Expression of Muscarinic and Adrenergic Receptors in Normal Human ...

Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium Amalia Enrı´quez de Salamanca,1 Karyn F. Siemasko,2 Yolanda Diebold,1 Margarita Calonge,1 Jianping Gao,2 Mo ´ nica Jua ´ rez-Campo,1 and Michael E. Stern2 PURPOSE. To study the presence of muscarinic and ␣- and ␤-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line. METHODS. Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M1-, M2-, and M3-muscarinic and to ␣1A-, ␣1B-, ␣1D-, ␣2A-, ␣2B-, ␣2C-, ␤1-, ␤2-, and ␤3-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-␣ and/or interferon (IFN)-␥. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS. By immunofluorescence microscopy, all receptor subtypes, except the ␣2C-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M2- and M3-muscarinic receptors and ␣1A-, ␣1B-, ␣1D-, ␣2A-, ␣2B-, ␣2C-, ␤1-, and ␤3-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M1-muscarinic and ␤2-adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M2 and M3-muscarinic and ␣1A-, ␣2A-, ␣2B-, ␤1- and ␤2-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M2-muscarinic and ␣1B- and ␣2B-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN␥ and/or TNF␣. CONCLUSIONS. The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation. (Invest Ophthalmol Vis Sci. 2005;46:504 –513) DOI:10.1167/iovs.04-0665

From the 1Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain; and 2Allergan Inc., Irvine, California. Presented in part at the annual meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2002 and May 2003. Supported by Ministry of Education and Science, Spain (FEDERCICYT MAT2004-03484-CO2-01,02) and Red Tema´tica de Investigacio ´ n Cooperativa Sanitaria C03/13, Spain. Submitted for publication June 8, 2004; revised September 23, 2004; accepted November 5, 2004. Disclosure: A. Enrı´quez de Salamanca, None; K.F. Siemasko, Allergan, Inc. (E); Y. Diebold, None; M. Calonge, None; J. Gao, Allergan, Inc. (E); M. Jua´rez-Campo, None; M.E. Stern, Allergan, Inc. (E) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Amalia Enrı´quez de Salamanca, IOBA-University of Valladolid, Ramo ´ n y Cajal 7, Valladolid E-47005, Spain; [email protected].

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I

nteraction between the nervous, immune, and endocrine systems plays an important role in inflammatory diseases. In addition to systemic neuroendocrine regulation, there is local regulation at the site of inflammation through the release of proinflammatory neuropeptides and neurohormones from peripheral nerves.1 These neurotransmitters can modulate the activity of both inflammatory and epithelial cells through specific receptors for them. There is growing evidence that neural alterations occur in several ocular surface diseases—for example, in dry eye syndrome2 and allergic disorders3 (Motterle L, et al., IOVS 2003; 44:ARVO;E-Abstract 3743). Dry eye syndrome is mediated by an immune-based inflammation in the components of the lacrimal functional unit2,4,5 in which the innervational loop between the lacrimal glands and the ocular surface becomes altered.6 In a murine model of Sjo ¨ gren’s syndrome, neurotransmitter release from the lacrimal and salivary gland nerves is impaired.7 Also, unresponsiveness to cholinergic stimuli8 and presence of autoantibodies against the M3-muscarinic receptor subtype9,10 have been reported in patients with Sjo ¨ gren’s syndrome. Neurotransmitters and neuropeptides have many ocular functions. Receptors for these substances are present in the ocular tissues, but the functional consequences of their activation have not always been fully characterized.11–20 Sensory, parasympathetic, and sympathetic nerves are present in the conjunctival stroma and epithelium of several species,21–25 but only parasympathetic and sympathetic nerves have been detected adjacent to rat conjunctival goblet cells.25 Cholinergic, adrenergic, and other receptors have also been reported in goblet and nongoblet stratified squamous epithelial cells in rat, mouse, and human conjunctiva21,22,26 –28 (Diebold Y, et al., manuscript submitted). These nerves and neurotransmitter receptors are important elements in the pathways that integrate the lacrimal functional unit.29 Thus, we compared the expression of muscarinic and adrenergic receptors in cultured IOBANHC cells derived from normal human conjunctiva30 with expression in vivo. We then assessed changes in receptors when cultured cells were exposed to inflammatory cytokines.

