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Middle ear epithelial mucin production in response to interleukin-6 exposure in vitro
www.elsevier.com/locate/jnlabr/ycyto Cytokine 26 (2004) 30e36
Middle ear epithelial mucin production in response to interleukin-6 exposure in vitro* Joseph E. Kerschnera,b,), Tanya K. Meyerb, Chris Yangb, Amy Burrowsb a
Division of Pediatric Otolaryngology, Medical College of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States b Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States Received 11 October 2003; accepted 7 December 2003
Abstract Objectives: To investigate the role of the inflammatory cytokine interleukin-6 (IL-6) in the regulation of mucin secretion by middle ear epithelia. Materials and methods: Primary chinchilla middle ear epithelial cultures were established and exposed to IL-6 in a dose- and timedependent manner. Mucin secretion was characterized by exclusion chromatography and liquid scintillation. Results: Epithelial cultures exposed to increasing doses of IL-6 demonstrated greater amounts of mucin secretion (p ¼ 0:018). Additionally, cultures exposed to IL-6 at 50 ng/ml showed significant increased secretion of mucin over control in time-dependent experiments at 6-, 15- and 24-h time points (p ¼ 0:003). Conclusions: IL-6 upregulates mucin secretion from cultured middle ear epithelial cells in a dose- and time-dependent manner. Elucidating the effect of specific cytokines on the regulation of mucin secretion is vital to understanding the pathophysiology of otitis media and the development of novel therapeutic strategies. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cell culture; Interleukin-6; Mucin; Otitis media
1. Introduction Otitis media is the most common diagnosis in pediatric patients who visit physicians for illness in the United States [1], causing an estimated 5 million annual episodes at a national cost of $3 to $6 billion [2]. Approximately 5e10% of acute otitis media progresses to chronic otitis media with effusion (COME), which is a leading cause of hearing loss in children. The most accepted treatment of COME is tympanostomy tube
*
Portions of this manuscript were presented at the AAO-HNSF/ ARO Research Forum, 23 September 2003, Orlando, FL. ) Corresponding author. Department of Otolaryngology and Communication Sciences, Children’s Hospital of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States. Tel.: C1-414-266-6476; fax: C1-414-266-6989. E-mail address: [email protected] (J.E. Kerschner). 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2003.12.006
insertion, which is now the most frequent pediatric surgical procedure requiring a general anesthetic [1]. Otitis media has significant potential for morbidity and presents increasing therapeutic challenges imposed by antimicrobial resistance. Despite this, much is still unknown about the cellular and molecular immunologic and inflammatory events in this disease process. Many recent investigations have demonstrated that the cytokine mediated inflammatory response is important in the pathophysiology of acute and chronic otitis media [3e7], and interleukin-6 (IL-6) has been identified as an inflammatory cytokine that plays a central role in this process [8,9]. The present study uses an in vitro model to investigate the effects of IL-6 on middle ear epithelium using mucin secretion, cellular proliferation, and cell viability as endpoints. This study was designed to examine the hypothesis that IL-6 causes increased mucin secretion by middle ear epithelial cells.
J.E. Kerschner et al. / Cytokine 26 (2004) 30e36
2. Results 2.1. Cell culture morphology Freshly harvested chinchilla middle ear epithelial cell suspensions contain clumps and sheets of cells, with approximately 5% of cells being ciliated. By day 3 after plating on collagen, cells had settled from the suspension, adhered to the plate, and begun to divide. They were 70e80% confluent with only scattered ciliated cells notable by light microscopy. Morphologically, the cells grow as a thin polygonal monolayer. Cells were experimented 3 days after reaching confluency, which was approximately day 8e10 after harvest. Immuno-staining for tissue specific markers was performed to ensure that the cultures consisted predominantly of epithelial cells. Cytokeratins are found in epithelial tissues, and staining with a-pancytokeratin showed greater than 95% positivity in our cell cultures (Fig. 1). Vimentins are intermediate filaments found in mesenchymal cells such as fibroblasts. Anti-vimentin staining showed less than 3% positivity in our cell cultures. Desmins are present in cells of smooth and striated muscle origin; there was no desmin staining in our cultures.
31
culture supernatant quantitated by fractionation with a Sepharose CL-4B column and liquid scintillation of appropriate fractions. IL-6 stimulated mucin secretion in a concentration-dependent manner, with an increase in mucin secretion observed with increased concentrations of IL-6 in the culture media (Fig. 2). All doses of IL-6 elicited significantly increased mucin secretion as compared to controls (p ¼ 0:018). No cytotoxic effects were observed at any concentration of IL-6. 2.3. Time dependency Paired sets of CMEE cultures were incubated with and without IL-6 at 50 ng/ml in growth media for 6 h, 15 h, and 24 h. Secretion of tritium labeled mucin in the cell culture supernatant was quantitated by fractionation and liquid scintillation. Control cultures (0 ng/ml IL-6) demonstrated a baseline secretion of mucin, which accumulated gradually over time. Cells exposed to IL-6 at 50 ng/ml increased secretion of mucin over controls in time-dependent experiments at 6-, 15- and 24-h time points (Fig. 3). Comparison data demonstrated statistically significant increases at 15- and 24-h time points and overall treated versus control cell populations (p ¼ 0:003). Experiments were performed twice in duplicate. No cytotoxic effects were observed at any time point.
2.2. Concentration dependency CMEEC were incubated with escalating concentrations of IL-6 (0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ ml) and secretion of tritium labeled mucin in the cell
2.4. Cell viability A constant rate of cell viability at O95% was seen in all cell cultures throughout this set of experiments as determined by Trypan blue dye exclusion. Each well within experiments contained the same number of cells within 3% as determined by trypsonization and counting with a hemocytometer. There were no toxic compounds used in any experiment.
