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Phenylethanolamine N-Methyl Transferase Expression in Mouse Thymus and Spleen
Brain, Behavior, and Immunity 16, 493–499 (2002) doi:10.1006/brbi.2001.0637
BRIEF COMMUNICATION Phenylethanolamine N-Methyl Transferase Expression in Mouse Thymus and Spleen Michelle D. Warthan,* Jessica G. Freeman,* Kathryn E. Loesser,† Carolene W. Lewis,* Min Hong,‡ Carolyn M. Conway,* and Jennifer K. Stewart*,1 *Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23284; †Department of Biology, Mary Washington College, Fredericksburg, Virginia 22401;and ‡Norman Bethune University of Medical Sciences, Chang Chun, Jilin Province, China Catecholamines usually are found in neurons and chromaffin cells of mammals. In this study, surprisingly high levels of the epinephrine synthesizing enzyme phenylethanolamine N-methyl transferase (PNMT) were detected in the thymus of young mice. Levels of PNMT activity in the thymus were comparable to levels in the brainstem and were suppressed by the PNMT inhibitor LY134046. PNMT mRNA was localized with in situ hybridization throughout the thymus, but levels were approximately twofold higher in the cortex than in the medulla. PNMT activity was barely detectable in the spleen, and only a few cells expressing PNMT mRNA were located in the marginal zone of the white pulp. These findings suggest that cells in the thymus of young mice have the ability to synthesize epinephrine. 2001 Elsevier Science (USA) Key Words: PNMT; epinephrine; thymus; spleen; catecholamines; neuroimmunology; lymphocytes.
INTRODUCTION
In mammals, catecholamines usually are found in neurons and chromaffin cells such as those in the adrenal medulla, but recently several laboratories have detected catecholamines in cells of the immune system (Bergquist, Tarkowski, Ekman, & Ewing, 1994; Cosentino, Marino, Bombelli, Ferrari, Lecchini, & Frigo, 1999; Knudsen, Christensen, & Bratholm, 1996; Marino, Cosentino, Bombelli, Ferrari, Lecchini, & Frigo, 1999; Musso, Brenchi, Setti, Indiveri, & Lotti, 1996; Spengler, Chensue, Giacherio, Blenk, & Kunkel, 1994). More than 20 years ago Pendleton and co-workers found that the rat spleen has N-methyl transferase activity with the same substrate specificity as the epinephrine synthesizing enzyme, phenylethanolamine N-methyl transferase (PNMT) (Pendleton, Gessner, & Sawyer, 1978). Our laboratory, using semiquantitative RT– PCR, confirmed that PNMT mRNA is present in rat spleen, and we detected lower levels of PNMT expression in rat thymus (Andreassi II, Eggleston, & Stewart, 1998). There is no information, however, on the distribution of PNMT mRNA in these organs, and PNMT activity has never been measured in thymus. PNMT activity in cells is considered to reflect the capacity to synthesize epinephrine. Even low levels of epinephrine in immune cells may be important because epinephrine is the most potent naturally occurring β2 agonist and many of the adrenergic actions on immune cells are β2 mediated (Sanders, 1995). For example, β2 agonists 1 To whom correspondence and reprint requests should be addressed. Fax: (804) 828-0503. E-mail: [email protected]. 493 0889-1591/01 $35.00
