Enhanced degradation of proteins of the basal lamina and stroma by matrix metalloproteinases from the salivary glands of Sjgren's syndrome patientsCorrelation with reduced structural integrity of acini and ducts.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 9, September 2003, pp 2573–2584 DOI 10.1002/art.11178 © 2003, American College of Rheumatology Enhanced Degradation of Proteins of the Basal Lamina and Stroma by Matrix Metalloproteinases From the Salivary Glands of Sjögren’s Syndrome Patients Correlation With Reduced Structural Integrity of Acini and Ducts Eduardo Goicovich, Claudio Molina, Paola Pérez, Sergio Aguilera, Juan Fernández, Nancy Olea, Cecilia Alliende, Cecilia Leyton, Rafael Romo, Lisette Leyton, and Marı́a-Julieta González the acinar and ductal basal lamina revealed abnormalities ranging from disorganization to disappearance of this ECM structure. These changes were paralleled by an important loss of microvilli on the apical surface, as well as decreased unstimulated salivary flow. Interestingly, the results were similar in LSGs from all SS patients, regardless of the proximity of infiltrating mononuclear cell foci. Conclusion. Our observation that the proteolytic action of MMPs toward ECM macromolecules is increased in SS patients provides a rationale for understanding the dramatic changes in the structural organization observed in the basal lamina and apical surface of acini in these patients. The results provide new evidence that acinar and ductal cells from the LSGs of SS patients display a molecular potential, with increased capacity to markedly disorganize their ECM environment and, thus, damage their architecture and functionality. Objective. To determine the effect of matrix metalloproteinase (MMP) activity from the labial salivary glands (LSGs) of Sjögren’s syndrome (SS) patients on proteins of the extracellular matrix (ECM) that form the basal lamina and stroma, and to compare this effect with the structural integrity of acini and ducts as well as the functionality of the LSGs. Methods. Gelatinase activity was determined by zymography. The digestion pattern of extracellular matrix (ECM) macromolecules was detected by gel electrophoresis and quantified by densitometry. The structural integrity of acini and ducts was evaluated by light and electron microscopy. Secretory function was evaluated by measuring unstimulated salivary flow and by scintigraphy. Results. LSG extracts showed increased levels of proteolytic activity toward purified proteins of the basal lamina (laminin and type IV collagen) and stroma (types I and III collagen and fibronectin). Enhanced degradation was most evident for fibronectin, laminin, and type IV collagen. Analysis of the ultrastructure of Important changes in acinar and ductal morphology and function, together with pronounced extracellular matrix (ECM) remodeling, are detectable in the labial salivary glands (LSGs) of Sjögren’s syndrome (SS) patients (1–3). The classic symptoms of this disease, xerostomia and keratoconjunctivitis sicca, have been related to the presence of autoantibodies and cytokines and to progressive denervation of the LSGs (4–7). Physiologic remodeling of ECM components depends on a balanced expression of proteolytic enzymes and their inhibitors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), respectively (8,9). Some pathologic processes are associated with excessive or insufficient ECM degra- Dr. González’ work was supported by a grant from the Fondecyt-Chile (grant 1020755). Dr. Pérez’ work was supported by postdoctoral fellowships from Conicyt, Mecesup-Postgraduate University of Chile (grant 99-03) and from the Laboratorio Tecno-FarmaChile. Eduardo Goicovich, Biochemist, Claudio Molina, DDS, MSc, Paola Pérez, Biochemist, Sergio Aguilera, MD, Juan Fernández, PhD, Nancy Olea, BSc, Cecilia Alliende, BSc, Cecilia Leyton, BSc, Rafael Romo, DDS, Lisette Leyton, PhD, Marı́a-Julieta González, MSc: University of Chile, Santiago, Chile. Address correspondence and reprint requests to Marı́aJulieta González, MSc, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Casilla 70061, Santiago 7, Chile. E-mail: email@example.com. Submitted for publication January 21, 2003; accepted in revised form May 1, 2003. 