3. Discussion
Fig. 1. Morphology and immunofluorescent staining of cultured chinchilla middle ear epithelia. Staining of cells with a-pancytokeratin using FITC conjugated secondary antibody (63!).
Despite the prevalence of otitis media, its potential for morbidity, and the enormous health care expenditures resulting from its treatment, much is still unknown about the immunologic and inflammatory events in this disease process at the cellular and molecular level. Medical management relies primarily on the use of antibiotic therapy whereas surgical management ranges from myringotomy with ventilation tube placement and adenoidectomy to mastoidectomy. However, increasing bacterial resistance to antibiotics coupled with the significant cost and potential morbidity associated with the surgical management of otitis media mandate exploration of novel approaches. Given these factors, a more thorough understanding of this disease process to
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Dose-Response IL6 Mouse 18000
5
CPM/5X10 cells
16000 14000 12000 10000 8000 6000 4000 2000 0 0 ng
50 ng
100 ng
200 ng
Dose IL6 - ng/ml Fig. 2. Graphic representation of the concentration-dependent effect of interleukin-6 on mucin secretion in chinchilla middle ear epithelial cells. Escalating concentrations of IL-6 causes increased MGP secretion in a dose-dependent manner. (Each condition was performed in triplicate and the experiment repeated twice giving 6 data points for each condition.)
facilitate development of innovative therapeutic strategies is needed. Variation in the quantity and character of middle ear secretions, specifically mucin is known to be important in the pathophysiologic mechanisms of otitis media [10,11]. Mucins are responsible for the high viscosity of middle ear effusions preventing normal mucociliary clearance [12,13], which predisposes to the development of chronic otitis media and subsequent hearing loss. Investigations in respiratory-type epithelium from areas
other than the middle ear, but with similar cellular makeup, have demonstrated that inflammatory cytokines are responsible for epithelial pathology by initiating changes in mucin production. Specific examples include bronchial and tracheal epithelium exposed to interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) with resulting increases in mucin production [14e16]. We have also recently reported that IL-1b stimulates mucin secretion from cultured middle ear epithelium in a dose- and time-dependent fashion [17].
Time-Response IL-6 4000
3000
IL-6
5
CPMx10e cells
Controls
2000
1000
0 6hr
15hr
24hr
Time Fig. 3. Graphic representation of the time-dependent effect of interleukin-6 on mucin secretion in chinchilla middle ear epithelial cells. IL-6 causes an increase in MGP secretion in a time-dependent manner. (Each condition was performed in duplicate and the experiment repeated twice giving 4 data points for each condition.)
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e11
Proteoglycans
e10
5
CPM / 1x10 cells
e9 e8
Mucins
e7 e6 e5 e4 e3 e2 0
5
10
15
20
25
30
35
Fraction Fig. 4. An example of the normalized radioactivity by fraction number of the culture supernatants after chondroitinase ABC digestion and separation through a Sepharose CL-4B column. Mucins are excluded from the column and are eluted first. The first peak represents the mucin containing fractions and the second peak represents digested proteoglycans. Mucin is quantitated by calculating the area under the first curve using Sigma Plot.
This current study is the first to demonstrate a dose- and time-dependent increase in mucin secretion from middle ear epithelium after exposure to the inflammatory cytokine IL-6. The results of this investigation provide further evidence of the importance of the inflammatory cytokine IL-6 in the pathophysiology of otitis media and direct evidence of its ability to regulate mucin secretion from middle ear epithelial cells. The cytokines IL-1b and TNF-a as initiators of the cytokine inflammatory cascade have generalized important effects including: enhancing T cell proliferation, macrophage activation, granulocyte influx to areas of inflammation, fever induction, and modulation of the coagulation cascade [18e20], which would naturally be expected to be important in otitis media. In fact, numerous clinical reports have outlined the importance of IL-1b and TNF-a in otitis media. These studies have identified a higher level of IL-1b in effusions of younger children and in purulent otitis [4,21,22], and have shown an increasing level of TNFa in effusions of older children, in chronic otitis, and in effusions of children who require multiple tympanostomy tubes [6,22]. Downstream from IL-1b and TNF-a, IL-6 has also been identified as having an important role in otitis media in both clinical reports and basic science models. These studies have suggested that IL-6 is an important regulator in both the early and late stages of OM with effusion and may be particularly important in leading to chronic forms of otitis media [8,9]. In addition, IL-6 has been demonstrated to be a sensitive marker of
inflammation in acute OM and has been shown to be more often present in bacterial OM than in nonbacterial OM [23]. It has also been suggested that with successful treatment and bacterial eradication in OM IL-6 levels decline more rapidly along with resolving inflammation [23]. Demonstration in this study that IL6 has the potential to stimulate middle ear epithelium to increase mucin secretion provides further insight into molecular and cellular processes that might be important as acute otitis media transitions to chronic disease. This study, along with previous work demonstrating that IL-1b and TNF-a also stimulate mucin secretion from middle ear epithelium [17,24] suggests that persistent inflammatory cytokines could be integral in the process of developing persisting middle ear fluid following OM. Previous thinking regarding the development of chronic OM has concentrated on eustachian tube dysfunction as the primary determinant in the development of this chronic disease and these findings may indicate that molecular factors as well as anatomic factors are important as chronic OM develops. The recent development of cytokine inhibitors and receptor antagonist has made it possible to inhibit the undesired effects of inflammatory cytokines such as IL1b, TNF-a and IL-6 in infectious and inflammatory disease processes. IL-1ra has been used in animal models to reduce shock and mortality in sepsis, reduce the inflammatory response in arthritis, reduce mortality and inflammation in pancreatitis and reduce the inflammatory response in inflammatory bowel disease [18,25e28].