2001 Elsevier Science (USA) All rights reserved.
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inhibit production of inflammatory regulators such as IL-12 and promote development of Th2 cells while inhibiting development of Th1 cells (Panina-Bordignon, Mazzeo, Di Lucia, D’Ambrosio, Lang, Fabbri, Self, & Sinigaglia, 1997; Vizi, 1998). Although mice are important models in immunological research, it is not known whether PNMT is expressed in murine lymphoid organs. The goal of this study was to determine if PNMT mRNA and activity are present in the thymus and spleen of young mice. MATERIALS AND METHODS
Outbred ICR mice (24–30 g, 6–7 weeks of age) were obtained from Harlan (Indianapolis, IN). Animals were killed with CO2 . Spleen, thymus, adrenal, and medulla oblongata were either frozen at ⫺80°C until homogenized or placed in plastic molds containing tissue freezing medium (Triangle Biomedical Sciences), frozen in liquid nitrogen, and then maintained at ⫺80°C until sectioned with a Lydia digital kryostat at 10 µm. Adjacent sections were collected for in situ hybridization and histological evaluation. For measurement of PNMT activity, frozen samples were homogenized in 5 mM Tris buffer (pH 8.6, 5°C). Homogenates were centrifuged at 30,000 g (4°C) for 25 min, and the supernatant was frozen at ⫺80°C until assayed for PNMT activity and total protein. Protein was measured according to Lowry et al. (Lowry, Rosebrough, Farr, & Randall, 1951). PNMT activity was assayed radioenzymatically as described previously (Chappell & Stewart, 1992). Blank tubes without the substrate phenylethanolamine were run for each tissue sample, and all reactions were performed in triplicate. PNMT activity is reported as radiolabeled product per milligram of tissue protein formed over 90 min and was linear with time and increasing amounts of sample protein. To provide evidence that activity measurements were due to PNMT rather than a nonspecific N-methyl transferase, aliquots of each homogenate were assayed in the presence of the PNMT inhibitor LY134046 at 10⫺6 M (Chappell & Stewart, 1992; Fuller, Hemrick-Luecke, Toomey, Horng, Ruffolo, Jr., & Malloy, 1981). RNA probes for in situ hybridization were synthesized from cloned rat PNMT cDNA obtained from Dr. Barry Kaplan (NIMH, Bethesda, MD). The rat PNMT cDNA consists of 905 nucleotides corresponding to nucleotides 1–850 of rat PNMT mRNA (EMB Accession No. X14211) plus 30 untranslated nucleotides inserted after nucleotide 5 and a polyA sequence. The clone exhibits 90% identity with regions of the mouse PNMT gene (GenBank Accession No. L12687) by BLAST analysis (www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast). We previously used the labeled rat cDNA probe for Southern blots of PNMT RT–PCR products from mouse brain and various rat organs (Andreassi II et al., 1998, and unpublished findings). The plasmid was linearized with BamHI to obtain the sense (T/3) strand and with EcoRV to obtain the antisense (T/7) strand. The linearized plasmid was purified with Genie Prep cartridges (Ambion Inc., Austin, TX). The RNA probes were synthesized with T/7-T/3 Maxiscript Kit (Ambion Inc.) and labeled with digoxigenin-labeled UTP (Boehringer-Mannheim) according to the manufacture’s protocol (Technical Bulletin 173). Sections for in situ hybridization were placed on silane adhesive-coated microscope slides and stored at ⫺80°C. In situ hybridization procedures (Braissant & W. Wahli, 1998) were modified as follows. Slides were placed in fresh 4% paraformalde-
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hyde in 1⫻ phosphate-buffered saline (PBS; anhydrous dibasic 12.5 mM Na2HPO4 , anhydrous monobasic 10 mM NaH2PO4 , and 0.9% NaCl) at 4°C for 10 min and washed twice in a freshly made solution of 1⫻ PBS with active 0.1% diethyl pyrocarbonate (DEPC) and then in 5⫻ saline sodium citrate (SSC; 0.75 M NaCl, 0.0075 M Na citrate, pH 7.0) for 15 min each. Sections were prehybridized with a fresh solution of 50% formamide, 50% 10⫻ SSC, and 40 µg/ml salmon-sperm-sheared DNA for 2 h at 58°C. Sections were hybridized overnight at 58°C with antisense and sense (negative control) probes diluted to a final concentration of 1 ng/µl (200 µl/slide). The slides were washed in 2⫻ SSC at room temperature for 30 min, 2⫻ SSC and 0.1⫻ SSC at 65°C for 1 h each, and 1⫻ Tris-buffered saline (TBS; 100 mM Tris, 150mM NaCl, pH 7.4–7.7) for 5 min and then incubated for 1 h in a blocking solution of 0.5% dry milk in 1⫻ TBS and for 2 h in alkaline phosphatase-coupled antidigoxigenin antibody (Boehringer-Mannheim) diluted 1:5000 in 0.5% dry milk in 1⫻ TBS. The slides were washed twice in 1⫻ TBS for 15 min each and twice in equilibration buffer (0.1 M Tris–HCl, 0.1 M NaCl, 0.05 M MgCl2 , pH 9.5) for 5 min. The alkaline phosphatase was detected with a freshly made color solution of 0.5 ml equilibration buffer containing 2.25 µl nitroblue tetrazolium [NBT; 75 mg/ml in 70% dimethylformamide (DMF)] and 1.75 µl 5-bromo-4-chloro-3-indolyl phosphate (BCIP; 50 mg /ml in 100% DMF) per slide. Color development was continued overnight at room temperature in a covered dark box and stopped with 1⫻ TE (10 mM Tris buffer and 1 mM EDTA, pH 7.4) for 10 min. Sections were dehydrated with a series of ethanol