Abstract
Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.
Keywords
P
The gene coding for rat Group II phospholipase A2 was cloned from the spleen (Ishizaki et al. 1989), platelets (Komada et al. 1990), liver (Van Schaik et al. 1993), and heart (De Windt et al. 1997). Southern blotting analysis of genomic DNA revealed that there is only a single copy in the rat genome (Komada et al. 1990). There are minimal differences between the reported cDNA sequences, obviously due to the study of different rat strains. The amino acid sequences of PLA2s from the spleen, platelets, and heart are identical, and in PLA2 from the liver there is only one amino acid difference compared to the PLA2s from other sources. The coding sequence of the rat PLA2 is 758 nucleotides, which predicts a 146-amino-acid protein with high homology (72%) to human PLA2 (Ishizaki et al. 1989). The rat PLA2 contains a 21-amino-acid N-terminal signal sequence, 14 cysteine residues, and a six-amino-acid C-terminal extension, which are characteristic of Group IIA phospholipase A2 (Tischfield 1997).
The purpose of this study was to investigate the distribution of PLA2 protein and mRNA in rat tissues. We produced recombinant rat PLA2 and polyclonal antibodies against it, and localized the PLA2 protein by immunohistochemistry and the mRNA of PLA2 by Northern and in situ hybridizations. Our results show that PLA2 is expressed in few specific cell types. The strongest expression of PLA2 is found in the Paneth cells of the intestinal mucosa.
Materials and Methods
Reagents
Rat Group II phospholipase A2 cDNA sequence cloned in the EcoRI and XbaI sites of pGEM4 (Promega; Madison, WI) was obtained from Shionogi Research Laboratories (Osaka, Japan). The bacterial expression vector pQE-60 was purchased from QIAGEN (Chatsworth, CA). Restriction enzymes were purchased from Boehringer Mannheim (Mannheim, Germany) except for BspHI, which was from New England Biolabs (Beverly, MA). DNA ligase was from Life Technologies, Gibco BRL (Paisley, UK). Primers were synthesized at the University of Kuopio (Kuopio, Finland). E. coli JM109 cells were obtained from Department of Microbiology (University of Turku, Turku, Finland) and M15 and SG13009 cells from QIAGEN. Unless otherwise noted, all other reagents were from Sigma (St Louis, MO).
PCR Amplification
Three oligonucleotide primers of sense and antisense orientations based on the sequence of rat PLA2 cDNA were synthesized. The forward primer sequences were (412) 5′-ATA TCC ATG GAT GAA GGT CCT CCT GTT GC-′3 and (551) 5′-ATA TTC ATG AGC CTT CTG GAG TTT GGG-′3 and the reverse sequence (413) 5′-ATA AGA TCT GCA ACT GGG CGT CTT CCC-′3. The region of rat PLA2 cDNA that encodes the signal peptide and the mature protein was amplified by PCR. Primer 412 has a unique NcoI site. Biotinylated primer 551 contained the sequence for BspHI and the initiating methionine. Primer 413 has a unique BglII site. Rat PLA2 contains the BspHI site in the protein coding sequence starting at nucleotide 258. PCR was carried out in 100-μl reaction volume containing 100 mM Tris-HCl (pH 8.3), 0.2 mM dNTP, 1 μM antisense primer, 1 μM sense primer, and 2.5 U Taq DNA polymerase (Finnzymes; Espoo, Finland). Conditions for 35 cycles of PCR were 95C/30 sec, 58C/60 sec, and 72C/45 sec.
Cloning and Expression of Rat Group II Phospholipase A2
Cloning of rat PLA2 was carried out in two steps (Figure 1). The first PCR was done with primers 412 and 413. The product was extracted from the gel and digested with NcoI and BglII. The digested product was ligated to pQE-60 vector previously digested with NcoI and BglII. This construct (pQE-60-PLA2-sham), which is not in frame, was used as the first step in cloning and later as a negative control. The second step was PCR with primers 551 and 413. The PCR product was attached to streptavidin-coated magnetic beads (Boehringer Mannheim). The product was digested with BamHI, washed, digested with BspHI, and recovered. The product was ligated and cloned to the pQE-60-PLA2-sham vector previously digested with NcoI and BamHI, resulting in the plasmid pQE-60-PLA2. Finally, successful construction was confirmed by sequencing.