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Our laboratory has also recently demonstrated that IL-1ra can lead to more rapid and complete resolution of otitis media when combined with antibiotics in an in vivo chinchilla model [7]. An improved understanding of the nature of regulation of mucin secretion under controlled conditions will provide insight into the normal biology and abnormal regulation in pathologic conditions such as chronic otitis media. Evidence that IL-6 increases mucin secretion by middle ear epithelial cells may provide potential future avenues for altering mucin secretion through cytokine modulation. Additional experiments examining the molecular pathways associated with cytokine modulation of mucin gene expression and protein secretion from middle ear epithelium and cytokine induced changes in epithelial cellular morphology are needed and are ongoing in our laboratory. The prevalence, cost, and morbidity of otitis media coupled with the declining efficacy of current treatment methods mandate a better understanding of the pathophysiology of otitis media and subsequent generation of novel treatment strategies. This study further demonstrates the importance of the inflammatory cytokine IL-6 in the pathophysiology of otitis media through its regulation of mucin secretion from chinchilla middle ear epithelial cell cultures. It also suggests the need for a more complete understanding of the mechanisms in which inflammatory cytokines contribute to acute and chronic otitis media.
4. Materials and methods
4.2. Primary cell culture Transbullar injection of 0.09% protease Type XIV (Sigma) in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 with 0.5% fetal bovine serum (FBS) containing 1% ITS (InsulineTransferrineSelenium), 50 ng/ml hydrocortisone (Sigma), antibiotic/antimycotic (ABX/AMX) solution (1000 U/ml penicillin G sulfate, 100 mg/ml streptomycin sulfate, and 250 ng/ml amphotericin B) was used to fill the middle ear cavity and bulla (components added to growth media obtained from Gibco unless otherwise indicated). The temporal bone was wrapped in Parafilm and placed at 4 (C for 16e20 h. Subsequently, the suspension within the middle ear containing the middle ear epithelial cells was aspirated. The middle ear was washed 3 times with washing solution consisting of DMEM/F-12 with 10% FBS, and ABX/AMX solution as above. Suspensions were pooled and centrifuged at 500g for 10 min. The pellet was gently disrupted and washed with fresh washing solution. Centrifugation and washing was repeated 3 times to completely remove the protease. The pellet was then resuspended in 1 ml of DMEM/F-12 with 0.5% FBS, 1% ITS, 50 ng/ml hydrocortisone, and ABX/AMX solution. The cells were plated in 24-well, collagen I coated plates (Becton Dickenson, Bedford, MA) at approximately 1!105 cells per cm2. The cells were grown in a humidified atmosphere at 37 (C containing 95% aire5% carbon dioxide. Growth medium was changed every second day. Cells were grown to confluency and then prepared for experimentation. Cell viability was assessed with Trypan blue staining. All cells in these experiments were from primary cultures, and were not passaged.
4.1. Animal donor Cells used in all cultures were harvested from mature (6e10 month old), 400e600 g mixed breed chinchillas (Moulton, Rochester, MN). Prior to tissue harvest, chinchillas were anesthetized with 50 mg/kg ketamine hydrochloride (Phoenix, St. Joseph, MO) and then euthanized with an intracardiac injection of 2 mg pentobarbital (Abbott Laboratories, North Chicago, IL) as approved by the Panel on Euthanasia of the American Veterinary Medical Association. The temporal bone, including tympanic membrane and middle ear cavity, was removed bilaterally. The tympanic membrane and middle ear cavity were examined closely to identify any evidence of infection. Animals were treated in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act; the animal use protocol was approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
4.3. Immunofluorescence Cells were fixed in fresh 4% paraformaldehyde in PBS at pH 7.4 for 20 min. Cells were permeabilized with 0.1% triton in PBS for 3 min on ice and then immediately incubated in 10% NGS/2% BSA in PBS for 30 min (normal goat serumdJackson Labs, BSAd Sigma). The cells were then incubated with the primary antibody at a dilution of 1:50 in 2% BSA/PBS for 45 min. The antibodies used were anti-pancytokeratin (Sigma, C2931), anti-vimentin (Santa Cruz, SC6260), and anti-desmin (Dako, D33). After 3 five-minute washes with 1! PBS, cells were incubated with FITC conjugated goat anti-mouse secondary antibody for 45 min at room temperature. Slides were washed with 1! PBS, coverslips mounted with Gelmount (Foster City, CA), and cells visualized using a fluorescent microscope (Zeiss Axioskop).
J.E. Kerschner et al. / Cytokine 26 (2004) 30e36
4.4. Metabolic labeling of glycoproteins Metabolic labeling with tritiated glucosamine (NEN Life Science, Boston) was performed on cell cultures to allow for mucin secretion analysis. Confluent CMEE cultures were incubated with 5 mCi 3H-glucosamine per ml of full growth media at 37 (C in 95% aire5% carbon dioxide for 24 h prior to assay. This medium was then aspirated and replaced with new media containing the appropriate experimental culture conditions. 4.5. Dose-dependency Following metabolic labeling, cell cultures were incubated with new full growth media containing scaled concentrations of IL-6 (R&D Systems, Minneapolis, MN) at 0, 50, 100 and 200 ng/ml. Labeled cells incubated only with full growth media and without cytokine exposure served as controls. After 16 h, 900 ml of media was aspirated and reserved for mucin quantification. Aspirates were stored at 80 (C for less than 2 weeks. Each condition was performed in triplicate, and the entire experiment repeated twice. Cell viability was assessed by Trypan blue exclusion. 4.6. Time-dependency Following metabolic labeling paired cell cultures were incubated with and without IL-6 at 50 ng/ml for 6 h, 15 h, and 24 h. After the appropriate time, 900 ml of media was aspirated and reserved for mucin quantification. Aspirates were stored at 80 (C for less than 2 weeks. Each condition was performed in duplicate, and the entire experiment repeated twice. Cell viability was assessed by Trypan blue exclusion.