2573 2574 dation (10–12), thereby highlighting the importance of the ECM for the maintenance of epithelial architecture and function (13,14). The basal lamina plays an important role in this maintenance function, since it lies at the interface between epithelial cell plasma membranes and the connective tissue. The ECM is also responsible for transmitting signals to cells via cell surface receptors, which include integrins and proteoglycans (3,5,15–20). Loss of adhesive interactions between epithelial cells and their microenvironment modifies gene expression and may lead to cell death (1,21). We recently demonstrated that LSGs from control subjects and SS patients showed gelatinolytic activity for MMP-2 and MMP-9 (3). Activation studies revealed that both enzymes were predominantly present in their latent form. The highest MMP-9 activity was detected in patients with severe, active primary SS. No differences in MMP-2 activity were observed between patients and controls. MMPs, as detected by immunolocalization, were found only in acinar and ductal cells and were homogeneously distributed throughout the LSGs. The expression of MMP-2 and MMP-9 paralleled their gelatinolytic activity. MMP-3, which was detectable only by immunologic methods, was absent in control subjects, but was abundantly expressed in SS patients. Moreover, levels of MMP protein in acinar and ductal cells were unrelated to the proximity of infiltrating mononuclear cells (3). Other investigators have reported the production of MMPs by mononuclear cells (22). However, our results suggested that the increased gelatinolytic activity of the LSGs from SS patients was due to the expression of some MMPs in acinar and ductal cells (3), and was not a consequence of the increased presence of mononuclear cells. Such increased MMP activity is directly related to greater damage of parenchymal glands, but is most likely not associated with the presence of infiltrating cells (3). The expression of MMPs is induced by cytokines, such as interleukin-1␣, interleukin-6, tumor necrosis factor ␣, and interferon-␥ (6,23). Studies of LSGs from patients with primary SS showed that these cytokines are expressed by infiltrating mononuclear cells and by acinar and ductal cells (24,25). Thus, MMPs could be induced in acinar and ductal cells by autocrine and paracrine pathways. Considering the mechanisms of induction and activation described for these enzymes and the cell types in which they are expressed (3,26,27), we hypothesized that MMPs could be secreted locally and degrade the basal lamina proteins of acini and ducts, as well as the stromal microenvironment. GOICOVICH ET AL Gelatin and casein are substrates that are frequently used to test the enzymatic activity of tissue MMPs in vitro. However, proteolysis of these substrates is not sufficient evidence to predict that these proteolytic enzymes would also degrade proteins of the ECM in vivo (28,29). Thus, a specific objective of the present study was to analyze the proteolytic activity of MMPs from extracts of LSGs obtained from SS patients toward purified proteins of the basal lamina (laminin and type IV collagen) and stroma (type I collagen, type III collagen, and fibronectin) and to compare this activity with that exerted toward in vivo substrates. We also evaluated the structural integrity of acini and ductal basal lamina, as well as the secretory properties of the LSGs. Analysis by gel electrophoresis and scanning densitometry revealed that extracts of LSGs from SS patients clearly contained increased proteolytic activity toward the proteins of interest. Degradation was most pronounced for fibronectin, laminin, and type IV collagen. Consistent with the findings of our biochemical analysis, the ultrastructure of acinar and ductal basal lamina showed abnormalities ranging from disorganization to disappearance of this ECM structure. Such changes were paralleled by a loss of microvilli on the apical surface, which suggests that inappropriate cytoskeletal reorganization could disfavor exocytosis of secretory granules. These results demonstrate that uncontrolled ECM remodeling of both the basal lamina and stroma due to excessive MMP activity may disrupt the architecture of parenchymal glands and thereby promote a loss of function. PATIENTS AND METHODS SS patients and control subjects. For this study, 15 patients with primary SS were selected and diagnosed according to the American–European Consensus Group criteria (30). Only patients with all the following signs and symptoms were included: keratoconjunctivitis sicca, xerostomia, low unstimulated whole salivary flow (ⱕ1.5 ml/15 minutes), abnormal scintigraphy curve (median or flat), presence of both anti-Ro and anti-La autoantibodies, and positive findings on LSG biopsy. Group A patients (n ⫽ 9) had an age range of 28–52 years (mean 42 years). Their focus scores ranged from 1 to 2, with 80–90% of remnant parenchyma present, and without fibrous or adipose tissue. LSGs from this group possessed MMP-9 gelatinolytic activity ranging from 1.4 to 2.9 (mean 2.4), expressed as follows: (兺 pixels area2) ⫻ 10–3. Group B patients (n ⫽ 6) had an age range of 51–70 years (mean 64 years). Their focus scores were ⬎2, with 20–60% of remnant parenchyma present, and with fibrous and/or adipose tissue. LSGs from this group displayed variable levels of MMP-9 gelatinolytic activity (0.6–2.4; mean 