solutions (25, 50, and 75%) and then placed in 95% ethanol for 4 h to remove nonspecific staining and cleared in xylene. Coverslips were mounted with Entellan rapid-mounting media. Adjacent sections stained with toluidine blue or hematoxylin and eosin and sections counterstained with eosin or methyl green were used to verify the regional distribution of labeling in 93 sections of spleen and 106 sections of thymus. Area and density of labeling in 340 µm2 areas in the cortical and medullary regions of the thymus were measured with the Bioquant System (R&M Biometrics, Inc., Nashville, TN) on two to four representative sections of thymus from each of five mice. Background labeling was subtracted for each analysis. Results on sections from each animal were averaged, and a paired t test was used to compare average labeling area and density in medulla with that in cortex of each animal. Statistical analyses were performed with GraphPad Software (San Diego, CA). RESULTS AND DISCUSSION
PNMT activity levels in murine thymus were approximately the same as in the medulla oblongata, and activity in the spleen was comparatively low, averaging only 37 fmol product/mg protein ⫻ 90 min ⫺1 (Fig. 1). but consistently above the detectable limits of the assay (10 fmol/mg protein ⫻ 90 min⫺1). The PNMT inhibitor LY134046 at a concentration of 1 µM decreased N-methyl transferase activity by 67–100% (Fig. 1), suggesting that the activity was due to PNMT rather than a nonspecific methyl transferase (Chappell & Stewart, 1992). In preliminary measurements, levels of PNMT activity in the spleen and thymus of three C57BL/6 mice obtained from Jackson Laboratory were almost identical to those in lymphoid organs of outbred mice (data not shown). Typical in situ hybridization results are shown in Fig. 2. PNMT mRNA was detected in both the cortical and medullary regions of the thymus (Fig. 2A, top and bottom panels). The area and density of labeling appeared to be higher in the cortex
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FIG. 1. PNMT activity in the medulla oblongata, thymus, and spleen of the mouse measured in the presence of the PNMT inhibitor LY134046 at 10⫺6 M or deionized water (vehicle). Solid bars represent the mean ⫾ SE activity in five to eight mice. The inhibitor reduced PNMT activity in three to six samples of each tissue (hatched bars, *p ⱕ .01 comparing vehicle to inhibitor by a paired t test). PNMT activity was lower in spleen than in the other tissues ( p ⬍ .001 by Dunnet’s multiple comparison test).
than in the medulla (Fig. 2A, bottom panel), and Bioquant analysis confirmed twoto fourfold higher labeling in the cortex (Fig. 3). Labeling was present in both the inner and outer cortex and was not associated with any particular structure such as blood vessels and was not present in the thymic capsule or septum. In each of the 93 sections of spleen examined, PNMT mRNA was observed in only 10–19 small clusters of labeling in the marginal zone of the white pulp (Fig. 2B). No PNMT mRNA was evident in sections hybridized with the sense probe (Fig. 2), indicating specific hybridization to the PNMT antisense probe. As expected for positive controls processed with each batch of slides, PNMT mRNA was abundant in the adrenal medulla but was not evident in the adrenal cortex (Fig. 2C). These observations indicate that the higher level of PNMT activity in the thymus compared to spleen is due to higher levels of expression of PNMT mRNA. In contrast, PNMT mRNA is more abundant in rat spleen than in thymus (Andreassi II et al., 1998). This difference may reflect either species or developmental differences because in previous studies adult rats were used (Pendleton et al., 1978; Andreassi II et al., 1998) and the young mice in this study were not yet sexually mature. Further investigation is needed to determine whether there are developmental changes in PNMT expression in the spleen and thymus, particularly because glucocorticoids are produced in thymic epithelial cells during fetal and postnatal development (Tolosa, King, & Ashwell, 1998; Vacchio, Papadopoulos, & Ashwell, 2000). It is well known that glucocorticoids from the adrenal cortex maintain PNMT expression in the adrenal medulla (Wong, Lesage, Siddall, & Funder, 1992), and it is conceivable that thymusderived glucocorticoids increase thymic levels of PNMT mRNA. Although we could not identify the specific cell types expressing PNMT mRNA, a subset of T cells is a good candidate. Tsao and colleagues detected immunoreactive tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthesis pathway, in murine thymocytes and enriched T cells from spleen (Tsao, Lin, & Cheng, 1998). Also, Josefsson et al. detected dopamine and norepinephrine in T-cell hybrido-
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FIG. 2. Mouse thymus (A), spleen (B), and adrenal (C) hybridized with PNMT RNA antisense or sense probes. Insets are adjacent sections stained with toluidine blue to localize regions of labeled probe, and arrows indicate regions of labeling. (A, top and bottom) The thymic cortex is to the left of the broken lines and medulla is to the right. Magnification is 483⫻ in A (top panel) and B, 176⫻ in A (bottom panel) and C, and 26⫻ in the insets.
