ARTICLE |
Correspondence to: Timo J. Nevalainen, Dept. of Pathology, Univ. of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
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Summary |
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Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group lI PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group ll PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group ll PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2. (J Histochem Cytochem 45:1109-1119, 1997)
Key Words: acute-phase response, immunohistochemistry, inflammation, in situ hybridization, mRNA, phospholipase A2, riboprobes, transgenic mice
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Introduction |
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Phospholipase A2 (PLA2) is a lipolytic enzyme that hydrolyzes phospholipids into free fatty acids and lysophospholipids. PLA2 plays an important role in inflammation by releasing arachidonic acid from membrane phospholipids for eicosanoid synthesis (
It has been proposed that group II PLA2 is an acute phase protein produced by hepatocytes (
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Materials and Methods |
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Production of Transgenic Mice
Transgenic mice were produced by standard methods as described in detail elsewhere (
Immunohistochemistry
Tissues from various organs of three transgenic mice and one control mouse were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were collected on polylysine-coated slides, reacted with an IgG fraction (4.5 mg/ml) of a rabbit anti-group II PLA2 antiserum (
In Situ Hybridization
In situ hybridization was performed on sections of formalin-fixed, paraffin-embedded tissues by probing with group II PLA2 anti-sense (test) and sense (control) single-stranded RNA-probes as described earlier (
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Results |
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Tissues from three transgenic mice (male, 6.5 months of age, line 854, #1300; female, 4.5 months of age, line 703, #2059; male, 3 weeks of age, line 854, #3532) and one control mouse (female, 4.5 months of age, #2063) were studied by immunohistochemistry (Table 1) for human group II PLA2 enzyme protein and by in situ hybridization (Table 2) for the mRNA of human group II PLA2.
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Liver
There was a strong immunoreaction in the portal areas of the liver lobules. Hepatocytes were intensely stained, whereas other cell types, including Kupffer cells, were unstained (Figure 1). There was no immunoreaction in the liver or other tissues of the non-transgenic control mouse (Figure 2). Similarly, there was no immunostaining when the primary antibody was replaced by preimmune rabbit serum. The in situ hybridization signal for the mRNA of group II PLA2 (anti-sense riboprobe) was evenly distributed over hepatocytes (Figure 3). In situ hybridization with the control (sense) riboprobe gave negative results in liver (Figure 4) and other tissues. In situ hybridization with both anti-sense and sense riboprobes gave negative results also in the tissues of the non-transgenic control animal.
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Urinary Organs
Bowman's capsule in a number of glomeruli of the transgenic animals showed positive immunoreaction (Figure 5). There was granular immunoreaction in the apical cytoplasm of proximal tubule cells (Figure 6). Epithelial cells of the renal pelvis, ureter, and urinary bladder were also strongly immunostained. In situ hybridization gave a clear signal in Bowman's capsule but proximal tubules were devoid of the mRNA of group II PLA2 (Figure 7 and Figure 8). The epithelium of the urinary bladder gave a strong in situ hybridization signal, and there was weaker positive signal for the mRNA of group II PLA2 in the smooth muscle cell layer of the bladder (Figure 9 and Figure 10).
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Skin
There were many strongly immunoreactive, elongated connective tissue cells in some sections of the dermis (Figure 11). Basal cells of the epidermis showed weak immunoreaction. There was a distinct in situ hybridization signal in the epidermis (Figure 12). Fibroblasts, histiocytes, and mast cells (identified by metachromasia in Toluidine blue-stained slides) were found in the dermis (Figure 13) in areas containing cells with positive immunoreaction for group II PLA2 (Figure 11). In these areas there was strong in situ hybridization signal for the mRNA of group II PLA2 (Figure 14). However, it was not possible to resolve unequivocally which cell type in the dermis contained the mRNA of group II PLA2. Fibroblasts in the loose connective tissue of the neck showed positive in situ hybridization signal for the mRNA of human group II PLA2 (Figure 15).
The spleen, pancreas, salivary glands, and Paneth cells of the small intestinal mucosa contained no immunoreactive material. The mRNA of human group II PLA2 was localized in the smooth muscle cells of the small intestinal wall by in situ hybridization, but Paneth cells were devoid of signal.
Reproductive Organs
The granulosa cells of the Graafian follicles of the ovary contained immunoreactive material (Figure 16). The Fallopian tube and uterus were devoid of immunoreaction. There was neither immunoreaction nor in situ hybridization signal in the testis or epididymis. More recently, in situ hybridization has been performed on other line 703 animals (a female, age 3 months and two males, age 2 months and 16 months). These results (data not shown) confirm the presence of the mRNA of human group II PLA2 in Graafian follicles and its absence in the testis.
