhorseradish peroxidase conjugate (1:400, 30 min, 20C) (Vector) (
RNA Isolation and RT-PCR Confirmation of Murine CD3, F4/80, CD20, and Glyceraldehyde-3-phosphate Dehydrogenase (G3PDH)
Total RNA was isolated from whole parotid gland and spleen preparations and from dispersed mouse parotid acinar cell preparations using the guanidine isothiocyanate method (
RT-PCR detection of CD3, F4/80, CD20, and G3PDH mRNAs was done using mouse-specific primers as previously described (
, and CD20 and 35 cycles for F4/80 were performed using a Perkin Elmer Cetus GeneAmp PCR System 2400 (Foster City, CA) with the following temperature profiles for G3PDH and F4/80 at 94C for 1 min, 60C for 1 min, and 72C for 1 min; for CD3
at 94C for 30 sec, 56C for 30 sec, and 72C for 1 min; and for CD20 at 94C for 1 min, 55C for 1 min, and 72C for 1 min. Mouse G3PDH primers (Gibco BRL Life Technologies; Gaithersburg, MD) included the sense primer: 26-mers 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3' from nucleotide position 5176 and the antisense primer: 24-mers 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3' from nucleotide position 10331010 (Clontech Laboratories; Palo Alto, CA). Mouse CD3
(generously donated by C. Fox and J. Danska; Hospital for Sick Children, Toronto) primers included the sense primer: 18-mers 5'-ACT GGA GCA AGA ATA GGA-3' from nucleotide position 473490 and the antisense primer: 18-mers 5'-AGG AGA GGA AAG GAA CTG-3' from nucleotide position 968951 (
DNA Assay
Acinar cells were washed off the filter paper and were freeze-thawed three times in distilled water to release total DNA from each preparation. DNA was stained with bisbenzamidetrihydrochloride (Sigma). Excitation and emission were measured at 360 and 450
respectively, in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi America; Princeton, NJ) according to the method of
ELISA
Il-1ß, IL-6, and TNF- determinations from lysates of unstimulated cells were made in a double-sandwich ELISA format using commercially available kits for mouse cytokines (Endogen; Cambridge, MA) as previously described (
Calculation of Total IL-1ß, IL-6, and TNF- Amounts in Parotid Acinar Cells
Cytokine concentrations (pg cytokine/µg DNA) were multiplied by the known amount of DNA in a rodent salivary gland cell (6.85 pg DNA/cell) (
Amylase and Lactate Dehydrogenase Determination
Amylase was determined using a 4,6-ethylidene (G7)-p-nitrophenyl (G1)- D-maltoheptaside substrate (Sigma) and LDH was determined using a pyruvate substrate (Sigma) in a centrifugal autoanalyzer (COBAS-BIO; Roche Diagnostic, Nutley, NJ) according to the method of
Statistical Methods
Differences among the percent amylase release and concentrations of IL-1ß, TNF-, and IL-6 in parotid acinar cells from BALB/c and NOD mice were examined for statistical significance using the one-tailed, two-sample t-test. 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, National Institutes of Health (Bethesda, MD).
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Results |
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Detection of T-cells and Macrophages in Mouse Spleen Parotid Gland and Parotid Acinar Cell Preparations
Histological examination of BALB/c mouse parotid glands revealed the presence of many well-circumscribed lymphoid nodules (i.e., 2002000 µm in diameter) (Fig 1) in close association with parotid gland lobules, as shown in Fig 1A for BALB/c. Hematoxylin and eosin staining of the lymphoid nodule showed typical germinal center-like architecture (Fig 1B), with abundant CD3+ T-lymphocytes in the periphery of the lymphoid nodules (Fig 1C).