mas, although they did not attempt to measure epinephrine in these cells (Josefsson, Bergquist, Ekman, & Tarkowski, 1996). Expression of both PNMT and tyrosine hydroxylase in thymic and splenic cells would suggest de novo synthesis of epinephrine in these cells. Alternatively, because lymphoid organs are innervated by sympathetic neurons (Felten, Felten, Ackerman, Bellinger, Madden, Carlson, & Livnat, 1990), it is possible that lymphoid cells take
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FIG. 3. Mean ⫾ SE area and density of labeling with the PNMT RNA probes in two to four sections of thymic medulla and cortex from each of five mice analyzed with a Bioquant system. *p ⬍ .05 by a paired t test compared to labeling in the medulla of the same animal.
up the sympathetic neurotransmitter norepinephrine and use PNMT to convert it to epinephrine. The affinity of epinephrine for β2 adrenergic receptors is approximately 100 times that of norepinephrine (Lands, Luduena, & Buzzo, 1967), and this conversion could enhance β2 actions on immune cells. Further investigation is needed to elucidate the role of PNMT in lymphoid organs. ACKNOWLEDGMENTS This study was supported by NSF Grant 9870382. C. W. Lewis is an undergraduate student supported by Grant 1E25GM56620-01 from the NIH Bridges to the Baccalaureate Program.
REFERENCES Andreassi, J. L., II, Eggleston, W. B., & Stewart, J. K. (1998). Phenylethanolamine N-methyltransferase mRNA in rat spleen and thymus. Neurosci. Lett. 241, 75–78. Bergquist, J., Tarkowski, A., Ekman, R., & Ewing, A. (1994). Discovery of endogenous catecholamines in lymphocytes and evidence for catecholamine regulation of lymphocyte function via an autocrine loop. Proc. Natl. Acad. Sci. USA 91, 12912–12916. Braissant, O., & Wahli, W. (1998). A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1, 10–16. Chappell, J. E., & Stewart, J. K. (1992). Soluble and particulate phenylethanolamine N-methyltransferase in hypothalamus of diabetic rats. Am. J. Physiol. 263, E335–E339. Cosentino, M., Marino, F., Bombelli, R., Ferrari, M., Lecchini, S., & Frigo, G. (1999). Endogenous catecholamine synthesis, metabolism, storage and uptake in human neutrophils. Life Sci. 64, 975– 981. Felten, D., Felten, S. Y., Ackerman, K. D., Bellinger, D. L., Madden, K., Carlson, S. L., & Livnat, S. (1990). Peripheral innervation of lymphoid tissue. In S. Freier (Ed.), The neuroendocrine-immune network, pp. 9–18. CRC Press: Boca Raton, FL. Fuller, R. W., Hemrick-Luecke, S., Toomey, R. E., Horng, J., Ruffolo, R. R., Jr., & Malloy, B. B. (1981). Properties of 8, 9-dichloro-2,3,4,5-tetrahydro-1H2-benzazepine, an inhibitor of norepinephrine Nmethyltransferase. Biochem. Pharmacol. 30, 1345–1352. Josefsson, E., Bergquist, J., Ekman, R., & Tarkowski, A. (1996). Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis. Immunology 88, 140–146.