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Circulatory Organs, Muscle, and Supporting Tissues
The intima and media of the aorta were strongly immunostained (Figure 17). However, there was no in situ hybridization signal in the aorta. The heart and skeletal muscle cells were devoid of immunoreactive material. Connective tissue fibers and tendon were occasionally weakly immunoreactive. The adipose tissue was devoid of immunostaining. Fibroblasts showed positive in situ hybridization signal (Figure 15). Articular cartilage and epiphyseal bone were immunoreactive (Figure 18). There was strong immunoreaction in the chondrocytes and a weaker reaction in the matrix of articular cartilage (Figure 19). Cortical bone was devoid of immunoreaction. In situ hybridization gave negative results in bone and cartilage.
Lung
Some bronchial epithelial cells contained immunoreactive material, whereas other cell types of the lung were devoid of immunoreaction. There was a weak in situ hybridization signal in occasional bronchial epithelial cells.
The cells of lymph nodes, bone marrow, and peripheral inflammatory cells were devoid of immunoreactive material. Neither the brain nor peripheral nerves showed immunoreaction. In situ hybridization gave negative results in bone marrow and neural tissues.
The immunohistochemical results are summarized in Table 1. The immunoreaction for group II PLA2 in hepatocytes was somewhat weaker in the younger animal (3 weeks, male) than in the older animal (6.5 months, male) of the same transgenic line. The in situ hybridization results are summarized in Table 2.
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Discussion |
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Phospholipase A2 has been implicated in the pathology of diseases involving inflammation (
The cellular source of circulating group II PLA2 has not been established unequivocally. Human hepatoma-derived cells were found to express increased amounts of group II PLA2 when stimulated in vitro by the cytokines interleukin 1, interleukin 6, and tumor necrosis factor (
Recently, we established transgenic mouse lines that express human group II PLA2 at serum levels that were 8- to 10-fold higher than serum levels in non-transgenic littermates (
In this study we investigated the distribution of human group II PLA2 and the expression of the human group II PLA2 gene in tissues of transgenic mice. By immunohistochemistry, the enzyme protein was localized in hepatocytes, Bowman's capsule of the kidney glomeruli, cytoplasmic granules of the proximal tubule cells of the kidney, and epithelial cells of the renal pelvis, ureter, and urinary bladder. By in situ hybridization, clear signals for group II PLA2 mRNA were found in hepatocytes, Bowman's capsule, epithelial and smooth muscle cells of the urinary bladder, smooth muscle cells of the small intestinal wall, epithelial cells of the epidermis, and dermal and connective tissue cells, including fibroblasts. However, there was no clear signal in kidney tubules, aorta, or cartilage by in situ hybridization. These results suggest that group II PLA2, although present in these structures, is not synthesized by these tissues but is derived from elsewhere, e.g., blood plasma. Alternatively, the expression levels in the kidney tubules, aorta, and cartilage may be below the level of detection of the current method of in situ hybridization.
A 6.2-KB HindIII fragment containing the entire human group II PLA2 gene was used as the transgene fragment to produce the current transgenic mice. In addition to the transcribed 4.2 KB, which consists of five exons and four introns, this fragment contains 1.6 KB upstream and 0.35 KB downstream of the PLA2 gene. Previously, it was shown by Northern analysis that RNA was expressed from this transgene fragment in a number of tissues (
In our earlier study, human group II PLA2 mRNA was found by Northern blotting in samples of the kidney of transgenic mice (
Articular chondrocytes and the matrix of cartilage of the transgenic animals contained immunoreactive human group II PLA2. However, we were unable to demonstrate unequivocally the presence of group II PLA2 mRNA in cartilage in the current transgenic animals by in situ hybridization. Human chondrocytes synthesize group II PLA2 and are believed to be a source of this enzyme found in synovial fluid in various arthropathies (
The intimal and medial layers of the aorta of the transgenic mice contained immunoreactive human group II PLA2. Rat aortic smooth muscle cells express group II PLA2 when stimulated by inflammatory mediators, including cytokines and endotoxin, both in vivo (
Transgenic mice expressing the human group II PLA2 gene have severe abnormalities in their skin. The animals lose hair and have marked epidermal and ad-nexal hyperplasia and hyperkeratosis (
Granulosa cells of the Graafian follicles of the ovary contained immunoreactive human group II PLA2, whereas immunostaining gave negative results in the testis. PLA2 activity has been detected in ovaries (
An interesting negative finding in the current study was that human group II PLA2 was not detected in the Paneth cells of the small intestinal mucosa of the transgenic mice. These cells are known to synthesize group II PLA2 in the human (
As indicated above, human group II PLA2 was expressed in many tissues of transgenic mice, and the catalytic activity of PLA2 in sera of these animals was markedly increased over that of the control mice. Despite the high levels of circulating PLA2 activity and expression of group II PLA2 in various vital organs, the animals showed no signs of generalized inflammation and were capable of reproducing (
In conclusion, the human group II PLA2 gene is expressed in various organs of transgenic mice. The enzyme is synthesized in hepatocytes, epidermis, dermis, fibroblasts of subcutaneous connective tissue, and epithelial and smooth muscle cells of the urinary bladder of these animals. The high local and circulating levels of group II PLA2 are not associated with compromised health of these animals, except for skin and fur abnormalities. Transgenic mice provide a useful tool with which to study the physiological and pathological roles and the regulation of the synthesis and metabolism of group II PLA2.