MATERIALS

AND

METHODS

All reagents were from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. Table 1 summarizes the information regarding clone, source, and dilution of primary and secondary antibodies. Human recombinant interferon (IFN)-␥ and tumor necrosis factor (TNF)-␣ were purchased from R&D Systems (Minneapolis, MN). Fluorescence antifade mounting medium (Vectashield) was from Vector Laboratories (Burlingame, CA). Propidium iodide (PI) was obtained from Molecular Probes (Leiden, The Netherlands). Bicinchoninic acid (BCA) protein-determination assay was from Pierce (Rockford, IL). Molecular markers (Rainbow) were from Amersham Biosciences (Buckinghamshire, UK), and unstained precision protein standards and StrepTactin-HRP solution were from Bio-Rad (Hercules, CA). All the other SDS-PAGE and Western blot reactives were obtained from Bio-Rad. Investigative Ophthalmology & Visual Science, February 2005, Vol. 46, No. 2 Copyright © Association for Research in Vision and Ophthalmology

Neurotransmitter Receptors in Conjunctival Epithelium

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TABLE 1. Antibodies Used for Flow Cytometry, Immunofluorescence, or Western Blot Analysis

Receptor* M1 M2 M3 ␣1A ␣1B ␣1D ␣2A ␣2B ␣2C ␤1 ␤2 ␤3

Clone

FC

Primary Antibody

Secondary Antibody

Dilution (␮g/mL)†

Source

IMF

WB

FC (FITC)

IMF (FITC)

Dilution (␮g/mL) WB (HRP)

FC IMF WB

AS-3701S Serum 1:33 Serum 1:1000 Serum 1:1000 BD-PharMigen Jackson ImmunoRes. Sta. Cruz Biotech 5 AS-3721S Serum 1:33 Serum 1:1000 Serum 1:800 BD-PharMigen Jackson ImmunoRes. Sta. Cruz Biotech 5 AS-3741S Serum 1:33 Serum 1:1000 Serum 1:500 BD-Pharmigen Jackson ImmunoRes. Sta. Cruz Biotech 5 (C-19) sc-1477 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (C-18) sc-1476 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (C-19) sc-9352 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (C-19) sc-1478 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (C-19) sc-1479 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (C-20) sc-1480 10 4 2 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15 (A-20) sc-567 10 2 1 BD-Pharmigen Jackson ImmunoRes. Sta. Cruz Biotech 5 (H-73) sc-9042 10 2 1 BD-Pharmigen Jackson ImmunoRes. Sta. Cruz Biotech 5 (C-20) sc-1472 10 2 1 Zymed Jackson ImmunoRes. Sta. Cruz Biotech 15

15 15 15 15 15 15 15 15 15 15 15 15

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

FC, flow cytometry; IMF, immunofluorescence; WB, Western blot; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase. * All primary antibodies were polyclonal and were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), except serum antibodies to M1-, M2- and M3-muscarinic receptors, which were obtained from R&D Antibodies (Benicia, CA). † Antibody concentration of muscarinic serum antibodies not provided by the manufacturer; serum dilution used is indicated instead.

Human and Animal Tissues Biopsy specimens from normal human conjunctiva (n ⫽ 4) were obtained, with informed consent, from healthy donors who were undergoing cataract surgery. Rat eyeballs, kidneys, and heart and mouse conjunctiva were surgically excised after the animals were euthanatized and were used as positive control specimens. Experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Declaration of Helsinki and were approved by the IOBA Research Committee.

Human Conjunctival Epithelial IOBA-NHC Cell Line and Culture Conditions The IOBA-NHC cell line derived from normal human conjunctiva was used.30 The normal culture medium was DMEM/F12 (Invitrogen-Gibco, Inchinnan, UK) supplemented with 2 ng/mL human epidermal growth factor (EGF), 0.1 ␮g/mL cholera toxin, and 1 ␮g/mL bovine pancreatic insulin, plus 10% fetal bovine serum (FBS), 5 ␮g/mL hydrocortisone, and antibiotics (50 U/mL penicillin, 50 mg/mL streptomycin, and 2.5 ␮g/mL amphotericin B). Cholera toxin- and hydrocortisone-free medium supplemented with 2% FBS was used when specified. The medium was changed every 2 days. Cells in passages 62 to 75 were used.

Flow Cytometry Assays Analysis of the Adrenergic and Muscarinic Receptor Expression in the IOBA-NHC Cell Line. IOBA-NHC cells, 1 ⫻ 106 cells/mL, were washed and resuspended in flow cytometry buffer composed of 1% bovine serum albumin (BSA) and 0.02% sodium azide in ice-cold phosphate-buffered saline (PBS). The cells were stained with anti-adrenergic or anti-muscarinic antibodies (Table 1) at 4°C for 20 minutes in the dark, washed, and incubated with secondary antibody. For intracellular staining, cells were fixed with 2% formaldehyde for 15 minutes at 4°C and washed, and primary and secondary antibodies prepared in buffer (0.5% saponin, 1% BSA, and 0.1% sodium azide in PBS) were added at 4°C. Negative control experiments included the omission of the primary antibodies. Samples were analyzed by flow cytometry (FACSCalibur and Cell Quest software; BD Biosciences).

