It is well known that the principal function of carotid body is to detect the change of arterial blood PO2 and modulate respiratory movement. The carotid body contains two distinct parenchyma cells: type I (glomus) and type II (sustentacular) cells. The dopaminergic type I cells in the carotid body are generally believed to be the chemoreceptors (Prabhakar,2000).
Although it has been widely accepted that the immune system influences the neuronal activity of central nervous system (CNS) during immune challenge (Ericsson et al.,1997; Watkins and Maier,1999; Dantzer et al.,2000; Roth and Souza,2001), how the immune information is transmitted to the brain is still unknown. It is generally believed that the informational molecules for immune-brain communication are proinflammatory cytokines originating in activated immune cells. Increasing evidence has indicated that the vagus plays an important role in transmitting immune information originating in the periphery to the brain (Goehler et al.,2000). However, the mechanism(s) that enables the immune cytokines, such as IL-1β, IL-6, and TNF-α, to stimulate the peripheral endings of sensory fibers in the vagus is still unclear. Goehler et al. (1997) reported that vagal paraganglia might play a possible role in the mechanism. We further found that the carotid body, like its peritoneal partner, had a strong expression of IL-1RI in the glomus cells (Wang et al.,2002), suggesting that the carotid body may also play a role in the sensation and transmission of immune information.
IL-6, a polyfunctional cytokine, is another important proinflammatory cytokine. It is a major factor for growth and differentiation of various cells and for the synthesis of acute phase protein in the liver, etc. (Naka et al.,2002). It is also proposed as an informational molecule for immune-to-brain communication (Hosoi et al.,2002). However, whether this proinflammatory cytokine influences the activity of the carotid body has not been investigated. The biological activity of IL-6 is initiated by its binding to the specific ligand-binding IL-6 receptor α chain (IL-6Rα). To study whether the carotid body plays a role in sensation and transmission of IL-6 information, we used immunohistochemistry, Western blots, and in situ hybridization methods to clarify the existence of IL-6Rα in the carotid body.
MATERIALS AND METHODS
Adult male Sprague-Dawley (SD) rats weighing 200–300 g (offered by Animal Center, Fourth Medical University) were used. The animals were housed under a 12:12 light/dark cycle and accessed to laboratory chow and water ad libitum.
The procedures for Western blots were used as reported previously (Wang et al.,2002). Briefly, carotid body from SD rats were homogenized in ice-cold 0.01 M phosphate-buffered saline (PBS) containing cold RIPA (Boehringer Mannheim, Mannheim, Germany) and 2% protease inhibitor (Boehringer Mannheim). The protein from resultant supernatant were subjected to electrophoresis on a 10% (wt/vol) polyacrylamide gel in SDS, and the gel was subsequently processed for electroblotting to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). The blotted nitrocellulose membrane was stained by Ponceau solution to show the protein belts of both marker and samples. First, the membrane containing sample proteins was blocked in a solution containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 for 1 hr at room temperature (RT). Then the membrane was sequentially incubated in rabbit anti rat IL-6Rα primary antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, biotinylated goat antirabbit antibody (1:200; Vector Lab, Burlingame, CA) for 2 hr at RT, and avidin-biotin-complex (ABC; 1:200; Vector Lab) for 1 hr at RT. After thorough washing, the positive bands were visualized with amine nickel sulfate-enhanced 3,3′-diaminobenzidine (DAB) method. The reaction products, black bands in the membrane, and the marker stained by Ponceau solution were scanned into the computer and processed by Photoshop 6.0 without changing the results.
Several controls were conducted for confirming the specificity of the primary antibody for IL-6Rα. First, preabsorption of IL-6Rα antibody with its corresponding antigen, IL-6Rα peptide (Santa Cruz Biotechnology) prior to the Western blot procedure resulted in a complete blockage of the specific signals. Second, by using Western blots, IL-6Rα protein was demonstrated in the hippocampus in which the existence of IL-6Rα has been reported (Vollenweider et al.,2003).
