ARTICLE |
Correspondence to: Alfredo Martínez, Cell and Cancer Biology Dept., NCI, NIH, 9610 Medical Center Drive, Rockville, MD 20850-3300.
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Summary |
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C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine -amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology. (J Histochem Cytochem 46:661668, 1998)
Key Words: thymus, stellate epithelial cells, amidation, oxytocin, immunohistochemistry, in situ hybridization, enzyme assays
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Introduction |
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C-terminal amidation is a post-translational process neccessary for the biological activity of many regulatory peptides (for review see -amidating monooxygenase (PAM) (
-hydroxylating monooxygenase (PHM), which catalyzes the conversion of glycine-extended peptides into peptidyl-
-hydroxyglycine intermediates. The second enzyme, peptidyl-
-hydroxyglycine
-amidating lyase (PAL), is contained in the middle third of the PAM precursor and converts the intermediate hydroxyglycine to the bioactive amidated peptide. The COOH-terminal third encodes a transmembrane domain and a hydrophilic cytoplasmic tail, which operates as a routing signal (
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PAM enzymes have been co-localized with amidated regulatory peptides inside secretory granules (
The thymus has a crucial role in the development and organization of the immune system. Maturation of primitive incompetent precursor cells to immunocompetent T-lymphocytes takes place in the thymus, and accumulating evidence points to the involvement of a secretory system of regulatory substances in this process (Nabarra and Adrianarison 1987;
A previous study (
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Materials and Methods |
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Twelve female Wistar rats weighing from 150200 g and four Balb/C mice were used in this study.
Northern Blot Analysis
Three rats and two mice were sacrificed by cervical dislocation and several tissues, including thymus, were dissected out and total mRNA was prepared, after homogenization, using the guanidine isothiocyanatecesium chloride method of PAM-1 (
Immunocytochemistry
Animals were anesthesized with an overdose of sodium pentobarbital and perfused through the left ventricle with PBS and 4% paraformaldehyde in PBS. The thymuses were excised and immersed for 3 additional hr in fixative. Tissues were embedded in paraffin or in epon.
Paraffin (4-µm-thick) and semithin plastic (1-µm-thick) sections were mounted on glass slides and immunocytochemical staining was performed using the avidinbiotin method (
Polyclonal antisera were raised against synthetic fragments of the predicted sequence of human PAM; CC (PAM 288310 with a Val for Ala conservative substitution at position 11) and PAL2 (PAM 527546) as previously described (
The sections were incubated with biotinylated secondary antibodies (Dakopatts; Glostrup, Denmark) and then with avidinbiotinperoxidase complex (Dakopatts). The bound antibodies were visualized with 3-3'-diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO). In some cases the reaction was enhanced with a nickel solution (
As specificity controls, antisera to PHM (CC) and PAL (PAL2) were preincubated with their respective synthetic antigens for 2 hr at RT at a concentration of 20 nmol of peptide/ml of optimally diluted antiserum, before application to tissue sections.
In Situ Hybridization
Paraffin-embedded thymus tissue was processed for in situ hybridization as previously described (
Visualization of digoxigenin was performed with a monoclonal antibody coupled to alkaline phosphatase (Boehringer) 1:500, acting for 2 hr at RT. Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (both from Sigma) were used as substrates for the alkaline phosphatase.
Controls included the use of the sense probe and digestion with RNase before the hybridization.
Cell Culture
Four thymic cell lines of mouse origin, previously characterized as epithelial stromal cells of the medulla (
Cell Extracts
Whole-cell extracts were prepared from approximately 5 x 106 cells homogenized in 2 ml of TMT (20 mM TES, pH 7.4, 10 mM mannitol, 1% Triton X-100) containing a protease inhibitor cocktail of phenylmethylsulfonylfluoride, bacitracin, aprotinin, benzamidine, and leupeptin. The homogenate was then sonicated at high power with a Branson ultrasonic probe and ultracentrifuged at 100,000 x g for 30 min. Media that had been conditioned for 24 hr by exponentially growing cells were also collected for enzymatic activity assays of secreted PAM.
PAM Enzyme Assay
This assay was done according to a procedure previously described (-amidated product acYF# (N-acetyl-tyrosyl-phenylalanylamide) by the sequential action of the PHM and PAL enzymes present in the sample. The assay mix contains concentrations of Cu2+ and ascorbate adjusted to yield maximal enzyme activity (a range of copper concentrations approximately 1 µM Cu2+ for each 0.5 µg of analyte protein, and 0.5 mM ascorbate). The enzyme activity is measured with a trace amount of radioiodinated substrate (20,000 cpm [125I-Tyr]-acYFG). Five copper concentrations were analyzed and the activity quoted was calculated from the percent conversion at the maximal concentration. Specific activities were calculated from enzyme activities and protein concentrations determined using the Pierce bicinchoninic acid reagent in the microtiter plate format. Modifications of the assay are used to determine individual PHM and PAL activities, as we have described (
RT-PCR Analysis
Total RNA was reverse-transcribed and amplified in a single solution as previously described (
Oxytocin Radioimmunoassay
The presence of oxytocin in the conditioned media was tested using the oxytocin RIA kit from Peninsula Laboratories (Belmont, CA) according to the manufacturer's specifications: 100 ml of conditioned medium was passed through a Sep-pak cartridge (Millipore; Bedford, MA), eluted in 3 ml of 60% acetonitrile0.1% trifluoroacetic acid, freeze-dried, and reconstituted in RIA buffer. A total of 100 µl of each sample was incubated overnight with 100 µl of antioxytocin antibody. The next day, [125I]-oxytocin was added to the solution, and on the third day 100 µl of goat anti-rabbit IgG and normal rabbit serum were added before centrifugation. The radioactivity left in the pellet was measured in a -counter and the cpm values were corrected according to the total counts and the nonspecific binding.