The plasmid pQE-60-PLA2 was transformed into E. coli strain JM 109. Transformants were selected on LB–agar plates supplemented with 100 μg/ml ampicillin. For induction of gene expression, E. coli JM109 cells containing pQE-60-PLA2 were grown at 30C in LB medium containing 100 μg/ml ampicillin. After reaching an OD550 = 1.0, isopropyl-β-
Preparation of Recombinant Rat Group II Phospholipase A2
Recombinant PLA2 was found exclusively in the inclusion bodies of E. coli. A cell pellet from 300 ml of bacterial culture (1.5 g wet weight) was thoroughly washed with buffer containing 100 mM Tris-HCl (pH 7.3) and 150 mM NaCl. The cells were lysed with an ultrasound sonicator (Labsonic L; Braun Diessel Biotech, Melsungen, Germany) at maximal power twice for 30 sec. Lysozyme was added to a final concentration of 1 mg/ml, incubated for 30 min at room temperature (RT) and sonicated. The pellet was harvested by centrifugation at 10,000 × g for 10 min and resuspended in 20 ml buffer containing 6 M guanidine hydrochloride and 100 mM Tris-HCl (pH 7.3). After incubation overnight at RT, silica was added to a final concentration of 1% and the cell lysate was pelleted by centrifugation. The supernatant was recovered and β-mercaptoethanol was added to a final concentration of 0.2%. The recombinant PLA2 was further purified by using nickel affinity chromatography. Briefly, a 20-ml sample was loaded onto an affinity column charged with Ni2+ and washed with 6 M guanidine hydrochloride, pH 7.6 and pH 5.3, and PLA2 was eluted with 6 M guanidine hydrochloride, pH 4.1. The eluate was dialyzed against water overnight at 4C. Amino-acid sequence analysis was performed with an Applied Biosystems (Foster City, CA) model 477A protein sequencer equipped with an on-line Applied Biosystems model 120A phenylthiohydantoin amino acid analyzer. The sample for mass spectrometry was mixed with sinapinic acid matrix and analyzed by a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS; Lasermat, Finnigan Mat, Bremen, Germany).
Cloning of rat Group II phospholipase A2.
Measurement of the Catalytic Activity of Phospholipase A2
The catalytic activity of phospholipase A2 was measured by a modified radiometric method (Schädlich et al. 1987) with L-α-1-palmitoyl-2-arachidonyl-containing phosphatidyl ethanolamine (NEN Life Science Products; Boston, MA) as a substrate.
Preparation of Antiserum
Three New Zealand White rabbits were immunized SC with 250 μg of purified recombinant rat PLA2 in Freund's complete adjuvant. Four booster injections of 200 μg of PLA2 in Freund's incomplete adjuvant were administered after 2, 4, 6, and 8 weeks. Blood was collected from each rabbit before each booster injection.
Western Blotting
Purified recombinant rat PLA2 and ileal lysates from Sprague–Dawley rats were separated in 4–15% SDS-PAGE gels (Phastsystem; Pharmacia, Uppsala, Sweden) and transferred to nitrocellulose filters (Schleicher & Schuell; Dassel, Germany). Anti-rat PLA2 antiserum diluted 1:5000 was used as the primary antibody. The immunoreaction was detected with a Vectastain avidin–biotin–peroxidase complex (ABC) kit (Vector Laboratories; Burlingame, CA) according to the manufacturer's instructions.
Immunohistochemistry
Tissues from various rat organs were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were incubated with several different dilutions ranging from 1:5000 to 1:20,000 of anti-rat PLA2 antiserum with 0.15 M NaCl in 0.05 M Tris buffer, pH 8.6, containing 1% bovine serum albumin. Rabbit anti-human von Willebrand factor (DAKO; Glostrup, Denmark) was diluted 1:1000. The immunoreaction was localized with a Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions. For control staining, the primary antibody was replaced by preimmune rabbit serum. The sections were counterstained with hematoxylin.