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Knudsen, J. H., Christensen, N. J., & Bratholm, P. (1996). Lymphocyte norepinephrine and epinephrine, but not plasma catecholamines predict lymphocyte cAMP production. Life Sci. 59, 639–647. Lands, A. M., Luduena, F. P., & Buzzo, H. J. (1967). Differentiation of receptors responsive to isoproterenol. Life Sci. 6, 2241–2249. Lowry, D. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with folin phenol reagent. J. Biochem. 193, 265–275. Marino, F., Cosentino, M., Bombelli, R., Ferrari, M., Lecchini, S., & Frigo, G. (1999). Endogenous catecholamine synthesis, metabolism, storage, and uptake in human peripheral blood mononuclear cells. Exp. Hematol. 27, 489–495. Musso, N. R., Brenchi, S., Setti, M., Indiveri, F., & Lotti, G. (1996). Catecholamine content and in vitro catecholamine synthesis in peripheral human lymphocytes. J. Clin. Endocrinol. Metab. 81, 3553– 3557. Panina-Bordignon, P., Mazzeo, D., Di Lucia, P., D’Ambrosio, D., Lang, R., Fabbri, L., Self, C., & Sinigaglia, F. (1997). Beta2-agonists prevent Th1 development by selective inhibition of interleukin 12. J. Clin. Invest. 100, 1513–1519. Pendleton, R. G., Gessner, G., & Sawyer, J. (1978). Studies on the distribution of phenylethanolamine N-methyltransferase and epinephrine in the rat. Res. Commun. Chem. Pathol. Pharmacol. 21, 315– 325. Sanders, V. M. (1995). The role of adrenoceptor-mediated signals in the modulation of lymphocyte function. Adv. Neuroimmunol. 5, 283–298. Spengler, R. N., Chensue, S. W., Giacherio, D. A., Blenk, N., & Kunkel, S. L. (1994). Endogenous norepinephrine regulates tumor necrosis factor-α production from macrophages in vitro. J. Immunol. 152, 3024–3031. Tolosa, E., King, L. B., & Ashwell, J. D. (1998). Thymocyte glucocorticoid resistance alters positive selection and inhibits autoimmunity and lymphoproliferative disease in MRL-lpr/lpr mice. Immunity 8, 67–76. Tsao, C.-W., Lin, Y.-S., & Cheng, J.-T. (1998). Inhibition of immune cell proliferation with haloperidol and relationship of tyrosine hydroxylase expression to immune cell growth. Life Sci. 62, 335–344. Vacchio, M. S., Papadopoulos, V., & Ashwell, J. D. (2000). Steroid production in the thymus: Implications for thymocyte selection. J. Exp. Med. 179, 1835–1846. Vizi, E. S. (1998). Receptor-mediated local fine-tuning by noradrenergic innervation of neuroendocrine and immune systems. Ann. N. Y. Acad. Sci. 851, 388–396. Wong, D. L., Lesage, A., Siddall, B., & Funder, J. W. (1992). Glucocorticoid regulation of phenylethanolamine N-methyl transferase in vivo. FASEB J. 6, 3310–3315. Received August 7, 2000; published online December 12, 2001
BRIEF COMMUNICATION Phenylethanolamine N-Methyl Transferase Expression in Mouse Thymus and Spleen Michelle D. Warthan,* Jessica G. Freeman,* Kathryn E. Loesser,† Carolene W. Lewis,* Min Hong,‡ Carolyn M. Conway,* and Jennifer K. Stewart*,1 *Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23284; †Department of Biology, Mary Washington College, Fredericksburg, Virginia 22401;and ‡Norman Bethune University of Medical Sciences, Chang Chun, Jilin Province, China Catecholamines usually are found in neurons and chromaffin cells of mammals. In this study, surprisingly high levels of the epinephrine synthesizing enzyme phenylethanolamine N-methyl transferase (PNMT) were detected in the thymus of young mice. Levels of PNMT activity in the thymus were comparable to levels in the brainstem and were suppressed by the PNMT inhibitor LY134046. PNMT mRNA was localized with in situ hybridization throughout the thymus, but levels were approximately twofold higher in the cortex than in the medulla. PNMT activity was barely detectable in the spleen, and only a few cells expressing PNMT mRNA were located in the marginal zone of the white pulp. These findings suggest that cells in the thymus of young mice have the ability to synthesize epinephrine. 2001 Elsevier Science (USA) Key Words: PNMT; epinephrine; thymus; spleen; catecholamines; neuroimmunology; lymphocytes.
