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Section on Neuroendocrinology, Office of the Scientific Director, NICHD, National Institutes of Health, Bethesda, Maryland 20892Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
* This study was supported by the Intramural Research Programs of NICHD (National Institutes of Health), NIAMS (National Institutes of Health), and NIDCR (National Institutes of Health), and the Lundbeck and Novo Nordisk Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org) contains supplemental data and supplemental Figs. 1–3. 1 Current address: The Centre for Genomic Application, 254, Ground Floor, Phase-III Okhla Industrial Estate, New Delhi, India 110020.
The high affinity immunoglobulin E receptor (FcϵRI) complex is dedicated to immunoglobulin E-mediated allergic responses. Expression of the FcϵRI receptor is thought to be relatively stable and limited to mast cells, basophils, eosinophils, monocytes, Langerhans cells, platelets, and neutrophils. We now report that the FcϵRIα and FcϵRIγ polypeptides are expressed in the pinealocyte, the melatonin-secreting cell of the pineal gland. Moreover, Fcer1a mRNA levels increased ∼100-fold at night to levels that were higher than in other tissues examined. Pineal FcϵRIα protein also increased markedly at night from nearly undetectable daytime levels. Our studies indicate that pineal Fcer1a mRNA levels are controlled by a well described neural pathway that controls pineal function. This pathway includes the master circadian oscillator in the suprachiasmatic nucleus and passes through central and peripheral structures. The circadian expression of FcϵRIα in the pineal gland is driven by this neural circuit via an adrenergic/cyclic AMP mechanism. Pineal FcϵRIα and FcϵRIγ may represent a previously unrealized molecular link between the neuroendocrine and immune systems.
A resonant theme in the initiation of an allergic reaction is the binding of allergens to immunoglobulin E (IgE) antibodies attached to their corresponding high affinity Fc receptors (FcϵRI)
The abbreviations used are: FcϵRI, high affinity immunoglobulin E receptor; NE, norepinephrine; ISO, isoproterenol; ZT, zeitgeber time; SCG, superior cervical ganglion; RBL, rat basophilic leukemia; SCN, suprachiasmatic nucleus; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol.
3The abbreviations used are: FcϵRI, high affinity immunoglobulin E receptor; NE, norepinephrine; ISO, isoproterenol; ZT, zeitgeber time; SCG, superior cervical ganglion; RBL, rat basophilic leukemia; SCN, suprachiasmatic nucleus; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol.
on mast cells or basophils. This event is essential for a broad range of events including immediate induction of signaling cascades leading to degranulation, which releases stored mediators including histamine, serotonin, and proteases, and increases the production of arachidonic acid. This results in the generation of symptoms characteristic of acute allergic reaction (
). The extracellular portion of α-subunit binds IgE molecules via the Fc domain. The β- and γ-subunits are not involved directly in IgE binding but are essential for membrane localization and signal transduction in that domains of both subunits are exposed to the cytoplasm (
). Signaling is triggered by the simultaneous binding of a single multivalent allergen to multiple IgE·FcR complexes leading to immunogen-induced cross-linking of the receptor on the cell surface. Tetrameric (αβγ2) and trimeric (αγ2) complexes are known to be active (
Expression of FcϵRI was originally thought to be limited to the effector cells of anaphylaxis, specifically mast cells and basophils. Later, it became apparent that the FcϵRI is expressed in antigen-presenting cells including human epidermal Langerhan cells, peripheral blood dendritic cells, monocytes, and eosinophils (
). Expression of FcϵRI has not been reported in neural, endocrine, or neuroendocrine cells.