1.2). PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS The control group was composed of 5 subjects who had consulted their physicians because of oral and ocular dryness, but who did not fulfill the criteria for SS. Their age range (40–50 years; mean 48 years) was similar to that of the SS group. Findings of serology and scintigraphy were normal, and unstimulated whole salivary flow was normal. Biopsied LSGs were normal, with scarce and scattered distribution of mononuclear cells, well-preserved parenchyma, and without fibrous or adipose tissue. MMP-9 gelatinolytic activity in these individuals was in the range of 0.5–0.7 (mean 0.6). LSGs were obtained in the morning (after at least 2 hours of fasting), using the technique described by Daniels (31). All patients gave their informed consent before undergoing LSG biopsy. The study protocol was approved by the Ethics Committee of the Faculty of Medicine, University of Chile. Preparation of LSG extracts. LSGs were homogenized in extraction buffer for ECM components (0.5M Tris HCl, pH 7.5, 1% Triton X-100, 10 mM CaCl2, 200 mM NaCl) at a ratio of 1:5 (weight/volume) at 4°C with a glass/glass conical homogenizer. The homogenate was then subjected to three 5-minute freeze–thaw cycles and centrifuged at 13,000g for 30 minutes at 4°C. The detergent-soluble supernatants were recovered and stored at –70°C for further analysis. All steps of the procedure were done in the presence of a protease inhibitor cocktail (4 mM phenylmethylsulfonyl fluoride, 100 g/ml of leupeptin, 2 mM benzamidine in DMSO). Protein concentrations in the extracts were determined as described elsewhere (32). Evaluation of gelatinolytic activity of MMPs in LSG extracts by zymography. Detergent extracts of glands from the study subjects were analyzed by zymography to determine the enzymatic activity of MMPs. Zymography was performed using the protocol previously described by our group (3). In order to activate proMMPs, the extracts were preincubated with 1 mM APMA in the presence of protease inhibitors for 2 hours at 37°C. Unlike other proteases, all members of the MMP family contain Zn2⫹ at the catalytic site (33); in addition, they require Ca2⫹ for stability and activity (34). To demonstrate that the observed proteolytic activity was due to the presence of MMPs, 4 mM EDTA was added to the incubation medium of the zymographs for 30 minutes at 37°C. Preparation of ECM macromolecules. Types I, III, and IV collagen (Sigma, St. Louis, MO) were dissolved at 2 mg/ml in 0.1M acetic acid and then dialyzed against 2 liters of MMP test buffer (150 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.02% NaN3) for 48 hours at 4°C, with changes of dialysis buffer every 12 hours. Each collagen preparation was stored in aliquots of 1 mg/ml at –70°C. Fibronectin (500 g; Sigma) was reconstituted in MMP test buffer to a final concentration of 1 mg/ml. A commercial solution of laminin (Sigma) was diluted to 1 mg/ml. These solutions were also stored as aliquots at –70°C. Digestion of ECM macromolecules. Aliquots (5 g) of proteins from each LSG extract were activated by 1 mM APMA for 2 hours at 37°C. Protein aliquots were incubated separately with 20 g of type I, III, or IV collagen or with 10 g of laminin or fibronectin, in 30-l total volume with an MMP test buffer containing a protease inhibitor cocktail. The samples were incubated at 37°C for 0–48 hours, and samples were taken every 12 hours. At time points shorter than 12 hours, no obvious changes were detectable. Purified proteins 2575 incubated without LSG extract at time 0 and at 24 hours or with LSG extract in the presence of EDTA for 24 hours served as controls. The reaction was stopped by the addition of 4 mM EDTA at specific time points. Samples were subsequently analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (35), and gels were stained with Coomassie blue R250. Densitometric analysis. Gels were scanned using Corel Photo-Paint (Corel, Ottawa, Ontario, Canada) on a gray scale at a resolution of 1,200 interpolated dots per inch. The intensity of the bands was analyzed using UN-SCAN-IT gel software, version 4.1 for Windows (Silk Scientific, Orem, UT). The intensity was expressed as follows: (兺 pixels area2) ⫻ 10–3. Morphologic studies. LSGs were processed for morphologic evaluation by either light microscopy or transmission electron microscopy (TEM). For light microscopy, samples were fixed with alcoholic Bouin’s fixative and embedded in Paraplast. Samples for TEM were fixed in Karnovsky’s fixative, postfixed in OsO4, and embedded in Epon (36). The samples were coded to ensure unbiased collection of data. Statistical analysis. The results of degradation analyses were expressed as the mean ⫾ SEM intensity. Differences in intensities were analyzed using the Mann-Whitney U test. P values less than 0.05 were considered