35
PBS containing 0.02% sodium azide, 0.9% sodium chloride, and 5 mM dithiothreitol (Sigma) at a constant flow rate of 0.5 ml/min collecting 2 ml fractions. Void volume fractions were mixed with 8 ml of Ecoscint A (National Diagnostics), and the radioactivity of fractions counted with a liquid scintillation system (United Technologies Packard, Tri-Carb 4530). Radioactivity at peak fractions was defined as the amount of mucin in the supernatant (Fig. 4). The radioactivity of each sample was divided by its own viable cell number, and the individual value of radioactivity, on an average, calculated. The calculated radioactivity was standardized by expressing the values as radioactivity per 5!105 viable cells. 4.8. Statistical analysis ANOVA models were used to assess dose- and timedependence data and Tukey adjustments were used for multiple comparisons. Interaction testing was done where appropriate. Least square means and confidence intervals were used to present the average response for each variable. Least square means estimate the marginal means over a balanced population, based on unbalanced data. The analysis was carried out using SAS statistical software (The SAS Institute, Cary, NC) and in consultation with Biostatistics Department at the Medical College of Wisconsin. Acknowledgements We acknowledge the kind assistance of Dan Eastwood, M.S., in the Biostatistics Department of the Medical College of Wisconsin. This study was supported by NIH grant NIDCD DC00192 (P.I. JEK).
4.7. Quantification of mucin Mucin secretion from the epithelial cells was analyzed and quantified using procedures previously described in our laboratory and a number of other laboratories [17,29e31]. These investigations have demonstrated that secretions from respiratory epithelium and middle ear epithelium contain high molecular weight glycoconjugates. The glycoconjugates resistant to digestion by chondroitinase ABC and excluded after Sepharose CL4B column chromatography have been identified as mucin. Stored aspirates were thawed and treated with 0.4 U/ ml of testicular chondroitinase ABC (Sigma) at 37 (C for 5 h to digest proteoglycans. After incubation, the digestion mixture was neutralized with 0.1 M acetic acid to a pH of 7.4 and applied to a Sepharose CL-4B column (0:7!50 cm) (Pharmacia) equilibrated with phosphate-buffered saline (PBS) containing 0.02% (wt/ vol) sodium azide (Sigma). Columns were eluted with
References [1] Bluestone CD, Klein JO. Otitis media, atelectasis, and eustacean tube dysfunction. In: Bluestone CD, Stool SE, Kenna MA, editors. Pediatric otolaryngology. Philadelphia: WB Saunders; 1996. p. 388e582. [2] Rosenfeld RM, Casselbrant ML, Hannley MT. Implications of the AHRQ evidence report on acute otitis media. Otolaryngol Head Neck Surg 2001;125(5):440e8. [3] Chonmaitree T, Patel JA, Sim T, Garofalo R, Uchida T, Sim T, et al. Role of leukotriene B4 and interleukin-8 in acute bacterial and viral otitis media. Ann Otol Rhinol Laryngol 1996;105(12):968e74. [4] Juhn SK, Garvis WJ, Lees CJ, Le CT, Kim CS. Determining otitis media severity from middle ear fluid analysis. Ann Otol Rhinol Laryngol Suppl 1994;163:43e5. [5] Maxwell KS, Fitzgerald JE, Burleson JA, Leonard G, Carpenter R, Kreutzer DL. Interleukin-8 expression in otitis media. Laryngoscope 1994;104(8 Pt 1):989e95. [6] Yellon RF, Doyle WJ, Whiteside TL, Diven WF, March AR, Fireman P. Cytokines, immunoglobulins, and bacterial pathogens in middle ear effusions. Arch Otolaryngol Head Neck Surg 1995;121(8):865e9.
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[7] Kerschner JE, Beste DJ, Lynch JB, Fox MC, Kehl MS. Interleukin-1 receptor antagonist as an adjunct in the treatment of Haemophilus influenzae otitis media in the chinchilla. Laryngoscope 2000;110(9):1457e61. [8] Yellon RF, Leonard G, Marucha P, Sidman J, Carpenter R, Burleson J, et al. Demonstration of interleukin-6 in middle ear effusion. Arch Otolaryngol Head Neck Surg 1992;118:745e8. [9] Sato K, Nonomura N, Kawana M, Nakano Y. Course of IL-1b IL-6, IL-8 and TNF-a in the middle ear fluid of the guinea pig otitis media model induced by nonviable Haemophilus influenza. Ann Otol Rhinol Laryngol 1999;108:559e63. [10] Brown DT, Litt M, Potsic WP. A study of mucus glycoproteins in secretory otitis media. Arch Otolaryngol 1985;111(10): 688e95. [11] Carrie S, Hutton DA, Birchall JP, Green GG, Pearson JP. Otitis media with effusion: components which contribute to the viscous properties. Acta Otolaryngol 1992;112(3):504e11. [12] FitzGerald JE, Green GG, Stafford FW, Birchall JP, Pearson JP. Characterization of human middle ear mucus glycoprotein in chronic secretory otitis media (CSOM). Clin Chim Acta 1987;169(2e3):281e97. [13] Hutton DA, Fogg FJ, Murty G, Birchall JP, Pearson JP. Preliminary characterization of mucin from effusions of cleft palate patients. Otolaryngol Head Neck Surg 1993; 109(6):1000e6. [14] Fischer BM, Krunkosky TM, Wright DT, Dolan-O’Keefe M, Adler KB. Tumor necrosis factor-alpha (TNF-alpha) stimulates mucin secretion and gene expression in airway epithelium in vitro. Chest 1995;107(3 Suppl):133Se5S. [15] Larivee P, Levine SJ, Martinez A, Wu T, Logun C, Shelhamer JH. Platelet-activating factor induces airway mucin release via activation of protein kinase C: evidence for translocation of protein kinase C to membranes. Am J Respir Cell Mol Biol 1994;11(2):199e205. [16] Levine SJ, Larivee P, Logun C, Angus CW, Ognibene FP, Shelhamer JH. Tumor necrosis factor-alpha induces mucin hypersecretion and MUC-2 gene expression by human airway epithelial cells. Am J Respir Cell Mol Biol 1995;12(2):196e204. [17] Kerschner JE, Meyer TK, Wohlfeil ER. Middle ear epithelial mucin production in response to interleukin-1b exposure in vitro. Otolaryngol Head Neck Surg 2003;129(1):128e35.