Northern Hybridization
RNA was isolated by the guanidine isothiocyanate/acid phenol method (Chomzynski and Sacchi 1987). Ten μg of total RNA was electrophoresed through a 1% agarose/formaldehyde gel and transferred to GeneScreen nitrocellulose membrane (NEN Life Science Products) with 10 × SSPE overnight. The RNA was crosslinked to the filter by UV irradiation and the filter was baked at 80C for 1 hr. A 10 × 15-cm filter was prehybridized for 4 hr at 42C in 7.5 ml Presoak (50% deionized formamide, 4 × SSPE, 5 × Denhardts solution, 1% SDS, 100 μg/ml denatured ssDNA) in a rotary incubator (Hybaid; Middlesex, UK). The prehybridization solution was replaced by 7.5 ml fresh Presoak containing the denatured probe, 32P-labeled EcoRI/XbaI fragment of PLA2/pGEM4 (Random Prime Labelling Kit; Boehringer Mannheim). The hybridization was performed in a rotary incubator at 42C overnight. The filter was washed twice for 15 min in 2 × SSPE/0.1% SDS at RT, 15 min in 2 × SSPE/0.1% SDS at 42C, 15 min in 0.1 × SSPE/0.1% SDS at RT, 15 min in 0.1 × SSPE/0.1% SDS at 42C, 15 min in 0.1 × SSPE/0.1% SDS at 56C, and 15 min in 0.1 × SSPE/0.1% SDS at 65C. The filter was exposed to Hyperfilm MP (Amersham; Poole, UK) with intensifying screens at −70C for 7 days. Several different exposures varying from 3 hr to 7 days were taken.
In Situ Hybridization (ISH)
ISH was performed on sections of formalin-fixed, paraffin-embedded tissues as described previously (Nevalainen et al. 1998), with slight modifications. Briefly, the 0.75-kb rat PLA2 cDNA insert was cloned into the XbaI and EcoRI sites of pGEM-4 vector (Ishizaki et al. 1989). Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription with T7 (sense) and SP6 (antisense) RNA polymerases, and the yields were estimated by using commercial kits (DIG RNA Labelling Kit and DIG Nucleic Acid Detection Kit; Boehringer Mannheim) as described in the kit protocols. Immediately before hybridization, the sections were rehydrated in PBS, treated with ficin (Zymed Digest-All; Zymed Laboratories, San Francisco, CA) for 10 min at 37C, washed in PBS, pH 7.4, and 2 × SSC (standard saline citrate; 1 × SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 5 min each. The hybridization solution contained either the antisense or sense digoxigenin-labeled cRNA probe at a final concentration of 5 ng/ml. Hybridization was performed overnight at 42C in a humidified chamber. After hybridization, the slides were rinsed in 2 × SSC and washed in 2 × SSC for 5 min at RT, three times for 5 min in 60% formamide at 60C, and twice for 5 min in 2 × SSC at RT. For the detection of digoxigenin-labeled cRNA probes, all incubations were performed at RT in a humidified lightproof chamber. The tissue sections were washed, blocked, and treated with a 1:2000 dilution of alkaline phosphatase-labeled anti-digoxigenin Fab fragments (Boehringer Mannheim). The slides were washed in TBS (100 mM Tris-HCl, 100 mM NaCl, pH 7.5) containing 0.05% Triton X-100 twice for 5 min and in TBS for 5 min, and rinsed in the development buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The tissue sections were covered with 500 μl substrate solution consisting of 0.18 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Boehringer Mannheim), 0.34 mg/ml 4-nitroblue tetrazolium chloride (NBT) (Boehringer Mannheim), and 0.24 mg/ml levamisole (Vector Laboratories) dissolved in the development buffer. The color reaction was allowed to develop for 10 hr at 4C and was stopped by washing twice for 5 min in 10 mM Tris, pH 8.0, containing 1 mM EDTA. The sections were counterstained with hematoxylin.
Results
Cloning and Expression of Rat Group II Phospholipase A2
PCR was used to amplify the protein coding sequence from rat PLA2 cDNA. The cDNA contains a BspHI site in the protein coding sequence at nucleotide 258. To avoid addition of extra amino acids to the N-terminus of the recombinant protein, PCR was done in two steps as described in Materials and Methods. A 378-bp DNA product was obtained (Figure 2). The DNA product was inserted into NcoI/BspHI and BglII sites of the pQE-60 expression vector to produce pQE-60-PLA2. This expression system produces a recombinant protein with six additional adjacent histidine residues at the C-terminus of the inserted gene. Plasmids were transformed into JM109 E. coli and selected bacteria were submitted to induction protocols. After the induction of plasmid pQE-60-PLA2 with IPTG, significant production of rat Group II phospholipase A2 recombinant protein could be achieved in JM109 E. coli, as visualized by the appearance of a protein band of approximately 18 kD in SDS-PAGE (Figure 3). We also found duplicates of recombinant PLA2 when more protein was loaded in SDS-PAGE (Figure 3). Recombinant PLA2 was found exclusively in the inclusion bodies of JM109 E. coli, whereas no recombinant protein was found in the soluble fraction of JM109 E. coli. The maximal rate of production of the recombinant protein was reached at 12 hr after the induction. For practical purposes, production was allowed to continue overnight. The induction of plasmid pQE-60-PLA2 was also tested in strains M15 and SG13009 of E. coli, but no production of the recombinant protein was seen in these bacteria.