significant. RESULTS Digestion of basal lamina molecules. To confirm the presence of MMPs, all LSG extracts were previously checked by zymography, as shown in Figure 1. The zymogram shows representative samples from a control subject and an SS patient. The 92-kd and 84-kd bands corresponded to proMMP-9 and active MMP-9, respec- Figure 1. Zymographic analysis of the gelatinolytic activity of matrix metalloproteinases (MMPs) from extracts of labial salivary glands (LSGs) obtained from Sjögren’s syndrome (SS) patients and controls. Shown are representative samples from an SS patient (lanes 4–6) and a control subject (lanes 1–3). Each lane was loaded with 30 g of protein derived from the LSG extracts and then subjected to zymography. Lanes 1 and 4, Untreated LSG extracts; lanes 2 and 5, LSG extracts treated with 1 mM APMA; lanes 3 and 6, LSG extracts treated with 4 mM EDTA. Results are representative of 3 independent experiments. 2576 GOICOVICH ET AL Figure 2. Analysis of laminin degradation by extracts of labial salivary glands (LSGs) from Sjögren’s syndrome (SS) patients and controls. A, Purified laminin (10 g) was incubated with LSG extracts (5 g) that had been preactivated with 1 mM APMA. Digestion time 1 ⫽ 12 hours; digestion time 2 ⫽ 24 hours. Lane 1, Purified laminin incubated for 24 hours without gland extract, showing 2 bands (␤/␥ and ␣) with apparent Mr of 220,000 and 400,000, respectively. All incubations were done in the presence of a protease inhibitor cocktail (see Patients and Methods). Proteins were separated on a 7% sodium dodecyl sulfate– polyacrylamide gel electrophoresis gel and stained with Coomassie blue R250. Results are representative of those obtained for 5 SS patients and 5 controls, each of which was analyzed in duplicate to confirm the reproducibility of the results. Lane St, Prestained molecular weight standards: myosin (212 kd) and myelin basic protein/␤-galactosidase (158 kd). B, Densitometric analysis of the gel shown in A. LM ⫽ laminin; E ⫽ LSG extract; t1 ⫽ 12 hours; t2 ⫽ 24 hours. tively. The 72-kd and 64-kd bands corresponded to proMMP-2 and active MMP-2, respectively. When extracts were incubated with APMA, lower Mr MMPs were detected; however, gelatinolytic activity was higher in patients than in controls. When the extracts were incubated in presence of EDTA, these bands were no longer observed. Laminin and type IV collagen are the main components of the basal lamina. The ability of MMPs from the LSGs of SS patients to degrade these physiologic substrates was examined and compared with that of control subjects. MMPs were preactivated with APMA, and the reaction was stopped by EDTA. The degradation products obtained after different incubation times were analyzed by SDS-PAGE and densitometry. The LSG extract loaded in each lane (5 g in each case) was carefully titrated to avoid any overlap of electrophoretic pattern between the bands in the glandular extracts and those of the ECM substrates. Using laminin, 2 different bands of apparent Mr 200,000 and Mr 400,000, corresponding to the laminin ␤/␥ and ␣ subunits, respectively, were detected (Figure 2A). Upon treatment with gland extracts, a decrease in intensity of these bands was observed. This effect was more evident in the patients than in the controls (Figures 2A and B). Purified laminin incubated in the absence of extract for 24 hours is also shown in Figures 2A and B for comparison. Laminin degradation by PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS 2577 Table 1. Comparison between laminin and fibronectin degradation at 24 hours and salivary function in SS patients and controls* Laminin, mean ⫾ SEM Control (n ⫽ 5) SS patients Group A (n ⫽ 9) Group B (n ⫽ 6) Laminin ⫹ LSG extract, mean ⫾ SEM Fibronectin ⫹ LSG extract, mean ⫾ SEM USF, mean (range) ␣ ␤␥ ␣ ␤␥ Fibronectin, mean ⫾ SEM 0.4 ⫾ 0.08 0.6 ⫾ 0.09 0.29 ⫾ 0.05 0.45 ⫾ 0.06 4.9 ⫾ 0.6 4.6 ⫾ 0.3 3.5 (2.6–4.6) 0.5 ⫾ 0.09 0.4 ⫾ 0.06 0.7 ⫾ 0.10 0.6 ⫾ 0.08 0.09 ⫾ 0.04† 0.17 ⫾ 0.07† 0.12 ⫾ 0.01† 0.28 ⫾ 0.14† 4.7 ⫾ 0.4 5.0 ⫾ 0.5 2.0 ⫾ 0.4† 2.9 ⫾ 0.7† 1.05 (0.8–1.3) 0.7 (0.5–1.1) Scintigraphy curve Normal Median Median (n ⫽ 3); flat (n ⫽ 3) * Degradation was evaluated as the decreased intensity of electrophoretic bands of laminin and fibronectin. Intensity was measured as (兺 pixels area2) ⫻ 10⫺3. Unstimulated salivary flow (USF) is expressed in milliliters per 15 minutes. Sjögren’s syndrome (SS) patient groups were classified as described in Patients and Methods. † P ⬍ 0.05. MMPs was not significantly enhanced by increasing the incubation time beyond 24 hours (Figure 2A, lanes 4 and 7, and Table 1). In patients (Figure 3B), protein bands characteristic of type IV collagen, with apparent Mr of 110,500 and 91,000 (lane 8) showed increased or decreased intensity, respectively (compare lanes 9 and 10 in Figure 3B with those