INTRODUCTION
In mammals, catecholamines usually are found in neurons and chromaffin cells such as those in the adrenal medulla, but recently several laboratories have detected catecholamines in cells of the immune system (Bergquist, Tarkowski, Ekman, & Ewing, 1994; Cosentino, Marino, Bombelli, Ferrari, Lecchini, & Frigo, 1999; Knudsen, Christensen, & Bratholm, 1996; Marino, Cosentino, Bombelli, Ferrari, Lecchini, & Frigo, 1999; Musso, Brenchi, Setti, Indiveri, & Lotti, 1996; Spengler, Chensue, Giacherio, Blenk, & Kunkel, 1994). More than 20 years ago Pendleton and co-workers found that the rat spleen has N-methyl transferase activity with the same substrate specificity as the epinephrine synthesizing enzyme, phenylethanolamine N-methyl transferase (PNMT) (Pendleton, Gessner, & Sawyer, 1978). Our laboratory, using semiquantitative RT– PCR, confirmed that PNMT mRNA is present in rat spleen, and we detected lower levels of PNMT expression in rat thymus (Andreassi II, Eggleston, & Stewart, 1998). There is no information, however, on the distribution of PNMT mRNA in these organs, and PNMT activity has never been measured in thymus. PNMT activity in cells is considered to reflect the capacity to synthesize epinephrine. Even low levels of epinephrine in immune cells may be important because epinephrine is the most potent naturally occurring β2 agonist and many of the adrenergic actions on immune cells are β2 mediated (Sanders, 1995). For example, β2 agonists 1 To whom correspondence and reprint requests should be addressed. Fax: (804) 828-0503. E-mail: [email protected]. 493 0889-1591/01 $35.00
2001 Elsevier Science (USA) All rights reserved.
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inhibit production of inflammatory regulators such as IL-12 and promote development of Th2 cells while inhibiting development of Th1 cells (Panina-Bordignon, Mazzeo, Di Lucia, D’Ambrosio, Lang, Fabbri, Self, & Sinigaglia, 1997; Vizi, 1998). Although mice are important models in immunological research, it is not known whether PNMT is expressed in murine lymphoid organs. The goal of this study was to determine if PNMT mRNA and activity are present in the thymus and spleen of young mice. MATERIALS AND METHODS
Outbred ICR mice (24–30 g, 6–7 weeks of age) were obtained from Harlan (Indianapolis, IN). Animals were killed with CO2 . Spleen, thymus, adrenal, and medulla oblongata were either frozen at ⫺80°C until homogenized or placed in plastic molds containing tissue freezing medium (Triangle Biomedical Sciences), frozen in liquid nitrogen, and then maintained at ⫺80°C until sectioned with a Lydia digital kryostat at 10 µm. Adjacent sections were collected for in situ hybridization and histological evaluation. For measurement of PNMT activity, frozen samples were homogenized in 5 mM Tris buffer (pH 8.6, 5°C). Homogenates were centrifuged at 30,000 g (4°C) for 25 min, and the supernatant was frozen at ⫺80°C until assayed for PNMT activity and total protein. Protein was measured according to Lowry et al. (Lowry, Rosebrough, Farr, & Randall, 1951). PNMT activity was assayed radioenzymatically as described previously (Chappell & Stewart, 1992). Blank tubes without the substrate phenylethanolamine were run for each tissue sample, and all reactions were performed in triplicate. PNMT activity is reported as radiolabeled product per milligram of tissue protein formed over 90 min and was linear with time and increasing amounts of sample protein. To provide evidence that activity measurements were due to PNMT rather than a nonspecific N-methyl transferase, aliquots of each homogenate were assayed in the presence of the PNMT inhibitor LY134046 at 10⫺6 M (Chappell & Stewart, 1992; Fuller, Hemrick-Luecke, Toomey, Horng, Ruffolo, Jr., & Malloy, 1981). RNA probes for in situ hybridization were synthesized from cloned rat PNMT cDNA obtained from Dr. Barry Kaplan (NIMH, Bethesda, MD). The rat PNMT cDNA consists of 905 nucleotides corresponding to nucleotides 1–850 of rat PNMT mRNA (EMB Accession No. X14211) plus 30 untranslated nucleotides inserted after nucleotide 5 and a polyA sequence. The clone exhibits 90% identity with regions of the mouse PNMT gene (GenBank Accession No. L12687) by BLAST analysis (www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast). We previously used the labeled rat cDNA probe for Southern blots of PNMT RT–PCR products from mouse brain and various rat organs (Andreassi II et al., 1998, and unpublished findings). The plasmid was linearized with BamHI to obtain the sense (T/3) strand and with EcoRV to obtain the antisense (T/7) strand. The linearized plasmid was purified with Genie Prep cartridges (Ambion Inc., Austin, TX). The RNA probes were synthesized with T/7-T/3 Maxiscript Kit (Ambion Inc.) and labeled with digoxigenin-labeled UTP (Boehringer-Mannheim) according to the manufacture’s protocol (Technical Bulletin 173). Sections for in situ hybridization were placed on silane adhesive-coated microscope slides and stored at ⫺80°C. In situ hybridization procedures (Braissant & W. Wahli, 1998) were modified as follows. Slides were placed in fresh 4% paraformalde-