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Acknowledgments |
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Supported by Turku University Foundation, by the Academy of Finland, and by the Cancer Foundation of Southwestern Finland.
We thank Ms Tuula Manninen, Ms Outi Asunta, and Mr Jaakko Liippo for technical assistance, Ms Maija Ahlholm for secretarial help, Drs Markku Kallajoki and Kai Nyman for methodologic advice, and Dr Mark Swanson for critical reading of the manuscript.
Received for publication February 20, 1997; accepted February 27, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersen S, Sjursen W, Laegreid A, Austgulen R, Johansen B (1994) lmmunohistologic detection of non-pancreatic phospholipase A2 (type II) in human placenta and its involvement in normal parturition at term. Prostaglandins Leukotrienes Essent Fatty Acids 51:19-26 [Medline]
Chen J, Engle SJ, Seilhamer JJ, Tischfield JA (1994) Cloning and recombinant expression of a novel human low molecular weight Ca2+-dependent phospholipase A2. J Biol Chem 269:2365-2368
Chock SP, Schmauder-Chock EA, Cordella-Miele E, Miele L, Mukherjee AB (1994) The localization of phospholipase A2 in the secretory granule. Biochem J 300:619-622 [Medline]
Clark JD, Lin L-L, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, Knopf JL (1991) A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65:1043-1051 [Medline]
Crowl RM, Stoller TJ, Conroy RR, Stoner CR (1991) Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem 266:2647-2651
Dennis EA (1994) Diversity of group types regulation and function of phospholipase A2. J Biol Chem 269:13057-13060
Di Marco S, Märki F, Hofstetter H, Schmit A, van Oostrum J, Grütter MG (1992) Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variant. J Biochem 112:350-354 [Abstract]
Ellies LG, Gupta AK, Aubin JE (1992) Differential regulation of phospholipase A2 by cytokines inhibiting bone formation and mineralization. Biochem Biophys Res Commun 188:1047-1053 [Medline]
Grass DS, Falkmer RH, Chiang M-Y, Wallace RE, Nevalainen TJ, Bennett CF, Swanson ME (1996) Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate. J Clin Invest 97:2233-2241
Harwig SS, Tan L, Qu XD, Cho Y, Eisenhauer PB, Lehrer RI (1995) Bactericidal properties of murine intestinal phospholipase A2. J Clin Invest 95:603-610 [Medline]
Hsu SM, Raine L, Fanger H (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580 [Abstract]
Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, Cromlish A (1995) A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains. J Biol Chem 270:22378-22385
Kiyohara H, Egami H, Shibata Y, Murata K, Ohshima S, Ogawa M (1992) Light microscopic immunohistochemical analysis of the distribution of group II phospholipase A2 in human digestive organs. J Histochem Cytochem 40:1659-1664
Kortekangas P, Aro HT, Nevalainen TJ (1994) Group II phospholipase A2 in synovial fluid and serum in acute arthritis. Scand J Rheumatol 21:283-288
Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow EP, Tizard R, Pepinsky RB (1989) Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 264:5768-5775
Martikainen P, Nyman K, Nevalainen TJ (1993) Toxic effects of human pancreatic and snake and bee venom phospholipases A2 on MCF-7 cells in culture. Toxicon 31:835-843 [Medline]
Morgan DW, Anderson GM, Meyers KP (1993) Recombinant human type II phospholipase A2 lacks edema producing activity in rat. Eur J Pharmacol 235:45-50 [Medline]
Murakami M, Kudo I, Inoue K (1993) Molecular nature of phospholipase A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells: possible participation of cytosolic and extracellular type II phospholipases A2. J Biol Chem 268:839-844