Immunohistochemistry for Light Microscopic Observation
Rats (n=10) were transcardially perfused with 4% paraformaldehyde, pH 7.4, at 4°C. After perfusion, the carotid bodies including the bifurcation of carotid artery were removed and put in 4% paraformaldehyde for 1 hr at 4°C before being moved into 20% sucrose at 4°C for cryoprotection overnight. Fifteen μm thick sections were cut with a cryostat microtome and mounted onto gelatinized slides. Sections were blocked with 3% normal goat serum and 1% BSA at RT for 50 min.
For double immunofluorescent staining, the sections were incubated with a mixture of rabbit anti–IL-6Rα (1:300) and mouse antityrosine hydroxylase (TH; a marker for glomus cells; 1:500; Sigma, St. Louis, MO) antibodies in 0.01 M PBS (pH 7.4) overnight at RT, then with a mixture of goat antirabbit IgG-Alexa 488 (1:400; Molecular Probes, Eugene, OR) and goat antimouse IgG-Texas Red (1:400; Molecular Probes). After 2-hr incubation at RT, the slides were washed and coverslipped with 50% glycerol. The fluorescent sections were observed and photographed with a confocal laser scanning microscope (CLSM; FV-300; Olympus, Tokyo, Japan).
Several controls were conducted for confirmation of the specificity of the primary antibodies of IL-6Rα and TH. Omission control was completed by replacing the primary anti IL-6Rα and TH antibodies with normal rabbit serum or 0.01 M PBS (pH 7.4), then incubating the sections with goat antirabbit IgG-Alexa 488 and goat antimouse IgG-Texas Red. To ensure no cross-reactivity between the primary antibodies and the unrelated second antibodies in double fluorescent staining, some sections were incubated in goat antirabbit IgG-Alexa 488 after the incubation in mouse anti-TH antibody or in goat antimouse IgG-Texas Red after the incubation in rabbit anti–IL-6Rα antibody. Specificity for IL-6Rα was further confirmed by preabsorbing the antibody with its corresponding antigen prior to the double immunofluorescent staining. In addition, hippocampus, the positive tissue for IL-6Rα, was stained with the IL-6Rα antibody.
In Situ Hybridization
Rats (n=6) were transcardially perfused with DEPC-4% paraformaldehyde, pH 7.4, at 4°C. Fifteen μm thick frozen sections of the carotid body were cut with a cryostat microtome and mounted onto gelatinized slides.
Complementary DNA (cDNA) probes were used for detection of mRNA for IL-6Rα. A 450 bp polymerase chain reaction (PCR) product with the template of the rat cDNA library was used to generate digoxigenin-labeled cDNA probe of IL-6Rα. PCR was performed according to the recommendation of the supplier (Clontech Laboratories, Palo Alto, CA). The cDNA with 450 bp was labeled with digoxigenin by using a DIG cDNA labeling kit (Boehrimger Mannheim).
For in situ hybridization histochemistry, cryostat sections were washed with 0.01 M PBS, then incubated in 0.3% TritonX100 and 0.2 mM hydrochloric acid (HCl) for 20 min. The sections were digested in 20 μg/ml proteinase K at 37°C for 15 min and stopped with 0.2% glycin at 37°C for 10 min, then dehydrated in alcohol gradients for 3 min, respectively. The hybridization was performed in a moist chamber at 42°C overnight. The sections were hybridized in the probe diluted in a hybridization solution containing 50% deionized formamide, 10% 20× standard saline citrate (SSC), 5% clupeine (10 mg/ml), 5% 50× dehardt's, and 20% dextran sulfate. After a thorough washing, sections were blocked in a solution containing 2% normal goat sheep and 0.3% TritonX100, then incubated in alkaline phosphatase-conjugated antidigoxigenin antibody (1:100; Boehrimger Mannheim) for 2 hr at RT. After a thorough washing, immobilized digoxigenin was revealed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solution. The positive signal was shown as blue-black granules. The sections were dehydrated in alcohol gradients, cleared in xylene, and mounted in gelatin.
Several controls were done: omitting the specific probe of IL-6Rα and conducting the in situ hybridyzation in the hippocampus, the known positive tissue for IL-6Rα mRNA.