The results presented in this report reflect a surprising observation made during the course of a preliminary microarray analysis of gene expression in the pineal gland, a tissue not known to be involved in the immune response. This tissue is recognized as being dedicated to the generation of the daily rhythm in circulating melatonin, a conserved feature of vertebrate biology (
). Our microarray results were surprising because they suggested that there is a daily rhythm in Fcer1a expression in the pineal gland and that nighttime levels are higher than in other tissues. This was especially intriguing when viewed against the evidence of highly restricted expression of FcϵRI and our current concept of the pineal gland as a melatonin factory. The potential importance of this discovery propelled us to confirm the preliminary finding of a 24-hour rhythm in Fcer1a mRNA and to extend this with the intention of obtaining a better understanding of FcϵRI biology in the pineal gland. Our findings provide justification to consider that FcϵRI might provide a unique molecular link between the neuroendocrine and immune systems.
Animals and Tissue Removal—Sprague-Dawley rats (Taconic Farms Inc., Germantown, NY) were used for all experiments. Animals were housed in a controlled lighting environment (14-h light and 10-h dark cycles, and for in situ hybridization studies light/dark 12:12). In experiments presented in Fig. 5A, animals were kept under constant darkness or constant light for 3 days. Surgical decentralization of the superior cervical ganglia (SCGdcn, Taconic Farms Inc.) was performed. Rats were allowed to recover and adjust to the light cycle for 2 weeks and then were sacrificed by CO2 asphyxiation and decapitated at the indicated zeitgeber time (ZT). During the dark period, animals were handled under red photographic safelights. Pineal glands and other tissues were removed immediately and frozen on dry ice. All procedures were done in accordance with National Institutes of Health animal use and care guidelines. For in situ hybridization studies, Wistar rats were used. Animals (Animal Section, Panum Institute, Copenhagen, Denmark) were housed for 2 weeks in a controlled lighting environment (12-h light and 12-h dark cycles). As indicated in Fig. 6A, animals were injected intraperitoneally with isoproterenol (ISO, 10 mg/ml phosphate-buffered saline, 5 mg/kg rat).
Cell and Organ Culture—Pinealocytes were prepared from rat pineal glands (
). Briefly, pineal glands were incubated (1 h, 37 °C) with 20 units/ml papain (Worthington) and 200 units/ml DNase I (Worthington) in Earle's balanced salt solution. Subsequently, glands were triturated, and the resulting preparation was passed through a 40-μm cell strainer (BD Falcon, Bedford, MA). The pinealocytes were harvested, washed, and suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (95% air, 5% CO2, 37 °C). Dispersed pinealocytes were cultured either in suspension (for Western blot analysis in Fig. 6C) or as an adherent monolayer (for immunocytochemical detection in Fig. 4) on slides precoated with polyornithine (Sigma). About 16 h following the cell preparation, the pinealocytes were treated with norepinephrine (NE, 1 μm), dibutyryl cyclic AMP (1 mm), or forskolin (10 μm) for 6 h.
The rat basophilic leukemia (RBL) cell line RBL-2H3 (ATCC, Manassas, VA) (
) was grown as an adherent monolayer in tissue culture flasks using Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum in 5% CO2 incubator at 37 °C. Pineal glands were cultured as described (Ref.
). S-antigen/arrestin was detected using a polyclonal rabbit anti-bovine S-antigen antiserum (NEI Z-02 from Dr. S. Zigler, NEI, National Institutes of Health). Mouse monoclonal anti-synaptophysin (Santa Cruz Biotechnology) and rabbit polyclonal anti-FcϵRIγ were purchased (Upstate Biotechnology).