in Figure 3A [controls]). Degradation products of type IV collagen with apparent Mr of 79,500 and 52,300 were observed. The former band was of comparable intensity in both patients and controls, whereas the latter band was detectable only in the patients (Figures 3A and B, lanes 9 and 10). Slight differences in type IV collagen digestion were apparent, depending on the incubation time (Figure 3C). Digestion of stroma molecules. Types I collagen, type III collagen, and fibronectin are important components of the gland stroma. We compared the MMP activity in extracts of LSGs from patients and control subjects with the use of these substrates. For types I and III collagen, a group of proteolytic fragments with apparent Mr ranging from 100,000 to 70,000 were detectable both in the control subjects (Figures 3A and B, lanes 3 and 4) and in the SS patients (Figures 3A and B, lanes 6 and 7). However, the intensity of these bands was higher in the SS patients. A second group of fragments (Mr range 60,000–45,000) was detected in the patients only when type I collagen was used as a substrate. The intensity of these bands was dependent on the incubation time. A third fragment that was observed when type III collagen was used as a substrate (Mr 54,000) was detected only in the SS patients (Figure 3B, lanes 6 and 7, bottom arrowhead). Two bands with apparent Mr of 212,000 and 142,000 showed decreased intensity (Figure 3B, lane 7, upper arrowheads) compared with the intensity of purified type III collagen (Figure 3B, lane 5). In samples from control subjects, fibronectin was the only protein that did not undergo important changes when exposed to gland extracts for different incubation times (Figure 4A, lanes 3 and 4). In the SS patients, however, the susceptibility of fibronectin to MMPmediated degradation was dependent on the incubation time (Figure 4A, lanes 6 and 7). An increase in the electrophoretic mobility of fibronectin was observed upon incubation with gland extracts from either group of subjects (Figure 4B). Analysis by gel electrophoresis and scanning densitometry revealed that extracts of LSGs from SS patients clearly contained increased proteolytic activity of MMPs toward the basal lamina and stromal substrates studied. Proteolytic activities were higher and less variable in group A patients than in group B patients (Table 1). Structural changes in acini and ducts. This study focused on alterations found in acini and ducts, changes that have previously been related to mononuclear cell infiltrates (31). In a histologic analysis of LSGs from control subjects, acinar and ductal cells displayed typical basal–apical polarity, whereby nuclei were basally located and secretion granules were visible at the apical pole. While mucous cells normally form a very tight acinar lumen (Figure 5A), a variety of modifications were observed in LSGs obtained from SS patients. These included dilated acinus lumen, loss of nuclear polarity (Figure 5B), detachment of acinar cells from the basal lamina (Figure 5C), and cellular debris in the lumen duct (Figure 5D). Morphologic changes observed both in the basal lamina and the apical pole of acinar cells were similar in both groups of SS patients, although the small amount of gland tissue present in patients of group B made the evaluation of these changes difficult in regions distant from mononuclear cell foci (data not shown). The acinar 2578 GOICOVICH ET AL Figure 3. Analysis of the degradation of types I, III, and IV collagen by extracts of labial salivary glands (LSGs) from A, a control subject and B, a patient with Sjögren’s syndrome (SS). Purified collagen (20 g) was incubated with LSG extracts (5 g) that had been preactivated with 1 mM APMA. Digestion time 1 ⫽ 12 hours; digestion time 2 ⫽ 24 hours. Proteins were separated on a 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and stained with Coomassie blue R250. Lane 1, LSG extract incubated for 24 hours. Lane St, Prestained molecular weight standards. Arrowheads at lane 7 indicate bands of apparent Mr of 212,000, 142,000, and 54,000. Braces at lanes 4 and 7 indicate groups of polypeptide fragments. Electrophoretic patterns are representative of all patients and controls. C, Densitometric analysis of the bands that showed intensity differences or new fragments in A and B, for both the control subject (open bars) and the SS patient (crosshatched bars). Values are the mean and SEM relative intensity. PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS 2579 Figure 4. Analysis of fibronectin degradation by extracts of labial salivary glands (LSGs) from Sjögren’s syndrome (SS) patients and controls. A, Purified fibronectin (10 g) was incubated with LSG extracts (5 g) that had been preactivated with 1 mM APMA. Digestion time 1 ⫽ 12 hours; digestion time 2 ⫽ 24 hours. Lane 