Effect of Inflammatory Cytokines on Adrenergic and Muscarinic Receptor Expression in the IOBA-NHC Cell Line. The effect of the inflammatory cytokines IFN-␥ and TNF-␣ on adrenergic and muscarinic receptor subtype expression level was an-

alyzed in IOBA-NHC cells by flow cytometry. Cells were plated at 1 ⫻ 105 cells/mL and incubated for 48 hours in the absence or the presence of IFN-␥ (500 U/mL), TNF-␣ (25, 50, and 100 ng/mL), and a combination of IFN-␥ (500 U/mL) and TNF-␣ (25 ng/mL). Untreated or stimulated cells were harvested after 48 hours, resuspended in buffer, and analyzed as described in the prior section.

Immunofluorescence Assays Normal human conjunctiva biopsy specimens and rodent conjunctiva, kidney, brain, and heart were fixed in 4% formaldehyde for at least 4 hours, rinsed in 5% sucrose dissolved in PBS, placed overnight at 4°C in 30% sucrose dissolved in PBS, embedded in optimal cutting temperature (OCT) compound, and frozen. Cryostat sections (7 ␮m) were collected in poly-L-lysine–treated slides and kept at ⫺80°C until used. IOBA-NHC cells in passages 62 to 65 were seeded onto glass coverslips. When confluence was reached, they were fixed in ice-cold methanol for 10 minutes, washed in PBS, and kept frozen until use. On the day of use, human conjunctiva, rodent tissue cryosections, and IOBA-NHC cell coverslips were hydrated for 30 minutes and blocked in PBS containing 1% BSA, 4% FBS, and 0.2% to 0.3% Triton X-100 for 1 hour. Antibodies to muscarinic and adrenergic receptor subtypes diluted in blocking buffer (Table 1) were incubated overnight at 4°C. Cells were washed three times. Secondary antibodies were incubated for 1 hour at room temperature. After they were washed, cells were counterstained with PI; 1:12,000), mounted, and viewed in a confocal laser scanning microscope (model LSM310; Carl Zeiss Meditec, Jena, Germany), equipped with a krypton-argon laser. FITC and PI were excited with 488- and 543-nm emission laser beams, respectively, and detected with a band-pass emission barrier filter. Digital images were taken. Negative control experiments included the omission of primary antibodies. All images were obtained using a 63⫻ objective, except Figure 2E, which was obtained with a 40⫻ objective.

Electrophoresis and Western Blot Analysis Cells and tissues were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA) plus protease inhibitors (100 ␮L/mL phenylmethylsulfonyl fluoride, 6 ␮L/mL aprotinin, and 100 nM sodium orthovanadate). After homogenization, samples were incubated for 30 minutes on ice and centrifuged at 14,000 rpm for 30 minutes at 4°C. Total cell protein in the supernatant was measured by the BCA method, which was compatible

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Enrı´quez de Salamanca et al.

with the buffer used for homogenization. Proteins in the homogenate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels according to the method of Laemmli.31 Proteins were transferred to nitrocellulose membranes according to Towbin32 and blocked in Tris-buffered saline (5% dried milk, 4% FBS, and 0.05% Tween-20 in Tris; TBS-T) overnight. Membranes were incubated with antibodies to muscarinic and adrenergic receptor subtypes for 1 hour at room temperature in blocking buffer. They were then washed three times with TBS-T buffer and incubated with secondary HRP-conjugated antibodies. StrepTactin-HRP solution (1:5000; Bio-Rad) was added for chemiluminescence protein standards detection. Membranes were incubated with HRP reagent according to the manufacturer’s protocol (ImmunoStar; Bio-Rad). Immunoreactive bands were visualized by the chemiluminescence method (ChemiDoc XRS; Bio-Rad), and images were analyzed on computer (Quantity One

IOVS, February 2005, Vol. 46, No. 2 software; Bio-Rad). Rat conjunctiva, kidney, aorta, and brain were used as control tissues.