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T-cells were rarely seen between parotid gland acinar cells in mice, regardless of age, including the parotid glands of the autoimmune NOD mouse (Fig 2A). Using immunohistochemical staining to detect murine F4/80 [a 160-dK macrophage transmembrane glycoprotein (
We next used RT-PCR and mouse primers specific for CD3, CD20, and F4/80 to detect the presence of CD3
, CD20, and F4/80 mRNAs in homogenized parotid gland, enzymatically dispersed acinar cell preparations, and spleen. CD3
could be detected only in mouse spleen and parotid gland (Fig 3, Lanes B and D). Dispersed acinar cell preparations from 5-week-old BALB-c (Fig 3, Lane F) and from 12-week-old NOD (Fig 3, Lane H) mice showed no CD3. The presence of G3PDH mRNA in the spleen, parotid gland, and in two acinar cell preparations (Fig 3, Lanes A,C,E,G) showed that our specimen handling methods yielded high-quality total cellular RNA. Controls in which the RT enzymes were omitted showed no reaction products for G3PDH, CD3
, of F4/80, indicating that our RNA preparations were free of genomic DNA contamination (data not shown).
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F4/80 could be detected only in mouse spleen and parotid gland (Fig 4, Lanes B and D). Dispersed parotid acinar cell preparations from 5-week-old BALB/c (Fig 4, Lane F), 4-week-old NOD (Fig 4, Lane H), and 12-week-old NOD (Fig 4, Lane J) mice showed no F4/80 mRNA. G3PDH mRNA was abundant in spleen, parotid gland, and in all acinar cell preparations (Fig 4, Lanes A,C,E,G,I).
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CD20 was detected in mouse spleen (Fig 5, Lane A) but not in dispersed parotid acinar cell preparations from 5-week-old BALB/c (Fig 5, Lane B) or from 12-week-old NOD (Fig 5, Lane C) mice. G3PDH mRNA was abundant in spleen and in acinar cell preparations (data not shown)
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Determination of Cell Number in Parotid Acinar Cell Harvests
Total cellular DNA in all acinar cell preparations was 13.9 ± 3 mg DNA/ml (n = 12 preparations). Next, using the published value of 6.85 pg DNA per rodent salivary gland acinar cell (
Determination of IL-1ß, IL-6, and TNF- Concentrations in Mouse Parotid Acinar Cell Preparations and Calculated Average Amounts in Individual Acinar Cells
We used ELISA and DNA assays to determine total cell concentrations of IL-1ß, IL-6, and TNF- in unstimulated, enzymatically dispersed mouse parotid acinar cell preparations harvested from 5-week- and 10-week-old BALB/c and NOD mice. Dispersed parotid acinar cells harvested from 10-week-old NOD mice showed a small but highly significant (p<0.003) increase in IL-ß and a large significant decrease (p< 0.008) in IL-6 compared to 5-week-old NOD mice (Table 1). In contrast, BALB/c mice showed a significant increase both in IL-1ß (p<0.03) and in IL-6 (p<0.005) with increasing age. In addition, the concentration of IL-6 in 10-week-old NOD mice was significantly lower (p<0.0007) than IL-6 in 10-week-old BALB/c mice. TNF-
showed no change in concentration with mouse strain or age (Table 1). Calculated average amounts of IL-1ß, IL-6, and TNF-
in parotid acinar cells from 5- and 10-week-old BALB/c and NOD mice are shown in Table 1.
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Amylase and LDH Release from Dispersed Parotid Acinar Cells Harvested from BALB/c and NOD mice
Amylase determinations from all partoid acinar cell preparations showed maximal release by 20 min after 10-5 M norepinephrine stimulation at 37C. The percent amylase released above basal levels for 5-week-old BALB/c and NOD mice (56 ± 12% and 58 ± 9%, respectively) was not statistically different from 10-week-old BALB/c and NOD mice (62 ± 4% and 66 ± 8%, respectively) (Fig 6A6D). Importantly, LDH determinations from the same acinar cell preparations were less than 5%, which showed that the concentration of norepinephrine used for stimulation did not cause acinar cell death (Fig 6A6D).