1, Purified fibronectin without gland extract incubated for 24 hours, showing a band with an apparent Mr of 400,000. All incubations were done in the presence of a protease inhibitor cocktail (see Patients and Methods). Proteins were separated on a 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and stained with Coomassie blue R250. Lane St, Prestained molecular weight standards. Electrophoretic patterns are representative of all SS patients and controls. B, Densitometric analysis of the gel shown in A. FN ⫽ fibronectin; E ⫽ LSG extract; t1 ⫽ 12 hours; t2 ⫽ 24 hours. and ductal cell images shown in this article were captured from gland regions distant from mononuclear cell foci. Ultrastructural changes in basal lamina acini and ducts. In control subjects, the acini and ducts of the basal lamina showed normal structural features (Figure 6A). However, in the SS patient, major modifications were observed, such as disorganization of the ultrastructural components of the basal lamina of acini and ducts and a massive infiltration of bundles of stromal collagen fibers (Figure 6B). The basal lamina was often found to be absent, and in this case, the collagen fibers were in direct contact with the basolateral plasma membrane (Figure 6D). Other changes observed in acinar cells included swollen cisternae of both the endoplasmic reticulum and mitochondria, nuclear vesiculation, and increased endosomal vesicles (Figures 6C and D). Ultrastructural changes in the apical region of acini. In control subjects, acini had a narrow lumen, with structural characteristics of normal gland epithelia, such as microvilli and caveolae. Secretion granules with low electron density and a well-defined structure were observed in the apical cytoplasm of acinar cells from the controls (Figure 7A). In SS patients, however, the acini showed a strongly dilated lumen, loss of microvilli, and loss of normal structure of the remaining microvilli (Figures 7B and C). In the lumen, abundant cellular debris and, unexpectedly, bundles of stromal collagen fibers were found (Figure 7D). The electron density of secretion granules and other cytoplasmic components 2580 GOICOVICH ET AL Figure 5. Morphologic changes in acini and ducts in labial salivary glands (LSGs) from patients with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject, showing normal architecture of acini. B, Acinus in an LSG section from an SS patient, showing 2 acinar cells that have lost nuclear polarity (arrow) and have a dilated lumen. C, Acinus in an LSG section from an SS patient, showing total detachment of acinar cells. Outlined area marks the limit where the basal lamina should be seen; however, this has been replaced by stromal fibers. D, Duct in an LSG section from an SS patient, showing cellular debris in the lumen (arrow). Images of acini and ducts represent regions distant from mononuclear cell foci. (Original magnification ⫻ 390 in A; ⫻ 900 in B, C, and D.) was higher in SS patients than in control subjects. Moreover, these granules were fused within the cells. Structures with the appearance of secretion granules were observed in the acinar lumen (Figures 7B and C). Consistent with the findings of our biochemical analysis, the ultrastructure of acinar and ductal basal lamina showed abnormalities ranging from disorganization to disappearance of this ECM structure. Such changes were paralleled by a loss of microvilli on the apical surface, suggesting that inappropriate cytoskeletal reorganization may impair exocytosis of secretory granules. Substrate degradation and secretory functionality. Table 1 shows the unstimulated salivary flow and scintigraphy data. The mean unstimulated salivary flow was 1.05 ml/15 minutes (range 0.8–1.3) in group A patients and 0.7 ml/15 minutes (range 0.5–1.1) in group B patients. Both these values are lower than the minimum accepted value for classification as SS (30). Scintigraphy curves were all in the median category for group A patients, while in group B patients, the curves were flat or median. Both parameters were normal in control subjects. Thus, low unstimulated salivary flow and abnormal scintigraphy curves correlated with higher substrate degradation as well as loss of ultrastructural integrity and functionality of the LSG. However, low proteolytic activity was observed in 3 of 6 group B patients, with low levels of residual gland tissue and high levels of mononuclear cells and fibrous tissue (Table 1). DISCUSSION In this study, we present the first evidence that MMP activity in LSGs from SS patients can degrade specific components of both the basal lamina and stroma with different intensities, laminin ⬎ fibronectin ⬎ type IV collagen ⬎ type III collagen ⬎ type I collagen (Figures 1–4). Moreover, we observed dramatic changes in the architecture of these structures, which suggests