ARTICLE |
Correspondence to: Gong Ju, Inst. of Neurosciences, The Fourth Military Medical University, Xi'an, P.R. China. E-mail: jugong@fmmu.edu.cn
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Summary |
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One of the unsolved key questions in neuroimmunomodulation is how peripheral immune signals are transmitted to the brain. It has been reported that the vagus might play a role in this regard. The underlying mechanism for this immune system-to-brain communication route is related to the binding of cytokines, such as interleukin (IL)-1ß originating from activated immune cells, to their receptors in glomus cells of the vagal paraganglia. The existence of IL-1 receptor type I (IL-1RI) in vagal paraganglia has been proved. On the basis of these studies, a hypothesis is raised that the carotid body, as the largest paraganglion, might play a similar role to that of its abdominal partner. In this study we examined the distribution of IL-1RI in the carotid body by immunohistochemistry (IHC) and Western blotting techniques. The IHC results showed that almost all glomus cells in the carotid body displayed strong IL-1RI immunoreactivity. The IL-1RI-immunoreactive products were localized in the cytoplasm, nucleus, and cell membrane of the glomus cells. The Western blotting results also confirmed the existence of IL-1RI in both membranous and cytoplasmic elements of the carotid body. These results imply that the carotid body not only serves as a chemoreceptor for modulation of cardiorespiratory performance, as traditionally recognized, but also acts as a cytokine chemorereceptor for sensing immune signals. (J Histochem Cytochem 50:16771684, 2002)
Key Words: carotid body, interleukin-1 receptor type I, immunocytochemistry, rat
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Introduction |
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Although it has been widely accepted that the immune system influences the neuronal activity of the central nervous system (CNS) during immune challenge (
It is generally believed that the informational molecules for immune-to-brain communication are proinflammatory cytokines originating from activated immune cells. Interleukin-1ß (IL-1ß) is one of the most likely mediators for immune-to-brain communication (
The mechanism(s) that enable the immune cytokines, such as IL-1ß, to stimulate the peripheral endings of sensory fibers in the vagus is still unclear. Recently,
According to anatomic criteria, the carotid body, which is well known for its chemoreceptive function, is the largest paraganglion in the body. The carotid body in mammals has a similar configuration to that of the abdominal paraganglion (
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Materials and Methods |
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Adult male SpragueDawley rats weighing 200300 g were used (n=29). The animals were housed under a 12:12 light:dark cycle and had access to laboratory chow and water ad libitum until they were deeply anesthetized with sodium pentobarbital (60 mg/kg IP).
Immunohistochemistry for Light Microscopy
Rats (n=14) were transcardially perfused with 100 ml of 0.9% saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, at 4C. After perfusion the carotid bodies, including the bifurcation of the carotid artery, were removed and put in 4% paraformaldehyde for 1 hr at 4C before being moved into 20% sucrose in 0.1 M PB overnight at 4C for cryoprotection. Fifteen-µm-thick frozen sections were cut with a cryostat and mounted on gelatinized slides. After being dried at room temperature (RT), the sections were blocked with 5% normal goat serum, 1% bovine serum albumin (BSA), and 0.3% Triton in 0.01 M PBS at RT for 50 min.
For double immunofluorescent staining, the sections were incubated with a mixture of anti-IL-1RI polyclonal antiserum raised from rabbit (1:300; Santa Cruz Biotechnology, Santa Cruz, CA; or 1:100, Research Diagnostic, Flanders, NJ) and anti-tyrosine hydroxylase (TH, a marker for glomus cells) (
Several controls were conducted to confirm the specificity of IL-1RI immunoreactivity. First, as noted above, the anti-IL-1RI polyclonal antiserum from different sources (Santa Cruz Biotechnology and Research Diagnostic) was used. Second, omission controls were performed. The primary anti IL-1RI antibody was replaced by normal rabbit serum or 0.01 M PBS (pH 7.4) containing 0.3% Triton and 1% normal goat serum, and then the sections were incubated with goat anti-rabbit IgGAlexa 488. The anti-TH MAb was replaced by normal mouse serum or 0.01 M PBS (pH 7.4) containing 0.3% Triton and 1% normal goat serum, and the sections were incubated with goat anti-mouse IgGTexas Red. Finally, to ensure that there was no crossreactivity between the primary antibodies and the non-related second antibodies in double fluorescent staining, some sections were incubated in goat anti-rabbit IgGAlexa 488 after the incubation in anti-TH MAb raised from mouse or in goat anti-mouse IgGTexas Red after the incubation in anti-IL-1RI polyclonal antiserum raised from rabbit.