[18] Fong Y, Moldawer LL, Shires GT, Lowry SF. The biologic characteristics of cytokines and their implication in surgical injury. Surg Gynecol Obstet 1990;170(4):363e78. [19] Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989;44:153e205. [20] Platanias LC, Vogelzang NJ. Interleukin-1: biology, pathophysiology, and clinical prospects. Am J Med 1990;89(5):621e9. [21] Himi T, Suzuki T, Kodama H, Takezawa H, Kataura A. Immunologic characteristics of cytokines in otitis media with effusion. Ann Otol Rhinol Laryngol Suppl 1992;157:21e5. [22] Yellon RF, Leonard G, Marucha PT, Craven R, Carpenter RJ, Lehman WB, et al. Characterization of cytokines present in middle ear effusions. Laryngoscope 1991;101(2):165e9. [23] Barzilai A, Dekel B, Dagan R, Leibovitz E. Middle ear effusion IL-6 concentration in bacterial and non-bacterial acute otitis media. Acta Paediatrica 2002;89:1068e71. [24] Lin J, Kim Y, Juhn SK. Increase of mucous glycoprotein secretion by tumor necrosis factor alpha via a protein kinase C-dependent mechanism in cultured chinchilla middle ear epithelial cells. Ann Otol Rhinol Laryngol 1998;107(3):213e9. [25] Dinarello CA, Thompson RC. Blocking IL-1: interleukin 1 receptor antagonist in vivo and in vitro. Immunol Today 1991; 12(11):404e10. [26] Norman J, Franz M, Messina J, Riker A, Fabri PJ, Rosemurgy AS, et al. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery 1995;117(6):648e55. [27] Arend WP. Interleukin 1 receptor antagonist. A new member of the interleukin 1 family. J Clin Invest 1991;88(5):1445e51. [28] Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991;77(8):1627e52. [29] Wu R, Nolan E, Turner C. Expression of tracheal differentiated functions in serum-free hormone-supplemented medium. J Cell Physiol 1985;125(2):167e81. [30] Adler KB, Cheng PW, Kim KC. Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: cellular composition and biochemical analysis of released glycoconjugates. Am J Respir Cell Mol Biol 1990;2(2):145e54. [31] Lin J, Kim Y, Lees C, Juhn SK. Effect of platelet-activating factor on secretion of mucous glycoprotein from chinchilla middle ear epithelial cells in vitro. Eur Arch Otorhinolaryngol 1995; 252(2):92e6.
Middle ear epithelial mucin production in response to interleukin-6 exposure in vitro* Joseph E. Kerschnera,b,), Tanya K. Meyerb, Chris Yangb, Amy Burrowsb a
Division of Pediatric Otolaryngology, Medical College of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States b Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States Received 11 October 2003; accepted 7 December 2003
Abstract Objectives: To investigate the role of the inflammatory cytokine interleukin-6 (IL-6) in the regulation of mucin secretion by middle ear epithelia. Materials and methods: Primary chinchilla middle ear epithelial cultures were established and exposed to IL-6 in a dose- and timedependent manner. Mucin secretion was characterized by exclusion chromatography and liquid scintillation. Results: Epithelial cultures exposed to increasing doses of IL-6 demonstrated greater amounts of mucin secretion (p ¼ 0:018). Additionally, cultures exposed to IL-6 at 50 ng/ml showed significant increased secretion of mucin over control in time-dependent experiments at 6-, 15- and 24-h time points (p ¼ 0:003). Conclusions: IL-6 upregulates mucin secretion from cultured middle ear epithelial cells in a dose- and time-dependent manner. Elucidating the effect of specific cytokines on the regulation of mucin secretion is vital to understanding the pathophysiology of otitis media and the development of novel therapeutic strategies. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cell culture; Interleukin-6; Mucin; Otitis media
1. Introduction Otitis media is the most common diagnosis in pediatric patients who visit physicians for illness in the United States [1], causing an estimated 5 million annual episodes at a national cost of $3 to $6 billion [2]. Approximately 5e10% of acute otitis media progresses to chronic otitis media with effusion (COME), which is a leading cause of hearing loss in children. The most accepted treatment of COME is tympanostomy tube
*
Portions of this manuscript were presented at the AAO-HNSF/ ARO Research Forum, 23 September 2003, Orlando, FL. ) Corresponding author. Department of Otolaryngology and Communication Sciences, Children’s Hospital of Wisconsin, 9000 West Wisconsin Avenue, Milwaukee, WI 53226, United States. Tel.: C1-414-266-6476; fax: C1-414-266-6989. E-mail address: [email protected] (J.E. Kerschner). 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2003.12.006
insertion, which is now the most frequent pediatric surgical procedure requiring a general anesthetic [1]. Otitis media has significant potential for morbidity and presents increasing therapeutic challenges imposed by antimicrobial resistance. Despite this, much is still unknown about the cellular and molecular immunologic and inflammatory events in this disease process. Many recent investigations have demonstrated that the cytokine mediated inflammatory response is important in the pathophysiology of acute and chronic otitis media [3e7], and interleukin-6 (IL-6) has been identified as an inflammatory cytokine that plays a central role in this process [8,9]. The present study uses an in vitro model to investigate the effects of IL-6 on middle ear epithelium using mucin secretion, cellular proliferation, and cell viability as endpoints. This study was designed to examine the hypothesis that IL-6 causes increased mucin secretion by middle ear epithelial cells.