that they might indeed be tissue-specific targets of these enzymes (Figures 5–7). This proteolytic activity was also correlated with the secretory function of the glands PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS 2581 Figure 6. Ultrastructural changes in the basal domain of acini in labial salivary glands (LSGs) from patients with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject, showing a normal appearance of the basal lamina (arrowheads). Note also the normal-appearing mitochondria (m). B, LSG section from an SS patient, showing a thin, but disorganized, basal lamina (arrowheads), with a swollen mitochondrion in the cytoplasm. C, LSG section from an SS patient, showing the relationship between acinar and myoepithelial cells (myo). There is a disorganized basal lamina, with a cotton-like appearance (arrowhead). Swollen mitochondria and dilated rough endoplasmic reticulum (rer) are visible in the acinar cell. D, LSG section from an SS patient, showing the absence of basal lamina. There are bundles of collagen fibers (col) in the intercellular space (arrow). Note the dilated rough endoplasmic reticulum and polymorphic nuclei (n) in the acinar cell. Dⴕ, Higher magnification view of boxed area in D, showing a myoepithelial cell process in tight contact with collagen fibers. Images of acini and ducts represent regions distant from mononuclear cell foci. Bars ⫽ 1 m. (Table 1). Group B patients had a higher content of inflammatory cells, lower amounts of parenchymal tissue, and lower levels of proteolytic activity, which were detected mainly in patients whose LSGs were enriched in fibrous/adipose tissue. The nature of this group explains the heterogeneity of results. However, despite these variations, findings in group B support our hypothesis that epithelial cells play a crucial role in the events that ultimately lead to gland destruction. Our previous findings provided evidence that expression of MMPs in acini and ducts was increased in SS patients (3), supporting the idea tested in the present study that MMPs may act locally on their basal lamina and thereby lead to the progressive gland destruction that is characteristic of SS. In this respect, it is important to note that the basal lamina architecture of other structures (e.g., blood vessels) did not show any apparent changes (data not shown). Lower molecular weight forms of MMPs were detected when extracts from patients and controls were treated with APMA. However, the gelatinolytic activity was more pronounced in patients (4-fold higher than in the controls) (Figure 1). High levels of TIMP-1 have been found in LSGs from all study subjects, patients as well as controls (Gonzalez J: unpublished observations), and as previously described (37), activation of some MMPs with trypsin or activation of MMP–TIMP-1 complexes with APMA or trypsin generates lower molecular 2582 GOICOVICH ET AL Figure 7. Ultrastructural changes in the apical domain of acini in labial salivary glands (LSGs) from patients with Sjögren’s syndrome (SS) as compared with controls. A, LSG section from a control subject, showing the normal appearance of the apical domain. Note the normal-appearing of microvilli (mi), secretion granules (sg), and a narrow lumen (L). B, LSG section from an SS patient, showing a very dilated lumen with a large amount of cellular debris. C, High magnification view of the apical pole of an acinar cell in an LSG section from an SS patient, showing few and abnormal microvilli. There is coalescence of secretion granules and abundant cellular debris in the lumen. D, LSG section from an SS patient, showing a lumen with bundles of collagen fibers (col; arrow). Note the differences in electron density and shape among secretion granules in B, C, and D, compared with those showed in A. Images of acini and ducts represent regions distant from mononuclear cell foci. Bars ⫽ 1 m. weight MMPs with decreased activity. This might explain the small effect observed with APMA in LSGs from control subjects (Figure 1, lane 2). However, if MMP–TIMP-1 complexes interact with other MMPs (such as MMP-3) before activation, the lower molecular weight MMP that is generated has a higher activity (38). In view of these observations, it is possible that the differences in APMA activation observed in samples from patients and controls (Figure 1, lanes 2 and 5) could be explained by the finding that MMP-3 is highly expressed in SS patients but not in controls (3). The disorganization observed in the basal lamina was accompanied by important changes in organelle morphology, particularly at the apical surface of acinar and ductal cells and in the lumen. Normally, adhesion molecules (e.g., integrins) connect components of the basal lamina with the inside of these cells by providing attachment sites for the cytoskeleton (3,18,39–42). This