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Discussion |
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These studies show that an enzymatic digestion method for dispersing secretory epithelial cells from the parotid gland also eliminates T-cells, B-cells, and macrophages below the level of detection by a highly sensitive RT-PCR method. Using standardized cytokine concentrations (i.e., pg cytokine/µg acinar cell DNA), we determined cytokine profiles in parotid acinar cells from BALB/c and NOD mice at precise time points during progression of autoimmune sialoadentis. The parotid gland was selected for these studies because it is the major saliva-producing gland in the oral cavity of humans (
Because of the recent emphasis on the role of acinar cells in the initiation and progression of autoimmune sialaodenitis (
In this study, it was necessary to determine if the enzymatic dispersion methods we used for harvesting parotid acinar cells resulted in cell preparations that were free of other cellular sources (i.e., T-cells, B-cells, macrophages) of these cytokines. Using RT-PCR, we showed the unequivocal absence of T-cell-, B-cell-, and macrophage-specific mRNA in all acinar cell preparations. Enzymatic dispersion methods similar to the one we used do not disrupt T-cell surface markers (
We considered the possibility that intracellular IL-1ß, TNF-, and IL-6 concentrations in isolated acinar cells might be altered secondary to the acinar cell dispersion procedure. Therefore, we used established cell dispersion methods (
. For example, media and dispersion solutions did not contain known inducers (e.g., endotoxin or serum factors) of IL-1 genes (
Although the precise role(s) of inflammatory cytokines in the pathophysiology of Sjögren's syndrome is unknown, evidence supporting their direct participation in initiating glandular dysfunction is mounting. For example, increased levels of cysteine proteases (e.g., interleukin-converting enzyme, ICE) were found by both an in vitro activity assay and Western blotting analysis of salivary glands from 20-week-old NOD mice compared with 8-week-old BALB/c and NOD mice (
It is interesting that whereas IL-1ß increased, TNF- concentrations in parotid acinar cells remained almost constant in BALB/c and NOD mice between 5 and 10 weeks. In support of our findings, others have shown in mononuclear leukocytes from aging NOD mice that IL-1 concentrations increased between 13 and 16 weeks but that TNF-
mRNA concentrations did not change (
mRNA was at the same level when measured at these two time pints (
may act synergistically with IL-1ß to induce expression of Fas ligand in secretory epithelial cells and to trigger apoptosis (
monoclonal antibody in NOD mice showed no reduction in the development of insulin-dependent diabetes mellitus, arguing against a crucial role of TNF-
in the induction of this autoimmune disease (
Several studies report a correlation between increased IL-6 concentrations and increased autoimmune disease activity (
In this study we demonstrated a surprising decrease in intracellular IL-6, specifically in parotid acinar cells from aging NOD mice. This result is interesting in light of two recent reports showing that recombinant mouse IL-6 suppresses both IL-1- and TNF--induced apoptosis in a cultured epithelial cell line (
An important contribution of this work was the estimation of cytokine quantities in individual mouse parotid acinar cells. We used a fluorescence assay to determine total DNA concentration for each cell preparation and ELISA assays to determine total cellular cytokines per milliliter of buffer. By using published values of DNA in rodent salivary gland acinar cells, we calculated the intracellular quantities of IL-1ß, IL-6, and TNF-. This approach has not been previously reported, to the best of our knowledge, but should be particularly valuable for cytokine determinations from glandular cells, which typically are difficult to separate from one another. Determination of cytoplasmic quantities of cytokines will be useful in testing recently proposed intracellular mechanisms of cytokine modulation of cell death pathways (
At the heart of our hypothesis is the idea that parotid acinar cells are important producers of the inflammatory cytokines that modulate their health and disease. We have now determined that mouse parotid acinar cells represent an independent source of abnormally expressed IL-6 and IL-1ß in sialoadenitis-prone animals. By mapping characteristic alterations in specific pro-inflammatory cytokines from salivary gland acinar cells, we ultimately hope to identity the particular micorenvironmental conditions that predispose the salivary gland to lymphocyte infiltration and acinar cell destruction. Ongoing studies of the intracellular signal pathways that control age-associated alterations in cytokine production by sialoadenitis-prone parotid acinar cells should help to clarify the role of inflammatory cytokine processing in the pathogenesis of Sjögren's syndrome.
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Acknowledgments |
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Supported by USPHS grants DE-10059 (GRL), DE12358 (GRL), DE10335 (DTW), and DE08680 (DTW).
Received for publication November 4, 1999; accepted March 29, 2000.
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