Salivary gland epithelial cells - Wiley Online Library

30 downloads 0 Views 155KB Size Report
Salivary Gland Epithelial Cells. A New Source of the Immunoregulatory Hormone Adiponectin. Stergios Katsiougiannis,1 Efstathia K. Kapsogeorgou,1 Menelaos ...
ARTHRITIS & RHEUMATISM Vol. 54, No. 7, July 2006, pp 2295–2299 DOI 10.1002/art.21944 © 2006, American College of Rheumatology

Salivary Gland Epithelial Cells A New Source of the Immunoregulatory Hormone Adiponectin Stergios Katsiougiannis,1 Efstathia K. Kapsogeorgou,1 Menelaos N. Manoussakis,1 and Fotini N. Skopouli2 pression by SGECs from patients with SS compared with controls. Conclusion. Our findings provide novel evidence that adiponectin is produced by SGECs. The high constitutive expression of adiponectin by SGECs from patients with primary SS is likely attributable to aberrant activation of these cells. Although the significance of adiponectin expression remains unknown, it is possible that adiponectin functions in an autocrine manner, as suggested by concurrent expression of the relevant receptors.

Objective. Adiponectin is an adipocytokine that displays insulin-sensitizing and immunoregulatory properties. Adipocyte development in association with fibrosis is frequently detected in primary Sjo ¨gren’s syndrome lesions, connoting a healing process. The aim of this study was to examine the expression of adiponectin in minor salivary gland biopsy specimens obtained from patients with primary SS and controls. Methods. The expression of adiponectin in minor salivary gland biopsy specimens and in long-term– cultured non-neoplastic salivary gland epithelial cell (SGEC) lines obtained from patients with primary SS and control subjects was examined, using immunohistochemistry and immunoblotting, respectively. The expression of adiponectin, adiponectin receptor 1 (AdipoR1), and AdipoR2 messenger RNA (mRNA) by SGECs was investigated by reverse transcription– polymerase chain reaction. Results. Immunohistochemical analysis for adiponectin revealed positive staining of adipocytes from primary SS lesions as well as ductal epithelial cells from both patients with primary SS and controls. All of the SGEC lines tested were shown to express adiponectin, AdipoR1, and AdipoR2 mRNA, whereas adiponectin protein expression was detected by immunoblotting in SGECs from patients with primary SS but not in those from controls. The analysis of concentrated culture supernatants also revealed increased adiponectin ex-

Adiponectin is an adipocytokine that was independently discovered by several groups of investigators, using different approaches (1). Although adiponectin was considered to be synthesized and secreted exclusively by adipocytes, recent data suggest that it is also produced by cells other than adipocytes (2–4). Adiponectin protein belongs to the soluble defense collagen superfamily and has structural homology with type VIII collagen, type X collagen, and complement factor C1q (1). This hormone has been shown to possess several biologic properties, ranging from an insulin-sensitizing function to an immunomodulatory function. Indeed, results of several studies suggest that the protein acts as an inflammation-modulating factor (1–6). In addition, a regulatory role of adiponectin in the apoptotic death of many types of cells has been suggested (7). The function of adiponectin is mediated by its receptors, adiponectin receptor 1 (AdipoR1) and AdipoR2, which have different patterns of expression. AdipoR1 is expressed predominantly in muscle, whereas AdipoR2 is expressed in the liver (1). High levels of expression of both receptors have also been reported in human and rat pancreatic beta cells, and, in the presence

1 Stergios Katsiougiannis, BSc, Efstathia K. Kapsogeorgou, PhD, Menelaos N. Manoussakis, MD: University of Athens, Athens, 2 Greece; Fotini N. Skopouli, MD: Harokopio University, Athens, Greece. Address correspondence and reprint requests to Fotini N. Skopouli, MD, Department of Dietetics and Nutritional Science, Harokopio University, El. Venizelou 70, 17671, Kallithea, Athens, Greece. E-mail: [email protected]. Submitted for publication January 23, 2006; accepted in revised form March 24, 2006.