RESULTS Flow Cytometry Analysis Flow cytometry of IOBA-NHC cells revealed constitutive expression on cell membranes of the M2- and M3-muscarinic receptors and the ␤1-, ␤3-, ␣1A-, ␣1B-, ␣1D-, ␣2A-, ␣2B-, and ␣2C-adrenergic receptors as well as expression in intracellular locations (Fig. 1A). M1-muscarinic and ␤2-adrenergic receptors were detected only intracellularly when normal culture medium was used (Fig. 1A). However, cell membrane expression of both these receptors was detected when cells were cultured

FIGURE 1. Flow cytometry analysis of muscarinic and adrenergic receptor subtypes in the IOBA-NHC cell line. Shaded traces: negative control; open traces: receptor expression. (A) Normal culture medium. (Left) Cells expressed detectable levels of all cell-membrane–bound muscarinic receptors except M1 and all adrenergic subtypes except ␤2. (Right) Cells expressed detectable intracellular levels of all muscarinic and adrenergic receptor subtypes. (B) When cultured for 72 hours in cholera toxin–and hydrocortisone-free medium, cells expressed detectable levels of cell membrane-bound M1-muscarinic (left) and ␤2-adrenergic (right) receptors. The primary antibody was omitted from negative control experiments. At least three independent experiments were performed. Each flow cytometry histogram corresponds to a representative experiment. FL1-H, relative fluorescence intensity; MFI, mean fluorescence intensity; CT, cholera toxin; H, hydrocortisone.



507

FIGURE 2. Confocal microscopy immunofluorescence detection of muscarinic and ␤-adrenergic receptor subtypes in the IOBA-NHC cell line. Cryosections of normal human conjunctiva biopsy specimens were used as control tissues. Both control (A–C, G–I) and IOBA-NHC (D–F, J–L) cells were double labeled with FITC-conjugated IgG secondary antibody to primary antibody for the M1-, M2-, and M3-muscarinic and ␤1-, ␤2-, and ␤3-adrenergic receptors (green) and with PI (red) to identify nuclei. Immunoreactivity to the M1-, M2-, and M3-muscarinic receptors was detected in all conjunctival epithelial cells in normal human conjunctiva (A–C). Immunoreactivity to the M2- and M3-muscarinic receptors was detected in IOBA-NHC cells, showing a cytosolic distribution (E, F). No detectable immunofluorescence was obtained for the M1-muscarinic receptor (D). Immunoreactivity to the ␤1-, ␤2-, and ␤3-adrenergic receptors was detected in human conjunctiva (G–I). Immunoreactivity to ␤1- and ␤2-adrenergic receptors was detected in IOBA-NHC cells, showing a cytosolic distribution (J, K). No detectable immunofluorescence was obtained for the ␤3-adrenergic receptor subtype in the IOBA-NHC cell line (L). Micrographs are representative of at least three different experiments. Magnification: (A–D, F–L) ⫻239; (E) ⫻204.

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FIGURE 3. Confocal microscopy immunofluorescence detection of ␣1- and ␣2-adrenergic receptor subtypes in the IOBA-NHC cell line. Cryosections of normal human conjunctiva biopsy specimens were used as control tissues. Both control (A–C, G–I) and IOBA-NHC (D–F, J–L) cells were double labeled with FITC-conjugated IgG secondary antibody to primary antibody for ␣1A-, ␣1B-, ␣1C-, ␣2A-, ␣2B- or ␣2C-adrenergic receptors (green) and with PI (red) to identify nuclei. Immunoreactivity to ␣1A-, ␣1B-, and ␣1D-adrenergic receptors was detected in human conjunctiva (A–C). Immunoreactivity to the ␣1A-adrenergic receptor was detected in IOBA-NHC cells, showing a specific intracellular location (D). Neither ␣1B- nor ␣1D-adrenergic receptor subtypes were detected in IOBA-NHC cells (E, F). Immunoreactivity to ␣2A- and ␣2B-adrenergic receptor subtypes was detected in human conjunctiva (G, H). The ␣2C-adrenergic receptor subtype was not detected in normal human conjunctiva (I). Immunoreactivity to ␣2A- and ␣2B-adrenergic receptors was detected in IOBA-NHC cells (J, K). The ␣2C-adrenergic receptor was not detected in the cell line (L). Micrographs are representative of at least three experiments. Magnification, ⫻239.



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A change in the distribution of immunofluorescence of the ␣1A- and ␣2C-adrenergic receptors was observed when cells were cultured for 48 hours in cholera toxin–and hydrocortisone-free medium supplemented with 2% FBS. A slight mobilization from perinuclear clusters toward the cell membrane occurred in the ␣1A-adrenergic receptor (Fig. 4A). The ␣2C-adrenergic receptor, which was not detected under the standard culture conditions, appeared in the cell membranes of IOBA-NHC cells in cholera toxin–and hydrocortisone-free medium supplemented with 2% FBS (Fig. 4B). No change in immunofluorescence distribution was observed for any of the other receptors studied (data not shown). FIGURE 4. Confocal microscopy immunofluorescence detection of ␣1Aand ␣2C-adrenergic receptors in the IOBA-NHC cell line cultured 72 hours in cholera toxin–and hydrocortisone-free medium. Cells were double labeled with FITC-conjugated IgG secondary antibody (green) to primary antibody for the ␣1A- or ␣2C-adrenergic receptors and with PI (red) to identify nuclei. (A) Immunoreactivity to the ␣1A-adrenergic receptor subtype was detected in IOBA-NHC cells, showing a slight mobilization from previous specific intracellular location shown in Figure 3D toward the cytosol. (B) The ␣2C-adrenergic receptor subtype was predominantly located in the cytosol. These micrographs are representative of at least three different experiments. Magnification, ⫻218.