For IHC staining with the avidinbiotin complex (ABC) method, the rabbit-raised primary antibodies against IL-1RI from both companies (Santa Cruz Biotechnology and Research Diagnostic) were used. The biotinylated goat anti-rabbit antiserum and Elite ABC kit were from Vector Labs (Burlingame, CA). The standard staining protocol for the ABC method was used and has been reported elsewhere (
Western Blotting
Rats (n=15) were transcardially perfused with 100 ml of 0.9% saline under deep anesthesia with sodium pentobarbital (60 mg/kg IP) to wash the blood out. Bilateral carotid bodies with some connective tissues were removed. At first, about 60 mg of tissue was lysed in 150 µl of ice-cold Tris-buffered saline (TBS) containing 2% protease inhibitors (Boehringer Mannheim; Mannheim, Germany) by using a glass homogenizer and was left to stand on ice for 30 min to release the cytoplasmic proteins. Insoluble materials were separated by centrifugation at 10,000 x g for 10 min. The supernatant fluid, the total cytoplasmic lysate, was collected. One hundred µl of cold RIPA containing 2% protease inhibitors was added to the insoluble materials. These membranous elements were lysed by using a glass homogenizer and were left to stand on ice for 30 min to release membranous cell proteins before clarification by centrifugation at 10,000 x g for 10 min. The supernatant fluid, the membrane lysate, was collected. Both supernatants were mixed with one-quarter volumes of 4 x SDS-PAGE sample buffer (Sigma) and heated at 100C for 5 min. Before electrophoresis, each sample was clarified again by centrifugation at 10,000 x g for 10 min. Electrophoresis was carried out by SDS-PAGE using 10% polyacrylamide according to standard protocols. Then the proteins in PAG were transferred to nitrocellulose membranes (Schleicher & Schuell; Dassel, Germany). Before membrane blocking and incubation in primary antibody, these membranes were stained with Ponceau solution to show the protein bands of both the marker and the samples. The following steps were performed according to the protocol of the BM chemiluminescence Western blotting kit (Roche Molecular Biochemicals; Mannheim, Germany). The nitrocellulose membranes were blocked in 1% blocking solution (Roche Molecular Biochemicals) under shaking for 1 hr at RT to block nonspecific binding of antibody after being washed twice with TBS. Next, we incubated the membranes for 1 hr at RT with rabbit anti rat IL-1RI primary antibody (Research Diagnostic) diluted in 0.5% blocking solution with gentle shaking. After being washed twice in TBST (TBS containing 0.05% Tween-20) for 10 min each and in 0.5% blocking solution for 10 min, the membranes were incubated with POD-labeled reconstitute second antibody (40 mU/ml; Roche Molecular Biochemicals) diluted with 0.5% blocking solution for 1 hr. The membranes were washed with TBST three times for 10 min each. After exposing the membranes to the detection reagents for 60 sec, we placed a sheet of X-ray film onto the membranes in an X-ray film cassette and exposed the film for 60 sec, in a dark room. Then we developed the exposed film immediately in developer and fixed it in fixing solution in tanks under a red safelight. The black bands in the film and the marker stained by Ponceau solution were scanned into the computer and processed by Photoshop without changing the results.
Omission controls were done in which the primary IL-1RI antibody was replaced by 0.5% blocking solution or normal rabbit serum.
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Results |
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Immunocytohistochemistry
In the fluorescent staining sections, the glomus cells of the carotid body were gathered in small cell groups and scattered in the connective tissue stroma (Fig 1D). There was strong IL-1R I-like immunoreactivity throughout the body (Fig 1A). Although the intensity of the immunostaining varied from cell to cell, almost all the glomus cells were intensely immunolabeled with IL-1RI (Fig 1E and Fig 1H). These type I cells were ovate and about 10 µm in diameter. The cell membrane and cytoplasm of the glomus cell were immunostained intensely. In addition, their nuclei were immunostained, although the staining was weak. There was also strong TH-like immunoreactivity throughout the carotid body (Fig 1B). TH-positive products appeared in the cytoplasm of glomus cells but not in the nucleus (Fig 1F1H). In double staining, all of the TH-positive cells were labeled with anti-IL-1RI antibody (Fig 1C, Fig 1G, and Fig 1H). Conversely, very few IL-1RI-positive glomus cells, judged from their morphology, were negative for TH (Fig 1G and Fig 1H). Blood vessels were stained only by IL-1RI (Fig 1G and Fig 1H). Some tissues between the glomus cells were labeled only with IL-1RI, but we could not identify them as sustentacular cells, connective tissue cells, or blood vessels under the present conditions. The staining results by the anti-IL-1RI antibodies from Santa Cruz Biotechnology (Fig 2A and Fig 2B) were the same as those from Research Diagnostic.
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No immunoreactive products were observed in the omission sections in which the primary anti IL-1RI antibody and anti-TH antibody were replaced by normal rabbit serum (Fig 1I), normal mouse serum (Fig 1K), or 0.01 M PBS containing 0.3% Triton and 1% normal goat serum (Fig 1J and Fig 1L). No crossreactivity was seen between rabbit-raised primary IL-1RI antibody and anti-mouse second antibody, and vice versa.
By using ABC methods, the same staining results by using the anti IL-1RI antibodies from Research Diagnostic were obtained (Fig 3A and Fig 3D). There was strong IL-1RI-like immunoreactivity throughout the body. The positive glomus cells clustered among the connective tissues. No immunoreactive products were observed in the omission control sections in which the primary IL-1RI antibody was replaced by normal rabbit serum (Fig 3B) or 0.01 M PBS containing 0.3% Triton and 1% normal goat serum (Fig 3C).
Western Blotting
In the cell membrane and cytoplasm lanes, specific bands were observed in the position of 80 kD, in agreement with the molecular weight of IL-1RI (Fig 4). There was no band in the corresponding position for the two omission controls. These results not only confirmed the specificity of the anti-IL-1RI antibody but also suggested that both the cytoplasm and the cell membrane contained IL-1RI.
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Discussion |
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The carotid body mainly contains two kinds of cells: type I cells (glomus cells) and type II cells (sustentacular cells) (
IL-1 is a prototype of the pro-inflammatory cytokines that induce expression of a variety of genes and the synthesis of several proteins which, in turn, induce acute and chronic inflammatory changes (
Anatomically, the carotid body is recognized as the largest paraganglion in mammals (
An interesting phenomenon in the present study is that the nucleus of the glomus cells was immunostained by the anti-IL-1RI antibody.
The mechanisms of origin of nerve impulses in the carotid body chemoreceptor are still unclear. Three structures have been proposed as the primary site for chemoreception: sustentacular cells, sensory terminals, and glomus cells (
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Acknowledgments |
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Supported by the Chinese National Nature Science Foundation (no. 39830130) and by the Key Teacher Supporting Program of the Chinese National Educational Administration.
Received for publication February 11, 2002; accepted June 19, 2002.
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