IL-6Rα Antibody Was Specific for 80 kD Protein
As a first step to study the expression of IL-6Rα protein in the carotid body, Western blot analysis was carried out on the homogenates of rat carotid body. Specific band was observed in the position of 80 kD in the nitrocellulose membrane, in agreement with the molecular weight of IL-6Rα (Fig. 1). There was no band in the corresponding position for the absorption control (data not shown). In the hippocampus tissue, a positive tissue control, a specific band in the position of 80 kD was also observed (Fig. 1).
IL-6Rα Colocalized With TH in Glomus Cells
The localization of IL-6Rα protein was investigated in situ by using double immunostaining (Fig. 2). The TH-positive glomus cells of the carotid body were gathered in cell clusters (Fig. 2A1, B1, and C1). There was strong IL-6Rα immunoreactivity throughout the body (Fig. 2A2 and B2). The stronger immunoreactive product for IL-6Rα protein was located mainly in the cell clusters of glomus cells. The cytoplasm of the glomus cells was intensely immunostained for IL-6Rα. These IL-6Rα–positive cells were ovate in shape. Although the intensity of the immunostaining varied from cell to cell, nearly all of the TH-positive glomus cells were immunolabeled with IL-6Rα (Fig. 2A3 and B3). Some tissues between the cell clusters of glomus cells, which probably were type II cells, blood vessel, and connective tissues, were also stained with the IL-6Rα (Fig. 2A2, A3, B2, and B3).
No positive products were observed in the omission controls (data not shown). No cross-reactivity was seen between rabbit-raised primary antibody and antimouse second antibody or vice versa (data not shown). No reactive products of IL-6Rα appeared in the absorption control (Fig. 2C2), in which the IL-6Rα antibody was preabsorbed with its corresponding peptide prior to double-staining the carotid body sections with TH. Only TH-positive signal was detected in this staining (Fig. 2C1). In the positive tissue control, IL-6Rα–positive products were seen in the pyramid cell layer of the hippocampus (Fig. 2C3).
IL-6Rα mRNA Was Detected in Glomus Cells
To identify the presence and localization of IL-6Rα mRNA in the carotid body, in situ hybridization was performed. The blue-black granules were recognized as positive signal. Strong positive signal was detected mainly in the clusters of the glomus cells in the carotid body (Fig. 3A), which was consistent with the result of the immunohistochemical result.
No positive signal was found in the sections without addition of the cDNA probe of IL-6Rα (Fig. 3B). In the hippocampus, the known positive tissue for IL-6Rα mRNA existence, the positive signal appeared mainly in the pyramid cell player and the granular cells of the dentate (data not shown).
In the present study, we demonstrated the presence and cellular localization of IL-6Rα protein and mRNA in the rat carotid body by using Western blots, immunofluorescent staining, and in situ hybridization. The results revealed that IL-6Rα protein and mRNA were mainly expressed in the glomus cells of the rat carotid body.
It is known that the biological activity of IL-6 is initiated by its binding to the IL-6R, which consists of two polypeptide chains, a specific ligand-binding chain (IL-6Rα) with molecular weight of 80 kD and a non–ligand-binding signal-transducing chain (gp130). IL-6 binds first to IL-6Rα with low affinity, then the complex binds to gp130 to form a high-affinity, functionally hexameric receptor complex composed of two IL-6, IL-6R, and gp130 heterotrimers to initiate intracellular signaling via the JAK/STAT, RAS/mitogen-activated protein kinase (MAPK), or other pathways (Toshio et al.,1997; Naka et al.,2002; Heinrich et al.,2003). Recently, we also demonstrated that gp130 was expressed and localized in the glomus cells of the rat carotid body by using Western blots and double immunostaining techniques (data not shown). Thus, the results of the present study provide part of morphological evidence for the functional role of IL-6 in carotid body.
We hypothesize that there are several possibilities for the role of IL-6 in the carotid body. First, stimulation of IL-6 might alter the excitability of glomus cells and change the electric activities in the sinus nerve. In this regard, the carotid body may be an alternative route, as the vagal paraganglia in abdomen, for transmission of the peripheral immune signals to the brain. Second, IL-6 may act as a modulator to glomus cell activity during PO2 detection. Third, IL-6 may modulate the survival, proliferation, and differentiation of glomus cells. Clearly, more physiological investigations need to be completed to verify the exact function of IL-6 in the carotid body.