Electrophoresis and Western Blot Analysis Western blot analysis of IOBA-NHC cell homogenates subjected to SDS-PAGE electrophoresis (Table 2) had immunoreactive bands for the M1-, M2-, and M3-muscarinic receptors (Fig. 5). A major 58-kDa band was detected for each. Weaker immunoreactive bands were also detected at 68 kDa. Immunopositive bands were also detected for the ␤1-, ␤2-, and ␤3-adrenergic receptors (Fig. 5). A major band at ⬃60 kDa was present for the ␤1-, ␤2- and ␤3-adrenergic receptor subtypes in IOBA-NHC cells, along with a weaker 70-kDa band.

in cholera toxin- and hydrocortisone-free medium supplemented with 2% FBS for 72 hours before analysis (Fig. 1B).

TABLE 2. Molecular Weight of Immunoreactive Bands

Immunofluorescence Assays

Receptor

Reported MW*

M1

55

120 68

M2

55

100 68

M3

55

109 68

␣1A

60

115 66

␣1B

67

120 68

␣1D

140 70

120 68

␣2A

64

115 67

␣2B

64

115 70 32

␣2C

55

␤1

50–55

115 67 120 70

␤2

68

100 70

␤3

65

120 70

The cellular localization of muscarinic and adrenergic receptor subtypes in normal human conjunctiva biopsy specimens and in IOBA-NHC cells was studied by confocal microscopy. Immunoreactivity to the M1-, M2- and M3-muscarinic receptors was detected in all epithelial cells in conjunctiva cryosections (Figs. 2A–C). Confocal microscopy revealed the presence of the M2and M3-muscarinic receptor subtypes in the cytosol of the IOBA-NHC cell line (Figs. 2E, 2F). No immunoreactivity was found for the M1-muscarinic receptor (Fig. 2D). The ␤1-, ␤2-, and ␤3-adrenergic receptor subtypes were detected in human conjunctiva biopsy specimens, as previously shown.22,33 All epithelial cells were positive (Figs. 2G–I). The ␤1- and ␤2-adrenergic receptor subtypes were also present in IOBA-NHC cells (Figs. 2J, 2K) and had a predominantly cytosolic localization. No immunoreactivity was found for the ␤3-adrenergic receptor subtype (Fig. 2L). Most of the ␣-adrenergic receptor subtypes were detected in normal human conjunctiva biopsy specimens (Figs. 3A–C, 3G, 3H), as previously described22 (Diebold Y, et al., 2003 Singapore Eye Research Institute/Association for Research in Vision and Ophthalmology meeting; Diebold Y, et al., manuscript submitted). Immunoreactivity for ␣1A-adrenergic receptors was detected in both goblet and nongoblet cells (Fig. 3A), and, in the apical cell layer, it was present in clusters. The ␣1B-adrenergic receptor was present in the basal epithelial cell layer (Fig. 3B). Immunoreactivity for the ␣1D-adrenergic receptor was detected more intensely in the goblet cells and was weaker in nongoblet cells (Fig. 3C). The ␣2A-adrenergic receptor was detected in all epithelial cells. In contrast, the ␣2Badrenergic receptor was clearly located in basal epithelial cells but only weakly in the rest of the epithelium (Figs. 3G, 3H). No immunoreactivity was detected for the ␣2C-adrenergic receptor in normal human conjunctiva (Fig. 3I). In the IOBA-NHC cell line, ␣1A-, ␣2A- and ␣2B-adrenergic receptors were detected. The ␣1A-adrenergic receptor subtype was clustered about the nucleus, whereas ␣2A- and ␣2B-adrenergic receptors were in the cell membranes and cytosol (Figs. 3D, 3J, 3K). No immunoreactivity was found for the ␣1B-, ␣1D-, and ␣2C-adrenergic receptors (Figs. 3E, 3F, 3L).

Cⴙ

IOBA-NHC Cell Line 120 68 58 100 68 58 114 68 58 115 67 55 120 67 60 120 67 59 115 67 58 115 67 57 32 115 58 114 67 58 100 80 67 60 116 67 60

Immunoreactivity of muscarinic and adrenergic receptor subtypes in the IOBA-NHC cell line was detected by Western blot analysis. * Data provided by the manufacturers, C⫹ (control tissue) for M1, M2, M3: rat conjunctiva; for ␣1A, ␣1B, ␣1D, ␣2A, ␣2B, ␤1 and ␤2: rat kidney; for ␤3: aorta; for ␣2C: rat brain.; MW, molecular weight.