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hyde in 1⫻ phosphate-buffered saline (PBS; anhydrous dibasic 12.5 mM Na2HPO4 , anhydrous monobasic 10 mM NaH2PO4 , and 0.9% NaCl) at 4°C for 10 min and washed twice in a freshly made solution of 1⫻ PBS with active 0.1% diethyl pyrocarbonate (DEPC) and then in 5⫻ saline sodium citrate (SSC; 0.75 M NaCl, 0.0075 M Na citrate, pH 7.0) for 15 min each. Sections were prehybridized with a fresh solution of 50% formamide, 50% 10⫻ SSC, and 40 µg/ml salmon-sperm-sheared DNA for 2 h at 58°C. Sections were hybridized overnight at 58°C with antisense and sense (negative control) probes diluted to a final concentration of 1 ng/µl (200 µl/slide). The slides were washed in 2⫻ SSC at room temperature for 30 min, 2⫻ SSC and 0.1⫻ SSC at 65°C for 1 h each, and 1⫻ Tris-buffered saline (TBS; 100 mM Tris, 150mM NaCl, pH 7.4–7.7) for 5 min and then incubated for 1 h in a blocking solution of 0.5% dry milk in 1⫻ TBS and for 2 h in alkaline phosphatase-coupled antidigoxigenin antibody (Boehringer-Mannheim) diluted 1:5000 in 0.5% dry milk in 1⫻ TBS. The slides were washed twice in 1⫻ TBS for 15 min each and twice in equilibration buffer (0.1 M Tris–HCl, 0.1 M NaCl, 0.05 M MgCl2 , pH 9.5) for 5 min. The alkaline phosphatase was detected with a freshly made color solution of 0.5 ml equilibration buffer containing 2.25 µl nitroblue tetrazolium [NBT; 75 mg/ml in 70% dimethylformamide (DMF)] and 1.75 µl 5-bromo-4-chloro-3-indolyl phosphate (BCIP; 50 mg /ml in 100% DMF) per slide. Color development was continued overnight at room temperature in a covered dark box and stopped with 1⫻ TE (10 mM Tris buffer and 1 mM EDTA, pH 7.4) for 10 min. Sections were dehydrated with a series of ethanol solutions (25, 50, and 75%) and then placed in 95% ethanol for 4 h to remove nonspecific staining and cleared in xylene. Coverslips were mounted with Entellan rapid-mounting media. Adjacent sections stained with toluidine blue or hematoxylin and eosin and sections counterstained with eosin or methyl green were used to verify the regional distribution of labeling in 93 sections of spleen and 106 sections of thymus. Area and density of labeling in 340 µm2 areas in the cortical and medullary regions of the thymus were measured with the Bioquant System (R&M Biometrics, Inc., Nashville, TN) on two to four representative sections of thymus from each of five mice. Background labeling was subtracted for each analysis. Results on sections from each animal were averaged, and a paired t test was used to compare average labeling area and density in medulla with that in cortex of each animal. Statistical analyses were performed with GraphPad Software (San Diego, CA). RESULTS AND DISCUSSION
PNMT activity levels in murine thymus were approximately the same as in the medulla oblongata, and activity in the spleen was comparatively low, averaging only 37 fmol product/mg protein ⫻ 90 min ⫺1 (Fig. 1). but consistently above the detectable limits of the assay (10 fmol/mg protein ⫻ 90 min⫺1). The PNMT inhibitor LY134046 at a concentration of 1 µM decreased N-methyl transferase activity by 67–100% (Fig. 1), suggesting that the activity was due to PNMT rather than a nonspecific methyl transferase (Chappell & Stewart, 1992). In preliminary measurements, levels of PNMT activity in the spleen and thymus of three C57BL/6 mice obtained from Jackson Laboratory were almost identical to those in lymphoid organs of outbred mice (data not shown). Typical in situ hybridization results are shown in Fig. 2. PNMT mRNA was detected in both the cortical and medullary regions of the thymus (Fig. 2A, top and bottom panels). The area and density of labeling appeared to be higher in the cortex
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FIG. 1. PNMT activity in the medulla oblongata, thymus, and spleen of the mouse measured in the presence of the PNMT inhibitor LY134046 at 10⫺6 M or deionized water (vehicle). Solid bars represent the mean ⫾ SE activity in five to eight mice. The inhibitor reduced PNMT activity in three to six samples of each tissue (hatched bars, *p ⱕ .01 comparing vehicle to inhibitor by a paired t test). PNMT activity was lower in spleen than in the other tissues ( p ⬍ .001 by Dunnet’s multiple comparison test).
