RAPID COMMUNICATION |
Correspondence to: Csaba Szabó, Div. of Critical Care, Childrens Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.
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Summary |
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Here we examined the changes in NADPH diaphorase (NADPHd) and inducible nitric oxide synthetase (iNOS) positivity in the medulla of the mouse thymus in response to treatment with the superantigen, Staphylococcal enterotoxin B (SEB). A few NADPHd+ and iNOS+ cells scattered in the medulla were detected in the thymi of naive mice. SEB induced the appearance of a large number of NADPHd+- and iNOS-immunoreactive cells in the thymic medulla. In the thymus of iNOS-deficient mice, a total absence of these NADPHd+ and iNOS+ medullary cells was found both under basal conditions and after SEB stimulation. With the NADPHd reaction, only endothelial staining was detected in the thymi of iNOS-deficient mice. Our data indicate that NADPHd+ cells in the thymic medulla express iNOS and that SEB induces iNOS expression in the mouse thymus. (J Histochem Cytochem 46:787791, 1998)
Key Words: NADPH diaphorase, nitric oxide synthetase, thymus, Staphylococcal enterotoxin B
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Introduction |
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Nitric oxide (NO) is a labile free radical which plays an important role in a variety of physiological and pathophysiological processes, ranging from vasodilatation, macrophage cytotoxicity, and neuronal signaling to cytotoxicity in various forms of inflammation (
Nitric oxide synthases have been shown to have NADPH diaphorase (NADPHd) activity, as evidenced by co-localization and co-precipitation of NADPHd and NOS activity (
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Materials and Methods |
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Materials
Anti-mouse iNOS antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The Vector Elite kit was obtained from Vector Laboratories (Burlingame, CA). All other reagents were from Sigma (St Louis, MO).
Treatment of Animals
Animal experiments conform with the "Guide for the Care and Use of Laboratory Animals" published by the US National Institute of Health and the treatment protocol was approved by the Institutional Animal Care and Use Committee. Male C57BL10 mice and Wistar rats were purchased from Jackson (Bar Harbor, ME) and Charles River Laboratories (Wilmington, MA), respectively. Male iNOS-deficient mice [on C57BL/10 background (
NADPH Diaphorase Histochemistry
The method was carried out essentially as described previously (
Immunohistochemistry
Cryostat sections (10 µm) were treated with 0.3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and then rinsed briefly in PBS. Nonspecific binding was blocked by incubating the slides for 1 hr in 2% goat serum (in PBS). To detect iNOS, rabbit polyclonal anti-iNOS antibody was applied at a dilution of 1:500 at 4C overnight. (Control sections were incubated in PBS without the primary antibody.) After extensive washing (five times for 5 min) with PBS, immunoreactivity was detected with a biotinylated goat anti-rabbit secondary antibody and the avidinbiotinperoxidase complex (ABC), both supplied in the Vector Elite kit. Color was developed using Ni-DAB substrate (95 mg diaminobenzidine, 1.6 g NaCl, 2 g nickel sulfate in 200 ml 0.1 M acetate buffer). Sections were counterstained with nuclear fast red for 2 min and mounted in Permount medium.
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Results |
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NADPHd Reveals iNOS-expressing Cells in the Medulla of Naive Mouse Thymus
The inducible isoform of nitric oxide synthetase (iNOS) is usually expressed in response to cytokine and/or bacterial stimuli. The thymus appears to be a unique organ in this respect, because a number of NADPHd+ medullary cells have been described in the resting chicken and rat thymus (
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SEB Induces iNOS Expression in Thymic Medullary Cells
NADPHd reaction after SEB administration revealed a massive increase in the number of positive cells in the mouse thymus (Figure 1C and Figure 1E) compared to the naive thymus. SEB induced a marked increase in the amount of immunohistochemical staining for iNOS, with a pattern identical to that of NADPHd (Figure 1D and Figure 1F). In the thymi of iNOS-deficient animals, neither NADPHd (Figure 1G) nor iNOS staining (Figure 1H) could be detected after SEB challenge (Figure 1G and Figure 1H). The lack of these NADPHd+ medullary cells in the thymocytes of iNOS knock-out mice (Figure 1G) strongly indicates that the NADPHd+ staining was indeed related to iNOS. We have observed some endothelial NADPHd staining in the iNOS knock-out thymi, which probably corresponds to eNOS expressed constitutively by endothelial cells (Figure 1G).
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Discussion |
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The key observations of this report can be summarized as follows. (a) We have provided evidence for NADPHd+ and iNOS+ cells in the naive mouse thymus. (b) We have demonstrated a massive increase in the number of NADPHd+ and iNOS+ cells in response to SEB exposure. (c) We have shown the complete absence of NADPHd+ and iNOS+ thymic medullary cells in the thymi of iNOS knock-out animals. Based on the parallel changes in the amount of NADPHd+ and iNOS+ medullary cells in response to SEB and on the absence of these cells in the thymi of iNOS-deficient mice, we propose that NADPHd staining primarily detects the presence of iNOS in the thymus, both under resting conditions and under conditions of immunostimulation.
We have detected the expression of iNOS in the medulla of the naive mouse thymus. Although this may seem contradictory to the term "inducible" NOS, we hypothesize that, as has been proposed by . Thymocyte-derived interferon-
, in turn, activates stromal cells leading to iNOS expression, NO secretion, and NO-mediated apoptosis of thymocytes (
prestimulated stromal cells (
The wide range of physiological and pathophysiological effects of NO and NO-derived reactive species set up a claim for simple and sensitive techniques to detect NOS activity in tissue sections. NADPHd histochemistry, a fast and simple reaction often used by neuroscientists to visualize the expression of bNOS in the nervous system, offered a good alternative to the expensive and time-consuming immunohistochemistry. Although many enzymes have diaphorase activity which may cause specificity problems, pretreatment of tissues with 4% p-formaldehyde has been shown to eliminate non-NOS-derived activity (
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