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IOVS, February 2005, Vol. 46, No. 2 The IOBA-NHC cell line contained immunoreactive bands for ␣1A-, ␣1B-, and ␣1D-adrenergic receptor subtypes (Fig. 5). Major bands were present at 55, 60 and 59 kDa for the ␣1A-, ␣1B-, and ␣1D-adrenergic receptor subtypes, respectively. Weaker bands were detected at 67 kDa in the three cases. The ␣2A-, ␣2B-, and ␣2C-adrenergic receptor subtypes were also present in the IOBA-NHC cell line (Fig. 5). Major bands were located at 58, 57 and 58 kDa, respectively. Weaker bands were again detected at 67 kDa for the ␣2A- and ␣2B-adrenergic receptors. For the ␣2B-adrenergic receptor, a strong band was detected at 32 kDa, which was also weakly present in rat kidney homogenates. For all receptors analyzed, immunoreactive bands at higher molecular weights (⬃110 to 120 kDa) were present. These may correspond to receptor dimers. Table 3 summarizes the results obtained with the three different techniques used: flow cytometry, immunofluorescence, and Western blot analysis.

Effect of Inflammatory Cytokines on Muscarinic and Adrenergic Receptor Expression in IOBA-NHC Cell Line

FIGURE 5. Western blot analysis of muscarinic and adrenergic receptors in IOBA-NHC cells. Cell homogenates were subjected to SDS-PAGE, and muscarinic and adrenergic receptors were detected by Western blot analyses with specific polyclonal antibodies. A major band of 58 kDa was present for each muscarinic receptor subtype. Major bands for the ␤1-, ␤2-, ␤3-, ␣1A-, ␣1B-, ␣1D-, ␣2A-, ␣2B-, and ␣2C-adrenergic receptors were obtained at 60, 60, 60, 55, 60, 58, 59, 57, and 58 kDa, respectively. Rat conjunctiva, kidney, and brain homogenates were used as positive controls (data not shown). A representative of at least three independent experiments is shown for each receptor. IOBA-NHC homogenates are shown in duplicate for each receptor. MM, molecular weight markers; Cells, IOBA-NHC cell homogenates.

To study the expression of muscarinic and adrenergic receptors under inflammatory conditions, we exposed the IOBANHC cells to a proinflammatory medium resembling a diseased ocular surface. IFN-␥ upregulated M2-muscarinic receptor expression (Fig. 6A) but had no effect on the other receptors examined (data not shown). TNF-␣ did not change the expression levels of either the muscarinic or adrenergic receptors (data not shown) at any of the doses tested. When cells were incubated with the combination of cytokines, ␣1B- and ␣2Badrenergic receptor cell surface expression was enhanced (Figs. 6B, 6C).

DISCUSSION This study demonstrates by flow cytometry, immunofluorescence microscopy, and Western blot analysis that IOBA-NHC cells express the muscarinic and adrenergic neurotransmitter

TABLE 3. Summary of Flow Cytometry, Immunofluorescence and Western Blot Results in the IOBA-NHC Cell Line Human Conjunctiva

IOBA-NHC Cell Line Flow cytometry

Receptor M1 M2 M3

␣1A ␣1B ␣1D ␣2A ␣2B ␣2C ␤1 ␤2 ␤3

Reported IMF Goblet and occasional nongoblet cells22 Goblet and occasional nongoblet cells22 Goblet and occasional nongoblet cells, basal epithelial cell layer22 All epithelial cells, in clusters in the apical layer* Basal epithelial cell layer* Goblet and stratified squamous epithelial cells22 All epithelial cells† Strong in basal epithelial cells† ⫺† Basal epithelial cell layer22 ⫹33 All epithelial cells22

Our Results IMF

Cell Membrane

Intracellular

IMF

WB

All epithelial cells

⫺/⫹‡

⫹

⫺

⫹


⫹

⫹

Cytosolic

⫹


⫹

⫹

Cytosolic

⫹

All epithelial cells, in clusters in the apical layer Basal epithelial cell layer All epithelial cells

⫹

⫹

⫹

⫹ ⫹

⫹ ⫹

Specific location near the nuclei in clusters ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫺/⫹‡ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

All epithelial cells Strong in basal cells ⫺ Basal epithelial cells All epithelial cells All epithelial cells

Cytosolic Cytosolic

⫺ Weak positive in cytosol Weak positive in cytosol ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

IMF, immunofluorescence; WB, Western blot analysis; ⫹, detected; ⫺ not detected; * Diebold et al, 2003 SERI-ARVO (Singapore Eye Research Institute/Association for Research in Vision and Ophthalmology meeting); † Diebold et al., manuscript submitted; ‡, detected when cells were cultured in a cholera toxin-/hydrocortisone-free medium.