J.E. Kerschner et al. / Cytokine 26 (2004) 30e36
2. Results 2.1. Cell culture morphology Freshly harvested chinchilla middle ear epithelial cell suspensions contain clumps and sheets of cells, with approximately 5% of cells being ciliated. By day 3 after plating on collagen, cells had settled from the suspension, adhered to the plate, and begun to divide. They were 70e80% confluent with only scattered ciliated cells notable by light microscopy. Morphologically, the cells grow as a thin polygonal monolayer. Cells were experimented 3 days after reaching confluency, which was approximately day 8e10 after harvest. Immuno-staining for tissue specific markers was performed to ensure that the cultures consisted predominantly of epithelial cells. Cytokeratins are found in epithelial tissues, and staining with a-pancytokeratin showed greater than 95% positivity in our cell cultures (Fig. 1). Vimentins are intermediate filaments found in mesenchymal cells such as fibroblasts. Anti-vimentin staining showed less than 3% positivity in our cell cultures. Desmins are present in cells of smooth and striated muscle origin; there was no desmin staining in our cultures.
31
culture supernatant quantitated by fractionation with a Sepharose CL-4B column and liquid scintillation of appropriate fractions. IL-6 stimulated mucin secretion in a concentration-dependent manner, with an increase in mucin secretion observed with increased concentrations of IL-6 in the culture media (Fig. 2). All doses of IL-6 elicited significantly increased mucin secretion as compared to controls (p ¼ 0:018). No cytotoxic effects were observed at any concentration of IL-6. 2.3. Time dependency Paired sets of CMEE cultures were incubated with and without IL-6 at 50 ng/ml in growth media for 6 h, 15 h, and 24 h. Secretion of tritium labeled mucin in the cell culture supernatant was quantitated by fractionation and liquid scintillation. Control cultures (0 ng/ml IL-6) demonstrated a baseline secretion of mucin, which accumulated gradually over time. Cells exposed to IL-6 at 50 ng/ml increased secretion of mucin over controls in time-dependent experiments at 6-, 15- and 24-h time points (Fig. 3). Comparison data demonstrated statistically significant increases at 15- and 24-h time points and overall treated versus control cell populations (p ¼ 0:003). Experiments were performed twice in duplicate. No cytotoxic effects were observed at any time point.
2.2. Concentration dependency CMEEC were incubated with escalating concentrations of IL-6 (0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ ml) and secretion of tritium labeled mucin in the cell
2.4. Cell viability A constant rate of cell viability at O95% was seen in all cell cultures throughout this set of experiments as determined by Trypan blue dye exclusion. Each well within experiments contained the same number of cells within 3% as determined by trypsonization and counting with a hemocytometer. There were no toxic compounds used in any experiment.
3. Discussion
Fig. 1. Morphology and immunofluorescent staining of cultured chinchilla middle ear epithelia. Staining of cells with a-pancytokeratin using FITC conjugated secondary antibody (63!).
Despite the prevalence of otitis media, its potential for morbidity, and the enormous health care expenditures resulting from its treatment, much is still unknown about the immunologic and inflammatory events in this disease process at the cellular and molecular level. Medical management relies primarily on the use of antibiotic therapy whereas surgical management ranges from myringotomy with ventilation tube placement and adenoidectomy to mastoidectomy. However, increasing bacterial resistance to antibiotics coupled with the significant cost and potential morbidity associated with the surgical management of otitis media mandate exploration of novel approaches. Given these factors, a more thorough understanding of this disease process to
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Dose-Response IL6 Mouse 18000
5
CPM/5X10 cells
16000 14000 12000 10000 8000 6000 4000 2000 0 0 ng
50 ng
100 ng
200 ng
Dose IL6 - ng/ml Fig. 2. Graphic representation of the concentration-dependent effect of interleukin-6 on mucin secretion in chinchilla middle ear epithelial cells. Escalating concentrations of IL-6 causes increased MGP secretion in a dose-dependent manner. (Each condition was performed in triplicate and the experiment repeated twice giving 6 data points for each condition.)
facilitate development of innovative therapeutic strategies is needed. Variation in the quantity and character of middle ear secretions, specifically mucin is known to be important in the pathophysiologic mechanisms of otitis media [10,11]. Mucins are responsible for the high viscosity of middle ear effusions preventing normal mucociliary clearance [12,13], which predisposes to the development of chronic otitis media and subsequent hearing loss. Investigations in respiratory-type epithelium from areas
other than the middle ear, but with similar cellular makeup, have demonstrated that inflammatory cytokines are responsible for epithelial pathology by initiating changes in mucin production. Specific examples include bronchial and tracheal epithelium exposed to interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) with resulting increases in mucin production [14e16]. We have also recently reported that IL-1b stimulates mucin secretion from cultured middle ear epithelium in a dose- and time-dependent fashion [17].
Time-Response IL-6 4000
3000
IL-6
5
CPMx10e cells
Controls
2000
1000
0 6hr
15hr
24hr
Time Fig. 3. Graphic representation of the time-dependent effect of interleukin-6 on mucin secretion in chinchilla middle ear epithelial cells. IL-6 causes an increase in MGP secretion in a time-dependent manner. (Each condition was performed in duplicate and the experiment repeated twice giving 4 data points for each condition.)