2295

2296

KATSIOUGIANNIS ET AL

of these receptors, bone-forming cells may play a role in modulating the effects of circulating adiponectin (1,2). Sjo ¨gren’s syndrome (SS) is an autoimmune disorder that is characterized by chronic dysfunction and destruction of the exocrine glands, mainly the salivary and lacrimal glands, leading to persistent dryness of the mucosa (8). Glandular epithelial cells, which are the main target of the inflammatory reactions in SS, are thought to play a central role in the development of autoimmune reactions (8). Destruction of the salivary glands is commonly accompanied by the development of adipose tissue and fibrotic tissue (9). The presence of adipocytes only in the setting of inflammation suggests their possible involvement in the defense or repair repertoire. Because there are no data concerning the role of adipocytes in SS lesions, we sought to study the expression of adipocyte-derived factors for which there is clear evidence of an immunoregulatory function, such as adiponectin. Therefore, the expression of adiponectin was investigated in minor salivary gland biopsy specimens obtained from patients with SS and controls. Subsequently, based on the observation that ductal epithelial cells displayed positive adiponectin staining in situ, we extended our study in salivary gland epithelial cell (SGEC) lines obtained from patients with SS and controls. In this study, we demonstrated for the first time that adiponectin is produced by long-term–cultured non-neoplastic human SGECs. SGECs derived from patients with SS secrete higher amounts of adiponectin compared with control SGECs. Additionally, we provide evidence that SGECs express adiponectin receptor messenger RNA (mRNA), possibly suggesting an autocrine function of adiponectin in SGECs. PATIENTS AND METHODS Tissue samples. Minor salivary gland specimens were obtained by biopsy (after informed consent was received) from 30 individuals undergoing diagnostic evaluation for sicca symptoms indicative of SS at the outpatient rheumatology clinic. In all 15 patients (14 women and 1 man) in this study, primary SS was diagnosed on the basis of the American–European Consensus criteria for primary SS (10). The disease control group comprised 15 patients (13 women and 2 men) who had subjective xerostomia and who did not fulfill the abovementioned criteria. None of the patients had evidence of lymphoma, sarcoidosis, essential mixed cryoglobulinemia, or infection by hepatitis B virus, hepatitis C virus, or human immunodeficiency virus type 1. Subcutaneous abdominal white adipose tissue was obtained, after informed consent was received, from patients

undergoing abdominal surgery at the Department of Surgery of Laiko Hospital (Athens, Greece). Immunohistochemical analysis. The in situ expression of adiponectin in paraffin-embedded minor salivary gland biopsy specimens was analyzed by standard technique, using the EnVision system (Dako, Glostrup, Denmark) and a specific anti-human adiponectin monoclonal antibody (Alexis, Lausen, Switzerland). Antigen retrieval was performed by microwaving in 10 mM citrate buffer (pH 6.0). Normal nonimmune fetal bovine serum and 0.5% H2O2 in methanol were used to block nonspecific antibody binding and endogenous peroxidase activity, respectively. Negative control stainings were performed by replacing primary antibodies with irrelevant isotype-matched antibodies. Staining was developed using diaminobenzidine tetrahydrochloride. All sections were counterstained with hematoxylin. Non-neoplastic SGEC lines. Primary long-term nonneoplastic SGEC lines were established by the explant outgrowth technique from minor salivary gland biopsy samples, which were obtained from some of the patients included in the above-described primary SS and control groups (4 women and 1 man in each), as previously described (11). The epithelial origin of the cultured SGEC lines was routinely verified by morphology, as well as by the uniform and consistent expression of epithelium-specific markers and the absence of markers indicative of lymphoid/monocytoid cells. The SS group included 5 patients with primary SS (4 women and 1 man). The control group included 4 women and 1 man. Reverse transcription–polymerase chain reaction (PCR). Total RNA and complementary DNA (cDNA) were prepared as previously described (11). The previously described primers (MWG Biotech, Ebersberg, Germany) and PCR conditions were applied for the amplification of adiponectin and of AdipoR1 and AdipoR2 (2). The housekeeping gene hypoxanthine guanine phosphoribosyltransferase was amplified to assess cDNA quality. The PCR products were run on 1% agarose gels containing ethidium bromide and were visualized on a Vilber Lourmat ultraviolet photo system (Marne La Valle´e, France). The amplified PCR products were isolated using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and were identified by automated sequencing (MWG Biotech). Cell culture supernatants. Three-day culture supernatants were collected from confluent cultures of the abovedescribed SGEC lines (using the same number of cells per SGEC line), cultivated in serum-free KBM medium, and immediately hypercentrifuged and filtered through a 0.2-mm filter (Millipore, Bedford, MA) to remove cellular debris. Subsequently, supernatants were subjected to 7-fold concentration by ultrafiltration using a YM-10 membrane (Amicon, Danvers, MA) and were stored at ⫺20°C until used. To ensure that the detected adiponectin did not originate from the epithelial culture medium, fresh complete KBM medium was routinely subjected to the above-described concentration procedure and analyzed for adiponectin expression. Immunoblotting. The adiponectin expressed or secreted by SGECs was analyzed in total cytoplasmic SGEC extracts (100 ␮g/lane) or concentrated SGEC culture supernatants (an equal quantity, derived from a similar number of cells), respectively, by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting, as previously