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Results |
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The Northern blot analysis showed a very faint, almost imperceptible band for the thymus compared with other tissues known to express high quantities of PAM mRNA (Figure 2).
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Immunocytochemical analyses demonstrated that thymus tissue reacted with antibodies directed against PHM but not PAL. The labeling was restricted to stromal stellate cells of the thymic medulla (Figure 3). These cells are characterized by a large, pale nucleus and the presence of several cytoplasmic processes (Figure 3A). The immunoreactivity was located in the cytoplasm of the medullary stellate cells displaying a patent granular pattern (Figure 3B), whereas thymocytes were absolutely devoid of staining. Preabsorption of the antibody with its synthetic peptide completely quenched the immunostaining.
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The expression of PAM mRNA in thymus was determined by in situ hybridization (Figure 4). Distribution of the positively hybridized cells was restricted to the thymic medulla and application of the sense probe gave no staining at all (Figure 4B). Staining for the mRNA was homogeneously distributed throughout the stellate cytoplasm and the number of positive cells was lower (around 20%) compared to the PHM-immunoreactive cells (compare Figure 3A to Figure 4A).
To further characterize the cell type producing PAM in the thymus, a study of four medullary thymic stromal lines was performed. Similar to the results of the in situ studies, cytoplasmic labeling of thymic epithelial cell lines was observed with anti-PHM but not with anti-PAL antiserum (Figure 5). Labeling of serial sections allowed a more complete characterization of the PAM-positive cells of the thymus (Figure 6). They were also immunoreactive to antibodies against cytokeratins, thus confirming their epithelial origin, and to B-lymphocyte associated antigens, a characteristic previously reported for stellate cells in human thymic medulla (
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Northern blot analysis showed that all of the medullary epithelial cell lines contained PAM mRNA (Figure 7). In all instances, PAM mRNA levels were lower than described previously for neuroendocrine lung tumor cell lines (
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RT-PCR analysis was performed to investigate the differences in expression between PHM and PAL. Other organs, including stomach, heart, and salivary glands, expressed both regions of the message. Conversely, the thymus extracts were positive only for PHM and not for PAL (Figure 8), indicating that the major spliced form of the PAM mRNA that is expressed in the thymus lacks the PAL domain.
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PAM enzymatic activity (Table 1) was lowest in the TE-71 epithelial cell line, highest in the Z172 line, and intermediate in the remaining two. The majority (95%) of either PHM or PAL activity recovered from stromal cell cultures was found in the conditioned medium, indicating the secretory nature of the processed PAM gene products.
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To identify amidated peptides in these cell lines, we performed a radioimmunoassay for oxytocin, one of the amidated peptides described in thymus (
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Discussion |
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This study shows for the first time that the amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla. The epithelial thymic cells form a meshwork in the organ parenchyma. Cortical epithelial cells are very reticular in shape, whereas the medullary cells comprise two types: stellate and large, vacuolated elements (
The low amount of mRNA revealed by Northern blot analyses of thymus RNA in this study and in a previous general survey (
The finding here that some PAM-producing cells are positive for the CC antiserum and not for PAL2 has been previously reported (
The lower number of positive cells found with the in situ hybridization method, as compared with immunohistochemistry, suggests that the expression of the PAM gene is not constitutive in the thymus but follows an intermitent pattern, possibly related to physiological conditions. A similar pattern has been observed for PAM expression in the human pituitary (
Over the past years, evidence has accumulated that the thymus secretes biologically active factors and that these factors are secreted by the epithelial cells of the thymic reticulum (Nabarra and Adrianarison 1987). The secreted substances reported in the literature include, among others, tumor necrosis factor (TNF), interferon
, several interleukins, the hormone thymulin, neurohypophysis-related peptides, and tachykinins (Nabarra and Adrianarison 1987;
It has been shown that several amidated regula-tory peptides, including substance P, cholecystokinin 8 (CCK8), and vasoactive inhibitory peptide (VIP), among others, regulate the growth and differentiation of T-lymphocytes (
In summary, PAM is expressed by medullary stellate epithelial cells of the thymus and is probably involved in the post-translational maturation of regulatory peptides secreted by these cells. This study opens a new avenue in the understanding of thymic regulation.
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Acknowledgments |
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Supported by a grant from Spain's Government (Ministerio de Educación y Ciencia, pf9316525626) to A.M. and by grants from the National Institutes of Health (Ag 04360, AI 24137) and the Department of Energy (DE-F006-86-ER60409) to A.F.
We gratefully thank Prof J. Engels for the gift of the PAM probes. This study was initiated by A. Martínez while at the Department of Cytology and Histology, University of Navarra, Pamplona, Spain.
Received for publication June 9, 1997; accepted December 11, 1997.
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