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e11
Proteoglycans
e10
5
CPM / 1x10 cells
e9 e8
Mucins
e7 e6 e5 e4 e3 e2 0
5
10
15
20
25
30
35
Fraction Fig. 4. An example of the normalized radioactivity by fraction number of the culture supernatants after chondroitinase ABC digestion and separation through a Sepharose CL-4B column. Mucins are excluded from the column and are eluted first. The first peak represents the mucin containing fractions and the second peak represents digested proteoglycans. Mucin is quantitated by calculating the area under the first curve using Sigma Plot.
This current study is the first to demonstrate a dose- and time-dependent increase in mucin secretion from middle ear epithelium after exposure to the inflammatory cytokine IL-6. The results of this investigation provide further evidence of the importance of the inflammatory cytokine IL-6 in the pathophysiology of otitis media and direct evidence of its ability to regulate mucin secretion from middle ear epithelial cells. The cytokines IL-1b and TNF-a as initiators of the cytokine inflammatory cascade have generalized important effects including: enhancing T cell proliferation, macrophage activation, granulocyte influx to areas of inflammation, fever induction, and modulation of the coagulation cascade [18e20], which would naturally be expected to be important in otitis media. In fact, numerous clinical reports have outlined the importance of IL-1b and TNF-a in otitis media. These studies have identified a higher level of IL-1b in effusions of younger children and in purulent otitis [4,21,22], and have shown an increasing level of TNFa in effusions of older children, in chronic otitis, and in effusions of children who require multiple tympanostomy tubes [6,22]. Downstream from IL-1b and TNF-a, IL-6 has also been identified as having an important role in otitis media in both clinical reports and basic science models. These studies have suggested that IL-6 is an important regulator in both the early and late stages of OM with effusion and may be particularly important in leading to chronic forms of otitis media [8,9]. In addition, IL-6 has been demonstrated to be a sensitive marker of
inflammation in acute OM and has been shown to be more often present in bacterial OM than in nonbacterial OM [23]. It has also been suggested that with successful treatment and bacterial eradication in OM IL-6 levels decline more rapidly along with resolving inflammation [23]. Demonstration in this study that IL6 has the potential to stimulate middle ear epithelium to increase mucin secretion provides further insight into molecular and cellular processes that might be important as acute otitis media transitions to chronic disease. This study, along with previous work demonstrating that IL-1b and TNF-a also stimulate mucin secretion from middle ear epithelium [17,24] suggests that persistent inflammatory cytokines could be integral in the process of developing persisting middle ear fluid following OM. Previous thinking regarding the development of chronic OM has concentrated on eustachian tube dysfunction as the primary determinant in the development of this chronic disease and these findings may indicate that molecular factors as well as anatomic factors are important as chronic OM develops. The recent development of cytokine inhibitors and receptor antagonist has made it possible to inhibit the undesired effects of inflammatory cytokines such as IL1b, TNF-a and IL-6 in infectious and inflammatory disease processes. IL-1ra has been used in animal models to reduce shock and mortality in sepsis, reduce the inflammatory response in arthritis, reduce mortality and inflammation in pancreatitis and reduce the inflammatory response in inflammatory bowel disease [18,25e28].
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Our laboratory has also recently demonstrated that IL-1ra can lead to more rapid and complete resolution of otitis media when combined with antibiotics in an in vivo chinchilla model [7]. An improved understanding of the nature of regulation of mucin secretion under controlled conditions will provide insight into the normal biology and abnormal regulation in pathologic conditions such as chronic otitis media. Evidence that IL-6 increases mucin secretion by middle ear epithelial cells may provide potential future avenues for altering mucin secretion through cytokine modulation. Additional experiments examining the molecular pathways associated with cytokine modulation of mucin gene expression and protein secretion from middle ear epithelium and cytokine induced changes in epithelial cellular morphology are needed and are ongoing in our laboratory. The prevalence, cost, and morbidity of otitis media coupled with the declining efficacy of current treatment methods mandate a better understanding of the pathophysiology of otitis media and subsequent generation of novel treatment strategies. This study further demonstrates the importance of the inflammatory cytokine IL-6 in the pathophysiology of otitis media through its regulation of mucin secretion from chinchilla middle ear epithelial cell cultures. It also suggests the need for a more complete understanding of the mechanisms in which inflammatory cytokines contribute to acute and chronic otitis media.
4. Materials and methods
4.2. Primary cell culture Transbullar injection of 0.09% protease Type XIV (Sigma) in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 with 0.5% fetal bovine serum (FBS) containing 1% ITS (InsulineTransferrineSelenium), 50 ng/ml hydrocortisone (Sigma), antibiotic/antimycotic (ABX/AMX) solution (1000 U/ml penicillin G sulfate, 100 mg/ml streptomycin sulfate, and 250 ng/ml amphotericin B) was used to fill the middle ear cavity and bulla (components added to growth media obtained from Gibco unless otherwise indicated). The temporal bone was wrapped in Parafilm and placed at 4 (C for 16e20 h. Subsequently, the suspension within the middle ear containing the middle ear epithelial cells was aspirated. The middle ear was washed 3 times with washing solution consisting of DMEM/F-12 with 10% FBS, and ABX/AMX solution as above. Suspensions were pooled and centrifuged at 500g for 10 min. The pellet was gently disrupted and washed with fresh washing solution. Centrifugation and washing was repeated 3 times to completely remove the protease. The pellet was then resuspended in 1 ml of DMEM/F-12 with 0.5% FBS, 1% ITS, 50 ng/ml hydrocortisone, and ABX/AMX solution. The cells were plated in 24-well, collagen I coated plates (Becton Dickenson, Bedford, MA) at approximately 1!105 cells per cm2. The cells were grown in a humidified atmosphere at 37 (C containing 95% aire5% carbon dioxide. Growth medium was changed every second day. Cells were grown to confluency and then prepared for experimentation. Cell viability was assessed with Trypan blue staining. All cells in these experiments were from primary cultures, and were not passaged.
