ARTICLE |
Correspondence to: Osamu Shimada, Dept. of Public Health, Gunma Univ. School of Medicine, Maebashi, Gunma 371, Japan.
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Summary |
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The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen. (J Histochem Cytochem 46:833840, 1998)
Key Words: deoxyribonuclease I, Paneth cell, small intestine, immunoelectron microscopy, immunofluorescence, in situ hybridization, human
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Introduction |
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Deoxyribonuclease I (DNase I: EC.3.1.21.1), an endonuclease preferentially degrading double-stranded DNA, was first considered to be an exocrine pancreatic enzyme but was later found to be localized in a number of other tissues (
Although Paneth cells in intestinal crypts have been studied extensively since their discovery by
In this study we used five different polyclonal antibodies and one monoclonal antibody (MAb) specific for human DNase I, raised by immunizing rabbits, chickens, or mice repeatedly with specific antigens. All six antibodies used yielded similar results and immunohistochemical, enzymological, and PCR analyses confirmed the presence of significant amounts of DNase I in Paneth cells.
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Materials and Methods |
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Tissue Preparation
Tissue samples were obtained from male human bodies during 10 postmortem examinations with the consent of the bereaved families. The patients, aged 3766 years, died within 2 weeks after having suffered a myocardial infarction (n = 6), angina (n = 1), a subarachonoidal hemorrhage (n = 2), and anaphylactic shock (n = 1). The biopsy specimens were obtained from two of the authors, a healthy man (OS) and a healthy woman (HTS), during gastrointestinal and colon endoscopic medical examinations.
Antibodies
We prepared five different polyclonal antibodies and one MAb specific for human DNase I. The characteristics of some of these antibodies have already been reported (
Immunofluorescence and In Situ Hybridization
As described previously (
For in situ hybridization, the oligonucleotide probe (5'-CTGAAGATCGCAGCCTTCAACATC-3'), corresponding to the N-terminal region of human DNase I (
Immunogold Electron Microscopy
For histological analysis, small pieces of tissue were fixed with 1% paraformaldehyde, 0.125% glutaraldehyde, and 0.1% picric acid in 0.01 M PBS, pH 7.2, for 24 hr at 4C. We found that the routinely used fixatives, particularly glutaraldehyde and osmium tetroxide, dramatically reduced the amounts of DNase I detected by all the antibodies; therefore, only weak fixation was feasible. After fixation the tissues were dehydrated with a graded ethanol series at 0C, embedded in Lowicryl K4M, and then cured for 3 days at -35C.
As described previously (
Assay of Tissue DNase I Activity
DNase I activity was determined using the single radial enzyme diffusion (SRED) method (
We determined the protein contents of the samples using a Bio-Rad protein assay kit (Bio-Rad; Hercules, CA) with BSA as the standard.
Detection of DNase I Gene Transcripts by PCR
The procedure used was previously described in detail (
Immunoblot Analysis
Each tissue specimen was homogenized in 100 times its volume of sample buffer containing 4% sodium dodecyl sulfate (SDS) and 10% 2-mercaptoethanol, incubated for 5 min at 90C, condensed several times using Centricell (Polyscience; Warrington, PA), and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% acrylamide gradient gel. The proteins were transferred to a nitrocellulose sheet and incubated with an antibody (1:1000 dilution). Finally, the antigens were detected with alkaline phosphatase-conjugated anti-rabbit, anti-chicken, or anti-mouse immunoglobulin (1:1000 dilution).
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Results |
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DNase I Activity in the Small Intestine
The specific DNase I activities in various parts (Paneth cell-rich, upper part of villi, smooth muscle fragments, and whole tissue) of the duodenum, jejunum, and ileum were determined by the SRED method and the results are summarized in Table 1. The specific activities in the extracts from the Paneth cell-rich fragments of every portion of the human small intestine were considerably higher than those in the other samples tested. The DNase I activities in smooth muscle fragments of the small intestine were very low. The detectable enzymatic activities were inhibited by 1 mM EDTA or monomeric (G) actin, and, most notably, were abolished by the anti-human DNase I antibodies.
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Distribution of 31-kD Protein in the Small Intestine
Immunoblot analysis showed high levels of DNase I-like immunoreactivity in the Paneth cell-rich fragments of the small intestine (Figure 1), whereas little or no DNase I immunoreactivity was detected in the smooth muscle fragments. We obtained similar results using other antibodies.
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DNase I Gene Transcripts by PCR
The presence of DNase I-specific mRNA was verified by PCR analysis of the total RNAs extracted from several human tissues. A unique 780 base pair (BP) fragment, corresponding to the region encoding the mature enzyme, could be amplified from the total RNAs of the human pancreas and the Paneth cell-rich fragment of the small intestine (Figure 2). No amplified products were obtained from the brain or the smooth muscle fragments.
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Immunofluorescence and In Situ Hybridization
Immunofluorescence revealed very strong immunoreactivity in the cytoplasm of Paneth cells at the bases of the crypts of Lieberkühn in all regions of the small intestine, including the duodenum, jejunum, and ileum (Figure 3). The immunoreactivity was widely distributed in the Paneth cell cytoplasm but the staining intensity was heterogeneous, with particularly strong immunoreactivity observed in the cellular apical regions. Interestingly, little or no immunoreactivity was found in the nuclear regions of Paneth cells. The staining intensities differed according to the antibodies we used, but the distribution patterns were basically the same with all six antibodies, confirming that human DNase I itself is concentrated in the cytoplasm of human Paneth cells.
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In situ hybridization showed high levels of specific human DNase I mRNA in the Paneth cells of duodenum, jejunum, and ileum (Figure 4). No fluorescence was observed in a control section. No fluorescence was observed in the control sections without a probe (Figure 7A).
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Immunogold Electron Microscopy
At the fine-structural level, immunogold electron microscopy demonstrated DNase I distributed along the exocrine secretory pathway of the Paneth cells (Figure 5 and Figure 6). The secretory granules were heavily labeled with anti-human DNase I, especially in their electron-lucent halo regions, whereas the electron-dense inner domains (the granular core regions) showed less intense DNase I labeling. The Golgi apparatus and rough endoplasmic reticulum (rER) appeared to be labeled with fewer but still considerable numbers of gold particles. The exocytotic granules in Paneth cells were also DNase I-immunopositive. We obtained similar results with other antibodies. No significant gold particles were detected in a control section in which excess human DNase I was added to the first antibody (Figure 7B).
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Discussion |
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The general structure of Paneth cells was almost identical to that described previously (
Although the epithelial cells in villous tips histologically showed no clear immunoreactivity like that in Paneth cells, the fragment containing the villous tips biochemically showed significant amounts of DNase I and its mRNA. The inconsistency between histological and biochemical results might be explained by sample contamination with blood cells and Paneth cells. The villi, particularly the upper part, were quite soft and easily flexible, so it was impossible to obtain only a selective portion of the villi. Actually, we found a number of blood cells and some Paneth cells contaminating the villous tip fragment under a light microscope. According to a number of previous reports, DNase I is secreted by exocrine glands, such as the pancreas and parotid gland, into the alimentary tract (
Paneth cells are generally considered to be involved in the regulation of intestinal flora because of their ability to secrete antimicrobial materials (
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Acknowledgments |
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We are very grateful to Prof Lee D. Peachey (Department of Biology, University of Pennsylvania, Philadelphia, PA) for revising this manuscript, and also to Drs Tomoyuki Kawada and Yoshiaki Sasazawa (Department of Public Health, Gunma University School of Medicine, Gunma, Japan) for valuable and practical advice.
Received for publication August 7, 1997; accepted February 25, 1998.
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