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FIGURE 6. Effect of proinflammatory cytokines on the neurotransmitter receptor expression in the IOBANHC cell line. Cells were incubated for 48 hours in the absence or presence of IFN-␥ (500 U/mL) or both IFN-␥ (500 U/mL) and TNF-␣ (25 ng/ mL). Shaded traces: negative control; black traces: receptor expression in the resting state; gray traces: receptor expression after IFN-␥ stimulation. (A) IFN-␥ treatment provoked an upregulation of the M2muscarinic receptor expression in IOBA-NHC cells. The primary antibody was omitted from negative controls. (B, C) INF-␥⫹TNF-␣ treatment provoked an upregulation of the ␣1B- and ␣2B-adrenergic receptor expression in IOBA-NHC cells. These results were reproduced for each of the cytokines tested in three independent experiments. FL1-H, relative fluorescence intensity; MFI, mean fluorescence intensity.

receptors that are also expressed in normal human conjunctiva epithelium (Table 3). The expression of some of these receptors can be upregulated when cells are treated with proinflammatory cytokines. The M1-muscarinic and ␣1B-, ␣1D-, ␣2C- or ␤3-adrenergic receptor subtypes were detected by flow cytometry but not by microscopy, perhaps because of the different sensitivities of the two techniques and/or the tissue fixation necessary for microscopy. We found M1-, M2- and M3-muscarinic receptors in all epithelial cells of the conjunctiva cryosections. Previously, we described these receptors to be localized in occasional epithelial cells throughout the human conjunctival epithelium, including the goblet cells, with M2- and M3-muscarinic receptors especially prominent in the basal epithelial cell layer.22 The differences between findings in that study and in the present one may be attributed to the use of cadaveric conjunctival tissues in the previous work, different localization of biopsy tissue, and different batches of antibodies. Western blot analysis of IOBA-NHC homogenates subjected to SDS-PAGE revealed the presence of immunoreactive bands for all the receptors studied (Fig. 5). The immunoreactive bands obtained with rat control tissues had a slightly higher molecular weight than previously reported (Table 2). Western blot analysis of the ␤2-, ␤3-, ␣1A-, ␣1B-, ␣1D-, ␣2A-, and ␣2Badrenergic receptors in the IOBA-NHC cell line showed the major immunoreactive bands at slightly lower molecular weight than that reported for these receptors. In addition, a weaker immunoreactive band was detected in all cases in the same position as that of the positive control (Fig. 5, Table 2). The differences observed in the molecular weight of the bands in control tissues and the IOBA-NHC cell line compared with the reported molecular weight of the receptors may be due to differences in the protein glycosylation/palmitoylation and/or phosphorylation pattern between different species and/or different tissues. In addition, many receptors, such as the ␣1A-34,35 and the ␤3-36 adrenergic receptors, occur in different isoforms. In the case of IOBA-NHC cells, another possibility is that nonmature or truncated forms of the receptors are expressed. It is possible that some of the bands detected by Western blot analysis correspond to the pool of intracellularly expressed receptors detected by flow cytometry and confocal microscopy that could correspond to partially degraded receptors. High-molecular-weight immunoreactive bands were observed in all cases, probably corresponding to dimers, as the formation of homodimers occurs for many receptors, including the ␤2-adrenergic and muscarinic receptors.37 The ␤2adrenergic receptor dimers are stable, even under the denaturing conditions applied for analysis by SDS gel electrophoresis.38 We detected neurotransmitter receptors in plasma membrane and at intracellular locations both by flow cytometry (Fig. 1) and by confocal microscopy (Figs. 2, 3). Although