Immunocytochemistry and Fluorescence Microscopy—All immunocytochemical analyses (see Fig. 4) were done with pinealocytes attached on multichambered slides (Sigma). Dispersed pinealocytes (1 × 106 cells/chamber) were transferred to polyornithine-coated chambers on the slide. Cells were allowed to attach during overnight incubation (95% O2, 5% CO2, 37 °C). On the following day, the cells were treated with NE (1 μm) for 6 h, washed twice in phosphate-buffered saline (PBS, pH 7.4), fixed (2% paraformaldehyde, 20 min, 24 °C), and permeabilized (0.05% saponin, 30 min, 24 °C). The permeabilized cells were rinsed with PBS and then in PBS containing 0.1 m glycine and blocked with 2% bovine serum albumin in PBS containing 5 μg/ml donkey IgG. The cells were then rinsed twice with PBS and incubated with primary antibodies. After incubation with primary antibodies against α- and γ-subunits of the FcϵRI receptor (10 and 5 μg/ml, respectively), cells were rinsed thoroughly in PBS and incubated with goat anti-mouse F(ab′)2 conjugated to Alexa 488 (for α-subunit) or goat anti-rabbit F(ab′)2 conjugated to Alexa 594 for γ-subunit (Molecular Probes, Eugene, OR). Finally, cells were rinsed in PBS and then in distilled water. Coverslips were mounted with Fluoromount G (EM Sciences, Fort Washington, PA). For negative controls, cells were treated identically and incubated only with secondary antibodies omitting primary antibodies.
Images were acquired by scanning confocal microscopy (Leica TCS NT, Heidelberg, Germany). The confocal parameters were established at the beginning of the study and remained constant throughout with an equal brightness setting and a constant confocal aperture pinhole optimized for the objective lens used.
In Situ Hybridization and Northern Blot—These methods were done using published techniques (
) as detailed in supplemental data. For in situ hybridization, two 38-mer oligonucleotide probes, IgE rec-1 and IgE rec-2 (derived from GenBank™ accession number M21622 antisense sequence), were used: 5′-TTG TTC AAA TAG CCT GTG CAG TGG TAG CTG CCA CTG TC-3′ (IgE rec-1) and 5′-TTG TCT AAG ACC ACG TCA GCA GAA GAT TGG AGC AGC AG-3′ (IgE rec-2). For Northern blot analysis, probes described previously (
) were used that were generated from the Fcer1a coding region nucleotides 177–600 (GenBank™ accession number M21622), Fcer1b coding nucleotides 2051–2470 (GenBank™ accession number M22923), and Fcer1g coding nucleotides 61–421(GenBank™ accession number L04306), respectively.
Real-time PCR and Quantification—RNA isolation, RNA processing, quantification, and cDNA synthesis were done as described previously (Ref.
and supplemental data). Primers used in the analysis are as follows: rat β-actin (V01217) 5′-ACG GTC AGG TCA TCA CTA TGG-3′ (sense); and 5′-AGC CAC CAA TCC A CA CAG A-3′ (antisense); rat Aanat (U38306) 5′-TCC TGT GGA GAT ACC TTC ACC A-3′ (sense) and 5′-CAG CTC AGT GAA GGT GAG AGA T-3′ (antisense); rat tryptophan hydroxylase 1 (XM_341862) 5′-CAA GGA GAA CAA AGA CCA TTC-3′ (sense) and 5′-ATT CAG CTG TTC TCG GTT GAT G-3′ (antisense); rat high affinity IgE receptor, α-subunit (M21622) 5′-TGT GTA CTT GAA CGT GAT GCA A-3′ (sense) and 5′-TGT CTA AGA CCA C GT CAG CAG-3′ (antisense); Fcer1 β-subunit (Fcer1b, M22923) 5′-TGT CCG AAA GGA AAA ACA CAC-3′ (sense) and 5′-CGA TGC TGC TGA CAA TGT TT-3′ (antisense); and Fcer1 γ-subunit (Fcer1g; L04306), primer 5′-CCC TGC TCT ACT GTC GAC TCA-3′ (sense) and primer 5′-TCT CAC GGC TGG CTA TGT CT-3′ (antisense).