EXPRESSION OF ADIPONECTIN BY SGECs

Figure 1. Representative examples of adiponectin expression by ductal epithelial cells in minor salivary gland biopsy specimens obtained from A, a patient with Sjo ¨gren’s syndrome and B, a control subject.

described (11). White adipose tissue was used as a positive control for adiponectin detection. A polyclonal rabbit antibody (1:5,000 dilution; Chemicon, Temecula, CA) was applied for the detection of adiponectin by immunoblotting. Normalization of the loaded proteins was evaluated by reprobing the membranes for the housekeeping protein actin, using a specific monoclonal antibody (Sigma, St. Louis, MO).

2297

mRNA, as identified by sequence analysis, thus confirming the expression of adiponectin mRNA by SGECs. Subsequently, the expression of adiponectin protein molecules was studied in all of the above-mentioned SGEC lines, by immunoblotting analysis. An ⬃30-kd protein band was detected in all of the SGEC lines obtained from patients with SS, but not in controls (Figure 3A). The secretion of adiponectin in SGEC culture supernatants was examined. Adiponectin was also detected in SGEC culture supernatants derived from both patients and controls (Figure 3B). A significantly higher amount of the protein (⬃12-fold higher, as estimated by ImageQuant software version 5.2 [Molecular Dynamics, Sunnyvale, CA]) was detected in the supernatants of SS SGECs compared with controls. Expression of adiponectin receptors in longterm–cultured non-neoplastic SGEC lines. To investigate whether SGECs are able to respond to adiponectin signaling, the expression of its receptors, AdipoR1 and AdipoR2, was examined. All SGEC lines tested were found to express AdipoR1 and AdipoR2 mRNA molecules (Figure 2). DISCUSSION

RESULTS

Adiponectin has been considered to be an adipose-specific hormone that is synthesized and se-

Expression of adiponectin in minor salivary gland biopsy specimens. Immunohistochemical analysis of adiponectin expression in minor salivary gland biopsy specimens revealed strong expression by the resident adipocytes. Intriguingly, definite positive staining was also detected in ductal epithelial cells (Figure 1). No differences were observed between patients with SS and controls. In addition, there were no differences between the 2 sexes or between premenopausal women and postmenopausal women. Finally, we did not observe any correlation between the degree of adipocyte infiltration and adiponectin expression by either adipocytes or epithelial cells (data not shown). Expression of adiponectin in long-term–cultured non-neoplastic SGEC lines. To examine whether the in situ–detected adiponectin staining of ductal epithelial cells was attributable to adsorption, the expression of adiponectin mRNA was investigated in long-term– cultured non-neoplastic SGEC lines obtained from patients with SS (n ⫽ 5) and controls (n ⫽ 5). An adiponectin–PCR product of the expected molecular weight (228 bp) was detected in all of the SGEC lines studied, as well as in positive control adipose tissue (Figure 2). This product was homologous to adiponectin

Figure 2. Representative examples of reverse transcription– polymerase chain reaction analysis showing mRNA expression of adiponectin (228-bp product), adiponectin receptor 2 (AdipoR2; 214-bp product), AdipoR1 (148-bp product), and the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT; 230-bp product). M ⫽ molecular weight marker; Sc ⫽ human subcutaneous adipose tissue (positive control); SS ⫽ salivary gland epithelial cells (SGECs) derived from 2 patients with Sjo ¨gren’s syndrome; Ct ⫽ SGECs obtained from 2 control patients; H2O ⫽ no cDNA template (negative control).