the functional significance of muscarinic and adrenergic receptors in the plasma membrane is well established, they are also present in subcellular locations in several types of cells.20,39 – 49 Modifications in the quantity of receptors at the plasma membrane help modulate responses to stimulation. Thus, intracellular locations may include both newly synthesized and recycled stores of receptors in amounts that vary with the rate of synthesis and the shedding and desensitization processes. Receptor desensitization controlled by PKA-dependent phosphorylation results in a generalized downregulation of all the receptors that regulate cAMP production, regardless of the state of receptor occupation.38 The presence of cholera toxin in the culture medium of IOBA-NHC cells could induce cAMP accumulation, resulting in the phosphorylation of receptor molecules that have the PKA substrate consensus motif. Furthermore, in polarized cells, the mobilizing of newly synthesized proteins to the apical surface is under the control of a cholera toxin-sensitive protein.50 These facts could account also for the intracellular localization observed for the receptors. When IOBANHC cells were cultured for 72 hours in cholera toxin– and hydrocortisone-free medium supplemented with 2% FBS, cell membrane receptor expression of the M1-muscarinic and ␤2-adrenergic receptors increased (Fig. 1B). In addition, some mobilization toward the cell cytosol of the ␣1A- and ␣2C-adrenergic receptors was observed by confocal microscopy (Fig. 4). Other mechanisms could also contribute to the intracellular presence of muscarinic and adrenergic receptors. For instance, the cell cholinergic environment,39,40 the presence of a pair of extracellular cysteine residues in the M3-muscarinic receptor,51 N-glycosylation of receptors,52 and temperature53 can also alter the distribution of cellular receptors. All these factors could work independently or in conjunction with cAMP-dependent redistribution of muscarinic and adrenergic receptors. Based on ligand binding and Western blot analyses, Hurt et al.48 proposed that the intracellular pool of ␣2C-adrenergic receptors in normal rat kidney cells have some functional significance. Also, a role in cellular growth regulation has been proposed20 for nuclei muscarinic binding sites in rabbit corneal cells. Intracellular receptors may have functions not related to signal transduction, as well. For instance, they can act as ligand buffers, as some of them bind ligand in a specific way.41 The autonomic nervous system is implicated in neural regulation of conjunctival cell functioning, both in electrolyte and water transport and in goblet cell protein and mucin secretion.21,27–29 More recently, the role of the autonomic nervous system and the endocrine system in inflammatory disorders has become apparent.1 There is growing evidence that neural alterations have a role in some inflammatory immune diseases, such as dry eye syndrome and allergic diseases2–10 (Motterle L,

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et al., IOVS 2003;44:ARVO E-Abstract 3743). In dry eye syndrome, excessive nervous stimulation can provoke the activation of T cells and subsequently the release of inflammatory cytokines into the lacrimal glands, tear film, and conjunctiva.54 During ocular allergic reactions several neurotransmitters are released from nerves at the ocular surface into the tear film.3 Epithelial cells from conjunctiva can also participate directly in inflammatory processes by secreting several cytokines after stimulation.55,56 In this study we showed that under a simulated inflammatory condition with the proinflammatory cytokine INF-␥, IOBANHC cells responded by upregulating M2-muscarinic receptors (Fig. 6A). In the presence of INF-␥⫹TNF-␣, the ␣1B- and ␣2Badrenergic receptors were upregulated (Figs. 6B, 6C). Both adrenergic and muscarinic receptors are present in lymphocytes and macrophages and can modulate immune functioning when activated.57– 62 Both IL-1␤ and TNF-␣ treatment provoke a downregulation of the ␣1a-adrenergic receptor mRNA expression in monocytes (THP-1) and in human umbilical endothelial cells (HUVECs).63 In the THP-1 cell line, the cytokines cause ␣1a-adrenergic receptor mRNA upregulation, whereas ␣1b-adrenergic receptor mRNA expression is not modified. In HUVECs, this treatment induces a downregulation of ␣1b-adrenergic mRNA expression.63 This cytokine-dependent regulation of ␣1-adrenergic subtype expression could play a role in the pathogenesis of inflammatory diseases, such as juvenile chronic arthritis.63 Investigators from this laboratory proposed that the increased TNF-␣ and/or IL-␤ production observed during chronic inflammation may be responsible for induction of ␣1A-adrenoceptor expression in cells of the immune system in these patients.63,64 Cytokine upregulation of M2-muscarinic and ␣1B and ␣2B-adrenergic receptor expression in our IOBA-NHC cells by INF-␥ and INF-␥⫹TNF-␣, respectively, suggests that cytokine-dependent regulation of neurotransmitter receptors in conjunctival epithelium plays a role in the pathogenesis of inflammatory ocular surface diseases. In summary, we have shown that the IOBA-NHC cell line expresses all the muscarinic and adrenergic receptor subtypes that are present in the human conjunctival epithelium in vivo and that proinflammatory cytokines upregulate the expression of some of them. More studies about the functionality of these receptors in IOBA-NHC cells, as well as in the normal human conjunctiva, are undoubtedly needed. Our findings support the use of the IOBA-NHC cell line as a tool for further study of the neural regulation of conjunctival epithelium and its possible role in neurogenic inflammation.

Acknowledgments The authors thank Jose M. Herreras, MD, for providing the human conjunctival biopsy specimens, Sagrario Callejo, PhD, for confocal microscopy assistance, Victoria Sae´z, and Carmen Garcıá-Va´zquez for excellent technical assistance, and Miguel Jarrıń, MSc, for animal handling.

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