dynamic framework regulates cytoskeletal organization and, as a consequence, the location of organelles (e.g., nuclear polarity), cellular volume, structure, and organization of the apical microvilli (13,43,44). In SS patients, different degrees of detachment were observed between the basal lamina and either acini or ducts, which correlated with a loss of cytoskeletal organization. Thus, both a complete lack of cellular architecture and structural disorganization of microvilli at the apical plasma membrane were detected (Figures 5–7). In acinar cells, maintenance of the integrity of the exocytotic machinery is essential to ensure that secretory granules fuse with the apical plasma membrane and release their content to the lumen (45,46). The alter- PROTEIN DEGRADATION BY MMPs FROM LSGs OF SS PATIENTS ations observed in our studies concerning the morphology of the apical compartment, including loss of microvilli, fusion, and accumulation of secretion granules, suggest that acinar secretion in LSGs is perturbed in SS patients (Figures 7). The alterations in the exocytic machinery caused by these structural changes and the disruption of the cytoskeleton could account for the reduction in unstimulated salivary flow and the modifications in the scintigraphy curves (Table 1). Aquaporin 5, a channel protein that regulates the movement of water across the apical membrane of salivary gland acinar cells, has been found by some researchers to accumulate in the cytoplasm of cells from SS patients (47,48), although other investigators have not identified such localization (49). Thus, based on our findings, the accumulation of aquaporin 5 in these patients may be attributed to a deficiency in vesicle sorting from the trans Golgi network to the plasma membrane. An important point here is that both the fusion of secretory granules with the plasma membrane and the sorting of molecules from the trans Golgi network to the apical surface are processes that are highly dependent on the integrity of cytoskeletal elements (3,50). Furthermore, other studies have demonstrated the presence of various alterations caused by a loss of interaction between the basal lamina and epithelial cells, such as changes in the movement of cell electrolytes and disruption of tight junctions between neighboring cells (51,52). The presence of bundles of collagen fibers in the acinar lumen of SS patients (Figure 7D) is indicative of a loss of tight interactions between cells. The disruption of these structural and functional barriers could allow components in the stroma and lumen that are normally separated to interact. Increased electron density in the cytoplasm of acinar cells and loss of cellular volume have been used as an indicator of changes in cell osmolarity and apoptosis (53). Thus, the modifications observed in the LSGs from SS patients might be the consequence of cell apoptosis. MMPs degrade ECM molecules, and it has been demonstrated that the resulting fragments of fibronectin and laminin induce the expression of MMP (54). Therefore, it is possible that a positive feedback loop is established that promotes gland damage. Stromal collagen is also a good inducer of MMPs (54). Interestingly, severe gland damage was found only in some cases, indicating the possible existence of a compensatory mechanism for replacing dying cells. In this respect, higher levels of cell proliferation using appropriate markers have been found in acinar and ductal cells of SS patients (Alliende C, et al: unpublished observations). 2583 So far, xerostomia has been mainly explained by imbalances in cytokine levels (6), production of autoantibodies against the M3 muscarinic receptor (7), desensitization to secretagogues (55), lack of sorting of aquaporin 5 (47–49), and mononuclear cell infiltration (1,2). Our study is the first to indicate that increased MMP activity may lead to both the basal and apical changes detected in acinar and ductal cells of LSGs from SS patients. Such changes could explain xerostomia at a molecular level, since the lack of communication between these cells and their microenvironment may lead to disorganization of the cytoskeleton and, as a consequence, deregulation of normal vesicle trafficking. ACKNOWLEDGMENTS We would like to dedicate this article to the late Michael Humphreys-Beher, MD (Department of Oral Biology, University of Florida, Gainesville) for his outstanding contribution to the current understanding of autoimmune exocrinopathy with the use of murine models. We thank Drs. Andrew Quest and Remigio López (Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile) for their support, discussions, and critical review of the manuscript. REFERENCES 1. Talal N. Sjögren’s syndrome: historical overview and clinical spectrum of disease. 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