2298

Figure 3. Immunoblot analysis of adiponectin (Adp) protein expression by salivary gland epithelial cells (SGECs), showing detection of adiponectin in total cytoplasmic SGEC extracts (ce) (A) and in 7-fold concentrated SGEC culture supernatants (spn) (B), using an antiadiponectin polyclonal or an isotype-control antibody. Lane 9 in A and lane 4 in B are negative controls. Whole cell lysates from human subcutaneous adipose tissue were used as positive control (lane 1 in A and lane 1 in B). A, Upper panel, Representative examples of adiponectin detection in SGEC cytoplasmic extracts derived from 4 patients with primary Sjo ¨gren’s syndrome (SS) (lanes 2–5) or 3 control subjects (lanes 6–8). Lower panel, ␤-actin blotting on the same membrane. B, Three-day culture supernatant obtained from SGEC lines derived from a control subject (lane 2) or a patient with primary SS (lane 3). Seven-fold concentrated fresh culture medium was used as negative control (lane 4).

creted exclusively by adipocytes. However, recent evidence indicated that adiponectin can be produced by cells other than adipocytes, such as bone-forming cells (2), fetal tissue (3), myocytes, and cardiomyocytes (4). Our findings further support the evidence that nonadipose cells can be a source of adiponectin. This study is the first to demonstrate that adiponectin is constitutively expressed and secreted by epithelia and, more specifically, by non-neoplastic SGECs. In patients with SS, minor SGECs are the target of the inflammatory processes. Moreover, SGECs are suitably equipped for the recruitment and activation of immune cells and possibly are key regulators of the SS autoimmune responses. As illustrated by the increased expression of various immunoregulatory and apoptosisrelated proteins, SGECs from patients with SS are aberrantly activated. This activation is sustained during long-term culture, likely indicating the operation of intrinsic activation mechanisms in these cells (8). In this context, the increased constitutive secretion of adiponectin by SGECs from patients with SS compared with control SGECs lends further support to the notion that epithelial cells from patients with SS are intrinsically activated. At present, the mechanisms underlying this activation remain unclear and possibly involve the action

KATSIOUGIANNIS ET AL

of soluble autocrine mediators (e.g., cytokines) and activation of strict intracellular pathways. We have previously provided evidence of the association between tissue fibrosis, adipocyte infiltration, and the number of mast cells in minor salivary gland lesions of patients with SS (9). It has been previously reported that in Crohn’s disease, the significantly increased production of adiponectin by adipocytes in hypertrophied mesenteric adipose tissue is likely involved in the regulation of intestinal inflammation (12). The importance of evaluating the interactions between adipocytes and lymphocytes in the context of immunity is also underlined by the observation that in mice, protective immunization against Helicobacter felis is related to up-regulation of adipocytokine genes as well as to the presence of, mostly, T lymphocytes in white adipose tissue surrounding the stomach (13). Consistent with these data, de novo adipogenesis within the minor salivary glands, with parallel adiponectin expression by adipocytes and SGECs, as well as fibrosis development, may be of central importance in terms of progress of the healing process in primary SS lesions. This process is mediated by several cytokines, possibly including adiponectin expressed locally by ductal epithelial cells and by adipocytes in the SS lesion. Recently, increasing attention has focused on the dual role of adiponectin in different types of immune responses. Adiponectin has been shown to induce the expression of antiinflammatory cytokines such as interleukin-10 (IL-10) and IL-1 receptor antagonist in myeloid cells (14) and to activate the release of proinflammatory cytokines in human placenta (15), suggesting that this hormone represents an important regulator of immunologic processes. However, there is no evidence of a direct action of adiponectin on T and B lymphocytes, which constitute the majority of the infiltrating cells in minor salivary gland lesions in patients with primary SS; we are currently investigating this property. The observed transcription of both adiponectin receptors in human SGECs indicates that this hormone may act in an autocrine manner in these cells. Although additional work is necessary to elucidate the pathophysiologic relevance of this finding, this may reveal a mechanism allowing responsiveness to high adiponectin production by primary SS SGECs. Beyond its function as an immunoregulatory adipocytokine, adiponectin is primarily known to participate in cell metabolism, stimulating glucose utilization through AMP-activated protein kinase (AMPK) (1,4). In the primary SS salivary gland lesion, autoimmune