Expression construct pQE-60-PLA2. Restriction enzyme sites used in the cloning are underlined. NcoI/BspHI is the ligation of Ncol (C′CATG_G) to BspHI (T′CATG_A). The partial pQE-60 vector sequence from EcoRI to HindIII (sites marked with dotted underlining) is in lower case. Amino acids not present in native rat Group II phospholipase A2 are marked with wavy underlining.
Large-scale Expression and Purification of Recombinant Rat Group II Phospholipase A2
JM109 E. coli harboring the plasmid pQE-60-PLA2 were grown in 1 liter of LB medium and induced with isopropyl-β-
SDS-PAGE of recombinant rat Group II phospholipase A2. Lane 1, molecular weight markers; Lane 2, 10 ng; Lane 3, 50 ng; Lane 4, 100 ng; Lane 5, 200 ng; and Lane 6, 400 ng of recombinant PLA2.
Production of Antibody
Once a sufficient amount of recombinant protein was available, antiserum was raised in rabbits against rat PLA2. To verify that the antibody produced to the recombinant protein would recognize native rat Group II phospholipase A2, an extract of rat ileum was analyzed by Western blotting. The antibody recognized a 14-kD protein (Figure 4). The antibody was used to detect the distribution of PLA2 protein in rat tissues by immunohistochemistry.
Crossreactivity of Anti-rat Group II Phospholipase A2 Antibody
Crossreactivity of the antibody was studied with sections of formalin-fixed, paraffin-embedded samples of human small intestine (Nevalainen et al. 1995) and tissues of human Group II phospholipase A2-transgenic mice (Nevalainen et al. 1997). Positive immunostaining was found in the Paneth cells of the human intestinal mucosa. In the human Group II phospholipase A2 transgenic mice, the same pattern of distribution of PLA2 was seen in various tissues as with the anti-human Group II phospholipase A2 antibody (data not shown).
Distribution of Group II Phospholipase A2 in Rat Tissues
Tissues from Sprague–Dawley rats of both sexes were studied by immunohistochemistry for the presence of PLA2 protein and by Northern and in situ hybridizations for the mRNA of PLA2. The results are summarized in Table 1.
Western blotting with anti-rat PLA2 antibody. Lane 1, 10 ng; Lane 2, 5 ng; Lane 3, 1 ng of recombinant group II PLA2; and Lane 4, extract of rat ileum.
Gastrointestinal Tract. The Paneth cells were strongly immunoreactive in the duodenum, jejunum, ileum, and cecum. The immunoreaction was localized in the secretory granules of Paneth cells (Figure 5). Many goblet cells showed moderate immunoreaction in the small intestine and colon. No immunoreaction was found in the walls of vessels in the lamina propria or in the smooth muscle cell layer. There was no immunoreaction in the esophageal mucosa, mucosa of the glandular stomach, or pancreas. Periportal and centrilobular hepatocytes were lightly stained, whereas other cell types in the liver were unstained.
Spleen. There were large multinuclear cells with strong immunoreaction in the red pulp area of the spleen (Figure 6). The same cells were immunoreactive for von Willebrand factor (Figure 7), suggesting that these cells were megakaryocytes.
Immunohistochemical localization and Northern and in situ hybridization of group II phospholipase A2 in rat tissues
–, no signal; +, weak signal or signal in occasional cells only; + +, moderate signal; + + +, strong signal; ns, tissues not studied.
Immunoreaction for rat Group II phospholipase A2 in the the small intestinal mucosa. The reaction is localized in the secretory granules of the Paneth cells. Anti-rat PLA2 antiserum 1:20,000, avidin-biotin-peroxidase complex (ABC). Bar = 50 μm.
Immunoreaction for PLA2 in megakaryocytes in the spleen. Anti-rat PLA2 antiserum 1:5000, ABC. Bar = 50 μm.
Immunoreaction for von Willebrand factor in megakaryocytes of the spleen. Anti-von Willebrand factor 1:1000, ABC. Bar = 50 μm.
