ARTICLE |
Correspondence to: Satoru Naruse, Department of Internal Medicine II, Nagoya U. School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8560, Japan. E-mail: snaruse@med.nagoya-u.ac.jp
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Summary |
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It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA2 isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.
(J Histochem Cytochem 49:777782)
Key Words: group IIA phospholipase A2, immunohistochemistry, in situ hybridization, rat, spleen, megakaryocyte, intestine, Paneth cell
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Introduction |
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PHOSPHOLIPASE A2 (PLA2) comprises a diverse family of enzymes that catalyzes the hydrolysis of glycerophospholipids at the sn-2 position to produce free fatty acids and lysophospholipids (
Group II PLA2 mRNA and protein were detected in homogenates of the spleen, lung, liver, and kidney of normal rats (
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Materials and Methods |
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Preparation of Rats
Male Wistar rats (180220 g) were anesthetized with an IP injection of pentobarbital sodium (50 mg/kg) and transcardially perfused with Tyrode's solution and then with 4% paraformaldehyde in 0.01 M PBS, pH 7.4. The jejunum, spleen, lung, liver, kidney, and pancreas were removed and postfixed in the same fixative for 3 hr. They were then immersed in a series of cold sucrose solutions (10%, 20%, 30% sucrose in PBS). Ten-µm-thick sections were cut on a cryostat microtome (Bright; Huntingdon, UK) and mounted on silanized slides (DAKO; Kyoto, Japan). The sections were dried on a hotplate at 37C and then kept frozen until use.
Digoxigenin-labeled RNA Probe
We used a 0.7 kilobase pair fragment of digoxigenin-labeled RNA probe in the present study. This RNA probe corresponds to bp 76748 of group IIA PLA2 mRNA, which covers about 90% of the whole mRNA. The NheI/EcoRI 0.7-kb pair fragment of rat group II PLA2 cDNA was excised from its full-length cDNA (
In Situ Hybridization
The sections were incubated in 0.3% Triton X-100 in PBS for 5 min and immersed in 10 µg/ml proteinase K (Boehringer Mannheim Biochemical; Indianapolis, IN) in PBS for partial proteolysis for 10 min at 37C. They were postfixed in 4% paraformaldehyde in PBS for 10 min and immersed in a solution containing 0.25% acetic acid anhydride (Nippon Gene; Toyama, Japan), 0.1 M triethanolamine for 20 min at room temperature (RT). They were then prehybridized in 50% formamide in 2 x SSC for 30 min at 42C. Hybridization was done with digoxigenin-labeled RNA antisense probe diluted to 1 µg/ml in hybridization buffer in a moist chamber for 16 hr at 42C. For negative control, digoxigenin-labeled RNA sense probe was hybridized to a section adjacent to the test section. After hybridization, the slides were washed in 50% formamide in 2 x SSC for 60 min at 42C and treated with 20 µg/ml RNase A in 0.5 M NaCl, 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) for 30 min at 37C. They were rinsed in the same buffer at 37C for 30 min, then in 0.1 x SSC for 60 min at 42C, and finally in PBS for 1 min at RT. For immunological detection of digoxigenin, the slides were preblocked in 1% skim milk fraction in PBS for 30 min at RT and then incubated with alkaline phosphatase-conjugated polyclonal anti-digoxigenin Fab fragments (Boehringer Mannheim) (1:500 dilution) for 90 min at RT. After incubation, the slides were washed in PBS and developed in a solution of nitroblue tetrazolium and X-phosphate for 16 hr at RT. The color reaction was stopped using 1 mM EDTA, 10 mM Tris-HCl. The sections were mounted in Vectashield (Vector Laboratories; Burlingame, CA).
Immunohistochemistry
Rabbit anti-rat group II PLA2 IgG raised against purified PLA2 released from thrombin-stimulated rat platelets was used for histochemistry. The slides were rinsed with PBS and nonspecific binding was blocked in 1% skim milk in PBS. Sections were incubated with rabbit anti-rat group II PLA2 antibody. (
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Results |
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Jejunum
In the jejunum, group IIA PLA2 mRNA-positive cells were present in the bottom of crypts (Fig 1a and Fig 1b). No significant signal was seen in the crypts in the negative control. Staining of the epithelial cells in the negative control indicated that it was due to the intrinsic alkaline phosphatase (Fig 1c). Group IIA PLA2 immunoreactivity was also observed in the bottom of crypts (Fig 2a2c). These cells were pyramidal, with a round or ovoid nucleus (Fig 2d2f). These characteristic features indicated that they were Paneth cells.
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Spleen
ISH revealed that signals of group IIA PLA2 mRNA were observed in large cells (3040 µm) with multilobular nuclei in the red pulp (Fig 3a and Fig 3b). Group IIA PLA2-positive cells were roughly spherical but often had blunt, irregular pseudopods on their surfaces. Their nuclei were extraordinarily elaborate, with multiple lobes of various sizes. The double-labeling study demonstrated that group IIA PLA2-positive cells were also positive for fibrinogen (Fig 4a4f). These morphological and immunohistochemical findings indicated that group IIA PLA2-positive cells in the spleen were megakaryocytes. Neither group IIA PLA2 mRNA nor immunoreactivity was observed in other types of cells in the spleen.
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The ED-1 immunohistological study revealed that ED1-positive cells were found in the red pulp. These cells were 10 µm in diameter, mononuclear, and rich in cytoplasm. Therefore, these ED1-positive cells in the red pulp appeared to be macrophages. A double-immunolabeling of group IIA PLA2 and ED1 revealed that group IIA PLA2 and ED1 were never co-localized in any cells. Thus, group IIA PLA2-positive cells were completely separated from ED1 positive cells (Fig 5a5f). These results indicated that megakaryocytes, but not macrophages, contained group IIA PLA2.
Other Organs
Group IIA PLA2 mRNA and immunoreactivity were not detected in any cells of the lung, liver, kidney, and pancreas.
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Discussion |
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In this study, using a specific RNA probe and an antibody for rat group IIA PLA2, we demonstrated that group IIA PLA2-producing cells were megakaryocytes in the spleen and Paneth cells in the small intestine in normal rats. The antibody used in the present study was raised against group IIA PLA2 isolated from the rat platelet (
IHC of smeared bone marrow cells indicated that megakaryocytes produced group II PLA2 (
The concentration of group II PLA2 was very low in human spleen (
Northern blotting analysis (
Because group II PLA2-specific activities, protein, or mRNA were present in the homogenate of the normal rat liver (
In conclusion, under normal conditions group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats. The induction of group IIA PLA2 in other types of cells under inflammatory conditions awaits further studies.
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Acknowledgments |
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Supported by a research grant for intractable pancreatic disease from the Ministry of Health and Welfare in Japan and by a grant from Pancreatic Research Foundation of Japan.
Received for publication December 28, 2000; accepted January 8, 2001.
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