Western Blot Analysis—To detect FcϵRIα protein, tissues or cells were homogenized in 0.1 mm Tris-HCl (pH 7.5) containing protease inhibitor mixture (Roche Applied Science) using a tissue grinder. The homogenate was sonicated (three 1-s pulses, Bronwill Scientific, Rochester, NY), boiled (Laemmli sample buffer in a nonreducing condition), and clarified (centrifugation 6,000 × g, 5 min). The proteins in the supernatant were separated by SDS-PAGE (12% precast gel, Invitrogen) and were then transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). FcϵRIα was detected with monoclonal ER antibody (10 μg/ml). The blots were stripped for subsequent detection with anti-actin antisera (1:1000 dilution, Sigma) to confirm equal loading (
). For FcϵRIγ protein detection, samples were run under nonreducing conditions in a 4–12% NuPage BisTris gel (Invitrogen), transferred to nitrocellulose membrane, and immunodetected with rabbit anti γ-subunit antisera (1:1000 dilution, Upstate Biotechnology).
Fcer1a mRNA and FcϵRIα Protein Occur in the Pineal Gland—Fcer1a mRNA and FcϵRIα protein were detected in pineal glands removed at midnight (Fig. 1, A and B) but not in those removed at noon. A similar night/day difference was obvious from in situ hybridization (Fig. 2A). Northern blot and 5′ rapid amplification of cDNA ends analysis (supplemental Fig. 1) indicated that the pineal transcript (∼1.3 kb) was identical in size and sequence to that expressed in RBL-2H3 cells (GenBank™ accession number M21622) (
). Western blot studies indicated that the immunopositive proteins in RBL cell and in the pineal gland were similar in size (∼50kDa, Fig. 1B). The maximal level of expression of the Fcer1a mRNA occurs 5 h after the lights are turned off (midnight, ZT19, Fig. 1, C and D), and elevated expression persists during the remainder of the night period.
For comparative purposes, the levels of transcripts of two well studied genes that are strongly expressed in the pineal gland are also presented, arylalkylamine N-acetyltransferase (Aanat) and tryptophan hydroxylase 1 (Tph1). The former is known to be highly rhythmic and the latter to be expressed at constant high levels (Fig. 1D). It was found that the 24-h patterns of Aanat and Fcer1a transcripts were quantitatively and qualitatively similar.
Pineal Fcer1a mRNA Levels Are Relatively High Compared with Other Tissues—The level of Fcer1a mRNA in the rat pineal gland was found to be greater than other tissues examined, as indicated by the results of in situ hybridization analysis (Fig. 2A) and Northern blot analysis (Fig. 2B). The latter included bone marrow, a source of mast cells in which the transcript was undetectable. Expression in the pineal gland was less intense than in the RBL-2H3 and in a culture of mouse bone marrow-derived mast cells. Fcer1a was also expressed in mouse pineal gland as demonstrated by end-point reverse transcription-PCR (supplemental Fig. 2), indicating that expression is not unique to the rat pineal gland.
The Pineal Gland Expresses FcϵRIγ but Not FcϵRIβ—The abundance of mRNA encoding the γ-chain of the FcϵRI receptor was similar in glands removed during the day or night (Fig. 1, A and D), although expression was somewhat lower than in bone marrow-derived mast cells and RBL cells (Fig. 3A). FcϵRIγ protein was also immunodetected at similar levels in the day and night pineal gland as a ∼25 kDa band using nonreducing conditions (Fig. 3B). However, pineal Fcer1b mRNA was not detected using Northern hybridization and quantitative real-time PCR methods that detect the transcript in RBL cells (data not shown).