Murakami M, Kudo I, Suwa Y, Inoue K (1992) Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process. Eur J Biochem 209:257-265 [Abstract]
Nakano T, Arita H (1990) Enhanced expression of group II phospholipase A2 gene in the tissues of endotoxin shock rats and its suppression by glucocorticoids. FEBS Lett 273:23-26 [Medline]
Nakano T, Ohara O, Teraoka H, Arita H (1990) Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells. FEBS Lett 261:171-174 [Medline]
Nevalainen TJ (1993) Serum phospholipases A2 in inflammatory diseases. Clin Chem 39:2453-2459
Nevalainen TJ, Aho HJ, Peuravuori H (1994) Secretion of group 2 phospholipase A2 by lacrimal gland. Invest Ophthalmol Vis Sci 32:417-421
Nevalainen TJ, Grönroos JM, Kallajoki M (1995) Expression of group II phospholipase A2 in human gastrointestinal tract. Lab Invest 72:201-208 [Medline]
Nevalainen TJ, Haapanen TJ (1993) Distribution of pancreatic (group I) and synovial-type (group II) phospholipases A2 in human tissues. Inflammation 17:453-464 [Medline]
Nevalainen TJ, Kallajoki M, Pesonen E, Andesson S, Kärkkäinen P, Höckerstedt K (1996) Origin of circulating group II phospholipase A2 in hepatocytes in a patient with epitheloid hemangioendothelioma of the liver. Lab Invest 74:585-591 [Medline]
Nevalainen TJ, Märki F, Kortesuo PT, Grütter M, Di Marco S, Schmitz A (1993) Synovial type (group II) phospholipase A2 in cartilage. J Rheumatol 20:325-330 [Medline]
Nguyen HT, Rice GE, Farrugia W, Wong M, Brennecke SP (1994) Bacterial endotoxin increases type II phospholipase A2 immunoreactive content and phospholipase A2 enzymatic activity in human decidua. Biol Reprod 50:526-534 [Abstract]
Nomura K, Fujita H, Arita H (1994) Gene expression of pancreatic-type phospholipase A2 in rat ovaries: stimulatory action on progesterone release. J Biol Chem 135:603-609
Oka S, Arita H (1991) Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. J Biol Chem 266:9956-9960
Okazaki T, Strauss JFI, Flickinger GL (1977) Lysosomal phospholipase A activities of rat ovarian tissue. Biochim Biophys Acta 487:343-353 [Medline]
Rosenthal MD, Gordon MN, Buescher ES, Slusser JH, Harris LK, Franson RC (1995) Human neutrophils store type II 14-kDa phospholipase A2 in granules and secrete active enzyme in response to soluble stimuli. Biochem Biophys Res Commun 208:650-656 [Medline]
Seilhamer JJ, Pruzanski W, Vadas P, Plant S, Miller JA, Kloss J, Johnson LK (1989) Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem 264:5335-5338
Seilhamer JJ, Randall TL, Yamanaka M, Johnson LK (1986) Pancreatic phospholipase A2: isolation of the human gene and cDNAs from porcine pancreas and human lung. DNA 5:519-527 [Medline]
Vadas P, Browning J, Edelson J, Pruzanski W (1993) Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states. J Lipid Mediators 8:1-30 [Medline]
van den Bosch H (1980) Intracellular phospholipases A. Biochim Biophys Acta 604:191-246 [Medline]
Veldhuis JD, Demers LM (1989) Mechanism(s) by which activation of protein kinase C is coupled to prostacyclin synthesis in granulosa cells. Endocrinology 63:219-226
Weinrauch Y, Elsbach P, Madsen LM, Foreman A, Weiss J (1996) The potent anti-Staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kD phospholipase A2. J Clin Invest 97:250-257
Wright GW, Ooi CE, Weiss J, Elsbach P (1990) Purification of a cellular (granulocyte) and an extracellular (serum) phospholipase A2 that participate in the destruction of Escherichia coli in a rabbit inflammatory exudate. J Biol Chem 265:6675-6681
Ying Z, Tojo H, Komatsubara T, Nakagawa M, Inada M, Kawata S, Matsuzawa Y, Okamoto M (1994) Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma. Biochim Biophys Acta 1226:201-205 [Medline]