Northern hybridization showed approximately an 800-base transcript in the esophagus, glandular stomach, duodenum, jejunum, ileum, cecum, colon, and spleen (Figure 8). The largest amount of PLA2 transcript was found in the ileum. By in situ hybridization, mRNA of PLA2 was found in the Paneth cells only (Figure 9). In situ hybridization with the control riboprobe gave negative results in the intestinal mucosa (Figure 10) and all other tissues studied.
Respiratory Organs and Cartilage. There was moderate immunoreaction in the chondrocytes and the matrix of the cartilage of the trachea and in the main bronchus. Costal cartilage was also immunostained. There was no immunoreaction in the other cell types of the lung. Northern blotting detected mRNA of PLA2 in the lung, but in situ hybridization gave negative results.
Heart and Circulatory System. There was no immunoreaction in the myocytes of the heart or the aortic wall. Northern blotting detected mRNA of PLA2 in the heart, whereas in situ hybridization gave negative results.
Urinary and Reproductive Organs. Neither immunoreaction or Northern blotting signal was observed in the kidney, prostate, seminal vesicle, testis, epididymis, ovary, Fallopian tube, and uterus. Urinary bladder gave a signal in Northern blotting but no immunoreactive material was observed.
Neural, Muscle, and Lymphoid tissues. Neural tissue and skeletal muscle were devoid of immunoreaction and Northern blotting signal. Neither lymph nodes nor adipose tissue contained immunoreactive material. Northern blotting gave negative results in the latter tissues as well.
Discussion
In this study we expressed rat Group II phospholipase A2 in E. coli and produced antibodies against the recombinant protein. Furthermore, we investigated the distribution of PLA2 protein and its mRNA in various rat organs and cell types.
Northern blotting of rat tissues for the mRNA of Group II phospholipase A2. Lane 1, cecum; Lane 2, liver; Lane 3, prostate; Lane 4, testis; Lane 5, pancreas; Lane 6, ventricle; Lane 7, lung; Lane 8, kidney; Lane 9, spleen; Lane 10, skin; Lane 11, cerebellum; Lane 12, adrenal gland; Lane 13, jejunum; Lane 14, lacrimal gland; Lane 15, adrenal gland; Lane 16, ileum; Lane 17, epididymis; Lane 18, parotid gland; Lane 19, heart; Lane 20, seminal vesicle; Lane 21, esophagus; Lane 22, duodenum; Lane 23, aorta; Lane 24, thymus; Lane 25, urinary bladder; Lane 26, skeletal muscle; Lane 27, colon; and Lane 28, brain. Each lane was loaded with 10 μg RNA and the autoradiograph was exposed for 4 days.
Positive in situ hybridization for the mRNA of Group II phospholipase A2 in rat ileum. The signal is localized in the Paneth cells. Anti-sense riboprobe. Bar = 50 μm.
Negative in situ hybridization for the mRNA of Group II phospholipase A2 in rat ileum. Control (sense) riboprobe. Bar = 50 μm.
The recombinant protein was generated by expressing the protein-coding sequence of rat PLA2 with an attached C-terminal histidine tag for rapid purification. Rat PLA2 cDNA was cloned and sequenced earlier (Ishizaki et al. 1989). It consists of 758 nucleotides coding for a 146-amino-acid protein. Rat group II PLA2 protein contains a 21-amino-acid N-terminal signal sequence, and the N-terminus of secreted PLA2 is serine at position 22 (Ono et al. 1988). We sequenced six N-terminal amino acids in the current recombinant rat PLA2 and found that, in 83% of the protein, the first amino acid was serine, as in native rat PLA2 (Ono et al. 1988). The recombinant PLA2 produced in the present study was catalytically active, which might be due to partial refolding caused by oxidation during the dialysis against water.
In this study we investigated the distribution of PLA2 and its mRNA in rat tissues. We used immunohistochemistry to detect the PLA2 protein and Northern and in situ hybridizations to detect the mRNA. As far as we are aware, the present study contains the first data on in situ hybridization of mRNA of Group II phospholipase A2 in the rat. We found the mRNA in the Paneth cells of the intestinal mucosa. Despite positive results in Northern blotting, no in situ hybridization signals were detected in other cell types. This finding indicates that the level of the gene expression of PLA2 in rat tissues may be below the detection limit of the current methods of tissue preservation and in situ hybridization.