FcϵRIα and FcϵRIγ Are Expressed in the Pinealocyte—The rat pineal gland is composed primarily of pinealocytes (>90%), and it also includes parenchymal interstitial/glial cells, phagocytes, vascular pericytes, endothelial cells, and smooth muscle cells. Nonresident transiently present cells include leuckocytes and immature mast cells (
). To investigate whether FcϵRIα and FcϵRIγ proteins are expressed specifically in the pinealocyte, immunocytochemical analysis was done using dispersed cells. Pinealocytes were positively identified using antisera against either of two pinealocyte marker proteins, S-antigen or synatophysin (
FcϵRIα protein was nearly undetectable in untreated cells. However, this was increased in nearly all cells following treatment with NE (1 μm, Fig. 4A), and most of the FcϵRIα positive cells were also positive for S-antigen. FcϵRIγ protein was detected in the synaptophysin-positive cells and in other cells (Fig. 4B). As with the immunoblot analysis, FcϵRIβ protein was not detected in the pinealocytes. Moreover, there was no binding to the cells by a monoclonal antibody (mAb BC4), which detects the cell surface expression of FcϵRI (data not shown). These results demonstrate that FcϵRIα and FcϵRIγ proteins occur in the pinealocyte albeit not on the cell surface.
Regulation of Fcer1a mRNA and Protein: Circadian and Photoneural Control—Circadian changes in pineal function are driven by the central circadian oscillator in the suprachiasmatic nucleus (SCN). It is hardwired to the pineal gland and generates pineal rhythms in animals in the absence of lighting cues in constant darkness. Here we found that the daily rhythm in Fcer1a mRNA persists in animals housed in constant darkness for 3 days (DD, Fig. 5A), indicating that the day/night rhythm in Fcer1a mRNA is circadian in nature and does not require light-dark transitions.
The transmission of signals from the SCN to the pineal gland is gated by light acting downstream of the SCN. To determine whether the Fcer1a mRNA levels were under photic control, animals were kept in constant light for 3 days. This blocked the nocturnal increase in Fcer1a mRNA (Fig. 5A, LL). In addition, a 30-min exposure to light at night (ZT19) rapidly reduced the abundance of Fcer1a mRNA (Fig. 1C and supplemental Fig. 3). Neural signals from the SCN to the pineal gland pass through the superior cervical ganglia (SCG), from which postganglionic processes send projections to the pineal gland (
). To determine whether neural stimulation via the SCG is required for rhythmic expression of Fcer1a in the pineal gland, the SCG was surgically decentralized. This blocked the nocturnal increase in pineal Fcer1a mRNA (Fig. 5B). Accordingly, these findings support the interpretation that the rhythm in expression of this gene is regulated by the same neural system that regulates melatonin production and many other elements of mammalian pineal function (
). To investigate whether β-adrenergic stimulation induces the Fcer1a mRNA, rats were injected with the β-adrenergic agonist ISO at ZT6 (midday). This increased Fcer1a mRNA in the pineal gland within 3 h (ZT9, Fig. 6A).
It was also found that in vitro adrenergic stimulation with NE elevated Fcer1a expression (Fig. 6B). To investigate whether the adrenergic stimulation of Fcer1a expression might involve cAMP, pineal glands were treated (6 h) with either the cAMP protagonist dibutyryl cyclic AMP (1 mm) or the adenylate cyclase activator forskolin (1 μm). Both treatments increased Fcer1a mRNA (Fig. 6B). NE or dibutyryl cyclic AMP treatment of isolated pinealocytes also increased FcϵRIα protein as detected by Western blot (Fig. 6C). Together with the known stimulatory effects of NE on pinealocyte cAMP, these observations are consistent with the conclusion that that Fcer1a expression in the pinealocyte is regulated by an adrenergic/cyclic AMP mechanism.
The discovery of high expression of both FcϵRIα and FcϵRIγ in the pineal gland provides tantalizing evidence that FcϵRI may have a physiological role in the pineal gland. Further support for the view that FcϵRI could function in the pineal gland comes from the finding that several molecules associated with FcϵRI signaling in the mast cell are also expressed in the pineal gland, including Lyn kinase and Fyn kinase.