than in the medulla (Fig. 2A, bottom panel), and Bioquant analysis confirmed twoto fourfold higher labeling in the cortex (Fig. 3). Labeling was present in both the inner and outer cortex and was not associated with any particular structure such as blood vessels and was not present in the thymic capsule or septum. In each of the 93 sections of spleen examined, PNMT mRNA was observed in only 10–19 small clusters of labeling in the marginal zone of the white pulp (Fig. 2B). No PNMT mRNA was evident in sections hybridized with the sense probe (Fig. 2), indicating specific hybridization to the PNMT antisense probe. As expected for positive controls processed with each batch of slides, PNMT mRNA was abundant in the adrenal medulla but was not evident in the adrenal cortex (Fig. 2C). These observations indicate that the higher level of PNMT activity in the thymus compared to spleen is due to higher levels of expression of PNMT mRNA. In contrast, PNMT mRNA is more abundant in rat spleen than in thymus (Andreassi II et al., 1998). This difference may reflect either species or developmental differences because in previous studies adult rats were used (Pendleton et al., 1978; Andreassi II et al., 1998) and the young mice in this study were not yet sexually mature. Further investigation is needed to determine whether there are developmental changes in PNMT expression in the spleen and thymus, particularly because glucocorticoids are produced in thymic epithelial cells during fetal and postnatal development (Tolosa, King, & Ashwell, 1998; Vacchio, Papadopoulos, & Ashwell, 2000). It is well known that glucocorticoids from the adrenal cortex maintain PNMT expression in the adrenal medulla (Wong, Lesage, Siddall, & Funder, 1992), and it is conceivable that thymusderived glucocorticoids increase thymic levels of PNMT mRNA. Although we could not identify the specific cell types expressing PNMT mRNA, a subset of T cells is a good candidate. Tsao and colleagues detected immunoreactive tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthesis pathway, in murine thymocytes and enriched T cells from spleen (Tsao, Lin, & Cheng, 1998). Also, Josefsson et al. detected dopamine and norepinephrine in T-cell hybrido-
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FIG. 2. Mouse thymus (A), spleen (B), and adrenal (C) hybridized with PNMT RNA antisense or sense probes. Insets are adjacent sections stained with toluidine blue to localize regions of labeled probe, and arrows indicate regions of labeling. (A, top and bottom) The thymic cortex is to the left of the broken lines and medulla is to the right. Magnification is 483⫻ in A (top panel) and B, 176⫻ in A (bottom panel) and C, and 26⫻ in the insets.
mas, although they did not attempt to measure epinephrine in these cells (Josefsson, Bergquist, Ekman, & Tarkowski, 1996). Expression of both PNMT and tyrosine hydroxylase in thymic and splenic cells would suggest de novo synthesis of epinephrine in these cells. Alternatively, because lymphoid organs are innervated by sympathetic neurons (Felten, Felten, Ackerman, Bellinger, Madden, Carlson, & Livnat, 1990), it is possible that lymphoid cells take
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FIG. 3. Mean ⫾ SE area and density of labeling with the PNMT RNA probes in two to four sections of thymic medulla and cortex from each of five mice analyzed with a Bioquant system. *p ⬍ .05 by a paired t test compared to labeling in the medulla of the same animal.
up the sympathetic neurotransmitter norepinephrine and use PNMT to convert it to epinephrine. The affinity of epinephrine for β2 adrenergic receptors is approximately 100 times that of norepinephrine (Lands, Luduena, & Buzzo, 1967), and this conversion could enhance β2 actions on immune cells. Further investigation is needed to elucidate the role of PNMT in lymphoid organs. ACKNOWLEDGMENTS This study was supported by NSF Grant 9870382. C. W. Lewis is an undergraduate student supported by Grant 1E25GM56620-01 from the NIH Bridges to the Baccalaureate Program.
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