Despite the high homology between human and rat Group II phospholipase A2 proteins, we have not previously found any crossreactivity between human anti-Group II phospholipase A2 antibody and rat PLA2 (unpublished data). In this study, we show crossreactivity between the anti-rat PLA2 antiserum and human Group II phospholipase A2 in immunohistochemistry. The Paneth cells of the human intestinal mucosa were immunostained with the anti-rat PLA2 antisera, and the latter crossreacted with the tissues containing human PLA2 in human Group II phospholipase A2-transgenic mice (Nevalainen et al. 1997).
Earlier, the distribution of PLA2 was studied in the gastrointestinal tract of humans (Kiyohara et al. 1992; Nevalainen et al. 1995) and rats (Senegas-Balas et al. 1984). The results have been divergent, probably due to the use of different antibodies. Synthesis of PLA2 was demonstrated in the Paneth cells of the intestinal mucosa by immunohistochemistry (Senegas-Balas et al. 1984; Kiyohara et al. 1992; Nevalainen et al. 1995) and by in situ hybridization (Nevalainen et al. 1995). In the present study, we found PLA2 protein and mRNA in the Paneth cells of rat intestine. Kiyohara et al. (1992) found PLA2 in the epithelial cells of the esophagus, stomach, duodenum, small intestine, and ascending colon. We found immunoreactive material in the goblet cells of the small intestine and colon but not in the epithelial cells of the esophagus or glandular stomach. On the other hand, we found mRNA of PLA2 by Northern blotting in the esophagus, glandular stomach, and colon.
It has been proposed that Group II phospholipase A2 has antibacterial effects in the gastrointestinal tract. PLA2 appears to be involved, together with other enzymes (e.g., lysozyme) and cryptidins in the protection of the small intestinal crypts against microbial invasion (Harwig et al. 1995). Kiyohara and others (1992) found PLA2 also in pancreatic acinar cells, Kupffer cells of the liver, and hepatocytes. Non-neoplastic hepatocytes of a patient suffering from an epithelioid hemangioendothelioma of the liver were found to express PLA2 (Nevalainen et al. 1996). In the current study, we found a slight immunostaining of some hepatocytes but no mRNA in the liver. Neither immunoreactive PLA2 nor mRNA of PLA2 was found in the pancreas.
Group II phospholipase A2 has been purified from rat spleen (Ono et al. 1988). The source of PLA2 in the spleen has been suggested to be splenic macrophages (Inada et al. 1991; Kiyohara et al. 1992). In the current study, mRNA of PLA2 was detected in the spleen by Northern blotting but in situ hybridization gave negative results. PLA2 was originally purified from platelets (Kramer et al. 1989). PLA2 of platelets is derived from megakaryocytes (Emadi et al. 1998). Here we show that PLA2 is located by immunohistochemistry in multinuclear giant cells in the spleen. These cells are also positive for von Willebrand factor, suggesting that they are megakaryocytes rather than monocyte macrophage-derived cells.
Recently, PLA2 was cloned from rat heart (De Windt et al. 1997) and PLA2 gene expression was observed in the heart, isolated ventricular myocytes, and cardiac-derived fibroblast-like cells by Northern blotting. PLA2 has been localized by immunohistochemistry in the ventricular myocytes of rat heart (Kriegsmann et al. 1993). However, in the current study, we did not find immunoreactive PLA2 in ventricular myocytes, although we detected mRNA of PLA2 in the heart by Northern blotting. This may be because there are very small amounts of the enzyme in myocytes.
Synovial fluid, chondrocytes, and the matrix of cartilage contain large amounts of PLA2 in the human (Nevalainen et al. 1993). Here, we found PLA2 in extra-articular cartilage (costal and tracheal cartilage). However, mRNA of PLA2 was not detected in chondrocytes by in situ hybridization.
In summary, we describe the production of large amounts of recombinant rat Group II phospholipase A2 and polyclonal rabbit antibodies against PLA2. Immunohistochemical, Northern blotting, and in situ hybridization analyses allowed a detailed characterization of the distribution of PLA2 in rat tissues. These methods provide useful tools for studies on the role of PLA2 in various experimental disease models.
Footnotes
Acknowledgements
Supported by the Turku University Hospital and the University of Turku Foundation.
We thank Dr Jun Ishizaki for providing the cDNA of rat Group II phospholipase A2, Dr Jukka Hellman for sequencing the recombinant PLA2, and Ms Sinikka Kollanus, Ms Anne Jokilammi–Siltanen, and Mr Jaakko Liippo for technical assistance.