4.1. Animal donor Cells used in all cultures were harvested from mature (6e10 month old), 400e600 g mixed breed chinchillas (Moulton, Rochester, MN). Prior to tissue harvest, chinchillas were anesthetized with 50 mg/kg ketamine hydrochloride (Phoenix, St. Joseph, MO) and then euthanized with an intracardiac injection of 2 mg pentobarbital (Abbott Laboratories, North Chicago, IL) as approved by the Panel on Euthanasia of the American Veterinary Medical Association. The temporal bone, including tympanic membrane and middle ear cavity, was removed bilaterally. The tympanic membrane and middle ear cavity were examined closely to identify any evidence of infection. Animals were treated in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act; the animal use protocol was approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
4.3. Immunofluorescence Cells were fixed in fresh 4% paraformaldehyde in PBS at pH 7.4 for 20 min. Cells were permeabilized with 0.1% triton in PBS for 3 min on ice and then immediately incubated in 10% NGS/2% BSA in PBS for 30 min (normal goat serumdJackson Labs, BSAd Sigma). The cells were then incubated with the primary antibody at a dilution of 1:50 in 2% BSA/PBS for 45 min. The antibodies used were anti-pancytokeratin (Sigma, C2931), anti-vimentin (Santa Cruz, SC6260), and anti-desmin (Dako, D33). After 3 five-minute washes with 1! PBS, cells were incubated with FITC conjugated goat anti-mouse secondary antibody for 45 min at room temperature. Slides were washed with 1! PBS, coverslips mounted with Gelmount (Foster City, CA), and cells visualized using a fluorescent microscope (Zeiss Axioskop).
J.E. Kerschner et al. / Cytokine 26 (2004) 30e36
4.4. Metabolic labeling of glycoproteins Metabolic labeling with tritiated glucosamine (NEN Life Science, Boston) was performed on cell cultures to allow for mucin secretion analysis. Confluent CMEE cultures were incubated with 5 mCi 3H-glucosamine per ml of full growth media at 37 (C in 95% aire5% carbon dioxide for 24 h prior to assay. This medium was then aspirated and replaced with new media containing the appropriate experimental culture conditions. 4.5. Dose-dependency Following metabolic labeling, cell cultures were incubated with new full growth media containing scaled concentrations of IL-6 (R&D Systems, Minneapolis, MN) at 0, 50, 100 and 200 ng/ml. Labeled cells incubated only with full growth media and without cytokine exposure served as controls. After 16 h, 900 ml of media was aspirated and reserved for mucin quantification. Aspirates were stored at 80 (C for less than 2 weeks. Each condition was performed in triplicate, and the entire experiment repeated twice. Cell viability was assessed by Trypan blue exclusion. 4.6. Time-dependency Following metabolic labeling paired cell cultures were incubated with and without IL-6 at 50 ng/ml for 6 h, 15 h, and 24 h. After the appropriate time, 900 ml of media was aspirated and reserved for mucin quantification. Aspirates were stored at 80 (C for less than 2 weeks. Each condition was performed in duplicate, and the entire experiment repeated twice. Cell viability was assessed by Trypan blue exclusion.
35
PBS containing 0.02% sodium azide, 0.9% sodium chloride, and 5 mM dithiothreitol (Sigma) at a constant flow rate of 0.5 ml/min collecting 2 ml fractions. Void volume fractions were mixed with 8 ml of Ecoscint A (National Diagnostics), and the radioactivity of fractions counted with a liquid scintillation system (United Technologies Packard, Tri-Carb 4530). Radioactivity at peak fractions was defined as the amount of mucin in the supernatant (Fig. 4). The radioactivity of each sample was divided by its own viable cell number, and the individual value of radioactivity, on an average, calculated. The calculated radioactivity was standardized by expressing the values as radioactivity per 5!105 viable cells. 4.8. Statistical analysis ANOVA models were used to assess dose- and timedependence data and Tukey adjustments were used for multiple comparisons. Interaction testing was done where appropriate. Least square means and confidence intervals were used to present the average response for each variable. Least square means estimate the marginal means over a balanced population, based on unbalanced data. The analysis was carried out using SAS statistical software (The SAS Institute, Cary, NC) and in consultation with Biostatistics Department at the Medical College of Wisconsin. Acknowledgements We acknowledge the kind assistance of Dan Eastwood, M.S., in the Biostatistics Department of the Medical College of Wisconsin. This study was supported by NIH grant NIDCD DC00192 (P.I. JEK).
4.7. Quantification of mucin Mucin secretion from the epithelial cells was analyzed and quantified using procedures previously described in our laboratory and a number of other laboratories [17,29e31]. These investigations have demonstrated that secretions from respiratory epithelium and middle ear epithelium contain high molecular weight glycoconjugates. The glycoconjugates resistant to digestion by chondroitinase ABC and excluded after Sepharose CL4B column chromatography have been identified as mucin. Stored aspirates were thawed and treated with 0.4 U/ ml of testicular chondroitinase ABC (Sigma) at 37 (C for 5 h to digest proteoglycans. After incubation, the digestion mixture was neutralized with 0.1 M acetic acid to a pH of 7.4 and applied to a Sepharose CL-4B column (0:7!50 cm) (Pharmacia) equilibrated with phosphate-buffered saline (PBS) containing 0.02% (wt/ vol) sodium azide (Sigma). Columns were eluted with
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