). This raises the possibility that the expression of this subunit is under independent control and that the functional tetrameric receptor formation is regulated by FcϵRIβ availability. For example, it might be regulated by a cytokine that is induced by immune stress. This hypothesis was tested either by treating pinealocytes with a mixture of cytokines or by initiating experimental pinealitis and uveitis (supplemental data). However, neither treatment induced expression of the β-chain. It is also possible that a functional trimeric (αγ2) intracellular FcϵRI complex forms as appears to be the case in eosinophils, monocytes, and antigen-presenting cells (
). These cells participate in IgE-dependent processes. Accordingly, a functional response could be generated if an IgE and allergen were translocated to the cytoplasm by the low affinity IgE receptor as occurs in the intestine (
). Based on these observations, it is not unreasonable to consider that allergens/IgE could enter the pinealocyte, triggering pineal responses through binding to soluble high affinity IgE receptor in the cytoplasm.
A striking feature of the expression of pineal Fcer1a expression is the large night/day difference that has not been reported previously in any cell. The large changes in transcript number appear to be driven by the neural system that controls melatonin production in the pineal gland (
). Whereas this is a pineal gland-specific control mechanism, cyclic AMP, the second messenger mediating control of Fcer1a, is ubiquitous. This is significant because cyclic AMP might impact Fcer1a expression in other cells.
The dynamic nature of the changes in Fcer1a expression not only reflects changes in cyclic AMP but is also dependent on undefined mechanisms that rapidly destroy the transcript after cyclic AMP-dependent ceases. This rhythmic pattern of transcript abundance appears to be translated immediately into a rhythm in the encoded protein. Moreover, the relatively rapid disappearance of the protein may reflect an inherent unstable nature of FcϵRIα protein in the cytosol and to rapid proteolysis, perhaps involving the proteasome as is the case with pineal arylalkylamine N-acetyltransferase (
); apparently, one or both events do not occur in the pinealocyte under the conditions examined in this study.
A daily rhythm in the production of FcϵRIα protein would provide the opportunity to generate new IgE·FcϵRI complexes every day, thereby providing an “up-to-date” reflection of circulating allergens and IgEs. The advantage of such a short memory immune response is not clear; however, it could mediate generation of a signal indicating that there has been a change in immune status because IgE·FcϵRI complexes on other cells are long lived.
In addition to being potentially linked to the resident phagocytes, a pineal IgE·FcϵRI system might initiate T-cell-mediated inflammatory response. It is known that T-cells are recruited into the chicken, mouse, and monkey pineal gland (
). Although the mechanism underlying this is unknown, it might reflect penetration through the vasculature and be influenced by the release of serotonin and cytokines from pinealocytes. T-cells in the pineal extracellular space would be exposed to high concentrations of pineal products, including serotonin, melatonin, and other serotonin derivatives. The effects of most of the serotonin derivatives on lymphocytes are not known. However, melatonin has been proposed to act as an immune modulator that enhances lymphocyte proliferation and cytokine production (
). The effects of exposure to high concentrations of pineal products are difficult to predict and deserve investigation.
Another potential set of signaling molecules in the pineal perivascular space are dopamine and NE, the transmitters of the sympathetic nervous system that are released at night. The latter is of some special interest because IgE production in B-lymphocytes is stimulated by NE and the mechanism involves a cAMP-p38MAPK pathway (
). Accordingly, B-lymphocytes invading the pineal gland might be induced to secrete IgE that would then be available at high local concentrations for interactions with pinealocytes. The effects of dopamine are not known.
A third set of molecules that could act on recruited lymphocytes may reside in pinealocyte dense core vesicles (
). The contents of these vesicles could be released via activation of the FcϵRI complex.
The discovery that pinealocytes express components of the high affinity IgE receptor regulated by circadian adrenergic/cAMP mechanism is of special value because it provides investigators with a molecular foundation to obtain a better understanding of the role of the pineal gland in the immune response, which could lead to novel drug targeting strategies in allergy treatments.
We thank Daniel T. Abebe for laboratory animal care and Dr. S. Zigler (NEI, National Institutes of Health) for the gift of anti-bovine S-antigen antiserum.