EXPRESSION OF ADIPONECTIN BY SGECs

attack and stress may create a high energy demand in SGECs, triggering activation of AMPK to fulfill the cell energy needs. In this case, increased constitutive adiponectin expression would be a pivotal regulator of SGEC metabolism in patients with primary SS, helping them to avoid low energy–induced programmed cell death. In conclusion, adiponectin is a hormone with many different functions. Apart from its metabolic role as an insulin sensitizer, it also seems to be a fundamental hormone that is involved in the immune functions of several biologic systems, with different properties each time. Considering this, the adiponectin produced by SGECs might be implicated in the regulation of local immune responses. The significance of the observed constitutive expression and secretion of adiponectin by SGEC needs to be elucidated. ACKNOWLEDGMENTS We thank Assistant Professor A. G. Tzioufas, MD, and Dr. J. Routsias for initiating one of us (SK) in the mystery of the immunology laboratory, and Professor H. M. Moutsopoulos for his continuing inspiration and guidance.

REFERENCES 1. Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev 2005;26:439–51. 2. Berner HS, Lyngstadaas SP, Spahr A, Monjo M, Thommesen L, Drevon CA, et al. Adiponectin and its receptors are expressed in bone-forming cells. Bone 2004;35:842–9. 3. Corbetta S, Bulfamante G, Cortelazzi D, Barresi V, Cetin I, Mantovani G, et al. Adiponectin expression in human fetal tissues during mid- and late gestation. J Clin Endocrinol Metab 2005;90: 2397–402. 4. Pineiro R, Iglesias MJ, Gallego R, Raghay K, Eiras S, Rubio J, et al. Adiponectin is synthesized and secreted by human and murine cardiomyocytes. FEBS Lett 2005;579:5163–9. 5. Wulster-Radcliffe MC, Ajuwon KM, Wang J, Christian JA, Spur-

2299

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

lock ME. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun 2004;316:924–9. Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2000; 96:1723–32. Staiger K, Stefan N, Staiger H, Brendel MD, Brandhorst D, Bretzel RG, et al. Adiponectin is functionally active in human islets but does not affect insulin secretory function or beta-cell lipoapoptosis. J Clin Endocrinol Metab 2005;90:6707–13. Manoussakis MN, Moutsopoulos HM. Sjogren’s syndrome: autoimmune epithelitis. Baillieres Best Pract Res Clin Rheumatol 2000;14:73–95. Skopouli FN, Li L, Boumba D, Stefanaki S, Hanel K, Moutsopoulos HM, et al. Association of mast cells with fibrosis and fatty infiltration in the minor salivary glands of patients with Sjogren’s syndrome. Clin Exp Rheumatol 1998;16:63–5. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al. Classification criteria for Sjogren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002;61:554–8. Kapsogeorgou EK, Moutsopoulos HM, Manoussakis MN. Functional expression of a costimulatory B7.2 (CD86) protein on human salivary gland epithelial cells that interacts with the CD28 receptor, but has reduced binding to CTLA4. J Immunol 2001; 166:3107–13. Yamamoto K, Kiyohara T, Murayama Y, Kihara S, OkamotoY, Funahashi T, et al. Production of adiponectin, an antiinflammatory protein, in mesenteric adipose tissue in Crohn’s disease. Gut 2005;54:789–96. Mueller A, O’Rourke J, Chu P, Kim CC, Sutton P, Lee A, et al. Protective immunity against Helicobacter is characterized by a unique transcriptional signature. Proc Natl Acad Sci U S A 2003;100:12289–94. Wolf AM, Wolf D, Rumpold H, Enrich B, Tilg H. Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes. Biochem Biophys Res Commun 2004;323: 630–5. Lappas M, Permezel M, Rice GE. Leptin and adiponectin stimulate the release of proinflammatory cytokines and prostaglandins from human placenta and maternal adipose tissue via nuclear factor-␬B, peroxisomal proliferator-activated receptor-␥ and extracellularly regulated kinase 1/2. Endocrinology 2005;146: 3334–42.